Plant Physiology Preview. Published on June 21, 2016, as DOI:10.1104/pp.16.00725
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Short title: Ethylene- and shade-induced stress escape
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Corresponding authors: Rashmi Sasidharan, Ronald Pierik
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Address: Padualaan 8, 3584 CH, Utrecht, The Netherlands
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Telephone number: +31 30 2536838
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Email:
[email protected],
[email protected]
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Ethylene- and shade-induced hypocotyl elongation share transcriptome
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patterns and functional regulators
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Das D.1, St. Onge K.R.1,2, Voesenek L.A.C.J.1, Pierik R.1* and Sasidharan R.1*
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*shared senior and corresponding authors
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1
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Netherlands
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Plant Ecophysiology, Institute of Environmental Biology, Utrecht University, 3584CH Utrecht, The Department of Biological Sciences, CW405 BioSci Building, University of Alberta, T6J2E9, Canada
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Summary: Ethylene and shade share a conserved set of molecular regulators to
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control plasticity of hypocotyl elongation in Arabidopsis.
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AUTHOR CONTRIBUTIONS
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R.P., R.S and L.A.C.J.V. conceived the original research plans and project. R.S.,
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R.P, L.A.C.J.V., K.R.S. supervised the experiments. D.D. performed most of the
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experiments. D.D., K.R.S., R.P. and R.S. designed the experiments. D.D. and
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K.R.S. analyzed the data. D.D. wrote the article with contributions of all the authors.
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FUNDING INFORMATION
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This research was supported by the Netherlands Organisation for Scientific
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Research (grant nos. ALW Ecogenomics 84410004 to L.A.C.J.V., ALW VENI
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86312013 and ALW 82201007 to R.S., ALW VIDI 86412003 to R.P.) and a Utrecht
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University Scholarship to D.D.
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CORRESPONDING AUTHOR EMAIL:
[email protected],
[email protected]
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1 Downloaded from www.plantphysiol.org on June 22, 2016 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Copyright 2016 by the American Society of Plant Biologists
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ABSTRACT
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Plants have evolved shoot elongation mechanisms to escape from diverse
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environmental stresses such as flooding and vegetative shade. The apparent
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similarity in growth responses suggests possible convergence of the signalling
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pathways. Shoot elongation is mediated by passive ethylene accumulating to high
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concentrations in flooded plant organs and by changes in light quality and quantity
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under vegetation shade. Here we study hypocotyl elongation as a proxy for shoot
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elongation and delineated Arabidopsis hypocotyl length kinetics in response to
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ethylene and shade. Based on these kinetics, we further investigated ethylene and
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shade-induced genome-wide gene expression changes in hypocotyls and cotyledons
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separately. Both treatments induced a more extensive transcriptome reconfiguration
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in the hypocotyls compared to the cotyledons. Bioinformatics analyses suggested
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contrasting regulation of growth promotion- and photosynthesis-related genes.
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These analyses also suggested an induction of auxin, brassinosteroid and gibberellin
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signatures and the involvement of several candidate regulators in the elongating
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hypocotyls. Pharmacological and mutant analyses confirmed the functional
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involvement of several of these candidate genes and physiological control points in
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regulating stress-escape responses to different environmental stimuli. We discuss
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how these signaling networks might be integrated and conclude that plants, when
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facing different stresses, utilise a conserved set of transcriptionally regulated genes
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to modulate and fine tune growth.
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INTRODUCTION
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All organisms, including plants, assess and respond to both biotic and abiotic factors
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in their environments (Pierik and De Wit, 2014; Pierik and Testerink, 2014; Franklin
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et al., 2011; Quint et al., 2016; Osakabe et al., 2014; Voesenek and Bailey-Serres,
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2015). However, unlike animals, plants, cannot move away from extremes in their
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surrounding environment but rather rely on various plastic morphological and
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metabolic responses. Such response traits include changes in plant architecture to
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escape the stress and optimize resource capture (Pierik and Testerink, 2014;
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Mickelbart et al., 2015). With energy reserves being invested in escape traits, plants
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often have lower plant biomass and crop yield (Casal, 2013). Molecular investigation
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of the different signalling pathways controlling these traits along with the
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characterization of underlying molecular components would not only enhance
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fundamental knowledge of stress-induced plasticity but also benefit crop
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improvement.
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Plants are highly sensitive to changes in their light environment. Young plants
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growing in a canopy experience changes in light quality and quantity due to
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neighbouring plants and compete to harvest optimum light (Pierik and De Wit, 2014;
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Casal et al., 2013). When a plant cannot outgrow its neighbours, it experiences
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complete vegetation shade (hereafter termed as “shade”) which, in addition to low
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red: far-red (R:FR), is marked by a significant decline in blue light and overall light
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quantity. These changes initiate so-called Shade Avoidance Syndrome (SAS)
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responses consisting of petiole, hypocotyl and stem elongation; reduction of
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cotyledon and leaf expansion; upward movement of leaves (hyponasty), decreased
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branching and increased apical dominance (Vandenbussche et al., 2005; Franklin,
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2008; Casal, 2012; Pierik and De Wit, 2014). Shade-induced elongation comprises a
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complex network of photoreceptor-regulated transcriptional and protein-level
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regulation involving BASIC HELIX LOOP HELIX (BHLH) and HOMEODOMAIN-
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LEUCINE ZIPPER, (HD-ZIP) transcription factors and auxin, gibberellin and
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brassinosteroid hormone genes (Casal, 2012; 2013). Flooding often leads to partial
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or complete submergence of plants. Water severely restricts gas diffusion and the
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consequent limited exchange of O2 and CO2 restricts respiration and photosynthesis.
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Another consequence is the rapid accumulation of the volatile hormone ethylene.
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Ethylene is considered an important regulator of adaptive responses to flooding,
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including accelerated shoot elongation responses that bring leaf tips from the water
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layer into the air (Sasidharan and Voesenek, 2015; Voesenek and Bailey-Serres,
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2015). In deepwater rice, this flooding-induced elongation response involves
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ethylene-mediated induction of members of the group VII Ethylene Response
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Factors (ERF-VII) family, a decline in active abscisic acid (ABA) and consequent
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increase in gibberellic acid (GA) responsiveness and promotion of GA biosynthesis
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(Hattori et al., 2009). In submerged Rumex palustris petioles, ethylene also rapidly
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stimulates cell wall acidification and transcriptional induction of cell wall modification
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proteins to facilitate rapid elongation (Voesenek and Bailey-Serres, 2015). Shade
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cues are reported to enhance ethylene production resulting in shade avoidance 3 Downloaded from www.plantphysiol.org on June 22, 2016 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
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phenotypes (Pierik et al., 2004). However, these responses are mediated by
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ethylene concentrations of a much lower magnitude than that occuring in flooded
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plant organs (1µl L-1) (Sasidharan and Voesenek, 2015).
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So far, it is largely unknown to what extent these growth responses to such highly
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diverse environmental stimuli, share physiological and molecular components
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through time. A preliminary study in R. palustris showed that GA is a common
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regulator of responses to both submergence and shade (Pierik et al. 2005). Although
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submergence is a compound stress, rapid ethylene accumulation is considered an
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early and reliable flooding signal triggering plant adaptive responses. High ethylene
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concentrations as occur within submerged plant organs, promote rapid shoot
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elongation (Voesenek and Bailey-Serres, 2015). This submergence response, which
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has been extensively characterised in rice and Rumex (Hattori et al. 2009; van Veen
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et al., 2013), can be almost completely mimicked by the application of saturating (1µl
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L-1) ethylene concentrations (Sasidharan and Voesenek, 2015). Saturating ethylene
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concentrations were therefore used here as a submergence mimic. Shade was given
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as true shade which combines the three known key signals that trigger elongation
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(red and blue light depletion with relative far-red enrichment).
