This is the accepted version of the article: Paulisic, Sandi; Qin, Wenting; Veraszto, Harshul Arora; [et al.]. «Adjustment of the PIF7-HFR1 transcriptional module activity controls plant shade adaptation». The EMBO Journal, Vol. 40, Issue 1 (January 2021), art. e104273. DOI 10.15252/embj.2019104273 This version is available at https://ddd.uab.cat/record/236284 under the terms of the license 1 Adjustment of the PIF7-HFR1 transcriptional module activity controls 2 plant shade adaptation 3 4 Sandi Paulišić1, Wenting Qin1, Harshul Arora Verasztó2, Christiane Then1a, 5 Benjamin Alary1, Fabien Nogue3, Miltos Tsiantis4, Michael Hothorn2, Jaime F. 6 Martínez-García1,5,6© 7 1 8 Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, 9 Cerdanyola del Vallès, Campus UAB, 08193 Barcelona, Spain. 2 10 Structural Plant Biology Laboratory, Department of Botany and Plant Biology, 11 Section of Biology, University of Geneva, 1211 Geneva, Switzerland. 3 12 Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, Université Paris- 13 Saclay, 78000, Versailles, France. 4 14 Department of Comparative Development and Genetics, Max Planck Institute 15 from Plant Breeding Research, 50829 Cologne, Germany. 4 16 Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, 17 Spain. 5 18 Institute for Plant Molecular and Cellular Biology (IBMCP), CSIC-UPV, 46022 19 València, Spain. 20 a 21 Current address, Department for Epidemiology and Pathogen Diagnostics, 22 Julius Kühn-Institut, Federal Research Institute for Cultivated Plants, 38104 23 Braunschweig, Germany. 24 Running title, PIF-HFR1 role in plant shade adaptation 25 1 26 ABSTRACT 27 Shade caused by the proximity of neighboring vegetation triggers a set of 28 acclimation responses to either avoid or tolerate shade. Comparative analyses 29 between the shade avoider Arabidopsis thaliana and the shade tolerant 30 Cardamine hirsuta, revealed a role for the atypical basic-helix-loop-helix LONG 31 HYPOCOTYL IN FR 1 (HFR1) in maintaining the shade-tolerance in C. hirsuta, 32 inhibiting hypocotyl elongation in shade and constraining expression profile of 33 shade induced genes. We showed that C. hirsuta HFR1 protein is more stable 34 than its A. thaliana counterpart, likely due to its lower binding affinity to 35 CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), contributing to enhance 36 its biological activity. The enhanced HFR1 total activity is accompanied by an 37 attenuated PHYTOCHROME INTERACTING FACTOR (PIF) activity in C. 38 hirsuta. As a result, the PIF-HFR1 module is differently balanced, causing a 39 reduced PIF activity and attenuating other PIF-mediated responses such as 40 warm temperature-induced hypocotyl elongation (thermomorphogenesis) and 41 dark-induced senescence. By this mechanism and that of the already-known of 42 phytochrome A photoreceptor, plants might ensure to properly adapt and thrive 43 in habitats with disparate light amounts. 44 45 Keywords, Cardamine hirsuta / HFR1 / PIFs / shade avoidance / shade 46 tolerance. 47 2 48 INTRODUCTION 49 Acclimation of plants to adjust their development to the changing 50 environment is of uttmost importance. This acclimation relies on the plant’s 51 ability to perceive many cues such as water, nutrients, temperature or light. 52 Conditions in nature often involve simultaneous changes in multiple light cues 53 leading to an interplay of various photoreceptors to adjust plant growth 54 appropriately (Ballare & Pierik, 2017; de Wit et al, 2016; Fiorucci & Fankhauser, 55 2017; Mazza & Ballare, 2015; Pierik & Testerink, 2014). Nearby vegetation can 56 impact both light quantity and quality. Under a canopy, light intensity is 57 decreased and its quality is changed as the overtopping green leaves strongly 58 absorb blue and red light (R) but reflect far-red light (FR). As a consequence, 59 plants growing in forest understories receive less light of a much lower R to FR 60 ratio (R:FR) than those growing in open spaces. In dense plant communities, 61 FR reflected by neighboring plants also decreases R:FR but typically without 62 changing light intensity. We refer to the first situation as canopy shade (very low 63 R:FR) and the second as proximity shade (low R:FR). In general, two strategies 64 have emerged to deal with shade: avoidance and tolerance (Gommers et al, 65 2013; Pierik & Testerink, 2014; Valladares & Niinemets, 2008). Shade avoiders 66 usually promote elongation of organs to outgrow the neighbors and avoid light 67 shortages, reduce the levels of photosynthetic pigments to cope to light 68 shortage, and accelerate flowering to ensure species survival (Casal, 2013). 69 The set of responses to acclimate to shade is collectively known as the shade 70 avoidance syndrome (SAS). In contrast, shade-tolerant species usually lack the 71 promotion of elongation growth in response to shade and have developed a 3 72 variety of traits to acclimate to low light conditions and optimize net carbon gain 73 (Smith, 1982; Valladares & Niinemets, 2008). 74 In Arabidopsis thaliana, a shade avoider plant, low R:FR is perceived by 75 phytochromes. Among them, phyA has a negative role in elongation, particularly 76 under canopy shade, whereas phyB inhibits elongation inactivating 77 PHYTOCHROME INTERACTING FACTORS (PIFs), members of the basic- 78 helix-loop-helix (bHLH) transcription factor family that promote elongation 79 growth. In particular, PIFs induce hypocotyl elongation by initiating an 80 expression cascade of genes involved in auxin biosynthesis and signaling [e.g., 81 YUCCA 8 (YUC8), YUC9, INDOLE-3-ACETIC ACID INDUCIBLE 19 (IAA19), 82 IAA29] and other processes related to cell elongation [e.g., XYLOGLUCAN 83 ENDOTRANSGLYCOSYLASE 7 (XTR7)]. Genetic analyses indicated that PIF7 84 is the key PIF regulator of the low R:FR-induced hypocotyl elongation with PIF4 85 and PIF5 having important contributions. Indeed, pif7 mutant responds poorly to 86 low R:FR compared to the pif4 pif5 double or pif1 pif3 pif4 pif5 quadruple (pifq) 87 mutants, but the triple pif4 pif5 pif7 mutant is almost unresponsive to low R:FR 88 (de Wit et al, 2016; Li et al, 2012; Lorrain et al, 2008; van Gelderen et al, 2018). 89 PhyB-mediated shade signaling involves other transcriptional regulators, such 90 as LONG HYPOCOTYL IN FR 1 (HFR1), PHYTOCHROME RAPIDLY 91 REGULATED 1 (PAR1), BIM1, ATHB4 or BBX factors, that either promote or 92 inhibit shade-induced hypocotyl elongation (Bou-Torrent et al, 2014; Cifuentes- 93 Esquivel et al, 2013; Gallemi et al, 2017; Roig-Villanova et al, 2007; Sasidharan 94 & Pierik, 2010; Sessa et al, 2005; Yang & Li, 2017). HFR1, a member of the 95 bHLH family, is structurally related to PIFs but lacks the phyB- and DNA-binding 96 ability that PIFs possess (Galstyan et al, 2011; Hornitschek et al, 2012). HFR1 4 97 inhibits PIF activity by heterodimerizing with them, as described for PIF1 (Shi et 98 al, 2013), PIF3 (Fairchild et al, 2000), PIF4 and PIF5 (Hornitschek et al, 2009), 99 Heterodimerization with HFR1 prevents PIFs from binding to the DNA and 100 altering gene expression. In this manner HFR1 acts as a transcriptional cofactor 101 that modulates SAS responses, e.g. it inhibits hypocotyl elongation in seedlings 102 in a PIF-dependent manner, forming the PIF-HFR1 transcriptional regulatory 103 module (Galstyan et al, 2011). 104 What mechanistic and regulatory adjustments in shade signaling are 105 made between species to adapt to plant shade is a topic that has not received 106 much attention until now. This question has been recently addressed 107 performing comparative analyses between phylogenetically related species. In 108 two related Geranium species that showed petioles with divergent elongation 109 responses to shade, transcriptomic analysis led to propose that differences in 110 expression of three factors, FERONIA, THESEUS1 and KIDARI, shown to 111 activate SAS elongation responses in A. thaliana, might be part of the 112 adjustments necessary to acquire a shade-avoiding or tolerant habit (Gommers 113 et al, 2017). When comparing two related mustard species that showed 114 divergent hypocotyl elongation response to shade, A. thaliana and Cardamine 115 hirsuta (Hay et al, 2014), molecular and genetic analyses indicated that phyA, 116 and to a lesser extent phyB, contributed to establish this divergent response. In 117 particular, the identification and characterization of the C. hirsuta phyA-deficient 118 slender in shade 1 (sis1) mutant indicated that differential features of this 119 photoreceptor in A. thaliana and C. hirsuta could explain their differential 120 response to shade. Thus, stronger phyA activity in C. hirsuta wild-type plants 121 resulted in a suppressed hypocotyl elongation response when exposed to low 5 122 or very low R:FR (Molina-Contreras et al, 2019). These approaches indicated 123 that the implementation of shade avoidance and shade tolerance involved the 124 participation of shared genetic components. They also suggest that other 125 responses co-regulated by these shared components will be accordingly 126 affected. 