Carolina State University in the interest of further elucidating the role of IP3 in plant ...... Benjamins, R., Galván-Ampudia, C.S., Hooykaas, P.J., and Offringa, ...
A CELLULAR AND MOLECULAR CHARACTERIZATION OF EARLY EVENTS IN PLANT GRAVITROPISM
BY EMILE C. BARNES
A Thesis
Submitted to the Division of Natural Sciences New College of Florida in partial fulfillment of the requirements for the degree Bachelor of Arts Under the sponsorship of Dr. Amy Clore
Sarasota, Florida January, 2017
ACKNOWLEDGMENTS
First and foremost, this thesis is the result of a lifetime of support from my parents in every one of my endeavors, no matter how frivolous; your unconditional love has always been matched in an unwavering faith in my ability to accomplish anything. Your faith has been my greatest motivator in this process. – Of all the things I have gained from my education at New College, the most meaningful and serendipitous of them has been to work under Dr. Clore, whose academic interests are so largely the same as my own, in a field in which I did not realize my own interest until well after my enrollment. Your mentorship throughout the writing of this thesis and throughout the majority of my time at New College has simply been invaluable. I would also like to thank Dr. Ryba and Dr. Walstrom for reading this thesis, participating in my baccalaureate examination, and for their time in doing so. – I could not imagine my time at New College without the remarkable peers I have had the joy to call my friends in the past four and half years. Their camaraderie and commiseration have truly defined my experience here.
ii
TABLE OF CONTENTS Acknowledgments …........................................................................................................ ii List of figures and tables …................................................................................................ v Abstract …........................................................................................................................ vii Chapter 1: Introduction ….................................................................................................. 1 1.1 Applications of gravitropism research …......................................................... 4 1.1.1.
Agronomic applications of gravitropism research …..................... 4
1.1.2.
Aerospace applications of gravitropism research …...................... 7
1.2 Methods in gravitropism research …............................................................... 9 1.2.1.
Reorientation ….............................................................................. 9
1.2.2.
Microgravity via clinostat/random positioning machine (RPM) ... 9
1.2.3.
Microgravity via low Earth orbit – the International Space Station …. 13
1.2.4.
Less common methods in plant gravitropism .............................. 15
1.3 The working model of plant gravitropism …................................................ 19 1.3.1.
Statolith sedimentation is a key early event …............................. 20
1.3.2.
Mechanotransductive ion channels confer a chemical signal ….. 23
1.3.3.
Altered auxin transport transfers signal to the site of response ... 26
1.3.4.
Auxin-mediated differential growth reorients the plant …........... 28
1.3.5.
Other theories exist that may not be mutually exclusive …......... 30
1.4 Conclusion …................................................................................................ 32 Chapter 2: Early signaling events and statolith sedimentation ….................................... 33 2.1 The cytoskeleton as a mediator and sensor of sedimentation …................... 34
iii
2.2 The endomembrane system …....................................................................... 45 2.3 Calcium-based signaling and inositol triphosphate ….................................. 53 2.4 Reactive oxygen species …........................................................................... 63 2.5 Cytosolic and apoplastic pH …..................................................................... 67 2.6 Alternate means of graviperception ….......................................................... 69 2.7 The TOC complex: a model of interconnectedness in gravitropic signaling …..... 74 2.8 Conclusion …................................................................................................ 77 Chapter 3: Commitment to response: from sedimentation to presentation time …......... 78 3.1 MAP kinase activity ….................................................................................. 78 3.2 Auxin asymmetry and lateral transport …..................................................... 80 3.3 Calcium and pH …........................................................................................ 89 3.4 ROS revisited …............................................................................................ 92 3.5 Conclusion …................................................................................................ 94 Chapter 4: Response, growth and reorientation …........................................................... 97 4.1 Hormone-mediated growth …....................................................................... 97 4.2 Cell wall changes ….................................................................................... 102 4.3 Coordination with other tropisms …........................................................... 106 4.4 Conclusion ….............................................................................................. 112 Chapter 5: A bioinformatics-based assessment of research targets in plant gravitropism ….... 114 5.1 A search for the amyloplast “actin workbench” …...................................... 114 5.2 Non-targeted gene expression analysis …................................................... 119 5.3 Conclusion ….............................................................................................. 130
iv
Chapter 6: Future directions and conclusions …............................................................ 132 Appendix A: Computational methods of gene expression analysis …........................... 141 Appendix B: Gene ontology visualization methods ….................................................. 146 References ….................................................................................................................. 150
LIST OF FIGURES AND TABLES Figure 1: Basic location and organization of gravitropic organs in model organisms ...... 6 Figure 2: The evolution of the clinostat …....................................................................... 11 Figure 3: Locations of graviresponsive bending cells …................................................. 20 Figure 4: Real-time amyloplast sedimentation in an Arabidopsis columella cell …........ 22 Figure 5: Auxin transporters redistribute upon gravistimulation … …..................…..... 27 Figure 6: The actin cytoskeleton of Arabidopsis endodermis cells …............................. 36 Figure 7: The actin cytoskeleton of flax columella cells …............................................ 38 Figure 8: CHUP1 function in chloroplast-actin interactions …....................................... 45 Figure 9: Transvacuolar strands and amyloplast sedimentation ….................................. 48 Figure 10: A model depicting ER-amyloplast interactions … ….................................... 52 Figure 11: Calcium signature of gravistimulation …....................................................... 55 Figure 12: ROS production in maize pulvini following gravistimulation … ….........… 65 Figure 13: Possible mechanisms for a TOC complex-ARG1 interaction … ….............. 76 Figure 14: Interactions between auxin, calcium and pH … ….................................…... 90 Figure 15: Interaction diagram of several components of gravitropic signaling …......... 96 Figure 16: AUX/IAA- and ARF-mediated, auxin-dependent gene regulation ….......... 100
v
Figure 17: Auxin-mediated cell expansion …................................................................ 104 Figure 18: Comparison of phototropic and gravitropic signaling … …....................... 107 Figure 19: Gene regulation in gravitational and mechanical signaling …..................... 112 Table 1: Searching for a CHUP1 functional equivalent in Arabidopsis ….................... 117 Table 2: Plastid-localized or actin-binding gene products showing differential expression after 2 h gravistimulation …............................................................................... 118 Table 3: Named gene products showing differential expression between gravistimulated shoot flanks ….................................................................................................... 123 Table 4: Unnamed gene products showing differential expression between gravistimulated shoot flanks ….......................................................................... 126 Table 5: Named/known gene products showing differential expression in both gravistimulated shoot flanks ….......................................................................... 129 Table 6: Unnamed genes showing differential expression in both gravistimulated shoot flanks ….............................................................................................................. 130
vi
A CELLULAR AND MOLECULAR CHARACTERIZATION OF EARLY EVENTS IN PLANT GRAVITROPISM Emile C. Barnes New College of Florida, 2017
ABSTRACT
For life on Earth, there is no way to skirt the influence of gravity. The phenomenon of gravitropism – translation of the mechanical force of gravity to a chemical signal and the appropriate reorientation or growth response – is observed in some sense in every organism, but is poorly understood, especially in initial stages of sensing and signal codification. The molecular mechanisms of gravitropism remain relatively poorly understood in plants, whose sessile lifestyle requires a thorough and highly dynamic response to the gravitational force as a growth signal. In addition to addressing a fundamental question of biology, work in this area may find use in agriculture; lodging, a horizontal reorientation of a crop relative to gravity due to wind or other factors, has been suggested to be potentially remediable by enhancement of the gravitropic pathway. Finally, an intimate understanding of the way plants perceive and respond to gravity will be essential for designing an ideal life support system required of any long-term space habitation mission. The plant gravitropic response is generally divided temporally into three parts: sensing the force or change in force of gravity; transduction of this physical signal into a chemical one, and subsequent transmission from the site of sensing to the site of vii
response; and the response phase, wherein differential growth, largely mediated by the growth hormone auxin, reorients the plant if necessary. The latter phase is the most wellunderstood, particularly the involvement of the auxin pathway, as well as the auxin transport system that typifies the later part of the transduction phase. These elements of plant gravitropism are extensively reviewed elsewhere and are only briefly summarized in this work, which highlights recent developments in the fields of gravity sensing and gravity-related mechanotransduction in higher plants. In addition to literature review focused on these early events, bioinformatics techniques are also employed in a preliminary fashion to identify potential subjects for future research.
____________________________ Amy M. Clore, Ph.D. Division of Natural Sciences
viii
CHAPTER 1: INTRODUCTION One fish asks to another, “How's the water today?”, and the fish replies, “...What is water?” So ubiquitous, so total is the liquid in the life of the fish. Even more pervasive than water to fish is gravity to life on our planet. Every member of Earth's biodiversity, from the very incipience of life, has evolved in the consideration of that constant, unchanging tug towards the hearth of our celestial home. Life on Earth is so completely imbued with the force of gravity that it is easy to take for granted the question of how, physically and mechanically, gravity alters the form of life, from the subcellular to the organismal level. How is this intangible, invariable force perceived by a cell and transduced into a chemical signal? How is that signal translated into a meaningful and appropriate response? These questions are most easily addressed in animals, where they have received the most extensive study and are the best understood. The most widely-employed and well-conserved method of gravity sensing in animals involves so-called statoliths in invertebrates or otoliths in vertebrates. Statoliths and otoliths are mineralized, generally calcified, granules usually found in larger structures, the statocyst and inner ear, respectively (Wiederhold et al., 1997). They are suspended in a fluid and confined to a small compartment lined with hair cells, microscopic protrusions from the wall of the sensing organ which are mechanosensitive and activate their associated nerve when deformed (Kingma, 2006). This information is then interpreted at the central nervous system and muscle firing reorients the animal, if needed.
1
Because plants are sessile and can only “move” through their environment through directional growth, and because they lack a nervous system to relay information throughout the organism, plants would clearly not be able to interpret or respond to gravitational force using the aformentioned mechanism. Environmental perception and cellular and organismal growth must be more closely integrated, more thoroughly intertwined than what is seen in animal systems. This feat is accomplished through highly dynamic and incredibly complex cellular signaling networks, many aspects of which remain elusive or contentious, particularly in the context of gravitropism, or growth in response to gravity. In most plants, changes in orientation relative to the gravity vector are sensed when starch-containing statoliths change position within specialized gravity-sensing cells, or statocytes (reviewed in Sack, 1991; Blancaflor & Masson, 2003). The response manifests most visibly in the shoot's growth in the direction opposite the gravity vector, while roots grow in its direction, such that these organs display negative and positive gravitropism, respectively. Gravitropism in the proper orientation is also referred to in both organs as orthogravitropism. The gravitropic response is particularly evident when a plant oriented upright is placed on its side: within minutes or hours, depending on species and age of the plant, the shoot will begin to “turn” (via differential cell growth, as motility is impossible) away from the ground, while the roots grow toward it. First named and described at the beginning of the 19th century (Knight, 1806), gravitropism first received extensive study from Cielieski (1872) and Darwin (1880), who identified the root cap as essential for gravitropic response in the root, and determined that differential
2
growth is the source of the response, and that the responsive growth occurs in a length of the root (currently termed the elongation zone) just behind the root tip. The incorporation of research facilities into spaceflight vessels, such as the 1967-68 Biosatellite II mission, Skylab, and several space shuttle flights, as well as other efforts by NASA and foreign space agencies, ushered in a modern era of research interest in field of gravitational biology (Paul et al., 2013). The paradigm of plant gravitropism put forth in the 19th and early 20th centuries has been continually expanded upon since. Comprehensively, the objective of the present work is to provide an ultimately thorough characterization of the state of research in plant gravitropism, much of which is performed as relatively discrete studies of specific aspects of the phenomenon. In this chapter, the importance of this field in agriculture and aerospace industries is summarized, and explanations of the experimental methods most commonly used in gravitropism research are given briefly. Finally, a chronological sequence of the canonical, widely agreed-upon aspects of the gravitropic response is given. Subsequent chapters focus on recent experimental results which demonstrate the more contentious, confusing, and conflicting aspects of this response, primarily focusing on early events including sensing and transduction. Final chapters report the preliminary results of bioformatics-based analyses to evaluate questions raised by recent literature using preexisting publicly available datasets, and suggest future lines of inquiry to resolve these questions.
3
1.1.
Applications of gravitropism research 1.1.1
Agronomic applications of gravitropism research
Other than growth in response to light, water and nutrients—all of which, unlike gravity, are essential to plant life—growth in response to gravity may be the tropism that contributes the most significantly to the form of a plant. The plant's height, the architecture of its root system, and the orientation of its photosynthetic organs are all informed by gravitational signals (Digby & Firn, 1995; Hangarter, 1997; Blancaflor & Masson, 2003). All of these traits impact the yield and productivity of crop plants. In woody plants, the formation of reaction wood, which keeps the tree canopy oriented upwards, is guided by the force of gravity (Du & Yamamoto, 2007). In gymnosperms, the formation of a form of reaction wood called compression wood poses problems for the wood industry as it produces less desirable timber and limits the tree's ability to be pulped due to high lignin content (Li et al., 2013). Root gravitropism pathways appear to be the most frequent target of crop improvement efforts involving gravitropic research (de Dorlodot et al., 2007). By enhancing gravitropic curvature and creating a deeper, more vertically-oriented root system, researchers hope to optimize uptake of water and nutrients through the soil, reduce root space limitations to crop density, and increase resistance to crop lodging, or tipping towards the horizontal (Meister et al., 2014; Rogers & Benfey, 2015; Uga et al., 2015). Uga and colleagues have identified the so-named Deeper rooting 1 (Dro1) gene through quantitative trait locus (QTL) mapping, and found that rice plants expressing a functional allele of Dro1 were more drought tolerant and wind resistant than rice plants
4
bearing a mutant allele, and additionally accumulated less heavy metal in their tissues when grown in cadmium-inundated soil (Uga et al., 2007; Uga et al., 2015). The DRO1 protein is reported to bear “no similarity to known proteins”, but fluorescent constructs revealed it is localized to the plasma membrane; since the gene product is not predicted to encode any transmembrane domains, it is thought that DRO1 may associate with an unknown membrane protein(s) (Uga et al., 2015). Although further analysis of the DRO1 gene and encoded protein will be necessary to determine its exact activity, it is thought to be involved in the auxin-mediated gravitropic growth response and is negatively regulated by auxin (a plant hormone) in the process (Uga et al., 2015). While DRO1 was identified in rice, most research into root gravitropism is performed in the model organism Arabidopsis thaliana, or thale cress, a tiny member of the cabbage family (Figure 1), partially because of its ease of study and partially because root gravitropism appears more universally similar among organisms than shoot gravitropism. While root gravitropism affects the susceptibility of a plant to lodging, shoot gravitropism acts to rescue it. In the shoots, gravitropic response is the basis of stem reorientation after plants have been lodged by heavy wind or rain. Since many lodged plants can become non-harvestable even after a partial gravitropic response (due to inaccessibility to harvesting equipment or exposure to soil microbes), increasing the gravitropic competency in crop shoots could significantly reduce crop losses (Chen et al., 1999; Clore, 2013). In maize shoots, as in several other grasses, gravisensing occurs in the pulvini, intermittent regions along the stem apical to each node which generally only grow to reorient the direction of stem growth as needed (Dayanandan et al., 1977;
5
Figure 1: Basic location and organization of gravitropic organs in model organisms. (A-D) A graviresponding maize stem (A) senses and responds to gravity at the pulvini (B-D). B, a cross-section of a maize pulvinus stained for starch with iodine shows the distribution of starch in the vascular bundle sheath (B), surrounding the xylem (X), phloem (Phl) and collenchyma (C). P = parenchyma. C, longitudinal pulvinus section showing the scattered distribution of vascular bundles and starch staining in the bundle sheaths. D, zoom of C. (EG) Columella cells in the root cap sense gravity in root gravitropism. E, cartoon of model eudicot Arabidopsis thaliana. F, the organization of the root cap. LRC cells in green: lateral root cap; E, blue: epidermis; C, dark purple: cortex; e, light purple: endodermis; unmarked, red: endodermis/cortex progenitor cell; unmarked, yellow: quiescent center; unmarked, dark orange: columella stem cells (CSCs) (immature columella cells); pale orange with asterisks: columella cells (statocytes). Asterisks represent amyloplasts (statoliths) in tiers S1-S3. G, The subcellular organization of a columella cell. The nucleus (N) is located at the “top” of the cell, with a heavy concentration of ER bodies (ER) distally (at the cell “bottom”). Amyloplasts (AM) occupy the central volume, from which a large central vacuole is conspicuously absent. Maize image modified from Clore, (2013); pulvinus micrographs reproduced from Johannes et al. (2001); Arabidopsis cartoon found at sites.dartmouth.edu/guerinot-lab/research; root cap micrograph belongs to author; columella cell micrograph reproduced from Lietz et al. (2009). All original works can be found in References.
Kaufman et al., 1987) (Figure 1). 6
Pulvini retain this ability even after the rest of the stem is mature and too extensively lignified for such growth (Clore, 2013). Clarifying how gravity and gravitropism modulate pulvinus growth should prove useful in optimizing yield within the Poaceae family of crops, which includes the three most extensively produced food crops in the world: maize, rice and wheat (Food and Agriculture Organization of the United Nations, 2016).
1.1.2
Aerospace applications of gravitropism research
“The chief problem for a manned mission [to space] is not getting there but learning how to survive after arrival. Surviving and making a home away from Earth are problems of biology rather than engineering. Any affordable program of manned exploration must be centered in biology. . . To make human space travel cheap, we will need advanced biotechnology in addition to advanced propulsion systems.” – Astrophysicist Freeman Dawson (1997)
Perhaps more obvious are applications of plant gravitropism research to plant cultivation on the Moon, Mars, or on spacecraft inhabited permanently or long-term, where fractional or negligible gravity (henceforth referred to as microgravity) is inescapable. Any space exploration or habitation missions of long-term scope (several years) will require a bioregenerative life support system (BLSS): a minimally wasteful, maximally efficient and maximally self-contained system able to provide astronauts with a continual supply of food, clean water, and clean air, and able to biologically recycle as much waste as possible from the environment (Barta & Henninger, 1994; Davies et al., 2003). The utility of plant life in such an environment is difficult to underestimate. Plants
7
convert exhaled carbon dioxide waste into organic matter and supply atmospheric oxygen. The biomass they produce, if not edible, may be used to feed insect or small animal farms, to enrich the soil, for fiber, or in several other applications. Plants and microbes can recycle organic waste back into fresh produce, and are capable of soil bioremediation. Finally, plants produce considerable amounts of clean water: an acre of corn, for example, transpires 3,000-4,000 gallons of water per day (Leopold & Langbein, 1960). The employment of plant life is certainly among the simplest, easiest, and most cost-effective ways to cultivate a habitable space environment. However, this solution is imperfect: for example, additional water, carbon dioxide, nitrogen, and other nutrient sources in large quantities will be needed to grow the plants, and the energy needed to provide enough light for the plants may be difficult to provide. Furthermore, as microgravity experiments or experiments at lunar or Martian gravity levels often yield unpredictable results regarding germination rates, fresh and dry weight, fruit/flower timing, and so on, it has been argued that the reliability and success of plant growth in reduced gravity should be very confidently known for accurate planning (Salisbury, 1991; Barta & Henninger, 1994; Salisbury, 1999; Davies et al., 2003). Clearly, overcoming these difficulties will require the work of cell biologists, crop scientists, microbiologists, biotechnologists, engineers, and many others. Understanding how plants perceive and respond to gravity, in the short and long term, and how altered gravity affects plant growth and productivity, would be an invaluable step towards this goal.
8
1.2.
Methods in gravitropism research 1.2.1
Reorientation
The simplest and easiest way to study gravitropism is to use Earth's gravity, rather than to attempt to compensate for or effectively alter it, as most other ground-based methods do. A gravitropic response is triggered by simply reorienting a plant with respect to the gravity vector; usually, a plant is tipped 90º into a horizontal orientation, although greater angles are commonly used, as well (see, e.g., Darwin, 1880, Wendt & Sievers, 1989, Taylor et al., 1996, Yamamoto & Kiss, 2002, Uga et al., 2013, and Zou et al., 2016). This method can be used with plants of virtually any age so long as their size is small enough to be easily tipped. Researchers can then assess changes in growth patterns, gene expression, cell signaling activity, cell physiology and so on at the desired time point or series of time points (e.g., Sedbrook et al., 1999; Yamamoto & Kiss, 2002; Kimbrough et al., 2004; Monshausen et al., 2011; Zhang et al., 2011). While this technique is ideal for researching gravitropism at 1 g, more complex methods must be used to simulate fractional and microgravity.
1.2.2
Microgravity via clinostat/random positioning machine (RPM)
A “classic” clinostat, a device designed to rotate a subject along its (typically long) axis so as to negate the downward force of gravity, was named and first used nearly 150 years ago (von Sachs, 1879). Such a clinostat that rotates in only one direction is known as a simple, horizontal, two-dimensional (2-D), or uni-axial clinostat, or a rotating wall vessel (Herranz et al., 2013). Clinostats that rotate about two or more axes are
9
termed 3-dimensional (3-D) clinostats or random positioning machines (RPM) (van Loon, 2007). Figure 2 (page 11) shows antiquated and modern 2-D clinostats alongside a fast-rotating clinostat (discussed below) and 3-D clinostat. The general effect of the clinostat arises from the speed of rotation, which is fast enough that the presentation time (the minimum length of a stimulus required to trigger a response) of the plant is not met in any position but slow enough that centrifugal force generated is minimal. Since the force of gravity acts evenly in all directions, changing too quickly to be perceived by the plant, it has no net direction when averaged over time and thus simulates the condition of microgravity (Dedolph & Dipert, 1971; Moore & Cogoli, 1996). Because the gravitational force still acts on the plant to some extent, the condition generated by the clinostat is often referred to as gravity compensation (used in the sense of counterbalance), gravity nullification, or omnilateral gravitational stimulation (Brown et al., 1976; Hensel & Sievers, 1980; Sievers & Hejnowicz, 1992; Moore & Cogoli, 1996; Hoson et al., 1997; Herranz et al., 2013). While these terms are preferred by some for greater technical accuracy, the more general, widely-accepted and easily-understood term “microgravity” will be used in the present work. The ability of this state of (frequently) omnilateral gravistimulation to faithfully replicate a state of weightlessness has been under question for nearly as long as the clinostat has existed. In September 1967, NASA launched BioSatellite II, carrying, among samples for various other experiments, six pepper plants whose leaf angles were measured every 10 minutes and compared to clinostated plants on Earth. Epinasty, a downward furling of leaf margins and drooping of stems, which has been long observed
10
Figure 2: The evolution of the clinostat. Clockwise from top left, (1) A drawing of Knight's "water wheel": a simple 2-D clinostat. (2) A modern-day uniaxial clinostat, also called a rotating wall vessel (RWV). A sample is affixed to the center wheel and movement is applied typically by motor force. (3) A fast rotating 2-D clinostat. Sample chambers – 6 are pictured here – are very small to minimize centrifugal force. (4) A 3-D clinostat, or random positioning machine (RPM), being prepared for use. The sample chamber and the platform to which it is affixed are rotated about two axes, each in random directions. (1) reproduced from van Loon (2007), originally from Davy (1813); (2) from United Nations Office for Outer Space Affairs via http://www.unoosa.org/oosa/en/ourwork/psa/hsti/capacitybuilding/zgip_3cycle.html; (3) from The German Aerospace Center (DLR) via http://www.grimm-space-research.com/RPM/RPM.html; (4) from Airbus Defence and Space Netherlands (Dutch Space).
11
in clinostated plants, was also seen in the satellite subjects, though the latter was more severe to a 1% statistical significance (p < 0.01) (Brown, et al., 1974). Plants from the satellite also recovered more slowly than clinostated plants. This experiment suggests that clinostat rotation merely attenuates, rather than wholly eliminates, the influence of gravity. Another experiment was performed wherein plant samples were rotated by a clinostat mounted to a centrifuge providing a specified g force experimentally varied from 1 to 10 g. The researchers proposed that, if clinorotation successfully nullifies gravitational force without otherwise affecting plant physiology, a series of physiological measurements taken from plants at differing g force levels would show minimal variability. Contrarily, the data collected were, in five of seven measurements, a function of the g force to which the sample was exposed (p < 0.0001), with the strongest correlation coefficient being -0.546 (Brown et al., 1976). The fidelity of the clinostat to actual microgravity can be improved by using instead what is called a fast-rotating clinostat. The mounted sample is rotated much more quickly so that the cellular components have very little opportunity to move, and thus cause less mechanical damage and stress (Sievers & Hejnowicz, 1992; Herranz et al., 2013). However, this limits the usable size of the sample chamber significantly, as there is a very small workable radius from the rotational axis before centrifugal forces become non-negligible (Hoson et al., 1997; Herranz et al., 2013) (Figure 2). The mounting case against the uni-axial clinostat (reviewed in Sievers & Hejnowicz, 1992 and Hoson et al., 1997) led to the development in the late 1980s of the 3-D clinostat designed for plant research (van Loon, 2016); in the time since, the 3-D
12
clinostat or random positioning machine (RPM) (Figure 2) has rendered its predecessor virtually obsolete as the standard for Earth-based simulations of weightlessness. When two- and three-dimensional clinostated plants were compared to equivalent spaceflight and ground controls regarding several microscopic measurements of root columella cells (statocytes of the root), it was found that the RPM produced samples of a statistically insignificant difference compared to spaceflight samples, whereas the two-dimensional clinostat samples varied significantly from all other test groups (Kraft et al., 2000). Additionally, RPM samples were devoid of undesirable artifacts which are known to occur from the use of the two-dimensional clinostat, including increased ethylene production (known to be a primary cause of epinasty [Abeles et al., 2012]) and deformation of the statocytes potentially resulting in cell death (Hensel & Iverson, 1980; Hensel & Sievers, 1981). A more recent study (Babbick, et al., 2007) found that, in terms of gene transcription, plant samples grown on a two-dimensional clinostat actually more closely resembled samples exposed to hypergravity (discussed below), whereas samples treated by RPM bore similar expression patterns to those subjected to magnetic levitation, another method of microgravity simulation discussed in Section 1.2.4. Despite some apparent flaws in the ability of a clinostat to replicate microgravity conditions, its use remains one of the most common methods in gravitropism research due to its relative ease and accessibility.