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A hypocotyl elongation assay in Arabidopsis thaliana (Col-0) was used as a proxy for
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shoot elongation under ethylene and shade in order to study to what extent ethylene
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and shade responses share molecular signalling components. Although ethylene
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suppresses Arabidopsis hypocotyl elongation in dark, high ethylene concentrations
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in light (as occurs during submergence) stimulate hypocotyl elongation in
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Arabidopsis (Smalle et al., 1997; Zhong et al., 2012). Also upon simulated shade,
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Arabidopsis demonstrates pronounced hypocotyl elongation (Morelli and Ruberti,
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2000). To capture early physiological responses and gene expression changes in
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response to ethylene and shade, Arabidopsis seedling hypocotyl elongation and
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cotyledon expansion were examined over time. The two treatments elicited
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characteristic hypocotyl growth kinetics. To uncover the transcriptomic changes
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regulating the elongation response to these signals, an organ-specific genome-wide
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investigation was carried out on hypocotyls and cotyledons separately at three time-
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points corresponding to distinct hypocotyl elongation phases. Clustering analyses in
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combination with biological-enrichment tests allowed identification of gene clusters
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with expression patterns matching the hypocotyl growth trends across the three time4 Downloaded from www.plantphysiol.org on June 22, 2016 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
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points in both the treatments. Correlation of genome-wide hypocotyl and cotyledon-
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specific transcriptomic changes to publicly available microarray data on hormone
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treatments identified enriched hormonal signatures of auxin, brassinosteroid and
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gibberellin in hypocotyl tissues and several potential growth regulatory candidate
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genes. Using hormone mutants and chemical inhibitors, we confirmed the combined
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involvement of these hormones and candidate regulators in the hypocotyl elongation
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response to ethylene and shade. We suggest that growth responses to diverse
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environmental stimuli like ethylene and shade converge on a common regulatory
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module consisting of both positive and negative regulatory proteins that interact with
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a hormonal triad to achieve a controlled fine-tuned growth response.
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RESULTS
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Delineation of hypocotyl elongation kinetics under ethylene and shade in
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Arabidopsis seedlings
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Exogenous application of ethylene (1 µl L-1) in light-grown seedlings resulted in thick,
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yet elongated hypocotyls and smaller cotyledons as compared to untreated controls.
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Shade, achieved by the use of a green filter, stimulated strong hypocotyl elongation
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in seedlings but resulted in mildly smaller cotyledons compared to controls (Fig. 1A).
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Hypocotyl length increments in the two treatments relative to control were around 2-
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fold under ethylene and greater than 3-fold under shade (Figs. 1, A and B). For both
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treatments growth stimulation was strongest in the first two days of treatment (Fig.
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1C) and faded out in the subsequent days. When combined, shade and ethylene
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exposure resulted in hypocotyl lengths that were intermediate to the individual
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treatments (Supplemental Fig. S1).
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To get a more detailed time-line of the early elongation kinetics, we performed a
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follow-up experiment with 3 h measurement intervals for the first 33 h (Fig. 1D).
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Ethylene-mediated stimulation of hypocotyl elongation started only in the middle of
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the dark period after 15 h. However, under shade, longer hypocotyls were recorded
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already after 3 h and this rapid stimulation continued until the start of the dark period.
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Interestingly, accelerated elongation was again observed at around 24 h after the
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start of the treatments (when the lights were switched on). Based on this time-line,
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we determined epidermal cell lengths at time-points 0, 3, 7.5, 15 and 27 h (Gendreau 5 Downloaded from www.plantphysiol.org on June 22, 2016 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
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et al., 1997; Fig. 1E), the latter four of which are either prior to start of accelerated
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growth or during it in response to ethylene and shade. The rapid stimulation of
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elongation under shade starts at the base of the hypocotyl (3 h) and then progresses
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all along the hypocotyl with maximum elongation occurring in the middle segment
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while under ethylene accelerated elongation is observed at the middle-bottom of
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hypocotyl (27 h).
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Organ-specific transcriptomics in hypocotyl and cotyledon under ethylene and
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shade
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The transcriptome response to ethylene and shade in hypocotyl and cotyledon
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tissues was characterised using Affymetrix Arabidopsis Gene 1.1 ST arrays at three
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time points of hypocotyl length kinetics (1.5 h, 13.5 h and 25.5 h) (Fig. 1D). Principal
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component analysis (PCA) (Abdi and Williams, 2010) of all replicate samples for
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hypocotyl and cotyledon exposed to control, ethylene or shade conditions showed
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that replicate samples generally clustered together (Fig. 2A). The first principal
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component (34.2%) separates tissue-specific samples, whereas the second principal
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component (13.0%) showed separate clustering of the 13.5h samples which falls
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during the dark period.
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Hierarchical clustering (HC) (Eisen et al., 1998) of mean absolute expression
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intensities for the different main samples (combination of 3 replicates) revealed
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similar trends (Fig. 2B). Fig. 2C shows the distribution of up- and down-regulated
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differentially expressed genes (DEGs) (genes with adjusted p-value ≤ 0.01) in
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hypocotyl and cotyledon for ethylene and shade at the three harvest time points 1.5
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h, 13.5 h, 25.5 h respectively. In both the conditions and tissues, the number of both
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up- and down-regulated DEGs increased with time. Ethylene regulated substantially
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more DEGs in the hypocotyl as compared to shade at all harvest time points (Fig.
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2C). Data analysis identified 6668 and 4741 genes (hereafter termed as “total
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DEGs”) that were differentially expressed in hypocotyl at one or more of the three
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tissue harvest time points by ethylene and shade respectively. Interestingly, in the
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cotyledon, at 1.5 h, the number of significant DEGs under ethylene was higher than
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under shade but at the subsequent two time points, shade regulated more genes. In
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the cotyledon, 1197 and 2173 DEGs were identified that were differentially
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expressed at one or more of the three tissue harvest time points by ethylene and
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shade respectively. 6 Downloaded from www.plantphysiol.org on June 22, 2016 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
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Interestingly, at 1.5 h, there was more transcriptional regulation in ethylene- than
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shade-exposed hypocotyls even though subsequent hypocotyl elongation was much
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more rapid in shade (Figs. 2C and 1D). For ethylene-specific downregulated DEGs
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at 1.5 h, the topmost enriched GO term was cell wall organization (containing 30
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genes), which suggested a repression of growth-promoting genes and possible lack
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of ethylene-mediated elongation at 1.5 h (Supplemental Fig. S2). We also found 6
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genes (AT1G65310, XYLOGLUCAN ENDOTRANSGLUCOSYLASE / HYDROLASE
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17 (XTH17); AT5G23870, pectin acetyl esterase; AT3G06770, glycoside hydrolase;
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AT5G46240, POTASSIUM CHANNEL IN ARABIDOPSIS THALIANA 1 (KAT1);
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AT1G29460, SMALL AUXIN UPREGULATED RNA 65 (SAUR65) and AT3G02170,
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LONGIFOLIA 2 (LNG2)) in the shade-specific upregulated 32 genes at 1.5 h, with
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implied cell expansion
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elongation response (Sasidharan et al., 2010; Philippar et al., 2004; Nozue et al.,
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2015; Chae et al., 2012; Lee et al., 2006).
roles and could possibly be associated with the rapid
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Different gene expression clusters contributing to hypocotyl growth in
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ethylene and shade
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In order to find specific genes regulating the elongation phenotype under both
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treatments, we used temporal clustering of DEGs based on expression values. Due
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to distinct hypocotyl length kinetics in response to ethylene and shade (Fig. 1D), we
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searched for a set of temporally co-expressed genes that could potentially contribute
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to this treatment-specific kinetics. Time-point based clustering was performed for the
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6668 ethylene and 4741 shade total DEGs based on the positive or negative
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magnitude of log2FC for DEGs at the three time points (Fig. 3, A and D). The gene
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expression patterns in clusters-1 and 5 across the three time points matched the
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ethylene hypocotyl growth kinetics closely (Fig. 3, B and C). Similarly, gene
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expression kinetics in clusters-1 and 3 matched the hypocotyl length kinetics in
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shade (Fig. 3, E and F). These growth pattern matching clusters were termed
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“positive”. All the clusters with mirror image of gene expression profiles to that of the
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positive clusters (clusters-8 and 4 in ethylene and clusters-8 and 6 in shade) were
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termed as “negative” clusters.
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Next, a hypergeometric over-representation test for selected MapMan bins (“stress”,
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“hormone”, “signalling”, “RNA.Regulation of transcription” and “cell wall”) was carried
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out for the temporal gene clusters (Fig. 3G). Interestingly, “cell wall”, “hormone” and
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“signalling” were highly co-enriched in positive clusters (cluster-1 and 5 for ethylene
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and cluster-1 for shade) which hints at co-regulation of genes mapped to these terms
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during transcriptomic response to ethylene and shade in the hypocotyl.
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To identify growth promotion related DEGs, we identified DEGs common to both the
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treatments in the clusters previously designated as ‘positive’ and ‘negative’ clusters
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(Fig. 3, H and I). 997 DEGs were obtained from a venn diagram between the
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treatment-specific positive clusters, upregulated at atleast two time-points, and
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hereafter called “Common Up”. Similarly, 824 DEGs were shared between ethylene
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and shade negative clusters, were downregulated at atleast two time points and
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hereafter called “Common Down”.
