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Plant Physiology Preview. Published on March 2, 2017, as DOI:10.1104/pp.17.00109 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Running head: RVE transcription factors inhibit plant growth The REVEILLE clock genes inhibit growth of juvenile and adult plants by control of cell size Authors: Jennifer A. Graya, Akiva Shalit-Kaneha, Dalena Nhu Chua, Polly Yingshan Hsua,b, Stacey L. Harmera Author affiliations: aDepartment of Plant Biology, College of Biological Sciences, University of California, Davis, CA 95616, USA One sentence summary: Myb-like transcription factors important for circadian clock function also inhibit cell expansion, affecting size of both juvenile and adult plants. FOOTNOTES Financial Source: This work was supported by the National Institutes of Health (grant no. GM069418 to SLH). Present address: bDepartment of Biology Duke University Durham, North Carolina, 27708, USA Corresponding Author: [email protected] Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. Copyright 2017 by the American Society of Plant Biologists 37 38 39 ABSTRACT 40 and fitness in a constantly changing environment. In Arabidopsis thaliana, the 41 clock is comprised of numerous regulatory feedback loops in which REVEILLE8 42 (RVE8) and its homologs RVE4 and RVE6 act in a partially redundant manner to 43 promote clock pace. Here, we report that the remaining members of the RVE8 44 clade, RVE3 and RVE5, play only minor roles in regulation of clock function. 45 However, we find that RVE8 clade proteins have unexpected functions in 46 modulation of light input to the clock and control of plant growth at multiple 47 stages of development. In seedlings, these proteins repress hypocotyl elongation 48 in a day-length and sucrose dependent manner. Strikingly, adult rve4 6 8 and 49 rve3 4 5 6 8 mutants are much larger than wild type, with both increased leaf 50 area and biomass. This size phenotype is associated with a faster growth rate 51 and larger cell size and is not simply due to a delay in the transition to flowering. 52 Gene expression and epistasis analysis reveal that the growth phenotypes of rve 53 mutants are due to misregulation of PHYTOCHROME INTERACTING FACTOR4 54 (PIF4) and PIF5 expression. Our results shows that even small changes in PIF 55 gene expression caused by perturbation of clock gene function can have large 56 effects on the growth of adult plants. The circadian clock is a complex regulatory network that enhances plant growth 57 2 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 58 59 60 INTRODUCTION Circadian rhythms are endogenous, biological rhythms that oscillate with 61 an approximately 24-hour period. These rhythms are observed in many 62 organisms throughout nature (Dunlap, 1999; Harmer et al., 2001; Harmer, 2009), 63 and are particularly vital for plants due to their sessile nature. Numerous studies 64 in plants have indicated that an impaired circadian oscillator can contribute to 65 diminished growth, impaired defense against herbivores and pathogens, and 66 even reduced fitness (Green et al., 2002; Dodd et al., 2005; Yerushalmi et al., 67 2011; Goodspeed et al., 2012; Ruts et al., 2012). 68 The molecular network of the Arabidopsis circadian clock is primarily 69 comprised of transcription factors that regulate each other’s as well as their own 70 expression (Hsu and Harmer, 2014; McClung, 2014). Two homologous single 71 MYB-domain transcription factors CIRCADIAN CLOCK ASSOCIATED1 (CCA1) 72 and ELONGATED HYPOCOTYL (LHY) (Schaffer et al., 1998; Wang and Tobin, 73 1998) have a circadian pattern of transcript peaking at dawn, and loss-of-function 74 mutants have a short free-running period in continuous light (Green and Tobin, 75 1999; Alabadi et al., 2002; Mizoguchi et al., 2002). CCA1 and LHY comprise a 76 negative feedback loop with TIMING OF CAB EXPRESSION (TOC1) in which 77 they bind to the evening element (EE) promoter motif of this and other evening- 78 phased clock genes to repress expression during the day (Alabadi et al., 2002; 79 Harmer and Kay, 2005; Nagel et al., 2015). TOC1 in turn regulates CCA1 and 80 LHY by repressing their expression in a time-of-day manner (Gendron et al., 81 2012; Huang et al., 2012; Pokhilko et al., 2012). 3 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 82 More recent work has characterized the role of the transcription factor 83 REVEILLE8 (RVE8), a homolog of CCA1 and LHY, in the circadian network 84 (Farinas and Mas, 2011; Rawat et al., 2011). These transcription factors are 85 deemed members of the core oscillator, as mutation of these genes results in a 86 free-running clock period that is either longer or shorter than wild type. 87 Specifically, while cca1 and lhy mutants have a short period, the rve8 loss-of- 88 function mutant has a long free-running period (Farinas and Mas, 2011; Rawat et 89 al., 2011). Although RVE8 binds to similar evening-phased gene targets as 90 CCA1 and LHY, this transcription factor activates instead of represses target 91 gene expression (Farinas and Mas, 2011; Rawat et al., 2011; Hsu et al., 2013), 92 explaining the opposite period phenotypes of these mutants. RVE8 forms an 93 additional feedback loop in the clock, in which it positively regulates the evening- 94 expressed transcription factor PSEUDO RESPONSE REGULATOR5 (PRR5), 95 whose protein in turn represses expression of RVE8 (Rawat et al., 2011). 