1.2.3
Microgravity via low Earth orbit – the International Space Station
The most informative gravitropism research has involved experiments which are
13
performed in actual, sustained microgravity. Research in this field since the 1970s has been performed by American and Soviet (later Russian) space agencies aboard space shuttle missions and as part of the Biosatellite, Skylab, Salyut, Soyuz, and Mir satellite programs (Zabel et al., 2016). These missions provided an essential proof-of-concept for the field, including the first “seed-to-seed” plant cultivation, i.e., plants which were germinated and successfully reproduced in space, aboard Mir in 1997 (Ivanova et al., 2001). However, since its construction in 1998, the International Space Station has housed the majority of plant spaceflight experiments. Currently, it contains more than ten modules designed for plant cultivation and experimentation with contributions from American, Russian and European space agencies (Stutte et al., 2016). The plant growth chambers employed aboard spacecraft in the modern era are generally rather small compartments (0.05-0.1 m2 growth area, on average) lit by fluorescent or LED lighting with artificial substrate and nutrient delivery systems (Stutte et al., 2016; Zabel et al., 2016). Some growth systems boast unique features. As examples, the European Modular Cultivation System (EMCS) houses two centrifuge rotors able to hold four experiment containers each, which can be set to different gravity levels up to 2 g (the only growth facility able to variably control g force) (Zabel et al., 2016). Meanwhile, the Advanced Biological Research System (ABRS) is equipped with a GFP (green fluorescent protein) Imaging System (GIS), allowing the live imaging of virtually any known protein in real time, primarily in the dimunitive model research plant Arabidopsis thaliana due to size constraints of the growth chamber (Stutte et al., 2016; Zabel et al., 2016).
14
Twenty-one plant growth chambers have been used in spaceflight, and all but one of them were specialized for research purposes (Stutte et al., 2016). NASA's VEGGIE Food Production System, launched in 2014, is the exception, designed to cultivate food for astronaut consumption, with more growth space (~45,000 cm 3) and stronger lighting than other growth systems (Stutte et al., 2016; Onate, 2016; Zabel et al., 2016). In August 2015, NASA astronauts ate their space-grown produce—lettuce—for the first time (Zabel et al., 2016). In light of VEGGIE's success, even larger and more sophisticated plant growth systems are scheduled to be incorporated into the ISS by 2020: namely, NASA's Advanced Plant Habitat (APH) and the European Space Agency's EDEN ISS ISPR (Massa et al., 2016; Morrow et al., 2016; Boscheri et al., 2016). The former will control the level, color composition and timing of light, humidity levels, water delivery, and temperature, and feature an exceptionally large growth volume (112,500 cm 3) equipped with multiple cameras (Massa et al., 2014; Onate, 2016). The latter is first being tested in Antarctica, and the crops grown in it will be used to supplement the diet of researchers stationed there (Schlacht, 2016; Boscheri et al., 2016).
1.2.4
Less common methods in plant gravitropism
Parabolic flight
Short of entering low-Earth orbit, parabolic flight is able to provide brief intervals of very low gravitational force (about an order of magnitude nearer to 0 g than is possible in laboratory settings), and has been used to study plant gravitropism (Volkmann et al., 1991; Moore & Cogoli, 1996; Limbach et al., 2005; Paul et al., 2011 and references 15
therein). Parabolic flight describes the general trajectory of an aircraft which gains and subsequently, rapidly loses some level of elevation. The change in elevation and the angle at which the aircraft attains this elevation determine the gravito-inertial acceleration (GIA, commonly g force, defined as the sum of linear acceleration due to gravity and inertial forces, wherein one g = 9.81 m/s2 at sea level) experienced by the passengers and payload of the aircraft throughout the parabola (Bryan et al., 2007). In simulating weightlessness, the aircraft climbs at a 45º pitch-up for 20 seconds before lowering the nose and entering a phase of (nearly) zero g for 25 seconds at an average angular momentum of 3 rad/sec, followed by a 45º pitch-down lasting 20 seconds (Karmali & Shelhamer, 2008). During pitch-up and pitch-down, a GIA of 1.8 g is felt. Typically, 3060 such parabolic maneuvers are performed during a parabolic flight (Paul et al., 2011). A major repercussion of parabolic flight is that brief periods of reduced gravity are interspersed among periods of hypergravity. The effects of these periods of altering gravity are difficult to parse out, and pose similar problems to those described in the following section regarding hypergravity via centrifugation.
Hypergravity via centrifugation The use of a centrifuge to accelerate plant samples beyond the gravitational force on Earth in order to potentially learn about their response in lowered gravity is based on the so-called “reduced gravity paradigm.” This paradigm includes the notions that the changes in physiology and gene expression seen when comparing plants in hypergravity to Earth's gravity will resemble those seen comparing Earth gravity to microgravity, and
16
is based on the premise that the relative difference between gravitational states is what determines change, rather than the absolute gravitational force experienced (van Loon, 2016). While the reduced gravity paradigm has rather limited supporting evidence in plants, experiments in animals corroborate the notion: for example, astronauts report similar symptoms after returning to 1 g from 3 g as when initially experiencing microgravity of free fall or low-Earth orbit (Nooij et al., 2007). Hypergravity experiments performed by centrifugation have the benefit of being simple, Earth-based procedures that do not require special equipment, unlike the other methods reviewed above. However, they are considerably limited in their power of inquiry. Most notably, the applicability of data gathered in this way to actual microgravity conditions relies on extrapolation which currently lacks a strong experimental justification. Furthermore, only very quickly-responding systems within the plant may be expected to show consistent and isolated effects from the change in gravity without the confounding effects of varying response to the specific, actual force of gravity (van Loon, 2016). Even these systems are subject to cross-regulation from intermediate- and slowresponse systems, further complicating the matter. Nonetheless, hypergravity conditions are able to restore gravitropism in reduced-response or agravitropic mutants, and the method retains research utility partly for this reason (Soga et al., 2004; Kiss, 2000). Determining how hypergravity has this effect may help clarify statolith-independent means of gravitropism, as discussed in Section 2.6.
Microgravity via magnetic levitation
The levitation of a biological sample via magnetic force was first accomplished in 17
1997 by two independent teams of researchers: A High Field Magnetic Laboratory-based team in Nijmegen, The Netherlands headed by Andre Geim levitated a live frog (Berry & Geim, 1997), while frog eggs were levitated at Brown University by Valles et al. (1997). Thus, magnetic levitation is among the newest microgravity simulation methods to be developed. The principle behind its use is that diamagnetic materials, such as water and consequently nearly all biological samples, are repelled by magnetic fields due to an induced field in the opposing direction. If the applied magnetic field is large enough, the generated force can be sufficient to relieve the sample of its weight; that is, to oppose and, as closely as possible, equal the gravitational force (Barry & Geim, 1997). The effective gravity of the sample is the sum of the diamagnetic and gravitational forces per unit mass. Diamagnetic force is supplied by a solenoid conductor containing a hollow, magnetic bore in its center; the sample is housed inside the bore (Berry & Geim, 1997; Valles et al., 1997). Calculations of the electromagnetic field assume a composition of pure water for these experiments; these calculations are then fine-tuned to optimize levitation of the sample (Herranz et al., 2013). For a sense of the strength of the magnet required for this technique, a 16-Tesla field was needed to suspend a frog (Berry & Geim, 1997); 7-Tesla fields are used in MRI scanners (Herranz et al., 2013), and the current world record for the strongest sustained magnetic field is 45 Tesla (Meet the 45 Tesla Hybrid Magnet, accessed 2016). That such a strong magnetic force may impart its own unique effects is not unfeasable (Herranz et al., 2013). While magnetic levitation has been successful using frogs and fruit flies, and may
18
prove useful with other small animals, the technique is of limited use in non-animal models such as Arabidopsis and the alga Chara (Herranz et al., 2013). Although the plant itself, most cellular components, and the cytosol are all levitated, the force applied by the magnet is insufficient to levitate the statoliths of plant cells (typically quite dense with starch) and thus does little to counteract gravistimulation and graviresponse. Despite this, levitated samples show several similarities with RPM and spaceflight samples, although they remain generally gravicompetent. These similarities include increases in cell proliferation, decreases in cyclin B2 expression (marking G2/M cell cycle progression), and abolition of polar auxin transport at effective gravity levels beyond 2 g (Manzano et al., 2013; Herranz et al., 2014). Since magnetic levitation is not capable of preventing statolith sedimentation, but has many similarities with RPM and spaceflight samples, this technology may be useful in identifying aspects of gravity perception and response that do not rely on statolith sedimentation.
1.3.
The working model of plant gravitropism
The following sections provide a brief description of the largely undisputed events of the plant gravitropic response, though they are surprisingly scant and often vague. While this depiction can be thought of as “textbook”, and is indeed found in textbooks (e.g., Hopkins & Huner, 2008, and Taiz & Zeiger, 2014), it is important to consider that this is a very limited view of the field. Many of the ambiguities alluded to in these sections are expanded upon in subsequent chapters, as are several controversies related to certain aspects of this generally widely-accepted model.
19
1.3.1
Statolith sedimentation is a key early event
It has been asserted for over a hundred years—nearly as long as the topic of plant gravitropism has been duly studied—that the main way plant cells perceive changes in gravity is as a consequence of the movement of statoliths (Darwin & Pertz, 1904). Generally, a statolith in plants is a very dense organelle that sediments rapidly and is involved in graviperception; in Figure 3: Locations of graviresponsive bending cells. A, the pulvinus of the Poaceae is the swollen amyloplasts, with cells specialized for gravity region responsible for the angle formed between flanking stem sensing bearing these organelles in the root and regions. B, a labeled Arabidopsis root showing the elongation zone, the graviresponsive site. The gravisensing shoot (Chen, et al., 1999). An exception occurs in columella cells are in the root cap at far left (CC).
nearly all cases, these organelles are starch-filled
the rhizoids of the alga Chara; its statoliths are
membrane-bound vesicles containing barium
A is reproduced from Clore (2013), originally from Sachs (1887); B is reproduced from Bargmann et al. (2013).
sulfate crystals in a carbohydrate and protein matrix (Wang-Cahill & Kiss, 1997). As mentioned before, a cell specialized for gravisensing is a statocyte. The columella cells of the root cap are the statocytes of higher plant roots, whereas statocytes best studied in the eudicotyledenous shoot are dispersed throughout the endodermis (a continuous layer of ground tissue surrounding the vascular bundle) and in the monocotyledonous shoot are found in the bundle sheath cells of the pulvini (Sack, 1991; Sievers, 1991; Johannes et al., 2001; Blancaflor & Masson, 2003; Clore, 2013) (Figures 1 & 3).
20
The ideas that, first, statolith sedimentation is among the most important means of plant graviperception, and, second, that starch-filled amyloplasts are the major statolith, are based on a very large body of experimental data. As mentioned above, it was shown as long ago as the 19th century that removal of the root cap, containing columella cells, destroys the gravitropic response, and it is restored when the root cap reforms (Darwin, 1880). More recently, laser ablation of the central columella cells of the Arabidopsis root cap caused a significantly reduced gravitational response; in the same study it was also noted that columella cells that contribute most significantly to gravisensing are those with the fastest amyloplast sedimentation velocities in the S2 and S3 files of the columella (Blancaflor, et al., 1998) (amyloplast sedimentation in such a columella cell is shown in Figure 4). Additionally, reduced-starch and starch-deficient mutants of Arabidopsis consistently show a gravitational sensitivity and response, in both the shoots and roots, that is proportional to starch content (Kiss et al., 1989; Sack, 1991; Kiss et al., 1996; Kiss et al., 1997; Hartwell, 2016). Similarly, the Amylomaize line of Zea mays has smaller amyloplasts which sediment more slowly, and this cultivar displays a slower and weaker response to gravistimulation (Sack, 1991). In 1998, a group of researchers from Kyoto and New York Universities showed that Arabidopsis mutants scarecrow (scr) and shortroot (shr), which display absent or severely malformed endodermis in the shoot and root, were severely shoot agravitropic but root gravitropic (Fukaki et al., 1998). A reduced shoot-gravitropic phenotype is seen in endodermal-amyloplastless 1 (eal1) mutants, which have endodermis but in which amyloplasts do not form (Fujihira et al., 2000). eal1 mutants show abnormal shoot growth, a greatly diminished curving response
21
Figure 4: Real-time amyloplast sedimentation in an Arabidopsis columella cell as shown by differential interference contrast microscopy performed by Leitz et al. (2009) accompanied by motion analysis (E-L). Note that neither the nucleus nor the ER show significant movement, and the length of time necessary for complete sedimentation. N, nucleus; AM, amyloplasts, ER, endoplasmic reticulum; CW, cell wall; ERi, ER interface (recreated as dotted lines in E-L); AMt, traced positions of amyloplasts; tR = time to (complete) reorientation (E); arrows labeled “g” represents the direction of gravity. Reproduced from Leitz et al. (2009).
22
in gravistimulated hypocotyls and total ablation of gravisensitivity in the inflorescence stem (noting that gravitropic curving in some other regions of the stem is still faintly present, suggesting an amyloplast-idependent pathway for gravisensing) (Fujihira et al., 2000). Like scr and shr mutants, eal1 roots have intact root caps and remain gravicompetent (Fujihira et al., 2000). These results strongly suggest that the endodermis and its amyloplasts are chiefly important for shoot, but not root, gravitropism. The extensive work identifying the root cap as the site of root gravisensing and its columella cells as the involved statocyte is reviewed by Volkmann & Sievers (1979), Sack (1991), Sievers (1991), Blancaflor et al. (1998), Kiss (2000), and Blancaflor & Masson (2003).
1.3.2
Mechanotransductive ion channels confer a chemical signal
Another more recently developed term for the statolith is the gravity susceptor – it is the body specifically susceptible to the influence of gravity. The effect of gravity on the susceptor is detected by the gravity receptor. The principal gravity receptor, if a singular principal receptor exists, has not been conclusively identified. Whatever the receptor structure may be, it is generally believed that, upon gravistimulation, it activates or allows the activation of mechanotransductive or stretch-activated ion channels, specifically those which transport ionic calcium (Masson et al., 2009 and references therein; Baldwin et al., 2013). This is based on results showing that inhibitors of stretchactivated calcium ion channels, such as lanthanum (La3+) and gadolinium (Gd3+), and calcium chelators all inhibit the gravitropic response (Chen et al., 1999; Blancaflor & Masson, 2003; Masson et al., 2009).
23
There are numerous organelles which could plausibly be site of these potential mechanotransductive receptors. Endoplasmic reticulum (ER) and vacuolar membranes appear to have a close relationship to sedimenting amyloplasts in root columella cells (Figure 4) and shoot endodermis, respectively, and may conceivably act as receptors. Additionally the disruption of the cytoskeleton and its tensegrity (balance of elastic and compressive forces [Ingber, 1993]) due to sedimenting amyoplasts convincingly seems to be an important part of the way the physical effect of gravity could be translated to a chemical signal (Blancaflor & Masson, 2003; Baldwin et al., 2013). In principal, microtubules and/or actin microfilaments, which are enmeshed to form a threedimensional structure throughout the cell that is anchored to the endo- or plasma membrane, are impacted by the weight of amyloplasts sedimenting on top, and transmit tensile force to mechanosensitive, or stretch-activated, ion channels at the plasma membrane. It is thought that this may be the cause of the rapid change in cytosolic pH and other ionic concentrations observed very briefly (seconds) after gravistimulation (Scott & Allen, 1999; Fasano et al., 2001; Boonsirichai et al., 2003; Toyota & Gilroy, 2013). The potential role of the cytoskeleton as a receptor or mediator of gravity sensing is revisited in Section 2.1. In Arabidopsis, there is experimental evidence that graviperception occurs less than one second after gravistimulation, as determined by experiments using very brief interruptions of clinorotation to provide equally brief stimuli and testing for graviresponse after (Hejnowicz et al., 1998; Blancaflor, 2002). This amount of time, based on average amyloplast sedimentation velocities, indicates amyloplast displacement
24
of only 0.5 µm can initiate graviresponse (Hejnowicz, et al., 1998; Blancaflor, 2002). This is incompatible with a model that requires the amyloplast actually reach the bottom of the cell, such as that wherein contact between the amyloplast and nodal ER networks (also known as distal ERs; clusters of smooth ER located in distal periphery of columella cells) initiates graviresponse. Of course, that amyloplast—ER contact contributes to graviresponse remains a possibility, and perhaps a likely one (Section 2.2). Interestingly, inhibition of actin polymerization actually seems to enhance the gravitropic response in some cases, though contradictory results exist (Blancaflor, 2002; Kiss, 2000). Therefore, the role of actin and the cytoskeleton as a whole in gravitropic sensing may be more complex than originally speculated. Nonetheless, it is not hard to imagine that a responsive network exists throughout the cytosol, such as the cytoskeleton, acting in part as a gravity receptor, as it would help explain the extent and nature of the cell's sensitivity to very slight or brief periods of gravistimulation. It is generally agreed that, somehow, these events lead to a redistribution of auxin transport proteins, particularly of auxin efflux carriers, towards the gravitational bottom of the cell, leading to lateral transport of the plant growth-modulating hormone auxin to the “bottom” of the gravistimulated tissue, as described below (Goto et al., 1987; Blancaflor & Masson, 2003; Masson et al., 2009; Blancaflor, 2013). The cell signaling events following the initial mechanotransductive step and what couples them to regulation of polar auxin transport remain somewhat mysterious; what we know of this process is discussed in Chapters 2 and 3.
25
1.3.3
Altered auxin transport changes growth at the site of response
As alluded to above, a crucial factor in transmission from sensing to response is auxin, and proteins that affect the movement of auxin through the cell. Its role in gravitropism, specifically gravitropic curvature, was first proposed as part of the Cholodny-Went theory in 1937, after the two researchers who independently arrived at the idea, and has remained central to research in the field ever since (Went, 1935; Lomax, 1997; Blancaflor & Masson, 2003; Baldwin, et al., 2013). Auxin's differential concentration between the upper and lower halves in the elongation region of the growth organ (root or shoot) is the cause of gravitational bending, but the concentration differential is not established at the site of initial perception (Baldwin et al., 2013). Rather, following perception by gravisensing cells, auxin is transported to epidermal and cortical cells in the elongation region (Figure 3) where it induces differential growth (Chen et al., 1999; Blancaflor & Masson, 2003; Harrison & Masson, 2008a). The establishment of the auxin gradient seems to begin with PIN proteins (so named for the the pin-like appearance of their mutants, which lack leaves and stem branching), also referred to as the AGR (AGravitropic Root) protein family, which localize auxin and move it out of the cell to neighboring cells (Sedbrook, 1997) (Figure 5). The protein family mainly responsible for auxin influx into cells is termed the AUX (AUXIN INSENSITIVE) protein family after its principal member, AUX1 (Blancaflor & Masson, 2003). PIN and AUX1 proteins are transmembrane proteins and are found in both the plasma and endomembranes, potentially providing a connection between the cell membrane and the membrane-bound organelles implicated in the gravitropic response
26
Figure 5: Auxin transporters redistribute upon gravistimulation to form auxin gradients. (A) In root columella cells, PIN3 and PIN7 redistribute to the cell bottom, establishing a gradient maintained by differential PIN2 activity. (B) In unstimulated shoot endodermis cells, PIN3 directs auxin inwards towards the shoot vasculature (left); following stimulation (right), PIN3 redirects to the bottom of the cell, directing auxin to the cortex and epidermis in the lower flank, where it will induce corrective growth. Reproduced from Petrášek & Friml (2009).
27
(Harrison & Masson, 2007; Baldwin et al., 2013). Upon gravistimulation, the PIN proteins, which are symmetrically dispersed in non-gravistimulated columella cells, are localized to the new gravitational bottom of the columella cells in root caps (Blancaflor & Masson, 2003; Harrison & Masson, 2007; Masson et al., 2009). AUX1 protein in flanking cells facilitate their auxin uptake; the cell file bordering the new 'top' of the gravistimulated cells experiences a relative deficit of auxin. In the 'bottom' of the gravistimulated organ, auxin is directed basipetally and radially to the epidermis of the elongation zone, the site of response, where it inhibits growth in roots and promotes it in shoots (Blancaflor & Masson, 2003; Harrison & Masson, 2007; Baldwin et al., 2013) (Figure 5).
1.3.4
Auxin-mediated differential growth reorients the plant
Auxins were the first plant hormone to be discovered; their effects on plant developmental and tropic growth, including gravitropism, were identified even before the class of phytohormones was named, isolated or chemically identified (Darwin, 1880; Went, 1935; Woodward & Bartel, 2005). The structure of IAA (indole acetic acid, the principal auxin and first to be isolated) was resolved only in 1977 (Thimann, 1977). In the root cap and elongation zone (Figures 3 & 5A), high auxin concentrations in the graviresponsive portion have an inhibitory effect. Auxin transport directed to the physical bottom of the root leads to supraoptimal concentrations which retard its growth, while the top half of the root grows at a normal rate, bending the root downward towards the gravity vector (Salisbury et al., 1988). In shoots, auxin translocation (Figure 5B) leads to
28
optimal concentrations that encourage cell growth, thus inciting the lower side of the stem to grow more quickly than the upper, again restoring the plant's proper orientation (Chen et al., 1999; Blancaflor & Masson, 2003). The importance of auxin signaling at the site of gravitropic response is supported by several experiments involving auxin inhibitors, fluorescent auxin reporters, exogenous application of auxin, and several other methods (Lomax, 1997; Woodward & Bartel, 2005). The primary effect of auxin at the site of response appears to be inducing specific gene expression patterns; many auxinresponsive proteins are transcription factors, as is discussed in Section 4.1 (Kimbrough et al., 2004). Principal targets of gene expression are cell wall-modifying proteins and genes relating to cell metabolism (Moseyko et al., 2002; Kimbrough et al., 2004) Ionic transport and crosstalk with other plant growth hormones, such as gibberellin and ethylene, are also known to affect gravity in this stage (Lomax, 1997; Blancaflor & Masson, 2003; Wolverton & Kiss, 2009; Zhang et al., 2011). Overall, the auxin-mediated growth response at this phase resembles the auxin-mediated response commonly seen in other tropisms, particularly phototropism (Okada & Shimura, 1992; Iino, 1994; Estelle, 1996; Vitha et al., 2000; Correll & Kiss, 2002; Nakamura et al., 2011). In research with null mutants in plant gravitropism, auxin-directed growth (endogenous or exogenously applied) is often tested as a control to determine if a particular factor is involved in the gravitropic pathway upstream of auxin redistribution and/or growth response (e.g., Yamamoto & Kiss, 2002; Stanga et al., 2009; Nakamura et al., 2011).
29
1.3.5
Other theories exist that may not be mutually exclusive
The perception and response pathway described above is a streamlined and abridged form of the most widely agreed-upon, experimentally-demonstrated series of events of gravitropism. In fact, there are many other theoretically- and empirically-based ideas regarding mechanisms of gravitropism, particularly in the sensing and, to a lesser extent, transduction phases. While many of these ideas were proposed and considered in the 1990s and even earlier as alternatives to the starch-statolith model, more sophisticated experimental methods have revealed that several of these mechanisms likely act in addition and secondarily to the clearly dominant mode of statolith sedimentation. As will be reviewed herein, alternative explanations of gravisensing have been pursued based on experimental knowledge that several genera of algae, mosses and ferns are gravicompetent without statolith sedimentation, and often entirely lacking any sort of statolith (Barlow, 1995; Kiss, 2000). Additionally, starch-deficient Arabidopsis plants show an attenuated but certainly present gravitropic response (Kiss et al., 1989; Kiss et al., 1997; Hartwell, 2016). Perhaps the most famous alternative model, the protoplast pressure model (or gravitational pressure model) proposes that gravity's deformational effect on the whole protoplast within the rigid cell wall is actually the first event of gravisensing, and thus the entire protoplast is the susceptor “organ”. Following this event, tension and compression forces between the plasma membrane and cell wall, and with the cytoskeleton, ultimately act on mechanotransductive ion channels in the membrane, resulting in the same gravitropic cascade thereafter that is proposed as part of the starch-statolith hypothesis
30
(Wayne et al., 1990; Staves et al., 1992; Staves, 1997). Many researchers note that the majority of evidence cited to support the gravitational pressure model comes from algae of the genus Chara and thus its clout in discussing gravitropism in higher plants is limited. However, the results of Staves et al. (1997b) in rice, Oryza sativa, and of several studies in starch-deficient mutants certainly allow for or even suggest the possibility of other means of gravitropic sensing beyond the simple starch-statolith hypothesis. An increasingly common position is that both the starch-statolith and protoplast pressure models—and potentially others, such as a possible statolith-independent gravitropic mechanism in root distal elongation cells or the possible role of other organelles as statoliths—are likely present in higher plants in an example of functional redundancy brought about by divergent evolution (Barlow, 1995; Sack, 1997; Masson et al., 2013). It is noted by Sack (1997) that such a divergent evolution would be a reasonable expectation, given the completely pervasive nature of gravitational force and its numerous and varied effects on cellular and whole-plant processes, and that similar tropisms in plants—namely phototropism—do indeed function by several mechanisms. Similarly, Barlow (1995) suggests that the continual evolutionary pressure of gravity has probably led to more basal mechanisms of gravitropism being retained with the development of more advanced mechanisms in higher plants. Finally, a statolithindependent means of gravisensing in higher plants is necessary to explain results showing gravitropic signals derived from the distal elongation cells of the Arabidopsis root, which lack amyloplasts (discussed in Section 2.6) (Ishikawa & Evans, 1993; Wolverton et al., 2002).
31
1.4.