127 With this frame of reference, we asked whether the phyB-dependent PIF- 128 HFR1 module was also relevant to shape the shade response habits in different 129 plant species. We found that C. hirsuta plants deficient in ChHFR1 gained a 130 capacity to elongate in response to shade. We also report that AtHFR1 and 131 ChHFR1 are expressed at different levels and encode proteins with different 132 protein stability, caused by their different binding affinities with CONSTITUTIVE 133 PHOTOMORPHOGENIC 1 (COP1), known to affect AtHFR1 stability under 134 shade (Pacin et al, 2016). We propose that adaptation to plant shade in A. 135 thaliana and C. hirsuta relies on the PIF-HFR1 regulatory module. As PIFs 136 regulates several other processes, we hypothesized that a set of responses co- 137 regulated by the PIF-HFR1 module are also affected and associated with the 138 shade-avoidance and shade-tolerant habits. After exploring this possibility, we 139 found that thermoregulation of hypocotyl elongation and dark-induced 140 senescence, two well-known PIF-regulated responses (Koini et al, 2009; 141 Sakuraba et al, 2014; Stavang et al, 2009), are consistently affected in C. 142 hirsuta. 143 6 144 RESULTS 145 HFR1 is required for the shade tolerance habit of C. hirsuta 146 First, we wanted to determine if HFR1 has a role in the shade-tolerance 147 habit of C. hirsuta, i.e., whether ChHFR1 contributes to inhibit hypocotyl 148 elongation when this species is exposed to shade. For this purpose, we 149 generated several C. hirsuta RNAi lines to downregulate HFR1 expression 150 (RNAi-HFR1 lines). As expected, ChHFR1 expression was attenuated in 151 seedlings of two RNAi-HFR1 selected lines (#01 and #21) compared to the wild 152 type (ChWT) (Fig EV1A). When growing under white light (W) of high R:FR 153 (>1.5), hypocotyl length of these two RNAi-HFR1 lines was undistinguishable 154 from ChWT (Fig 1A). By contrast, under W supplemented with increasing 155 amounts of FR (W+FR) resulting in moderate (0.09), low (0.05-0.06) and very 156 low (0.02) R:FR (that simulated proximity and canopy shade) (Martinez-Garcia 157 et al, 2014), the hypocotyl elongation of RNAi-HFR1 seedlings was significantly 158 promoted compared to ChWT, which was unresponsive (Fig 1A). 159 Using CRISPR-Cas9, we obtained two mutant lines of ChHFR1 (named 160 chfr1-1 and chfr1-2) with a single nucleotide insertion in their sequence leading 161 to a premature stop codon (Fig EV1C). These mutants showed a non-significant 162 decrease of ChHFR1 expression in W-grown seedlings (Fig EV1B). Similar to 163 the RNAi-HFR1 lines, their hypocotyls were undistinguishable from ChWT under 164 W but elongated strongly in response to W+FR exposure (Fig 1B), showing a 165 slender in shade (sis) phenotype. Together, we concluded that HFR1 represses 166 hypocotyl elongation in response to shade in C. hirsuta. 167 Exposure of A. thaliana wild-type (AtWT) and ChWT seedlings to low R:FR 168 induces a rapid increase in the expression of various direct target genes of 7 169 PIFs, including PIF3-LIKE 1 (PIL1), YUC8 and XTR7 (Fig 1C, D) (Ciolfi et al, 170 2013; Hersch et al, 2014; Molina-Contreras et al, 2019). The shade-induced 171 expression of these genes was significantly higher in RNAi-HFR1 and chfr1 172 mutant lines compared to ChWT (Fig 1C, D), indicating that ChHFR1 might 173 repress shade-triggered hypocotyl elongation in part by down-regulating the 174 rapid shade-induced expression of these genes in C. hirsuta, as it was 175 observed with AtHFR1 in A. thaliana seedlings (Hornitschek et al, 2009). 176 177 HFR1 expression is higher in C. hirsuta than in A. thaliana seedlings 178 To test if the lack of elongation of ChWT hypocotyls in response to shade 179 was caused by higher levels of ChHFR1 expression in this species, we used 180 primer pairs that amplify HFR1 (Fig EV2A) and three housekeeping genes 181 (EF1α, SPC25, YLS8) in both species (Molina-Contreras et al, 2019). As 182 expected, expression of HFR1 was induced in shade-treated seedlings of both 183 species, in agreement with the presence of canonical PIF-binding sites (G-box, 184 CACGTG) in the HFR1 promoters (Hornitschek et al, 2009; Martinez-Garcia et 185 al, 2000) (Fig EV3A). More importantly, ChHFR1 transcript levels were always 186 higher than those of AtHFR1 during the whole period analyzed (from days 3 to 187 7) (Fig 2). Because HFR1 is part of the PIF-HFR1 regulatory module, we next 188 compared transcript levels of PIF genes in both species. PIF7 expression was 189 significantly lower in C. hirsuta than in A. thaliana in either W or W+FR during 190 the period analyzed (Fig 2). By contrast, PIF4 expression was higher in C. 191 hirsuta than in A. thaliana, whereas that of PIF5 was similar in both species (Fig 192 EV2B). Together, these results indicated that whereas HFR1 expression is 193 enhanced, that of PIF7 is globally attenuated in ChWT compared to AtWT 8 194 seedlings. As a consequence, the PIF-HFR1 transcriptional module might be 195 differently balanced in these species, with HFR1 imposing a stronger 196 suppression on the PIF7-driven hypocotyl elongation in the shade-tolerant C. 197 hirsuta seedlings. 198 199 ChHFR1 protein is more stable than AtHFR1 200 A higher specific activity of ChHFR1 compared to its orthologue AtHFR1 201 might also contribute to the role of this transcriptional cofactor in maintaining the 202 shade tolerance habit of C. hirsuta. To test this possibility, we transformed A. 203 thaliana hfr1-5 plants with constructs to express either AtHFR1 or ChHFR1 204 fused to the 3x hemagglutinin tag (3xHA). These genes were expressed under 205 the transcriptional control of the 2 kb of the AtHFR1 promoter (pAt), generating 206 hfr1>pAt:ChHFR1 and hfr1>pAt:AtHFR1 lines (Fig 3A). Fusion of pAt to the 207 GUS reporter gene resulted in GUS activity in cotyledons and roots of 208 transgenic lines, with increased levels in hypocotyls of seedlings exposed for 2- 209 4 h to W+FR (Fig EV3B). Several independent transgenic lines of each 210 construct were analyzed for hypocotyl length (Appendix Fig S1), HFR1 211 transcript levels and 3xHA-tagged protein abundance. In these lines, HFR1 212 biological activity was estimated as the difference in hypocotyl length of 213 seedlings grown under W+FR (HypW+FR) and W (HypW) (HypW+FR-HypW) 214 (Molina-Contreras et al, 2019). The potential to suppress the hypocotyl 215 elongation in shade below that of hfr1-5 seedlings would depend on the 216 transcript level of HFR1 and/or its protein levels. The hfr1>pAt:ChHFR1 lines 217 had shorter hypocotyls in shade (i.e., stronger global HFR1 activity) compared 218 to hfr1>pAt:AtHFR1 lines of similar HFR1 expression levels (Figs 3B, C, EV3C), 9 219 suggesting that total HFR1 activity was higher in hfr1>pAt:ChHFR1 than in 220 hfr1>pAt:AtHFR1 lines. However we observed much higher abundance of 221 HFR1-3xHA protein after shade exposure in hfr1>pAt:ChHFR1 lines than in 222 hfr1>pAt:AtHFR1 lines with comparable levels of HFR1 expression (Fig 3D), 223 suggesting that the ChHFR1 protein might be much more stable. Together, 224 these results point to differences in protein stability (rather than in specific 225 activity) as the main cause for the enhanced HFR1 total activity of ChHFR1 226 compared to AtHFR1 in complemented lines. 227 AtHFR1 stability is affected by light conditions. In etiolated seedlings, 228 exposure to W promotes stabilization and accumulation of AtHFR1, whereas in 229 W-grown seedlings, high intensity of W increases its abundance (Duek et al, 230 2004; Yang et al, 2005). Importantly, AtHFR1 stability has a strong impact on its 231 biological activity as overexpression of stable forms of this protein leads to 232 phenotypes resulting from enhanced HFR1 activity (Galstyan et al, 2011; Yang 233 et al, 2005). As AtHFR1 and ChHFR1 primary structures are globally similar 234 (Fig EV4A), we aimed to test if ChHFR1 stability is also light-dependent. We 235 first examined ChHFR1 protein accumulation in response to different W 236 intensities in seedlings of an A. thaliana hfr1-5 line that constitutively express 237 ChHFR1 (hfr1>35S:ChHFR1) (Fig EV4B). When grown in our normal W 238 conditions (~20 µmol m-2 s-1), these seedlings accumulated low but detectable 239 levels of ChHFR1; when transferred to higher W intensity (~100 µmol m-2 s-1), 240 ChHFR1 levels increased 10-fold (Fig EV4C). As ChHFR1 is expressed under 241 the constitutive 35S promoter, these results indicate that ChHFR1 protein 242 accumulation is induced by high W intensity, as it has been described for 243 AtHFR1 (Yang et al, 2005). This prompted us to pretreat W-grown seedlings 10 244 with 3 h of high W intensity in all our subsequent experiments to analyze 245 ChHFR1 levels. 