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Enriched functional categories in the different gene sets from Venn diagrams of
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positive and negative clusters, were identified using the GeneCodis tool (Tabas-
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Madrid et al., 2012) (Fig. 4). In the Common Up set, we found a variety of growth
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associated GO categories including cell wall modification, hormone (auxin and
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brassinosteroid) signaling and metabolism, transport processes, tropisms, response
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to abiotic stimuli and signal transduction. The ethylene-specific set for positive
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clusters was enriched for ethylene-associated terms as expected, but also for
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various sugar metabolic, endoplasmic reticulum (ER)-related and protein post-
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translational modification-related processes. Some of the enriched GO terms in the
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ethylene-specific set for positive clusters were also found in the Common Up set, but
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caused by different genes in the same GO category, including those associated with
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growth, hormones and transport processes. The shade-specific set for positive
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clusters showed only few clear GO enrichments such as trehalose metabolism,
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secondary cell wall biogenesis and amino acid metabolism, but also shared some
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with the Common Up set like shade avoidance and protein phosphorylation and with
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both Common Up and ethylene-specific set for positive clusters, such as response to
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auxin and unidimensional growth.
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In the Common Down set, GO terms associated with photosynthesis, primary and
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secondary metabolism, response to biotic and abiotic stress, as well as
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photomorphogenesis were enriched. The latter is striking given that ethylene does
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not alter the light environment. The ethylene-specific set for negative clusters
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included strong enrichment of circadian rhythm and a variety of photosynthesis-and
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chloroplast-associated GO terms, partially shared with the Common Down set. In the
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shade-specific set for negative clusters, flavonoid/anthocyanin biosynthesis and
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response to UV-B and heat were enriched. The shade-specific set for negative
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clusters shared terms from defence-associated GO categories and cadmium, karrikin
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response with Common Down set. Terms common to all three sets of negative
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clusters were related to photomorphogenesis and metabolic process.
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Functional characterization: shared components in ethylene -and shade-
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mediated regulation of hypocotyl length
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We classified Common Up and Down set genes into transcriptional regulators,
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hormone metabolism genes, signalling genes and cell wall genes and also applied a
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logFC filter (see “Materials and Methods” sub-section “LogFC Filter and Gene
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Classification”) to obtain a final list of 53 and 8 genes in the two sets respectively
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(Fig. 5, A and B). We selected a subset of candidate regulators for functional testing
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and made sure to include two transcription factors since these may be relatively
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upstream in the convergence of signaling pathways. Obvious targets for these
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regulators would be different plant hormones, and before zooming in on these, we
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ran a hormonometer analysis with our transcriptome data to further subset our
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candidate regulators.
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Transcription factor candidates:
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ARABIDOPSIS THALIANA HOMEOBOX PROTEIN 28 (ATHB28) is a zinc-finger
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homeodomain (ZF-HD) transcription factor that showed upto 2-fold induction in the
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hypocotyls in ethylene and shade respectively. A homozygous null mutant
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(Supplemental Fig. S3, A-D), “athb28”, showed a significant reduction in hypocotyl
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lengths under both ethylene and shade (Fig. 6A), consistent with its induction upon
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both the treatments.
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Both shade and ethylene also significantly induced transcript levels of the bHLH
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transcription factor INCREASED LEAF INCLINATION1 BINDING bHLH1 like-1
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(IBL1) Arabidopsis hypocotyls. However, the ibl1 (SALK T-DNA insertion line)
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showed wild-type responses to the treatments (Fig. 6B). The bHLH transcription
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factors IBL1 and its homolog IBH1 have been implicated in repressing BR-mediated
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cellular elongation. In an ibl1 mutant, IBH1 is still present and may negatively
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regulate cell elongation independently of IBL1. The 35S overexpression line, IBL1OE
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(Zhiponova et al., 2014), had shorter hypocotyls than the wild-type under control
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conditions. IBL1OE also lacked ethylene and shade-induced hypocotyl elongation
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implying an inhibitory role for IBL1 (Fig. 6C).
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Hormone candidates: auxin, brassinosteroid and gibberellin
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To further investigate the significant hormone related changes amongst the growth
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related DEGs we analysed our data using Hormonometer (Volodarsky et al., 2009).
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For both treatments, the hormonal signatures across the three time-points for BR
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and GA most closely matched the hypocotyl elongation kinetics (Figs. 1D and 7A).
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The analysis also showed significant correlations with auxin responses for all data
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sets.
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In the Common Up set 49 genes were present that were all also auxin-regulated,
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whereas there were 14 that were also BR-regulated. In the Common Down set 16
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genes were present that can be regulated by auxin, 16 that can also be ABA-
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regulated and 13 genes that are also JA-regulated. Interestingly, there were no
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genes for BR in the Common Down set. In addition, genes involved in auxin-
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conjugation genes (GRETCHEN HAGEN 3 FAMILY PROTEIN 3.17 (GH3.17) and
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AT5G13370), GA catabolising genes (GIBBERELLIN 2 OXIDASE (GA2OX) 2,
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GA2OX4 and GA2OX7) and JA augmenting genes (LIPOXYGENASE (LOX) 1,
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LOX2 and LOX3) were down-regulated.
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In order to test the possible role of auxin, GA and BR, in mediating shade and
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ethylene-induced hypocotyl elongation, we first tested the effects of pharmacological
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inhibitors of these hormones on shade and ethylene-induced hypocotyl elongation.
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To visualize the auxin effect we treated the pIAA19:GUS auxin response marker line
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with the auxin transport inhibitor, 1-N-Naphthylphthalamic acid (NPA). As shown in 10 Downloaded from www.plantphysiol.org on June 22, 2016 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
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Fig. 7B, application of NPA (25 µM) inhibited hypocotyl elongation and also strongly
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reduced staining in the hypocotyl region. This inhibition was rescued by IAA (10 µM).
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The auxin perception inhibitor, α-(phenylethyl-2-one)-indole-3-acetic acid, PEO-IAA
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(100 µM), strongly reduced staining in the whole pIAA19:GUS seedling and inhibited
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elongation in response to both treatments. The significant inhibition of ethylene and
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shade-induced hypocotyl elongation by the different auxin inhibitors is quantitated in
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Fig. 7,C-E. The addition of NPA, yucasin (auxin biosynthesis inhibitor) and α-
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(phenylethyl-2-one)-indole-3-acetic acid (PEO-IAA; auxin antagonist) inhibited
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hypocotyl elongation under both the treatments confirming that all three aspects of
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auxin are required for ethylene- and shade-induced hypocotyl elongation. BR
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biosynthesis inhibitor brassinazole (BRZ) and GA biosynthesis inhibitor paclobutrazol
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(PBZ) fully inhibited these elongation responses as well (Fig. 7, F and G).
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To further validate the involvement of auxin, BR and GA in ethylene and shade-
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induced hypocotyl elongation we tested hypocotyl elongation responses in a variety
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of hormone mutants, including mutants for candidate genes from Figure 5.
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Both the auxin receptor (tir1-1) and biosynthesis (wei8-1) mutants showed
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significantly impaired hypocotyl elongation responses compared to the wild type
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ethylene and shade response (Fig. 8, A and B). A similar effect was seen in the
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auxin transport mutant pin3pin4pin7 which had severely reduced hypocotyl
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elongation in both treatments (Fig. 8C).
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The GA biosynthesis (ga1-3) and insensitive (gai) mutant both showed a complete
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lack of hypocotyl elongation in both treatments (Fig. 8, D and E). We also tested the
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GA biosynthesis mutant ga20ox1-3, since it was identified as a ‘common Up’ gene,
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induced in response to both treatments (Fig. 5A). The ga20ox1-3 mutant showed a
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significantly reduced elongation phenotype in both treatments compared to the wild
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type response (Fig. 8F).
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The BR receptor (bri1-116) and biosynthesis (dwf4-1) mutants both showed severe
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hypocotyl elongation phenotypes, and did not respond to either treatment (Fig. 8, G
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and I). In another biosynthesis mutant, rot3-1, while the ethylene elongation
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response was absent there was a severely reduced shade response (Fig. 8H). Two
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BR-metabolism related genes were identified in the common Up set (Fig. 5A):
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BR6OX1 and BAS1. The bas1-2 mutant showed constitutive elongation in all
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treatments ((Fig. 8J) confirming a negative role of BR catabolism through BAS1 in 11 Downloaded from www.plantphysiol.org on June 22, 2016 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
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hypocotyl elongation control. Although the BR biosynthetic mutant br6ox1 (cyp85a1-
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2) mutant did not show any phenotypic alteration (Fig. 8K), a double mutant of
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BR6OX1 and BR6OX2 (cyp85a1cyp85a2) showed a complete lack of elongation in
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response to both ethylene and shade (Fig. 8L).