Conclusion
Plant gravitropism is a field of plant biology which has applications in agricultural and aerospace industries. This field has received extensive and continual study not only for this reason, but also because of the continual aspects of mystery, contention, and surprise researchers have enjoyed for more than 200 years, beginning with Knight (1806). To closely and comprehensively analyze some of the more contentious and leastunderstood aspects is the goal of this thesis. As such, the less-understood earlier phases of gravisensing and signal transduction as well as tissue polarity establishment, will be reviewed and discussed at length in Chapters 2 and 3. When considering these questions, however, it is necessary to refer to the canonical model, which will now be summarized again. Plant cells perceive changes of the strength or direction of the gravity vector relative to their position in specialized cells through the sedimentation of dense, starchfilled amyloplasts, although other means may contribute to gravity sensing as well. The physical force of gravity is then transformed into a chemical signal via mechanotransductive ion channels. Local and whole-cell changes in ionic concentrations due to activation (or, theoretically, deactivation) of these channels result in the relocation of auxin transport proteins to the gravitational bottom of the gravisensing cells and tissues. Lateral auxin transport from gravisensing cells to cortical and epidermal cells in the elongation region results in differential auxin concentrations, which acts with other growth factors to reorient the organ with respect to gravity.
32
CHAPTER 2: EARLY SIGNALING EVENTS AND STATOLITH SEDIMENTATION More so than the growth-response phase or the tissue-level signaling that unites them, the earliest phases of gravisensing and early signal transduction remain the most mysterious of all within the study of plant gravitropism. There are a few factors contributing to this confusion. One is that these events begin immediately upon gravistimulation, and consistent and reliable analysis within this time frame has historically been challenging. Another is that differences in the gravitropic pathway between different organisms appear to be the most divergent at this phase, whereas further-downstream components, such as lateral auxin transport, seem to be very common amongst several organisms that have been investigated. Yet another challenge is the scale of this response. Many factors active in this phase of gravitropic signaling need to be clearly resolved at a high spatiotemporal resolution for their function to be understood, in contrast to tissue or whole-organ level dynamics that mediate and dictate the later growth response. Finally, these nascent signals can be closely intertwined with each other as well as with cellular components, often making it rather difficult to pin any particular result to a particular signaling pathway without extensive controls and follow-up experiments. All of these factors will be discussed regarding specific experimental challenges in studying the early players identified in this chapter: the cytoskeleton, the endomembrane system, calcium-based signaling, reactive oxygen species, and pH and proton distribution. Despite these significant challenges, remarkable insights have been made in the past (approximately) twenty years, owing in large part to the proliferation of next-
33
generation sequencing methods, and show no sign of slowing in pace. In fact, flurries of (sometimes contradictory) results are being generated more quickly than sense can be made of them. An effort to closely analyze these results and form a cohesive idea of the earliest phases of gravisensing and signal transduction will not only help to contextualize and clarify existing results, but will inform the basis of future experiments as well. Such an effort is made here.
2.1.
The cytoskeleton as a mediator and sensor of sedimentation
The plant cytoskeleton is a complex network principally composed of microtubules and actin filaments (F-actin, or microfilaments), tangled throughout the cell and including the proteins that bind these filamentous elements (noting that plant cells generally lack the intermediate filaments found in other eukaryotic cells) (Volkmann & Baluška, 1999; Blancaflor, 2002). The cytoskeletal elements, which traverse the cell to form molecular routes that interconnect organelles to each other and to the plasma membrane, are highly responsive to a plurality of developmental and environmental cues, mediating cellular processes including cell division, tropic growth, morphogenesis, stimulus response and cell signaling (Volkmann & Baluška, 1999; Blancaflor, 2002; Blancaflor & Masson, 2003; Blancaflor, 2013). The cytoskeleton is known to be involved in response to other mechanical stimuli (e.g., wind and touch), and is capable transmitting mechanical force throughout the cell (Ingber, 1993; Esmon et al., 2005). Based on this characteristics, it has been extensively investigated as having a potential role in plant gravitropism, particularly in the early phases of perception and transduction (Hejnowicz
34
& Sievers, 1981; Sack, 1991; Baluška & Hasenstein, 1997; Volkmann & Baluška, 1999; Kiss, 2000; Blancaflor, 2013). While experiments with inhibitors and hyperstabilizers of microtubules and actin suggest that the cytoskeleton is somehow involved in the gravitropic response, results and conclusions describing exactly how it is involved are far from conclusory; more often, they appear contradictory, sometimes even in the same organism (Blancaflor, 2013). Before these results can be examined, the role of the cytoskeleton in maintaining the cellular architecture of gravisensing cells, particularly columella cells of the root cap, must be described first. Columella cells display a unique polar organization in ungravistimulated roots: the nucleus is localized to the “top” of these cells (the area basipetal/proximal to the root meristem), while amyloplasts typically rest at the “bottom” of the cell (the acropetal/distal region) atop ER bodies concentrated cortically; centrally, several smaller vacuoles are seen in place of the large central vacuole generally observed in most plant cells (Sack, 1991) (Figures 1 & 4). Abiding by the principle that form follows function, it is thought that this organization of the columella cell, which leaves the center volume of the cell relatively unobstructed, allows amyloplasts to sediment more quickly and with an improved signal-to-noise ratio upon gravistimulation (Sack, 1997). When vertical roots are clinostated or treated with cytoskeletal inhibitors, mature columella cell organellar polarity is disrupted, an effect not seen in immature columella cells, and plant gravitropism is reduced. These findings suggest that this cellular polarity is regulated by both the cytoskeleton and gravitropism and is important for the columella's role in gravisensing (Hensel & Sievers, 1980; Hensel, 1989; Wendt & Sievers, 1989; Sack,
35
1991). Rather than the very thick, cable-like actin microfilament bundles usually seen streaking across cells that are not gravisensitive, columella cells display a much more diffuse network of actin focused in structures described as resembling “hammocks” surrounding organelles, namely the nucleus, amyloplasts, and distal ER bodies (Sievers, 1991; Yoder et al., 2001; Blancaflor, 2002) (Figures 6 & 7, page 38). These actin structures appear to anchor the nucleus and ER bodies in their positions, and to slow the sedimentation rate of the amyloplasts, most strongly in cortical regions of the cell where these actin structures are most abundant and amyloplast sedimentation is slowest (Hensel & Sievers, 1980; Sievers, 1991; Leitz et al., 2009). These actin “hammocks” are able to tether the organelles they surround because they are connected to the plasma membrane via association with larger transcellular actin filaments (Perbal & Driss-Ecole, 2003). Although endodermis and bundle sheath cells, the gravity-sensing cell types of the shoot, do not share the polar organization of root columella cells, their actin
Figure 6: The actin cytoskeleton of Arabidopsis endodermis cells show transcellular cables as well as diffuse "hammocks" around amyloplasts (arrows). Reproduced from Blancaflor (2013).
36
cytoskeletons are similarly diffuse and bundled around organelles, but also retain the transcellular cable-like actin seen in most of the plant (Blancaflor, 2013) (Figure 6). Because of its peculiar distribution in gravisensitive cells and because of its ability in other systems to transmit mechanical force on the time scale at which gravisensing is known to occur, the actin cytoskeleton has often been proposed as a promising potential gravitropic receptor (Baluška & Hasenstein, 1997; Perbal & Driss-Ecole, 2003; Nakamura et al., 2011; Blancaflor, 2013). The role of the actin cytoskeleton as a putative receptor is proposed to occur in one of two ways: restrained or unrestrained gravity sensing (Baluška & Hasenstein, 1997). In the case of restrained gravity sensing, movements of the amyloplasts impart a tensional force on the cytoskeleton which activates or inactivates unknown receptors linked to the cytoskeleton. In unrestrained gravity sensing, the actin cytoskeleton anchoring amyloplasts to the cell membrane is broken by the force with which the amyloplast sediments, and the weight of the amyloplasts sedimenting onto structures at the gravitational bottom of the cell initiates the gravitropic signaling cascade (Sievers et al., 1991; Baluška & Hasenstein, 1997; Blancaflor, 2002). Generally, the restrained model of gravity sensing has more experimental support. Importantly, gravisensing is seen to occur before amyloplasts are completely sedimented and in fact does not require complete sedimentation at all – displacement of amyloplasts by less than a micrometer appears sufficient to trigger graviresponse, based on the minimum time to trigger a response and average amyloplast sedimentation speeds (Hejnowicz et al., 1998; Blancaflor, 2002). While the unrestrained gravity sensing model is not the primary or exclusive method of gravity sensing used by
37
Figure 7: The actin cytoskeleton of flax columella cells. Light (A) and fluorescent (B) micrographs of columella cells labeled with anti-actin antibodies and Alexa-fluorphalloidin show amyloplasts (a) present in columella cells (c) and absent in neighboring peripheral cap cells (pc). The differences in the actin cytoskeletons of these cell types is apparent in B, particularly the brighter and more fibrous appearance of the peripheral cap cytoskeleton (due to transcytosolic actin cables absent in the columella). C, a optical section of three columella cells shows fine fibers surrounding amyloplasts (asterisks). Bars = 10 μm. Modified from Blancaflor (2002).
38
plants, it is entirely possible that interactions between fully sedimented amyloplasts and cortical ER bodies play some further-downstream role in gravitropism (Sack, 1997; Blancaflor, 2002). The involvement of the endomembrane system in gravisensing is discussed in the next section. Despite these initial clues, experimental attempts to clarify the role of the actin cytoskeleton in gravisensing suggest it is more complex than originally assumed, and integrated with other cellular components in equally complex ways. Were the cytoskeleton, particularly actin, the primary receptor of the mechanical stimulus of amyloplast sedimentation, actin inhibitors/destabilizers would be expected to impair gravitropic response, perhaps entirely. It is not so simple: several studies show enhancement of gravitropism after treatment with actin inhibitors (Yamamoto & Kiss, 2002; Hou et al., 2003; Palmieri & Kiss, 2005). Cytochalasins (B and D) and latrunculins (B) are the most commonly used actin inhibitors in such studies. Low concentrations (1020 μM) of cytochalasin D were not only reported to cause no visible effect on gravitropic curvature in maize, rice, and Lepidum roots, but to improve the agravitropic defects of decapped maize roots, as well (Blancaflor & Hasenstein, 1997; Staves et al., 1997c; Mancuso et al., 2006). A concentration of 50 μM of cytochalasin B in maize roots was found to cause a weakly-improved gravitropic response compared to untreated controls, whereas a 10 μM treatment of cytochalasin D significantly reduced curvature (Blancaflor & Hasenstein, 1997). Latrunculins, more powerful inhibitors of actin polymerization than cytochalasins, have been found to promote gravitropism in Arabidopsis shoots but inhibit it in roots when used at concentrations between 0.02-2 μM, in a dose-dependent manner
39
in both organs (Yamamoto & Kiss, 2002). Like cytochalasin D, latrunculin B does not affect the growth angle of gravistimulated maize roots but does lessen the impacts of decapping on roots, actually more significantly than cytochalasin D (Mancuso et al., 2006). Concentrations of 40-42 μM of cytochalasin D were shown to inhibit gravitropic bending in the flowering stems of Antirrhinum (snapdragon) (Friedman et al., 2003). A 12 μM treatment of latrunculin B reduced gravitropic curvature in snapdragon shoots, though much less so than cytochalasins (Friedman et al., 2003). These apparently conflicting results obtained from Antirrhinum explants may be a result of the exceptionally high concentrations of cytochalasin D and latrunculin B used by Friedman et al (2003) (compared to, e.g., Yamamoto & Kiss, 2002; Mancuso et al., 2006). Overall, it is difficult to discern a consistent pattern among these results, and an effort should be made to perform a more comprehensive and comparative investigation of the involvement of actin in the earlier phases of plant gravitropism. On the whole, these results speak strongly against the role of the actin cytoskeleton as the primary gravireceptor: its stability is not necessary for gravitropism, and in fact its dissolution can improve gravitropic response (Staves et al., 1997; Blancaflor, 2003). These findings go against the initial suppositions of many researchers within the field imagining a simplistic role of the cytoskeleton, but recently have been considered as reconciliable with a tensegrity model wherein the actin cytoskeleton finetunes amyloplast sedimentation through negatively regulating it, i.e., by providing a physical barrier to sedimentation (Volkmann & Sievers, 1979; Sack, 1991; Blancaflor, 2013). Its role as a negative regulator, however, is clearly not a passive one. Several actin
40
inhibitor studies have shown that these cytoskeletal elements mediate the staltatory movements of amyloplasts (non-gravitational and non-Brownian localized motions triggered by gravistimulation), and although the exact function of these movements remain unknown, they do serve to slow sedimentation time, consistent with the idea of negative regulation by the actin cytoskeleton (Saito, 2005; Morita, 2010). This may be useful to ensure that a sufficiently strong and sustained change in the gravity vector is present before initiating the entire growth response cascade, so that the plant is not expending energy responding to transient stimuli that will not have significant effects on its growth. Also consistent with this reasoning is the result that latrunculin B treatment causes Arabidopsis stems to overshoot their growth—to exceed the gravitational set point angle (GSA), the angle with respect to the gravity vector at which a plant organ maintains its growth—in the direction opposite to the stimulus in response to gravistimulation (Digby & Fern, 1995; Yamamoto & Kiss, 2002; Nakamura et al., 2011). Additionally, when Zea mays roots were treated with jasplakinolide and phalloidin, stabilizers of Factin that respectively enhance nucleation and prevent depolymerization, gravitropism was significantly reduced (Mancuso et al., 2006). A potential molecular basis for the role of the cytoskeleton in gravisensing and signal transduction was first described by Sedbrook et al. (1998), who identified ARG1 (ALTERED RESPONSE TO GRAVITY1) in Arabidopsis. arg1 mutants show weakly reduced root gravitropism, but accumulate amyloplasts which sediment normally and mutants respond to phototropic stimuli or exogenous auxins as normal; thus, ARG1 is concluded to function in gravitropic signal transduction (Sedbrook et al., 1998). This is
41
supported by the findings that arg1 mutants do not exhibit the rapid cytoplasmic alkalinization that typically follows gravistimulation, and additionally fail to relocalize PIN3 (discussed in Section 2.5 and in Chapter 3) (Boonsirichai et al., 2003). Endodermis amyloplasts in arg1 mutant lines, in contrast to the phenotypically normal amyloplasts of the root, show reduced sedimentation and staltatory movement, suggesting a more central role for the protein in shoot gravitropism (Kumar et al., 2008; Blancaflor, 2013). ARG1 is a small (410 aa), ubiquitously-expressed protein bearing the J domain of DnaJ-like proteins, which are molecular co-chaperones that cooperate with HSC70 and are frequently involved in signal transduction (Sedbrook et al., 1998). The protein also has a coiled-coil domain near its C-terminus, a common feature of cytoskeletal-interacting proteins, as well as a transmembrane domain near the J domain at the N-terminus (Sedbrook et al., 1998). Although it was originally supposed that ARG1/ARL2 may interact directly with statocytes, Harrison & Masson (2008b) showed that their function is likely amyloplast-independent (Boonsirichai, 2003). Sedbrook et al. (1998) note that the weak phenotype of arg1 roots may belie the function of ARG1 due to possible functional similarity with its paralogous protein ARL2 (ARG1-LIKE2). Harrison and Masson (2008b) showed via coprecipitation and cosedimentation assays that ARG1 and ARL2 associate in vivo with HSC70. Although ARL2, like ARG1, contains a J domain near its N-terminus, it appears that ARG1 alone associates with actin whereas ARL2 does not; however, it is possible that association was not detected due to rather low expression levels of ARL2 (Harrison & Masson, 2008b). Exactly how the ARG1-ARL2 complex interacts with the actin cytoskeleton to contribute
42
to gravitropic signal transduction remains to be determined; its role in auxin redistribution and lateral auxin transport is discussed in Section 3.1. For a graphic representing the role of the ARG/ARL complex and the proteins by themselves, see Figure 16 in Section 3.4. In 2011, Nakamura and others identified a RING-type E3 ligase of the SGR (SHOOT GRAVITROPISM) protein family in Arabidopsis, SGR9. This protein expressed in the endodermis and root cap, and its mutants show only mild phenotypes during normal growth (nearly identical to wild-type except that lateral shoots bend upward at a lesser angle) and attain maximal curvature angles of only 50-60º upon reorientation (Nakamura et al., 2011). Since the sgr9 mutation does not affect growth rates or phototropic curvature (actually improving the latter), and the gravitropic deficiency of the mutant is restored by expression of SGR9 as driven by the endodermisspecific SCR (SCARECROW) promoter, the researchers conclude that SGR9 is involved in shoot gravitropism. The formation of an SGR9-GFP fusion protein revealed that SGR9 localizes to amyloplasts within endodermis cells (Nakamura et al., 2011). In the sgr9 mutant, amyloplasts were found to show more dynamic movement upon reorientation, but sedimentation towards the gravitational bottom of the cell was reduced; these effects were ablated by the disruption of the actin cytoskeleton, as tested by treatment with latrunculin B and the formation of an sgr9 double mutant line with fiz1, a mutation impairing actin filament formation (Nakamura et al., 2011). Based on these results, it appears that SGR9 interacts with the actin cytoskeleton to mediate the movement of amyloplasts in the Arabidopsis endodermis. Nakamura et al. (2011) propose that these
43
amyloplasts display two basic modes of movement based on live cell imaging results: rapid saltatory movements when actin-bound, and a slower “sedimentable mode” in the direction of the gravity vector when amyloplasts are released by actin. In such a system, SGR9 is postulated to assist in releasing amyloplasts from actin cables and thus allowing them to enter the sedimentable mode (Nakamura et al., 2011). Mutated SGR9, featuring the substitution of a conserved amino acid within its RING finger domain, did not rescue the gravitropic phenotype of sgr9, implying that the ubiquitin ligase activity of the protein is critical to its role in gravitropism (Nakamura et al., 2011). Ubiquitin E3 ligases such as SGR9, part of the proteosome-ubiquitin complex to which 5% of the Arabidopsis genome is dedicated, function as a nexus in cell signaling, particularly in response to abiotic stress in plants (Mazzucotelli et al., 2006; Lee & Kim, 2011). Another E3 ligase, WAV3, may function in an opposite way, as wav3 mutants show enhanced gravitropism (Sakai et al., 2012). Since E3 ligases are generally involved in substrate recognition in the ubiquitination pathway, identifying the targets of SGR9 and WAV3 should prove useful in clarifying the role of the actin cytoskeleton in mediating amyloplast sedimentation in shoot gravitropism (Lee & Kim, 2011). SGR9 and WAV3 are also depicted in Figure 16 (Section 3.4). Comparatively more light has been shed on the role of F-actin in the phototropically-driven movement of chloroplasts, and consequently this system has been looked to for insight into the gravitropic system. CHUP1 (CHLOROPLAST UNUSUAL POSITIONING1) has been identified as an actin-binding protein involved in chloroplast positioning (Oikawa et al., 2003; Schmidt von Braun & Schleiff, 2008). In addition to the
44
actin-binding domain, CHUP1 contains a profilin-binding domain and is found to associate in vivo with profilins, actin monomer-binding proteins partially controlling the rates of polymerization and depolymerization of actin (Staiger et al., 1997; Schmidt von Braun & Schleiff, 2008). These results are consistent with a model, such as put forth by Wada and Suetsugu (2004), wherein CHUP1 serves as a “work bench” of actin dynamics anchored to the outer membrane of the chloroplast and controlling its positioning (Figure 8). Interestingly, chup1 mutants show abnormal plastid positioning similar to the abnormal amyloplast positioning seen in sgr9 mutants (Kadota et al., 2009; Nakamura et al., 2011; Blancaflor, 2013). To determine the similarity between plastid movement mediation by SGR9 and CHUP1 will require more extensive study of the function of SGR9, and to this end analogous experiments should be performed with SGR9 as were done with CHUP1 (Blancaflor, 2013).
Figure 8: CHUP1 function in chloroplast-actin interactions. Could a similar system be at work at amyloplast membranes? Reproduced from Schmidt von Braun & Schleiff (2008).
2.2.
The endomembrane system
The endomembrane system is a collective term applied to organelles including the endoplasmic reticulum, Golgi bodies, endosomes, the nuclear envelope, and the plasma 45
membrane (Otegui & Reyes, 2010; Morita & Shimada, 2014). Generally conserved across Eukarya, the endomembrane system is a very dynamic network directing the movement of certain proteins, lipids and waste products through the cell. This system, like many of the plants' most dynamic, is implicated in a wide array of cellular and organismal processes, including gravitropism (Morita et al., 2002; Morita & Shimada, 2014). As mentioned in Chapter 1, the proper formation of the endodermis is critical for shoot gravitropism in eudicots (Fukaki et al., 1998). SGR2 and ZIG (Zig-zag, in reference to the growth form of the stems of the mutant) are two genes whose mutated forms cause phenotypes of abnormal amyloplast sedimentation, reduced shoot gravitropism, and vacuolar misformation in Arabidopsis (Fukaki et al., 1996b). While the corresponding proteins are found throughout the plant, driving either wild-type gene under the SCR endodermis-specific promoter rescues their respective mutant phenotypes in terms of gravitropism (Morita et al., 2002). An SGR2-GFP fusion containing the ubiquitous 35S promoter or endogenous SGR2 promoter revealed that the SGR2 protein is localized to vacuolar and small-organelle membranes (Morita et al., 2002). ZIG, identified by phenotypic analysis and gene mapping, had been previously reported as AtVTI11, the Arabidopsis homolog to yeast VTI1 that encodes a v-SNARE protein involved in vesicular transport and membrane fusion (Zheng et al., 1999; Kato et al., 2002; Lipka et al., 2007). SGR2 encodes a phospholipase-protein similar to bovine phosphatidic acidpreferring phospholipase A1 (PL-PLA1) (Kato et al., 2002; The Arabidopsis Information Resource [TAIR], accessed 19 November 2016). An interacting partner of AtVTI11,
46
AtVAM3, encoded by SGR3 and involved in membrane trafficking like AtVTI11 and particularly in vacuolar assembly, was also found to cause a reduced gravitropic phenotype when mutated (Sato et al., 1997; Yano et al., 2003). These results strongly suggest the involvement of the vacuole, vacuolar membrane, and vesicle trafficking in the gravitropic response. Again, mutants in vacuolar biogenesis and vesicle trafficking show abnormal patterns of amyloplast sedimentation; there is evidence to suggest that interactions between amyloplasts and vacuoles are important for shoot gravitropism. sgr2 and zig mutants display amyloplast sedimentation to the bottom of endodermis cells at proportions of only 60.4% and 43.4% of total amyloplasts, respectively, compared to 92.1% of shoot amyloplasts in wild-type plants (Morita et al., 2002). Unlike columella cells, the gravisensing cells of the endodermis feature the large central vacuole typically associated with most plant cells (Morita, 2010; Hashiguchi et al., 2012). In these statocytes, the amyloplasts generally move through invaginations of the vacuolar membrane which form very narrow cytoplasmic channels through the vacuole called transvacuolar strands (Morita, 2010; Hashiguchi et al., 2012) (Figure 9). In zig mutants, amyloplasts are generally seen in the cytosolic space between the plasma membrane and the vacuole, rather than in transvacuolar strands; rescue of the zig phenotype by mutation of ZIG REPRESSOR (ZIP) (via formation of a zip-zig double mutant) is accompanied by relocalization of amyloplasts to transvacuolar strands and to the gravitropic bottom of the cell, but an only partial rescue of the agravitropic phenotype (Morita et al., 2002; Yano et al., 2003; Morita, 2010; Hashiguchi et al., 2012).
47
Figure 9: Transvacuolar strands and amyloplast sedimentation. Depiction of amyloplast sedimentation through transvacuolar strands immediately after inversion (a), throughout the time course of amyloplast sedimentation (b-e), and after sedimentation is complete (f). Each numbered circle represents an individual amyloplast; v = vacuole; ts = transvacuolar strand; sv = secondary vacuole; pc = peripheral cytoplasm. Drawings are based on 15-20 transmission electron micrographs taken per time point at 0 (a), 2 (b), 4 (c), 6 (d), 8 (e), and 10 (f) min after gravistimulation. Reproduced from Clifford et al. (1989).