246 Next, we exposed hfr1>pAt:ChHFR1 (line #22) and hfr1>pAt:AtHFR1 247 (line #13) seedlings to W+FR (Fig 4A). Although HFR1 expression in both lines 248 was similarly induced after 3 h of W+FR, hfr1>pAt:ChHFR1 line displayed 249 higher levels of recombinant HFR1 protein compared to hfr1>pAt:AtHFR1 line 250 after 3-6 h of W+FR exposure (Fig 4A), suggesting a higher stability of the C. 251 hirsuta protein compared to the A. thaliana orthologue. ChHFR1 protein is more 252 abundant than AtHFR1 also when transiently expressed to comparable levels in 253 Nicotiana benthamiana (tobacco) leaves (Fig 4B, C). This indicates that the 254 higher abundance of ChHFR1 is an intrinsic property of the protein that resides 255 in its primary structure. 256 AtHFR1 is known to be targeted for degradation via the 26S proteasome 257 in dark-grown seedlings. Shade also promotes AtHFR1 degradation compared 258 to non-shade treatments (Pacin et al, 2016). Hence, ChHFR1 abundance might 259 be similarly targeted, and the increased ChHFR1 protein stability might be due 260 to differences in degradation kinetics, likely by the 26S proteasome. We 261 addressed this possibility by treating tobacco leaf discs overexpressing 262 ChHFR1 and AtHFR1 with the protein synthesis inhibitor cycloheximide (CHX) 263 combined with shade (Fig 4D). This treatment resulted in a decrease in 264 ChHFR1 and AtHFR1 protein levels. However, ChHFR1 degradation was 265 significantly slower than that of AtHFR1 (Fig 4D), supporting that changes in 266 degradation kinetics likely contribute to the observed differences in stability 267 between ChHFR1 and AtHFR1. 11 268 Light- and shade-regulated degradation of AtHFR1 requires binding to 269 COP1 and the COP1 E3 ubiquitin ligase activity. Binding to COP1 results in 270 HFR1 ubiquitination, which targets HFR1 for degradation via the 26S 271 proteasome (Jang et al, 2005; Pacin et al, 2016; Yang et al, 2005). COP1- 272 interacting proteins harbor sequence-divergent Val-Pro (VP) motifs that bind the 273 COP1 WD40 domain with different affinities (Lau et al, 2019). 274 Inspection of the COP1 WD40 - AtHFR1 complex structure (Lau et al, 275 2019) revealed that sequence differences between AtHFR1 and ChHFR1 map 276 to the N-terminus of the VP peptide involved in the interaction with COP1 (Fig 277 5A). We hypothesized that these sequence variations between HFR1 species 278 may result in different COP1 binding affinities, affecting targeting and 279 subsequent degradation of the two HFR1 orthologues. We thus quantified the 280 interaction of synthetic AtHFR1 and ChHFR1 VP peptides with COP1 using 281 microscale thermophoresis (MST, see Methods). AtHFR1 bound the COP1 282 WD40 domain with a dissociation constant (kD) of ∼120 µM (Fig 5B, EV5). The 283 ChHFR1 VP peptide showed only weak binding to COP1 WD40, with a kD in the 284 millimolar range (Fig 5B, EV5). Importantly, a second putative VP sequence in 285 At/ChHFR1 showed no detectable binding, while the previously characterized A. 286 thaliana cryptochrome 1 (AtCRY1) and the human HsTRIB1 VP sequences 287 bound COP1 WD40 with a kD in the ∼1 µM range, in good agreement with 288 earlier isothermal titration calorimetry binding assays (Figs 5B, EV5) (Lau et al, 289 2019). Taken together, AtHFR1 VP peptide interacted more strongly with COP1 290 WD40, suggesting that AtHFR1 may represent a better substrate for COP1 than 291 ChHFR1. 12 292 Next we aimed to explore if these differences in COP1 affinity had an 293 impact in the subsequent degradation of AtHFR1 and ChHFR1 proteins. To test 294 this possibility, we generated chimeric HFR1 genes in which the VP region was 295 swapped, named as ChHFR1* and AtHFR1* (Fig 5C). ChHFR1* differed from 296 ChHFR1 in the VP region, that was substituted for the AtHFR1-VP1. 297 Reciprocally, AtHFR1* contained the ChHFR1-VP region. Like the wild-type 298 versions, these HFR1 derivative genes were fused to the 3xHA and placed 299 under the control of the 35S promoter (Fig 5C). When transiently expressed in 300 tobacco leaves, ChHFR1* was now less abundant than AtHFR1*, suggesting 301 that the VP regions contain enough information to determine the pattern of 302 stability of the resulting HFR1 protein (Fig 5D). Because AtHFR1-VP1 binds to 303 COP1 WD40 domain with higher affinity than ChHFR1-VP1, these results 304 indicate a negative correlation of the binding affinity to COP1 with the 305 accumulation (i.e., the higher the affinity the lower the accumulation). Hence, 306 we concluded that in the HFR1 context, a stronger binding to COP1 results in 307 lower abundance. 308 309 HFR1 interacts with PIF7 310 AtHFR1 has been shown to interact with all the members of the 311 photolabile AtPIF quartet (PIF1, PIF3, PIF4 and PIF5). Using a yeast two-hybrid 312 (Y2H) assay, we observed that AtHFR1 homodimerized, which indicated that its 313 HLH domain is functional in this assay (Fig 6A). In the same assay, AtHFR1 314 was also shown to interact with AtPIF7 (Fig 6A). These results agree with 315 recent data (Zhang et al, 2019). Because AtPIF7 is the main PIF in A. thaliana 316 promoting hypocotyl elongation in response to low R:FR (Li et al, 2012), we 13 317 aimed to address whether HFR1 also interacts genetically with PIF7. First, we 318 analyzed the genetic interaction between AtHFR1 and AtPIF7. After crossing A. 319 thaliana hfr1-5 with pif7-1 and pif7-2 mutants, we analyzed the hypocotyl 320 response of the obtained double mutants in different low R:FR conditions. As 321 expected, hfr1 hypocotyls were longer and those of pif7 mutants were shorter 322 compared to AtWT under both W+FR conditions used (Fig 6B). In W and low 323 R:FR (0.06), double pif7 hfr1 mutant seedlings behaved mostly as pif7 single 324 mutants. However, under very low R:FR (0.02), they elongated similar to AtWT 325 hypocotyls (Fig 6B). Together, these results indicate that pif7 is epistatic over 326 hfr1 under low R:FR, whereas it seems additive under very low R:FR, two 327 conditions that we speculate as mimicking proximity and canopy shade, 328 respectively (Martinez-Garcia et al, 2014). 329 To further analyze the HFR1-PIF7 interaction, we aimed to test if HFR1 330 overexpression will interfere with PIF7 overexpression and impede its effects. 331 For HFR1, we used a line overexpressing a stable but truncated form of the 332 protein (missing the N-terminal, 35S:GFP-ΔNt-HFR1, line #03) that strongly 333 inhibits shade-induced hypocotyl elongation in A. thaliana without affecting 334 other aspects of the seedling development (Galstyan et al, 2011) (Fig 6C, D). 335 For PIF7 we used two available 35S:PIF7-CFP lines (#1 and #2) (Leivar et al, 336 2008) that were almost unresponsive to W+FR (Fig 6C) and smaller and less 337 developed than the AtWT in W (Fig 6D). The inhibition of shade-induced 338 elongation observed in the 35S:PIF7-CFP lines contrasts with the positive effect 339 of growth observed by several other authors when overexpressing PIF7 fused 340 to smaller tags (Flash-tag peptide) (Li et al, 2012), likely caused by toxic or 341 squelching effects caused by high levels of the PIF7-CFP protein. In W, 14 342 35S:GFP-ΔNt-HFR1 35S:PIF7-CFP double transgenic seedlings (#1 and #2) 343 did not differ in hypocotyl length and general aspect with AtWT; interestingly they 344 did elongate clearly in low and very low R:FR (Fig 6C, D). The recovery of the 345 shade-induced hypocotyl elongation and size of the seedlings took place even 346 though HFR1 transcript levels were significantly lower than in the 35S:GFP-ΔNt- 347 HFR1 parental line. PIF7 transcript levels were not significantly different in the 348 double transgenic seedlings than in their respective parental lines (Appendix Fig 349 S2). Therefore, the inhibitory effect of PIF7-CFP overexpression appeared to be 350 counteracted by the overexpression of the truncated HFR1, further supporting 351 the genetic interaction between HFR1 and PIF7 (Fig 6C, D). 352 Altogether, these analyses support that HFR1 and PIF7 interaction is 353 important for the regulation of hypocotyl elongation in response to shade. These 354 results are consistent with HFR1 functioning as a suppressor of PIF7. 355 356 HFR1 restrains PIF activity in C. hirsuta 357 The similarity between shade-induced and warm temperature-induced 358 hypocotyl elongation (thermomorphogenesis) suggests common underlying 359 mechanisms. In A. thaliana, the increased activity of HFR1 at warm 360 temperatures was previously shown to provide an important restraint on PIF4 361 action that drives elongation growth (Foreman et al, 2011). Similarly, we 362 hypothesized that the increased activity of HFR1 in C. hirsuta might restrain PIF 363 activity more efficiently and consequently alter thermomorphogenesis (Fig 7A). 