Increased levels of reactive oxygen species and ... - Wiley Online Library

Plant, Cell and Environment (2008) 31, 144–158

doi: 10.1111/j.1365-3040.2007.01744.x

Increased levels of reactive oxygen species and expression of a cytoplasmic aconitase/iron regulatory protein 1 homolog during the early response of maize pulvini to gravistimulation A. M. CLORE, S. M. DOORE & S. M. N. TINNIRELLO

Division of Natural Sciences, New College of Florida, Sarasota, FL 34243, USA

ABSTRACT

INTRODUCTION

The maize (Zea mays L.) stem pulvinus is a disc of tissue located apical to each node that functions to return a tipped stem to a more upright position via increased cell elongation on its lower side. We investigated the possibility that reactive oxygen species (ROS) and hydrogen peroxide (H2O2), in particular, are involved in the gravitropic response of the pulvinus prior to initiation of the growth response by employing the cytochemical stain 3,3⬘diaminobenzidine (DAB). DAB polymers were found in the bundle sheath cells of gravistimulated pulvini in association with amyloplasts after 1 min of gravistimulation, and the signal spread throughout the cytosol of these cells by 30 min. Furthermore, treatment of maize stem explants containing pulvini with 1 mM H2O2 on their upper sides caused reversal of bending polarity. Similar, though less dramatic, results were obtained via application of 1 mM ascorbic acid to the lower side of the explants. In addition, we determined that a maize cytoplasmic aconitase/iron regulatory protein 1 (IRP1) homolog is up-regulated in the pulvinus bundle sheath cells after gravistimulation using suppressive subtractive hybridization PCR (SSH PCR), real-time RT-PCR and in situ hybridization. Although we do not yet know the role of the IRP1 homolog in the pulvinus, the protein is known to be a redox sensor in other systems. Collectively, our results point to an increase in ROS quite early in the gravitropic signalling pathway and its possible role in determining the direction of bending of the pulvini. We speculate that an ROS burst may serve to link the physical phenomenon of amyloplast sedimentation to the changes in cellular biochemistry and gene expression that facilitate directional growth.

The ability of a plant to maintain its vertical orientation is crucial for its survival and can also be important for harvestability. In maize (Zea mays L.), the phenomenon of lodging, in which the plants are tipped by such factors as strong winds, hail or pathogen attack, results in major losses for growers (Carter & Hudelson 1988; Nelson 2002). Maize plants that are lodged can usually only partially recover via ‘goose-necking’, in which the stem curves upwards because of a growth response that occurs at periodic sites along the stem. Unfortunately, the stem usually does not reach full vertical height, resulting in plants that grow out into the aisles of the field or under adjacent vertical plants. Such growth leads to photosynthetic stress caused by mutual shading (Nelson 2002), reduced grain yield (Carter & Hudelson 1988; Nelson 2002) and also to difficulties during mechanical harvesting (Nelson 2002). The sites along many grass stems that respond to changes in orientation with respect to the gravity vector are called pulvini. Pulvini are found just apical to each node and function to return a tipped/gravistimulated plant to vertical by greater cell elongation on their lower sides, but do not grow in the absence of stimulation (Kaufman et al. 1987; Collings et al. 1998). The increased asymmetric growth of multiple pulvini acting in concert along a reoriented stem results in upward curvature as described by Collings et al. (1998). Grass stem pulvini have proven to be a valuable system in which to study the signalling events and changes in gene expression that allow plants to detect changes in stem orientation and direct altered growth (Kaufman et al. 1987; Collings et al. 1998; Perera, Heilmann & Boss 1999; Chang & Kaufman 2000; Heilmann et al. 2001; Johannes et al. 2001; Perera et al. 2001; Long et al. 2002; Clore et al. 2003; Yun et al. 2006). However, much work remains to be done before we can fully understand how the pulvinus carries out this function. We aimed to determine those early events that are critical for the response, but that occur prior to visible changes in growth and may be involved in the re-establishment of polarity. The earliest event already reported to occur in the pulvinus after gravistimulation is a transient fivefold increase in

Key-words: Zea mays L.; amyloplasts; 3,3′diaminobenzidine (DAB); gravitropism; hydrogen peroxide; pulvinus; real-time RT-PCR.

Correspondence: A. M. Clore. Fax: +19414874396; e-mail: [email protected] 144

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd

Reactive oxygen species and cytoplasmic aconitase expression in gravistimulated maize pulvini 145 inositol 1,4,5-trisphosphate (IP3) levels in the lower half after 10 s of gravistimulation (Perera et al. 1999). This transient increase is followed by fluctuations in levels of IP3 that occur between the upper and lower pulvini halves with a period of approximately 90 s for at least the first 30 min after tipping. Furthermore, within 30 min of tipping, cytoplasmic pH changes occur in the bundle sheath cells of the pulvini, with slight alkalinization occurring at the new ‘bottom’ regions of the cells and acidification occurring at the sides (Johannes et al. 2001). We previously reported that changes in mitogen-activated protein kinase (MAPK) activity that resemble oscillations occur in the upper and lower halves of pulvini following gravistimulation, and that this activity is important to the normal bending response because inhibition of MAPK activation using the drug U0126 inhibited upward bending by approximately 65% (Clore et al. 2003). We showed that a statistically significant alternation of higher levels of activity occurred between the upper and lower halves such that at 75 min after gravistimulation, MAPK activity was higher in the upper half; at 90 and 105 min, the activity was higher in the lower half; and from 2 to 3 h, the activity in the upper half was again higher than the lower (and two- to threefold higher than vertical) (Clore et al. 2003).Therefore, the fluctuation we observed in MAPK activity appears to follow the fluctuations in IP3 levels documented by Perera et al. (1999). Furthermore, both signals stabilize such that eventually, IP3 levels remain higher in the lower half and MAPK activity levels remain higher in the upper half during the early hours after tipping (Perera et al. 1999; Clore et al. 2003). The nature of the relationship between these two signalling pathways is not yet known, nor is it understood how these biochemical asymmetries are generated across the whole tissue. A role for these reciprocal changes between the two halves has been hypothesized, however. We speculated that such oscillations in biochemical activities may help to distinguish between transient minor perturbations in plant orientation, such as would occur because of mild wind, and more significant long-term changes, such as lodging (Clore et al. 2003). Furthermore, the biochemical asymmetry that occurs across the tissue once the fluctuations stabilize may help to determine the direction of bending (Clore et al. 2003). Because the work of Joo, Bae & Lee (2001) implicated reactive oxygen species (ROS) [and particularly hydrogen peroxide (H2O2)] in the gravitropic response of maize roots, and because ROS signalling has been shown to precede MAPK activation in many systems (as reviewed in Guzik, Korbut & Adamek-Guzik 2003 for animal systems, and e.g. Kovtun et al. 2000; Nakagami, Kiegerl & Hirt 2004; Rentel et al. 2004 for plant systems), we tested the hypothesis that ROS may be involved in both the early and late responses of the pulvinus to gravistimulation. This work extends the information obtained by Joo et al. (2001) in roots because it includes an investigation of a potential role for ROS in stem gravitropism, incorporates the study of very early portions of the response and because we sought to determine the subcellular location of any ROS produced. To accomplish the latter, we stained gravistimulated pulvini using 3,3′-

