This is the accepted version of the article: Molina-Contreras, Maria José; Paulišić, Sandi; Then, Christiane; [et al.]. «Pho- toreceptor Activity Contributes to Contrasting Responses to Shade in Car- damine and Arabidopsis Seedlings». The Plant Cell, (September 2019). DOI 10.1105/tpc.19.00275 This version is avaible at https://ddd.uab.cat/record/214181 under the terms of the license Plant Cell Advance Publication. Published on September 17, 2019, doi:10.1105/tpc.19.00275 1 RESEARCH ARTICLE 2 3 Photoreceptor Activity Contributes to Contrasting Responses 4 to Shade in Cardamine and Arabidopsis Seedlings 5 6 Maria Jose Molina-Contrerasa1, Sandi Paulišića1, Christiane Thena1,2, Jordi 7 Moreno-Romeroa, Pedro Pastor-Andreua, Luca Morellia, Irma Roig-Villanovaa3, 8 Huw Jenkinsb, Asis Hallabc4, Xiangchao Ganc, Aurelio Gomez-Cadenasd, Miltos 9 Tsiantisc, Manuel Rodríguez-Concepcióna, Jaime F. Martínez-Garcíaa,e,g 10 a 11 Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, 12 08193 Barcelona, Spain. b 13 Department of Plant Sciences, University of Oxford, Oxford OX1 3BR, United 14 Kingdom. c 15 Department of Comparative Development and Genetics, Max Planck Institute 16 from Plant Breeding Research, 50829 Cologne, Germany. d 17 Departament de Ciències Agràries i del Medi Natural, Universitat Jaume I, 18 12071 Castellò de la Plana, Spain. e 19 Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, 20 Spain. g 21 corresponding author, jaume.martinez@cragenomica.es 1 22 These authors contributed equally to this work and are shown in alphabetical 23 order. 2 24 Current address, Institute for Epidemiology and Pathogen Diagnostics, Julius 25 Kühn-Institut, Federal Research Institute for Cultivated Plants, 38104 26 Braunschweig, Germany. 3 27 Current address, Escola Superior d'Agricultura de Barcelona, Universitat 28 Politècnica de Catalunya, Campus Baix Llobregat, Castelldefels 08860 29 Barcelona, Spain. 4 30 Current address, IBG-2 Plant Sciences, Forschungszentrum Jülich, Jülich, 31 Germany 32 33 Short title: Genetic regulation of shade tolerance 34 35 One-sentence summary: The lack of a shade-induced hypocotyl elongation 36 response in Cardamine hirsuta results from the enhanced repressor activity of 37 the phytochrome A photoreceptor. 38 39 The author responsible for distribution of materials integral to the findings 40 presented in this article in accordance with the policy described in the 41 Instructions for Authors (www.plantcell.org) is: Jaime F. Martinez-Garcia 42 (jaume.martinez@cragenomica.es). 43 44 45 1 ©2019 American Society of Plant Biologists. All Rights Reserved 46 ABSTRACT 47 48 Plants have evolved two major ways to deal with nearby vegetation or shade: 49 avoidance and tolerance. Moreover, some plants respond to shade in different 50 ways; for example, Arabidopsis thaliana undergoes an avoidance response to 51 shade produced by vegetation, but its close relative Cardamine hirsuta tolerates 52 shade. How plants adopt opposite strategies to respond to the same 53 environmental challenge is unknown. Here, using a genetic strategy, we 54 identified the C. hirsuta slender in shade1 (sis1) mutants, which produce 55 strongly elongated hypocotyls in response to shade. These mutants lack the 56 phytochrome A (phyA) photoreceptor. Our findings suggest that C. hirsuta has 57 evolved a highly efficient phyA-dependent pathway that suppresses hypocotyl 58 elongation when challenged by shade from nearby vegetation. This suppression 59 relies, at least in part, on stronger phyA activity in C. hirsuta; this is achieved by 60 increased ChPHYA expression and protein accumulation combined with a 61 stronger specific intrinsic repressor activity. We suggest that modulation of 62 photoreceptor activity is a powerful mechanism in nature to achieve 63 physiological variation (shade tolerance vs. avoidance) for species to colonize 64 different habitats. 65 66 INTRODUCTION 67 Understanding how plants colonize different habitats requires identifying 68 the genetic differences underlying physiological variation between species. In 69 this work, we focus on angiosperm responses to changes in light produced by 70 nearby vegetation, perception of which alerts the plant to potential resource 71 competition by other plants. Nearby vegetation is perceived as changes in light 72 parameters: whereas sunlight has a high red (R) to far-red light (FR) ratio 73 (R:FR, >1.1), proximity to vegetation lowers this ratio (Smith, 1982). Because 74 vegetation specifically reflects FR, proximity to other plants initially results in a 75 mild reduction in R:FR (<0.7) due to the FR enrichment. Eventually, when the 76 vegetation canopy closes, sunlight is filtered by photosynthetic tissues, strongly 77 reducing the intensity of the photosynthetic active radiation (PAR, between 400 78 to 700 nm, which includes blue and R) while marginally affecting FR. As a 79 result, R:FR resulting from natural canopy shade typically drops to lower values 80 (<0.05) (Casal, 2012; de Wit et al., 2016; Martinez-Garcia et al., 2014; Smith, 81 1982). In the laboratory, both vegetation proximity and canopy shade can be 82 simulated by providing plants grown under white light (W, high R:FR) varying 83 amounts of supplemental FR (W+FR; low or very low R:FR) while maintaining 2 84 total PAR, a treatment known as simulated shade (Casal, 2012; Roig-Villanova 85 and Martinez-Garcia, 2016). 86 Plants have two main strategies to acclimate to vegetation proximity and 87 shade: avoidance or tolerance. In the early stages of development, shade- 88 avoider species invest energy into promoting elongation to overgrow their 89 neighbors as part of the so-called shade avoidance syndrome (SAS). By 90 contrast, shade-tolerant plants adopt other physiological and metabolic 91 responses to adapt to a highly conservative utilization of resources, commonly 92 accompanied by very low growth rates, i.e., do not involve promotion of 93 elongation growth (Smith, 1982; Valladares and Niinemets, 2008). 94 Analyses of the shade-avoider Arabidopsis thaliana laid the basis for our 95 knowledge of the genetic components and mechanisms involved in the 96 regulation of the SAS (Casal, 2012; Martinez-Garcia et al., 2010; Roig-Villanova 97 and Martinez-Garcia, 2016). The shade signal is perceived by the phytochrome 98 photoreceptors: phytochrome B (phyB) and phyA have major and antagonistic 99 roles (respectively) in hypocotyl elongation, the most conspicuous A. thaliana 100 response to low R:FR (Casal, 2012; Mathews, 2010). Lowering the R:FR to 101 resemble either vegetation proximity or canopy shading, deactivates phyB in 102 wild-type seedlings, resulting in the hypocotyl elongation promotion. By 103 contrast, phyA accumulates and is strongly activated under very low R:FR to 104 prevent excessive seedling elongation (Martinez-Garcia et al., 2014; Yang et 105 al., 2018). Consistent with this, A. thaliana phyB-deficient mutants display 106 constitutive shade responses under high R:FR whereas phyA mutant seedlings 107 show enhanced hypocotyl elongation only under very low R:FR conditions, 108 which indicates that phyA antagonizes phyB activity under these specific 109 canopy shade conditions (Casal et al., 2014; Martinez-Garcia et al., 2014; Yang 110 et al., 2018; Yanovsky et al., 1995). 111 SAS responses are mainly initiated because of the interaction of active 112 phytochromes with PHYTOCHROME INTERACTING FACTORs (PIFs), 113 eventually triggering rapid changes in the expression of dozens of genes that 114 implement the SAS responses. Genetic analyses in A. thaliana indicate that 115 PIFs, which are basic-helix-loop-helix transcription factors, have a role in 116 positively regulating the shade-triggered hypocotyl elongation. The active form 117 of phyB interacts with PIFs and inhibits their transcriptional activity (Casal, 3 118 2012; Martinez-Garcia et al., 2010). After exposure to shade, the proportion of 119 active phyB decreases and PIF activity increases. Enhanced PIF binding to G- 120 boxes of auxin biosynthetic genes (e.g. YUCCA genes) then promotes their 121 expression, which results in a rapid (1-4 h) increase in free IAA that is required 122 for the promotion of shade-induced hypocotyl elongation (Bou-Torrent et al., 123 2014; Hornitschek et al., 2012; Li et al., 2012; Tao et al., 2008). In addition, 124 nuclear-pore complex components and chloroplast-derived signals also prevent 125 an excessive response to shade, providing additional regulatory levels of this 126 response (Gallemi et al., 2016; Ortiz-Alcaide et al., 2019). 127 There are, however, still major gaps in understanding the genetic and 128 molecular regulation of SAS and, by extension, shade-tolerance traits. 129 Comparative analyses using shade-avoiding and shade-tolerant species is 130 expected to identify regulators of traits associated with shade tolerance habits 131 (Gommers et al., 2013). Indeed, a comparative transcriptomic approach using 132 two Geranium species with divergent petiole responses to shade unveiled 133 components that might suppress growth in the shade-tolerant species 134 (Gommers et al., 2017; Gommers et al., 2018). The use of related species but 135 amenable for genetic analyses is expected to push this effort further to find 136 regulatory components used in nature to modulate these divergent responses. 