96 RVE8 is one of eleven homologous MYB-like transcription factors in 97 Arabidopsis (Rawat et al., 2009). RVE8 acts in a partially redundant manner with 98 REVEILLE4 (RVE4) and REVEILLE6 (RVE6), two closely related genes, to 99 promote clock pace (Hsu et al., 2013). Two remaining members of the RVE8 100 clade, REVEILLE3 (RVE3) and REVEILLE5 (RVE5), were found to associate 101 with the EE promoter motif both in vitro and in vivo (Gong et al., 2008; Rawat et 102 al., 2011). However, their role in the clock has not been previously characterized. 103 Additional key clock proteins include the transcription factors EARLY 104 FLOWERING3 (ELF3), EARLY FLOWERING4 (ELF4), and LUX ARRYTHMO 4 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 105 (LUX), which together comprise the tripartite evening complex (EC) (Nusinow et 106 al., 2011). Each component of the EC is necessary for its function, as the lux, 107 elf3, and elf4 single mutants are all arrhythmic in continuous light (Hicks et al., 108 2001; Doyle et al., 2002; Hazen et al., 2005). The EC forms in the early evening 109 and acts to regulate numerous downstream genes as well as expression of other 110 clock components (Dixon et al., 2011; Helfer et al., 2011; Nusinow et al., 2011; 111 Mizuno et al., 2014). 112 Approximately one-third of the Arabidopsis transcriptome is under 113 circadian control (Covington et al., 2008), and consistent with this many aspects 114 of plant growth and development are influenced by the circadian clock (Farre, 115 2012; Song et al., 2013). Hypocotyl and root elongation and leaf growth undergo 116 daily rhythms, and the phases of peak growth rates are shifted but not abolished 117 in constant environment conditions (Nozue et al., 2007; Poire et al., 2010; Iijima 118 and Matsushita, 2011; Yazdanbakhsh et al., 2011). 119 The clock is vital for growth rhythms in multiple organs, as the leaves and 120 roots of arrhythmic circadian mutants CCA1-ox and prr9 7 5 exhibit perturbed 121 growth (Ruts et al., 2012). The clock also modulates the timing of the transition 122 from vegetative to reproductive growth by controlling rhythmic oscillations of key 123 transcriptional regulators that promote flowering (Song et al., 2013). Flowering 124 time is also regulated by light signaling pathways, which modulate stability of 125 clock-regulated proteins to control their activity in a day length-dependent 126 manner (Song et al., 2013). 5 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 127 Many other developmental pathways are regulated not only by the clock 128 but also by environmental stimuli. Hypocotyl elongation in Arabidopsis is 129 influenced by changes in light and temperature as well as by the clock (Nozue et 130 al., 2007; Nomoto et al., 2012). Two key regulators of hypocotyl elongation are 131 the basic helix-loop-helix (bHLH) transcription factors PHYTOCHROME 132 INTERACTING FACTOR 4 (PIF4) and PIF5 (Leivar et al., 2008; Lorrain et al., 133 2008; Hornitschek et al., 2012; Leivar et al., 2012). The differential patterns of 134 hypocotyl growth in constant light and in light-dark cycles are due to the EC- 135 mediated circadian regulation of PIF4 and PIF5 expression (Nusinow et al., 136 2011) combined with the post-translational regulation of PIF protein stability by 137 red light-activated phytochrome (Nozue et al., 2007; Lorrain et al., 2008). In 138 addition, PIF4 and PIF5 influence the rhythmic growth of leaves (Dornbusch et al., 139 2014). PIF proteins have also been shown to act in many other response 140 pathways, such as auxin (Franklin et al., 2011; Kunihiro et al., 2011; Nozue et al., 141 2011; Hornitschek et al., 2012; Sun et al., 2012), gibberellin (de Lucas et al., 142 2008), high temperature (Koini et al., 2009; Stavang et al., 2009) and sucrose 143 (Liu et al., 2011; Stewart et al., 2011). 144 Although the roles of the circadian clock and PIFs in regulation of 145 hypocotyl elongation in Arabidopsis have been thoroughly examined, clock 146 modulation of growth in adult plants is much less understood. Here, we 147 demonstrate that loss of RVE function alters PIF gene expression. Surprisingly, 148 this perturbation causes greatly enhanced growth at both juvenile and mature 149 stages of development, resulting in increased cell size and greater aerial 6 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 150 biomass in rve mutants than in wild-type controls. These data provide a rare 151 example in which alteration of circadian clock function promotes instead of 152 inhibits plant growth, potentially opening up novel agricultural applications. 