diaminobenzidine (DAB).Thordal-Christensen et al. (1997) showed that this compound can be taken up by living plant tissue and used to indicate H2O2 production in the presence of peroxidase activity, and many other groups have subsequently used this reagent to test for the presence of H2O2 production in plant tissues (Vanacker, Carver & Foyer 2000; Pellinen et al. 2002; Ruuhola & Yang 2006; DeWitte et al. 2007, to name a few). We also treated maize half-stem explants (containing pulvini) with H2O2, the ROS scavenger ascorbic acid and the flavoenzyme inhibitor, diphenylene iodonium (DPI), and assayed their ability to respond to gravistimulation. DPI inhibits one possible source of H2O2, NADPH oxidase (O’Donnell et al. 1993), and has been used in plant studies to study H2O2 signalling (e.g. OrozcoCárdenas, Narváez-Vásquez & Ryan 2001; Rodriguez, Grunberg & Taleisnik 2002; Qin, Lan & Yang 2004, among others). In addition, we used suppressive subtractive hybridization PCR (SSH PCR), which exponentially and specifically amplifies differentially expressed genes (Diatchenko et al. 1996) to see if we could identify any ROS-related genes as being differentially expressed in gravistimulated pulvini. Indeed, we found that a cytoplasmic aconitase/iron regulatory protein 1 (IRP1) homolog is up-regulated following gravistimulation. The expression of this homolog was investigated in detail because the IRP1 protein is known to function as a redox sensor that couples H2O2 signalling to post-transcriptional changes in gene expression in animal systems (Caltagirone, Weiss & Pantopoulos 2001; Mutze et al. 2003). Therefore, we further tested the hypothesis that this gene is involved in the gravity response through the use of real-time RT-PCR and in situ hybridization to determine the kinetics and localization of its expression, respectively.

MATERIALS AND METHODS Chemicals and plant material All chemicals were purchased from Sigma (St Louis, MO, USA) unless otherwise indicated. Maize plants (Z. mays L., W64A+, courtesy of B. Larkins, University of Arizona) were grown in soil at a density of 3–4 plants per 20 cm pot in a greenhouse, and fertilized with a modified Hoagland’s solution three times weekly. Five- to six-week-old plants were used for experiments conducted in the summer, and 7- to 8-week-old plants were used in the fall and winter because of slower growth during those times. The P3 pulvinus (as described by Collings et al. 1998) was used for most experiments, including those incorporating DAB, DPI, ascorbic acid and H2O2, and for in situ hybridization and was verified to be responsive for each batch of plants prior to each experiment. For the real-time RT-PCR experiments, upper and lower halves of the P3 and P4 pulvini, or the P4 and P5 pulvini, depending upon which pair was most responsive for a given set of plants, were pooled together prior to RNA isolation to allow for sufficient yield.

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 144–158

146 A. M. Clore et al.

DAB staining Pulvini were removed from vertical plants using a razor blade following the peeling back of surrounding leaf sheaths. Care was taken to keep the pulvini in their native planar (vertical) position during placement into solutions as follows. Pulvini were pretreated with either 300 mL of 1 mg mL–1 DAB or nanopure water (negative control) for 10 min, after which each pulvinus was moved into a gravistimulated position by upending it, thereby balancing it on its new lower side. The pulvinus remained in the gravistimulated position for either 1 or 30 min. During the longer time period, the pulvinus was refreshed with its respective solution every 5 min to prevent dessication. For a subset of samples, after stimulation was complete, the pulvini were chemically fixed (while still in the gravistimulated position) in 600 mL of 4% w/v formaldehyde (Ted Pella, Redding, CA, USA) in buffer [10 mm MgCl2, 2 mm ethylene glycol tetraacetic acid (EGTA) and 1 mm phenylmethanesulphonyl fluoride (PMSF) in 5 mm N-2-hydroxyethylpiperazineN′-2-ethanesulphonic acid (HEPES), pH 7.5] for 15 min. The pulvini (fixed and non-fixed) were washed twice with 1¥ phosphate-buffered saline (PBS) for 3 min and examined under a microscope as a whole wet mount. The distributions of DAB staining were found to be identical whether the tissue was fixed or not fixed (data not shown), so only unfixed pulvini are shown in the micrographs. For staining of pulvini later in the response, pots were tipped to gravistimulate whole plants for either 5 or 72 h after which the pulvini were harvested and stained with DAB. To determine the extent of DAB staining as a result of wounding and basic manipulation alone, we placed some DAB and nanopure water-pretreated pulvini into a freshly dispensed droplet of DAB while keeping them flat (and therefore, ungravistimulated) for various periods of time.To test the hypothesis that the DAB staining resulted from ROS, 10 mm of the ROS scavenger, ascorbic acid, was added to the DAB solution used to pretreat control pulvini prior to gravistimulation. For each treatment at each time point, at least three samples from three different plants were tested, and representative micrographs are shown in the figures. To allow comparison of the DAB staining patterns to the distributions of starch and chlorophyll, separate pulvini were either stained with 0.2% (w/v) iodine in 5% (w/v) potassium iodide and viewed with a light microscope, or were viewed using a Leitz (Wetzlar, Germany) Orthoplan 2 fluorescence microscope with a rhodamine filter set, respectively.

Treatment of stem explants with DPI For treatment via the transpiration stream, stem explants, each about 5 cm in length and containing a pulvinus, were first pretreated with 10 mm DPI in 2-[N-morpholino] ethanesulphonic acid (MES) nutrient solution (5 mm MES, pH 5.5; 100 mm sucrose) or in 50 mm dimethyl sulphoxide (DMSO) (the solvent for the DPI) in nutrient solution as a control. Such treatment was accomplished by placement of

the explant (i.e. basal cut end down) in a scintillation vial containing 20 mL of the appropriate solution. Then, each stem segment was kept vertical while it was longitudinally bisected.The resulting half-stem explants were placed into a horizontal position on moistened paper towels in a humidity chamber at time 0, and a volume of treatment solution sufficient to cover the surface (approximately 400 mL) was puddled onto the longitudinal (upper) cut surface and covered with a piece of parafilm (as described in Clore et al. 2003). Other samples were not pretreated with DPI, but instead, the DPI was simply puddled on the upper surface at time 0 as described earlier. All of the samples were kept in a Powers Scientific, Inc. (Pipersville, PA, USA) growth chamber at 25 °C under a 16 h light/8 h dark cycle for 40 h at which time the angles of gravitropic bending were measured using a protractor, and pictures were taken using a digital camera. In addition, DAB staining (as described earlier) was conducted on cross-sections of pulvini that had been pretreated with DPI and gravistimulated for 15 min to see if the drug had an effect on DAB staining.