360 361 362
Discussion
363
Accelerated shoot elongation is a common mode of stress escape that allows plants
364
to grow away from stressful conditions (Pierik and Testerink, 2014). Stress escape
365
does, however, come at an energetic cost and is only beneficial if improved
366
conditions are achieved. Here our goal was to establish to what extent shade and
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ethylene elicit similar responses through shared or distinct molecular pathways. In
368
our study we found distinct elongation kinetics in ethylene and shade for Arabidopsis
369
hypocotyls, differing in both temporal regulation and the degree of response. Shade
370
treatment evoked a rapid, strong and persisting hypocotyl elongation, whereas
371
ethylene initially inhibited elongation and only in the first night period started to
372
promote hypocotyl length (Fig. 1). In both treatments, hypocotyl growth involved
373
enhanced epidermal cell elongation. Previous studies have shown that low R:FR
374
induces ethylene biosynthesis in Arabidopsis (Pierik et al., 2009) and it could be
375
argued that the shade response might act through ethylene. However, ethylene
376
marker genes were not induced in shade, and the ethylene-insensitive ein3eil1
377
mutant retained a full response to shade (Supplemental Fig. S4), ruling out a role for
378
ethylene in the shade response. Interestingly, combining shade and ethylene
379
treatments did not lead to an additive response and instead dampened shade-
380
induced hypocotyl elongation (Supplemental Fig. S1). The growth inhibitory effect of
381
ethylene under shaded conditions could function similar to its effects in limiting
382
hypocotyl elongation in the dark i.e. via induction of negative growth regulators such
383
as ERF1 (Zhong et al., 2012). Transcriptome characterisation of the elongating
384
hypocotyl upon exposure to single shade and ethylene stresses indicated
385
considerable overlap between the two treatments. Thus, a large portion of DEGs
386
under both treatments may contribute to similar processes implying that they target
387
shared genetic components but have treatment-specific upstream regulatory factors.
388
12 Downloaded from www.plantphysiol.org on June 22, 2016 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
389
Hypocotyl
growth
390
concurrently under ethylene and shade
391
By identifying gene clusters with expression patterns closely matching the distinct
392
ethylene and shade growth kinetics, we identified positive and negative clusters for
393
the respective treatments. These clusters were also among the bigger clusters that
394
contributed to most of the transcriptomic changes, suggesting that a large part of
395
transcriptomic response are associated with the hypocotyl growth and concurrent
396
biological processes. Functional enrichment analysis for the Common Up set (shared
397
between positive clusters of ethylene and shade) (Fig. 4), suggested an involvement
398
of growth promoting genes. Cell wall genes are all involved in mediating cellular
399
expansion in growing hypocotyls. However, they need to be controlled by either the
400
environmental signal directly or by upstream factors in the signal transduction
401
pathway. The Common Down set (shared between negative clusters of ethylene and
402
shade) (Fig. 4), was highly enriched in photosynthesis-related terms and proteins.
403
The effects of ethylene on photosynthesis can be positive or negative depending on
404
the context (Iqbal et al., 2012; Tholen et al., 2007). Low R:FR treated stems of
405
tomato showed reduced expression of photosynthetic genes (Cagnola et al., 2012).
406
This reduction was mainly due to a decrease in expression of Calvin cycle genes,
407
which we also observed for our Common Down set (Supplemental Table S1). In
408
addition, under ethylene specifically, Photosystem II and I genes were mostly
409
repressed. Thus acceleration of hypocotyl elongation is accompanied by repression
410
of
411
photosynthesis. This was also shown to be true for low R:FR treated elongating
412
stems of tomato (Cagnola et al., 2012). Light capture and carbon fixation are
413
minimized and energy is apparently invested in stimulating growth (Sulpice et al.,
414
2014; Henriques et al., 2014; Lilley et al., 2012). It would be interesting to investigate
415
how photomorphogenic responses are associated with and influence photosynthesis
416
and growth promotion.
genes associated
promotion
with
and
photosynthesis
non-elongation
processes
repression
like
occur
metabolism
and
417 418
Convergence of signaling pathways in response to ethylene and shade in
419
control of hypocotyl elongation
420
In shade avoidance responses, photoreceptors like phyB and cry1/2 would regulate
421
the elongation phenotype via control of Phytochrome Interacting Factor (PIF) levels.
422
However, since these are photoreceptors, it seems unlikely that these proteins 13 Downloaded from www.plantphysiol.org on June 22, 2016 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
423
themselves would integrate information from the ethylene pathway as well (Park et
424
al., 2012; Li et al., 2012). It was shown by van Veen et al. (2013), that in the
425
submerged petioles of R. palustris (which displays petiole elongation under complete
426
submergence), early molecular components of light signalling (KIDARI, COP1, PIFs,
427
HD-ZIP IIs) are induced by ethylene independently of any change in light quality.
428
Over expression of PIF5 on the other hand leads to increased ethylene production in
429
etiolated Arabidopsis seedlings, causing inhibition of hypocotyl length (Khanna et al.,
430
2007). Interestingly, the downstream ethylene signal transduction protein EIN3 was
431
shown to physically interact with PIF3 (Zhong et al., 2012). We, therefore, suggest
432
that ethylene and shade might both induce this shared gene pool by, for example,
433
targeting (different) members of the PIF family of transcription factors. Since different
434
PIFs likely regulate the expression of at least partly shared target genes (Leivar and
435
Monte, 2014), this would explain our observed partial overlap in the transcriptional
436
response to shade and ethylene. PIFs are also known to directly bind and regulate
437
expression of other transcription factors like homeodomain (HD) TFs (Capella et al.,
438
2015; Kunihiro et al. 2011) in the control of shade avoidance responses. Indeed, our
439
TAIR motif analysis hinted towards the presence of significantly enriched binding
440
signatures of PIF/MYC proteins (CACATG) as well as HD proteins (TAATTA) in the
441
upstream promoter sequences of the Common Up set genes (Pfeiffer et al., 2014;
442
Kazan and Manners, 2013; Supplemental Fig. S5).
443
Several potential transcriptional regulators were identified in the narrowed down
444
Common Up gene set. A growth-promoting role of KIDARI in regulating elongation in
445
response to shade and ethylene was suggested previously (Hyun and Lee 2006; van
446
Veen et al., 2013). Upregulation of another bHLH encoding gene and a negative
447
regulator of elongation, IBL1 was observed for both treatments (Fig. 6C)). While
448
PIF4 induces IBH1 and IBL1, IBH1 represses PIF4 targets (Zhiponova et al., 2014).
449
IBH1 and its homolog IBL1 collectively regulate expression of a large number of BR-,
450
GA- and PIF4-regulated genes and this might their mode of action in shade- and
451
ethylene-induced hypocotyl elongation. In addition to these bHLH proteins, we show
452
that the ZF-HD transcription factor ATHB28 is also involved in regulating hypocotyl
453
elongation under ethylene and shade (Fig. 6A). Hong et al. (2011) showed that
454
another ZF-HD protein, MIF1 interacts strongly with four other ZF-HD proteins
455
including ATHB33 and ATHB28. This leads to non-functional MIF1-ATHB
456
heterodimers and inhibition of e.g. ATHB33-regulated expression and growth 14 Downloaded from www.plantphysiol.org on June 22, 2016 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
457
promotion.
Transcriptomics
data
for
458
phenotype), shows downregulation of auxin, BR and GA responsive genes and
459
upregulation of ABA genes (Hu and Ma, 2006). We can speculate that MIF1 on one
460
hand and ATHB33 and ATHB28 on the other hand, might target the same set of
461
hormone genes, but in an opposite manner, to control growth. What remains to be
462
studied is how ethylene and shade regulate ZF-HD transcription factors and this will
463
be an important topic for future studies.
464
Well-established targets for above-mentioned PIFs and HD TFs are various aspects
465
of auxin signalling and homeostasis, such as YUCCA biosynthetic enzymes and
466
AUX/IAA proteins for signalling (Kunihiro et al. 2011; Li et al., 2012; Sun et al., 2012;
467
De Smet et al., 2013). Our list of candidate genes (Fig. 5) contained auxin-
468
responsive transcriptional regulator IAA3 and many of the auxin-responsive SAUR
469
genes which have been shown to positively modulate hypocotyl elongation (Kim et
470
al., 1998; Sun et al., 2012; Chae et al., 2012; Spartz et al., 2012) and may act
471
individually or in concert to regulate the phenotype. With reference to elongation
472
responses under shade in Arabidopsis seedlings, auxin seems to play a major role.