364 We analyzed this response by growing seedlings constantly at 22ºC, 28ºC, or 365 transferred from 22ºC to 28ºC after day 2 (Fig 7B). Whereas warm temperature 366 promoted hypocotyl elongation of AtWT seedlings compared to those growing at 15 367 22ºC, pifq and pif7-2 mutant seedlings were almost unresponsive to 28ºC, in 368 accordance with the role of PIF4, PIF5 and PIF7 in thermomorphogenesis 369 (Fiorucci et al, 2020; Franklin et al, 2011; Stavang et al, 2009). Unlike the hfr1-5 370 mutant, which was slightly but significantly more responsive than AtWT, A. 371 thaliana seedlings that overexpress a stable form of HFR1 (35S:GFP-ΔNt- 372 HFR1, ΔNtHFR1) were almost unresponsive to 28ºC (Fig 7C), indicating that 373 HFR1 activity impacts this PIF-dependent response. A lack of hypocotyl 374 elongation was also observed in ChWT at 28ºC, a response that was recovered 375 in the C. hirsuta chfr1 mutant seedlings (Fig 7C). These results support our 376 hypothesis that a strong suppression of PIFs by the enhanced HFR1 activity is 377 responsible for the lack of hypocotyl elongation at 28ºC of ChWT seedlings (Fig 378 7A). Together, our results suggest that the activity of the PIF-HFR1 regulatory 379 module might be a general mechanism to coordinate the hypocotyl elongation in 380 response to both W+FR exposure and 28ºC. 381 We also studied dark-induced senescence (DIS), another PIF-dependent 382 process (Fig 7D). In A. thaliana, DIS can be induced by transferring light grown 383 seedlings to complete darkness, a process in which PIF4 and PIF5 have major 384 roles (Liebsch & Keech, 2016; Sakuraba et al, 2014; Song et al, 2014). DIS 385 results in a degradation of chlorophylls, which can be quantified as markers of 386 senescence progression (Sakuraba et al, 2014; Song et al, 2014). To examine 387 DIS, we transferred light-grown AtWT, pifq and ChWT seedlings to total darkness 388 for up to 20 days (Fig 7E). After DIS was activated, AtWT seedlings became pale 389 and eventually died. After just 5 days of darkness, chlorophyll levels dropped, 390 and longer dark treatments resulted in pronounced differences between the 391 three genotypes. AtWT seedlings became visibly yellow at day 10, accompanied 16 392 by a strong reduction of chlorophyll levels that dropped to less than 10% (Fig 393 7F). DIS was delayed in 35S:GFP-ΔNt-HFR1 seedlings, supporting that a stable 394 HFR1 form can interfere with PIF activity in regulating this trait. However, DIS in 395 was not advanced in hfr1 mutants (Fig 7E). In ChWT seedlings, chlorophyll 396 levels declined more slowly and seedlings were still green after 20 days of 397 darkness, just like pifq (Fig 7E). The observed delay in the DIS in C. hirsuta was 398 not affected in chfr1 mutants, suggesting that HFR1 does not regulate this trait 399 in any of the two species. It also pointed to a reduced PIF activity as the main 400 cause for the delayed DIS in this species (Fig 7D-F). As HFR1 is very unstable, 401 particularly in dark-grown conditions (Duek et al, 2004; Yang et al, 2005), it 402 seems plausible that HFR1 does not accumulate in seedlings when transferred 403 to the dark. Despite this attenuation of PIF activity, ChWT seedlings showed an 404 etiolated phenotype similar to that of AtWT when grown in the dark, in contrast to 405 A. thaliana pifq and 35S:GFP-ΔNt-HFR1 seedlings (Fig 7G), suggesting the PIF 406 activity is high enough in C. hirsuta to induce the normal skotomorphogenic 407 development. 408 409 DISCUSSION 410 It is currently unknown whether the switch between shade avoidance and 411 tolerance strategies is an easily adjustable trait in plants. The existence of 412 closely related species with divergent strategies to acclimate to shade provides 413 a good opportunity to study the genetic and molecular basis for adapting to this 414 environmental cue. To this goal, we performed comparative analyses of the 415 hypocotyl response to shade in young seedlings of two related Brassicaceae: A. 416 thaliana and C. hirsuta. A. thaliana, a model broadly used to study the SAS 17 417 hypocotyl response, is well characterized on a physiological, genetic and 418 molecular level. C. hirsuta was previously described as a shade tolerant species 419 whose hypocotyls are unresponsive to simulated shade (Hay et al, 2014; 420 Molina-Contreras et al, 2019). Recent work showed that phyA is a major 421 contributor to the suppression of hypocotyl elongation of C. hirsuta seedlings in 422 response to shade, mainly due to the stronger phyA activity in this species 423 compared to the shade-avoider A. thaliana (Molina-Contreras et al, 2019). 424 Importantly, an enhanced phyA activity was not enough to explain the lack of 425 shade-induced hypocotyl elongation in C. hirsuta, pointing to additional 426 components that contribute to this response. Our aim to fill this gap led us to 427 uncover a role for HFR1 in this response. 428 In C. hirsuta, removal of HFR1 function resulted in a strong slender in 429 shade (sis) phenotype but milder than that of sis1 plants, deficient in the phyA 430 photoreceptor (Molina-Contreras et al, 2019), providing genetic evidence for the 431 role of HFR1 in restraining the C. hirsuta hypocotyl elongation in shade (Fig 1A, 432 B). This indicates that, like phyA, HFR1 contributes to implement the shade 433 tolerant habit in C. hirsuta seedlings. Because of the sis phenotype of chfr1 and 434 RNAi-HFR1 seedlings (Fig 1) we hypothesized that HFR1 activity is higher in C. 435 hirsuta than in A. thaliana. Consistently, transcript levels of HFR1 were 436 significantly higher in ChWT than AtWT seedlings in both W and W+FR (Fig 2). 437 Higher HFR1 levels in C. hirsuta may not be relevant in W because of the 438 expected lower abundance and activity of PIFs, but a higher pool of ChHFR1 439 ready to suppress early ChPIF action in shade could provide a fast and 440 sustained repression of the elongation response. Indeed, the shade-induced 441 expression of PIL1, YUC8 and XTR7, known to be direct PIF target genes in A. 18 442 thaliana, was strongly and rapidly enhanced in chfr1 and RNAi-HFR1 seedlings 443 (Fig 1C, D). More importantly, rapid shade-induced expression was globally 444 attenuated in ChWT compared to AtWT seedlings (Molina-Contreras et al, 2019). 445 In addition to changes in gene expression, a higher HFR1 activity in C. 446 hirsuta could also result from post-translational regulation affecting protein 447 stability. Our immunoblot analyses indicated that HFR1 proteins rapidly 448 accumulate in response to simulated shade (W+FR), likely as a consequence of 449 the strong shade-induced responsiveness of the promoter (Fig 4A). These 450 results support that regulation of HFR1 protein abundance in low R:FR occurs 451 mainly at the transcriptional level, as suggested (de Wit et al, 2016). More 452 importantly, ChHFR1 accumulates significantly more when expressed under the 453 control of a constitutive promoter either under W or W+FR (Fig 4B-D) 454 suggesting that intrinsic differences in post-translational stability between these 455 proteins play a role in their contrasting accumulation. 456 AtHFR1 protein abundance is modified post-translationally by 457 phosphorylation (Park et al, 2008) and ubiquitination in a light- and COP1- 458 dependent manner (Jang et al, 2005; Yang et al, 2005). Canopy shade 459 promotes nuclear accumulation of COP1 (Pacin et al, 2013; Pacin et al, 2016) 460 allowing it to directly interact with and polyubiquitinate AtHFR1, leading to its 461 degradation by the 26S proteasome (Huang et al, 2014; Jang et al, 2005; Yang 462 et al, 2005). AtHFR1, like ChHFR1, contains two putative COP1 binding sites 463 (VP motifs) on its N-terminal half (Fig EV4A), although only one binds COP1 464 (Figs 5A, EV5) (Lau et al, 2019). Deletion of AtHFR1 Nt leads to its stabilization 465 in the dark and light (Duek et al, 2004), and results in a stronger biological 466 activity (Galstyan et al, 2011; Jang et al, 2005; Yang et al, 2005), highlighting 19 467 the importance of the COP1-interacting domain for light regulation of AtHFR1 468 stability. Our MST binding assays showed that AtHFR1 binds to COP1 about 469 100 times more weakly than other plant COP1 substrates do (Lau et al, 2019), 470 and ChHFR1 even more weakly than AtHFR1 (Fig 5A, B). AtHFR1 and 471 ChHFR1 primary structures are similar, including the putative COP1-interacting 472 domain (Jang et al, 2005), except for the addition of 30 amino acids at the N- 473 terminal part of ChHFR1 and a 9-amino acid insertion in the C-terminal part of 474 AtHFR1 (Fig EV4A). We cannot discount the possibility that protein sequence 475 and/or structural differences other than the VP motifs could also contribute to 476 the affinity of the full-length HFR1 orthologues for COP1 and account for the 477 difference in abundance between C. hirsuta and A. thaliana HFR1. However, 478 the strong impact of swapping the VP region between ChHFR1 and AtHFR1 on 479 the abundance of the resulting HFR1* proteins (Fig 5C, D) further points to the 480 binding affinity of COP1 for its substrates as a main determinant of the stability 481 of the two HFR1 orthologues. Together, our results point to (1) the regulation of 482 affinity for COP1 as impacting HFR1 stability; and (2) HFR1 stability as a 483 mechanism to control global HFR1 activity to modulate adaptation of different 484 plant species to vegetation proximity and shade. 