Asymmetric treatment of stem slice explants with H2O2 and ascorbic acid Slices of stem, each approximately 7 mm thick and containing tissue from one pulvinus, were removed while the stem remained vertical. These slices allowed for treatment of the explants on either their upper or lower sides during gravistimulation. The resulting explants were placed horizontally with the longitudinal cut that had been made closest to the epidermis facing down onto moistened paper towels in a humidity chamber at time 0. In the case of those explants treated on the upper surface, approximately 400 mL of the appropriate solution (either 1 mm H2O2 or 1 mm ascorbic acid) was immediately dispensed onto the upper side and covered with a piece of parafilm to prevent evaporation. For those treated on the lower side, the solution was first dispensed in the shape of an oblong droplet onto a piece of parafilm placed on the paper towel lining the bottom of the humidity chamber and the explant placed onto the droplet. Angles of bending of all samples were measured periodically (at time 0 as well as at 3, 5, 20 and 24 h using a protractor. All explants were kept in the aforementioned growth chamber during the experiment.

cDNA synthesis Total RNA was extracted from whole pulvini that had either been gravistimulated for 1 h and then frozen in liquid nitrogen or from frozen vertical controls using the Qiagen RNeasy Plant Mini kit (Qiagen, Valencia, CA, USA) as per the instructions of the manufacturer. Quality and yield were assessed using gel electrophoresis and UV spectrophotometry. For SSH PCR, polyA RNA was isolated using the PolyATract system (Promega, Madison, WI, USA) as per the manufacturer’s instructions. Reverse transcription was carried out using 1.7 mg of both vertical and re-oriented


Reactive oxygen species and cytoplasmic aconitase expression in gravistimulated maize pulvini 147 polyA RNAs using the SMART cDNA synthesis kit (Clontech, Mountain View, CA, USA) as per the provided instructions. cDNA used for real-time RT-PCR was made using total RNA that had been isolated from vertical pulvini halves as well as from upper and lower halves of those gravistimulated for various amounts of time (at 10, 20, 30, 60, 90, 105, 180, 240 and 300 min), again using the RNeasy Plant Mini Kit (Qiagen). cDNA was also synthesized from vertical pulvini harvested at comparable times of day as the gravistimulated pulvini as well as from internode tissue after 10, 30, 120 and 180 min of gravistimulation, and the leaf sheath immediately surrounding individual pulvini after 30 min (see Fig. 10 legend for details). In all cases, a quantity of 1 ng of RNA was used in a 20 mL reaction using 125 ng of random hexamers (Operon Biotechnologies, Huntsville, AL, USA) and Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) for first-strand cDNA synthesis as per the instructions of the enzyme manufacturer.

SSH PCR The amplified cDNAs from gravistimulated and vertical pulvini were used as tester and driver, respectively, in SSH PCR using the PCR-Select cDNA subtraction kit (Clontech) according to the instructions provided with the kit. The final amplification step using nested primers gave rise to four discrete bands, which were gel purified using Qiagen’s Qiaquick system. The purified cDNAs were sent to the North Carolina State University Forest Biotechnology Sequencing Facility for sequencing.

Preparation of probes for Northern blots and in situ hybridization Digoxigenin-labelled probes for the in situ hybridization study were synthesized from the IRP1/cytoplasmic aconitase cDNA fragment that had been obtained using SSH PCR. The probe was made using the Roche PCR DIG Probe Synthesis Kit (Roche Diagnostics, Mannheim, Germany) following the instructions of the manufacturer. A 1.2 kb fragment was amplified using the nested primers 1 and 2R from the PCR Select kit (Clontech), and was predicted to hybridize to the middle portion of the endogenous sequence. As a positive control, probes were generated against calmodulin (CaM), which is known to be up-regulated in gravistimulated pulvini (Heilmann et al. 2001).

Northern blotting A quantity of 30 mg of total RNA, from pulvini harvested from vertical controls and from plants that had been reoriented for 1 h, was loaded into each lane of a 1.2% (w/v) formaldehyde–agarose gel. The RNA was transferred to a positively charged nylon membrane (Roche Diagnostics), and the membrane was hybridized overnight at 65 °C in buffer [5¥ saline sodium citrate (SSC), 0.02% (w/v) sodium

dodecyl sulphate (SDS), 0.1% N-lauroyl sarcosine, 2% (w/v) blocking reagent (Roche Diagnostics) and 50% formamide] containing 150 ng digoxigenin-labelled probe specific for the IRP1 homolog fragment. The membrane was washed twice for 5 min each with 2¥ SSC containing 0.1% (w/v) SDS at room temperature followed by two 15 min washes in 0.1¥ SSC, 0.1% (w/v) SDS at 65 °C. The signal was detected using anti-digoxigenin antibodies, CSPD chemiluminescent substrate (Roche Diagnostics) and a 30 min exposure to X-ray film.

In situ hybridization For the monitoring of cytoplasmic aconitase/IRP1 expression by in situ hybridization, plants were first gravistimulated by tipping of the pots on their sides for 30 min, at which point the pulvini were harvested. Control pulvini were harvested from vertical plants. In all cases, the third pulvinus from the base of the stem was removed and separated into upper and lower (relative to their horizontal position) cross-sectional halves using razor blades. The halves were fixed at room temperature in 4% (v/v) formaldehyde and 2% (v/v) glutaraldehyde in PBS (0.01 m phosphate buffer, containing 2.7 mm KCl and 137 mm NaCl at pH 7.4) while rotating in a Pelco Infiltron rotator (Pelco International, Redding, CA, USA) at low speed for 3 h. The tissue was dehydrated in a graded ethanol series (10, 20, 30 and 40%, for 15 min each) followed by substitution with tertiary butyl alcohol (TBA), infiltration with Paraplast Plus-saturated TBA and embedding in 100% (w/v) Paraplast Plus (Electron Microscopy Sciences, Fort Washington, PA, USA). Sections 10 mm thick were cut and mounted on poly-d-lysine-coated slides, which were then stored at 4 °C, and the slides were baked vertically at 50 °C overnight prior to continuation of the procedure. Following the removal of paraffin using a clearing solvent (Electron Microscopy Sciences), the sections were rehydrated, incubated in 0.2 n HCl for 20 min, then in prewarmed 2¥ SSC for 15 min at 70 °C and finally, rinsed briefly in PBS. Prior to hybridization, the sections were treated with 1 mg mL–1 proteinase K [in 100 mm tris(hydroxymethyl)aminomethane (Tris), pH 7.5; 50 mm ethylenediaminetetraacetic acid (EDTA)] at 37 °C for 25 min and rinsed in PBS containing 0.1% Tween 20 (PBT) and post-fixed for 20 min in 4% (v/v) formaldehyde in PBS at room temperature followed by a final rinse in PBT. Following ethanol dehydration, the slides were air-dried and stored in a desiccator overnight at 4 °C. The sections were then treated with preheated prehybridization solution (110 mL solution pooled on each slide and covered with parafilm), consisting of 50% (v/v) formamide, 5¥ SSC, 50 mg mL–1 heparin, 100 mg mL–1 denatured salmon sperm DNA and 0.1% (v/v) Tween 20, at 45 °C in a humidity chamber. Digoxigenin-labelled probe (55 ng per slide) was heat denatured and sheared by boiling for 60 min, then added to a new batch of prewarmed prehybridization solution to give a total volume of 110 mL of hybridization