137 This is what we are addressing in this work. 138 Comparing A. thaliana and its close relative Cardamine hirsuta to 139 understand the genetic basis for trait diversification between species is a 140 powerful strategy to understand the evolution of morphological traits. Key to this 141 approach is the wide morphological and physiological diversity between these 142 species, such as differences in leaf morphology and seed dispersal mechanism 143 among others (Barkoulas et al., 2008; Hay et al., 2014; Hofhuis et al., 2016; 144 Vlad et al., 2014; Vuolo et al., 2016). Like A. thaliana, C. hirsuta has a short 145 generation time, small size, inbreeding habit, abundant progeny and ease of 146 large scale cultivation (Hay and Tsiantis, 2016; Hay et al., 2014). It is a diploid 147 species with a small genome and eight chromosomes that has been completely 148 sequenced (Gan et al., 2016). Genetic transformation by floral dipping, a dense 149 genetic map and chemically mutagenized populations, provide the tools to 150 identify the genetic components and molecular mechanisms underlying 151 diversification or morphology and response to environment (Hay and Tsiantis, 4 152 2016). C. hirsuta is an invasive herbaceous plant that can grow in open sun but 153 it is often found in shaded or semi-shaded areas. Indeed, C. hirsuta does not 154 need much light to grow and their stems become purplish (likely to prevent 155 oxidative damage) in strong sun 156 (http://edis.ifas.ufl.edu/pdffiles/EP/EP51100.pdf; 157 http://practicalplants.org/wiki/Cardamine_hirsuta; 158 http://www.asturnatura.com/especie/cardamine-hirsuta.html; 159 http://dnr.wi.gov/topic/Invasives/documents/classification/LR_Cardamine_hirsut 160 a.pdf; https://www.wildfooduk.com/edible-wild-plants/hairy-bittercress/). These 161 observations are consistent with C. hirsuta being shade-tolerant (Bealey and 162 Robertson, 1992). In agreement, whereas seedlings of A. thaliana elongate in 163 response to shade, those of C. hirsuta are unresponsive to the same stimulus 164 (Hay et al., 2014). 165 The divergent hypocotyl response to shade of A. thaliana and C. hirsuta 166 species led us to take a comparative approach to understand the genetic basis 167 of the evolution of this physiological trait. We found that C. hirsuta has acquired 168 a highly-efficient phyA-dependent pathway that represses hypocotyl elongation 169 and other SAS-associated responses when exposed to simulated shade. After 170 complementing A. thaliana phyA mutant plants with endogenous or C. hirsuta 171 phyA molecules we concluded that these two photoreceptors are not 172 exchangeable. Differences in phyA intrinsic activity hence contribute to a 173 different response of C. hirsuta and A. thaliana to shade exposure. 174 175 RESULTS 176 C. hirsuta seedlings perceive low R:FR but do not elongate 177 A recent study revealed that different species of the Tradescantia genus 178 with divergent tolerance to shade showed clear differences in maximum 179 quantum efficiency of photosystem II (Fv/Fm) upon variations of the growth light 180 (Benkov et al., 2019). In particular, the sun-resistant T. sillamontana (a 181 succulent growing in semi-desert regions of Mexico and Peru, hence adapted to 182 high light intensities) was more tolerant to changes in irradiation intensity (i.e. 183 showed a more constant Fv/Fm) than the shade-tolerant T. fluminensis 184 (habitant of tropical rainforests and other shaded areas in south-eastern Brazil 185 and hence adapted to grow under low light intensities). 5 186 Using a similar experimental system, we aimed to confirm whether C. 187 hirsuta is a shade-tolerant plant compared to A. thaliana (a broadly accepted 188 shade-avoider). Indeed, when wild-type seedlings of these two species (ChWT 189 and AtWT) were transferred from normal white light (W) to conditions in which 190 PAR was first increased 10-fold (high light, HL) and then reduced 5-fold relative 191 to W (low light, LL) or viceversa, Fv/Fm changes were much more pronounced 192 in ChWT (Supplemental Figure 1A). The lower capacity of ChWT to adapt to 193 intense irradiation was confirmed by the bleaching symptoms (e.g., lower 194 chlorophyll contents) observed in ChWT (but not in AtWT) upon transferring to HL 195 (Supplemental Figure 1B). ChWT seedlings only showed a better performance 196 than AtWT when transferred from W to LL. Rapid light curve (RLC) analysis 197 confirmed that ChWT was better able to maintain its level of photosynthetic 198 activity under LL conditions than AtWT (Supplemental Figure 1C), as expected 199 for a shade-tolerant plant (Han et al., 2015). 200 Besides differentially responding to decreased light quantity, plant 201 species from open habitats show a stronger elongation response to reduced 202 R:FR (i.e. light quality) compared to those from woodland shade habitats 203 (Gommers et al., 2017; Smith, 1982). Further supporting the conclusion that C. 204 hirsuta tolerates shade, ChWT failed to elongate their hypocotyls when exposed 205 to a range of low R:FR treatments (i.e., W+FR), that mimic vegetation proximity 206 (intermediate or low R:FR; 0.09 - 0.07) and canopy shade (very low R:FR; 0.02) 207 (Supplemental Figure 2, Figure 1). W-grown ChWT hypocotyls, as well as 208 cotyledons, are substantially longer than those of AtWT growing under the same 209 conditions. ChWT hypocotyls were also longer than those of AtWT when growing 210 in the dark (Figure 1C), indicating that C. hirsuta is overall bigger than A. 211 thaliana. More importantly, when treated with growth stimulants, such as 212 gibberellic acid (Hay et al., 2014) or picloram (PIC, a synthetic auxin), 213 hypocotyls of both species elongate (Figure 1D). We therefore concluded that 214 the elongation of C. hirsuta hypocotyls is not generally compromised, arguing 215 against the possibility that this species displays a constitutive SAS phenotype. 216 In A. thaliana, exposure to simulated shade also triggers the elongation 217 of leaf petioles. We quantified the elongation response of the petiole and rachis 218 in 2-week-old ChWT and AtWT plants subjected to 7 days of high (W) or low R:FR 219 (W+FR). In agreement with previous studies (de Wit et al., 2015; Kozuka et al., 6 220 2010; Sasidharan et al., 2010), shade-treated AtWT leaves showed substantially 221 longer petioles than those of plants grown under W. Petiole and rachis length in 222 ChWT, however, was similar in leaves from plants grown under W or W+FR 223 (Figure 1E, Supplemental Figure 3). These results together suggest that 224 elongation responses to low R:FR are dramatically arrested in C. hirsuta plants. 225 226 C. hirsuta shows other attenuated responses to shade 227 Beyond elongation responses, low R:FR triggers a reduction in the levels 228 of photosynthetic pigments, i.e., carotenoids and chlorophylls (Bou-Torrent et 229 al., 2015; Cagnola et al., 2012; Roig-Villanova et al., 2007). While these 230 pigments were also significantly reduced in shade-treated ChWT seedlings 231 (Figure 1F), the decrease was less prominent than in AtWT. These results 232 indicated that not all SAS responses are equally compromised in C. hirsuta. 233 We next used RNA sequencing (RNA-seq) to compare the genome wide 234 expression patterns of 7-day-old AtWT and ChWT whole seedlings in W versus 1 235 h of simulated shade (W+FR) (Figure 2). Incorporating knowledge about gene 236 orthology, 432 differentially expressed genes (DEGs) were categorized as 237 rapidly regulated by shade in one species or in both. Plotting the W+FR vs. W 238 fold-change in C. hirsuta against the same ratio in A. thaliana resulted in a 239 linear regression equation with a slope of 0.54 (Figure 2B), which supported 240 that shade-modulated changes in gene expression are also attenuated in C. 241 hirsuta compared to A. thaliana. In A. thaliana, shade treatment induced 246 242 (fold change>1.5, p<0.05,) and repressed 58 genes (fold change<-1.5, p<0.05,). 243 In C. hirsuta, this same treatment induced 181 and repressed 54 genes 244 (Supplemental Figure 4A, Supplemental Data Sets 1–4). From the set of 245 induced DEGs, 102 responded in both species. They included several of the 246 well-known shade-marker genes in A. thaliana and other species, such as 247 ARABIDOPSIS THALIANA HOMEOBOX PROTEIN 2 (ATHB2), 248 BRASSINOSTEROID-ENHANCED EXPRESSION 1 (BEE1), BES1- 249 INTERACTING MYC-LIKE1 (BIM1), LONG HYPOCOTYL IN FR 1 (HFR1) or 250 XYLOGLUCAN ENDOTRANSGLYCOSYLASE 7 (XTR7) (Cifuentes-Esquivel et 251 al., 2013; Karve et al., 2012; Procko et al., 2014; Ueoka-Nakanishi et al., 2011). 252 Gene ontology (GO) and MapMan-Bin (MMB) functional prediction of 253 these up-regulated gene group indicated that terms related to auxin were 7 254 significantly overrepresented (Supplemental Data Sets 5 and 6), suggesting an 255 early role for auxins in both A. thaliana and C. hirsuta. Indeed, W+FR treatment 256 for 1 h increased auxin (IAA) levels not only in AtWT, as published (Bou-Torrent 257 et al., 2014; Hersch et al., 2014; Hornitschek et al., 2012; Tao et al., 2008), but 258 also in whole ChWT seedlings (Figure 2C). 259 Using public transcriptomic data, we identified a group of 13 genes 260 whose expression was induced in A. thaliana wild-type seedlings but not in 261 mutants that do not accumulate auxins (shade avoidance 3-2, (sav3-2) and pif7- 262 1) after 1 h of shade treatment (Bou-Torrent et al., 2014; Li et al., 2012; Tao et 263 al., 2008). Based on our RNAseq data, the expression of these genes was 264 significantly upregulated in AtWT and, to a lower extent, ChWT seedlings 265 (Supplemental Figure 5), consistent with the observed increase in IAA content 266 in both species. Since only A. thaliana elongates in response to shade 267 exposure, either the observed early changes in gene expression and auxin 268 levels are not reflecting the differences in hypocotyl growth between these 269 species, or the elongation is consequence of differential later events. 270 In our RNAseq analyses, 55 and 49 DEGs were specifically repressed in 271 either AtWT or ChWT seedlings, respectively, and just 3 genes were repressed in 272 both species. Regarding up-regulated genes, 142 and 79 DEGs were 273 specifically induced either in AtWT or in ChWT, respectively (Supplemental Figure 274 4A). GO and MMB functional prediction of the 142 DEGs specific for AtWT 275 showed genes related to several aspects of plant development, whereas the 79 276 DEGs specifically induced in ChWT showed enrichment for genes related with 277 the photosynthetic machinery. Particularly, C. hirsuta rapidly responds by 278 inducing the expression of genes encoding components of both photosystems I 279 and II, the NADH dehydrogenase-like complex (involved in chlororespiration) 280 and both small and large subunits of plastidial ribosomes (Supplemental Figure 281 5B, Supplemental Data Sets 5 and 6). Whether these rapid changes are 282 maintained after prolonged exposure to shade or have any functional relevance 283 is unknown. Nonetheless, these transcriptome differences support that the two 284 mustard species employ alternative strategies to adapt to plant proximity and 285 shade that go further from the modulation of elongation growth. 