473
An increase in free auxin levels and its transport towards epidermal cells in hypocotyl
474
is necessary for low R:FR-mediated hypocotyl elongation (Tao et al., 2008;
475
Keuskamp et al., 2010; Zheng et al., 2016). The importance of YUCCAs and TAA1 in
476
low R:FR responses has been previously demonstrated (Li et al., 2012). It is
477
generally assumed that auxin synthesized in the cotyledons is required to regulate
478
hypocotyl elongation in response to e.g. low R:FR light conditions (Procko et al.
479
2014). Indeed, cotyledons are key regulators of hypocotyl elongation in a
480
phytochrome-dependent way (Endo et al., 2005; Warnasooriya and Montgomery,
481
2009; Estelle, 1998; Tanaka et al., 2002). In our data, hormonometer analysis
482
identified strong induction of auxin-associated genes in the cotyledons in both
483
treatments (Fig. 7). We speculate that the physiological regulation of hypocotyl
484
elongation in our study depends on cotyledons via auxin dynamics. However, we
485
also show that auxin is certainly not the only shared physiological regulator between
486
the ethylene and shade response.
487
GA20OX1 and BR6OX1 expression were up-regulated in patterns that closely
488
matched the hypocotyl elongation profiles (Fig. 5A), and hormonometer analysis also
489
revealed enrichment of GA and BR hormonal signatures in ethylene and shade
35S:MIF1
(displaying
short
hypocotyl
15 Downloaded from www.plantphysiol.org on June 22, 2016 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
490
exposed hypocotyls. (Fig. 7). The positive role of GA in flooding-associated shoot
491
elongation (Voesenek and Bailey-Serres, 2015) and shade avoidance (Djakovic-
492
Petrovic et al., 2007) is well established. It is well known that GA20OX1 specifically
493
affects plant height without having any other major phenotypic effects (Barboza et
494
al., 2013; Rieu et al., 2008), and it has been shown to be involved in shade
495
avoidance (Nozue et al. 2015; Hisamatsu et al. 2005). In our data, ga20ox1 knockout
496
showed reduced elongation to shade as well as ethylene, extending its function from
497
controlling shade avoidance to ethylene mediated elongation responses (Fig. 8F).
498
GA biosynthesis via GA20OX1 can be induced by brassinosteroids, suggesting a
499
possible cross-talk between the two growth-promoting hormones (Unterholzner et
500
al., 2015). Although future studies are needed to establish if this crosstalk occurs
501
under the conditions tested here, we do confirm that BR is an important hormone
502
involved in both the responses since several BR mutants showed disturbed
503
elongation responses to ethylene and shade (Fig. 8G-8L). Interestingly, also auxin
504
and brassinosteroids have partially overlapping roles in hypocotyl elongation control
505
(Chapman et al., 2012; Nemhauser et al., 2004), further extending the crosstalk
506
towards a tripartite network. Among the BR mutants tested is the cyp85a1cyp85a2
507
mutant, encoding a double mutant for BR6OX2 and BR6OX1 which showed a
508
complete lack of elongation to both treatments (Fig. 8L) similar to a BRZ treatment
509
(Figs. 7F). BR6OX1 was one of the direct candidate genes identified from the
510
transcriptomics analysis (Figure 5A). A tripartite bHLH transcription factor module
511
consisting of IBH1, PRE and HBI1, has been previously implicated in regulating cell
512
elongation in response to hormonal and environmental signals (Bai et al., 2012).
513
Several BR biosynthesis and signaling genes are direct targets of HBI1, including
514
BR6OX1 (Fan et al. 2014), indicating the possible involvement of this bHLH
515
regulatory module in promoting BR responses during shade and ethylene exposure.
516
Why is there be such an elaborate network of regulators and even hormones
517
involved in controlling unidrectional cell expansion in hypocotyl growth responses?
518
To achieve a controlled growth, feedback loops are likely required, and crosstalk
519
between different routes are probably a necessity to deal with multiple environmental
520
inputs simultaneously. We found BAS1 transcriptional upregulation in hypocotyls in
521
response to ethylene and shade. BAS1 may act to balance the hypocotyl growth
522
promotion mediated by brassinosteroids (castasterone (CS) and brassinolide (BL)) 16 Downloaded from www.plantphysiol.org on June 22, 2016 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
523
as it inactivates both CS and BL (Neff et al., 1999; Turk et al., 2005). In shade
524
avoidance research, likewise, HFR1 is induced by PIFs to suppress the growth
525
promotion induced the same PIF proteins, and putative control of DELLAs would
526
modulate GA responses.
527
Conclusion
528
Hypocotyl elongation in response to ethylene and shade treatments is likely
529
regulated at the upstream level by (a) a bHLH module consisting of positive growth
530
regulators, PIFs (PIF3 in ethylene and PIF4 and PIF5 in shade) and inhibitory factors
531
like IBL1 and (b) a homeodomain module, where ZF-HD TFs like ATHB28 may
532
either act in parallel to the bHLH TFs or are regulated by PIFs (similar to induction of
533
HD-ZIP TFs) to transcriptionally target genes related to the growth promoting
534
hormone module (auxin, BR and GA), as hinted by promoter motif analysis. We
535
hypothesize that in Arabidopsis seedlings, shade and ethylene stimulate auxin
536
synthesis in the cotyledons, which is then transported to the hypocotyl to epidermal
537
cell layers where it interacts with both GA and BR to co-ordinately induce hypocotyl
538
elongation. This increased auxin response, indicated by elevated SAUR levels, and
539
likely increased levels of GA and BR as indicated by increased GA20OX1 and
540
BR6OX expression in the hypocotyl, likely act to induce unidirectional epidermal cell
541
wall elongation via upregulation of genes encoding cell-wall modifying proteins,
542
which promote cellular expansion leading to hypocotyl elongation.
543 544
MATERIALS AND METHODS
545
Plant Material and Growth Conditions
546
Around 30 Arabidopsis thaliana (Col-0) and mutant seeds were sown per agar plate
547
containing 1.1 g L-1 Murashige-Skoog (1/4 MS) and 8 g L-1 Plant-agar (0.8%w/v)
548
(both Duchefa Biochemie, The Netherlands). Mutants or overexpression lines used
549
in this work were: pin3pin4pin7 (Blilou et al., 2005); wei8-1 (Stepanova et al., 2008),
550
tir1-1 (Ruegger et al., 1998), dwf4-1 (Azpiroz et al., 1998), rot3-1 (Kim et al., 1998),
551
ga1-3 (Wilson et al., 1992), gai (Talon et al., 1990), ein3eil1 (Binder et al., 2004);
552
bri1-116 (van Esse et al., 2012); ibl1 and IBL1OE (Zhiponova et al., 2014);
553
cyp85a1/cyp85a2 and cyp85a1-2 (Nomura et al., 2005); bas1-2 (Turk et al., 2005);
554
ga20ox1-3 (Hisamatsu et al., 2005). 17 Downloaded from www.plantphysiol.org on June 22, 2016 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
555
ibl1 (N657437), cyp85a1 (N681535), wei8-1 (N16407) and ga20ox1-3 (N669422)
556
were obtained from NASC seed-stock centre; bri1-116 was kindly provided by Sacco
557
de Vries Lab, Wageningen University while cyp85a1/cyp85a2 and dwf4-1 were
558
kindly provided by Sunghwa Choe Lab, Seoul National University. IBL1OE
559
overexpression lines were kindly provided by the Jenny Russinova lab (VIB Ghent,
560
Belgium). bas1-2 mutant was kindly provided by Michael Neff lab (Washington State
561
University, USA). Some GA mutants used were in the Ler background. All other
562
mutants were in the Col-0 background. After stratification at 4°C for 3 d in the dark,
563
seeds were transferred for 2 h to control light conditions (see below) and then kept in
564
dark (at 20°C) again for another 15 h. Subsequently seedlings were allowed a period
565
of 24 h growth under control light conditions in Short-Day photoperiod conditions (15
566
h dark/9h light) before being transferred to 22.4 L glass desiccators with air-tight lids
567
for specific treatments. Col-0 genotypes were grown at 21 ± 1 °C. Ler genotypes
568
were grown at 19 ± 1 °C (Supplemental Figure S6).