148 A. M. Clore et al. solution per slide. Hybridization took place at 45 °C overnight in the humidity chamber. The slides were then rinsed twice for 15 min with prehybridization solution at 45 °C, followed by treatment with a series consisting of the following prehybridization and PBT solutions (three parts prehybridization solution to two parts PBT; one part prehybidization solution to four parts PBT; 100% PBT; each for 15 min at 45 °C). Following a 15 min wash in 0.1% (w/v) bovine serum albumin (BSA) at room temperature, the probes were detected using antidigoxigenin antibodies (Roche Diagnostics) conjugated to alkaline phosphatase and visualized by color development with nitroblue tetrazolium (NBT) and bromochloroindolyl phosphate (BCIP) (both from Enzo, Farmingdale, NY, USA) overnight. After cessation of the color reaction via PBT and nanopure H2O rinses, the slides were dehydrated in an ethanol series followed by treatment with xylene and mounting with Permount (Fisher Scientific International, Hampton, NH, USA).

Real-time RT-PCR For each time point tested, a 0.5 mL aliquot was taken from each 20 mL cDNA reaction (described earlier) for use in 40 mL reactions containing SyBR Green Master Mix (Applied Biosystems, Foster City, CA, USA), which contained ROX as a passive reference dye. The reactions were run using an Applied Biosystems 7500 Real-Time PCR System. Each pulvinus half sample was amplified in quadruplicate reactions (technical replicates) during each run, and multiple biological replicates (upper and lower halves from multiple plants harvested on different days) were analysed for each time point (three to four replicates for 10, 30, 60 and 105 min samples, and two biological replicates for 20, 90, 180, 240 and 300 min samples). Negative controls lacking template or containing total RNA as template were included and did not yield product, and denaturation curves and agarose gels were run to verify single product amplification (data not shown). Control reactions were also run using cDNA made from vertical pulvini that had been harvested at times of day comparable to the gravistimulation time course, and from internode and leaf sheath tissue from gravistimulated plants as described in further detail in the legend of Fig. 10. The primers (IRP1F1: 5′-CGGCCATGAGATACAAAT CTGA-3′; IRP1R1: 5′-CGAGAACTGCCGCTTCCAT3′; 18SF2: 5′-TGCCTAGTAAGCGCGAGTCAT-3′; and 18SR2: 5′-ACGGGCGGTGTGTACAAAG-3′) were designed using Primer Express 3.0 Software (Applied Biosystems) based upon the appropriate sequences from GenBank (accession number AY104770 for the IRP1/ cytoplasmic aconitase homolog in maize, and AF168884 for 18S rRNA). The cycling parameters were as follows: 95 °C for 10 min followed by 50 cycles of 95 °C for 15 s and 60 °C for 1 min. For each set of primers, dilution series were also run such that cDNA made from various amounts of starting total RNA was used to create a standard curve that verified good primer efficiency in all cases (slope –3.3 ⫾ 0.3).

The experimental results were normalized against 18S rRNA, which was set as the endogenous control, and vertical samples were used as calibrators. The Applied Biosystems 7500 System Software version 1.3.1 was used to calculate relative levels of expression versus vertical to give fold changes in expression. Averages were then taken across runs and used to calculate mean fold changes and SE values across biological replicates (see the following section).

Digital imaging, graphing and statistics A compound microscope was used to examine all slides and tissue, with the exception of the fluorescent imaging of chlorophyll mentioned earlier. A Canon G3 camera equipped with a Max View Plus microscope adapter (Scopetronix, Cape Coral, FL, USA) was used with the light microscope to take the micrographs. Brightness and contrast of the micrographs were adjusted using Adobe Photoshop Elements version 2.0. The fluorescent micrograph was taken using a Leitz Orthoplan microscope equipped with epifluorescence and the Canon G3 camera with scope adapter mentioned earlier. In addition, images of stem explants were taken with the Canon G3 digital camera alone. The graph in Fig. 5 was made using KaleidaGraph 4.0 (Synergy Software), while those in Figs 8 and 10 were made using Microsoft Excel 2003. SE and Tukey–Kramer honestly significant difference (HSD) calculations were performed using SAS JMP version 4.0 (a = 0.05) (SAS Institute, Cary, NC, USA).

RESULTS We have tested the hypothesis that ROS/H2O2 play a role in both the early and later phases of the gravity response of maize pulvini by using DAB staining, treatment with DPI and asymmetric application of H2O2 and the ROS scavenger ascorbic acid, and offer the following observations.

DAB staining levels increase rapidly following gravistimulation When we performed DAB staining on samples that had been harvested from gravistimulated explants, we observed brown color in the regions of the pulvinus corresponding to the bundle sheath cells, particularly those surrounding the peripheral bundles near the epidermis (Fig. 1a). Staining was less prevalent in the bundle sheath cells found towards the center of the stem (towards the top of Fig. 1a). The apoplast in general was somewhat stained, with the xylem cell walls being particularly dark (Fig. 1a). In contrast, in pulvini that had remained in the native vertical (planar) position after harvesting, diffuse brown staining was visualized but neither the starch grains nor the xylem walls were labelled (Fig. 1b). It is likely that this diffuse staining was


Reactive oxygen species and cytoplasmic aconitase expression in gravistimulated maize pulvini 149

(a)

(b)

(c)

(d)

Figure 1. 3,3′-Diaminobenzidine (DAB) staining in typical gravistimulated (a) and non-gravistimulated (b & c) pulvini in the absence (a & b) and presence (c) of ascorbic acid. (a) Low magnification image of the new lower edge of a pulvinus that was gravistimulated for 30 min and then stained with DAB. Brown precipitate was seen in a punctate pattern in the bundle sheath cells surrounding mainly the first few bundle layers in from the epidermis. A similar pattern was observed around the entire circumference of the pulvinus (not shown). It should be noted that in the very outermost layer of bundles (bottom of micrograph), the bundle sheaths of adjacent bundles are essentially confluent, whereas successively larger intervening regions of parenchyma are found internally (see also Fig. 3b). The apoplast in general was somewhat stained, with the xylem cell walls being markedly dark as compared to ungravistimulated controls. (b) Low magnification image of an edge of a pulvinus that was maintained in its normal planar position after harvesting for 30 min and then stained with DAB. Unlike the punctate pattern seen in gravistimulated samples, diffuse brown staining was found in and between the first few bundles in from the epidermis. The xylem cell walls were not stained as in (a). (c) Low magnification image of an edge of a pulvinus that remained in its normal planar position after harvesting and was stained with DAB in the presence of the hydrogen peroxide (H2O2) scavenger, ascorbic acid. (d) Gravistimulated samples treated with water in lieu of DAB contained no brown deposits. Each experiment was replicated at least three times with similar results. X, xylem; P, parenchyma; Ph, phloem; C, collenchyma. Downward grey and white arrows (in a & d, respectively) indicate the direction of the gravity vector. Bar = 200 mm.

caused by a wound response because the tissue was cut and manipulated. The addition of the ROS scavenger ascorbic acid to the DAB solution effectively prevented the formation of the brown color in gravistimulated pulvini (Fig. 1c), and samples treated with water in lieu of DAB contained no brown deposits (Fig. 1d). Visualization of the DAB-stained samples at high magnification revealed the staining to be in oval structures within the bundle sheath cells at 1 min, and to have spread to throughout the cytoplasm of the bundle sheath cells by 30 min (Fig. 2a,b, respectively). This was the case around the entire circumference of the pulvini, although only the new lower edge is shown in the figure for the sake of simplicity. The pattern of distribution of the brown oval structures in Figs 1a and 2a did not appear to match that of chloroplasts (Fig. 3a), which were found throughout the parenchyma cells between the peripheral vascular bundles. Instead, their distribution was similar to the distribution of starch as indicated by the dark staining in pulvini stained

with iodine (Fig. 3b), in that they were confined to the layers of cells in close association with the bundles.