286 Comparative approaches have been used before to investigate the 287 differential response to shade of related species. Transcriptomic analyses using 8 288 two Geranium species that display divergent shade-induced petiole elongation 289 (G. pyrenaicum as a shade avoider or responsive, and G. robertianum as a 290 shade tolerant) identified a series of 31 up-regulated genes that included a 291 number of candidate regulators of differential shade avoidance (Gommers et al., 292 2017). In these two species, putatively orthologous transcript groups (OMCL) 293 were defined, and the best BLAST hit with the A. thaliana transcriptome was 294 used to name Geranium OMCL groups (Gommers et al., 2017). When we 295 compared our lists of shade-regulated genes with the Geranium OMCLs 296 differentially regulated after 2 h of low R:FR in the petioles, we found that the 297 number of genes up-regulated in both shade-tolerant and shade-avoider 298 species was higher for the AtWT/ChWT pair than between the Geranium species 299 (Supplemental Figure 4C; Supplemental Data Sets 7 and 8). GO analyses did 300 not identify any function from the lists of genes specifically induced in either G. 301 pyrenaicum or G. robertianum. Overlap was very limited between the sets of 302 repressed genes. Together, the contrasting rapid shade-induced gene 303 expression changes might either support differences in the early molecular 304 mechanisms between the Geranium and mustard groups, or just reflect the 305 differences in tissues (whole seedlings vs. leaf petioles) and/or shade and 306 growth conditions (continuous light vs. photoperiod) between experiments. 307 We also analyzed the changes in gene expression of PIF3-LIKE 1 (PIL1) 308 and ATHB2, two typical shade-marker genes, in response to longer (up to 8 h) 309 exposure to low R:FR. Expression of PIL1 and ATHB2 were rapidly induced in 310 both mustard seedlings after simulated shade exposure. However, the relative 311 induction of the expression of these genes was attenuated in ChWT compared to 312 AtWT (Figure 2D). Together, our results indicate that C. hirsuta seedlings sense 313 plant proximity and respond molecularly and metabolically to it; however, this 314 signal does not promote hypocotyl elongation in C. hirsuta as it does in the 315 shade-avoider A. thaliana. 316 317 Shade-induced elongation in C. hirsuta is repressed 318 To explain the hypocotyl elongation differences between A. thaliana and 319 C. hirsuta, we hypothesized two mutually exclusive mechanisms: (i) 320 uncoupling: shade perception is specifically unplugged from the endogenous 321 mechanisms of control of hypocotyl elongation, or (ii) suppression: there are 9 322 mechanisms that strongly suppress the shade-induced elongation of 323 hypocotyls. To distinguish between these possibilities, a genetic screening 324 looking for C. hirsuta seedlings with long hypocotyls under simulated shade (> 6 325 mm long) was carried out, using an EMS-mutagenized population (Vlad et al., 326 2014). If suppression mechanisms exist, then loss-of-function mutants that 327 unleash shade-induced hypocotyl elongation might be recovered. Indeed, from 328 the various long hypocotyl seedlings identified we focused in two slender in 329 shade (sis) mutants, shown to be recessive and allelic. After backcrossing these 330 mutants twice with the ChWT plants, homozygous mutants had slightly longer 331 hypocotyls in W than the wild type, and very long hypocotyls under W+FR. We 332 named the mutants as sis1-1 and sis1-2 (Figure 3). These results indicated that 333 (1) loss-of-function (recessive) mutations support the “suppression” 334 mechanisms in C. hirsuta to establish shade-tolerance; and (2) a single gene, 335 SIS1, is able to repress the elongation response to shade in C. hirsuta. 336 As a first step to explore SIS1 identity, we determined whether light 337 perception was altered in sis1 mutants by analyzing hypocotyl length after de- 338 etiolation under monochromatic lights. We noticed that ChWT seedlings were 339 quite hyposensitive to R compared to AtWT (Figure 3B), suggesting that an 340 attenuated phyB signaling might result in a constitutive SAS hypocotyl 341 response, causing the observed suppression of the shade-induced hypocotyl 342 elongation. Considering the relationship between the attenuated 343 responsiveness to R and the strength of the shade-induced hypocotyl 344 elongation of the weak phyB-4 and strong phyB-1 A. thaliana mutant seedlings 345 (Figure 3C,D), the hyposensitivity to R observed in ChWT might contribute but is 346 not enough to fully suppress the shade-induced hypocotyl elongation in this 347 species. Therefore, additional components are required to establish the shade- 348 tolerant hypocotyl habit in C. hirsuta. Indeed, mutant sis1 seedlings, although 349 slightly hyposensitive to R and blue light, were fully blind to FR compared to 350 ChWT seedlings (Figure 3B). 351 A very similar pattern of response was also shown by A. thaliana phyA- 352 deficient phyA-501 seedlings (Figure 3B) (Li et al., 2011), which suggested that 353 sis1 seedlings might be deficient in phyA activity or signaling. Sequencing of the 354 C. hirsuta PHYA (ChPHYA) gene from sis1-1 and sis1-2 plants showed point 355 mutations (transitions) that introduced either a nonsense mutation in Gln935 (in 10 356 sis1-1) or a missense mutation in the conserved Gly913 (in sis1-2) (Figure 3E, 357 Supplemental Figure 6A). Immunoblot analyses using a specific monoclonal 358 antibody against phyA (073D), indicated that only sis1-1 was lacking phyA 359 (Figure 3D). Consistent with this, C. hirsuta lines with reduced activity of phyA 360 by overexpressing an RNA interference construct directed towards the ChPHYA 361 gene (lines 35S:RNAi-ChPHYA) also resulted in a sis phenotype (Supplemental 362 Figure 6B-D). Together, these results indicated that sis1 are C. hirsuta phyA 363 deficient mutants (for clarity, we will keep the sis1 mutant name along the 364 manuscript to distinguish it from the phyA mutants from A. thaliana). They also 365 suggested that shade tolerance in C. hirsuta might be caused by the existence 366 of a phyA-dependent suppression mechanism that represses the hypocotyl 367 elongation response to shade. 368 Molecular analyses showed that the relative induction of PIL1 and 369 ATHB2 expression was enhanced in both sis1 mutants compared to ChWT 370 seedlings after more than 4 h of simulated shade exposure (Figure 4). This 371 relatively late effect of ChPHYA absence (sis1) on gene expression is 372 consistent with what was observed in A. thaliana phyA mutants (Ciolfi et al., 373 2013). We also measured the levels of photosynthetic pigments (carotenoids 374 and chlorophylls) after long-term exposure to low R:FR, in wild-type and phyA- 375 deficient A. thaliana and C. hirsuta seedlings. Simulated shade triggered a 376 stronger decrease in the accumulation of these pigments in phyA-501, sis1-1 377 and sis1-2 seedlings compared to wild-type controls (Figure 4B), hence 378 indicating that phyA represses this trait in both species, likely to avoid 379 exaggerated losses of photosynthetic pigments in response to vegetation 380 proximity and shade. 381 Phytochrome A represses the shade-induced hypocotyl elongation in A. 382 thaliana caused by the deactivation of phyB only under conditions that mimic 383 closed canopies, i.e., under very low R:FR (Casal et al., 2014; Martinez-Garcia 384 et al., 2014; Yanovsky et al., 1995). Indeed, A. thaliana phyA deficient mutants 385 behaved almost like AtWT seedlings under various shade mimicking conditions 386 except for the lowest R:FR tested (Figure 4C). By contrast, C. hirsuta sis1 387 mutants behaved differently than its ChWT under all the low R:FR applied 388 (Figure 4D), indicating that phyA has a broader role in suppressing the shade- 389 induced hypocotyl elongation in C. hirsuta than in A. thaliana. 11 390 391 C. hirsuta has higher phyA activity than A. thaliana 392 Our results suggested the possibility that phyA activity is higher in the 393 shade-tolerant C. hirsuta than in the shade-avoider A. thaliana. Higher phyA 394 activity can be achieved by at least two alternative and non-exclusive ways: 395 higher phyA levels and/or higher specific (intrinsic) activity of the photoreceptor. 396 To analyze these possibilities, we first aimed to compare PHYA expression 397 levels in AtWT and ChWT seedlings. Data extracted from our RNAseq experiment 398 indicated that the expression of several commonly-used reference genes, such 399 as EF1α or YLS8 (Gallemi et al., 2017b; Hornitschek et al., 2009; Kohnen et al., 400 2016), was within the same range (Supplemental Table 1). Then, we quantified 401 PHYA expression levels in AtWT and ChWT seedlings growing under W or W+FR 402 (Figure 5) using primers that recognize the sequence of the target gene (PHYA) 403 and three normalizer genes (EF1, SPC25, YLS8) in both species 404 (Supplemental Figure 7). Expression of PHYA was significantly higher in C. 405 hirsuta than in A. thaliana seedlings (two-way ANOVA tests, p<0.05) in 406 seedlings of different ages grown under W or W+FR conditions (Figure 5B). 407 Higher expression of PHYA in C. hirsuta might result in higher phyA 408 protein levels, contributing to an increased phyA activity in this species. Our 409 immunoblot analyses showed that PHYA protein levels were significantly higher 410 in C. hirsuta than A. thaliana etiolated seedlings (Figure 5D). More importantly, 411 whereas PHYA levels almost disappear after 6 h of W exposure in both species, 412 C. hirsuta seedlings maintained higher PHYA levels than A. thaliana when 413 exposed to W+FR for 6-10 h (Figure 5C-D). Together, these results support that 414 PHYA levels in C. hirsuta are generally higher than in A. thaliana seedlings, 415 even under shade conditions. This observation is consistent with the strongest 416 difference in hypocotyl length under W of wild-type and phyA-deficient seedlings 417 from C. hirsuta compared to A. thaliana (Figure 4C-D, Supplemental Figure 6D) 418 (Martinez-Garcia et al., 2014). Furthermore, transgenic overexpression of PHYA 419 has been shown to attenuate shade-triggered hypocotyl elongation in A. 420 thaliana seedlings and stem elongation in other species (Heyer et al., 1995; 421 Robson et al., 1996; Roig-Villanova et al., 2006). 