Altogether, proper sedimentation of endodermis amyloplasts to trigger a gravitropic response appears to be mediated in a significant way by the vacuole and its membrane, the tonoplast. Whether this mediation is an indirect effect, based purely 48
physical obstruction/interaction, or if the vacuole mobilizes its stores of signaling molecules such as ionic calcium and protons upon gravistimulation is, as of yet, unclear. Interestingly, movement of amyloplasts through the transvacuolar strands is mediated by cable-like actin bundles running through them, supporting a model of gravitropism that may involve aspects of the tensegrity model, which incorporates amyloplast sedimentation with important roles of the cytoskeleton, plasma membrane and endomembrane system (Haswell, 2003; Saito, 2005; Morita, 2010) (Section 2.6). More recently, Hashiguchi et al. (2014) reported a new member of the SGR family, SGR6, a HEAT-repeat-containing protein which acts in the endodermis and contributes to gravitropism in Arabidopsis. The sgr6 mutant shows reduced amyloplast sedimentation compared to wild-type by 20 and 40 min after reorientation and requires approximately 50% more time to attain 90º reorientation compared to wild-type (Hashiguchi et al., 2014). Hashiguchi et al. propose that SGR6 may mediate vacuolar strand formation and amyloplast sedimentation, perhaps in coordination with F-actin and an unknown membrane protein, based on the following: the vacuole of the sgr6 mutant does not form vacuolar strands; the HEAT-repeat domain found in SGR6 is often involved in protein-protein interactions; and SGR6 lacks a transmembrane domain, but localizes to the tonoplast with high fidelity, remaining membrane-bound even after treatment with detergent (Hashiguchi et al., 2014). Remarkably, the close relationship between amyloplasts and vacuoles in the endodermis represents a fundamental difference in the mechanism of graviperception in root and shoot gravitropism. Amyloplasts of the columella cells sediment through the
49
cytoplasm, and none the vacuolar protein mutants identified as producing reduced shoot gravitropic phenotypes affect root gravitropism (Saito et al., 2005). In columella cells, more attention has been given to the endoplasmic reticulum, whose cell type-specific positioning has been postulated to be related to the gravisensing function of the columella (Volkmann & Sievers, 1979; Sack, 1991). Specifically, many propose that the intracellular ion stores held in the ER are released upon gravistimulation as an initial signaling event. This is thought to occur via stretch-activated ion channels becoming activated by the force of newly sedimenting amyloplasts, and/or due to the relief of pressure experienced by those ER bodies upon which the amyloplasts were settled before gravistimulation (Perbal & Driss-Ecole, 2003; Leitz et al., 2009; Morita, 2010; Hashiguchi et al., 2012). The cortical ER of columella cells is also proposed to act as a physical barrier to slow sedimenting amyloplasts and prevent them from harmfully deforming the plasma membrane (Leitz et al., 2009). No evidence to date directly supports these roles for the ER in gravisensing, and the ER is most likely not the principal receptor because contact between amyloplasts and the ER has been reported to be unnecessary for gravicompetence (Sack, 1991; Morita, 2010). However, certain results do support some relationship between the ER and gravitropism (Surpin et al., 2005). One such early finding was that the distal clustering of ER bodies is ablated upon clinorotation of vertically-grown roots, but not in roots germinated and continually grown during clinorotation (or in untreated roots), suggesting their polar positioning is responsive to changes in gravity rather than passively dictated by forces within in cell (Hensel & Sievers, 1981). Additionally, Sievers and Busch (1992)
50
showed that an ER-specific Ca2+-ATPase inhibitor, cyclopiazonic acid (CPA), apparently limits gravitropic bending to roughly 20º in Lepidium sativum roots. Since the general growth rate of these roots was not affected, the researchers conclude that the chemical affects gravitropism during the signaling or transduction phase. Recently, it was shown via electron microscopy that amyloplasts and the endoplasmic reticulum come in near enough contact to facilitate protein-protein interactions (~30 nm) and that sedimenting amyloplasts cause significant deformations (~200 nm indentations) in the ER membrane (Leitz et al., 2009). The same study held amyloplasts against the cortical ER using optical tweezers and reports a significant elastic force upon release; in fact, if the amyloplasts were not released but continuously pressed nearer to the distal membrane against the cortical ER, the opposing force from the ER membrane was strong enough to force the amyloplasts from the optical tweezers (Leitz et al., 2009). These results demonstrate the possibility of the endoplasmic reticulum acting as a sensor to amyloplast sedimentation in not only a purely mechanosensitive fashion but also mediated by protein interactions, an idea which will be revisited at the end of this chapter. These results persuasively argue in favor of contributions from the ER to early gravitropic signaling in columella cells. Importantly, however, several signaling events have necessarily already occurred before these amyloplast-ER interactions, because the time to sedimentation (3-5 min in Arabidopsis) is much longer than the earliest reported changes due to gravistimulation (within 1-10 s in Arabidopsis) (Sack, 1991; Blancaflor et al., 1998; Leitz et al., 2009). The authors of Leitz et al. (2009) suggest that the elastic
51
force supplied by ER membranes to amyloplasts resting above them could perhaps enhance the sensitivity of the membrane to changes in orientation relative to the gravity vector by rendering the force equilibrium more complex and responsive to subtle changes, theoretically allowing for essentially immediate detection of the gravistimulus at this membrane-amyloplast interface (Leitz et al., 2009). While such an interaction is theoretically possible (Figure 10), the role of the ER as a gravity sensor will require much more study to enter the realm of practical understanding.
Figure 10: A model depicting how ER-amyloplast interactions could contribute to gravitropic sensing both immediately (B) and following amyloplast sedimentation (C-D). While only the latter scenario has traditionally been considered, recent research suggests the possibility of that as seen in (B). (A) represents a columella cell prior to gravistimulation. Reproduced from Leitz et al. (2009).
52
2.3.
Calcium-based signaling and inositol triphosphate
Calcium and calcium binding proteins have very diverse roles as signaling molecules in plant biology as in all eukaryotic biology, often implicated in dynamic cell signaling events such as the detection of and response to biotic and abiotic stresses, hormonal growth regulation, cell cycle progression and ion homeostasis (Bush, 1995; Sanders et al., 2002; Schulz et al., 2013). Generally, Ca2+ channels, including pumps, carriers, and passive calcium-permeable channels, show a response to the stimulus in question, resulting in distinct “waves” of ionic calcium in the cell. The waves of calcium are often termed monophasic, biphasic or oscillatory based on their pattern; the duration and intensity of the signal impart specificity, altogether referred to as the “calcium signature” (Tuteja, 2009; Sanders et al., 1999). While animal systems do utilize changes in cytosolic calcium levels as messengers in cell signaling, the calcium signature in its full extent of complexity is a uniquely plant phenomenon (Berridge et al., 2000; Edel & Kudla, 2014). The calcium signature of a signal is interpreted by calcium-binding proteins, including calmodulins (CaMs), calmodulin-like proteins (CMLs), and calciumdependent protein kinases (CDPKs), among other protein families (Ranty et al., 2006; Schulz et al., 2013). These proteins generally feature conserved calcium-binding and EFhand domains, although the latter are absent in some calcium-binding proteins (e.g., calreticulin) (Tuteja, 2009). Often, these proteins undergo a conformational change upon calcium binding that relieves autoinhibitory features of the protein and allows enzymatic action (Ranty et al., 2006; Schulz et al., 2013). A role of calcium in plant gravitropism has been speculated based on several
53
studies showing that calcium chelators, as well as inhibitors of calcium ion channels or calcium-based signaling proteins, inhibit gravitropic response (Masson et al., 2009). Inhibition of Ca2+-ATPase channels, in particular, has been shown to consistently inhibit gravitropic growth (Björkman & Leopold, 1987; Sievers & Busch, 1992; Busch & Sievers, 1993). Additionally, the use of calcium-specific chelator BAPTA (1,2-bis(2aminophenoxy)ethane-N,N,N',N'-tetracetic acid) also inhibited gravitropic growth (Björkman & Cleland, 1991). Similar results were reported with the use of divalent metal ion chelators EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid) and EDTA (ethylenediaminetetraacetic acid) (Lee et al., 1983; Daye et al., 1984). Calcium is proposed to potentially act as a second messenger after gravisensing upon its release from intra- and extracellular stores including from the endoplasmic reticulum, vacuole, and apoplast through calcium ion channels, some of which are mechanotransductive (Sievers & Busch, 1992; Masson et al., 2009; Boonsirichai et al., 2002). Rapid bursts of calcium in the cytosol of cells in the gravistimulated root tip have been reported in Arabidopsis (Plieth & Trewavas, 2002; Toyota et al., 2008) (Figure 11), although imaging this ion at cellular and subcellular levels has been notoriously difficult, especially on the very rapid time scale at which calcium signaling is thought to occur after gravistimulation (Masson et al., 2009; Clore et al., 2013). A rather thorough study performed by Legué and others (1997) using confocal and vertical-stage fluorescence microscopy reported no transient or sustained increase in cytoplasmic calcium levels at the whole-organ, tissue or cellular level in Arabidopsis roots. Their results do not preclude the possibility of subcellular fluctuations in cytoplasmic calcium concentration
54
Figure 11: Calcium signature of gravistimulation. Fluorescence ratios of cytosolic calcium in response to gravitational stimulation as reported by Plieth & Trewavas (2002). Initial reorientation at 135° causes a sharp spike and relatively long-term shoulder response (A). (B), reorienting the plant and immediately returning it to a vertical position elicits a singular spike similar to that seen after a 360° (C) and common to mechanical stimuli (e.g., wind) (D). Reproduced from Plieth & Trewavas (2002).
([Ca2+]c), or that gravitropic calcium signaling functions at such low concentrations that cellular changes were imperceptible with the methods used (Fasano et al., 2002; Clore, 2013). Indeed, the high levels of calmodulin present in gravisensitive tissues would render them responsive to levels of calcium so low that detection would require a reporter more sensitive to lower concentrations and smaller fluxes in cytoplasmic calcium concentration than the indo-1 probe used by Legué and others (1997) (Sinclair et al., 1996; Fasano et al., 2002). In an attempt made to create such a reporter, Knight et al. (1991) generated a more sensitive imaging system using the calcium-activated fluorescent protein aequorin
55
to analyze calcium transients in response to gravistimulation. Aequorin is an isolate from Aequorea victoria (the same jelly in which GFP was discovered) formed by the association of apoaequorin, a polypeptide of ~22 kDa, and coelenterazine (CTZ), the small hydrophobic molecule which aequorin oxidizes in a calcium-dependent manner into the fluorescent coelenteramide (Shimomura et al., 1974). Generally, to analyze cytoplasmic calcium in plants, apoaequorin is driven under the desired promoter, and whole plants or tissues are incubated with coelenterazine (Knight et al., 1991; Sedbrook et al., 1996). Although attempts to image [Ca2+]c in gravitropism using native CTZ have been unsuccessful, Plieth and Trewavas were able to produce reliable results using cyclopentyl (cp)-CTZ, a conjugated form of CTZ which greatly increases the calcium sensitivity of aequorin (Plieth and Trewavas, 2002). They reported a sharp initial spike in [Ca2+]c within 5 s of gravistimulation in Arabidopsis seedlings followed by a less-intense response “shoulder” peaking around 90 s and lasting about 15 min (Figure 11). The spike did not change in intensity with varying degrees of reorientation or adapt to sequential reorientation events, whereas the shoulder reached a maximum intensity when the plant was reoriented 135º and diminishes with each subsequent reorientation (Plieth & Trewavas, 2002). This initial spike is also characteristic of mechanical stimuli – reorientation of the plant by 360º in a mock simulation generated a singular calcium spike like that seen during wind or touch response but did not reproduce the shoulder phase (Knight et al., 1992; Plieth & Trewavas, 2002) (Figure 11). Thus, the researchers propose that the spike and shoulder may have distinct causes, i.e., the spike is purely a function of mechanical stimulation while the shoulder is specifically elicited in response to
56
gravitational stimulation (Plieth & Trewavas, 2002). The duration of the shoulder phase corresponds roughly to the presentation time in Arabidopsis, perhaps relatedly (Plieth & Trewavas, 2002). Whether the duration of this second [Ca 2+]c increase is related to presentation time may be revealed by an experiment measuring [Ca 2+]c upon gravistimulation in a starch-deficient Arabidopsis line like the pgm mutant used by Kiss et al. (1989). These mutants show a significantly increased presentation time, so a longer and potentially less intense shoulder phase would be expected if the relationship between complete amyloplast sedimentation time and the length of the shoulder response is causal. The duration of the shoulder phase of the calcium signature also corresponds to the timing of complete amyloplast sedimentation (Plieth & Trewavas, 2002). However, the relationship between the gravi-induced calcium phase and amyloplast sedimentation is uncertain, as discussed below. Another effort to demystify the calcium response in gravitropism was made by Toyota et al. (2008), who used a photon-counting camera to image cytoplasmic calcium concentrations via aequorin in Arabidopsis seedlings. The group found gravity-induced changes in [Ca2+]c in hypocotyls and petioles, but not in cotyledons or roots; the researchers propose that this may be due to incomplete reassembly of aequorin in the case of the root, as the reporter did not respond to exogenously applied calcium chloride in this organ as it did in the cotyledon. In hypocotyls and petioles, they reported a biphasic increase in [Ca2+]c, with a ~6-fold spike increase within the first ten seconds following reorientation, followed by a return to near-basal levels around 20 s, then followed by a broader ~2- to 3-fold increase from basal levels peaking around ~40 s, finally tapering
57
back to basal levels by 2 min. To analyze this biphasic [Ca 2+]c more fully, Toyota et al. (2008) compared relative aequorin luminesence levels through time after gravistimulation at differing rotational angles and speeds. They found that the initial spike in calcium levels increases its signal strength with increasing clinostat speed, suggesting this phase of the response is correlated to the strength of the gravitational stimulus; the angle of reorientation, however, did not appear to significantly impact the strength of the first phase of the response. Contrarily, the intensity of the second phase of the response was related to rotational angle, reaching a maximum at 135°, but was not changed significantly with varying rotational speeds. They propose that the second wave may respond to deviations from the gravitropic set-point angle. Taken together, Toyota et al. (2008) reached the same conclusion regarding the nature of the biphasic calcium transient as did Plieth and Trewavas (2002): that the initial spike is a signal generated in response to mechanical stimuli, and that the second transient, which they term gravi-induced, is responsive to changes in orientation relative to the gravity vector. Finally, Toyota et al. used inhibitor analyses to study the subcellular origins of the separate phases of the calcium response believed to have different sources. Their results show that the calciumspecific chelator BAPTA completely inhibited both phases of the calcium response at a concentration of 5 mM (Toyota et al., 2008). Additionally, plasma membrane-specific mechanotransductive ion channel inhibitors gadolinium (Gd 3+) and lanthanum (La3+) (both in their free ionic form) dampen the initial phase and significantly weaken the shoulder phase in a dose-dependent manner (Toyota et al., 2008). The endomembrane Ca2+-permeable channel inhibitor, ruthenium red (RR), did not affect the initial phase, but
58
completely ablated the shoulder phase at 0.1 and 1 mM concentrations (Toyota et al., 2008). In light of the the fact that actin inhibitors cytochalasin B and latrunculin B also reduced the second transient, the results of Toyota et al. (2008) suggest that the actin cytoskeleton may contribute to the calcium response of gravitropism by potentially activating mechanotransductive ion channels at the plasma and/or endomembranes. Perhaps surprisingly, amyloplasts do not seem to be the cause of this effect, as eal1 mutants were found to show nearly identical calcium response to reorientation as wildtype seedlings (Toyota et al., 2008). Alternatively, the calcium signal may be related to the activity of inositol triphosphate. Inositol triphosphate (IP3) is a small signaling molecule generated equivalently with diacylglycerol (DAG) from the cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C (Tuteja, 2009). In animal cells, IP 3 is known to trigger the release of calcium stores from the ER and extracellular space by activating Ca 2+-selective ion channels found therein via a phenomenon termed IP 3-induced Ca2+ release (IICR) (Krinke et al., 2006). Similar results have been reported in plants: Schumaker & Sze (1987) reported the release of ionic calcium from the tonoplast in oat following IP 3 application, a result corraborated by Alexandre et al. (1990) in Beta vulgaris roots. In addition to IICR at the tonoplast, Muir and Sanders (1997) demonstrated that plant cell plasma and/or ER membranes also respond to exogenous IP3 in this way. Finally, IP3related calcium signaling has been observed as a response to several abiotic stressors, including cold shock, salt stress, and gravity (Sanders et al., 1999; DeWald et al., 2001; Perera et al., 2006; Im et al., 2010). Among the most rapidly detected changes in
59
gravistimulated cells are increases in levels of IP 3 in grass pulvini within 10 seconds of stimulation, which again implicates calcium-based signaling in the gravitropic response, with this molecule playing an early role (Perera et al., 1999, 2001; Clore, 2013). Furthermore, inhibition of IP3 generation by phospholipase C attenuated the gravitropic response (Perera et al., 2001). These findings in monocots (oat, wheat, and maize) are bolstered by those in Arabidopsis, which shows inhibited gravitropism when expressing a foreign IP3-hydrolyzing enzyme, human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase) (Perera et al., 2006). Several insightful results have been obtained by the Perera-Boss group of North Carolina State University in the interest of further elucidating the role of IP 3 in plant gravitropism, principally in grass pulvinus systems. They have shown that within 10 s of reorientation, a 5- to 6-fold increase in IP3 distinguishes the lower halves of the gravistimulated pulvini from the upper halves, followed by a rapid fall to roughly initial levels (from ~10 s to ~60 s) and then a modest increase (from ~60 s to ~90s), generally mimicking the biphasic calcium response to reorientation observed in Arabidopsis (Perera et al., 1999; Plieth & Trewavas, 2002; Toyota et al., 2008). Unlike the calcium response, this pattern was found to continue to oscillate for at least 30 min after stimulation (Perera et al., 1999). The group also showed that, in the lower half only of the pulvinus, synthesis of IP3 precursor phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphorylation of PIP by PIP 5-kinase shows a large initial spike, limited by bioavailability of PIP, within 10 min of stimulation, then a decrease below initial activity by 30 min, followed by rapid increases through 110 min (Perera et al., 1999). Similar results were shown in oat (Perera
60
et al., 2001). In both cases, the rapid oscillatory changes in IP3 levels are followed by a sustained increase in only the lower halves throughout presentation time roughly until responsive bending begins (Perera et al., 1999, 2001). In each study, the internodal stem regions of the plants tested did not display the fluctuations of IP 3 seen in the pulvini, which also have higher basal levels of the molecule, implying a specificity of this response to gravitropic organs (Perera et al., 1999, 2001). The IP3 response is thought to have a function in early phases of gravitropism based on experiments with cold-treated seedlings. In several plants, gravistimulation in the cold does not induce a response while still in cold temperatures, presumably due to the inhibition of auxin transport at low temperatures; however, when returned to room temperature and vertical orientation, a growth response occurs as determined by the stimulus made in low temperatures in angle and duration (Fukaki et al., 1996a; Wyatt et al., 2002). Thus, gravistimulating plants in the cold is a useful method to separate study of gravitropic sensing and response. Transgenic Arabidopsis seedlings expressing an IP3hydrolyzing enzyme reoriented in cold treatment showed a reduced gravitropic response when returned to room temperature with growth reduced 50-60% compared to wild-type, showing that IP3 functions upstream of auxin in gravitropism (Perera et al., 2006). This is in fact a stronger inhibition than the 30% lessening of gravitropic growth of these transgenic plants when reoriented at room temperature (Perera et al., 2006). Results in wild-type oat stem seedlings show that IP3 fluctuations prompted by gravistimulation occur as usual at 4° C, as would be expected for elements of gravitropic sensing (Perera et al., 2001). While IP3 shows a sustained increase well into the later phases of sensing
61
and signal transduction, these experimental results suggest that the function of IP 3 is likely limited to the early phases of sensing and functions upstream of polar auxin transport (Perera et al., 2006). Despite the numerous results implicating IP3 in not only gravitropic signaling but also in response to several other stimuli, efforts to identify a plant IP 3 receptor have been unsuccessful (Krinke et al., 2006; Im et al., 2010). Genetic screens for homologs of the animal IP3 receptor (IP3-R) in Arabidopsis yielded some potential candidates, but their sequences do not suggest transmembrane or ion-pumping features of a putative IP 3activated Ca2+ ion channel (Lin et al., 2004; Krinke et al., 2006). Most likely, this search has been troubled by a lack of homology between the plant and animal receptors for this molecule. Although more difficult and tedious, a protein-based assay identifying and characterizing as many binding partners of IP3 as necessary, including (receptor-like) proteins and small molecules, may be a more useful approach than genetic screens in this case. Identifying and analyzing the IP3 receptor in plants should help clarify its part in gravisensing and signal transduction. Another subject worthy of further pursuit that may help clarify these questions regarding phosphoinositol-based signaling is the potential role of DAG (I. Perera, personal communication to A. Clore), which again is produced equivalently with the generation of IP3 from PIP2 from PLC. Calcium and calcium-based signaling are further implicated in early phases of gravitropism by the participation of calmodulin (CaM). First, calmodulin protein levels in Arabidopsis roots are highest in the root cap and meristem, and transcription of calmodulin is increased upon gravistimulation (Allan & Trewavas, 1985; Stinemetz et
62
al., 1987; Sinclair et al., 1996). Additionally, calmodulin inhibitors weaken gravitropic growth in maize and Arabidopsis roots (Björkman & Leopold, 1987; Stinemetz et al., 1992; Sinclair et al., 1996). Calmodulin functions as a molecular sensor of calcium concentration, and its importance as a signaling element in all eukaryotic cells can hardly be overstated (Berridge et al., 2005). In plants, more than 50 proteins known as calmodulin-related proteins (CMLs) and several isoforms of calmodulin itself (in Arabidopsis, four; tobacco, three) are genetically diverse and create a very specific and varied Ca2+/calmodulin signaling network (Ranty et al., 2006; Bender & Snedden, 2013). Insufficient work has been done to identify specific CMLs and their interacting partners that are likely responsive to gravitropism; to do so is the logical next step given gravitropism-related enrichment of calmodulin expression, and should be pursued. Overall, the participation of calcium signaling in early gravitropism appears eminent, which is consistent with its widespread function as a second messenger. Determining the subcellular localization of cytosolic calcium, IP 3, and calmodulin within gravistimulated cells may help elucidate some very early events in gravisensing and signal transduction.
2.4.
Reactive oxygen species
Reactive oxygen species, including the superoxide radical ( •O2-), hydrogen peroxide (H2O2), the hydroxyl radical (•OH), and singlet oxygen (1O2), have traditionally been regarded as the result of malfunctions in electron transport chains in the mitochondria and chloroplasts and as toxic stressors known to cause oxidative damage in
63
cells (Joo et al., 2001; Mori & Schroeder, 2004; Wrzaczek et al., 2013). More recently, ROS have also been have also been realized in plant and animal systems to be tightly regulated as signaling molecules, including their production for the purpose of signaling, often connecting environmental stressors to downstream changes in metabolism and gene expression (Joo et al., 2001; Mittler et al., 2001; Choudhury et al., 2016; Dietz et al., 2016). Consistent with its part in regulating other tropisms and potentially mechanotransductive events, rapid ROS signaling has been reported in response to gravistimulation in Zea mays roots and pulvini (Joo et al., 2001; Mori & Schroeder, 2004; Clore et al., 2008). These signaling events include the proliferation of ROS species, particularly H2O2, and the enrichment of expression of oxidative stress-related genes (Joo et al., 2001; Moseyko et al., 2002; Clore et al., 2008). One such upregulated element is an IRP1 (Iron Regulatory Protein1) homolog, the transcript of which found to be localized to bundle sheath cells of the pulvinus upon gravistimulation (Clore et al., 2008). Interestingly, IRP1 is known to function as something of a redox sensor in animal systems (Pantopoulos & Hentze, 1998; Mütze et al., 2003). In maize stem explants containing pulvini, gravitropic curvature was inhibited or even reversed when H 2O2 was applied to the upper flank or when the ROS scavenger ascorbic acid was applied to the lower flank (Clore et al., 2008). One interpretation of these results is that a ROS gradient may inform the direction of responsive bending (Clore et al., 2008). Consistent with this general notion, Joo et al. (2001) observed bending towards a hydrogen peroxide source in vertical roots and inhibition of root curvature following the application of antioxidant Nacetyl-cysteine (NAC) (Joo et al., 2001). The effect of H2O2 is not simply one of
64
Figure 12: ROS production in maize pulvinus bundle sheath cells following gravistimulation is correlated to starch content. A cross-section from the lower pulvinus half of a maize plant depleted of starch (amyloplasts) by growth in dark (left) and a control (light-grown) maize plant (right) were stained for ROS production after 30 min gravistimulation by reorientation. Note the well-defined concentration of fluorescence in the basal portions of many more bundle sheath cells (examples indicated by arrowheads) at right surrounding the vascular bundles (which show mild autofluorescence in the sample at left). Reproduced from Hartwell (2016).
enhancement or inhibition, as pulvini bend away from endogenous peroxide sources while roots bend towards them, regardless of the orthogravitropic direction (Clore et al., 2008). Finally, visualization of ROS generation in maize pulvini using 3,3'diaminobenzidine (DAB) shows a distribution suggestive of amyloplast outlines (i.e., immediately surrounding what appear to be amyloplasts) within 1 min following gravistimulation (Clore et al., 2008) and is correlated to amyloplast starch content (Hartwell, 2016) (Figure 12). In at least the pulvinus, the ROS production in response to gravistimulation is characterized by both short (prior to polar auxin transport)- and longer-term fluctuations, with production evident around bundle sheath amyloplasts by 1 min and asymmetry between upper and lower halves of the pulvinus apparent at 30 min, 5 h and 72 h after gravistimulation (Clore et al., 2008; Hartwell, 2016). In maize roots, 65
ROS
production
can
be
auxin-induced
and
dependent
on
the
action
of
phosphatidylinositol 3-kinase, suggesting an interesting crosstalk between these different signaling pathways (Joo et al., 2001, 2005). Auxin-ROS crosstalk is established in later phases of gravitropism, but the earlier bursts of ROS described in the literature and recapitulated here are prior to auxin redistribution, and their function remains to be shown (Clore et al., 2008; Clore, 2013). Recently, a new brood of ROS-sensing reagents have been employed for plant use which offer more sensitive and less invasive methods of live cell imaging (Swanson et al., 2011). Some of these are based on fluorescein, including Singlet Oxygen Sensor Green sensitive to 1O2 and OxyBurst Green, which reports H2O2 (Swanson et al., 2011; Hartwell, 2016; Zhang et al., 2016) (Figure 12). Others are genetic elements based on GFP and related fluorophores (i.e., YFP and RFP), such as HyPer and HyPerRed (Swanson et al., 2011; Chiu et al., 2014; Ermakova et al., 2014). These sensors have in a few cases been employed in gravitropism research, and have provided high-quality imaging and resolution of ROS generation (Hartwell, 2016). Their continued use in the field should certainly help to clarify the subcellular origin of gravistimulus-generated ROS, their interactions with other cellular components, and their function in this signaling pathway. Unfortunately, employment of such genetically-encoded sensors will be delayed in the model monocot maize, which remains much more difficult to transform than Arabidopsis, tobacco, and other eudicotylenous plants.
66
2.5.