Treatment of stem slice explants with DPI failed to prevent bending or DAB staining One possible source of ROS in plants is via the enzyme NADPH oxidase (reviewed in Sagi & Fluhr 2006). Several researchers have successfully used the compound DPI, which inhibits flavin-containing enzymes, to inhibit this enzyme and affect a reduction of ROS production in vivo (e.g. Rodriguez et al. 2002; Liszkay, van der Zalm & Schopfer 2004). However, our treatment of gravistimulated pulvini with DPI using two different techniques failed to result in an inhibition of bending (Fig. 4a) or DAB staining (Fig. 4b). There were no statistically significant differences in the extent of bending between the treated explants and the controls, or between the groups of explants treated with DPI by each of the two different methods (statistics not shown).



(a)

(b)

Figure 2. High-magnification images of vascular bundles from pulvini pretreated with 1 mg mL–1 3,3′-diaminobenzidine (DAB) for 10 min prior to gravistimulation for 1 min (a) or 30 min (b). By 1 min of stimulation, staining was seen associated with oval structures (arrows) that are consistent in terms of size and distribution with amyloplasts (see Fig. 3b). After 30 min of gravistimulation, staining was seen throughout the cytosol of the bundle sheath cells (arrows). Phl, phloem; X, xylem; P, parenchyma; C, collenchyma portion of the bundle, which surrounds the vascular cells of pulvini (as also shown in Johannes et al. 2001, albeit in younger pulvini with fewer layers of collenchyma). White arrows (lower left) indicate direction of the gravity vector. Bar = 200 mm.

Directional treatment of stem explants with H2O2 and ascorbic acid dramatically alters the bending response When the upper versus lower sides of stem slice explants were treated with 1 mm H2O2 or 1 mm ascorbic acid, the direction and extent of bending were sometimes altered dramatically, depending upon the side of application (Fig. 5). While application of either H2O2 on the lower side or ascorbic acid on the upper side failed to have a significant effect on bending, the reciprocal experiments, in which H2O2 was deposited on the upper side and ascorbic acid on the lower, caused the explants to be positively gravitropic (Fig. 5). The latter effect was more marked for the explants treated with

(a)

H2O2 than those treated with ascorbic acid, but the shapes of the curves were similar (Fig. 5). In addition, the shapes of the curves representing the reciprocal treatments (i.e. H2O2 on the lower side and ascorbic acid on the upper versus H2O2 on the upper side and ascorbic acid on the lower) were also similar, particularly during the later times tested.

DAB staining at later time points indicates asymmetry The results we obtained of pulvini with H2O2 and investigate the distribution points in the response. We

from asymmetric treatment ascorbic acid led us to also of DAB staining at later time studied pulvini that had been

(b)

Figure 3. Fluorescent (a) and light (b) micrographs showing autofluorescence and starch distribution, respectively, at the edge of cross-sections of unstimulated maize pulvini. (a) Image taken using a rhodamine filter set. The red fluorescence is largely because of the chloroplasts, which are found in abundance in the first several parenchyma layers in from the epidermis, while the dark voids contain the vascular bundles. (b) Light micrograph showing a cross-section of a pulvinus stained with 0.2% (w/v) iodine in 5% (w/v) potassium iodide. In contrast to the chloroplasts, amyloplasts are largely confined to the bundle sheath cells (arrows), which exist in a layer approximately one to three cells thick surrounding each bundle. This pattern is reminiscent of that seen in 3,3′-diaminobenzidine (DAB)-stained sections of gravistimulated pulvini (cf. Figs 1a, 2 and 3b). Note that near the edge of the pulvinus, the bundle sheaths surrounding each bundle are directly adjacent to one another. X, xylem; P, parenchyma; C, collenchyma; Ph, phloem. Bar = 200 mm. © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 144–158

Reactive oxygen species and cytoplasmic aconitase expression in gravistimulated maize pulvini 151

(b)

Figure 4. Treatment of gravistimulated maize pulvini with diphenylene iodonium (DPI). (a) Maize stem half explants were treated with 50 mm DPI (left four explants) or dimethyl sulphoxide (DMSO) (right four explants). Both compounds were either introduced via the transpiration stream or via puddling of solution onto the cut surface as indicated. Parafilm was overlaid onto the explants to prevent dessication. This photograph was taken after 40 h of bending. Angles of bending were not statistically significant between the groups (data not shown). (b) Micrograph of a pulvinus pretreated with DPI via the transpiration stream, then placed in 3,3′-diaminobenzidine (DAB) stain and gravistimulated for 15 min. Downward black arrow (lower left) indicates direction of the gravity vector. Pretreatment with DPI prevented neither bending nor hydrogen peroxide (H2O2) production in two replicate experiments (one shown here), and each incorporating two different methods of DPI treatment. Bar = 150 mm.

gravistimulated for both 5 h (just after the presentation time as measured by Perera et al. 1999), as well as those harvested well into the growth phase (after 72 h of stimulation). At the 5 h time point, bundle sheath labelling was again seen in both halves, but with perhaps lower levels occurring in the upper half than in the lower (Fig. 6a,b, respectively). A more obvious asymmetry, which was actually visible by eye (data not shown), was observed at the 72 h time point (as seen in micrographs in Fig. 6c,d). At this time, DAB staining was visible throughout the pulvini in both bundle sheath and parenchyma cells, and the intensity was reproducibly stronger in the lower half.