422 To compare AtphyA and ChphyA specific (intrinsic) activities, 423 complementation analyses of the A. thaliana phyA-501 mutant were carried out 12 424 with the AtPHYA or ChPHYA genes under the control of the endogenous 425 promoter of AtPHYA (pAtPHYA:AtPHYA or pAtPHYA:ChPHYA, respectively). 426 The resulting lines were named as phyA>AtPHYA and phyA>ChPHYA (Figure 427 6). We obtained a total of 5 independent phyA>AtPHYA lines and 7 428 independent phyA>ChPHYA lines with different transcript and protein levels 429 (Supplemental Figure 8). To estimate PHYA protein levels we used etiolated 430 seedlings, as phyA is photolabile. Because PHYA expression is repressed by 431 light via phyA and phyB (Canton and Quail, 1999), RNA was extracted from 432 seedlings either grown in the dark or under W+FR (Supplemental Figure 8A). 433 PHYA expression in seedlings grown in these two conditions correlated 434 positively in both phyA>AtPHYA (R2=0.79) and phyA>ChPHYA lines (R2=0.79) 435 (Supplemental Figure 8B). The slope of these equations, however, was 436 significantly higher (p<0.05) for phyA>AtPHYA (7.49) than phyA>ChPHYA 437 (2.81) lines. Specifically, phyA>AtPHYA and phyA>ChPHYA lines with 438 comparable PHYA expression levels in the dark showed lower PHYA 439 expression under simulated shade when complemented by ChPHYA 440 (phyA>ChPHYA) compared to AtPHYA (phyA>AtPHYA). These results pointed 441 to a stronger activity for the ChphyA protein in repressing its own (PHYA) 442 expression. 443 For the comparison of AtphyA and ChphyA activities, we initially studied 444 their effect on the promotion of the shade-induced hypocotyl elongation in 445 transgenic lines. AtWT and phyA-501 seedlings were incorporated as controls. In 446 these experiments, the difference in hypocotyl length between seedlings grown 447 under W+FR vs. W (HypW+FR-HypW) provided values indicative of the 448 complementation level (or phyA biological activity) for the response analyzed. 449 Consequently, in these analyses, the lower the HypW+FR-HypW value, the higher 450 the phyA activity. Opposite to that observed with transcript levels (Supplemental 451 Figure 8C), HypW+FR-HypW correlated well with ChPHYA but not with AtPHYA 452 protein levels (Supplemental Figures 8D). These results together indicate that 453 the two photoreceptors are not fully exchangeable and suggest different intrinsic 454 qualities (i.e., biological activity) between the phyA receptors of A. thaliana and 455 C. hirsuta. 456 When lines with comparable PHYA protein levels were selected (Figure 457 6B), the response to shade (HypW+FR-HypW) was more strongly attenuated by 13 458 ChPHYA (Figure 6C-D). As an additional way to test for phyA activity, we 459 estimated hypocotyl elongation in seedlings etiolated (HypD) and deetiolated 460 under monochromatic FR (HypFR). In this case, the higher the difference 461 between these two values (HypD-HypFR), the stronger the activity of phyA. 462 Similar to the shade response analyses, ChphyA showed a stronger activity 463 than AtphyA in deetiolating seedlings under FR (Figure 6E-F). A good 464 correlation between these two phyA-mediated responses was also found when 465 all the lines were considered together (Supplemental Figure 8E), reinforcing our 466 interpretation that ChphyA is intrinsically more active than AtphyA. 467 The expression of dozens of auxin-responsive genes is repressed by 468 phyA after just 1 h of very low R:FR treatment (Yang et al., 2018). As an 469 additional and complementary test of phyA biological activity different from 470 hypocotyl elongation we evaluated the repressive effect of AtphyA and ChphyA 471 on the expression of these genes. First, we selected 1-AMINO- 472 CYCLOPROPANE-1-CARBOXYLATE SYNTHASE 8 (ACS8), GRETCHEN 473 HAGEN 3.3 (GH3.3), INDOLE-3-ACETIC ACID INDUCIBLE 19 (IAA19) and 474 IAA29, four auxin-responsive genes described as repressed phyA targets (Yang 475 et al., 2018). As expected, the shade-induced expression of these genes was 476 attenuated in AtWT compared to phyA-501 seedlings, but under our shade 477 conditions the differences were most obvious after long exposure to W+FR 478 (Figure 7). 479 The expression of the same genes was next quantified in seedlings from 480 the various phyA>AtPHYA and phyA>ChPHYA lines grown for 24 h under 481 W+FR. When plotting transcript levels of phyA target genes as a function of 482 PHYA expression in these lines, the clouds of data corresponding to 483 phyA>ChPHYA lines (red) were separated from that of phyA>AtPHYA lines 484 (blue) (Figure 7B). Importantly, the expression of all phyA target genes tested 485 was overall lower in phyA>ChPHYA than phyA>AtPHYA lines, indicating that 486 ChphyA repressed more efficiently gene expression than AtphyA (Figure 7B). 487 Consistent with this conclusion, the expression of these and other phyA target 488 genes (Yang et al., 2018) was attenuated in shade-induced seedlings of ChWT 489 compared to AtWT (Supplemental Figure 9). Together, these data further support 490 that ChphyA is intrinsically more active than AtphyA. 491 14 492 DISCUSSION 493 Currently, the genetic basis of shade tolerance is poorly understood. To 494 address this open question, we have focused on comparative analyses of the 495 hypocotyl response to shade in young seedlings of two related mustards, A. 496 thaliana and C. hirsuta. Shade avoidance and tolerance are ecological concepts 497 originated from the natural habitats of plants species (Callahan et al., 1997). 498 Hence, defining the shade habit of a species is difficult because shade 499 tolerance is not an absolute value but a relative concept; indeed, plants may 500 exhibit different strategies during the juvenile and adult phases of their lives 501 (Valladares and Niinemets, 2008). Despite the uncertainty, A. thaliana is 502 generally considered as shade avoider and it is a model broadly used to study 503 the SAS hypocotyl response, but there is little information referring to its 504 physiological shade-responsiveness habit. C. hirsuta, by contrast, has been 505 previously described as a shade tolerant species whose hypocotyls are 506 unresponsive to shade (Bealey and Robertson, 1992; Hay et al., 2014), but little 507 is known about other shade response mechanisms. Here we confirm that, as 508 expected for a shade-tolerant species, C. hirsuta showed a much better 509 capacity to acclimate to LL than to HL compared to A. thaliana (Supplemental 510 Figure 1). Most strikingly, C. hirsuta seedlings failed to elongate in response to 511 simulated proximity or canopy shade (Figure 1). Such a dramatic hypocotyl 512 elongation response compared to A. thaliana makes these two related species 513 good candidates for comparative analyses of divergent responses to shade. 514 Our comparative and genetic analyses suggest that the absence of a 515 shade-induced hypocotyl elongation in C. hirsuta is not caused by defects on 516 the rapid biosynthesis of auxin in seedlings (Figure 2). Although we cannot 517 exclude local defects in auxin biosynthesis (e.g., in hypocotyls) that might be 518 masked by collecting whole seedlings, our conclusion is consistent with the lack 519 of effect of phyA on the rapid shade-induced biosynthesis of auxin (Yang et al., 520 2018). On the contrary, we favor that the differences in hypocotyl elongation 521 between these species is the result of a suppression mechanisms sustained by 522 the stronger activity of the ChphyA photoreceptor, likely enhanced by the 523 attenuated ChphyB activity (Figure 3B). A stronger intrinsic (specific) repressor 524 activity of ChphyA would result in a strong suppression of the elongation of C. 525 hirsuta seedlings when exposed to shade (Figure 8). The underlying 15 526 mechanism likely relies, at least partly, upon suppression of auxin signaling via 527 phyA directly binding and stabilizing AUX/IAA proteins, as it has been shown in 528 A. thaliana (Yang et al., 2018). In this scenario, ChphyA seems to suppress not 529 auxin biosynthesis but signaling more strongly than AtphyA, as deduced from 530 the results with transgenic lines (Figure 7B) but also from the stronger 531 repression in shade of auxin-responsive genes with a putative role in auxin- 532 signaling (e.g., several IAA and SAUR genes) detected in ChWT compared to 533 AtWT (Supplemental Figure 9). 534 AtphyA and ChphyA might achieve different activities by changes in 535 particular residues that could alter susceptibility to post-translational 536 modifications. For instance, phyA stability, Pfr to Pr reversion rate upon shade 537 treatment or/and interaction with protein partners (e.g., PIF1/PIF3, FHY1/FHL, 538 AUX/IAA) affect phyA activity in A. thaliana (Dieterle et al., 2005; Genoud et al., 539 2008; Kim et al., 2004; Oka et al., 2012; Seo et al., 2004; Sheerin et al., 2015). 540 These intrinsic differences might be also enhanced by changes in protein 541 abundance of phyA or/and other components in its signaling pathway 542 specifically acting in light-grown seedlings (see below). Comparison of the 543 amino acid sequences of AtphyA and ChphyA, however, did not point to any 544 obvious specific residue or region that could be responsible for the observed 545 intrinsic differences in activity (Supplemental Figure 10). This is an issue that 546 would need future research. 547 The genetic mechanisms underlying physiological evolution remain 548 largely unknown, but changes in the timing, location and levels of gene 549 expression (i.e., cis-regulatory evolution of key genes) have caused much of 550 morphological evolution changes (Carroll, 2008). Our data on PHYA expression 551 and PHYA protein levels (Figure 5) agree with this view, but they go a step 552 beyond by showing that differences in protein (ChphyA and AtphyA) intrinsic 553 activities also contribute to differential responses to shade (Figure 6-7). As both 554 components (levels vs. intrinsic activity) are intimately connected (e.g., phyA 555 represses its own expression in a light-dependent manner), at this stage it is 556 difficult to quantify the specific contribution of each one. Moreover, additional 557 components might contribute: while we show that phyA is a central component 558 of a range of regulators that can be modulated in nature to implement shade 559 tolerance, the observation that none of the phyA>ChPHYA lines display a 16 560 shade-tolerant habit (Supplemental Figure 8D) strongly suggests that additional 561 downstream components of the shade-regulatory network are also participating 562 in suppressing this response in C. hirsuta (e.g., differences in phyB activity). 