153 154 155 RESULTS 156 The RVEs act in a partially redundant manner to promote the pace of the 157 clock 158 159 Previous work has demonstrated that multiple members of the RVE family of 160 transcription factors help control the pace of the circadian clock. Whereas 161 mutations in the MYB-like transcription factors CCA1 and LHY shorten clock 162 pace (Green and Tobin, 1999; Alabadi et al., 2002; Mizoguchi et al., 2002), 163 mutations in their homolog RVE8 and two closely related genes, RVE4 and 164 RVE6, lengthen circadian period (Farinas and Mas, 2011; Rawat et al., 2011; 165 Hsu et al., 2013). 166 RVE3 and RVE5 are the remaining members of the RVE8 clade. To 167 investigate their role in clock function, we first identified plants with T-DNA 168 insertions within these two genes. Neither RVE3 nor RVE5 was detectably 169 expressed in the rve3-1 (SALK_001480C) or rve5-1 (SAIL_769_A09) mutants, 170 respectively (Supplemental Fig. 1). As neither single mutant has a detectable 171 circadian clock phenotype (data not shown), we examined the free-running 172 circadian period in rve3-1 rve5-1 (rve3 5) mutants harboring a clock-regulated 173 luciferase reporter construct (CCR2::LUC). In contrast to the long-period 174 phenotype of the rve4-1 rve6-1 rve8-1 (rve4 6 8) triple mutant (Hsu et al., 2013), 7 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 175 the rve3 5 double mutant displays a slightly shorter free-running period than wild 176 type (Col = 23.95±0.09; rve4 6 8 = 27.83±0.7; rve3 5 = 23.63±0.04) (Fig. 1). 177 Although the period of the rve3 5 double mutant is statistically significantly 178 different than wild type, the subtle difference in clock pace compared to wild type 179 suggests that RVE3 and RVE5 don’t play a major role in clock function. 180 We next examined whether RVE3 and RVE5 play a partially redundant 181 role in the clock with their homologs RVE4, RVE6, and RVE8. In contrast to the 182 slightly shorter period seen in rve3 5 double mutants when compared to wild type, 183 plants mutant for all five RVE8-related transcription factors (rve3 4 5 6 8) have a 184 slightly longer free-running period than the rve4 6 8 triple mutant (rve4 6 8 = 185 27.83±0.7; rve3 4 5 6 8 = 28.33±0.09). The difference in period between the rve 186 triple and quintuple mutants is small, although statistically significant (Fig. 1). 187 This result further indicates a modest role for RVE3 and RVE5 in the control of 188 clock pace. 189 We next examined whether the sensitivity of the circadian clock to light is 190 altered in rve mutants. For this, we examined the effects of different fluence 191 rates of monochromatic red and blue light on free-running circadian period. 192 Mutations in light-signaling components have previously been shown to alter the 193 relationship between free-running period and fluence rate (Somers et al., 1998; 194 Devlin and Kay, 2000). We found that the shortening of period in response to 195 high fluence rates of monochromatic red light seen in Col and rve3 5 is less 196 pronounced in rve4 6 8 and rve3 4 5 6 8 (Fig. 1), suggesting reduced sensitivity 197 of the clock to red light in these mutants. Surprisingly, rve triple and quintuple 8 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 198 mutants in monochromatic blue light have longer free-running periods at higher 199 than at lower fluence rates, the opposite of the trend seen in wild type (Fig. 1). 200 Thus clock responsiveness to blue light is fundamentally different in the rve 201 mutants than in control plants. Together, these data indicate that the RVEs affect 202 both red and blue light signaling to the circadian clock, but in a distinct manner. 203 204 Flowering time is modestly delayed in the rve mutants 205 The circadian clock plays an important role in the day-length dependent control 206 of the transition from vegetative to reproductive growth (Song et al., 2013). To 207 determine whether the RVEs play an important role in the regulation of flowering 208 time, we examined both leaf number and days to bolting in Col, rve4 6 8, and 209 rve3 4 5 6 8 mutants grown in long-day (LD) (16 hr light:8 hr dark) conditions. In 210 our growth conditions, wild-type plants bolt after approximately 24 days, while 211 flowering time in the rve4 6 8 triple mutant is delayed by an average of three 212 days. The rve3 4 5 6 8 quintuple mutant flowers significantly later than the triple 213 mutant, at approximately 29 days (Fig. 2). These slight delays in the timing of 214 the transition to flowering are mirrored by changes in