Increases in expression of a cytoplasmic aconitase/IRP1 homolog occur in gravistimulated pulvini By performing SSH PCR using cDNA derived from polyA RNA isolated from pulvini that had been gravistimulated for 1 h as tester and cDNA derived from vertical pulvini as driver, we obtained four fragments corresponding to potentially up-regulated genes (Fig. 7). Digoxigenin-labelled

probes made from these fragments hybridized to discrete bands on Northern blots containing 30 mg of total pulvinus RNA (Fig. 7). The probe corresponding to the largest fragment hybridized to a band approximately 2.7 kb in size, while both of the probes corresponding to the intermediatesized fragments hybridized to what seemed to be the same single band (when the same blot was stripped and reprobed), which was approximately 0.7 kb in size (Fig. 7). Hybridization of the probe from the smallest fragment could not be detected on our blots. The signal corresponding to the largest fragment was reproducibly (albeit modestly) more intense in the lane containing the RNA isolated from pulvini that had been gravistimulated for 1 h as compared to the lane containing RNA extracted from vertical pulvini (Fig. 7). Sequencing of the fragments followed by BLAST (Altschul et al. 1990) searches revealed that the largest fragment had a high sequence similarity along its length to IRP1, a.k.a. cytoplasmic aconitase. Specifically, it showed 90– 100% similarity to plant cytoplasmic aconitases/IRP1s, and 70–80% similarity to mammalian IRP1s. In addition, the partial sequence we have obtained thus far appears to be identical to the comparable region (bases 1459–1869), of a particular mRNA (PCO074909) in Z. mays (accession number AY104770), which, when electronically translated,

10 MES H2O2 up H2O2 L ASC up ASC L

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Figure 5. Hydrogen peroxide (H2O2) and ascorbic acid were applied to the cut surfaces of maize half-stem explants, each containing a pulvinus. The angles of bending were determined after various times of gravistimulation. Both application of H2O2 to the top side of the explant and ascorbic acid to the lower side caused a general reversal in the direction of bending (inset: H2O2 treated), with the H2O2 treatment having the most dramatic effect. Values are the mean changes in angle after time zero minus SE for a minimum of three independent experiments. Statistical analysis using the Tukey–Kramer honestly significant difference (HSD) test indicated that the angles of bending for the H2O2 L treatment were significantly different from all of the other treatments from the 5 h time point onwards (a = 0.05).



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Figure 6. 3,3′-Diaminobenzidine (DAB) staining of upper (a & c) and lower (b & d) halves of pulvini that had been gravistimulated for 5 h (a & b) and 72 h (c & d). After 5 h, staining was seen in the bundle sheath cells (arrowheads) in both halves, perhaps more so in the lower. During the growth phase (after 72 h of gravistimulation as shown in c & d), DAB staining was present in both parenchyma and bundle sheath cells of both halves, but the staining was reproducibly more intense in the lower half than in the upper. X, xylem; P, parenchyma; Ph, phloem; C, collenchyma. Downward grey arrows indicate the direction of the gravity vector. Bar = 200 mm.

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also has very high similarity to plant and animal cytoplasmic aconitases/IRP1s. The ~150 kb fragment obtained using SSH PCR showed some sequence similarity to DnaJ proteins (data not shown). Interestingly, Patrick Masson and colleagues have already been working extensively on the role of DnaJ-like proteins in gravitropism in Arabidopsis seedlings (Sedbrook, Chen & Masson 1999; Boonsirichai et al. 2003; Guan et al. 2003). The other two fragments we isolated, on the other hand, did not show similarity to any known sequence.We therefore chose to focus our efforts for further research on the IRP1 homolog in part because of the documented relationship between the IRP1 protein and redox sensing in other systems (Caltagirone et al. 2001; Mutze et al. 2003). We wanted to further test the hypothesis that the expression of the cytoplasmic aconitase/IRP1 homolog in maize is involved in the gravity response because the increases in intensity on RNA blots were modest. Therefore, we

Purified cDNA used as probes

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? Figure 7. Northern blot to verify expression of genes identified using suppressive subtractive hybridization PCR (SSH PCR). (a) The four cDNA fragments isolated from gravistimulated pulvini using SSH PCR are shown in an ethidium bromide-stained agarose gel (lane 1, 1 kb ladder). (b) The fragments were purified and labelled with digoxigenin and used to probe blots containing 30 mg of total RNA (1 h, RNA from pulvini re-oriented for 1 h; V, RNA from vertical pulvini) per lane. The probe from the largest fragment hybridized to a ~2.5 kb band, while the two intermediate probes both hybridized to what seems to be a single band ~0.7 kb (same blot stripped and reprobed). Hybridization of the probe from the smallest fragment could not be detected on these blots (symbolized by the question mark). © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 144–158

Reactive oxygen species and cytoplasmic aconitase expression in gravistimulated maize pulvini 153 10

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Figure 8. Changes in expression of the cytoplasmic aconitase/iron regulatory protein 1 (IRP1) homolog in upper and lower pulvini halves along a gravistimulation time course. Real-time RT-PCR was used to determine fold changes in cytoplasmic aconitase/IRP1 mRNA levels versus vertical with 18S rRNA levels used for normalization as described in the Materials and methods section. Dotted lines were added to emphasize that time points were not taken as frequently after 105 min, so the detailed kinetics of expression during the later period are less certain. Data are presented as mean fold changes + SE for upper samples and – SE for lower samples. Note that the error bars for the 10 min lower, 90 min lower and 180 min upper time points are too small to be visible on the graph (= 0.03, 0.06 and 0.13, respectively). Beta tubulin mRNA levels were assayed in single whole pulvini at three early time points as a control (lowest curve).

employed real-time RT-PCR to investigate the kinetics of the expression of this gene in a quantitative fashion. The results, which are shown in Fig. 8, indicate that levels of the corresponding mRNA change in a dynamic fashion following the onset of gravistimulation. Although the sizes of the error bars are large in some cases, it is notable that the curves are quite parallel between the two halves, and with the possible exception of the 10 min time point, the levels of the transcript in the upper versus lower halves are relatively consistent with one another (Fig. 8). In contrast to the levels of the cytoplasmic aconitase/IRP1 homolog, beta tubulin mRNA levels were not seen to increase at the time points tested during the first 30 min of the response (Fig. 8).

In situ hybridization studies revealed expression of the cytoplasmic aconitase/IRP1 homolog in the bundle sheath cells of gravistimulated pulvini To determine the cellular localization of the expression of the IRP1 homolog, we employed the technique of in situ hybridization. Digoxigenin-labelled probes were hybridized to pulvini that had been gravistimulated for 30 min, as well as to vertical controls. The 30 min time point was chosen because of its high level of expression, as indicated by the real-time RT-PCR results (Fig. 8). Furthermore, as a positive control, we investigated the distribution of CaM, which Heilmann et al. (2001) had previously shown to be up-regulated in gravistimulated pulvini. In sections made from plants that had been gravistimulated for 30 min, signal corresponding to the IRP1 homolog was found in randomly distributed bundle sheath

cells (~1–4 per bundle) of centrally located bundles (Fig. 9a,b). There did not appear to be any discernable pattern in any of these samples as to which side of each bundle (upper versus lower with respect to the gravity vector) the labelling occurred, and bundle sheath cells were labelled in both the upper and lower halves (note that a lower half is shown in Fig. 9, but the appearance was similar in the upper half; data not shown). In contrast, labelling was diffuse and weak in the vertical control halves probed for IRP1 (Fig. 9c). Hybridization of the probe we used against CaM gave rise to reasonably strong labelling in multiple cell types (including parenchyma cells and bundle sheath cells surrounding both central and peripheral bundles), particularly in the lower half (data not shown).