563 Indeed, it cannot be excluded that the mutant screen, despite identifying an 564 important regulator, did not establish the causal difference between the two 565 species in terms of shade-induced hypocotyl elongation. Nonetheless, our 566 results unveil the importance of modulating photoreceptor activity as a powerful 567 evolutionary mechanism in nature to achieve physiological variation between 568 species, hence enabling the colonization of new, different habitats. In addition, 569 searching for variability in phyA function could provide a suitable tool to modify 570 the impact of neighbors’ cues in crops to minimize yield losses. 571 572 MATERIALS AND METHODS 573 Plant material and plant growth conditions 574 Plants of Arabidopsis thaliana Col-0 (AtWT) phyA-501 (in Col-0 background), 575 phyB-1, phyB-4 (both phyB deficient lines are in Ler background), and 576 Cardamine hirsuta, of the reference Oxford (Ox) accession (ChWT), have been 577 described (Hay et al., 2014; Martinez-Garcia et al., 2014; Reed et al., 1993). 578 Plant growth conditions have been described elsewhere (Gallemi et al., 2016; 579 Martinez-Garcia et al., 2014). Normal light conditions refer to W produced by 580 cool-white vertical fluorescent tubes (PAR of 20-24 µmol·m-2·s-1). Low and high 581 light conditions corresponded to PAR values of 4 and 200 µmol·m-2·s-1, 582 respectively. Shade treatments in seedlings were provided by enriching W 583 (R:FR of 2.5) with different intensities of FR LEDs (730-nm peak; Philips 584 Greenpower Research modules) to produce the indicated R:FR (0.091 to 0.021) 585 without altering PAR. Light spectra are presented in Supplemental Figure 2. For 586 estimating petiole and rachis length, rosette plants were grown under long day 587 (LD, 16 h light, 8 h dark) photoperiods, in which W was generated by cool-white 588 horizontal fluorescent tubes (PAR of 100 µmol m-2 s-1, R:FR of 3.0); for shade 589 treatments, W was supplemented with FR (W+FR, PAR of 100 µmol m-2 s-1, 590 R:FR of 0.05). Fluence rates were measured with a Spectrosense2 meter 591 associated with a 4-channel sensor (Skye instruments Ltd., 592 www.skyinstruments.com) which measures PAR (400-700 nm) and 10 nm 593 windows in the blue (464-473 nm), R (664-673 nm) and FR (725-734 nm) 17 594 regions (Gallemi et al., 2017b). Light spectra were generated using a Flame 595 Model Spectrometer with Sony Detector (FLAME-S) (https://oceanoptics.com). 596 597 Hypocotyl, petiole and rachis measurements 598 For hypocotyl measurement, about 30 seeds of each genotype were 599 germinated on the plates for observing the seedling phenotype and at least 20 600 seedlings were measured for quantification of hypocotyl length. All experiments 601 were repeated at least three times with consistent results. Hypocotyl 602 measurements from all the different experiments were averaged. For petiole 603 measurement, about 30 seeds of each genotype were germinated under 604 continuous W. One week later, 20 seedlings in a similar stage of development 605 were transferred to individual pots and moved to a LD growth chamber (R:FR of 606 3.0). After one week, half of the rosette plants stayed under W and the other 607 half were moved to a W+FR shelf (R:FR of 0.05). After one week of differential 608 R:FR treatment, leaves were harvested and petiole was measured; in the case 609 of complex leaves from C. hirsuta, rachises were measured, covering the 610 distance from the base of the leaf until the base of the main leaflet 611 (Supplemental Figure 3). At least 8 leaves were measured for quantification of 612 petiole and rachis length for each leaf number. Experiments were repeated four 613 times with consistent results. Petiole and rachis measurements from all four 614 experiments were averaged. 615 616 Photosynthetic pigment quantification and chlorophyll fluorescence. 617 Whole 7-day-old seedlings of the indicated genotypes and grown under W or 618 W+FR (Figures 1 and 4) or transferred to HL conditions (Supplemental Figure 619 1B) were harvested, ground in liquid nitrogen, and the resulting powder was 620 used for quantification of chlorophylls and carotenoids spectrophotometrically or 621 by HPLC, as described (Bou-Torrent et al., 2015). 622 Fluorescence measurements were carried out on seedlings grown under 623 different light regimes using a MAXI-PAM fluorometer (Heinz Walz GmbH). For 624 every measurement the whole cotyledons of 7 seedlings were considered. 625 Maximum quantum yield of photosystem II (PSII), Fv/Fm, was calculated as 626 (Fm-Fo)/Fm, where Fm and Fo are respectively the maximum and the minimum 627 fluorescence of dark-adapted samples. For dark acclimation, plates were 18 628 incubated for at least 30 minutes in darkness to allow the full relaxation of 629 photosystems. Rapid light curves (RLCs) were constructed with 10 incremental 630 steps of actinic irradiance (E; 0, 1, 21, 56, 111, 186, 281, 396, 531, 701 μmol 631 photons m-2 s-1). For each step, the effective quantum yield of PSII (ΔF/Fm’) 632 was monitored every min and relative electron transport rate (rETR) was 633 calculated as E×ΔF/Fm’. The light response was characterized by fitting 634 iteratively, using MS Excel Solver, the model of Platt (1980) to rETR versus E 635 curves. The fit was very good in all the cases (r>0.98). 636 637 Expression analyses by RT-qPCR and RNA-seq 638 RNA was extracted from whole seedlings of A. thaliana and C. hirsuta (grown 639 as detailed in each experiment, three biological replicates per time point, each 640 biological replicate composed of 30-40 seedlings) using commercial kits 641 (RNAeasy Plant Mini kit; Qiagen; www.qiagen.com; or the semi-automatic 642 Maxwell SimplyRNA kit; Promega; www.promega.com). For real-time qPCR 643 analysis, two micrograms of RNA were reverse-transcribed using the M-MLV 644 Reverse Transcriptase (Invitrogen, www.lifetechnologies.com) or Transcriptor 645 First Strand cDNA synthesis (Roche, lifescience.roche.com). Reference genes 646 used were UBQ10, EF1α, SPC25 or/and YLS8. 647 For RNA-seq analyses, quantification of gene expression was performed 648 as indicated elsewhere (Gan et al., 2016) and detailed as Supplemental 649 information. From the lists of genes, we selected as differentially expressed 650 those whose fold change was significantly (p adjusted < 0.05) and higher than 651 1.5 (Supplemental Data Sets 1 and 3) or lower than 0.67 (Supplemental Data 652 Sets 2 and 4) in seedlings treated for 1 h with W+FR compared to those grown 653 under W in either C. hirsuta (Supplemental Data Sets 1 and 2) or A. thaliana 654 (Supplemental Data Sets 3 and 4). 655 656 Gene Ontology (GO) and MapMan analysis 657 A strict synteny based approach was used to identify conserved orthologs 658 between the two species. The A. thaliana orthologs of the C. hirsuta genes were 659 used for getting the GO term annotations and MapMan-Bins. The GO term 660 annotations for A. thaliana genes, used as a reference, were obtained from The 661 Gene Ontology Consortium (http://www.geneontology.org/) (Ashburner et al., 19 662 2000). The results are presented as Supplemental Data Set 5. For the 663 MapMan-Bin analyses, each list of genes were submitted to the “Mercator” 664 gene function prediction pipeline (Lohse et al., 2014), that annotates the query 665 genes with the hierarchical ontology MapMan-Bins (Klie and Nikoloski, 2012; 666 Thimm et al., 2004). Based on these MMB annotations, exact Fischer tests for 667 function enrichment within the six groups of differentially expressed genes were 668 carried out and interpreted (Supplemental Data Set 6). 669 670 Protein extraction and immunoblot analysis 671 Methods for extracting and detecting phyA protein levels in A. thaliana or C. 672 hirsuta seedlings (Gallemi et al., 2017b; Martinez-Garcia et al., 1999) are 673 detailed as follows. Protein extracts from C. hirsuta seedlings analyzed in 674 Figure 3 and Supplemental Figure 6 were prepared following the direct extract 675 protocol (Martinez-Garcia et al., 1999) with the modifications described below. 676 Extracts were prepared from ChWT, sis1 and RNAi-ChPHYA seedlings 677 germinated and grown in the dark for 4 days. Ten seedlings per genotype were 678 harvested in the dark and extracted in 1.5 mL microfuge tubes containing 300 679 µL of Laemmli buffer supplemented with protease inhibitors (10 µg/mL 680 Aprotinin, 1 µg/mL E-64, 10 µg/mL Leupeptin, 1 µg/mL Pepstatin A, 100 µM 681 PMSF). These extracts were prepared in duplicate and similar results were 682 observed. Plant material was ground using disposable grinders in the 683 Eppendorf tube at room temperature until the mixture was homogeneous 684 (usually less than 15 s). Once all the samples were prepared, tubes were 685 placed in boiling water for 3 minutes. Tubes were centrifuged in a microfuge at 686 maximum speed (13000 g, 10 min) immediately before loading. Fifteen µL of 687 each extract, equivalent to about 0.5 seedlings, were loaded per lane in an SDS 688 - 8% PAGE. 689 Protein extracts analyzed in Figure 5 were prepared from AtWT and ChWT 690 seedlings grown as indicated in the figure legend. Extracts were obtained from 691 four biological replicates. Protein extracts analyzed in Figure 6 were prepared 692 from AtWT, phyA-501, phyA>AtPHYA and phyA>ChPHYA seedlings germinated 693 and grown in the dark for 4 days, as described (Gallemi et al., 2017a). Extracts 694 were obtained from three biological replicates. Each biological replicate was 695 obtained from about 100 seedlings. Protein concentration in these extracts was 20 696 determined using the Pierce BCA Protein Assay kit (Cat no. 23225; 697 www.thermofisher.com). Five or 7.5 µg of each extract were loaded per lane in 698 an SDS - 8% PAGE. 699 Immunoblot analyses of PHYA and TUB were performed at the same 700 time with the antibodies (073D, commercial anti-TUB) and dilutions indicated 701 elsewhere (Martinez-Garcia et al., 2014). Anti-mouse horseradish peroxidase- 702 conjugated antibody (www.promega.com) was used as a secondary antibody. 