leaf number at bolting, with 215 the rve4 6 8 mutant generating an average of two more and rve3 4 5 6 8 an 216 average of three more rosette leaves than wild type (Fig. 2). Thus the RVE8 217 clade genes play a minor role in the control of flowering time. 218 219 Day-length dependence of RVE mutant hypocotyl phenotypes 9 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 220 The circadian clock drives rhythmic oscillations of hypocotyl elongation in 221 Arabidopsis, and mutations in many clock genes results in seedlings with 222 perturbed hypocotyl growth patterns (Farre, 2012). Since RVE8 clade genes 223 affect clock pace, we investigated the hypocotyl phenotypes of the rve triple and 224 rve quintuple mutants at various intensities of white light and under different day 225 lengths. 226 When grown in short-day (SD) conditions (8 hr light : 16 hr dark), both 227 wild-type and rve mutant plants show inhibition of hypocotyl elongation in 228 response to increasing fluence rates of light (Fig. 3). However, the hypocotyl 229 lengths of the rve triple and rve quintuple mutants are significantly longer than 230 wild type at all fluence rates tested, with the maximal difference between the 231 mutants and wild type occurring at the highest fluence rate tested (100 μmol m-2 232 s-1 white light) (Fig. 3). In LD conditions, hypocotyl lengths of the rve triple and 233 quintuple mutants are significantly different from wild type at mid-to-high light 234 intensities (7.5-50 μmol m-2 s-1), but not at the lowest (0.6 μmol m-2 s-1) or highest 235 (100 μmol m-2 s-1) fluence rates tested (Fig. 3). Similar to LD conditions, 236 hypocotyls of the rve4 6 8 and rve3 4 5 6 8 mutants are longer than wild-type at 237 mid-range intensities (7.5-25 μmol m-2 s-1) of continuous white light (LL), but are 238 no different from the control at very low (0.6 μmol m-2 s-1) or high (50-80 μmol m-2 239 s-1) fluence rates (Fig. 3). The hypocotyl lengths of rve4 6 8 and rve3 4 5 6 8 are 240 not significantly different from each other under any of the conditions tested. 241 Taken together, these data indicate that RVE4, RVE6, and RVE8 contribute to 242 regulation of hypocotyl length in a fluence-rate and day-length dependent 10 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 243 manner, and that RVE3 and RVE5 do not significantly contribute to this 244 phenotype in the conditions tested. 245 246 The RVEs repress growth in adult plants 247 Since we found that the RVEs influence growth at the seedling stage, we 248 investigated if this is also true at later stages of development by measuring the 249 rosette diameter of six-week old plants grown in LD conditions. We found that 250 both rve4 6 8 and rve3 4 5 6 8 are approximately 1.5 fold larger in diameter than 251 wild type (Fig. 4). To determine if this rosette size difference is associated with 252 increased biomass, we measured the dry weight of aerial tissues of plants 253 harvested at six weeks of age. Strikingly, the rve triple and rve quintuple mutants 254 exhibit an approximately 30% increase in aerial dry weight compared to wild type 255 (Fig. 4). There is no significant difference in aerial biomass between the rve 256 triple and rve quintuple mutants, indicating that RVE3 and RVE5 do not 257 significantly contribute to growth regulation in LD conditions (Fig. 4). 258 To determine whether the extra rosette leaves made by the rve mutants 259 before the transition to flowering (Fig. 2) could account for the increased growth 260 of these mutants, we assessed rosette diameter by measuring the distance 261 between leaves 8 and 9, which were produced by all genotypes. As measured 262 this way, the rosette diameter of rve4 6 8 and rve3 4 5 6 8 mutants is 263 approximately 25% larger than the wild-type controls (Fig. 4), demonstrating a 264 growth phenotype in these plants independent of the flowering time phenotype. 11 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 265 We next measured blade area, blade length, and petiole length of leaves 8 266 and 9 in the rve mutants to determine what aspects of leaf development are 267 affected. Blade area of both leaves 8 and 9 is increased approximately 20-30% 268 in the triple and quintuple rve mutants compared to wild type (Fig. 4). Average 269 blade length of leaf 8 is also increased by about 20%, being ~7mm longer (p- 270 value = 6.4x10-4) in the rve triple and ~9mm longer (p-value = 4.8x10-5) in the rve 271 quintuple mutant relative to wild type. Likewise, blade length in leaf 9 is 272 increased by ~8mm (p-value = 1.6x10-4) and ~10mm (p-value = 5.6x10-6) in the 273 rve triple and quintuple mutants, respectively, compared to wild type (Fig. 4). 274 The difference in average petiole length is similar between the two mutants and 275 wild type, with an increase in petiole length of ~7-8mm (p-value < 5x10-4) in rve4 276 6 8 and ~9-10mm (p-value < 5x10-3) in rve3 4 5 6 8 relative to controls (Fig. 4). 