Cytosolic and apoplastic pH
Because it can affect enzymatic efficiency, protein conformation, protein-protein interactions and enzyme-substrate binding, it is perhaps unsurprising that pH serves to regulate or act as a second messenger to several cellular processes and signaling pathways in plants and animals alike (Scott & Allen, 1999). The relation between apoplastic (extracellular) pH and root growth was first described in auxin-mediated cell elongation as the acid growth theory, which states that auxin triggers H + release from affected cells into the apoplast, where the lowered pH allows cell wall loosening and elongation (Rayle & Cleland, 1970). As a result, most earlier studies probing the relationship between hydrogen ions and gravitropism focused on the effect of cell wall pH on the graviresponse phase (Taylor et al., 1996). More recently, it has been discovered that rapid changes in cytosolic pH specific to columella cells, particularly tier 2 and 3 columella cells, are found upon reorientation in Arabidopsis roots (Scott & Allen, 1999; Fasano et al., 2001). Notably, tier 2 and 3 cells are the columella cells most responsible for gravisensing (Blancaflor et al., 1998). Treatment of the root cap with acidic or basic solution enhanced or inhibited gravitropic curvature, respectively (Scott & Allen, 1999). A directional gradient was reported across tier 2 cells with lower cells more alkaline for the first ~5 min after reorientation, after which the gradient dissipates (Scott & Allen, 1999). However, Fasano et al. (2001) were unable to replicate that result; they found that individual cells of both tiers show an increase of cytoplasmic pH by 0.3-0.4 units within 2 min. In each study, the pH changes reported were correlated to amyloplast content and sedimentation by microscopy-based
67
analysis and through the use of the starchless pgm1 mutant (Scott & Allen, 1999; Fasano et al., 2001). In pgm1 mutants, the rapid pH changes prompted by reorientation in wildtype roots are completely absent (Fasano et al., 2001). This suggests that the pH changes seen in statocytes after gravistimulation is likely to be causally related to amyloplast sedimentation, or closely accompanying events such as cytoskeletal disruption (Hashiguchi et al., 2013). Comparable findings were found with arg1 mutants, which, as discussed in section 2.1, are deficient in a protein speculatively identified as an actin- and possibly plastid-binding partner involved in early gravitropic signaling (Sedbrook et al., 1998; Boonsirichai et al., 2002, 2003; Harrison & Masson, 2008b). Thus, the function of pH changes in gravitropism may not be a direct consequence of sedimentation per se, but perhaps of cytoskeletal perturbations that may be triggered by amyloplast sedimentation. Similar to results indicating changes in pH in collumella cells of Arabidopsis roots, Johannes et al. (2001) reported pH changes in maize pulvinal cells. Specifically, they saw an alkalinization in the lower half of individual bundle sheath cells and alkalinization of the sides of the cells 5-10 min after gravistimulation, and that these effects were absent in neighboring, agravitropic parenchyma cells (Johannes et al., 2001). The exact function of these cytosolic pH changes remains unclear, partially due to the ubiquitous relevance of pH in cell and molecular biology. It appears that protons are actively involved in cell signaling in the early phases of gravitropism, and that their role is perhaps related to those of [Ca2+]c, IP3, and the actin cytoskeleton; however, the exact roles of these signal transducers will only be able to be clearly delineated with the extensive use of more sophisticated imaging techniques for each of them (Boonsirichai et
68
al., 2003; Toyota et al., 2008; Monshausen et al., 2011; Swanson et al., 2011).
2.6.
Alternate means of graviperception
As alluded to in Chapter 1, the starch-statolith hypothesis has not been sufficient to explain all aspects of plant gravitropism which have been observed. Thus, several alternative means of graviperception have been theorized, including the protoplast pressure and tensegrity models, as well as the possibility of nuclei or other nonamyloplasts acting as statoliths, and the occurrence of graviperception in nonspecialized cells. Moreover, it is increasingly commonly being considered possible and in fact likely that any number of these mechanisms of gravisensing may be employed in the full breadth of gravitropism in higher plants (and perhaps even within the same plant or same cell), the integration of which remains far from well-understood (Barlow, 1995; Baldwin et al., 2013). The most commonly theorized alternative means of gravisensing, known by several names (the gravitational/protoplast/hydrostatic pressure model, static/passive sensing, and “autosensing” among them), proposes that the plant cell perceives changes relative to the gravity vector by the weight and/or buoyancy of the whole cell, via deformations and tension/compression forces acting on the plasma membrane and/or cytoskeleton, rather than by the sedimentation of dense organelles. The model has been theorized as possible since the nineteenth century (reviewed by Staves, 1997), but in the modern era has found a principal advocate in a group of researchers at Cornell University (Wayne et al., 1990; Staves, 1997; Staves et al., 1997a, 1997b, 1997c). These researchers
69
showed that gravistimulation alters the polarity of cytoplasmic streaming in characean algae Nitellopsis and that cell suspension in adequately dense media destroys or inverts the orientation of cytoplasmic streaming (Wayne et al., 1990). Additionally, laser ablation of plasmodesmata (narrow cytoplasmic channels extending through cell walls and thereby connecting neighboring plant cells) or ultraviolet irradation of either end of internodal cells (to disrupt plasma membrane-cell wall connections) eliminated gravitropic sensitivity (Wayne et al., 1990). Thus, Wayne et al. concluded that characaean cells are normally able to sense the weight of their protoplast, that this ability allows graviperception and is affected by the medium suspending these cells, and that the statoliths of higher plants simply act as ballasts to increase the weight and density of the statocyte so as to increase the signal-to-noise ratio of the falling protoplast against the thermodynamic noise of the cell (Wayne et al, 1990). Their tryptych of 1997 publications report that: treating characaean algae with media of equal or greater density to the cytoplasm ceases or reverses the cytoplasmic streaming of the algal cells which is oriented around the gravity vector in the cell; that growing rice plants in increasingly dense media results in an increasingly weak gravitropic response; and that actin polymerization inhibitors do not inhibit gravitropism in rice, corn, or Arabidopsis (Staves et al., 1997a, 1997b, 1997c, respectively). Regarding the first two results, the researchers underscore that gravitropism is altered when the density of the surrounding medium is altered, although cytoplasm density and amyloplast sedimentation rates are not affected themselves. This is argued as evidence for the notion that gravitropic cells are able to sense their own weight relative to their surroundings and
70
that this informs gravisensing, at least in part (Staves et al., 1997a, 1997b). In the third publication, the researchers argue that contact between sedimenting amyloplasts and actin microfilaments of the cytoskeleton cannot be an essential event of gravitropism in light of their results (Staves et al., 1997c). Incorporating principles from both the protoplast pressure and the starch-statolith models, the tensegrity model is a more recently-developed theory of gravisensing based on the aforementioned principal of tensegrity put forth by Ingber (1993). This model proposes that a fine mesh of actin throughout the cell, connected to microtubules and to membranous organelles, transmits mechanical force to stretch-sensitive receptors in the plasma and/or endomembranes (Staehelin et al., 2000). Disruption or repositioning of any organelle(s) within the cell, and/or of the plasma membrane and connected actin cytoskeleton, would thus result in gravitropic signal transduction (Staehelin et al., 2000). Importantly, both amyloplasts and the whole protoplast, including its other constituent organelles, could function as the susceptor organ in this model. The nodal ER complexes concentrated at the distal and side walls of gravisensing cells are proposed to act as a physical protective barrier against harmful membrane deformation caused by the force of amyloplast sedimentation, and additionally may help to specify a directionality to the gravitropic signal (Zheng & Staehelin, 2001). Overall, the extensive amyloplastcytoskeleton-endomembrane interconnection and mutual responsibility in graviperception put forth in the tensegrity model is supported by some experimental evidence as reviewed in Sections 2.1 and 2.2. However, the model as it was originally suggested includes that amyloplasts are untethered to the actin cytoskeleton or plasma membrane and thus favors
71
an unrestrained mode of gravisensing, whereas recent studies have shown a potentially extensive and certainly active role of actin in amyloplast sedimentation, as discussed in Section 2.1. Additionally, the originally-posed tensegrity model of gravisensing casts actin as a gravireceptor, which has repeatedly been shown to be contrary to the abundance of experimental results which suggest that actin actually dampens gravitropic sensing and signaling, acting instead as a fine-tuning negative-regulatory mechanism (Blancaflor, 2013). Despite these flaws, the tensegrity model clearly has a place in gravitropism research as the first model to unite sometimes contentious schools of thought regarding the mechanism of graviperception, namely the starch-statolith and protoplast pressure models. Some results have demonstrated the potential for some level of graviperception in the root to occur outside of the root cap based on early centrifugation experiments by Piccard (1904) and Haberlandt (1908) and similar experiments that have been performed since (Haberlandt, 1914; Poff & Martin, 1989). Additionally, evidence identifying the root cap as the principal site of graviperception does not necessarily show that it is the only site (Juniper et al., 1966; Poff & Martin, 1989; Blancaflor et al., 1998). Thus, Wolverton et al. (2002) constructed a live imaging device, “ROTATO”, featuring a rotating platform able to maintain root caps in a constant orientation while gravistimulating other parts of Arabidopsis roots at a specified orientation. Using the ROTATO system, they found that the elongation zone contributes up to 20% of the gravitropic stimulus, which is generally consistent with the finding by Kiss et al. (1989) that starchless Arabidopsis seedlings are about 30% as gravicompetent as wild-type roots
72
(Wolverton et al., 2002). Additionally, the reduced graviresponse in these starchless pgm1 mutants happens at a continual 8 degrees per hour regardless of the root cap angle, unlike the root cap-angle-dependent rate of wild-type gravitropic growth (Wolverton et al., 2011). This suggests that the reduced gravitropism in these starchless mutants, and perhaps in the amyloplastless cells of the elongation zone, may function via an altogether different mechanism than those speculated to mediate statolith-based gravitropism in statocytes (Wolverton et al., 2011; Baldwin et al., 2013). A final interesting, though insufficiently probed, possibility is that other organelles may sediment and act as susceptors. The nucleus has been identified as a potential statolith, as it is among the densest organelles following amyloplasts (1.14 g/cm3 and 1.44 g/cm3, respectively), with the nucleolus being particularly dense (1.5g/cm3) (Sack, 1991). The nuclei of gravisensitive cells generally remain in position upon gravistimulation, in large part because they are tethered in position by the cytoskeleton (Hensel, 1989). Previously, this fact was used to argue against the role of the nucleus as a susceptor under the principle that the gravisensitive susceptor should show consistent relocation upon gravistimulation (Sack, 1991). However, more recent results showing that rather slight organellar movements can trigger gravisensing reopens the possibility that the nucleus and extensive actin network holding it in place may contribute to gravisensing as secondary a secondary susceptor. The changes in size and shape of the nucleus upon gravistimulation noted by Kordyum and Guikema (2001) could, in theory, have relevance to gravisensing or signal transduction, as well. Amyloplasts clearly are the most important gravity susceptor in many plant
73
systems. However, it is also clear that the sedimentation of amyloplasts cannot be the singular event that begins the gravitropic signaling cascade. Similarly, it seems increasingly likely that the canonical gravitropic signaling pathway as triggered by amyloplast sedimentation may not be the only means by which plants have come to adapt to this ever-present stimulus (Barlow, 1995; Perbal, 1999; Baldwin et al., 2013). A complete understanding of this phenomenon will require renewed and continual probing of these proposed alternative mechanisms to determine which of them actually function in planta, and whether these alternative signaling events converge on the same transduction/response cascade or act through an entirely different form of gravitropism.
2.7.
The TOC complex: a model of interconnectedness in gravitropic signaling
An extraordinary example of the intricate connection of these cellular elements in early-stage gravitropism is represented by the role of the TOC (Translocon at the Outer envelope membrane of the Chloroplast) complex in gravitropism. The TOC complex is an assembly of a pore protein (Toc75) and one member each of two receptor protein families (the Toc159 and Toc34 families) (Strohm et al., 2015). These complexes form in the outer membrane of plastids (first discovered in chloroplasts, as the name suggests) and allow the transport of nuclear-encoded proteins into the plastid (Stanga et al., 2009) (Figure 14). While null mutants of the pore protein complex are embryo-lethal (Baldwin et al., 2005; Stranga et al. 2009) successfully cultivated two defective-protein mutants, mar1 and mar2, formed by the substitution of a conserved amino acid in the pore-
74
forming region of Toc75 and by a premature stop codon in Toc132 (of the Toc159 family), respectively (Strohm et al., 2015). Interestingly, mar1 and mar2 were first identified as mutant phenotypes severely worsening the agravitropic phenotype of arg1 mutants by Boonsirichai et al. (2002). These mutants show a gravitropic response indistinguishable from wild-type as single mutants, but in arg1 backgrounds form a completely agravitropic phenotype; arg1 single mutants show a weakened but present gravitropic response in the roots and shoots (Boonsirichai et al., 2003; Stanga et al., 2009). Interestingly, Toc75 and ARG1 do not colocalize within the cell: Toc75 is confined to plastid membranes while ARG1 is found in several components of the endomembrane system, primarily found membrane-associated rather than free in the cytosol and sometimes also associated with the cytoskeleton (Sedbrook et al., 1998; Baldwin et al., 2005; Stanga et al., 2009; Strohm et al., 2015). Precisely how these proteins interact to mediate the gravitropic response remains unclear. One possibility is that the TOC complex controls the uptake of an unknown gravitropic signal and its integration into the amyloplast outer membrane, where either the signal itself (Figure 13A) or TOC132 (Figure 13B) perhaps come into contact with ARG1 in endomembranes upon gravistimulation and sedimentation (Stanga et al., 2009; Strohm et al., 2015). These preliminary results and hypotheses point to the potentially extensive interconnected nature of several cellular components during early-phrase gravitropism, including the cytoskeleton, ER, plasma membrane, and amyloplasts. Notably, this type of system would allow for an active role of amyloplasts in gravity sensing or transduction, rather
75
than simply being controlled by gravity and the actin cytoskeleton. The TOC complex could also represent a ligand-receptor form of gravisensing in higher plants as described in characean algae (wherein statoliths must make contact with specific sites at the distal plasma membrane), potentially uniting two realms of gravitropism research often considered difficult to reconcile (Braun, 2002; Limbach et al., 2005; Masson et al., 2009; Stanga et al., 2009). Clearly, further study of the TOC complex and its interacting partners during early gravitropism will provide valuable insight into the nature of the gravistimulus and the mechanism of signal transduction.
Figure 13: Possible mechanisms for a TOC complex—ARG1 interaction function in gravitropism. (A), the TOC complex regulates the uptake of a receptor, Y, which is incorporated into the amyloplast membrane and interacts with ligand X, whose position is determined by ARG1. (B) The TOC complex itself (particularly TOC132) directly acts as the receptor to ligand X, again localized under the control of ARG1. Reproduced from Stanga et al. (2009).
76
2.8.
Conclusion
When considering the many aspects of gravitropic sensing and signal transduction, it is not only difficult but counterproductive to consider them in isolation. It is becoming increasingly apparent that the cytoskeletal and endomembrane systems form a dynamic and specialized network throughout the cell which is likely able to sense gravity through several means, not the least of which is amyloplast-dependent. This network is rapidly able to induce subcellular changes in pH, ion concentrations and ion species profiles to signify gravitational direction and establish a basis for gravitropic response. Though the finer molecular workings of the earliest events of gravitropism are still far from well-understood, this increasingly clear picture emerging of the unique nature of gravisensitive cells and how this nature informs and allows gravity sensing and signal transduction is developing rapidly. The factor connecting these highly sensitive elements to the earliest phases of signal transduction remains unknown, but it is likely that it functions via a tensegrity-based stimulus.
77
CHAPTER 3: COMMITMENT TO RESPONSE: FROM SEDIMENTATION TO PRESENTATION TIME As discussed in Chapter 1, perception of changes in position relative to the gravity vector can be detected by plants cells extremely quickly and very early signaling events follow within 10 sec, and likely earlier. Presentation time, the time length required of a gravitropic stimulus to cause a growth response, is on the order of 10-30 sec in Arabidopsis roots and shoots and ~2-4 h in maize stems (Kiss et al., 1989; Perera et al., 1999). It is only a bit later that the plant begins to show responsive growth – reaction time begins at about 15 minutes in Arabidopsis, and 8 hours in maize (Moore & Cogoli, 1996). Between these time points, a cellular or subcellular signal generated in the statocytes is sustained past the time of sensing, often transported a distance to the site of response (with the pulvinus system being an example where the response site is the same as the site of sensing), and transmitted into a differential growth response. This is accomplished through diverse and widely-implicated signal transduction pathways, such as MAP kinase activity, Ca2+/calmodulin-signaling, the formation of ROS gradients, and cell-to-cell translocation of factors like auxin. These means to the establishment of tissue polarity starting from subcellular signals are the subject of the present chapter.
3.1.
MAP kinase activity
IP3-, calmodulin-, and ROS-based signaling pathways are identified in Chapter 2 as among the earliest apparent components of gravitropism. The gravitropic function of
78
these pathways may also be related in that they all can stimulate activity in MAP kinase signaling in other plant systems (Clore et al., 2003; as reviewed in Mittler et al., 2011). The necessity of MAP kinase activity for maize shoot gravitropism was shown by Clore et al. (2003) using the MAPK kinase (MEK) chemical inhibitor U0126, which reduced gravitropic curvature by nearly 65% in half-stem and whole maize explants containing graviresponsive pulvini. MEK inhibitors also prevent the fluctuations of MAP kinase activity between upper and lower pulvini halves that are observed in chemically untreated gravistimulated stems (Clore et al., 2003). These fluctuations appear to vary in pattern within the first hour following stimulation, becoming more significant at 75 min and resulting in a sustained increase of MAPK activity in the upper half of gravistimulated pulvini over the lower half from 2-3 h (Clore et al., 2003). This transition from variable fluctuation to a dramatic and well-established gravitationally-oriented gradient in MAP kinase activity may serve as a way for the maize pulvinus system to differentiate between transient stimuli and sustained reorientation requiring full response (Clore et al., 2003). It is roughly at the end of this time frame that adequately gravistimulated (4.5+ h) maize pulvini begin to show significant asymmetry of tissue IP 3 at sustained levels (rather than the transient fluctuations shown within 10 s), with lower halves showing a maximal ~5fold increase over the upper halves peaking at 6-8 h, again lasting roughly until bending begins (Perera et al., 1999). Use of another chemical MEK inhibitor, PD98059, on the lower half of gravistimulated maize roots significantly inhibited responsive curvature while application to the upper half enhanced gravitropic response; asymmetrical application to unstimulated roots did not affect growth (Liu et al., 2009). These results
79
point again to a role for MAP kinase signaling in maize gravitropism. Considering the time frame of their invocation in gravitropic signaling, their role in establishing whole-tissue gradients in response to gravistimulation, and their generally diverse and wide-reaching effects on cell signaling and physiology, it seems both likely and logical that MAP kinase activity may contribute an important transition from a subcellular gravisensing phase to a whole-organ response phase.
3.2.
Auxin asymmetry and lateral transport
Auxin signaling and the factors that contribute to it could readily comprise the entire volume of the present work; it is probably the single-most extensively researched topic in plant gravitropism. To detail the evolution of the field or to provide a fully comprehensive view of everything known about the subject is well beyond the purview of this thesis. Consistent with the goals of this work, this section will focus on the recent developments in the literature that remain inconclusive. Much of the work fitting this criteria seeks to elucidate the underlying molecular bases for phenomena that are often long-known and well-established, e.g., the accumulation of auxin in the basal side of gravistimulated organs. As was explained in Chapter 1, auxin is the principal factor that transports the gravitropic signal from statocytes to the site of response and drives the differential growth response during reorientation. The chemiosmotic hypothesis argues that the movement of auxin requires influx and efflux proteins, and has been supported by several molecular and genetic analyses performed since then (Rubery & Sheldrake, 1974; Raven, 1975;
80
Boonsirichai et al., 2002). Supporting results show that the initially symmetrical distribution of auxin efflux carriers in the statocytes is rearranged upon gravistimulation to favor the lower flank of the cell, and in neighboring cells the relocalization of auxin influx and efflux carriers directs movement of the hormone to the site of response (basipetally in roots, or away from the root cap and towards the root/shoot interface) (Boonsirichai et al., 2002; Friml et al., 2002; Young et al., 2006; Geisler et al., 2013). The integrity of this auxin influx-efflux carrier system is critical for full gravitropic response (Friml & Palme, 2002; Friml et al, 2002; Friml, 2003; Swarup et al. 2005). In short, PIN3 and PIN7 are thought to begin the auxin translocation process by transporting the hormone out of statocytes (Friml et al., 2002; Feraru et al., 2015). PIN2 is responsible for moving auxin from lateral root cap cells to the cortex and epidermis cells of the elongation zone (Sato et al., 2015). AUX1 and the three other members of its gene family, LAX (LIKE AUXIN)1-3, appear to be the principal influx carrier regulating gravitropic auxin signaling throughout the length of the root (Geisler et al., 2013; Feraru et al., 2015). Inward direction of auxin via PIN proteins (towards the vascular core) in the elongation region epidermis and cortex cells and the disruption of the directional auxin gradient once 40º of bending growth has been achieved later serves to restore symmetric auxin and vertical growth (Blilou et al., 2005; Band et al., 2012). PIN3 is uniquely expressed in columella cells in Arabidopsis root caps, where its initially symmetric distibution in the plasma membrane changes into a highly asymmetric distribution favoring the bottom flank of the cell within 2-5 min post-stimulation (Friml et al., 2002). The relocalization of PIN3 upon gravistimulation is regulated by and
81
requires the function of ARG1 and ARL2, as shown by knockout studies performed by Harrison and Masson (2008a). In arg1 and arg2 mutants, PIN3 redistribution to the 'bottom' flank of gravistimulated statocytes is severely impaired, and auxin movement out of the statocytes is similarly reduced (Harrison & Masson, 2008). Curiously, auxin levels in columella cells increase in these mutants, and the columella cell identity appears to be assumed by the neighboring lateral cell flanks, based on auxin and starch levels in these neighboring cells (Harrison & Masson, 2008a). Finally, arg1 and arl2 mutants show normal photoresponsive growth (unlike pin3 mutants), and columella-specific expression of their respective proteins rescues the root gravitropic phenotype, suggesting that the ARG1-ARL2-PIN3 pathway is a specifically gravitropic one (Harrison & Masson, 2008a). Because a functional redundancy between PIN3 and PIN7 is suggested by their colocalization in vivo and the greater agravitropic severity of pin3 pin7 double mutants compared to either single mutant, another ARG-ARL-PIN7 complex may also contribute to signal transduction (Friml et al., 2002; Sato et al., 2015). The initial relocalization of PIN3 and PIN7 to the gravitational bottom of the cell is also mediated by the vesicle trafficking proteins which control their continual recycling through the plasma and endomembranes (Kleine-Vehn et al., 2010). The ARP (ACTIN RELATED PROTEIN)2/3 complex has been implicated in not only actin polymerization, but also in vesicle trafficking and tropic responses; the ARP3 subunit, in particular, has been proposed to potentially regulate auxin signaling via vesicle trafficking (Reboulet et al., 2010; Rotty et al., 2013). Zou et al. (2016) investigated the role of ARP3 (also known as DIS1) in gravitropic signaling, and found that it seems to, in fact, contribute to
82
multiple phases of gravitropism. First, interaction between ARP3 and actin appears to regulate amyloplast movement in statocytes, and arp3 null mutation causes cable-like actin to form in the columella cells from which it is usually absent (Zou et al., 2016). Interestingly, the application of latrunculin B restores gravitropic curvature in arp3 mutants to wild-type, or to pgm single mutant levels in arp3 pgm double mutants. Latrunculin B also restored asymmetric auxin distribution in arp3 mutants, perhaps by disrupting the cable-like actin induced by the mutation (Zou et al., 2016). Additionally, arp3 mutants show defects in endocytosis and aberrant PIN2, PIN3 and PIN7 localization and aggregation (Zou et al., 2016). Once auxin is moved radially outward from the columella cells of the root cap, its shootward transport is mediated by PIN2 (also known as AGR1, EIR1, and WAV6) and AUX1 (Boonsirichai et al., 2002; Blilou et al., 2005; Geisler et al., 2013; Sato et al., 2015). The differential auxin gradient across the upper and lower halves of the root generated at the columella appears to be maintained by differential regulation of PIN2 by proteosome and vesicle trafficking proteins, as shown by Abas et al. (2006). Clathrinmediated endocytosis appears to be the primary form of PIN protein endocytosis in Arabidopsis (Dhonukshe et al., 2010). In the upper half of gravistimulated Arabidopsis roots, PIN2 is maintained at lower concentrations than in the lower half, promoting supraoptimal auxin concentrations known to inhibit growth to accumulate in the lower half (Blilou et al., 2005; Abas et al., 2006). When roots are treated with vesicle trafficking inhibitor brefeldin A (BFA) or proteosome inhibitors (e.g., MG132 and clastolastacystin-β-lactone (LAC)), wild-type PIN2 and ubiquitously-driven PIN2 in an eir1-4
83
35S:PIN2 double mutant line both showed increased levels as determined by western blot and fluorescence analyses, although transcription levels were unaffected (Abas et al., 2006). Treatment with these chemicals also prevented localization of PIN2 to the plasma membrane and inhibited root gravitropism (Abas et al., 2006). These results are consistent with previous results from Geldner et al. (2001) showing that BFA caused similar defects in PIN1 turnover and distribution and inhibited gravitropism. Another PIN2 mutant, wav6-52, bears a hyperstabilized form of PIN2 and also shows a severe root agravitropic phenotype, further highlighting the importance of PIN2 endocytosis and degradation in gravitropism (Abas et al., 2006). Other factors that affect PIN2 cycling and localization in Arabidopsis are the small secretory gloven (glv) peptides (Whitford et al., 2012). The GLV peptide family has also been identified as the ROOT GROWTH FACTOR (RGF) family, although not all GLV/RGF proteins are found in the root (Whitford et al., 2012). Overexpression of GLV1, GLV2 or GLV3 resulted in agravitropic phenotypes in roots and reduced curvature in shoots, and application of synthetic GLV1, GLV2 and GLV3 peptides prevented the establishment of a PIN2 and thus an auxin gradient (Whitford et al., 2012). This appears to function by the inhibition of endocytosis from the plasma membrane and thereby preventing the differential turnover of PIN2 that leads to auxin asymmetry (Whitford et al., 2012). Like PIN2 itself, some GLV peptides are responsive to auxin, showing rapid transcriptional upregulation after auxin treatment (Whitford et al., 2012). The trafficking modulator TENin1 (trafficking and endocytosis inhibitor1/TE1) was identified in a large chemical screen as producing inhibited gravitropism in
84
hypocotyls (Surpin et al., 2005). Later, it was found by Paudyal et al. (2014) to cause increased endocytic accumulation of membrane proteins, including PIN2 as well as brassinosteroid receptor BRI1 and aquaporin subunit PIP2a. TE1 appears to promote the retention of PIN2 in the pre-vacuolar complex (PVC) and plasma membrane (particularly the PVC) rather than its recycling back to the trans-Golgi network (TGN) (i.e., endocytosis) whereas its exocytosis to the PVC, vacuole, and plasma membrane appear uninhibited (Paudyal et al., 2014). Therefore, the form of endocytosis disruption apparently caused by TE1 differs from those caused by other small-molecule endomembrane trafficking inhibitors such as BFA and wortmannin, which primarily affect traffic to and from the plasma membrane (Hicks & Raikhel, 2010; Paudyal et al., 2014). Paudyal et al. (2014) also identified two Arabidopsis ecotypes that are resistant to the gravitropism-inhibiting effects of TE1, which may be used to further determine the molecular basis for the chemical's effect on PIN trafficking and gravitropism. Because RAC/ROP (Rho of Plants) GTPases have been implicated as components of auxin signaling in general in root hair development and epidermal pavement cell patterning in particular, Lin et al. (2012) sought to determine if ROP GTPases mediate PIN distribution in polar auxin transport (Zheng & Yang, 2000; Wu et al., 2011). To do so, they performed a genetic screen for mutant Arabidopsis lines in which ROP6 overexpression causes more extreme phenotypes than wild-type and identified spk1-4, a partial loss-of-function mutation of SPK1 (SPIKE1) (Basu et al., 2008; Lin et al., 2012). Indeed, spk1-4 mutants appear to be defective in PIN2 polar localization and endocytosis (Lin et al., 2012). spk1-1 pin2 double mutants show identical root phenotypes to either
85
spk1-1 or pin2 single mutants, and spk1-1 single mutants show decreased auxin levels outsite of the root tip compared to wild-type, failure to generate an asymmetric auxin gradient in the root elongation zone, and decreased gravitropic curvature (Lin et al., 2012). The GFP::PIN2 fluorescent signal is weaker in the plasma membrane and stronger in the cytosol in spk1 backgrounds compared to wild-type, suggesting that SPK1 promotes PIN2 retention to the plasma membrane (Lin et al., 2012). SPK1 is known as a Rho guanine nucleotide exchange factor (GEF) of ROPs (Basu et al., 2008). Consequently, Lin et al. (2012) compared rop phenotypes to the spk1 phenotype to identify the likely target of SPK1. They found that the rop6 phenotype was the most similar, bearing several of the pleiotropic effects of the spk1-4 mutation. Like spk1 mutants, rop6 show a greater level of PIN2 internalization upon BFA treatment, and ROP6 was shown to colocalize with SPK1 to the plasma membrane (Lin et al., 2012). Thus, it appears that PIN2 localization and internalization is in part controlled by ROP6 via SPK1. RIC1 is an effector of ROP6 whose null and overexpression mutants show phenotypes similar to equivalent lines of ROP6 and SPK1. A combinatorial series of experiments testing PIN2 localization in ric1 and RIC1OE mutants in the presence or absence of an actin inhibitor (latrunculin B) or hyperstabilizer (jasplakinolide) revealed that the ROP6-RIC1 pathway promotes the retention of PIN2 at the plasma membrane by the hyperstabilization of actin microfilaments, similar to the ROP2-RIC4 pathway's control over PIN1 localization in pavement cell patterning (Wu et al., 2011; Lin et al., 2012). This is consistent with the findings by Basu et al. (2008) which showed that SPK1 activates the actin-nucleating ARP2/3 complex. Finally, Lin et al. (2012) showed that
86
auxin treatment increases ROP6 activity in vivo and that the auxin-induced inhibition of PIN2 internalization is absent in spk1, rop6 and ric1 mutants. This suggests a role for the SPK1-ROP6-RIC1 pathway in this autoregulatory aspect of auxin signaling. Beyond protein localization and movement throughout the cell, PIN proteins and their
gravitropic
involvement
are
modified
post-translationally,
e.g.,
through
phosphorylation (Sukumar et al., 2009; Huang et al., 2010; Ganguly et al., 2012; Barbosa et al., 2014). The phosphorylation status of PIN2 in gravitropic basipetal auxin transport appears to be maintained by PINOID kinase (PID) and the ROOTS CURL IN NAPHTHYLPHTHALMIC ACID1 (RCN1) protein (an isoform of the regulatory A subunit of Protein Phosphatase 2A (PP2A)), responsible for phosphorylation and dephosphorylation, respectively (Deruère et al., 1999; Michniewicz et al., 2007; Sukumar et al., 2009). Root gravitropism is impaired by pid or rcn1 loss-of-function mutations, PID overexpression, general protein kinase inhibition or PP2A inhibition due to changes in PIN2 trafficking and auxin transport (Sukumar et al., 2009). These results are consistent with similar findings with PIN1 and PIN3 in Arabidopsis (Huang et al., 2010; Ganguly et al., 2012). Currently, it is unknown how the phosphorylation state of PIN proteins is interpreted into downstream response (Ganguly et al., 2012). Like PIN proteins, D6 PROTEIN KINASE (D6PK) localizes to the basal plasma membrane and undergoes rapid endocytic cycling in vivo (Barbosa et al., 2014). Mutation of D6PK or its removal from the plasma membrane results in decreased PIN phosphorylation, basipetal auxin transport and root gravitropism (Barbosa et al., 2014). Based on these results and others in a study parsing the activity of D6PK in the plant cell, particularly in response to
87
tropic growth, Barbosa et al. (2014) argue that D6PK phosphorylates PIN proteins directly. To further determine the relevance of PID and D6PK to root gravitropism will require more direct study of the interactions between these kinases and PIN proteins. Additionally, auxin itself appears to regulate PIN proteins. In Arabidopsis roots, a higher concentration of auxin in the lower half of the gravistimulated root tip stabilizes the localization of PIN2 to the plasma membrane, whereas a lower concentration in the upper half promotes its internalization and degradation (Paciorek et al., 2005; Abas et al., 2006; Masson et al., 2011; Sato et al., 2015). Auxin inhibition of clathrin-mediated endocytosis of PIN proteins (namely PIN2) appears to be a function of AUXIN BINDING PROTEIN1 (ABP1), an auxin receptor with H +-ATPase function that activates upon auxin binding (Robert et al., 2010). PINOID kinase activity is also auxinresponsive, showing increased expression after auxin treatment and enhancing polar auxin transport (Benjamins et al., 2001). These positive feedback loops likely serve to amplify and accelerate the gravitropic signal and response. Despite them, the auxin gradient across the elongation zone of the root is completely dissipated by 6 h after gravistimulation in Arabidopsis (Abas et al., 2006). Once the auxin signal is generated in the statocyte and reaches the elongation region, how is it terminated? The answer to this question is not yet clear, but has begun to be resolved. Blilou et al. (2005) showed that PIN3, PIN7 and PIN2 redirect auxin movement at the elongation zone back into the vascular core of the root and recycled back towards the root tip in a manner they describe as an 'auxin reflux' model. Band et al. (2012) showed, using auxin reporter domain II (DII)-VENUS, that the PIN3/PIN7
88
asymmetry generated rapidly after gravistimulation is rapidly dissipated once root tips have reoriented about 40º. In addition to observing this apparent trend in normal gravitropic response, the researchers were able to prolong or attenuate the lateral auxin gradient by manually maintaining the roots above or below this threshold value. The molecular basis of this so-called tipping point mechanism is unknown, but Band et al. (2012) propose that the tipping point corresponds to a resettling of the amyloplasts back towards the original bottom wall of the columella after having sedimented on a lateral wall.