703 ECL or ECL-plus chemiluminescence kits (www3.gehealthcare.com) were used 704 for detection. Signal was visualized and quantified using the ChemiDoc Touch 705 Imaging System (www.bio-red.com). 706 707 Hormone analyses 708 Hormone extraction and analysis were carried out as described (Durgbanshi et 709 al., 2005) with a few modifications. Briefly, 0.02 g of dry tissue (about 150 AtWT 710 seedlings and 100 ChWT seedlings) was extracted in 1 mL of ultrapure water 711 after spiking with 50 ng of [2H2]-IAA, in a ball mill (MillMix20, Domel, Železniki, 712 Slovenija). After centrifugation at 4000 g at 4ºC for 10 min, supernatants were 713 recovered and pH adjusted to 3 with 30% acetic acid. The water extract was 714 partitioned twice against 2 mL of diethyl ether and the organic layer recovered 715 and evaporated under vacuum in a centrifuge concentrator (Speed Vac, Jouan, 716 Saint Herblain Cedex, France). Once dried, the residue was resuspended in a 717 10:90 MeOH:H2O solution by gentle sonication. The resulting solution was 718 filtered through 0.22 µm polytetrafluoroethylene membrane syringe filters (Albet 719 S.A., Barcelona, Spain) and directly injected into an ultra performance LC 720 system (Acquity SDS, Waters Corp., Milford, MA, USA). Chromatographic 721 separations were carried out on a reversed-phase C18 column (Gravity, 50 × 722 2.1 mm, 1.8-µm particle size, Macherey-Nagel GmbH, Germany) using a 723 MeOH:H2O (both supplemented with 0.1% acetic acid) gradient at a flow rate of 724 300 µL min−1. IAA was quantified with a TQS triple quadrupole mass 725 spectrometer (Micromass, Manchester, UK) connected online to the output of 726 the column though an orthogonal Z-spray electrospray ion source. 727 728 Data availability 21 729 The Illumina RNA-seq reads are available from the website 730 http://chi.mpipz.mpg.de/assembly. Source code of BAMLINK is available at 731 http://chi.mpipz.mpg.de/software. The data that support the findings of this 732 study are also available from the corresponding author on request. 733 734 Accession Numbers 735 Sequence data from this article can be found in the Arabidopsis Genome 736 Initiative or the C. hirsuta (http://chi.mpipz.mpg.de/assembly) databases under 737 the following accession numbers: AtATHB2 (At4g16780), ChATHB2 738 (CARHR223400), AtPIL1 (At2g46970), ChPIL1 (CARHR142340), AtUBQ10 739 (At4g05320), AtPHYA (At1g09570), ChPHYA (CARHR009540), ACS8 740 (At4g37770), GH3.3 (At2g23170), IAA19 (At3g15540), IAA29 (At3g15540), 741 AtEF1α (At5g60390), ChEF1α (CARHR274060 and CARHR274080), SPC25 742 (At2g39960), ChSPC25 (CARHR134880 and CARHR134890), YLS8 743 (At5g08290) and ChYLS8 (CARHR204840). 744 745 Supplemental Data 746 Supplemental Figure 1. Photosynthetic-related responses of A. thaliana and 747 C. hirsuta seedlings to changing light conditions. 748 749 Supplemental Figure 2. Light spectra of the treatments used in this study. 750 751 Supplemental Figure 3. Longitudinal length of A. thaliana and C. hirsuta 752 leaves respond differently to simulated shade. 753 754 Supplemental Figure 4. A. thaliana and C. hirsuta seedlings change gene 755 expression differently in response to simulated shade. 756 757 Supplemental Figure 5. The expression of a set of shade-induced but auxin- 758 dependent genes, identified in A. thaliana, is also shade-induced in C. hirsuta. 759 760 Supplemental Figure 6. Reduction of phyA activity in C. hirsuta seedlings 761 results in a sis phenotype. 762 22 763 Supplemental Figure 7. Partial alignment of ChPHYA/AtPHYA, 764 ChEF1/AtEF1, ChSPC25/AtSPC25 and ChYLS8/AtYLS8 sequences. 765 766 Supplemental Figure 8. Strategies to compare biological activity between 767 AtphyA and ChphyA in transgenic lines. 768 769 Supplemental Figure 9. The expression of a set of shade-induced phyA- 770 repressed genes, identified in A. thaliana, is attenuated in C. hirsuta. 771 772 Supplemental Figure 10. Alignment of C. hirsuta and A. thaliana phyA amino 773 acid sequences. 774 775 Supplemental Table 1. RPKM of eight genes commonly used for normalizing 776 in RT-qPCR analyses. 777 778 Supplemental Table 2. Primers used in this work. 779 780 Supplemental Data Set 1. Bioset of up-regulated genes in C. hirsuta seedlings 781 in response to simulated shade. 782 783 Supplemental Data Set 2. Bioset of down-regulated genes in C. hirsuta 784 seedlings in response to simulated shade. 785 786 Supplemental Data Set 3. Bioset of up-regulated genes in A. thaliana 787 seedlings in response to simulated shade. 788 789 Supplemental Data Set 4. Bioset of down-regulated genes in A. thaliana 790 seedlings in response to simulated shade. 791 792 Supplemental Data Set 5. Results of Venn Diagrams of the GO categorization. 793 794 Supplemental Data Set 6. Functional enrichment groups based on the 795 MapMan-Bin analyses. 796 23 797 Supplemental Data Set 7. Bioset of shade-regulated OMCL groups in 798 Geranium pyrenaicum petioles in response to simulated shade. 799 800 Supplemental Data Set 8. Bioset of shade-regulated OMCL groups in 801 Geranium robertianum petioles in response to simulated shade. 802 803 Supplemental Data Set 9. Summary of statistical tests. 804 805 806 ACKNOWLEDGEMENTS 807 We thank Ester Botterweg and Mª Rosa Rodríguez (CRAG) for their technical 808 support; Victor González and Martí Bernardo (Bioinformatics Core unit, CRAG) 809 for help in statistical analyses; Peter Quail (PGEC, Albany, CA, USA) for 810 providing the anti-phyA antibody; Fernando Valladares (National Museum of 811 Natural History, Madrid, Spain) for discussions about the shade habit of A. 812 thaliana and C. hirsuta; and to Charlotte Gommers (CRAG) for comments on 813 the manuscript. MJM-C, SP and LC received predoctoral fellowships from the 814 Spanish Ministerio de Economía y Competitividad (MINECO, FPI program), the 815 Agència d’Ajuts Universitaris i de Recerca (AGAUR - Generalitat de Catalunya, 816 FI program) and La Caixa Foundation (INPhINIT fellowship 817 LCF/BQ/IN18/11660004), respectively. JM-R received a International CRAG 818 “Severo Ochoa” postdoctoral program fellowship and a postdoctoral contract 819 (H2020-MSCA-IF-2017 – Proposal 797473) funded by the European 820 Commission. CT received a Marie Curie postdoctoral contract (FP7-PEOPLE- 821 IEF-2008 – Proposal 237492) funded by the European Commission and a 822 CRAG short-term fellowship. Our research is supported by grants from BBSRC 823 (BB/H006974/1) and Max Planck Society (core grant) to MT, and from 824 MINECO-FEDER (BIO2017-85316-R, and BIO2017-84041-P) and AGAUR 825 (2017-SGR1211, 2017-SGR710 and Xarba) to JFM-G and MRC. We also 826 acknowledge the support of the MINECO for the “Centro de Excelencia Severo 827 Ochoa 2016-2019” award SEV-2015-0533 and by the CERCA Programme / 828 Generalitat de Catalunya. 829 830 AUTHOR CONTRIBUTIONS 24 831 JFM-G conceived the original research plan, and directed and coordinated the 832 study. AH, HJ, XG and MT performed RNAseq and analyzed the data. AG-C 833 analyzed auxin levels; LM and MR-C measured and analyzed photosynthetic 834 parameters and pigment levels; MJM-C, SP, CT, JM-R, PP-A and IR-V 835 performed all the other experiments. All authors analyzed their data and 836 discussed the results. JFM-G wrote the paper with revisions of MR-C and 837 contributions or/and comments of all other authors. 838 839 COMPETING INTERESTS 840 The authors declare no competing interests. 841 842 FIGURE LEGENDS 843 844 Figure 1. A. thaliana and C. hirsuta differ in the hypocotyl elongation 845 response to neighboring vegetation. (A) Phenotype of representative 846 seedlings of wild-type A. thaliana (AtWT) and C. hirsuta (ChWT) after 3 days 847 grown in W and retained in W (left panels) or transferred to W+FR (R:FR of 848 0.02; right panels) until day 7 (d7). Scale bar, 5 mm. (B) Hypocotyl length of d7 849 AtWT and ChWT seedlings grown for the last 4 days under the indicated R:FR. 850 (C) Hypocotyl length of d4 AtWT and ChWT seedlings grown in darkness. (D) 851 Hypocotyl length of d7 AtWT and ChWT seedlings grown under W in media 852 supplemented with increasing concentrations of picloram (PIC). (E) Petiole and 853 rachis length of 3-week-old leaves of AtWT and ChWT plants grown for the last 7 854 days under the indicated R:FR. (F) Carotenoid (CRT) and chlorophyll (CHL) 855 levels of AtWT and ChWT seedlings grown in W and W+FR (as detailed in A). 856 Values are means and s.e.m. of three to five independent samples. Asterisks 857 indicate significant differences (**p<0.01) relative to W-grown plants. 858 859 Figure 2. A. thaliana and C. hirsuta seedlings respond to neighboring 860 vegetation by altering gene expression. (A) RNA-seq was performed with 861 RNA extracted from AtWT and ChWT seedlings that were grown in W for 7 days 862 (d7) and then treated for 1 h with W+FR (R:FR = 0.02). White circles indicate 863 the moment of harvesting for RNA extraction. Three independent biological 864 replicates were used for each genotype and treatment. (B) Correlation between 25 865 log-transformed fold-change of 432 DEGs in AtWT and ChWT. The estimated 866 regression equation is shown at the top of the graph. (C) IAA content in AtWT 867 and ChWT seedlings grown and harvested as indicated in A. Whole seedlings 868 were collected and lyophilized to measure IAA levels. Data are presented as the 869 means and s.e.m. of three (AtWT) or four (ChWT) biological replicates. DW, dry 870 weight. (D) Effect of W+FR treatment on PIL1 and ATHB2 expression in AtWT 871 and ChWT seedlings (R:FR = 0.02). W-grown d7 seedlings of Col-0 and Ox were 872 treated for 0, 1, 4 and 8 h with W+FR. Transcript abundance, normalized to 873 EF1 is shown. Values are means and s.e.m. of three independent RT-qPCR 874 biological replicates relative to values at 0 h for each species. In C and D, 875 asterisks indicate significant differences (**p<0.01, *p<0.05) relative to 0 h 876 samples. 877 878 Figure 3. Mutant sis1 seedlings of C. hirsuta are deficient in phyA activity. 879 (A) Phenotype of representative seedlings of ChWT, sis1-1 and sis1-2 after 3 880 days grown in W and retained in W (white panels) or transferred to W+FR 881 (R:FR of 0.02; pink panels) until day 7 (d7). All panels are to the same scale. 882 (B) Hypocotyl length of AtWT, phyA-501 (A. thaliana), ChWT, sis1-1 and sis1-2 883 (C. hirsuta) lines grown for 4 days in darkness (Dark) or under monochromatic 884 FR (2.6 µmol·m-2·s-1), R (38.9 µmol·m-2·s-1) and blue (B, 1.9 µmol·m-2·s-1) light. 