569
For athb28 (GK-326G12), lines were obtained from NASC (UK). Genotyping was
570
performed
571
(CTAAGTACCGGGAATGTCAGAAG);
572
AGCTA) and LB primer o8474: (ATAATAACGCTG CGGA CATCTACATTTT). For
573
verifying transcript levels, ATHB28_fwd (GGAGAAGATGAAGGAATTTGCA) and
574
ATHB28_rev (TGTTTCTCTTCA TTGCTTGCT) were used.
using
the
following
primers: athb28_rev
for
athb28,
athb28_fwd
(TAACCAACTGAGCTATTCC
575 576
Treatments
577
Control
578
(Photosynthetically Active Radiation, PAR = 140 μmol m-2 s-1, blue light (400 nm-500
579
nm) = 29 μmol m-2 s-1 and R:FR = 2.1). Ethylene treatments were started by injecting
580
ethylene into the desiccators (with 1 µl L-1 final concentration in the dessicator) and
581
levels were verified with a Gas Chromatograph (GC955; Synspec, The Netherlands).
582
Shade treatment was started by putting desiccators under a single layer of Lee Fern
583
Green Filter (Lee Hampshire, UK) (PAR = 40 μmol m-2s-1, blue light (400 nm-500
584
nm) = 3 μmol m-2 s-1 and R:FR = 0.45). For growth curve experiments, two plates
585
with 15 seedlings per treatment per genotype distributed over two desiccators were
586
used. For mutant analyses, one plate with 15 seedlings per treatment per genotype
587
was used.
and
ethylene
desiccators
were
kept
in
control
light
conditions
588 18 Downloaded from www.plantphysiol.org on June 22, 2016 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
589
Imaging and Hypocotyl Length Measurements
590
For hypocotyl elongation assays, experiments were replicated twice. Seedling plates
591
were collected from the dessicators. Seedlings were flattened on the agar plates to
592
reveal the full extent of their hypocotyl, and images of the seedlings were obtained
593
by scanning the plates using EPSON Perfection v370 Photo scanner (Epson Europe
594
BV, The Netherlands). Hypocotyl lengths were measured from these images using
595
ImageJ (http://rsbweb.nih.gov/ij/) and values (per data point) were obtained for n ≥
596
30-60 seedlings. Final values for the data was obtained by taking mean ± S.E. for
597
values from the two independent experiments.
598 599
Epidermal Cell Length Measurements
600
Seedlings were mounted on microscopic slides and covered with a cover slip.
601
Hypocotyl epidermal cells were imaged using Olympus AX70 (20x objective, Nikon
602
DXM1200 camera), after which cell lengths were measured using ImageJ software
603
tool (http://rsbweb.nih.gov/ij/).
604 605
Microarray Tissue Harvest, RNA Isolation and Array Hybridization
606
Seedlings were dissected using BD PrecisionGlide Hypodermic 27 Gauge 1 1/4"
607
Grey Needle with outer diameter = 0.41 mm (Becton Dickinson B.V., The
608
Netherlands) to separately harvest the hypocotyl and cotyledon + shoot apical
609
meristem (hereafter termed “cotyledon”). The roots were discarded. Samples were
610
harvested at 1.5 h, 13.5 h and 25.5 h after the start of the treatments. For 13.5 h time
611
point, which occurs during the dark period, dissection was carried out under low
612
intensity green safelight (≈ 5 μmol m-2 s-1). For minimizing the effects of green light
613
on gene expression, seedlings were kept in the dark until dissected and dissection
614
was carried out in several rounds with each round involving maximum 2-3 seedlings.
615
In total, 3 replicate experiments were carried out. In each replicate experiment,
616
tissues were harvested from two independent technical replicates (each with 25
617
seedlings from the two technical replicate plates as mentioned in “Treatments”
618
section above). Harvested material was immediately frozen in liquid nitrogen and
619
stored at -80°C until further use.
620
Frozen tissue was ground using tissue lyser and total RNA was isolated using
621
RNeasy Mini Kit (QIAGEN, Germany). QIAGEN RNase-Free DNase set was used to
622
eliminate Genomic DNA contamination by performing on-column DNase digestion. 19 Downloaded from www.plantphysiol.org on June 22, 2016 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
623
Extracted RNA was verified (for quality check) and quantified using NanoDropTM ND-
624
1000 Spectrophotometer (Isogen Life Science, De Meern, The Netherlands).
625
RNA samples were sent to AROS (AROS Applied Biotechnology A/S, Aarhus,
626
Denmark). RNA was repurified on low-elution QIAGEN RNeasy columns, re-
627
quantified with NanoDropTM 8000 UV-Vis Spectrophotometer and checked for quality
628
with Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). RNA samples
629
with RNA Integrity Number (RIN) value of > 7.5 were considered for further use. 50
630
ng RNA from each of the two independent technical replicates was mixed in the ratio
631
1:1 and the pooled sample was considered as one biological replicate for
632
hybridization experiments. Thus, 3 biological replicates were obtained for the 3
633
replicate experiments. 100 ng of RNA sample was processed for cDNA synthesis,
634
fragmentation and labelling was carried out for the RNA samples. The samples were
635
hybridized to the Affymetrix Gene 1.1 ST Arabidopsis Array Plate and washed on an
636
Affymetrix GeneAtlas system followed by scanning of arrays at AROS Applied
637
Biotechnology (http://arosab.com/services/microarrays/gene-expression/).
638 639
Microarray Data Analysis
640
Scanned arrays in the form of .cel files (provided by AROS) were checked for quality
641
control using Affymetrix Expression Console Software and an in-house script in R
642
and
643
(Bioconductor “oligo” and “pd.aragene.1.1.st” ). Bioconductor was used for Robust
644
Multi-array Average (RMA) normalization of raw data at Gene level to obtain
645
summarized signal intensity values for all genes present on the array (log2 format).
646
Principal component analysis was carried out using Affymetrix Expression Console
647
Software (http://www.affymetrix.com/) and dendrogram of all microarray samples
648
according to the mean signal intensity values was generated using R (“plot”
649
package). Bioconductor (“Limma” package) was used for carrying out differential
650
expression analysis.
Bioconductor
(http://www.r-project.org./;
http://www.bioconductor.org/)
651 652
Temporal Clustering And Bioinformatics Analysis
653
We clustered the list of total DEGs (defined as number of DEGs that were regulated
654
at atleast one of the three time points) under ethylene and shade based on positive
655
or negative regulation at each of the three time-points. With three time-points and
656
two directions of expression (positive or negative), 23 = 8 possible trends can occur 20 Downloaded from www.plantphysiol.org on June 22, 2016 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
657
and accordingly as many clusters were obtained. DEGs were clustered temporally
658
based on log2FC.
659 660
MapMan bin overrepresentation using hypergeometric test was done using R (“stats”
661
package) and adjusted p-values for the statistical significance of enrichment were
662
converted into negative logarithm score and plotted as a heatmap. A score above
663
1.3 for this score was considered significant.
664
GeneCodis (http://genecodis.cnb.csic.es/compareanalysis) webtool was used for GO
665
analysis of different sets obtained from venn of Positive clusters and of Negative
666
Clusters. A score above 1.3 for negative log of adjusted p-values was considered
667
significant.
668 669
LogFC Filter and Gene Classification
670
In order to narrow down the genes for functional characterization (from the list of
671
classified genes), we utilised a logFC filter. To narrow down the Common Up set, a
672
filter of log2FC < 0.5 at 1.5 h and log2FC ≥ 0.5 at both 13.5 h and 25.5 h for ethylene
673
and log2FC ≥ 0.5 at both 1.5 h and 25.5 h for shade was applied. To narrow down
674
the Common Down set, we applied a filter of log2FC > - 0.5 at 1.5 h and log2FC ≤ -
675
0.5 at both 13.5 h and 25.5 h under ethylene and of log2FC ≤ - 0.5 at both 1.5 h and
676
25.5 h under shade. These resulting group of genes were then classified based on
677
MapMan based classification for the terms: “RNA.regulation of transcription”, “Cell
678
wall”,
679
(http://planttfdb.cbi.pku.edu.cn/index.php?sp=Ath)
680
(http://plntfdb.bio.uni-potsdam.de/v3.0/index.php?sp_id=ATH) were also included as
681
a source for gene-classification to select for additional transcriptional regulators.
“Signalling”
and
“Hormone
metabolism”.