3.3.
Calcium and pH
As was discussed in the previous chapter, calcium-based signaling is thought to play a role in very early phases of gravitropic signaling. However, its influence continues well past this time, in fact exerting some level control of the nearly-final cell wall loosening beginning gravitropic growth (see Chapter 4). Once auxin signaling and transport have begun around the time that presentation time has been met, essentially every component of gravitropic signaling is affected by it. Calcium signaling is no exception. Auxin is observed to elicit waves of shootward calcium movement dependent on Ca2+ channels in Arabidopsis, and similarly the inhibition of auxin channels prevents the gravitropic movement of calcium (dela Fuente & Leopold, 1973; Lee et al., 1984; Evans et al., 1992; Monshausen et al., 2011). Gravitropically-induced changes in cytosolic and extracellular pH also seem to be intertwined with auxin and calcium signaling (Monshausen et al., 2011) (Figure 14).
89
Figure 14: The interactions between auxin, calcium, and pH in later-phase root gravitropism. (1), IAA- is pumped by PIN proteins from a cell into the cell wall, where a lower pH allows its interconversion to and from its protonated form (IAAH). IAAH is free to diffuse into neighboring cells, whereas IAA - is actively transported by AUX1 (2). Increases in cytoplasmic auxin concentrations act on an unknown receptor (3) to stimulate the activity of plasma membrane Ca 2+ channels (4). Increases in [Ca2+]c result in changes in activity of H+/OH- channels (5) so that, in the lower half gravistimulated roots, alkalinization of the apoplast occurs (6). Note that this figure depicts the flank of a gravitropic organ in which growth is inhibited (the lower root flank); in the opposite flank, cytoplasmic calcium concentrations cause proton transport and acidification of the cell wall. Reproduced from Monshausen et al., 2011.
In Arabidopsis seedlings, pretreatment with auxin transport inhibitors N-(1Naphthyl) phthalamic acid (NPA) or 2,3,5-triiodobenzoic acid (TIBA) significantly dampens both phases of the gravitropic calcium signature, the spike and the shoulder (Plieth & Trewavas, 2002). [Ca2+]c increases following external auxin application are mimicked, but significantly lesser, with non-hormonal auxin analogs sodium butyrate and butyrate-methyl-ester (Plieth & Trewavas, 2002). This suggests that the effect of auxin on
90
cytoplasmic calcium may partially be due to its weak acidity, but its role as a signaling molecule is more relevant (Plieth & Trewavas, 2002). Conversely, calcium and the calcium binding proteins TOUCH3 (TCH3) and PINOID BINDING PROTEIN1 (PBP1) have been shown to have a role in regulation of polar auxin transport via interaction with PID kinase (Robert & Offringa, 2008). TCH3 and PBP1 act as repressors and enhancers of PID, respectively, in a calcium-dependent fashion (Benjamins et al., 2003). These preliminary results relating auxin, calcium, pH and gravitropic signaling were significantly expanded upon nearly a decade later in a study performed by Monshausen and others (2011), described in greater detail below. Loss of function of the principal auxin influx carrier in aux1 mutants abolishes the long-term, whole-organ changes in pH at the surface of gravistimulated Arabiopsis roots (Monshausen et al., 2011). Exogenous application of auxin supplied by an IAAimpregnated agar block making contact with the root cap caused an alkalinization of the root elongation zone surface within 60 s which progressed shootward with roughly the same velocity as auxin movement (Monshausen et al., 2011). This effect, too, is lost in aux1 mutants. When exogenous IAA is applied to the whole plant, elongation zone alkalinization does occur in aux1 mutants but within a smaller range and to a lesser extent than in wild-type roots (Monshausen et al., 2011). Either of these treatments of auxin also cause a rapid, shootward-directed wave of [Ca2+]c that increases with similar dynamics to the observed pH changes. Treatment with calcium channel blocker La 3+ not only prevented the auxin-induced cytosolic calcium wave, but the equivalent changes in pH, as well, suggesting that calcium signaling mediates these auxin-dependent pH changes
91
(Monshausen et al., 2011). While auxin may be considered upstream of calcium and pH in this scenario, there is reciprocal influence from the latter elements on the former (Figure 14). Influx of auxin is affected by apoplastic pH: in its protonated form (IAAH), it is able to diffuse through the cell wall in what was previously thought to be the singular or most important form of auxin influx (Friml & Palme, 2002; Monshausen et al., 2011). The active influx by AUX1 only occurs as a symport between IAA- and H+ and is in fact the principal means of auxin influx (Friml & Palme, 2002). The increase in cytoplasmic calcium concentrations following auxin influx in turn alters proton motility across the plasma membrane favoring alkalinization of the apoplast. Thus, the auxin-induced changes in intra- and extracellular ion concentrations increases the favorability of active influx over diffusion; paired with tight regulation of AUX1, this may serve to provide more carefully-controlled auxin transport in gravistimulated roots than in vertically-growing roots (Monshausen et al., 2011). In the upper half of gravistimulated roots, auxin-dependent changes in [Ca2+]c acidify the apoplast, increasing cell elongation rates (Mullen et al., 1998). Importantly, these changes help to transform the rapid signaling described in Chaper 2 to the auxininduced cell wall loosening and cell expansion that define gravitropic response and allow corrective growth.
3.4.
ROS revisited
As discussed in the previous chapter, ROS seem to help establish gravitational direction in earlier phases of gravitropism; in later phases, they help to establish the tissue
92
polarity that allows differential growth. In maize pulvini, a dramatic asymmetry of ROS (favoring the lower half) was found at 72 h following gravistimulation as indicated by DAB staining (Clore et al., 2008; Hartwell, 2016). Results from Joo et al. (2001, 2005) provided early insight into the function of ROS in later root gravitropism, particularly of auxin-induced ROS. They showed that gravistimulation induces ROS production in Arabidopsis roots, treating roots with ROS scavengers (NAC, N-acetyl-cysteine) greatly depletes response, and that auxin also triggers ROS generation (Joo et al., 2001). They also
showed
that
auxin-induced
ROS
generation
requires
the
activity
of
phosphatidylinositol 3-kinase (PI3K), and one of its products, PIP, is required for a ROS response to auxin (Joo et al., 2005). This presents the potential for an interesting connection between auxin-induced ROS production and IP3 and thus calcium signaling. In root hair growth, ROS signaling modulates the activity of a plasma membrane Ca 2+ channel; the possibility of a similar effect, perhaps mediated by IP 3, in gravitropism is not an unreasonable one (Swanson & Gilroy, 2009). While gravitropic ROS production in the maize pulvinus seems to be independent of NAPDH oxidase as shown by diphenyleneiodium (DPI) treatment, NAPDH oxidase-generated ROS activity is wellknown to affect several cell signaling pathways and represents another precedent for the potential interaction between ROS- and calcium-based signaling (Clore et al., 2008; Mazars et al., 2010; Saed-Moucheshi et al., 2014). It has been noted by Swanson and Gilroy (2010) that the effects of hydrogen peroxide treatment on gravitropic bending performed as part of the elucidation of ROS signaling in maize root and shoot systems may be directly related to effects on the cell
93
wall; however, results from others point to a more causal relationship. In favor of a signaling function, MAPK signaling seems to be downstream of early ROS signaling in gravitropism, as pretreatment with PD98059 precluded the previously-documented effects of H2O2 on gravitropic curvature (Liu et al., 2009). Studies performed by Barjaktarović et al. (2007, 2008, 2009) demonstrate the differential phosphorylation of proteins related to ROS metabolism and MAP kinases/MEKs in response to gravistimulation. While ROS signaling pathways are, of course, not the sole activator of MEKs, it remains entirely possible that graviresponsive ROS and perhaps PI-based sigalling converge on this famously diverse signaling pathway. While ROS, IP3 and the MAP kinase pathway all appear to be involved in the gravitropic response, the apparent connections between them are less evident. Nonetheless, the potential for these signals to provide a more unified image of gravitropism when considered together is very exciting. To the extent of the author's knowledge, no work to date has been performed to further clarify the roles of all three of these signaling-molecule families in relation to each other within the context of plant gravitropism. Regardless of whether these pathways do all interact directly, to perform such studies should provide much-needed insight into the signaling and transduction phases of gravitropism.
3.5.
Conclusion
Between the very early events preceding, mediating and immediately responsive to statolith sedimentation and the eventual differential growth response, sub-cellular and
94
cellular signals are transformed into gradients across whole tissues (including asymmetries in IP3 and ROS levels and MAP kinase activity leading to an auxin gradient) that establish the physiological environment necessary to allow the gravitropic cell expansion described in the following chapter. In the midst of the rapid signaling preceding it and the more gradual growth response that follows, this intermediary transduction phase involves many of the same dynamic pathways acting early in the gravitropic response (ROS, pH, calcium, etc.), all modulating and eventually being modulating by auxin and polar auxin transport. While this phase of gravitropism is, compared to sensing and early transduction, somewhat well-understood, significant strides stand to be made in our understanding before it may be described as confidently as later phases of the response. An advantage to future studies attempting to elucidate the events that occur within this time frame is that knowledge of the preceding and following phases may be used to help solve the problem “from both ends” to provide a continuous, rather than fragmented, idea of gravitropism. While the challenge of this task is still significant, many important developments have been made, and have highlighted in this chapter and the previous chapter. Many aspects of these developments are summarized in Figure 15.
95
Figure 15: The cytoskeleton, endomembrane system and plastids coordinate with an array of signaling molecules to confer the gravitropic signal. Several key molecular components have been identified, as discussed in Chapters 2 and 3, and are shown here including ARG1(/ARL2), ARP, SGR6, SGR9, WAV3, PIN proteins, the TOC complex, MAP kinases, calcium, and IP3-related compounds and enzymes. The unlabeled blue shapes spanning the upper plasma membrane represent currently unidentified proteins providing connections between the cytoskeleton, plasma membrane and cell wall similar to animal integrins, and/or mechanosensitive stretchactivated ion channels. ROS are a notable exclusion from this figure, but would logically be placed upstream of MAPK signaling, and depending on the system at hand, potentially downstream of auxin. Modified from Kolesnikov et al. (2016).
96
CHAPTER 4: RESPONSE, GROWTH AND REORIENTATION Auxin-mediated growth, as a part of gravitropism and more generally, is a famously well-studied phenomenon. Volumes upon volumes could be filled—and, indeed, have been—on the subject, from the long history of its research, its function in stimulus response as well as developmental organo- and morphogenesis, to its direct and indirect effects on cell biology. In keeping with the intentions of this work, the present chapter focuses on the more-recently characterized molecular and genetic bases for this response as it pertains to gravitropism. Still, an exhaustive description of individual elements involved in this response is beyond the scope of this thesis. Rather, the more broadly-defined pathways that these elements invoke are described. Being at the end of a long and highly involved signaling pathway, it is inevitable that auxin-mediated growth is partially regulated by extensive crosstalk from other growth signals, such as (non-auxin) hormones and other tropic stimuli. This crosstalk is also discussed in this chapter.
4.1.
Hormone-mediated growth
As has been stated, asymmetrical auxin concentrations in the elongation region of a root or stem are responsible for differential growth that reorients the plant. The essence of this fact has been known for a very long time (Darwin, 1880). However, the molecular and genetic bases for this growth response have only begun to be unraveled rather recently (Leyser, 2002). This largely began with the identification of five stable Arabidopsis lines with lessened sensitivity to auxin, the AUXIN RESISTANT (AXR)
97
mutants (Estelle & Somerville, 1987; Lincoln et al., 1990; Wilson et al., 1990). AXR1 is a subunit of the ubiquitin-like RUB (RELATED TO UBIQUITIN) protein and is involved protein ubiquitination (del Pozo et al., 2002). AXR2 and AXR3, also known as IAA7 and IAA17, are transcription regulators that colocalize with SCF complex and proteasome components. AXR4, or RGR1 (REDUCED ROOT GRAVITROPISM1) is not well understood; it is an integral membrane component found in ER and vacuolar membranes and in the nucleus and mitochondria (TAIR, accessed January 2017). AXR5 (IAA1) is a transcription-regulating protein like AXR2 and AXR3, but has received less study than the two (TAIR, accessed January 2017). The alternate (IAA) gene names for AXR2, AXR3 and AXR5 indicate that they belong to the IAA/AUX family of auxin influx transporters; in total, there are 29 IAA-family proteins (Sato et al., 2015). Similar to some AUX/IAA proteins, auxin response factors (ARFs) are a class of 22 transcription coregulators in Arabidopsis identified by their ability to bind auxin response element (AuxRE) transcription factors (Ulmasov et al., 1997; Li et al., 2016). ARFs homodimerize in the presence of auxin to trigger auxin-mediated gene expression; IAAs and ARFs will dimerize together in the absence of auxin, repressing the activity of ARFs via the corepressor TOPLESS (TPL) (Leyser, 2002; Mockaitis & Estelle, 2008; Li et al., 2016) (Figure 16). In addition to acting as transcription factors, many auxin-responsive elements are involved in the E3 SCF (Skp1-Cullin-F-box, named for its component proteins) ubiquitin protein ligase complex (Mockaitis & Estelle, 2008). AXR1 forms RUB, which binds to the CUL1/AXR6 (cullin) component and affects its availability to certain proteins and the
98
availability of the E3 complex to the ubiquitin-E2 components that complete the protein ubiquitination machinery (Kerscher et al., 2006). The F-box component is also an auxinresponsive protein, TIR1; this specific SCF complex is referred to as SCF TIR1 (Ruegger et al., 1998). The SCF complex provides protein target specificity to the ubiquitin proteasome pathway, particularly from the F-box component. AUX/IAAs are the targets of SCF TIR1; how TIR1-AUX/IAA interactions are regulated is unknown (Mockaitis & Estelle, 2008). IAA proteins are degraded following polyubiquitination by SCF TIR1 in response to high auxin concentrations (Blancaflor & Masson, 2003; Mockaitis & Estelle, 2008). TIR1 seems to be the auxin receptor within the complex; based on digital simulations, auxin appears to improve hydrophobic interactions between TIR1 and IAAs, embedded within the TIR1 protein, likely until the ubiquitinated IAA protein is removed from the complex to the proteasome (Mockaitis & Estelle, 2008). Thus, sufficiently high auxin concentrations release repressor IAA proteins from the AuxRE-promoting ARFs, allowing auxin-mediated gene transcription to occur (Figure 16). Finally, auxin-mediated gene expression causes production of proteins which principally effect cell wall homeostasis and metabolism. The specific class of auxinresponsive genes invoked by a stimulus is determined by the ARF/IAAs regulating them; the ARFs ARF7 and ARF19 and the IAA proteins AXR2 and AXR3 seem to be most important for gravitropic response, and they regulate a number of genes promoting growth such as cell wall-related XTHs and osmoregulatory ion channels, as discussed in the next section (Okushima et al., 2005; Sato et al., 2015).
99
Figure 16: AUX/IAA- and ARF-mediated, auxin-dependent gene regulation of auxin-responsive genes. In the absence of auxin, ARF and AUX/IAA proteins dimerize, and a co-repressor that complexes to IAA proteins prevents the action of ARF as a positive regulator of auxin-responsive gene transcription. When auxin is present, the SCFTIR1 ubiquitin E3 ligase complex marks AUX/IAA proteins for degradation, and the corepressor leaves concommitantly. ARFs then dimerize with each other and activate transcription of auxin-responsive genes. Reproduced from Chapman & Estelle, 2009.
While auxin-mediated growth is very much known as the principal driving force for gravitropic growth, the influence of other hormones is evident, as well, with potential crosstalk occurring particularly between auxin and ethylene, cytokinins (CKs), gibberellic acid (GA), and abscisic acid (ABA) (Zhang et al., 2011; Clore et al., 2013; Schüler, 2015). The interaction between auxin and CK is particularly important in root patterning and development (Moubayidin et al., 2009). Two CK oxidase genes showed upregulation, one in a transient manner only in the lower half of the pulvinus while the other first increased globally with a sustained increase only in the lower half (Zhang et
100
al., 2011). Generally, cytokinins promote cell division over elongation, so enrichment in the oxidase proteins that convert CKs to inactive compounds seems reasonable. CKdegrading proteins show a similar increase in transcription; however, the significant of these results is unclear considering that active CK levels are not found to vary significantly between the halves gravistimulated maize pulvini (Zhang et al., 2011). A GA 20-oxidase gene showed an extended (> 24 h) increase in transcription in the lower half of maize pulvini, and unlike the CK oxidase genes actually result in increased active GA levels in the lower half upon gravistimulation (Zhang et al., 2011). GA is known to encourage stem elongation, so it is unsurprising to see these results in maize pulvini (Zhang et al., 2011; Clore, 2013). GA appears to increase the rate of growth response by promoting PIN protein retention at the plasma membrane, and removal of GA from the response site helps more quickly restore symmetrical auxin distribution (Band et al., 2012; Löfke et al., 2013; Schüler et al., 2015). An ABA hydroxylase showed increased transcription in both pulvinus halves (Zhang et al., 2011). ABA usually is associated with inhibited cell elongation, but is upregulated in the lower pulvinus (Zhang et al., 2011). It is possible that the role of ABA in gravitropic growth bears relevance as a mediator of GA- or ethylene-based signaling (Zhang et al., 2011). Historically, study of the relationship between ethylene, auxin and gravitropism has yielded contradictory experimental results and their interactions remain overall unclear (Kramer et al., 2003; Muday et al., 2012). However, Kramer et al. (2003) showed that ethylene sensitivity is necessary for full gravicompetence, consistent with findings that ethylene-responsive protein binding factors are transcriptionally enriched in
101
gravitropism in Arabidopsis (Moseyko et al., 2002). Ethylene seems to serve as a negative regulator of auxin signaling in gravitropism, reducing responsive growth (Muday et al., 2012), perhaps to prevent “overshooting” of the gravitropic set-point angle. Additionally, ethylene-insensitive mutants (etr1, ein2 and alh1) show enhanced root gravitropism in Arabidopsis (Muday et al., 2012). Ethylene appears to modulate polar auxin transport and inhibit both shoot and root gravitropism (Muday et al., 2012). The effect of ethylene on gravitropism may be due to its induction of flavonoid synthesis; tt4 mutants do not produce flavonoids and are not sensitive to the effects of ethylene on root gravitropism (Buer et al., 2006).
4.2.