485 AtHFR1 was previously shown to interact with all the AtPIFQ members 486 and to form non-DNA-binding heterodimers (Fairchild et al, 2000; Hornitschek et 487 al, 2012; Shi et al, 2013). Our genetic and Y2H experiments extended the list of 488 AtHFR1 interactors to AtPIF7, the major SAS-promoting PIF (Fig 6). If ChHFR1 489 maintains similar PIF-binding abilities, the reduced expression of ChPIF7 (Fig 2) 490 might further contribute to imbalance the PIF-HFR1 module in favor of the 491 negative HFR1 activity in C. hirsuta compared to A. thaliana. Because of the 20 492 higher stability of ChHFR1 over AtHFR1 in shade (Fig 4), an even stronger 493 repression of global PIF activity in C. hirsuta would contribute to the 494 unresponsiveness of hypocotyls to shade. The attenuation of the warm 495 temperature-induced hypocotyl elongation in C. hirsuta, which is a PIF- 496 regulated process in A. thaliana (Fiorucci et al, 2020; Hayes et al, 2017; Koini et 497 al, 2009; Stavang et al, 2009) and HFR1-dependent in both species (Figs 7A- 498 C), further agrees with our proposal of an enhanced activity of HFR1 in C. 499 hirsuta compared to A. thaliana. On the other hand, the delayed DIS observed 500 in C. hirsuta, shown to be PIF-regulated in A. thaliana (Sakuraba et al, 2014; 501 Song et al, 2014) but unaffected by HFR1 in the two species analyzed (Figs 7D, 502 E), suggests that PIF activity is globally lower per se in C. hirsuta than in A. 503 thaliana. Together, our results indicate that PIF-HFR1 module is balanced 504 differently in C. hirsuta by the combination of (1) an attenuated global PIF 505 activity and PIF7 expression compared to A. thaliana, and (2) the increased 506 levels of ChHFR1 in light and shade conditions, resulting in the repression of 507 PIF-regulated processes in C. hirsuta (Fig 8). Importantly, although attenuated, 508 PIF activity in C. hirsuta is enough to provide a functional and effective etiolation 509 response (Fig 7G) for seedlings survival during germination in the dark. 510 Activity of HFR1 and phyA (Molina-Contreras et al, 2019) appears to be 511 increased in C. hirsuta to maintain unresponsiveness of hypocotyls to shade. 512 An aspect shared by both negative regulators is that their expression and/or 513 stability are strongly affected by light conditions. Expression of both PHYA and 514 HFR1 is induced by simulated shade in de-etiolated seedlings. By contrast, 515 whereas the stability of the photolabile phyA is reduced by light but enhanced 516 by shade, that of AtHFR1 is promoted by light and decreased by shade (Casal 21 517 et al, 2014; Ciolfi et al, 2013; Duek et al, 2004; Kircher et al, 1999; Martinez- 518 Garcia et al, 2014; Pacin et al, 2016; Park et al, 2008; Yang et al, 2018). 519 Although expression of both PHYA and HFR1 is higher in C. hirsuta than in A. 520 thaliana, different mechanisms might contribute to their increased activity in C. 521 hirsuta. Indeed, enhanced ChphyA repression was achieved by its stronger 522 specific intrinsic activity (Molina-Contreras et al, 2019). By contrast, enhanced 523 ChHFR1 repression was accomplished through its higher gene expression and 524 protein stability coupled with an attenuated PIF7 activity. Altogether this could 525 provide a more repressive state of the C. hirsuta PIF-HFR1 module. Because of 526 the temporal differences downregulating many of the shade marker genes 527 between phyA (observed after 4-8 hours of shade exposure) (Molina-Contreras 528 et al, 2019) and HFR1 (rapidly detected after just 1 h of shade exposure) (Fig 529 1C, D), it seems likely that ChHFR1 and ChphyA suppressor mechanisms of 530 shade response in C. hirsuta act independently, as it was reported for A. 531 thaliana (Ciolfi et al, 2013; Ortiz-Alcaide et al, 2019). Therefore, the concerted 532 activity of these two independent suppressor mechanisms seems to 533 coordinately prevent the shade-induced hypocotyl elongation in C. hirsuta. 534 Whether other shade tolerant species employ the same adaptive principles is 535 something we aim to explore in the future. 536 537 MATERIALS AND METHODS 538 Plant material and growth conditions 539 Arabidopsis thaliana hfr1-5, pif7-1, pif7-2 and pifq mutants, 35S:PIF7- 540 CFP and 35S:GFP-ΔNt-HFR1 lines (in the Col-0 background, AtWT) and 541 Cardamine hirsuta (Oxford ecotype, Ox, ChWT) plants have been described 22 542 before (Galstyan et al, 2011; Hay et al, 2014; Leivar et al, 2008; Yang et al, 543 2005). Plants were grown in the greenhouse under long-day photoperiods (16 h 544 light and 8 h dark) to produce seeds, as described (Gallemi et al, 2016; Gallemi 545 et al, 2017; Martinez-Garcia et al, 2014). For transient expression assays, 546 Nicotiana benthamiana plants were grown in the greenhouse under long-day 547 photoperiods (16 h light and 8 h dark). 548 For hypocotyl assays, seeds were surface-sterilized and sown on solid 549 growth medium without sucrose (0.5xGM–). For gene expression analyses, 550 immunoblot experiments and pigment quantification, seeds were sown on a 551 sterilized nylon membrane placed on top of the solid 0.5xGM– medium. After 552 stratification (dark at 4ºC) of 3-6 days, plates with seeds were incubated in plant 553 chambers at 22ºC under continuous white light (W) for at least 2 h to break 554 dormancy and synchronize germination (Paulisic et al, 2017; Roig-Villanova et 555 al, 2019). 556 W was emitted from cool fluorescent tubes that provided from 20 to 100 557 µmol m-2 s-1 of photosynthetically active radiation (PAR) with a red (R) to far-red 558 light (FR) ratio (R:FR) from 1.3-3.3. The different simulated shade treatments 559 were produced by supplementing W with increasing amounts of FR (W+FR). FR 560 was emitted from GreenPower LED module HF far-red (Philips), providing R:FR 561 of 0.02-0.09. Light fluence rates were measured with a Spectrosense2 meter 562 (Skye Instruments Ltd), which measures PAR (400-700 nm), and 10 nm 563 windows of R (664-674 nm) and FR (725-735 nm) regions (Martinez-Garcia et 564 al, 2014). Details of the resulting light spectra have been described before 565 (Molina-Contreras et al, 2019). 23 566 Temperature induced hypocotyl elongation assays were done by placing 567 the plates with seeds under the indicated light conditions in growth chambers at 568 22ºC or 28ºC. 569 570 Measurement of hypocotyl length 571 Hypocotyl length was measured as described (Paulisic et al, 2017; Roig- 572 Villanova et al, 2019). Experiments were repeated at least three times with 573 more than 10 seedlings per genotype and/or treatment, providing consistent 574 results. Hypocotyl measurements from the different experiments were 575 averaged. 576 577 Generation of transgenic lines, mutants and crosses 578 A. thaliana hfr1-5 plants were transformed to express AtHFR1 and 579 ChHFR1 under the promoters of 35S or AtHFR1 (pAt). The obtained lines were 580 named as hfr1>35S:ChHFR1, hfr1>pAt:AtHFR1 and hfr1>pAt:ChHFR1. 581 Transgenic RNAi-HFR1 and mutant chfr1-1 and chfr1-2 lines are in ChWT 582 background. Details of the constructs used for the generation of these lines 583 (Morineau et al, 2017) are provided as Appendix Supplementary Methods. 584 585 Gene expression analyses 586 Real-time qPCR analyses were performed using biological triplicates, as 587 indicated (Gallemi et al, 2017). Total RNA was extracted from seedlings, treated 588 as indicated, using commercial kits (Maxwell® SimplyRNA and Maxwell® RSC 589 Plant RNA Kits; www.promega.com). 2 µg of RNA was reverse-transcribed with 590 Transcriptor First Strand cDNA synthesis Kit (Roche, www.roche.com). The A. 24 591 thaliana UBIQUITIN 10 (UBQ10) was used for normalization in A. thaliana hfr1- 592 5 lines expressing AtHFR1 or ChHFR1. The ELONGATION FACTOR 1α 593 (EF1α), YELLOW-LEAF-SPECIFIC GENE 8 (YLS8) and SPC25 (AT2G39960) 594 were used for normalizing and comparing the levels of HFR1 and PIF7 between 595 A. thaliana and C. hirsuta (Molina-Contreras et al, 2019). Primers sequences for 596 qPCR analyses are provided in Appendix Table S1. 