277 Thus many aspects of leaf growth are enhanced in rve mutants. 278 We also measured these growth parameters at three weeks of age, before 279 the transition to bolting in both wild-type and rve mutant plants. Similar to later 280 stages of growth, rve triple and rve quintuple mutants both display significant 281 increases in blade area, blade length, and petiole length compared to wild type 282 when grown in LD (Supplemental Fig. 2). In contrast, blade area and length are 283 not significantly different between the rve mutants and wild type grown in SD, 284 although petiole length in the mutants is greater than in the controls in both SD 285 and LD. 286 and rve3 4 5 6 8. However, the rve3 4 5 6 8 mutant has a significantly longer 287 petiole than rve4 6 8 in SD but not LD (Supplemental Fig. 2). This suggests that Most leaf parameters are not significantly different between rve4 6 8 12 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 288 the roles of RVE3 and RVE5 are influenced by environmental factors. Together, 289 our data show that members of the RVE8 clade are inhibitors of plant growth at 290 both the seedling and adult stages of development and across different 291 environmental conditions. 292 293 294 Growth rate is accelerated in rve mutants To better understand why rve mutants are larger than wild type, we 295 investigated if the rve leaves are expanding at a faster rate or growing for a 296 longer period of time than in control plants. To avoid the confounding effects of 297 differences in flowering time, we measured rosette diameter as the distance 298 between leaves 8 and 9, which are produced by all genotypes. Significant 299 differences in rosette diameter are evident for both the rve triple and quintuple 300 mutants throughout all stages of growth, long before the transition to flowering 301 (Fig. 5). Both mutant and wild-type plants ceased rosette expansion 302 approximately 30 days after germination, indicating that the larger size of rve 303 mutants is due to a faster growth rate rather than an extended period of leaf 304 growth. These data demonstrate that the larger rosette size in adult rve mutants 305 is partly caused by an enhanced growth rate of individual leaves and not just a 306 delay in flowering time. 307 We examined whether this increased growth rate is due to larger average 308 cell size or more cells per organ in the mutant compared to control. We found 309 that the average area of mesophyll cells in the leaves of six-week old rve4 6 8 310 mutants is increased by approximately 30% compared to wild type (Col = 13 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 311 7.01x103 μm2; rve4 6 8 = 9.27x103 μm2; p-value = 5.3x10-36) (Fig. 5). This 312 increase in cell size is similar to the increase in total blade area in the mutant (Fig. 313 4), suggesting that larger average cell size is primarily responsible for the 314 increased leaf size in rve mutants. 315 316 rve mutants display an enhanced sensitivity to sucrose 317 Sugars are well-known regulators of plant growth (Lastdrager et al., 2014). We 318 therefore investigated whether rve mutants have an altered response to sucrose. 319 We grew rve4 6 8, rve3 4 5 6 8, and Col seedlings on various concentrations of 320 sucrose in 12 hr light:12 hr dark conditions and measured hypocotyl lengths. 321 The maximal enhancement of hypocotyl elongation for all genotypes occurred at 322 2% sucrose, and diminished at higher concentrations (Fig. 6). We next examined 323 responsiveness to sucrose by comparing the degree of sucrose-mediated growth 324 enhancement relative to the no-sucrose controls for each genotype (Fig. 6). 325 Notably, both rve triple and quintuple mutants displayed an enhanced response 326 to sucrose compared to Col at concentration of sucrose between 0.5 and 3% (Fig. 327 6). The quintuple rve mutant demonstrated an even greater response to sucrose 328 than the rve triple at 2% and 6% sucrose (Fig. 6), suggesting that RVE3, RVE5, 329 or both act in a partially redundant manner with RVE4, RVE6, and RVE8 to 330 repress sucrose growth responses. 331 332 Expression of PIF transcription factors is altered in rve mutants 14 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 333 Previous studies have revealed a role for the clock-regulated transcription factors 334 PIF4 and PIF5 in the promotion of hypocotyl elongation in response to sucrose 335 (Liu et al., 2011; Stewart et al., 2011). Since the rve mutants are hyper- 336 responsive to sucrose (Fig. 6), we investigated whether PIF expression patterns 337 are altered in rve mutants in either constant light and/or light/dark cycles. First, 338 wild-type and mutant seedlings were grown in light-dark cycles and then 339 transferred to constant light and temperature conditions at dawn (Zeitgeber Time 340 0; ZT 0). After 32 hours in constant conditions, plants were harvested at three- 341 hour intervals and gene expression was determined using qRT-PCR. As 342 expected given the long period phenotype of rve4 6 8 mutants (Fig. 1), the 343 circadian patterns of expression of both PIF4 and PIF5 show obvious phase 344 delays (Fig. 7 and Supplemental Fig. 3). Notably, the maximal abundance of 345 PIF4 and PIF5 is not appreciably different from wild type in these conditions. 