Cell wall changes
Gravitropic growth is a result not of cell division, but of cell expansion (PerrotRechenmann, 2010). The ultimate result of gravitropic signaling is to allow this process: the extracellular matrix must become more plastic, and the protoplast must increase in volume to expand the wall, allowing for cellular elongation. Eventually, the wall rigidity is restored. The plant cell wall is very heterogeneous and complex, but is essentially formed by a network of cellulose fibers in a matrix primarily made of polysaccharides (Darley et al., 2001). Pectins and glycans are the principal polysaccharides; in most plants, xyloglucan is the “matrix glycan”, or glycan cross-linking the cellulose fibers, although Poaceae family members (cereal grasses including maize and oat) frequently exhibit glucuronoarabinoxylan (GAX) as their matrix glycan (Gibeaut et al., 1990; Darley et al.,
102
2001). Pectins in the cell wall provide water retention to form a hydrated matrix. Finally, it is important to consider the presence of proteins involved in signaling, such as arabinogalactan proteins (AGPs), in the cell wall (Gaspar et al., 2001). Within minutes of auxin treatment (i.e., on a pre-genomic time scale), cell elongation begins. Auxin has been shown to activate H+-ATPases at the plasma membrane, forcing protons out of the cell (Frias et al., 1996; Perrot-Rechenmann, 2010). In addition to directly acidifying the cell wall and contributing to acid growth, this proton pumping hyperpolarizes the plasma membrane, which then leads to the activation of voltage-dependent ion channels, allowing the flux of cations into the cell (Rayle & Cleland, 1992; Philippar et al., 1999; Perrot-Rechenmann, 2010) (Figure 17). The influx of ions, notably potassium, contributes to increasing water influx and thus total protoplasmic volume (via the influence of osmotic pressure), which is necessary for cell expansion. The auxin-mediated activation of these H +-ATPases seems to rely, at least in shoots, on AUXIN BINDING PROTEIN1 (ABP1), an auxin receptor shown to facilitate protoplast swelling in stems of maize and Arabidopsis (Rück et al., 1993; Steffens et al., 2001; Perrot-Rechenmann, 2010). The role of ABP1 in roots is less clear, but involves inhibition of PIN endocytosis as mentioned in Section 3.1. Protons in the cell wall have diverse effects which generally favor the weakening of non-covalent interactions between matrix components and thus loosening of the cell wall (Perrot-Rechenmann, 2010) (Figure 17). After the more immediate effects on ion/pH-dependent crosslinking of matrix proteins and wall integrity as a result of changes in ion homeostasis described above, cell
103
Figure 17: Auxin-mediated cell expansion. Beginning with auxin (green pentagon) binding ABP1, plasma membrane H +-ATPases are activated, promoting loosening of the cell wall and influx of cations (primarily potassium ions). Protoplast swelling occurs as a result of these osmotic changes. Action of auxin in the nucleus activates regulation of cell wall proteins and polymers as well as plasma membrane ion channels. Reproduced from Perrot-Rechenmann (2010).
wall changes after gravistimulation also occur as the result of a significant genomic phase (Figure 17). Beta-expansins and xyloglucan endotransglucosylase/hydrolases (XTHs) are two cell wall remodeling enzyme classes whose genes are differentially expressed in the gravitropic differential growth response and are often auxin-responsive (Perrot-
104
Rechenmann, 2010; Sato et al., 2015). XTHs act on xyloglucans in the extracellular matrix, while another class of auxin-responsive proteins, β-glucanases reduce the integrity of the cell wall by hydrolizing cellulosic polysaccharides (Fry et al., 1992; Zhang et al., 2011). XTHs help to “rebuild” the cell wall by depositing small xyloglucan residues onto the longer xyloglucan components of the expanding wall (PerrotRechenmann, 2010). Contrarily, β-glucanases reduce the integrity of the cell wall by hydrolizing cellulosic polysaccharides (Perrot-Rechenmann, 2010). B-expansins bind to cellulose microfibrils and weaken the connections between apoplastic cellulose and other extracellular elements when activated by low pH (4.5-6) (McQueen-Mason & Cosgrove, 1995; Perrot-Rechenmann, 2010; Zhang et al., 2011). Changes in the sugar composition of the cell wall are also seen to follow gravitropic stimulation. Zhang et al. (2011) found that the lower halves of gravistimulated maize pulvini show a significant enrichment in arabinoxylan content, which may have a function in increasing wall strength to support the mechanical stress to which it is subjected during elongation. In oat, β-ᴅ-glucan synthase is significantly upregulated and the proportion of the glucan in wall composition is significantly (but slightly) increased in the lower half of gravistimulated pulvini, which also had significantly greater levels of invertase enzymatic activity (Gibeaut et al., 1990). More so than a particular function for glucan in gravitropic growth, it is thought that its enrichment in the cell wall and the increased invertase activity in the lower half of gravistimulated oat pulvini may represent the extensive deposition of soluble sugars in the cell wall further driving osmotic water influx and thus expediting gravitropic
105
curvature (Gibeaut et al., 1990).
4.3.
Coordination with other tropisms
Phototropism Particularly in aerial shoots, the roles of phototropism and gravitropism are much more closely linked than they are often treated in laboratory settings, and it can remain difficult to separate gravitropic and phototropic effects even when controls are used to minimize their interference (Molas & Kiss, 2009). Both responses cue differential tropic growth in many of the same ways, involving the uses of calcium, ethylene, and principally auxin and auxin-related proteins (Correll & Kiss, 2002) (Figure 18). How, then, do they differ from and interfere with each other during their sensing and signal transduction phases? First, it is helpful to classify the phototropic effect on gravitropism into two fundamentally different cases: tonic effects and vector effects. Tonic effects are induced by the detection of light regardless of intensity or direction, while vector effects are determined by these latter factors (Correll & Kiss, 2002). Tonic interactions between phototropism and gravitropism have received rather little study, namely in a work conducted by Grolig et al. (2000) showing that omnilateral blue light enhances gravitropism in Phycomyces sporangiophores compared to dark in a manner downstream of statolith sedimentation. Similar studies in higher plants are generally lacking. Most study of the interaction between these tropisms describes vector effects; a notable distinction here is the differing effects of blue and red light, which involve
106
different photoreceptors – the PHOTOTROPIN (PHOT) and CRYPTOCHROME (CRY) families, and the PHYTOCHROME (PHY) family, respectively (noting that red light can also, to a lesser extent, activate cryptochromes) (Figure 18). Unidirectional red light has been shown to have differing effects on plant gravitropism. In pea, sesame and rice, red light enhances gravitropic response (Britz & Galston, 1982; Woitzik & Mohr, 1988; Yoshihara & Iino, 2007). In other plants, such as tomato, maize and Arabidopsis, red light reduces gravitropism, with the effect in Arabidopsis being the most well-studied (Lu et al., 1996; Poppe et al., 1996; Behringer & Lomax, 1999). In Arabidopsis, plants grown in red light show agravitropic, randomly-
Figure 18: Differences and similarities between components of the phototropic and gravitropic signaling pathways. The shaded box includes elements of signal transduction and response common to both tropisms. Reproduced from Correll & Kiss (2002).
107
oriented growth, compared to proper (orthogravitropic) root and shoot orientation in dark- or white light-grown seedlings (Liscum & Hangarter, 1993; Poppe et al., 1996; Soh et al., 1998; Fairchild et al., 2000). The molecular basis for this red-light interference with gravitropism has been partially clarified by knockout studies implicating PHYA and PHYB as well as CRY1 and CRY2 (Robson & Smith, 1996; Soh et al., 1998; Behringer & Lomax, 1999; Fairchild et al., 2000; Ohgishi et al., 2004). It appears that phytochromes actively inhibit negative gravitropism in red-lit hypocotyls (the portion of the stem closest to the roots), as this effect is absent in phyA mutants (Fairchild et al., 2000). Cryptochromes apparently serve rather to simply enhance the strength of the phototropic response towards red light, although they induce a similar random-orientation growth pattern in blue light (Ohgishi et al., 2004). Other than photoreceptors, several other proteins have been identified for which a responsibility in this agravitropic effect is suggested by knockout studies, such as HFR1 (Long Hypocotyls in Far Red 1) and FIN2 (Far-red Insensitive 2), SHY2 (Supressor of Hy5) and GIL1 (Gravitropic in the Light 1) (i.e., hfr1, fin2, shy2 and gil1 mutants do not show this loss of gravitropism when grown in red light) (Soh et al., 1998; Fairchild et al., 2000; Halliday & Fankhauser, 2003; Allen et al., 2006). HFR1 forms a complex with its homolog PIF3, a nuclear DNA-binding protein; the PIF3-HFR1 complex binds activated PHYA and allows transcription of PHYA-specific genes (Fairchild et al., 2000). Like HFR1, SHY2 can bind PHYA, is localized to the nucleus, and is transcriptionally regulated by light (Halliday & Fankhauser, 2003). The roles of GIL1 and FIN2 are not
108
known, other than that their elimination ablates red-light-dependent randomization of hypocotyl growth and that they function downstream of PHYA. Contrasting with these nuclear interactions between PHYA and other proteins in response to red light, cytosolic PHYA, PHYB and PHOT1 also interact with phytochrome kinase subtrate (PKS) proteins in blue light (Lariguet et al., 2006; Boccalandro et al., 2008). PKS1 is one such PKS, and has dual functions in phototropism and gravitropism: it is a positive regulator of hypocotyl phototropism, a positive regulator of negative root phototropism in blue light, and a negative regulator of positive root gravitropism (Lariguet et al., 2006; Boccalandro et al., 2008). Thus, PKS1 and potentially other members of the PKS family serve an important function in determining the shape and growth form of shoots and roots alike given input related to orientation relative to both gravity and light. The role of PKS1 in root gravitropism/phototropism is reliant on PHYA and is proposed to function downstream of this photoreceptor, and upstream of the convergence of phototropic and gravitropic signaling pathways (Boccalandro et al., 2008). The specific role of cytoplasmic PHYA has been clarified by double mutants of two nuclear PHYA translocating proteins, FHY1 and FHL (Rösler et al., 2007). While many of the typical functions of PHYA are lost in these mutants and thus mediated by nuclear PHYA, the inhibition of gravitropism occurred as usual, showing that cytosolic PHYA and PKS function together in a perhaps entirely different light-mediated signaling pathway than is seen from the more commonly invoked, nuclear pool of PHYA (Rosler et al., 2007; Molas & Kiss, 2009). The connection between phototropism and gravitropism is supremely important
109
for plant form and survival; without both functioning properly, inadequate levels of light may be available for photosynthesis and roots may not penetrate the soil deep enough to supply adequate water and nutrients. While a reasonable start has been made to better understand how these tropisms antagonize each other and work together to ensure survival of the plant, a significant amount of study remains to be done to this end.
Thigmotropism and other mechanical stimuli While gravitropism and phototropism are intertwined and overlapping in many ways, gravitropism is perhaps more similar to a different form of tropism: thigmotropism, which refers to changes in growth responsive to touch and other mechanical stimuli. Other than gravitational force, mechanical sensing is also employed in response to wind and rain, soil impedance, touch (e.g., in vines and carnivorous plants), and weight sensing (e.g., the perception and maintenance of a heavy fruit hanging from a tree branch) (Monshausen et al., 2008). Gravitropism is, in fact, often considered an adapted (sometimes called “inside-out”) mode of mechanosensing (Pickard & Ding, 1992; Knight et al., 1992). Like gravitropism, touch has repreatedly been shown to elicit calcium transients in Arabiopsis, maize, tobacco and several other plants (Braam & Davies, 1990; Trewavas & Knight, 1994; Plieth & Trewavas, 2002 Toyota & Gilroy, 2013). Similar results arise from a sufficiently strong wind force (Knight et al., 1992; Trewavas & Knight, 1994; Toyota & Gilroy, 2013). Unlike gravitropism and as discussed in Chapter 2, the calcium transients from touch and wind force only feature a single spike without a sustained
110
shoulder phase. Another contrast to gravitropism is that touch- and wind-induced calcium spikes are inhibited only by ER and mitochondrial calcium channel blocker ruthenium red (RR), and not by plasma membrane and stretch-activated channel blockers La 3+ and Gd3+ (Knight et al., 1992; Trewavas & Knight, 1994). Furthermore, Braam and Davies (1990) identified the TOUCH family of five genes by their significant upregulation following mechanical stimulus; TCH1 is calmodulin, and two are calmodulin-like isoforms (TCH2 & TCH3 – the latter is is discussed briefly in Section 3.2). When gravitropic stimuli and other thigmotropic stimuli act on a plant at the same time, the influence of the thigmotropic stimulus dominates cellular activity (Pickard, 2007). Since an acute touch is more likely to represent an immediate endangerment to a plant than a skew against its GSA, this results make reasonable sense. However, these responses are far from mutually exclusive: Kimbrough et al. (2004) found that gravistimulation and mechanical stimulation caused differential (P < 0.01) gene expression in similar proportions of the genome (7.6% and 7.5%, respectively) and more remarkably, only 1.5 – 3.8% of the genes differentially expressed after these stimuli are unique to the stimulus rather than common between them (gravitropism with the higher share of uniquely responsive genes). Of the 1,665 genes differentially expressed in both conditions (compared to unstimulated controls), 1,641 show up- or downregulation similarly in both conditions, while 24 are upregulated in one and downregulated in the other (Kimbrough et al., 2004). Identifying the functions of these 24 genes should provide insight into elements that are important to each response, but define their differences, which will certainly be interesting. Nearly 23.4% of mutually differentially
111
expressed genes are classified as functioning in gene transcription or cell wall regulation (Figure 19), consistent with later phases of gravitropic response (Kimbrough et al., 2004). Clearly, understanding mechanical sensing and signaling and understanding gravitropic sensing and signaling are, to some extent, the same pursuits.
Figure 19: Transcription factors (left) and cell wall-related (right) gene families that are differentially regulated in both gravitational and mechanical stress signaling. Reproduced from Kimbrough et al. (2004).
4.4.
Conclusion
In the gravitationally responsive site of differential growth, auxin stimulates immediate and long-term changes in cell wall structure and composition that favor cell expansion and thus allow differential growth to occur. This is accomplished through regulation of intracellular and apoplastic ion homeostasis and regulation of auxin112
responsive genes. Other pathways controlling plant growth and form, i.e. other hormones and tropisms, interfere both positively and negatively in a largely genomic fashion.
113
CHAPTER 5: A PRELIMINARY BIOINFORMATICS-BASED ASSESSMENT OF RESEARCH TARGETS IN PLANT GRAVITROPISM Hypocotyl mRNA sequencing data (NCBI GEO Accession GDS1689; Esmon et al., 2006) from gravistimulated seedlings grouped into lower flank and upper flank samples were used to determine differential gene expression patterns and gene ontology patterns in these samples. Unstimulated control samples are used for comparison. Genes that appear show significant up- or down-regulation in both flanks, one of two, or one compared to the other are analyzed herein. It is important to note that gene expression data is of very limited utility in elucidating the more controversial and confusing aspects of plant gravitropism which generally precede changes in gene expression, and the results gained from this prospective work most likely help to clarify regulation of gravitropism well after the very fast, very interesting events seen within a few minutes of gravistimulation. Since the nature of this chapter is meant to be explorative rather than descriptive, rigorous statistical analysis is foregone in favor of casting a broader interrogative net. See Appendix for further details on how, computationally, the information in this chapter was obtained.
5.1
A search for the amyloplast “actin workbench”
Research into gravitropic amyloplast movement is at an interesting juncture. Strides in molecular genetics and live cell imaging have allowed for far greater
114
understanding of this process than was possible twenty years ago, and have raised such interesting questions that our understanding feels lesser than ever. A major lesson from these recent developments is that the process is very actively regulated, and in large part by the actin cytoskeleton. Experiments on ARG1, ARL2, and SGR9 have begun to clarify how the molecular superstructure linking actin filaments to amyloplasts and regulating the movement of the latter may look or behave, but its actual components have not conclusively been identified. However, such a system has been identified and characterized with chloroplasts – namely, the CHUP1 protein. Since chloroplasts, like amyloplasts, are membrane-bound plastids that show tropic movements mediated by actin, it makes sense that the protein(s) mediating amyloplast movement may bear some similarity, in structure, expression, localization or post-translational modification, to CHUP1. Since sgr9 mutants show aberrancies in amyloplast localization and movement similar to chup1 chloroplasts, proteins that colocalize with SGR9, potentially with similar gene expression patterns, also follow as potential subjects. Based on this reasoning, a list of BLAST (Basic Local Alignment Search Tool, NCBI) hits for CHUP1 with E values of less than 0.05 was compiled alongside a list of (putatively) transmembrane proteins predicted to localize to plastids and a list of actin-binding proteins by the Gene Ontology Consortium1. The genes encoding this combined list of proteins were searched for significant (p < 0.05) differential expression in Arabidopsis hypocotyl top and bottom flanks, as well as between the two flanks, after 2 h of gravistimulation. The results of this search are shown 1: An objective by the National Human Genome Research Institute to provide gene annotations for function, whole-plant and subcellular localization, molecular function, etc., to the genomes of several model organisms. 115
in Table 1. In total, nine of the target genes identified were found to show differential gene expression after 2 h gravistimulation. Four came from the list of predicted transmembrane plastid proteins, while five were actin-binding proteins. Six of these eight proteins showed differential regulation between the bottom and top halves of gravistimulated shoots (p between 0.01 and 0.05), and of those one also showed differential regulation in the top flank compared to unstimulated controls. The seventh and eighth proteins showed differential expression in the top and/or bottom flanks compared to unstimulated controls, but not between the two flanks. More details describing the eight proteins identified are given in Table 2.
116
Table 1: Searching for a CHUP1 functional equivalent in Arabidopsis gravitropism† Gene location
Predicted plastid transmembrane proteins (PPDB)
CHUP1 BLAST E < 0.05 (NCBI)
EGY3 AT1G22850 AT1G44920 AT1G50020 Chr. 1 AT1G54350 AT1G54520 EMB1303 ABA4 AT1G75690
AT1G06740 AT1G07120 AT1G42350 AT1G48280 AT1G52080 AT1G54180 AT1G61080
AT2G03140 AT2G20920 Chr. 2 AT2G21960 EMB2410 AT2G26840 CDS4
AT2G36650
Chr. 3
AT3G02900 FAD5 AT3G15900 AT3G17830 AT3G43520 AT3G56010 AT3G56320 CDS5 AT3G61870
AT3G25690
Chr. 4
AT4G13500 AT4G24090 CLT2 EMB1923 AT4G31040 MRL1
AT4G04980 AT4G05220 AT4G18570 AT4G21810
Chr. 5
AT5G02160 AT5G06130 AT5G13720 AT5G16640 AT5G43745 LUT2 AT5G60750 AT5G63040 AT5G65250
Actin-binding (GO:0003779) AT1G10200 AT1G13180 AT1G20260 AT1G20450 AT1G01780 AT1G01750 AT1G31810 AT1G42980 AT1G42980 AT1G49340 AT1G52080 AT1G59910 AT70140 AT1G71790 AT1G76030 AT1G28630 AT2G16700 AT2G19760 AT2G19770 AT2G25050 AT2G26770 AT2G29820 AT2G30910 AT2G31200 AT2G31300 AT2G34150 AT2G38440 AT2G39900 AT2G41740 AT2G43800 AT2G45800 AT3G05470 AT3G22790 AT3G25500 AT3G32400 AT3G45990 AT3G46000 AT3G46010 AT3G54870 AT3G57410 AT3G61230 AT3G15200 AT4G15200 AT4G19400 AT4G19410 AT4G00680 AT4G01710 AT4G25590 AT4G26700 AT4G29340 AT4G29350 AT4G30160 AT4G34490 AT4G34970 AT4G38510 AT5G07650 AT5G07740 AT5G07760 AT5G07770 AT5G07780 AT5G35700 AT5G48360 AT5G52360 AT5G54650 AT5G55400 AT5G56600 AT5G57320 AT5G58160 AT5G59880 AT5G59890 AT5G67470
Logfold change (abs. value) p < 0.05 (2 hr)1
AT1G44920 (b/t -19.97)
AT2G26770 (b/t 12.73); AT2G39900 (b/t 19.56)
AT3G56010 (b/t 17.59)
AT4G00680 (t 1.53); AT4G13500 (b -2.18, t -1.99); MRL1 (t 2.22, b/t 18.98)
AT5G07740 (b/t 34.47); AT5G58160 (t 1.93)
†
: mRNA sequencing data from Arabidopsis hypocotyls gravistimulated for 2 h was used to identify differentially expressed genes encoding for proteins (predicted to be) localized to plastid envelopes, bearing a significant sequence similarity to CHUP1, or (predicted to be) actin-binding, in an attempt to identify potential functional equivalents of CHUP1 in the gravitropic movements of amyloplasts. 1
: b = differential expression in lower stem flank; t = differential expression in upper stem flank; b/t = differential expression between bottom and top halves. For b and t, following numbers indicate logfold expression change from unstimulated control; for b/t, following numbers indicates ratio of expression values between bottom and top flanks (high numbers indicate that gene expression heavily favors the lower half of the gravistimulated shoot). Changes in logfold expression values at which p < 0.05: b = ±1.43; t = ±1.43; bottom/top = -5.11, +6.24. Gene expression data collected by Esmon et al. (2006) and analyzed using RStudio. 117
Table 2: Plastid-localized or actin-binding gene products showing differential regulation after 2 h gravistimulation† TAIR ID
Target source
Description
Notes
AT1G44920
Plastid-localized
Unknown
–
AT2G26770
Actin-binding
Plectin-related
AKA SCAB1 (STOMATAL CLOSURERELATED ACTIN B); coexpressed with IAA12
AT2G39900
Actin-binding
Zinc finger transcription factor family protein
Involved in actin filament bundle assembly, regulation of hormone levels, cell wall organization
AT3G56010
Plastid-localized
Unknown
Coexpressed with ATTERC, a protein involved in chloroplast relocation
AT4G13500
Plastid-localized
Unknown
–
AT4G00680
Actin-binding
Actin depolymerization factor 8
MRL1
Plastid-localized
Pentatricopeptide repeat superfamily
Involved in chloroplast relocation
AT5G07740
Actin-binding
Unknown
–
AT5G58160
Actin-binding
Unknown
Actin filament and cytoskeleton organization
: Differentially expressed genes in Arabidopsis hypocotyls after 2 h of gravistimulation that bind actin or are transmembrane proteins (predicted to be) localized to the plastid. Information from ATTED-II. Gene expression data collected by Esmon et al. (2006), analyzed using RStudio. †
While far from conclusive, this preliminary search has identified eight proteins potentially involved in amyloplast-actin interactions. These proteins show some level of differential expression in Arabidopsis shoot following 2 h of gravistimulation and are either actin-binding or transmembrane proteins localized at plastid membranes. No protein meets both of the latter criteria, perhaps suggesting a multiple-part mechanism. Two of the identified proteins are involved in chloroplast relocation; whether they may function in a mechanism common to all plastids or a chloroplast-specific complex will require further inquiry. Another is involved in hormone level regulation and cell wall organization, two reasonable ontologies to show enrichment 2 h after gravistimulation 118
and consistent with our understanding of later-phase gravitropism. Finally, another is coexpressed with a member of the AUX/IAA family, known to be relevant to gravitropism. The roles of these proteins in gravitropism, if any, is certainly not made clear by the very preliminary work shown here; however, they may represent useful places to begin or augment the search of an actin-binding complex that mediates amyloplast movements in plant gravitropism. A notable shortcoming of this analysis is that the gene expression data used were taken after 2 h of gravistimulation, very long after amyloplasts have resettled in Arabidopsis. If the molecular complex being pursued here does show differential regulation in vivo, one might predict it may no longer occur at 2 h. Additionally, such a complex which is necessarily highly responsive to quick stimuli might not show a significant level of transcriptional regulation, perhaps instead being dictated by posttranslational regulation, such as protein modification. Nonetheless, the eight proteins identified here do fit a profile suggestive of an actin-based function in gravitropism and may merit further investigation.
5.2
Non-targeted gene expression analysis
For each flank of the gravistimulated hypocotyl, 1,138 genes showed a logfold change with a significance threshold of 0.05. A total of 363 genes appear in both of these lists. Since each list itself, as well as the list of commonly differentially expressed genes, are all too large to consider on an individual basis, they will instead by analyzed using gene ontology (GO), which clusters results by biological process, cellular component, or
119
molecular function. Ontologies of genes differentially expressed in both flanks First, the 363 genes that show significant differential expression in both flanks were submitted to AgriGO, a GO analysis tool tailored for use in plant biology that provides more complete annotation for Arabidopsis than other, generally more common GO tools (e.g., PANTHER, DAVID or GOrilla gene analysis tools). The GO map generated and given in Appendix B shows that differential gene expression common to the upper and lower flanks (in each flank, compared to unstimulated controls) largely converges on stress response, gene expression and regulation, protein metabolism and modification, and protein transport and localization. Ontologies of genes differentially expressed only in one flank (versus vertical) The 779 genes that show differential expression in the lower flank only were then analyzed using GO, as well as the 779 genes differentially expressed in only in the upper flank. The biological process ontologies in each of these gene sets are quite similar to each other, and additionally similar to the ontology of the 363 mutually enriched genes. The greatest difference is the relative level of enrichment. In both cases, posttranslational protein modification and all the processes directly upstream from it show dramatic enrichment. Overall, the biological process ontology for the lower-flank gene set is more similar to the mutually-expressed ontology, with moderate enrichment in transcription, translation and gene regulation. The lower-flank ontology features enrichment in chromosome/chromatin organization and apoptosis compared to vertical
120
controls. The upper-flank ontology also shows enrichment in the same processes, but at much more significant levels across the board; additionally, protein folding, ribosome biogenesis, and cell surface receptor-linked signaling all show unique upregulation in the upper flank of gravistimulated hypocotyls. In terms of molecular function, the gene ontologies of the upper and lower flank, both show significant enrichment in binding protein families. Zinc, calcium, nucleosides/nucleotides and DNA- binding characteristic of transcription factors are all upregulated to similar levels. The lower flank uniquely shows upregulation of FAD binding proteins and more intense enrichment of heme binding, while the upper flank has a greater tendency towards expression of proteins involved in lipid binding. The upper flank shows mild to moderate enrichment in expression suggestive of oxidoreducase, tyrosine kinase, endopeptidase, carboxylesterase and ATPase activity. The lower flank shows similar but more significant patterns as well as also showing particular differential expression of UDP-glycosyltransferase, monooxygenase and electron carriers. Both flanks show similar levels of enrichment in signal transduction pathway-associated transcripts. Both gene lists result in differential gene expression primarily affecting the nucleus, nuclear components (i.e., DNA and related proteins), and the endomembrane system. The lower flank also shows mild enrichment in microtubule organizing center (MTOC) proteins.