597 598 Expression of HFR1 derivatives in Nicotiana benthamiana 599 N. benthamiana plants were agroinfiltrated with A. tumefaciens strains 600 transformed with the plasmids to express the various HFR1 derivatives, and 601 kept in the greenhouse under long-day photoperiods. Samples (leaf circles 602 obtained from infiltrated areas) were taken 3 days after agroinfiltration and 603 frozen immediately. In Fig 4D, prior freezing, leaf circles were incubated in Petri 604 dishes with 10 mL of the ±CHX solution for the indicated times and conditions. 605 Each biological sample contained about 75 mg of leaf tissue from the same leaf. 606 Additional details of the preparation of the plasmids used are provided in 607 Appendix Supplementary Methods. 608 609 Protein extraction and immunoblotting analyses 610 To detect and quantify transgenic AtHFR1 and ChHFR1, proteins were 611 extracted from ~50 mg of 7-day old seedlings (grown as indicated) or from 50- 612 75 mg of agroinfiltrated N. benthamiana leaves. Plant material was frozen in 613 liquid nitrogen, ground to powder and total proteins were extracted using an 614 SDS-containing extraction buffer (1.5 µL per mg of fresh weight), as described 615 (Gallemi et al, 2017). Protein concentration was estimated using Pierce™ BCA 25 616 Protein Assay Kit (Thermo Scientific, www.thermofisher.com). Proteins (45 - 50 617 µg per lane) were resolved on a 10% SDS-PAGE gel, transferred to a PVDF 618 membrane and immunoblotted with rat monoclonal anti-HA (High Affinity, clone 619 3F10, Roche; 1:2000 dilution) or mouse monoclonal anti-GFP (monoclonal mix, 620 clones 7.1 + 13.1, Roche; 1:2000 dilution). Secondary antibodies used were 621 horseradish peroxidase (HRP) conjugated goat anti-rat (Polyclonal, A9037, 622 Sigma, www.sigmaaldrich.com; 1:5000 dilution) and HRP conjugated sheep 623 anti-mouse (Promega; 1:10000 dilution). Development of blots was carried out 624 in ChemiDocTM Touch Imaging System (Bio-Rad, www.bio-rad.com) using ECL 625 Prime Western Blotting Detection Reagent (GE Healthcare, RPN2236). Relative 626 protein levels of three to four biological replicates were quantified using Image 627 Lab™ Software (Bio-Rad, www.bio-rad.com). 628 629 Yeast 2 Hybrid (Y2H) assays 630 For Y2H assays we employed a cell mating system, as described 631 (Gallemi et al, 2017). The leucine (Leu) auxotroph YM4271a yeast strain was 632 transformed with the AD-derived constructs and the tryptophan (Trp) auxotroph 633 pJ694α strain with the BD-derived constructs. Colonies were selected on 634 synthetic defined medium (SD) lacking Leu (SD-L) or Trp (SD-W), grown in 635 liquid medium and set to mate by mixing equal volumes of transformed cells. 636 Dilutions of the mated cells were selected on SD-LW and protein interactions 637 were tested on SD-LW medium lacking histidine (SD-HLW). Details of the yeast 638 constructs used are provided as Appendix Supplementary Methods. 639 640 Expression of AtCOP1 WD40 protein and purification 26 641 AtCOP1 WD40 (residues 349-675) was expressed in Spodoptera 642 frugiperda Sf9 cells (Thermofisher) and purified as described previously (Lau et 643 al, 2019). Details of the procedure are provided as Appendix Supplementary 644 Methods. 645 646 Protein labeling and Microscale thermophoresis (MST) 647 COP1 WD40 was labeled using Monolith Protein Labeling Kit RED-NHS 648 2nd Generation Amine Reactive kit (MO-L011, Nanotemper Technologies, 649 Munich, Germany). After the TEV cleavage, COP1 WD40 was in buffer A 650 containing 2 mM β-ME, which is incompatible with the labeling procedure. 651 Therefore, prior to labeling, the buffer was exchanged using labeling buffer NHS 652 provided in the kit. In the last step, the protein was purified from the free dye, in 653 the assay buffer 20 mM Hepes pH 7.5, 150 mM NaCl, 2 mM TCEP and 0.05% 654 [v/v] Tween-20 in 12 to 15 different fractions. The absorbance of each sample 655 was measured at 280 nm and 650 nm. The Degree of Labeling (DOL) was 656 calculated using the formula provided in the manual. Aliquots containing 2000 to 657 8000 nM concentration of proteins and DOL of >0.5 were flash frozen for the 658 use in the assay. 659 Peptide solutions were freshly prepared in the assay buffer at desired 660 concentrations. For each independent replicate, 10 μL of peptide solution was 661 serially diluted 1:1 using assay buffer, in 16 PCR tubes. 10 μL of solution was 662 discarded from the 16th tube, thus each tube contained 10 μL of peptide 663 solution. Each dilution step was mixed with 10 μL of 150 nM of COP1 WD40 664 and transferred into Monolith NT.115 Premium Capillaries (MO-K025). The 665 samples were measured with the Monolith NT.115 instrument at a 25% LED 27 666 Power and 20% MST power. The resulting thermophoresis data were analyzed 667 with the MOAffinityAnalysis software (Nanotemper Technologies). 668 669 DATA AVAILABILITY 670 This study includes no data deposited in external repositories. 671 672 ACKNOWLEDGEMENTS 673 We are grateful to Peter Quail (PGEC, Albany, CA, USA) for providing 674 35S:PIF7-CFP seeds; and to Manuel Rodriguez-Concepción (CRAG) for 675 comments on the manuscript. SP received predoctoral fellowships from the 676 Agència d’Ajuts Universitaris i de Recerca (AGAUR - Generalitat de Catalunya, 677 FI program). WQ is a recipient of a predoctoral Chinese Scholarship Council 678 (CSC) fellowship. CT received a Marie Curie IEF postdoctoral contract funded 679 by the European Commission and a CRAG short-term fellowship. We also 680 acknowledge the support of the MINECO for the “Centro de Excelencia Severo 681 Ochoa 2016-2019” award SEV-2015-0533 and by the CERCA Programme / 682 Generalitat de Catalunya. FN research at the IJPB benefits from the support of 683 the LabEx Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS). Our 684 research is supported by grants from BBSRC (BB/H006974/1) and Max Planck 685 Society (core grant) to MT, and from MINECO-FEDER (BIO2017-85316-R) and 686 AGAUR (2017-SGR1211 and Xarba) to JFM-G. MH is an International 687 Research Scholar by the Howard Hughes Medical Institute. 688 689 AUTHOR CONTRIBUTIONS 28 690 JFM-G conceived the original research plan, and directed and coordinated the 691 study. SP, WQ, CT, BA and FN designed and/or carried out experiments using 692 A. thaliana and C. hirsuta. MT and FN fundamentally contributed to design the 693 constructs to obtain C. hirsuta transgenic and mutant lines. 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Front Plant Sci 875 8: 1527 876 Yang C, Xie F, Jiang Y, Li Z, Huang X, Li L (2018) Phytochrome A Negatively 877 Regulates the Shade Avoidance Response by Increasing Auxin/Indole 878 Acidic Acid Protein Stability. Dev Cell 44: 29-41 e24 879 Yang J, Lin R, Sullivan J, Hoecker U, Liu B, Xu L, Deng XW, Wang H (2005) 880 Light regulates COP1-mediated degradation of HFR1, a transcription 881 factor essential for light signaling in Arabidopsis. Plant Cell 17: 804-821 36 882 Zhang R, Yang C, Jiang Y, Li L (2019) A PIF7-CONSTANS-Centered Molecular 883 Regulatory Network Underlying Shade-Accelerated Flowering. Mol Plant 884 12: 1587-1597 885 886 FIGURE LEGENDS 887 888 Figure 1. Hypocotyls of C. hirsuta seedlings with reduced levels of 889 ChHFR1 strongly elongate in response to simulated shade. 890 A, B Hypocotyl length of ChWT, (A) RNAi-ChHFR1 transgenic and (B) chfr1 891 mutant seedlings grown under different R:FR. Seedlings were grown for 7 days 892 in continuous W (R:FR>1.5) or for 3 days in W then transferred to W 893 supplemented with increasing amounts of FR (W+FR) for 4 more days, 894 producing various R:FR. Aspect of representative 7-day old ChWT, RNAi-HFR1 895 and chfr1-1 seedlings grown in W or W+FR (R:FR, 0.02), as indicated, is shown 896 in lower panel. 897 C, D Effect of W+FR exposure on the expression of PIL1, YUC8 and XTR7 898 genes in seedlings of ChWT, (C) RNAi-HFR1 and (D) chfr1 mutant lines. 899 Expression was analyzed in 7-day old W-grown seedlings transferred to W+FR 900 (R:FR, 0.02) for 0, 1, 4, 8 and 12 h. Transcript abundance is normalized to 901 EF1α levels. 902 Data information: Values are the means ± SE of three independent biological 903 replicates relative to ChWT value at 0 h. Asterisks mark significant differences 904 (Student t-test: ** p-value <0.01; * p-value <0.05) relative to ChWT value at the 905 same time point. 906 37 907 Figure 2. Levels of HFR1 transcript are higher in C. hirsuta than A. 908 thaliana seedlings. 909 Seedlings of ChWT and AtWT were grown for 3 days in W then either kept under 910 the same conditions or transferred to W+FR (R:FR, 0.02) for the indicated 911 times. Plant material was harvested every 24 h. Transcript abundance of HFR1 912 and PIF7 was normalized to three reference genes (EF1α, SPC25, and YLS8). 913 Expression values are the means ± SE of three independent biological 914 replicates relative to the data of AtWT grown in continuous W at day 3. Asterisks 915 mark significant differences (2-way ANOVA: ** p-value <0.01, *** p-value 916 <0.001) between ChWT and AtWT when grown under W (black asterisks) or 917 W+FR (red asterisks). 