346 However, the waveforms of PIF gene expression are altered in rve4 6 8 such that 347 trough levels of PIF gene expression are increased during the subjective night 348 (ZT 36 – 48) and the peaks of gene expression are broader (Fig. 7). This 349 suggests that overall PIF expression levels may be higher in the mutant than in 350 wild type. Indeed, quantification of PIF4 and PIF5 expression across the entire 351 LL time series, determined by computing the area under the curves using natural 352 cubic spline interpolation, reveals significantly greater expression levels in the rve 353 mutant compared to wild type (Fig. 7). These data suggest that the growth 354 phenotypes of rve mutants maintained in constant light (Fig. 3) might be due to 355 increased overall levels of PIF gene expression. 15 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 356 We next examined PIF expression in seedlings grown in LD and SD 357 cycles (Fig. 7 and Supplemental Fig. 3). Similar to the LL data, we saw no 358 difference in peak levels of PIF4 and PIF5 expression but did see an increase in 359 trough levels. Quantification of PIF gene expression across the entire LD and 360 SD time courses did not reveal significant differences between the rve mutant 361 and wild type (data not shown). However, since PIF4 and PIF5 proteins are 362 destabilized in the light (Nozue et al., 2007; Lorrain et al., 2008), differences in 363 transcript levels at night are likely to be of greatest biological significance. We 364 therefore calculated PIF mRNA expression levels integrated across only the dark 365 portions of the LD and SD time series. We found significantly higher nighttime 366 levels of PIF4 and PIF5 expression in LD in the rve mutant compared to wild type. 367 In SD, the differences in PIF expression between the genotypes were smaller, 368 reaching statistical significance for PIF4 but not PIF5 (Fig. 7). These data 369 suggest that the enhanced growth phenotypes of rve mutants in light/dark cycles 370 (Fig. 3, Fig. 4, and Fig. 5) might be due to increased levels of PIF expression 371 during the night. 372 Expression of PIF4 and PIF5 is directly repressed by multiple clock 373 components, including the EC (Nusinow et al., 2011), PRR5 (Kunihiro et al., 374 2011; Nakamichi et al., 2012), and TOC1 (Huang et al., 2012). Since we 375 previously reported that RVE8 directly induces expression of PRR5, TOC1, and 376 LUX, a member of the EC, (Hsu et al., 2013), we examined transcript levels of 377 these genes in rve4 6 8 mutants and wild-type plants grown in LL, LD, and SD 378 (Supplemental Fig. 4 and 5). As expected, in LL all three evening genes display 16 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 379 a phase delay in the rve mutant (Supplemental Fig. 4 and 5). Both PRR5 and 380 TOC1 have significantly decreased expression levels in the rve mutant when 381 values are integrated across the LL time series (Supplemental Fig. 4). However, 382 only PRR5 shows an obvious decrease in peak levels of expression in rve4 6 8 383 (Supplemental Fig. 4). We did not observe a significant difference in LUX 384 expression levels in rve4 6 8 (Supplemental Fig. 4). Thus the increased overall 385 increased levels of PIF4 and PIF5 expression in the rve4 6 8 mutant in LL (Fig. 7) 386 may be due to lower levels of PRR5 and/or TOC1. 387 We next examined PRR5, TOC1, and LUX expression in LD and SD. 388 Especially in LD, there are clear differences in the waveforms and phases of 389 expression of these evening genes in rve4 6 8 (Supplemental Fig. 4 and Fig. 5). 390 However, quantification of their expression did not reveal significant decreases in 391 expression in the rve mutant compared to wild type when assessed across the 392 entire LD and SD time series (Supplemental Fig. 4). Surprisingly, overall TOC1 393 expression in LD is slightly elevated in the mutant compared to wild type 394 (Supplemental Fig. 4). These data suggest that the night-specific, but not overall, 395 increase in PIF4 and PIF5 expression in rve4 6 8 in LD and SD (Fig. 7) may be 396 due to changes in phase of evening-gene expression but are not attributable to 397 changes in their overall expression levels. Together, these data suggest the 398 RVEs have a key role in setting the appropriate phase of gene expression in 399 light/dark cycles and overall target gene expression levels in constant 400 environmental conditions. 