121
Ontologies of genes differentially expressed between flanks A total of 1,138 genes were found to show differential expression between the two gravistimulated shoot flanks (at p < 0.05). The only GO term for biological process that showed significant enrichment between the two flanks, according to AgriGO, is lipid localization, and the endomembrane system is the only cell component showing significant enrichment, as well. The biological process ontology is also much less complex than in the other three cases, only showing enrichment in heme binding, ATP binding, monooxygenase activity, electron transport, and protein serine/threonine kinase activity. These somewhat opaque results may indicate that genes differentially expressed in both halves of a gravistimulated hypocotyl span a wide profile of biological role, molecular function, cellular localization, or potentially that a large number of these genes are not currently annotated. In this case, it may be more useful to consider differentially expressed genes individually. The genes with the forty highest ratios of logfold expression change in bottom halves versus top halves of gravistimulated hypocotyls (p < 1.76 × 10-3) were found for further analysis. Of these, 22 have an assigned name (official gene symbol) while 18 are identified by their TAIR ID in ThaleMine. The named genes are discussed first, and are presented in Table 3.
122
Table 3: Genes showing differential expression between upper and lower flanks of gravistimulated Arabidopsis hypocotyls (2 hr)† Gene Gene ID Description Notes Description Notes ID Involved in IP3 Myo-inositol monophosphatase precursor synthesis, CORI3 like 1 signal transduction
IMPL1
Homeodomain-like superfamily protein
PRS
Natural resistance-
NRAMP associated macrophage protein 3 3
Non-photochemical quenching 1
NPQ1
Gluthathione S-
GSTU14 transferase tau 14 APRL4
APR-like 4
Regulates lateral axis-dependent development Metal ion transmembrane transporter (vacuolar) Violaxanthin deepoxidase
IQ-domain 10
Calmodulin binding
GuILO6
D-arabinono-1,4lactone oxidase family protein
–
Nucleoside 2
Receptor-like protein 6
Defense response
MPK20 MAP kinase 20
–
IAA28 IAA-inducible 28
Negative regulator of lateral root formation
Redox homeostasis; TRZ4 ER-localized
IQD10
NDPK2 diphosphate kinase
Salinity, hormone, wounding, microbe stress responses
Electron carrier; ENODL Early nodulin-like 9 protein 9 membrane component
–
RNA-binding RNA binding; DNA (RRM/RBP/RNP demethylation motifs) family protein
ROS3
RLP6
Tyrosine transaminase family protein
Chloroplast protein; UV, oxidative stress
PPa2 Pyrophosphorylase 2 Redox homeostasis
tRNAse Z4
tRNA 3'-end processing
ARM repeat SHR-interacting superfamily embryo-lethal protein DNA binding, NF- Nuclear factor Y, YC11 subunit C11 transcription regulation Nodulin UMAMI MtN21/EamA-like Transmembrane acid T41 transporter family transporter protein Cell wall organization; Glucan synthaseGSL09 integral membrane like 9 component Voltage-gated anion SLAC1 SLAH1 channel; auxinhomologue 1 regulated SIEL
†
: Twenty-two genes of forty showing the greatest ratio in bottom-flank log-fold gene expression change compared to the top flank in Arabidopsis hypocotyls after 2 h of gravistimulation. Gene expression data collected by Esmon et al. (2006), analyzed using RStudio.
Trends in the gene descriptions and notes of the characterized proteins listed in Table 3 are generally consistent with expectations for this phase in gravitropism. Calcium and IP3 signaling, stress response, cell wall reorganization, gene (transcription) regulation,
123
auxin response, MAP kinase activity and oxidative pathways are all represented. Some of these genes stand out as particularly interesting. Two, PRS (PRESSED FLOWER/WOX3) and SIEL (SHORTROOT-INTERACTING EMBRYO LETHAL), are involved in tissue patterning. PRS, along with WOX1 (WUSCHEL-RELATED HOMEOBOX1), regulates lateral-axis development of flowers and adaxial-abaxial (topbottom) patterning of leaves (Nakata et al., 2012). SIEL is endomembrane-associated and interacts with the microtubule skeleton to mobilize the SHR transcription factor to affect radial pattern formation in roots (Koizumi et al., 2011; Wu & Gallagher, 2013, 2014). Interestingly, SIEL specifically facilitates the movement of SHR from cell to cell (via plasmodesmata, which are cytoplasmic channels through the walls of neighboring cells) and tissue to tissue (Wu & Gallagher, 2014). If SIEL does in fact serve a purpose in gravitropism, what may its cargo be? ATTED-II is a database cataloging gene expression data and algorithmically curating co-expression patterns, assigning a Mutual Rank (MR) value that increases with less similar expression (so that each gene has a MR of 0 with itself). For SIEL, ATTED-II lists some interesting associations—including PI5K, a calmodulin-interacting protein, and a tetratricopeptide repeat-like superfamily protein, members of which are known to be involved in hormone signaling including auxin (Schapire et al., 2006)—but no obvious answer to this question of molecular cargo exists without further research. Additional interesting genes from Table 3 include MAP Kinase 20 and calmodulin-binding IQ-Domain 10, a family member to IQD2 which shows coexpression with MAPK20. IAA28, like MAPK20 and IQD10, is a member a protein family with biological function known to be involved in the gravitropic pathway; these
124
singly-identified elements may help clarify the molecular bases MAP kinase-, calmodulin- and auxin-dependent signaling pathways in gravitropism. Finally, NRAMP3 is a transmembrane tonoplast (vacuolar membrane) transporter that translocated several different metal ions and is predicted by gene ontology to be a positive regulator of ROS metabolism. Next, unnamed genes differentially expressed between gravistimulated hypocotyl flanks are considered. They are listed below in Table 4.
125
Table 4: Genes showing differential expression between upper and lower flanks of gravistimulated Arabidopsis hypocotyls (2 hr)† Gene ID
Description Pentatricopeptide AT1G06580 repeat superfamily protein AT1G103390
Nucleoporin autopeptidase
Notes
Gene ID
Description
Notes
–
AT1G13050
Proline-rich receptorlike kinase
–
Nuclear mRNA export; shade avoidance
AT4G24330
Hypothetical protein (DUF1682)
–
NeuroblastomaGeranyl- and Retrograde vesicleTerpenoid synthases AT5G24350 amplified sequence AT3G20160 farnesyltransfermediated transport superfamily protein protein ase AT5G12010
nuclease
Polyketide cyclase/dehydrase AT4G23680 and lipid transport protein
AT1G79090
Topoisomerase IIassociated protein
mRNA processing
Defense against AT1G78010 biological stress
tRNA modification GTPase
–
Salt stress
GDSL-like AT5G22810 lipase/acylhydrolase superfamily protein
–
AT5G12220
las1-like family protein
–
AT5G16110 Hypothetical protein
–
AT3G11500
Small nuclear ribonucleoprotein family
–
Myelin transcription factor
–
F-box and associated Integral vacuolar AT3G16580 interaction domainsmembrane protein containing protein
AT5G01910
Cyclophilin-like AT3G15520 peptidyl-prolyl cistrans isomerase
Chloroplast membranes; ubiquitinated
AT5G63460
SAP domaincontaining protein
Pollen germination & tube growth
†
: Of the forty genes showing the greatest ratio in bottom-flank log-fold gene expression change compared to the top flank in Arabidopsis hypocotyls after 2 h of gravistimulation, the eighteen without assigned gene symbols are listed above. Gene expression data collected by Esmon et al. (2006), analyzed using RStudio.
Overall, the potential gravitropic functions of the less-extensively characterized genes in Table 4 are less clear than those in Table 3, due in some part to the fact that functions in general are not well-understood. Related to the tetratricopeptide repeat-like proteins mentioned above are pentatricopeptide repeat-like proteins, which are RNA-
126
binding proteins that assist in localizing their binding partner RNAs to organelles – all PPRs with known localization to date target mitochondria or chloroplasts (SchmitzLinneweber & Small, 2008). The recurrence of tricopeptide repeat-like proteins in differentially expressed gene lists and the potential for their involvement in gravitropism is previously unreported, and may be of potential research interest. Genes involved in other stress responses are also represented, which again is a reasonable expectation that may represent avenues to extend our understanding of the relationship between gravitropism and other stress responses. The F-box associated domain-containing protein and the chloroplast membrane protein that is a target of ubiquitination follow this trend as well. A similar gene-by-gene analysis was applied to the genes included in the 115 most strongly differentially-expressed genes in both the top and bottom flanks (versus vertical controls), rather than between the two flanks. These 28 genes are listed with brief descriptions and/or notes in Tables 5 and 6. Like Tables 3 and 4, several functional patterns are evident that are consistent with our current expectations of later-phase gravitropism. Among these are cell wall-related genes, proton pumps, stress and auxin response, and ubiquitination (Tables 5 and 6). Some curious results are also revealed, among these an abundance of transposons/transposable elements and a protein identified as involved in body weightinduced secondary growth in Arabidopsis (Ko et al., 2004) (Table 6). Like the gravitropic stimulus, the secondary growth stimulus initiated by body weight was identified as upstream of auxin-mediated growth by Ko et al. applying artificial body weight to
127
Arabidopsis seedlings (2004). The gene identified in this response and by the gravitropism analysis in the present work is similar to an AAA+ (ATPase Associated in Diverse Ceullar Activities) in Medicago, belonging to a widely diverse class of proteins serving functions as varied as metal chelation, DNA repair, and even acting as molecular motors (noting that dynein is an AAA+) (Snider et al., 2008).
128
Table 5: Genes showing differential expression in both upper and lower flanks of gravistimulated Arabidopsis hypocotyls (2 hr)† Gene ID Protein Known functions Gene ID Protein Family Known Functions Family UDP-glucosylFlavonol 7-OGlycoside XTH24 Cell wall loosening UGT73B2 hydrolase transferase glucosylation Chloroplast Chloroplast ribsomal Seed storage Endomembrane lipid SESA2 RPL14 ribosomal albumin transport; seeds proteins L14 & L16 complex Transducin family / Cysteine-rich Defense response to Cell division CRK21 receptor-like CDC20.4 1 WD-40 repeat family pathogens cycle protein kinase protein Transcription G2-like + Na /H+ and Li+/H+ BOA transcription regulator; circadian NHX8 Antiporter antiporter factor clock Auxin response; Fatty acid Fatty acid Small acidic SMAP2 AT1G06360 found in floral biosynthesis; protein desaturase structures oxidation-reduction PRMT1A
MEF25
AT5G08570
NHX6
Arginine methyltransferase
NDF6
Tentratricopep- Mitochondrial RNA tide repeat-like editing factor
AHG2
Methyltransferase
Pyruvate kinase
K+ and Mg+ binding
Antiporter
Na+/H+ antiporter; osmotic stress response
LEA4-5
NAD(P)H Photosystem I dehydrogenase electron transport Poly(A) ribonuclease
ABA and stress response
Late embryogenesis Drought response abundant
†
Genes of known function that show strong differential regulation (p < 5 × 10-3) in both the top and bottom flanks (versus vertical controls) of Arabidopsis hypocotyls gravistimulated for 2h. Gene names, protein families and known functions were gathered with TAIR. 1 : Interestingly, CDC20.4 is an intron-less isoform of CDC20, but is identified as a transducin family / WD-40 repeat family protein. Gene expression data collected by Esmon et al. (2006), analyzed using RStudio.
The identification of AT5G37110 in both body weight-responsive secondary growth and gravitropism in Arabidopsis is an interesting result, but not necessarily surprising, considering the large extent of overlap between differential gene expression in gravitropic and mechanical stimuli as shown by Kimbrough et al. (2004). Unfortunately, almost nothing else is known about this protein; further research into its general function and specific role in mechanotransductive stimuli should be conducted. 129
Table 6: Genes showing differential expression in both upper and lower flanks of gravistimulated Arabidopsis hypocotyls (2 hr)† Gene ID Notes Gene ID Notes AT1G17900 AT3G31900
Transposable element
AT5G35510
ATP-dependent helicase family; DNA AT1G52460 binding at SNF2-related domain
Mitochondrial localization Α/β hydrolase superfamily; cytoplasmic localization
Contains a domain usually associated with another kinase domain, but lacks the latter Involved in N-terminal protein CACTA-like transposase family; best AT4G03710 match is maize transposon “Shooter” AT5G52500 myristoylation; integral membrane component Transposon similar to Medicago AT1G41797 Athila gypsy-like retrotransposon AT5G37110 AAA ATPase; identified in body weight-responsive secondary growth AT2G15190
Ulp1 protease family; transposable element; psuedogene
AT5G14540
Ubiquitin-associated/translation elongation factor EF1B
AT3G22060
†
: Hypothetical, poorly described, and unknown-function genes that show differential regulation in both the top and bottom flanks of Arabidopsis hypocotyls gravistimulated for 2h (versus vertical controls). Gene names, protein families and known functions were gathered with TAIR. Gene expression data collected by Esmon et al. (2006), analyzed using RStudio.
5.3
Conclusion
A genomics approach was employed to identify genes potentially involved in actin-amyloplast interactions in gravistimulated Arabidopsis based on relevant criteria. Differentially expressed genes were identified in a NCBI GEO dataset of mRNA data in lower and upper halves of Arabidopsis hypocotyls gravistimulated for 2 h compared to vertical controls in each half. Genes differentially expressed in only one flank or the other were analyzed by gene ontology. Genes differentially expressed in both flanks (versus vertical) were analyzed by gene ontology and gene-by-gene analysis. Genes differentially expressed between the two flanks were analyzed on a gene-by-gene basis. Many results returned by this analysis are consistent with elements known or thought to be involved in
130
gravitropic signaling, while the potential roles of other genes identified are less clear. In both cases, further analysis may provide insight into the molecular basis of gravitropic signaling.
131
CHAPTER 6: FUTURE DIRECTIONS AND CONCLUSIONS Plant gravitropism has fascinated researchers for over two hundred years (Knight, 1806). The advent of the space era and the realization of the relevance of gravitropism to agriculture in the early and mid-20th century gave a practical relevance to this field of biology which has only continued to grow since that time. Recent strides in biochemistry and biotechnology methods allow the investigation of this phenomenon with unprecedented depth and insight to the underlying cellular and molecular biology, which is highlighted in this work. The gravitropic pathway can be generally divided into three phases of gravitational sensing, signal transduction, and growth response. In the first phase, the physical force of gravity is sensed by cellular components and/or whole cells and transduced into a chemical signal by poorly-understood mechanisms. Chemical signaling begins almost instantaneously after gravistimulation and continues throughout reorientation, involving a wide array of notoriously diverse transductive factors (e.g., calcium, ROS, MAP kinases, etc.). Ultimately, these signals lead to changes in polar auxin transport resulting in a differential concentration gradient of auxin in the responding part of the organ that triggers growth reorienting the plant. While many aspects of the early phases of gravitational sensing and signal transduction were previously assumed to proceed in singular, discrete, linear manners, research has revealed quite the opposite. Several different means of graviperception seem to exist, and it seems increasingly likely that higher plants may utilize any number of
132
these in addition to the canonical starch-statolith model. Rather than a passive process purely mediated by gravitational force, statolith sedimentation is now understood to be a highly involved process controlled by the cytoskeleton and endomembrane system. Even the signal transduction elements function in more closely-related ways than originally anticipated. Auxin-mediated growth has been realized to be determined by these very elements often first identified due to their downstream relation to auxin; they are also crucial to gravity sensing. Finally, there is confluence of several signaling pathways at the response phase. Despite the wealth of information that informs this astoundingly interconnected reimagining of how gravitropism functions, what still remains unknown is staggering. Perhaps it would be useful to reconsider how we approach experimental questions and evaluate their results. While several careful examinations of different methods of microgravity simulation have been performed, assumption of general accuracy is the default, regardless of method used. The methods commonly used are agreed upon as generally faithful for research purposes; however, this practice can be dismissive of the differences between these methods. These differences should be reconsidered and specifically investigated as useful tools to parse out fine details of gravitropic mechanisms. That is, the subtle differences in successful ways to accomplish the goal of nullifying the gravity stimulus provide a relatively simple way (via transcriptional or proteomic analysis, for example) to identify components that are involved in detection of, or early response to, the stimulus. As an example, magnetic levitation can be used to levitate the body of a small plant and successfully levitates the cytosol and most
133
organelles, but fails to levitate the dense statoliths within gravisensing cells. Despite that statolith sedimentation is not affected, plants levitated in this way clearly show a cell physiological and transcriptional profile that bears resemblance to plants exposed to microgravity simulated in other ways (Babbick et al., 2007; Manzano et al., 2013; Herranz et al., 2014). Determining exactly how this unique method of microgravity simulation differs from others would highlight the difference between amyloplastdependent and -independent means of graviperception. Additionally, magnetic levitation could potentially be used to ask if other various aspects of the gravitropic pathway are reliant on amyloplast sedimentation. Comparison of starchless mutants to wild-type treated with simulated microgravity conditions by magnetic levitation or by other means (say, 3-D clinostatting) may also provide useful insight into various sensing pathways that do and do not begin with amyloplast sedimentation. Similar arguments can be made for the increased mechanical stimulation that is generally seen with clinorotation, or the rapid changes in gravitational force that accompany parabolic flight. Another factor of the basic mindset of gravitropism research that may be complicating our understanding is the disconnect between research into root and shoot gravitropism. Since root and shoot gravitropism employ many of the same mechanisms for sensing and signal transduction, but result in completely opposite growth patterns, it is generally unclear if and how a result in either roots or shoots will translate to the other. Furthermore, the possibility of communication of gravitropic signaling and response between shoots and stems, rather than the process occuring completely independently in different organs, has hardly, if at all, been addressed. To further complicate the issue of
134
comparing different gravitropic systems, pulvinus tissues are a tissue specifically adapted for shoot gravitropism whereas the endodermis is a continuous cell layer found throughout the plant and with varied functions. Reconciling these results between shoot systems can often be difficult, and differences that arise between them beg the question of whether they are tissue-specific (e.g., specific to pulvinus systems), class-specific (e.g., specific to grasses), or species-specific peculiarities, and which results may be applied to a general model. Species-specific differences in root gravitropism also raise difficulties; the lack a meaningful comparison of the basic response between different model plants (e.g., Arabidopsis, maize, tobacco, oat, soybean) often precludes an understanding of differences in results obtained even within the same gravitropic system. A systematic comparison of the basic components of gravitropism between as many plants as possible, ideally initially performed within the same lab (with comprehensive results being compared between several labs), would be greatly beneficial in beginning to address these fundamental concerns. To address these issues, the field of gravitropism would benefit immensely from a compendium of comparative data involving gravitropic tissue (i.e., root, endodermis, pulvinus, protoplast, etc.), cell lineage (i.e., columella, pulvinus, endodermis, elongation zone cortex, etc.), and species. Exactly how such a database may be assembled and how these aspects should be compared in a standardized way is unclear. Generating averages of gene expression data by species and gravitropic tissue/lineage to catalog average expression of individual genes and gene ontologies may be a useful point of departure. Such a database would be useful in future studies and for retroactive comparison of
135
existing work. As for specific topics meriting further study, the involvement of the actin cytoskeleton in amyloplast sedimentation, and more generally in gravity sensing, and early phases of signal transduction poses many interesting questions. It will be particularly interesting to further determine the roles of ARG1, ARL2 and SGR9, which have been identified as proteins that interact with actin and are involved in gravitropic sensing. ARG1 and ARL2 are homologous proteins that are thought to potentially form a plasma membrane-associated complex in columella cells that forms with actin and HSC70, a J-domain co-chaperone that binds to the J-domain of ARL2 (Harrison & Masson, 2008b). As was mentioned in Section 2.1, ARG1/ARL2 likely contribute to gravitropic sensing in a way could be independent of amyloplasts. Their relation to plastid-localized TOC75 and TOC132 in the context of gravitropism, however, speaks strongly to an indirect connection between ARG1/ARL2 function and amyloplast sedimentation, perhaps via regulation of an unknown receptor activated by an unknown ligand localized at amyloplast membrane via TOC complex. The molecular connection between ARG1/ARL2 and the TOC complex proteins will provide valuable insight into gravitropic sensing and early signal transduction. Co-immunoprecipitation assays performed with ARG/ARL and TOC antibodies may allow for the identification of the element(s) underlying this connection. Unlike ARG1 and ARL2, SGR9 localizes to the amyloplasts and is directly implicated in their sedimentation. SGR9 is a ubiquitin E3 ligase that appears to facilitate the release of amyloplasts from F-actin cables. Therefore, the target of ubiquitination by
136
SGR9 is probably a molecule that helps to physically link amyloplasts to the actin cytoskeleton and potentially mediates their movement along the cytoskeleton. Ubiquitination assays using SGR9 followed by anti-ubiquitin immunoblotting, as described by Choo & Zhang (2009), could be a useful way to identify SGR9's substrate in vivo. WAV3 is another ubiquitin ligase apparently related to gravitropism as wav3 mutants show an enhanced root gravitropism phenotype featuring altered amyloplast dynamics similar to those seen following actin disruption. Similar ubiquitination assays with WAV3 may be valuable. Finally, the nine genes identified in Chapter 5 as differentially expressed following 2 h of gravistimulation in Arabidopsis hypocotyls and as putatively plastid-localized or actin-binding are generally only minimally characterized, if at all; their further study may help address the topic of cytoskeletal influence on amyloplast sedimentation. Another aspect of gravitropic signaling and signal transduction worth further experimental pursuit is the role of calcium. Recent improvements in calcium imaging techniques have shown a reproducable calcium signature that has been visualized in hypocotyls and petioles, but resolution has not surpassed the organ level. A greater temporospatial resolution of the calcium response will be necessary to understand it fully. Ideally, resolution at a subcellular level would be extremely useful in clarifying the early involvement of calcium in gravity sensing. While adequately precise techniques in calcium imaging have been demonstrated (Swanson et al., 2011; Costa et al., 2013), it is not clear if such an effort has been made in gravitropism research – studies visualizing or visually quantified gravity-induced cytoplasmic calcium fluctuations have mainly used
137
whole seedlings with luminescence measured using a photomultiplier tube (Plieth & Trewavas, 2002; Toyota et al., 2008, 2013). It would be very interesting to attempt to visualize calcium transients at the subcellular level in real time following gravistimulation using a vertical-stage confocal microscope. Additionally, reconstitution of coelenterazine or aequorin in root tissues still represents a major challenge to microscopy-based calcium signaling research. Short of subcellular visualization, driving calcium reporters under endodermis- or columella-specific promoters and measuring for the gravitropic calcium transient by photomultiplier tube technology would help to confirm or deny the involvement of these tissues in the calcium signature. The results from Toyota et al. (2008) that endodermis-amyloplastless1 (eal1) mutants show a nearly identical gravitropic calcium signature to wild-type are interesting, and may point to an amyloplast-independent means of gravity sensing as predicted by the protoplast-pressure or tensegrity models. More work with different mutants affecting amyloplast formation and sedimentation would make the relationship between gravity sensing and calcium response more clear. Beyond measures of calcium itself, other aspects of calcium-related signaling in gravitropism will be interesting to study further. Protein and small molecule interacting partners with IP3 should be searched in pursuit of a putative receptor, perhaps by a form of small-molecule immunoprecipitation as has been used in animal systems (Ong et al., 2009; Lomenick et al., 2011). Identifying the specific calmodulins and calciumdependent protein kinases involved in gravitropism and their downstream targets will help characterize how transient signals are transformed into a chemical signaling cascade
138
eventually leading to differential growth. The same principle applies to the MAP kinase family proteins involved in gravitropism and their phosphorylation targets, as well. Both MAP kinases and calmodulin-like/calmodulin-binding proteins were identified in Chapter 5 as differentially regulated in shoot gravitropism in Arabidopsis; again, the specific proteins identified therein may be useful places to continue further study. While calcium and auxin are known to be coregulatory and mutually involved in gravitropism (see Figure 15), the way auxin induces increases in cytosolic calcium is not entirely understood. It seems that auxin-induced changes in membrane potential activate cation channels allowing influx of Ca2+ and K+; however, this is not completely able to explain the extent or nature of auxin-induced calcium signals (Vanneste & Friml, 2013). Since auxin is known to induce ROS production, and ROS signaling is known to relate to calcium signaling, it may be of interest to investigate a potential role for ROS in mediating auxin-induced calcium transients. Auxin-induced ROS production requires the activity of PI3K and the presence of one of its products, PIP (Joo et al., 2005); PIP is phosphorylated (also by PI3K) in vivo to PIP2, the precursor to IP3. Therefore, IP3induced calcium release provides a means for auxin-induced ROS production to affect cytosolic calcium. Pursuing this possibility may help more fully explain the coordination between ROS and Ca2+ signaling pathways to regulate auxin-mediated signaling and differential growth (Blancaflor & Masson, 2003). A place to begin may be to analyze ROS-induced calcium release in transgenic Arabidopsis expressing in IP3- or PIP2hydrolyzing enzymes, as constructed by Perera et al. (2006). It would also be interesting to probe a potential function of DAG, e.g., by comparing overexpression lines and
139
knockout mutants of DAG kinase, as DAG is produced concurrently with IP 3 and is known to act as a signaling molecule in the phosphoinositol-based pathway (Perera et al., 2006). The suggestions made here as to future research topics and methods represent only a very abridged volume of the research that will be necessary to answer some of our most pressing issues in gravitropic research. Many of the genes identified by bioinformatics-based analysis in Chapter 5 may represent avenues to begin to address some of these questions. From E.B. Blancaflor and P.H. Masson (2003), an apt summary of the state of research in the field: These are promising and very exciting times in a research field that has long been underpopulated despite the importance and complexity of the biological processes that contribute to these fascinating growth behaviors!
140
APPENDIX A: COMPUTATIONAL METHODS OF GENE EXPRESSION ANALYSIS Identifying, accessing, and manipulating datasets The National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) was searched for microarray datasets performed on plants: 95 results were returned for Arabidopsis thaliana, 16 for rice, 11 for soybean, 3 for maize, 3 for barley, 2 for tomato, and one each for a number of plants (e.g., potato, pepper, tobacco, petunia, pine). Of these, only one pertains directly to gravitropism (GDS 1689) and was the dataset used in Chapter 5. GEO datasets are imported into RStudio using the GEOquery package from Bioconductor: GDS1689