885 (C) Hypocotyl length of A. thaliana Ler, phyB-4 and phyB-1 seedlings grown for 886 4 days in darkness (Dark) or under monochromatic R (40.6 µmol·m-2·s-1) light. 887 (D) Hypocotyl length of A. thaliana Ler, phyB-4 and phyB-1 seedlings under the 888 indicated R:FR. Seedlings were grown for 2 days in W (R:FR > 2.5) and then 889 kept in W (R:FR > 2.5) or transferred to W+FR (R:FR of 0.06 or 0.02) until day 7 890 (d7). (E) Schematic diagram of the lesions found in the ChPHYA gene in the 891 sis1-1 and sis1-2 alleles compared to the wild-type sequence (ChWT) and the 892 predicted changes in the amino acid sequence. (F) Immunoblot detection of 893 phyA and tubulin with mouse monoclonal anti-phyA (073D) and anti-TUB 894 antibodies in extracts of etiolated seedlings of ChWT, sis1-1 and sis1-2 lines. 895 896 Figure 4. C. hirsuta sis1 seedlings are impaired in their tolerance to plant 897 proximity. (A) Effect of W+FR treatment on PIL1 and ATHB2 expression in 898 ChWT sis1-1- and sis1-2 seedlings. Seedlings were grown as in Figure 2D. 26 899 Transcript abundance, normalized to EF1 is shown. Values are means and 900 s.e.m. of three independent RT-qPCR biological replicates relative to values at 901 0 h for each genotype. Asterisks indicate significant differences (**p<0.01) 902 relative to 0 h samples. (B) Carotenoid (CRT) and chlorophyll (CHL) levels of 903 AtWT and phyA-501 A. thaliana and ChWT, sis1-1 and sis1-2 C. hirsuta seedlings 904 grown in W and W+FR (as detailed in Figure 1A). Values are means and s.e.m. 905 of five independent samples. Asterisks indicate significant differences 906 (**p<0.01) relative to W-grown plants. (C,D) Hypocotyl length of d7 AtWT, phyA- 907 501 (A. thaliana) (C) and ChWT, sis1-1, sis1-2 (C. hirsuta) (D) seedlings grown 908 for the last 4 days under the indicated R:FR. Asterisks indicate significant 909 differences (*p<0.05, **p<0.01) relative to the corresponding wild-type plant 910 grown under the same R:FR. In D, asterisks apply for both sis1 mutants. 911 912 Figure 5. C. hirsuta seedlings have higher phyA levels than those of A. 913 thaliana. (A) Cartoon showing the design of the experiment. Wild-type 914 seedlings of A. thaliana (AtWT) and C. hirsuta (ChWT), grown as in Figure 1A, 915 were harvested at the indicated times of W or W+FR treatments (asterisks) for 916 RNA extraction. (B) Evolution of PHYA transcript levels in A. thaliana and C. 917 hirsuta wild-type seedlings grown as detailed in A. Primers used (Supplemental 918 Figure 8A) allow quantifying and comparing expression levels by RT-qPCR 919 between both species. PHYA transcript abundance was normalized to three 920 reference genes (EF1, SPC25 and YLS8). Values are means and s.e.m. of 921 three independent RT-qPCR biological replicates relative to PHYA transcript 922 levels of d3 A. thaliana seedlings. Two-way ANOVA showed that PHYA levels 923 are significantly different (**p<0.01) between species under either W or W+FR. 924 (C) Immunoblot detection of phyA and tubulin with the antibodies indicated in 925 Figure 3C in extracts of AtWT and ChWT seedlings grown as detailed at the top of 926 the section: 5-day-old etiolated seedlings were exposed to W light and material 927 was harvested before and after 6 h of W-exposure (arrows). (D) Evolution of 928 relative phyA protein levels (PHYA:TUB) in AtWT and ChWT seedlings exposed to 929 simulated shade, as detailed at the top of the section: 5-day-old etiolated 930 seedlings were exposed to W+FR light and material was harvested before and 931 after 6, 8 and 10 h of simulated shade exposure (arrows). Values are means 932 and s.e.m. of four independent biological replicates relative to PHYA:TUB levels 27 933 of etiolated AtWT seedlings. Two way ANOVA showed that relative PHYA levels 934 under W+FR are significantly increased (**p<0.01) in C. hirsuta than A. thaliana. 935 936 Figure 6. ChphyA has a stronger activity than AtphyA in repressing 937 shade-induced hypocotyl elongation. (A) Cartoon detailing the constructs 938 used to complement A. thaliana phyA-501 mutant plants. (B) Relative 939 PHYA:TUB in etiolated seedlings of AtWT, phyA-501, and selected 940 phyA>AtPHYA (blue bars) and phyA>ChPHYA (red bars) complementation 941 lines. Seedlings were grown as indicated in Supplemental Figure 8. Values are 942 means and s.e.m. of four independent biological replicates relative to 943 PHYA:TUB levels of etiolated AtWT seedlings. (C) Cartoon illustrating how phyA 944 activity in simulated shade was established as differences in hypocotyl length 945 between simulated shade- and the W-grown seedlings (HypW+FR-HypW). 946 Seedlings were grown for 2 days under W then for 5 additional days under W or 947 W+FR (R:FR = 0.02), when hypocotyls were measured. (D) HypW+FR-HypW in 948 seedlings of AtWT, phyA-501, and selected phyA>AtPHYA (blue bars) and 949 phyA>ChPHYA (red bars) complementation lines. (E) Cartoon illustrating how 950 phyA activity in de-etiolation was established as differences in hypocotyl length 951 between dark- and FR-grown seedlings (HypD-HypFR). Seedlings were grown as 952 indicated in Figure 3B. (F) HypD-HypFR in seedlings of AtWT, phyA-501, and 953 selected phyA>AtPHYA (blue bars) and phyA>ChPHYA (red bars) 954 complementation lines. In C and E, mutant phyA-501 seedlings have no phyA 955 activity. 956 957 Figure 7. ChphyA has a stronger activity than AtphyA in repressing 958 shade-induced expression of ACS8, GH3.3, IAA19 and IAA29 genes. (A) 959 Effect of phyA in the shade-induced expression of ACS8, GH3.3, IAA19 and 960 IAA29. W-grown d5 seedlings of AtWT and phyA-501 were treated for 0, 1, 8 and 961 24 h with W+FR (R:FR = 0.02), when material was harvested for RNA 962 extraction, as indicated at the top of the panel. Transcript abundance, 963 normalized to EF1 is shown. Values are means and s.e.m. of three 964 independent RT-qPCR biological replicates relative to values at 0 h for AtWT. 965 Asterisks indicate significant differences (**p<0.01, *p<0.05) between phyA-501 966 and AtWT seedlings exposed for the same time to W+FR. (B) Correlation 28 967 between ACS8, GH3.3, IAA19 and IAA29 expression and relative levels of 968 PHYA protein in the seedlings of AtWT, phyA-501, phyA>AtPHYA (blue lines and 969 dots) and phyA>ChPHYA (red lines and dots) complementation lines. Gene 970 expression was quantified in W-grown d5 seedlings exposed to W+FR 971 (R:FR=0.02) during 24 h, as indicated at the top of the panel. Transcript 972 abundance was normalized to EF1. Relative phyA protein levels (PHYA:TUB, 973 data already shown in Supplemental Figure 8) were estimated in etiolated 974 seedlings. Values are means and s.e.m. of three independent RT-qPCR 975 biological replicates relative to values of AtWT. The estimated regression lines 976 for the phyA>AtPHYA (blue line) and phyA>ChPHYA (red line) 977 complementation lines are shown for each correlation. 978 979 Figure 8. Model of how increased phyA activity in C. hirsuta might 980 implement the shade tolerance of hypocotyl elongation. 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(1995) Phytochrome A, 1239 phytochrome B and HY4 are invovled in hypocotyl growth responses to 1240 natural radiation in Arabidopsis: Weak de-etiolation of the phyA mutant 1241 under dense canopies. Plant Cell Environ, 18, 788-794. 1242 1243 34 A W W+FR B AtWT ChWT 7 Hypocotyl length (mm) 6 AtWT 5 4 3 2 ChWT 1 0 >1.6 0.09 0.07 0.02 R:FR ratio C 25 D 0 μM 5.0 μM PIC Hypocotyl length (mm) 0.5 μM PIC 10.0 μM PIC 6 Hypocotyl length (mm) 20 5 15 4 10 3 5 2 0 1 AtWT ChWT 0 AtWT ChWT E A. thaliana C. hirsuta W W+FR 25 50 (AtWT) ** (ChWT) ** Petiole & rachis 20 ** 40 length (mm) 15 30 10 20 5 10 0 0 4 5 6 3 4 5 Leaf number Leaf number F 120 CRT CHL W 100 ** ** Relative levels ** ** W+FR 80 60 40 20 0 AtWT ChWT AtWT ChWT Figure 1. A. thaliana and C. hirsuta differ in the hypocotyl elongation response to neighboring vegetation. (A) Phenotype of representative seedlings of wild-type A. thaliana (AtWT) and C. hirsuta (ChWT) after 3 days grown in W and retained in W (left panels) or transferred to W+FR (R:FR of 0.02; right panels) until day 7 (d7). Scale bar, 5 mm. (B) Hypocotyl length of d7 AtWT and ChWT seedlings grown for the last 4 days under the indicated R:FR. (C) Hypocotyl length of d4 AtWT and ChWT seedlings grown in darkness. (D) Hypocotyl length of d7 AtWT and ChWT seedlings grown under W in media supplemented with increasing concentrations of picloram (PIC). (E) Petiole and rachis length of 3-week-old leaves of AtWT and ChWT plants grown for the last 7 days under the indicated R:FR. (F) Carotenoid (CRT) and chlorophyll (CHL) levels of AtWT and ChWT seedlings grown in W and W+FR (as detailed in A). Values are means and s.e.m. of three to five independent samples. Asterisks indicate significant differences (**p<0.01) relative to W-grown plants. A C 450 IAA content (ng/g DW) ChWT * W 400 350 W+FR AtWT 300 0h 1h (W) (W+FR) 250 200 150 100 ** (7 days, W) 50 0 AtWT ChWT D 0h 1h 4h 8h 70 ** B y = 0.54x + 0.12 60 50 * PIL1 10 40 6 Relative transcript levels 30 ** Fold-change in ChWT * * 6 20 ** 10 4 0 2 20 ** 0 16 ATHB2 -2 12 ** * ** -4 8 * 0 2 4 6 6 -4 -2 10 4 * Fold-change in AtWT 0 AtWT Arabidopsis ChWT Cardamine Figure 2. A. thaliana and C. hirsuta seedlings respond to neighboring vegetation by altering gene expression. (A) RNA-seq was performed with RNA extracted from AtWT and ChWT seedlings that were grown in W for 7 days (d7) and then treated for 1 h with W+FR (R:FR = 0.02). White circles indicate the moment of harvesting for RNA extraction. Three independent biological replicates were used for each genotype and treatment. (B) Correlation between log-transformed fold-change of 432 DEGs in AtWT and ChWT. The estimated regression equation is shown at the top of the graph. (C) IAA content in AtWT and ChWT seedlings grown and harvested as indicated in A. Whole seedlings were collected and lyophilized to measure IAA levels. Data are presented as the means and s.e.m. of three (AtWT) or four (ChWT) biological replicates. DW, dry weight. (D) Effect of W+FR treatment on PIL1 and ATHB2 expression in AtWT and ChWT seedlings (R:FR = 0.02). W-grown d7 seedlings of Col-0 and Ox were treated for 0, 1, 4 and 8 h with W+FR. Transcript abundance, normalized to EF1D is shown. Values are means and s.e.m. of three independent RT-qPCR biological replicates relative to values at 0 h for each species. In C and D, asterisks indicate significant differences (**p<0.01, *p<0.05) relative to 0 h samples. A W W+FR W W+FR W W+FR ChWT sis1-1 sis1-2 B Dark FR Dark R Dark B 20 20 20 d d d dd d f d f d d Hypocotyl length (mm) 16 16 16 a a a a a aa 12 12 g 12 h e 8 8 8 e e e 4 b 4 c 4 c bc b b 0 0 0 AtWT sis1-1 sis1-2 AtWT AtWT phyA-501 sis1-1 sis1-2 sis1-1 sis1-2 phyA-501 WT phyA-501 WT WT OX sis1-1 sis1-2 OX sis1-1 sis1-2 Col-0 phyA-501 Col-0 phyA-501 OX sis1-1 sis1-2 Col-0 phyA-501 Ch Ch Ch C 120 Dark R E Hypocotyl length (mm) C2803T (Q935*) 100 ChWT: ...ATT-TTG-CAA-ACT-AGC... ... I L Q T S ... 