918 919 Figure 3. The activity of ChHFR1 is higher than that of AtHFR1 in A. 920 thaliana seedlings. 921 A Cartoon of constructs containing ChHFR1 or AtHFR1 under the HFR1 922 promoter of A. thaliana (pAtHFR1) used to complement hfr1-5 mutant of A. 923 thaliana (At hfr1-5). 924 B Relative expression of HFR1 in seedlings of AtWT, At hfr1-5, 925 hfr1>pAt:ChHFR1 (in blue) and hfr1>pAt:AtHFR1 (in red) lines grown under 926 W+FR (R:FR, 0.02). Expression values are the means ± SE of three 927 independent biological replicates relative to the data of 7 days old AtWT. 928 Transcript abundance is normalized to UBQ10 levels. 929 C Elongation response of seedlings of the indicated lines grown for 7 days in 930 continuous W or 2 days in W then transferred for 5 days to W+FR (R:FR, 0.02). 931 The mean hypocotyl length in W (HypW) and W+FR (HypW+FR) of at least four 38 932 biological replicates was used to calculate HypW+FR-HypW. Error bars represent 933 SE. 934 D Relative HFR1 protein levels in seedlings of the indicated lines, normalized to 935 actin protein levels, are the means ± SE of three independent biological 936 replicates relative to hfr1>pAt:ChHFR1 line #22 that is taken as 1. Seedlings 937 were grown for 7 days in continuous W (~20 µmol m-2 s-1) after which they were 938 incubated for 3 h in high W (~100 µmol m-2 s-1) and transferred to W+FR (R:FR, 939 0.06) for 3 h. 940 Data information: Different letters denote significant differences (one-way 941 ANOVA with Tukey test, p-value <0.05) among means. 942 943 Figure 4. ChHFR1 and AtHFR1 proteins show different stability in shade. 944 A Expression of HFR1 and protein levels of HFR1-3xHA in seedlings of 945 hfr1>pAt:ChHFR1 (line #22) and hfr1>pAt:AtHFR1 (line #13). Seedlings were 946 grown for 7 days in continuous W (~20 µmol m-2 s-1) after which they were 947 incubated for 3 h in high W (~100 µmol m-2 s-1) and then either kept at high W or 948 transferred to W+FR (R:FR, 0.06) for 3 or 6 h, as indicated in the cartoon at the 949 top. Relative HFR1 transcript levels, normalized to UBQ10, are the means ± SE 950 of three independent biological replicates relative to hfr1>pAt:ChHFR1 #22 951 grown for 3 h under W+FR. Relative protein levels, normalized to actin, are the 952 means ± SE of three independent biological replicates relative to 953 hfr1>pAt:ChHFR1 #22. Samples were collected at data points marked in the 954 cartoon with asterisks. 955 B Cartoon of constructs containing ChHFR1 or AtHFR1 under the 35S promoter 956 used for transient expression of transgenes in N. benthamiana leaves. 39 957 C Relative HFR1 transcript levels transiently expressed in tobacco leaves, 958 normalized to the GFP, are the means ± SE of three independent biological 959 replicates (left). Relative HFR1 protein levels, normalized to the GFP levels, are 960 the means ± SE of four independent biological replicates (right). In A and C, 961 asterisks mark significant differences (Student t-test: * p-value <0.05, ** p-value 962 <0.01) between the indicated pairs. 963 D Degradation of ChHFR1 (35S:ChHFR1) and AtHFR1 (35S:AtHFR1) in 964 tobacco leaf discs treated with cycloheximide (CHX, 100 µM) for the indicated 965 times. Tobacco plants were kept under high W (~200 µmol m-2 s-1) for 3 days 966 after agroinfiltration and then leaf circles were treated with W+FR (R:FR, 0.2) 967 and CHX. Relative HFR1 protein levels (ChHFR1, blue bars; AtHFR1, red bars), 968 normalized to the GFP levels, are the means ± SE of four biological replicates 969 relative to data point 0, taken as 1 for each line. Asterisks mark significant 970 differences (2-way ANOVA: * p-value <0.05) between ChHFR1 and AtHFR1 at 971 the same time point. 972 973 Figure 5. AtHFR1 interacts more strongly than ChHFR1 with the WD40 974 domain of COP1. 975 A Overview of the COP1 WD40-AtHFR1 complex (PDB ID 6QTV). The COP1 976 WD40 domain and the AtHFR1 VP peptide are shown in surface representation 977 and colored in blue and orange, respectively. The N-terminus of HFR1 VP 978 peptide, the amino acid of which differs between AtHFR1 and ChHFR1, is 979 highlighted in magenta. 980 B Table summaries of the microscale thermophoresis binding assay (see Fig 981 EV5). The sequence of the respective synthetic peptides is indicated. 40 982 C Cartoon of constructs containing ChHFR1, AtHFR1, ChHFR1* and AtHFR1* 983 derivatives under the 35S promoter used for transient expression of transgenes 984 in N. benthamiana leaves. 985 D Relative HFR1 protein levels, normalized to the GFP levels, are the means ± 986 SE of four independent biological replicates. Asterisks mark significant 987 differences (Student t-test: * p-value <0.05, ** p-value <0.01) between the 988 indicated pairs. 989 990 Figure 6. AtHFR1 interacts with AtPIF7. 991 A Y2H growth assay showing the interaction between AtHFR1 and AtPIF7. The 992 BD- and the AD- derivative constructs used in the assay are shown on the left 993 side of the panel. SD-LW or SD-HLW refer to the selective medium (plated as 994 drops in dilutions of 1, 1:10 and 1:100) indicative of transformed cells or 995 interaction between the hybrid proteins, respectively. Truncated forms of murine 996 p53 (BD-fused) and SV40 large T-antigen (AD-fused), known to interact, were 997 used as a positive control. Empty vectors (/) were used as negative controls. 998 B, C Hypocotyl length of seedlings of AtWT, (B) pif7-1, hfr1-5, pif7-1 hfr1-5 (top 999 graph), pif7-2, hfr1-5 and pif7-2 hfr1-5 (bottom graph) mutants, and (C) 1000 transgenic 35S:GFP-ΔNt-HFR1 (35S:ΔNt-HFR1), two lines of 35S:PIF7-CFP 1001 (35S:PIF7 #1 and #2), and 35S:GFP-ΔNt-HFR1 35S:PIF7-CFP double 1002 transgenic (35S:ΔNt-HFR1 x 35S:PIF7 #1 and #2) seedlings grown under 1003 different R:FR. Seedlings were grown in W (R:FR > 1.5) for 7 days or for 2 days 1004 in W and then transferred to two W+FR treatments (R:FR 0.06 or 0.02) for 5 1005 additional days. Values of hypocotyl length are the means ± SE of three 1006 independent biological replicates (at least 10 seedlings per replica). 41 1007 D Aspect of representative 7-day-old W-grown seedlings shown in C. Scale bar 1008 is 1 cm. 1009 1010 Figure 7. C. hirsuta has an attenuated hypocotyl elongation at warm 1011 temperature and delayed dark-induced senescence (DIS). 1012 A In AtWT, PIFs promote hypocotyl elongation as a response to warm 1013 temperature (28ºC). High ChHFR1 activity is expected to inhibit this response 1014 by repressing PIFs more effectively in ChWT and attenuate hypocotyl elongation 1015 at 28ºC. 1016 B Seedlings were grown for 7 days in W at either 22ºC, 2 days at 22ºC then 1017 transferred to 28ºC for additional 5 days (22ºC > 28ºC) or for 7 days at 28ºC, as 1018 represented in the panel. 1019 C Hypocotyl length of seedlings of (left) AtWT, pifq, pif7-2, ChWT, (middle) 1020 35S:GFP-ΔNt-HFR1 (ΔNtHFR1), hfr1-5 and (right) chfr1-1 and chfr1-2 lines 1021 grown at warm temperatures. Hypocotyl lengths are the means ± SE of three 1022 biological replicates. Asterisks mark significant differences (Student t-test: * p- 1023 value <0.05, ** p-value <0.01) relative to the same genotype grown at 22ºC (left 1024 and right graphs, black asterisks), and between the indicated pairs (middle 1025 graph, red asterisks). 1026 D In AtWT, PIF-mediated DIS involves a reduction of chlorophyll levels. HFR1 1027 activity might inhibit DIS through repression of PIFs. If PIF activity is attenuated 1028 in ChWT, DIS would be delayed in this species compared to AtWT. 1029 E Seedlings were grown for 7 days in W and then transferred to total darkness 1030 for several days to induce senescence, as illustrated at the right panel. 42 1031 F Relative chlorophylls levels of (left) AtWT, pifq, ChWT, (middle) ΔNtHFR1, hfr1- 1032 5 and (right) chfr1-1 and chfr1-2 lines after DIS was promoted for the indicated 1033 time. For each genotype, values are relative to pigment levels at time 0 (7 days 1034 in W). Data are the means ± SE of four independent biological replicates. 1035 G Aspect of 4-day old dark-grown seedlings of AtWT, pifq, pif7-2, hfr1-5 and ΔNt- 1036 HFR1 (left panel), and AtWT, ChWT and chfr1-1 (right panel). 1037 1038 Figure 8. Model summarizing how PIF-HFR1 transcriptional module is 1039 differently balanced in A. thaliana and C. hirsuta. 1040 Shade (low R:FR) displaces phytochrome photoequilibrium towards the inactive 1041 form, allowing PIFs to promote the expression of shade avoidance related 1042 genes, such as HFR1. PIF transcript or/and protein levels are induced in 1043 response to warm temperatures, resulting in enhanced expression of growth- 1044 promoting genes. HFR1 abundance is also increased by warm temperature. 