Genes
from
Plant
and
Potsdam
TFDB TFDB
682 683
Hormone Correlational Analysis
684
Hormonometer software (http://genome.weizmann.ac.il/hormonometer/) was used to
685
evaluate transcriptional similarities between the transcriptome data obtained here
686
and the published, indexed list of those elicited by exogenous application of plant
687
hormones. Arabidopsis gene locus IDs were converted to Affymetrix GeneChip
688
identifiers using the “at to AGI converter” tool (The Bio-Analytic Resource for Plant
689
Biology, http://bar.utoronto.ca/). We used the new Affymetrix aragen1.1st arrays (28k
690
genes) for transcriptomics but the hormonometer data is based on 3’ ATH1 arrays 21 Downloaded from www.plantphysiol.org on June 22, 2016 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
691
(22k genes). Accordingly, many locus IDs could not be included (those which were
692
newly incorporated in the aragene1.1st arrays) in this analysis. In few other cases
693
where multiple ATH1 GeneChip IDs for one locus ID was obtained, all IDs were
694
retained.
695 696
Pharmacological Treatments
697
Auxin transport was inhibited by use of 25 µM NPA (Duchefa Biochemie, The
698
Netherlands) (Petrásek et al., 2003). Auxin perception was blocked by use of 100 µM
699
PEO-IAA (Hayashi et al., 2008). Auxin biosynthesis was blocked by use of 50 µM
700
yucasin (Nishimura et al., 2014). 2 µM brassinazole (TCI Europe, Japan) was used
701
to inhibit BR biosynthesis (Asami et al., 2000). 2 µM paclobutrazol (Duchefa
702
Biochemie, The Netherlands) was used to inhibit GA biosynthesis (Rademacher,
703
2000). All chemicals were dissolved in respective solvents (DMSO or ETOH) with
704
final solvent concentration in media < 0.1% to prevent toxicity due to solvents. All
705
chemicals were applied by pipetting 150 µl of chemical solutions or mock solvents as
706
a thin film over the MS-agar media in the petri plates and then allowing the solution
707
to diffuse through the medium before starting the treatments.
708 709
GUS Staining and Imaging
710
For GUS assays, seedlings were transferred immediately from treatments to a GUS
711
staining solution (1 mM X-Gluc (5-bromo-4-chloro-3-indolyl-D-glucoronide) (Duchefa
712
Biochem, The Netherlands) in 100 mM Sodium phosphate buffer (pH=7.0) along with
713
0.1 % Triton X-100, 0.5 mM each of Potassium ferrocyanide (K4Fe(CN)6 and
714
Potassium ferricyanide (K3Fe(CN)6 and 10 mM of EDTA (Merck Darmstadt,
715
Germany)) and kept at 37°C overnight. Seedlings were bleached in 70% ethanol for
716
1 day before capturing images.
717 718
Statistical Analysis and Graphing
719
1-way ANOVAs followed by Tukey's HSD Post-Hoc tests were performed on the
720
measurements obtained in hypocotyl length/cotyledon area kinetics to assess
721
statistically significant differences between mean hypocotyl length/cotyledon area
722
under ethylene or shade relative to control at the same time point.
723
A two-way ANOVA followed by a Tukey's HSD Post-Hoc test was used for pairwise
724
multiple comparison. For hypocotyl elongation assays, statistical significance was 22 Downloaded from www.plantphysiol.org on June 22, 2016 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
725
indicated by the use of different letters. All statistical analyses were done in the R
726
software environment. Graphs were plotted using Prism 6 software (GraphPad
727
Software, USA).
728 729
Accession numbers
730
.CEL files utilised in the organ-specific transcriptomics for hypocotyl and cotyledon
731
tissues are posted with the GEO accession series GSE83212.
732 733
Supplemental data
734
The following materials are available in the online version of this article.
735
Supplemental Figure S1. Hypocotyl elongation response in wild-type Col-0 under
736
control, ethylene, shade and combination (ethylene + shade) treatments.
737
Supplemental Figure S2. Venn diagram of gene intersection between up- and
738
down- regulated DEGs of ethylene and shade separately at 1.5 h.
739
Supplemental Figure S3. Genotyping and transcript level verification for (A)-(D)
740
athb28.
741
Supplemental Figure S4. Hypocotyl elongation response in ethylene signalling
742
mutant, ein3eil1 under ethylene and shade.
743
Supplemental Figure S5. TAIR motif analysis for Common Up and Down genesets.
744
Supplemental Figure S6. Hypocotyl length of Ler under control and ethylene
745
conditions when grown at 220 C day / 200 C night and 200 C day / 180 C night
746
temperature regime.
747
Supplemental Table S1. Photosynthesis gene proportion in genesets from Venn of
748
negative clusters.
749
ACKNOWLEDGMENTS
750
We would like to thank all the group members of Plant Ecophysiology, Utrecht
751
University, The Netherlands for their help in the harvest for the transcriptomics
752
experiment.
23 Downloaded from www.plantphysiol.org on June 22, 2016 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
753 754
FIGURE LEGENDS
755
Figure 1. Physiological responses, hypocotyl length and epidermal cell length
756
kinetics under ethylene and shade in Arabidopsis (Col-0) seedlings. 1-day old
757
seedlings were exposed to control conditions (PAR = 140 μmol m-2 s-1, blue light =
758
29 μmol m-2 s-1and R:FR = 2.1), elevated ethylene (1 µl L-1, PAR = 140 μmol m-2 s-1,
759
blue light = 29 μmol m-2 s-1 and R:FR = 2.1) or shade (PAR = 40 μmol m-2 s-1, blue
760
light = 3 μmol m-2 s-1 and R:FR = 0.45). A, Figure shows representative seedlings
761
displaying typical phenotypes after 96 h of exposure to control, ethylene or shade
762
conditions. B, Mean hypocotyl lengths at 0 h, 24 h, 48 h, 72 h and 96 h under control
763
(open circle), ethylene (closed circle) and shade (open triangle) are shown. C, Rate
764
of increase in hypocotyl length. Differences between mean hypocotyl lengths of
765
subsequent time points averaged over 1 d time interval for control, ethylene and
766
shade are shown. D, Detailed hypocotyl length kinetics. 1-day old seedlings were
767
exposed to control (open circle), ethylene (closed circle) and shade (open triangle)
768
and measured at 3 h time intervals (s, e: first time point at which shade (s) or
769
ethylene (e) treatment lead to significantly longer hypocotyls). Data are mean ± S.E.
770
(n=60) for (A)-(D). Shaded area denotes the 15 h dark period in the 15 h dark/9 h
771
light photoperiodic growth condition. e and s denote the first point of statistically
772
significant differences in hypocotyl length or cotyledon area relative to control for
773
ethylene and shade respectively. E, Epidermal cell length kinetics. 1-day old
774
seedlings were exposed to control, ethylene or shade. Mean cell length ± S.E. (n ≥
775
10) for epidermal cells of the Arabidopsis hypocotyl at 0 h control (black line) and at
776
3 h, 7.5 h, 15 h and 27 h control (orange line), ethylene (blue line) and shade (green
777
line) is shown. Apex denotes the hypocotyl-cotyledon junction and base denotes the
778
hypocotyl-root junction.
779
Figure 2. Overall description of microarray data. A, Principal component analysis
780
(PCA) and hierarchical clustering (HC) was used to describe the structure in the
781
microarray data. Expression intensities for all genes on the array for all 54 hypocotyl
782
and cotyledon samples (3 time points, 3 treatments and 3 replicates) were projected
783
onto the first three principal components. B, Hierarchical clustering was used to
784
group 18 main samples (according to mean expression intensity of 3 replicates for
24 Downloaded from www.plantphysiol.org on June 22, 2016 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
785
each main sample) into a dendrogram. C, Distribution of differentially expressed
786
genes (DEGs) in hypocotyl and cotyledon samples at three time-points in response
787
to ethylene and shade. DEGs obtained for each time-point were plotted separately
788
as up-regulated, down-regulated (adjusted p-value ≤ 0.01 and log2FC > or < 0) or
789
non-significant (adjusted p-value > 0.01). Bar length denotes total DEGs obtained
790
after combining DEGs from all 3 time points.
791
Figure 3. Temporal gene expression clusters for ethylene (A) and shade (D);
792
hypocotyl growth curve for ethylene (B) and shade (E); clusters with gene expression
793
matching hypocotyl growth kinetics in ethylene (C) and shade (F). Heatmap for
794
temporal clusters (A, D) based on the log2FC at the three microarray time points
795
(grey box represents dark; arrows indicate heatmap time-points and treatments
796
include control (open circle), ethylene (closed circle), shade (open triangle)). Yellow
797
denotes up-regulation and blue denotes down-regulation. Two gene expression
798
clusters (C, F) with mean log2FC temporal pattern resembling the hypocotyl length
799
kinetics (B, E) were named positive clusters. G, Heatmap for hypergeometric
800
enrichment of selected MapMan bins for temporal clusters under ethylene and
801
shade. Horizontal-axis denotes the cluster number. More intense colours indicate
802
higher statistical significance. Grey color indicates non-significant score or absence
803
of genes in the bin. H, Venn intersection for positive clusters (clusters with gene
804
expression pattern matching the hypocotyl length kinetics) from ethylene and shade
805
to obtain “Common Up” genes. I, Venn intersection for negative clusters (mirror
806
image to Positive clusters) from ethylene and shade to obtain “Common Down”
807
genes.