80 sis1-1: ... I L * 60 G2738A (G913E) 40 ChWT: ...CTG-TCC-GGG-ATC-ATG... 20 ... L S G I M ... sis1-2: ... L S E I M ... 0 Ler phyB-4 phyB-1 sis1-1 sis1-2 ChWT 10 Hypocotyl length (mm) D Ler F 8 phyB-4 phyB-1 6 120 α-PHYA 4 85 2 0 α-TUB 50 >2.5 0.06 0.02 R:FR ratio Figure 3. Mutant sis1 seedlings of C. hirsuta are deficient in phyA activity. (A) Phenotype of representative seedlings of ChWT, sis1-1 and sis1-2 after 3 days grown in W and retained in W (white panels) or transferred to W+FR (R:FR of 0.02; pink panels) until day 7 (d7). All panels are to the same scale. (B) Hypocotyl length of AtWT, phyA-501 (A. thaliana), ChWT, sis1-1 and sis1-2 (C. hirsuta) lines grown for 4 days in darkness (Dark) or under monochromatic FR (2.6 µmol·m-2·s-1), R (38.9 µmol·m-2·s-1) and blue (B, 1.9 µmol·m-2·s-1) light. (C) Hypocotyl length of A. thaliana Ler, phyB-4 and phyB-1 seedlings grown for 4 days in darkness (Dark) or under monochromatic R (40.6 µmol·m -2·s-1) light. (D) Hypocotyl length of A. thaliana Ler, phyB-4 and phyB-1 seedlings under the indicated R:FR. Seedlings were grown for 2 days in W (R:FR > 2.5) and then kept in W (R:FR > 2.5) or transferred to W+FR (R:FR of 0.06 or 0.02) until day 7 (d7). (E) Schematic diagram of the lesions found in the ChPHYA gene in the sis1-1 and sis1-2 alleles compared to the wild-type sequence (ChWT) and the predicted changes in the amino acid sequence. (F) Immunoblot detection of phyA and tubulin with mouse monoclonal anti-phyA (073D) and anti-TUB antibodies in extracts of etiolated seedlings of ChWT, sis1-1 and sis1-2 lines. A 0h 1h 4h 8h B W W+FR 120 120 PIL1 ** 100 100 * ** ** 80 80 ** ** CRT 60 ** 60 Relative transcript levels * 40 40 Relative levels 20 20 0 0 35 ATHB2 ** * 30 100 ** 25 80 ** ** ** CHL 20 ** 60 15 ** 40 10 5 20 0 0 sis1-1 sis1-2 phyA-501 AtWT ChWT ChWT sis1-1 sis1-2 ChWT OX C AtWT Col-0 D sis1-1 phyA-501 sis1-2 8 12 Hypocotyl length (mm) Hypocotyl length (mm) ** ** 10 6 ** 8 ** 4 6 * 4 2 2 0 0 >2.5 >2.5 0.091 0.050 0.027 0.091 0.050 0.027 R:FR ratio R:FR ratio Figure 4. C. hirsuta sis1 seedlings are impaired in their tolerance to plant proximity. (A) Effect of W+FR treatment on PIL1 and ATHB2 expression in ChWT sis1-1- and sis1-2 seedlings. Seedlings were grown as in Figure 2D. Transcript abundance, normalized to EF1α is shown. Values are means and s.e.m. of three independent RT-qPCR biological replicates relative to values at 0 h for each genotype. Asterisks indicate significant differences (**p<0.01) relative to 0 h samples. (B) Carotenoid (CRT) and chlorophyll (CHL) levels of AtWT and phyA-501 A. thaliana and ChWT, sis1-1 and sis1-2 C. hirsuta seedlings grown in W and W+FR (as detailed in Figure 1A). Values are means and s.e.m. of five independent samples. Asterisks indicate significant differences (**p<0.01) relative to W-grown plants. (C,D) Hypocotyl length of d7 AtWT, phyA-501 (A. thaliana) (C) and ChWT, sis1-1, sis1-2 (C. hirsuta) (D) seedlings grown for the last 4 days under the indicated R:FR. Asterisks indicate significant differences (*p<0.05, **p<0.01) relative to the corresponding wild-type plant grown under the same R:FR. In D, asterisks apply for both sis1 mutants. A C d5 0 6 * ** ** ** ** AtWT ChWT time (h): 0 6 0 6 α-PHYA AtWT (W) ChWT (W) B AtWT (W+FR) ChWT (W+FR) α-TUB 6 W 5 4 D Relative PHYA transcript levels 3 d5 ** 0 6 8 10 2 1 AtWT (W+FR) 0 ChWT (W+FR) Relative PHYA:TUB protein 6 3.5 W+FR 5 3.0 4 2.5 2.0 3 1.5 ** 2 ** 1.0 1 0.5 0 0 3 4 5 6 7 0 6 8 10 day time under W+FR (h) Figure 5. C. hirsuta seedlings have higher phyA levels than those of A. thaliana. (A) Cartoon showing the design of the experiment. Wild-type seedlings of A. thaliana (AtWT) and C. hirsuta (ChWT), grown as in Figure 1A, were harvested at the indicated times of W or W+FR treatments (asterisks) for RNA extraction. (B) Evolution of PHYA transcript levels in A. thaliana and C. hirsuta wild-type seedlings grown as detailed in A. Primers used (Supplemental Figure 6A) allow quantifying and comparing expression levels by RT-qPCR between both species. PHYA transcript abundance was normalized to three reference genes (EF1α, SPC25 and YLS8). Values are means and s.e.m. of three independent RT-qPCR biological replicates relative to PHYA transcript levels of d3 A. thaliana seedlings. Two-way ANOVA showed that PHYA levels are significantly different (**p<0.01) between species under either W or W+FR. (C) Immunoblot detection of phyA and tubulin with the antibodies indicated in Figure 3C in extracts of AtWT and ChWT seedlings grown as detailed at the top of the section: 5-day-old etiolated seedlings were exposed to W light and material was harvested before and after 6 h of W-exposure (arrows). (D) Evolution of relative phyA protein levels (PHYA:TUB) in AtWT and ChWT seedlings exposed to simulated shade, as detailed at the top of the section: 5- day-old etiolated seedlings were exposed to W+FR light and material was harvested before and after 6, 8 and 10 h of simulated shade exposure (arrows). Values are means and s.e.m. of four independent biological replicates relative to PHYA:TUB levels of etiolated AtWT seedlings. Two way ANOVA showed that relative PHYA levels under W+FR are significantly increased (**p<0.01) in C. hirsuta than A. thaliana. A B 1.2 Relative PHYA:TUBD 1.0 pAtPHYA AtPHYA protein levels in phyA-501 [phyA>AtPHYA lines] 0.8 0.6 pAtPHYA ChPHYA 0.4 in phyA-501 [phyA>ChPHYA lines] 0.2 0 #02 #19 #06 #16 #11 #20 phyA AtWT Col-0 phyA-501 C D 10 HypW+FR – HypW (*) HypW+FR – HypW (mm) 8 6 (*) (*) 4 2 W W+FR W W+FR 0 #02 #19 #06 #16 #11 #20 phyA AtWT Col-0 phyA-501 E HypD – HypFR (*) (*) F 12 HypD – HypFR (mm) 10 8 (*) 6 4 2 0 #02 #19 #06 #16 #11 #20 phyA AtWT D FR D FR Figure 6. ChphyA has a stronger activity than AtphyA in repressing shade-induced hypocotyl elongation. (A) Cartoon detailing the constructs used to complement A. thaliana phyA-501 mutant plants. (B) Relative PHYA:TUB in etiolated seedlings of AtWT, phyA-501, and selected phyA>AtPHYA (blue bars) and phyA>ChPHYA (red bars) complementation lines. Seedlings ere grown as indicated in Supplemental Figure 8. Values are means and s.e.m. of four independent biological replicates relative to PHYA:TUB levels of etiolated AtWT seedlings. (C) Cartoon illustrating how phyA activity in simulated shade was established as differences in hypocotyl length between simulated shade- and the W-grown seedlings (HypW+FR-HypW). Seedlings were grown for 2 days under W then for 5 additional days under W or W+FR (R:FR = 0.02), when hypocotyls were measured. (D) HypW+FR-HypW in seedlings of AtWT, phyA-501, and selected phyA>AtPHYA (blue bars) and phyA>ChPHYA (red bars) complementation lines. (E) Cartoon illustrating how phyA activity in de-etiolation was established as differences in hypocotyl length between dark- and FR-grown seedlings (HypD-HypFR). Seedlings were grown as indicated in Figure 3B. (F) HypD-HypFR in seedlings of AtWT, phyA- 501, and selected phyA>AtPHYA (blue bars) and phyA>ChPHYA (red bars) complementation lines. In C and E, mutant phyA-501 seedlings have no phyA activity. A 0 24 AtWT 5 days under W W+FR phyA-501 500 8 ACS8 GH3.3 400 ** 6 * 300 ** 4 Relative transcript levels 200 ** 2 100 0 0 25 70 IAA19 IAA29 60 20 50 15 40 10 30 * ** 20 5 ** 10 0 0 0 1 8 24 0 1 8 24 Time under W+FR (h) B 24 h AtWT 5 days under W W+FR phyA-501 phyA>AtPHYA lines phyA>ChPHYA lines 200 2.5 ACS8 GH3.3 150 2.0 100 1.5 Relative transcript levels 50 1.0 0 0.5 0 6 2.5 IAA19 IAA29 5 2.0 4 1.5 3 1.0 2 1 0.5 0 0 0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 Relative PHYA:TUBD protein levels Figure 7. ChphyA has a stronger activity than AtphyA in repressing shade-induced expression of ACS8, GH3.3, IAA19 and IAA29 genes. (A) Effect of phyA in the shade-induced expression of ACS8, GH3.3, IAA19 and IAA29. W- grown d5 seedlings of AtWT and phyA-501 were treated for 0, 1, 8 and 24 h with W+FR (R:FR = 0.02), when material was harvested for RNA extraction, as indicated at the top of the panel. Transcript abundance, normalized to EF1α is shown. Values are means and s.e.m. of three independent RT-qPCR biological replicates relative to values at 0 h for AtWT. Asterisks indicate significant differences (**p<0.01, *p<0.05) between phyA-501 and AtWT seedlings exposed for the same time to W+FR. (B) Correlation between ACS8, GH3.3, IAA19 and IAA29 expression and relative levels of PHYA protein in the seedlings of AtWT, phyA-501, phyA>AtPHYA (blue lines and dots) and phyA>ChPHYA (red lines and dots) complementation lines. Gene expression was quantified in W-grown d5 seedlings exposed to W+FR (R:FR=0.02) during 24 h, as indicated at the top of the panel. Transcript abundance was normalized to EF1α. Relative phyA protein levels (PHYA:TUB, data already shown in Supplemental Figure 8) were estimated in etiolated seedlings. Values are means and s.e.m. of three independent RT-qPCR biological replicates relative to values of AtWT. 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Martinez-Garcia Plant Cell; originally published online September 17, 2019; DOI 10.1105/tpc.19.00275 This information is current as of October 11, 2019 Supplemental Data /content/suppl/2019/09/17/tpc.19.00275.DC1.html Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298 X eTOCs Sign up for eTOCs at: http://www.plantcell.org/cgi/alerts/ctmain CiteTrack Alerts Sign up for CiteTrack Alerts at: http://www.plantcell.org/cgi/alerts/ctmain Subscription Information Subscription Information for The Plant Cell and Plant Physiology is available at: http://www.aspb.org/publications/subscriptions.cfm © American Society of Plant Biologists ADVANCING THE SCIENCE OF PLANT BIOLOGY