Ethylene- and shade-induced hypocotyl elongation

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

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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

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hypocotyls, differing in both temporal regulation and the degree of response. Shade

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treatment evoked a rapid, strong and persisting hypocotyl elongation, whereas

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ethylene initially inhibited elongation and only in the first night period started to

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promote hypocotyl length (Fig. 1). In both treatments, hypocotyl growth involved

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enhanced epidermal cell elongation. Previous studies have shown that low R:FR

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induces ethylene biosynthesis in Arabidopsis (Pierik et al., 2009) and it could be

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argued that the shade response might act through ethylene. However, ethylene

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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


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


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.


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


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,


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)


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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