401 17 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 402 The PIFs are epistatic to the RVEs at multiple growth stages 403 The increased levels of PIF4 and PIF5 transcripts in rve4 6 8 overall in LL and 404 specifically at night in LD and SD (Fig. 7) suggest increased PIF activity might 405 contribute to the enhanced growth phenotypes in this mutant. We therefore 406 generated a quintuple rve4 6 8 pif 4 5 mutant to investigate whether the PIFs are 407 required for the large size phenotype of the rve mutants. As expected (Nozue et 408 al., 2007; Lorrain et al., 2008), pif4 5 double mutants have shorter hypocotyls 409 than wild type (Fig. 8). Notably, while the hypocotyls of the rve4 6 8 mutant are 410 almost 50% longer than those of wild type in LL and LD, the hypocotyls of the 411 quintuple rve4 6 8 pif4 5 mutant are almost the same length as those of the pif4 5 412 mutant in these conditions (Fig. 8). A similar suppression of the rve hypocotyl 413 phenotype is observed in plants grown in 12 hr light:12 hr dark conditions, both 414 with and without sucrose in the growth media (Supplemental Fig. 6). 415 Interestingly, we did not observe epistasis when seedlings were maintained in 416 SD (Fig. 8). Loss of rve4 6 8 in these conditions caused an approximately 50% 417 increase in hypocotyl length both in the Col and the pif4 5 background. Thus 418 when seedlings are grown in LL, LD, or 12:12 L:D conditions, PIF4 and/or PIF5 419 are largely responsible for the effects of RVE4, RVE6, and RVE8 on hypocotyl 420 growth; however, a different mechanism appears to cause the long hypocotyls of 421 rve4 6 8 seedlings grown in SD. 422 We next sought to determine whether the PIFs are responsible for the 423 large adult rosette phenotype in rve mutants. We therefore examined rosette 424 and leaf size phenotypes of long-day grown quintuple rve4 6 8 pif4 5 mutants 18 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 425 and the appropriate controls at three weeks of age. Strikingly, the pif4 5 and 426 rve4 6 8 pif4 5 mutants have very similar rosette diameters (Fig. 8). Consistent 427 with this overall appearance, the blade areas, blade lengths, and petiole lengths 428 of these mutants are not significantly different from each other (Fig. 8). Thus the 429 pif4 5 mutations suppress both the seedling and adult growth phenotypes of rve4 430 6 8 mutants, indicating a role for the RVEs in modulation of PIF gene expression 431 and the control of growth throughout plant development. 432 433 434 435 436 DISCUSSION 437 CCA1/LHY/RVE transcription factors in the plant circadian network (Wang and 438 Tobin, 1998; Green and Tobin, 1999, 2002; Rawat et al., 2009; Farinas and Mas, 439 2011; Rawat et al., 2011; Hsu et al., 2013) Remarkably, different members of this 440 small gene family play contrasting roles: while CCA1 and LHY lengthen (Green 441 and Tobin, 1999; Alabadi et al., 2002; Mizoguchi et al., 2002) and RVE4, RVE6, 442 and RVE8 shorten circadian period (Farinas and Mas, 2011; Rawat et al., 2011; 443 Hsu et al., 2013), RVE1 does not affect clock pace but instead mediates 444 circadian regulation of the auxin pathway (Rawat et al., 2009). With this range of 445 functions in mind, we phenotyped plants mutant for RVE3 and RVE5, the last two 446 uncharacterized members of this Myb-like transcription factor family. 447 Numerous previous studies have highlighted the importance of the RVE8 and its close homologs RVE4 and RVE6 were previously shown to 448 control circadian clock pace in a partially redundant manner (Hsu et al., 2013). 449 We now show that the two remaining homologs of the RVE8 clade, RVE3 and 19 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 450 RVE5, play only a minor role in controlling clock pace. The rve3 5 double mutant 451 has a free-running circadian period very similar to wild type (Fig. 1). Additionally, 452 although the free running period of the rve3 4 5 6 8 mutant is slightly longer than 453 that of the triple rve4 6 8 mutant in red plus blue light, the period differences are 454 small (Fig. 1). Taken together, our results suggest that RVE3 and RVE5 only 455 promote clock pace in conjunction with RVE4, RVE6, and RVE8, and that their 456 role the clock is subtle. It is important to note that the rve6 allele used in this 457 study is not a complete knockout, retaining approximately ∼30% of normal RVE6 458 transcript levels (Hsu et al., 2013). Further studies with a null rve6 allele would 459 reveal if complete loss of all RVE8 clade members affects rhythmic robustness in 460 addition to causing a long period phenotype. 