Investigation of the levels of the cytoplasmic aconitase/IRP1 homolog mRNA over time in vertical pulvini and in other tissues following gravistimulation When real-time RT-PCR analysis was carried out on samples isolated from vertical pulvini that had been harvested at comparable times of day as the gravistimulated pulvini, very little change in levels of the cytoplasmic aconitase/IRP1 mRNA homolog was noted (Fig. 10a). In addition, only a slight decline in level was measured in internode tissue harvested from the upper and lower sides of stems that had been gravistimulated for 10, 30, 120 and 180 min (Fig. 10b). In contrast, the levels increased appreciably in leaf sheath tissue harvested after 30 min of gravistimulation from the region immediately surrounding the pulvinus (Fig. 10c).



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Figure 9. Results of in situ hybridization experiments to locate cytoplasmic aconitase/iron regulatory protein 1 (IRP1) expression in re-oriented pulvini. Low (a) and high (b) magnification images of cross-sections of pulvinus tissue harvested after 30 min of re-orientation and treated with probe against the cytoplasmic aconitase/IRP1 homolog mRNA. (c) Typical section of pulvinus tissue that remained vertical and was treated with the same probe. Multiple bundle sheath cells (black arrows) were stained in the sections taken from gravistimulated pulvini (a) and (b), but only diffuse, weak signalling was observed in vertical sections (c). Results shown are representative of six independent experiments per treatment. X, xylem; P, parenchyma; Phl, phloem; C, collenchyma. Grey arrows indicate the direction of the gravity vector. Bars = 150 mm.

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ROS levels increase rapidly in gravistimulated maize pulvini

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The results of our DAB staining experiments indicate that increases in ROS, and perhaps specifically H2O2, are associated with the amyloplasts immediately following gravistimulation, and that the ROS spread throughout the bundle sheath cells by 30 min post-stimulation. It should be noted, however, that although the staining may be indicative of the presence of H2O2 and is generally interpreted in this manner, according to some recent references, the stain is

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Figure 10. Expression of cytoplasmic aconitase/iron regulatory protein 1 (IRP1) in vertical pulvini over time and in non-pulvinus tissues. (a) To determine the extent to which cytoplasmic aconitase/IRP1 mRNA levels change over time in vertical pulvini, pulvinus tissue was harvested at comparable times of day as the gravistimulated pulvini over the first three hours of the time course. All samples were normalized using 18S rRNA levels prior to calculation of fold changes over the levels in vertical pulvini harvested at time 0 (used as the calibrator). Y-scale set to be comparable to that in Fig. 8 to allow comparison. Error bars indicate SE; n = 2–3. (b) Investigation of levels of cytoplasmic aconitase/IRP1 mRNA in upper versus lower halves of internode tissue from gravistimulated maize stems. All samples were normalized using 18S rRNA levels prior to calculation of fold changes over the levels in vertical, unstimulated control internode (V, used as the calibrator). Error bars indicate SE; n = 3. (c) Levels of cytoplasmic aconitase/IRP1 mRNA in leaf sheath tissue surrounding vertical (V leaf) versus gravistimulated/tipped (T leaf) stem pulvini after 30 min of stimulation. All samples were normalized using 18S rRNA levels prior to calculation of fold changes over the levels in vertical, unstimulated pulvini (used as the calibrator). Error bars indicate SE; n = 3.


Reactive oxygen species and cytoplasmic aconitase expression in gravistimulated maize pulvini 155 not always specific for H2O2, but may indicate the presence of ROS in general (Halliwell & Whiteman 2004; Shulaev & Oliver 2006). The results of our control experiments suggest that the DAB staining is, in fact, because of the presence of ROS, because the addition of the ROS scavenger, ascorbic acid, prevented DAB staining, and no brown structures were seen in unstained gravistimulated controls. Our experiments do not tell us whether the ROS is being generated by the amyloplasts themselves, although there is precedent for ROS production by plastids. For example, chloroplasts (as reviewed in Mullineaux, Karpinski & Baker 2006) and root plastids (Mittova et al. 2002) have been shown to produce ROS under certain conditions. The size, distribution and abundance of the DAB-stained structures we observed in the stimulated pulvini were consistent with those of amyloplasts and not chloroplasts, for the latter appeared to be widely distributed and more abundant than the former. Another interesting and potentially relevant finding by Balmer et al. (2006) is the existence of a ferrodoxin/thioredoxin system in the amyloplasts of wheat starchy endosperm.

Both ROS accumulation and the bending response appear to occur independently of flavin-containing oxidases Our observation that both re-orientation and DAB staining occurred in the presence of DPI may suggest that the ROS is produced by a non-flavin-containing oxidase, or by a DPIresistant flavin-containing oxidase. Another logical possibility, which we intend to test in the future, is that the apparent increase in ROS is because of a decrease in ROS scavenging in gravistimulated pulvini.

ROS may provide directional cues during the gravitropic response Consistent with the notion that ROS signalling may play a role in gravitropism in the pulvinus is the fact that gravitropic bending was essentially reversed when H2O2 was applied to the upper side of the pulvini or ascorbic acid was applied to the lower. The similarity of the shapes of the bending curves of explants treated with either H2O2 on the upper side or ascorbic acid on the lower indicates that the two compounds may be impacting the same processes. We speculate that a gradient of H2O2 may provide directional information such that the pulvinus bends away from the area of highest concentration. When the peroxide is applied to the upper side of the pulvinus, the directionality of the endogenous gradient may become inverted. A similar inversion may also occur when endogenous peroxide is scavenged from the lower side by ascorbic acid, such that a new maximum concentration would result on the upper side. One possible explanation for the fact that application of excess peroxide to the lower side did not have an effect on bending may be that the endogenous levels that occur

following gravistimulation are already saturating.We did not note any dramatic asymmetries in ROS distribution in our DAB staining of pulvini gravistimulated for 1 and 30 min, but did note a possible asymmetry at 5 h and a clear asymmetry at 72 h, at which time there was a significantly higher level of staining in the lower half. However, this histological technique is not quantitative in nature, so we intend to further investigate the levels of ROS over a gravistimulation time course in the future using additional methods. The work of Joo et al. (2001) addressed the role of ROS in root gravitropism and focused on the phase of the response following auxin redistribution. The authors did not provide information as to the subcellular distribution of the ROS produced, but they did report that the increase occurred in the lower cortex within 30 min of gravistimulation and in both the upper and lower cortex after prolonged stimulation (Joo et al. 2001). They concluded that this increase in ROS was downstream of auxin distribution because the application of H2O2 was able to cause curvature in the presence of an auxin transport inhibitor. Our DAB staining results indicate that in the maize pulvinus, the initial increase in ROS precedes auxin redistribution because Long et al. (2002) found that no asymmetry in free indole acetic acid exists across this tissue prior to 2 h poststimulation. We propose that ROS may act both before and after auxin translocation in gravitropism, but further work will be required to determine if this is the case. Additional experiments are planned to further address this hypothesis, including the spectrophotometric quantitation of H2O2 in extracts made from the two halves of the pulvini along a gravistimulation time course and in plants in which starch sedimentation is reduced (e.g. via starch depletion). We previously reported that MAPK activation appears to be important for the pulvinus bending response, and oscillations in MAPK activity occur initially but resolve such that higher levels of activity persist on the upper side during the early hours after gravistimulation (Clore et al. 2003). The ROS that appears to accumulate early in the response may be related to initial MAPK activation because ROS has been shown to induce MAPK activation in both plant (Kovtun et al. 2000; Nakagami et al. 2004; Rentel et al. 2004) and animal systems (as reviewed in Guzik et al. 2003). It is conceivable that both ROS and MAPK activation could both facilitate asymmetric growth in multiple ways. For example, increased MAPK activity has been correlated with a loss of auxin responsiveness in ascorbate oxidase (AO) antisense lines of tobacco (Pignocchi et al. 2006). Perhaps the increased level of MAPK activation on the upper side of the pulvinus serves to prevent auxin responsiveness as well, which could lead to growth inhibition. ROS themselves have been associated with both growth inhibition (typically through cell wall cross-linking) as well as enhancement (through cell wall loosening) as described in a recent review by Gapper & Dolan (2006). In the case of the gravity response of the pulvinus, the latter seems to be more likely because the pulvini bent away from the site of H2O2 application, and because more intense DAB staining was