1045 HFR1 modulates these responses by heterodimerizing with PIFs and inhibiting 1046 their DNA-binding ability. As a result, HFR1 attenuates hypocotyl elongation of 1047 A. thaliana seedlings in response to shade or warm temperature. In C. hirsuta, 1048 higher HFR1 activity inhibits more effectively PIF action than in A. thaliana. In 1049 addition, PIF abundance is attenuated in C. hirsuta. Both changes alter the PIF- 1050 HFR1 balance in C. hirsuta, resulting in lower PIF transcriptional activity. As a 1051 consequence, shade- and warm temperature-induced hypocotyl elongation are 1052 repressed and DIS is delayed in this species. 1053 1054 EXPANDED VIEW FIGURE LEGENDS 1055 43 1056 Figure EV1. Characterization of RNAi-HFR1 and chfr1 mutants in C. 1057 hirsuta. 1058 A, B Relative expression levels of ChHFR1 gene, normalized to EF1α in ChWT, 1059 (A) two RNAi-HFR1 lines (#01 and #21) and (B) the two chfr1 mutants of C. 1060 hirsuta. Seedlings were grown for 7 days in W. Expression values are the mean 1061 ± SE of three independent biological replicates relative to ChWT. Asterisks mark 1062 significant differences (Student t-test: ** p-value <0.01) relative to ChWT. 1063 C The two identified chfr1-1 and chfr1-2 mutants have one nucleotide insertion 1064 at position 420 of the ChHFR1 ORF, which leads to a frame shift and a 1065 premature stop codon. 1066 1067 Figure EV2. Alignments of HFR1, PIF4, PIF5 and PIF7 partial DNA 1068 sequences in A. thaliana and C. hirsuta 1069 A Location of shared primers and amplicons used for comparison of expression 1070 levels by RT-qPCR between species. 1071 B Transcript abundance of PIF4 and PIF5, normalized to YLS8, SPC25 and 1072 EF1α in ChWT and AtWT grown as in Fig 2. Expression values are the means ± 1073 SE of three independent biological replicates relative to the data of AtWT grown 1074 in continuous W at day 3. Asterisks mark significant differences (2-way ANOVA: 1075 ** p-value <0.01, *** p-value <0.001) between ChWT and AtWT when grown 1076 under W (black asterisks) or W+FR (red asterisks). 1077 1078 Figure EV3. ChHFR1 and AtHFR1 complement the A. thaliana hfr1-5 1079 mutant long hypocotyl phenotype. 44 1080 A Cartoon of HFR1 promoters from A. thaliana (pAtHFR1) and C. hirsuta 1081 (pChHFR1). These promoters cover 2000 bp from the beginning of the 1082 translation start of the two HFR1 genes. The positions of G-boxes (CACGTG) 1083 are indicated with arrows. 1084 B GUS staining of representative A. thaliana seedlings expressing GUS under 1085 the pAtHFR1 (line #03). Seven-day-old W-grown seedlings were treated with 1086 W+FR for the indicated amount of time. 1087 C Correlation between HypW+FR-HypW (means ± SE of at least four biological 1088 replicates, data shown in Fig 3C) and relative levels of ChHFR1 or AtHFR1 1089 expression (means ± SE of three biological replicates, data shown in Fig 3B). 1090 The estimated regression equations and the R2 values are shown for each plot. 1091 1092 Figure EV4. ChHFR1 protein accumulates in high W. 1093 A Alignment of AtHFR1 and ChHFR1 protein sequences. Putative COP1 1094 interacting motifs, defined in AtHFR1, are highlighted with a light grey box. VP 1095 motifs are highlighted with blue letters. Amino acid sequences inside the blue 1096 line rectangles correspond to the synthetic AtHFR1, ChHFR1 and At/ChHFR1 1097 VP peptides used in the microscale thermophoresis assays (Appendix Table 1098 S3). 1099 B Cartoon representing the light treatments given to seedlings to estimate 1100 relative HFR1-3xHA levels. Seedlings grown for 7 d in low W (~20 µmol m-2 s-1, 1101 R:FR≈6.4) were first moved to high W (~100 µmol m-2 s-1, R:FR≈3.9) for 3 h and 1102 then either transferred to high W (control) or high W+FR (R:FR≈0.06) for 3 h. 1103 Seedling samples were collected at the time points indicated with asterisks. 45 1104 C Relative HFR1-3xHA protein levels of hfr1>35S:ChHFR1 seedlings (line #16) 1105 grown as indicated in B, with a representative immunoblot in a lower panel. 1106 Relative protein levels are the mean ± SE of three independent biological 1107 replicates relative to the data point of 0 h in high W (0 h W). Asterisks mark 1108 significant differences in protein levels (Student t-test: ** p-value <0.01; * p- 1109 value <0.05) relative to the 0 h W value. 1110 1111 Figure EV5. Microscale thermophoresis (MST) experimental traces and 1112 analysis. 1113 A-F Raw MST traces and analysis of AtCOP1 WD40 with different peptides in 1114 triplicates (duplicates for At/ChHFR1 VP). The concentration of AtCOP1 WD40 1115 is fixed at 0.15 μM mixed with 16 serially diluted peptide concentrations at 1:1 1116 ratio. Panels A, C and E show the normalized MST traces. The blue box area 1117 illustrates the fluorescence before activation of the infrared- (IR-) laser and red 1118 box area illustrates average fluorescence after activation of the IR-laser. 1119 Average values ± SD (error bars) were subsequently used for fluorescence 1120 normalization. kD fit displaying fraction bound as a function of ligand 1121 concentration is shown in adjacent right panels B, D and F. 1122 A Raw MST traces for AtHFR1 (in blue) and ChHFR1 (in light-brown) VP 1123 peptides. Individual concentrations that showed slight aggregation or 1124 precipitation are shown in gray and were excluded from the kD fit calculation. 1125 B Fitted data over a concentration range from 0.032 to 500 μM for AtHFR1 VP 1126 (blue dots) and 0.032 to 1000 μM for ChHFR1 VP (light-brown dots) were used 1127 to derive the corresponding dissociation constant kD. 46 1128 C Raw MST traces for At/ChHFR1 VP peptide (in orange). One concentration 1129 that showed slight precipitation or aggregation is shown in gray. A concentration 1130 range of 0.0154 to 506 μM was used for the At/ChHFR1 VP. 1131 D No kD was determined, as no binding between COP1 WD40 and the 1132 At/ChHFR1 VP peptide (orange dots) was detected. 1133 E A concentration range from 0.0076 to 250 μM for HsTRIB1 (in red) and 1134 AtCRY1 (in green) peptides was used. Raw MST traces show no aggregation or 1135 precipitation effects during this binding. One AtCRY1 VP outlier is shown in 1136 gray. 1137 F The kD for HsTRIB1 (brown dots) and AtCRY1 (green dots) VP peptides was 1138 calculated using the normalized traces. 1139 47 1 APPENDIX PDF 2 3 Adaptation to plant shade relies on rebalancing the transcriptional activity of 4 the PIF7-HFR1 regulatory module. 5 6 Sandi Paulišić, Wenting Qin, Harshul Arora Verasztó, Christiane Then, Benjamin 7 Alary, Fabien Nogue, Miltos Tsiantis, Michael Hothorn, Jaime F. Martínez-García 8 9 APPENDIX TABLE OF CONTENTS 10 1. Appendix Figure S1. 11 2. Appendix Figure S2. 12 3. Appendix Supplementary Methods. 13 4. Appendix Table S1. 14 5. Appendix Table S2. 15 6. Appendix Table S3. 16 7. Appendix References. 17 18 19 1 1 1. APPENDIX FIGURE S1. 2 W+FR W In A. thaliana hfr1-5 Line: 3 pAtHFR1 ChHFR1-3xHA hfr1>pAt:ChHFR1 4 pAtHFR1 AtHFR1-3xHA hfr1>pAt:AtHFR1 A. thaliana wild-type Col-0 At WT 5 A. thaliana hfr1-5 At hfr1-5 7 6 6 7 5 8 Hypocotyl length (mm) 4 9 3 10 2 11 1 0 12 #24 #32 #22 #11 #13 #21 At At WT hfr1-5 Col-0 hfr1-5 hfr1>pAt:ChHFR1 hfr1>pAt:ChHFR1 hfr1>pAt:AtHFR1 hfr1>pAt:AtHFR1 13 14 Appendix Figure S1. ChHFR1 and AtHFR1 complement the A. thaliana hfr1-5 15 mutant long hypocotyl phenotype. Hypocotyl length of the shown lines grown as 16 indicated in Fig 3C. Values were used to generate data on Fig 3C. 17 2 1 2. APPENDIX FIGURE S2. 2 3 ** ** 250 PIF7 4 240 HFR1 230 5 Relative expression 220 210 6 200 50 40 7 30 20 8 10 9 0 At WT Col-0 35S:ΔNt- 35S:ΔNt- 35S:PIF7 35S:PIF7 35S:PIF7 (1) 35S:PIF7 (2) 35S:ΔNt- 35S:ΔNt- 35S:ΔNt- 35S:ΔNt- #1 #2 HFR1 x HFR1 x 10 HFR1 35S:PIF7 35S:PIF7 35S:PIF7 (1) 35S:PIF7 (2) #1 #2 11 12 13 Appendix Figure S2. Relative expression levels of AtHFR1 and AtPIF7 genes 14 in transgenic lines overexpressing GFP-ΔNt-HFR1 and/or PIF7-CFP. Relative 15 expression, normalized to UBQ10, was estimated in seedlings grown for 7 days in 16 W. Expression values are the mean ± SE of three independent biological replicates 17 relative to AtWT. Asterisks mark significant differences in expression (Student t-test: 18 ** p-value <0.01; * p-value <0.05) relative to 35S:GFP-ΔNt-HFR1-GFP or 19 35S:PIF7-CFP values. 20 21 3 1 3. APPENDIX SUPPLEMENTARY METHODS. 2 3 Generation of RNAi-HFR1 plants of C. hirsuta 4 To generate an RNAi construct for silencing of the endogenous ChHFR1, a 5 fragment of 222 bp was PCR amplified using primers CTO35 + CTO36 (Appendix 6 Table S2) and cDNA of 7-day old C. hirsuta seedlings grown 1 h under W+FR. This 7 partial fragment of ChHFR1 (ptChHFR1) was cloned into pCRII-TOPO (Invitrogen, 8 www.thermofisher.com) to generate pCT17, which was confirmed by sequencing. 9 An EcoRI fragment of pCT17 was subcloned into pENTR3C vector (Invitrogen), to 10 create the Gateway entry clone pCT19 (to have ptChHFR1 flanked with attL1 and 11 attL2, attL1