808
Figure 4. Gene Ontology (GO) enrichment analysis using GeneCodis for positive
809
and negative clusters. Adjusted p-values for statistical significance of GO enrichment
810
were converted into negative logarithm score (base10) (> 1.3 are considered
811
significant). Heatmap colours denote this score and more intense colours indicate
812
higher statistical significance. White color indicates non-significant score or absence
813
of genes in the GO terms.
814 815
Figure 5. Heatmap of (A) Common Up and (B) Common Down set of genes
816
classified into the categories: transcriptional regulator, hormone metabolism,
25 Downloaded from www.plantphysiol.org on June 22, 2016 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
817
signalling and cell wall genes after application of a log2fold-change (log2FC) filter.
818
Log2FC at the three time-points 1.5 h, 13.5 h, 25.5 h for ethylene and shade
819
microarray dataset identified in the color scheme of the heatmap. Genes were
820
categorized according to Mapman bin gene classification (shown on left side).
821 822
Figure 6. Hypocotyl length measurements for (A) athb28 (B) ibl1 (C) IBL1-OE
823
following 96 h of control, ethylene and shade treatments. Data represents mean ±
824
S.E. (n=30 seedlings). Different letters above the bars indicate significant differences
825
(2-way ANOVA followed by Tukey’s HSD Post-Hoc pairwise comparison).
826 827
Figure 7. A, Identification of enriched hormonal signatures in the ethylene- and
828
shade-induced Arabidopsis transcriptome. Ethylene and shade-induced hypocotyl
829
and cotyledon transcriptomes were analyzed for hormonal signatures using the tool
830
Hormonometer (Valdorsky et al., 2009) to establish correlations with expression data
831
in an established hormonal transcriptome database. Positive correlations were
832
colored yellow and negative correlations blue. Significant correlations were identified
833
with absolute correlation values of 0.3 and higher. Numbers in the cells represent the
834
exact correlation values. Rows denote hormone treatments that are indicated by the
835
name of the hormone and the duration of hormone treatment. Columns denote
836
ethylene and shade transcriptome in the hypocotyl and cotyledon at the three time-
837
points of tissue harvest. The magnitude of correlation in gene expression is indicated
838
by the color scale at top right side. B, Effect of auxin transport inhibitor NPA (25 µM),
839
IAA (10 µM), NPA (25 µM) + IAA (10 µM) and auxin perception inhibitor PEO-IAA
840
(100 µM) on GUS staining of the pIAA19:GUS lines. For NPA and PEO-IAA effect in
841
GUS assay, seedlings were exposed to 2 days of treatment conditions. Arabidopsis
842
(Col-0) seedlings were treated with chemical inhibitors for (C) auxin transport (NPA),
843
(D)
844
brassinosteroid biosynthesis (BRZ) and (G) gibberellin biosynthesis (PBZ) at the
845
indicated concentrations in the legend and length was measured following 96h of
846
ethylene and shade. Hypocotyl length was measured following 96 h of ethylene and
847
shade. Mean ± S.E. was calculated for 30 seedlings. Different letters above the bars
848
indicate significant differences from a 2-way ANOVA followed by Tukey’s HSD Post-
849
Hoc pairwise comparison. Abbreviations: PEO-IAA is α-(phenylethyl-2-one)-indole-3-
auxin
biosynthesis
(Yucasin),
(E)
auxin
perception
(PEO-IAA),
(F)
26 Downloaded from www.plantphysiol.org on June 22, 2016 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
850
acetic acid; NPA is 1-N-Naphthylphthalamic acid; BRZ is Brassinazole; PBZ is
851
Paclobutrazol.
852 853
Figure 8. Hypocotyl elongation response in response to ethylene and shade in (B)–
854
(D) auxin, (E)–(G) gibberellin (GA) and (H)–(J) brassinosteroid (BR) mutants. Mean
855
± S.E. was calculated for 30 seedlings. Different letters above the bars indicate
856
significant differences from a 2-way ANOVA followed by Tukey’s HSD Post-Hoc
857
pairwise comparison.
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SUPPLEMENTAL METHODS Functional Enrichment Analysis g:Profiler (http://biit.cs.ut.ee/gprofiler/) webtool was used for finding enriched GO terms in the DEGs at 1.5 hrs with Best per parent (moderate filtering) setting from g:Profiler for “Hierarchical filtering” option in order to select only the most statistically significant term from a particular hierarchical group. A score above 1.3 for negative log of adjusted p-values was considered significant. Motif Enrichment Analysis Webtool (https://www.arabidopsis.org/tools/bulk/motiffinder/index.jsp) was used for identifying motif overrepresentation (using hypergeometric test) within the 1000 bp upstream sequences (relative to Transcription Start Site) of genes. MapMan Photosynthesis Genes Mappings excel sheet “Ath_AFFY_ATH1_TAIR10” at (http://mapman.gabipd.org/ web/guest/mapmanstore) was used to assign photosynthesis genes to the different categories in Supplemental Table S1.
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SUPPLEMENTAL FIGURES AND TABLES
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Supplemental Figure S1. Effect of 7 d control, ethylene, shade and combination (ethylene + shade) treatments on hypocotyl length of Arabidopsis seedlings in short day photoperiod (9 h light / 15 h dark). Data represents mean ± S.E. (n=30-50). Different letters above the bars indicate significant differences (1way ANOVA followed by Post-Hoc pairwise comparison).
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Supplemental Figure S2. g:Profiler analysis for enriched Gene ontology (GO) IDs (adj. p-value ≤ 0.05) or genes for DEGs (A) up- or (B) down- regulated at 1.5 hrs in the hypocotyl in response to ethylene or shade. Biological process (BP) terms corresponding to enriched GO IDs have been tabulated on the right side.
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Supplemental Figure S3. Genotyping and transcript level verification. A, T-DNA insertion position in GABIKAT line GK-326G12 (athb28) B, Genotyping of athb28, (C) and (D) Null transcript and downregulation level of ATHB28 in athb28 (vs. Col-0). Abbreviations: In (B) LP: left primer, RP: right primer, LBo8474: GABI-KAT sequencing primer.
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Supplemental Figure S4. Ethylene signalling mutant ein3eil1 shows hypocotyl elongation response similar to wild-type in response to shade but totally lacks elongation in response to ethylene. Hypocotyl length was measured following 96 h of ethylene and shade. Mean ± S.E. was calculated for 60 seedlings. Different letters indicate significant differences from a 2-way ANOVA followed by Tukey’s HSD Post-Hoc pairwise comparison.
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Supplemental Figure S5. TAIR motif analysis and functional validation of PIFs and MYCs in hypocotyl elongation. TAIR motif overrepresentation (Lamesch et al., 2012). Top 2 hexamer motifs are shown for each set along with the name of motifs (relevance). % Microarray and % Genome indicate percentage of genes with the motif relative to the total number of genes in the gene set and in the genome respectively.
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Supplemental Figure S6. Hypocotyl length of Landsberg erecta ecotype of Arabidopsis thaliana under control and 1 µlL-1 ethylene conditions when grown at 22⁰C day / 20⁰C night and 20⁰C day / 18⁰C night temperature regime.
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Supplemental Table S1. Proportion of photosynthesis genes in different gene sets from the Venn diagram of negative clusters. Numbers in the table denote the % of total Mapman-assigned genes for the different photosynthetic processes. Bold numbers indicate top 3 photosynthetic categories.
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LITERATURE CITED
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J. Reimand, M. Kull, H. Peterson, J. Hansen, J. Vilo (2007). g:Profiler - a web-based toolset for functional profiling of gene lists from large-scale experiments NAR. 35: W193-W200
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Nagel, A., Thimm, O., Redestig, H., Blaesing, O.E., Steinhauser, D., Gibon, Y., Morcuende, R., Weicht, D., Meyer, S., and Stitt, M. (2005). Extension of the Visualization Tool MapMan to Allow Statistical Analysis of Arrays, Display of Corresponding Genes, and Comparison with Known Responses. Plant Physiol. 138: 1195–1204.
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