156 A. M. Clore et al. observed in the lower (i.e. elongating) half of the pulvini after 72 h of gravistimulation.

A cytoplasmic aconitase/IRP1 homolog is up-regulated in gravistimulated pulvini and is expressed in the bundle sheath cells Evidence from this study indicating that a cytoplasmic aconitase/IRP1 homolog is involved in gravitropism in the maize pulvinus includes the following findings. Firstly, a cDNA fragment with high sequence similarity to mammalian IRP1 was amplified using SSH PCR to identify genes differentially expressed after 1 h of tipping. Secondly, a single band was detected when digoxigenin-labelled probes against IRP1 were hybridized to Northern blots and the bands were more intense (albeit modestly so) in gravistimulated pulvini samples than in vertical control samples. Thirdly, the results of real-time RT-PCR analysis indicate that the gene is dynamically regulated in gravistimulated pulvini, with repeated rises and falls in expression occurring in both halves over the first few hours of gravistimulation. This pattern of expression is different than that found for beta tubulin transcripts, which changed little during the first 30 min of gravistimulation and CaM transcripts, which were shown to increase steadily over the first several hours in both halves (Heilmann et al. 2001). Heilmann et al. (2001) also reported the steady recruitment of mRNAs into polysomes over the first 24 h after an initial decrease during the first 15 min. Therefore, we do not think that dynamic changes in IRP1 expression are because of any sort of universal rises and falls in gene expression. Finally, the fourth line of evidence that IRP1 may be involved in the gravity response is the fact that it is expressed in the bundle sheath cells of gravistimulated pulvini which are thought to be the sites of gravity perception because they contain sedimentable amyloplasts (Collings et al. 1998).

Levels of cytoplasmic aconitase/IRP1 homolog mRNA did not increase in vertical pulvini harvested throughout the day or in gravistimulated internode tissue, but did increase in gravistimulated leaf sheath tissue near the pulvinus To ensure that the levels of the cytoplasmic aconitase/IRP1 homolog did not undergo oscillatory changes in vertical pulvini over time, we conducted real-time RT-PCR analysis of its levels in vertical pulvini harvested at comparable times of day as the gravistimulated pulvini. A lack of significant changes in expression suggests that an endogenous rhythm unrelated to gravistimulation is probably not responsible for the changes measured in gravistimulated samples. To test whether the changes in expression were specific to the pulvinus, we tested internodes of gravistimulated stems over time. Only a slight decrease in expression was noted, indicating that there is some level of specificity. However, leaf sheath tissue harvested from the area

adjacent to the pulvinus after 30 min of gravistimulation did show a reproducible increase in expression. Although the leaf sheath is not thought to play an active role in maize stem gravitropism (Collings et al. 1998), we have observed that the region of the leaf sheath immediately adjacent to the pulvinus contains abundant starch near the vascular bundles (data not shown). In addition, we have observed that this region of the sheath appears to somehow form an ‘outpocketing’ to accommodate the growing pulvinus. Therefore, we speculate that this portion of the leaf sheath may also be gravisresponsive in maize. We do not yet know the role of the cytoplasmic aconitase/ IRP1 homolog in the gravity response, but we speculate that it may couple the ROS signalling to post-transcriptional changes in gene expression that may be important for establishing asymmetric growth. Interestingly, Moeder et al. (2007) have recently reported a potential role for a cytoplasmic aconitase/IRP1 homolog in the regulation of resistance to oxidative stress and cell death in Arabidopsis and Nicotiana. In the same paper, they also reported evidence that the protein binds to a particular superoxide dismutase mRNA in chloroplasts. We intend to further pursue the role of this protein in the gravitropic response of maize in the future.

SUMMARY AND CONCLUSIONS ROS levels in bundle sheath cells of maize stem pulvini increase very soon after the onset of gravistimulation. Perhaps the increases in ROS that occur in or near the amyloplasts within 1 min of stimulation could serve to couple the sedimentation of these organelles to downstream intracellular changes that lead to altered growth. Application of H2O2 to the upper side and ascorbic acid to the lower side of gravistimulated pulvini reversed the directionality of the response, which may suggest a role for an H2O2 gradient in determining polarity. Both the increase in ROS and the gravitropic response are DPI insensitive, suggesting that they are not dependent upon flavin-containing oxidases. Finally, a cytoplasmic aconitase/IRP1 homolog is expressed in the bundle sheath cells of gravistimulated pulvini further implicating ROS signalling in the pathway. Future experiments will investigate the kinetics and potential sources of the ROS increase, including the possibility that ROS scavenging ability may be reduced in gravistimulated pulvini. We also intend to further investigate the potential relationship between amyloplast sedimentation and ROS generation, and to ultimately elucidate the role of the cytoplasmic aconitase/IRP1 homolog in the pathway.

ACKNOWLEDGMENTS The SSH PCR was conducted by A.M.C. when she was a North Carolina State NSCORT postdoctoral fellow under Dr Ross Whetten, and was supported by NASA NSCORT grant NAGW-4984. All of the remaining work was conducted at New College of Florida by A.M.C. in collaboration with undergraduate students S.M.D. and S.M.N.T. with


Reactive oxygen species and cytoplasmic aconitase expression in gravistimulated maize pulvini 157 the generous support of the New College of Florida Foundation, Alumni Association, and the Division of Natural Sciences. We also wish to thank the National Science Foundation for the funds with which to purchase the real-time PCR machine (MRI award DBI-0520607), and Dr Ingo Heilmann for providing us with a cDNA fragment corresponding to CaM and for associated primers. We wish to acknowledge Ms Deborah Schmidt for her assistance with maize cultivation and manuscript editing, and Dr Katherine Walstrom for valuable discussions. Finally, we thank students Ms Raleigh Gardiner and Ms Barbara Kahn for their assistance with preliminary real-time RT-PCR experiments, and Mr Eric Gars for assistance with starch staining.

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