461 In addition to longer free-running rhythms, light regulation of clock function 462 is altered in plants mutant for the RVEs (Fig. 1). Typically, the free-running 463 circadian period of diurnal organisms is shorter at higher light intensities and 464 longer at lower light intensities (Aschoff, 1960; Somers et al., 1998). In contrast 465 to wild-type Arabidopsis, the circadian pace in rve triple and quintuple mutants is 466 relatively insensitive to increasing fluence rates of red light (Fig. 1). Similar 467 insensitivity to fluence rate is observed in plants expressing a constitutively 468 activated phytochrome photoreceptor (Jones et al., 2015), suggesting that there 469 are changes in red light input to the clock in rve mutants. Surprisingly, increasing 470 intensities of continuous blue light results in progressive lengthening of free- 471 running period in the triple and quintuple rve mutants (Fig. 1). A similar 472 lengthening of circadian period in response to increasing intensities of red (but 20 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 473 not blue) light has been reported in plants mutant for LNK1 or LNK2 (Xie et al., 474 2014), which encode transcriptional co-activators that work with RVE proteins to 475 control expression of central clock genes (Rugnone et al., 2013; Xie et al., 2014). 476 The differential sensitivities of lnk and rve mutants to red and blue light are 477 therefore at first surprising. However, LNK1 and LNK2 affect clock function in a 478 partially redundant manner (Rugnone et al., 2013; Xie et al., 2014) and the effect 479 of light on clock pace in lnk1 lnk2 mutants has not yet been reported. In addition, 480 LNK proteins have been shown to antagonize RVE8 function in at least one clock 481 output pathway (Perez-Garcia et al., 2015). Finally, LNK1 and LNK2 interact with 482 CCA1 and LHY in addition to RVE4 and RVE8 (Xie et al., 2014), suggesting they 483 may affect the clock independent of RVE8-clade proteins. The relationship 484 between the Myb-like transcription factors and the LNK proteins is therefore 485 complex, making mutant phenotypes difficult to predict. 486 The most obvious phenotype in the rve triple and quintuple mutants is 487 their enhanced size relative to wild type at both seedling and adult stages of 488 development (Figs. 3 and 4). RVE3 and RVE5 appear to play a minor role in 489 growth regulation, as the triple and quintuple rve mutants have similar growth 490 phenotypes. One exception is the longer petiole lengths of rve3 4 5 6 8 plants 491 relative to rve4 6 8 plants at 3 weeks of age; interestingly, this difference is 492 observed for plants grown in SD but not those grown in LD (Supplemental Fig. 2). 493 RVE3 and RVE5 therefore play both a photoperiod and tissue-specific role in 494 growth regulation. 21 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. Epistasis experiments also suggest that RVE4, RVE6, and RVE8 regulate 495 496 plant growth via distinct mechanisms depending upon the photoperiod. PIF4 497 and/or PIF5 are required for the enhanced growth of rve triple mutants in most 498 photoperiods, but not in SD (Fig. 8 and Supplemental Fig. 6). It is possible that 499 the enhanced growth of rve4 6 8 mutants in SD is instead due to misregulation of 500 the related transcription factors PIF1 and/or PIF3. Both of these proteins have 501 been implicated in the promotion of hypocotyl elongation in response to SD (Soy 502 et al., 2012; Soy et al., 2014). Moreover, when plants are grown in SD, TOC1 503 and PIF3 co-regulate expression of growth-related genes, with TOC1 inhibiting 504 PIF transactivation activity at the promoters of common target genes in the first 505 part of the night (Soy et al., 2016). It is possible that in rve4 6 8 plants grown in 506 SD, there is a delay in the phase of TOC1 protein accumulation such that the 507 normal inhibition of PIF growth-promoting activity during the early night does not 508 occur. 509 In contrast to plants grown in SD, the enhanced growth of rve4 6 8 510 mutants maintained in LL, LD, or 12 hr light:12 hr dark conditions depends on 511 functional PIF4 and PIF5 genes (Fig. 8 and Supplemental Fig. 6). In addition, the 512 overall expression levels of both PIF4 and PIF5 in the rve mutant are significantly 513 elevated relative to wild-type plants when examined over a 1.5 day time course 514 collected in LL (Fig. 7). RVE8 directly promotes expression of PRR5 and TOC1 515 (Rawat et al., 2011; Hsu et al., 2013), two known repressors of PIF4 and PIF5 516 expression (Kunihiro et al., 2011; Huang et al., 2012; Nakamichi et al., 2012). 517 Notably, we find that PRR5 and TOC1 expression is significantly reduced in rve4 22 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 518 6 8 plants grown in LL (Supplemental Fig. 4), potentially explaining the enhanced 519 expression of PIF4 and PIF5 in this condition. 520 The ability of the pif4 pif5 mutations to suppress the long-hypocotyl 521 phenotypes of rve mutants in LL (Fig. 8) is at first surprising given the relatively 522 small increases in PIF4 and PIF5 transcript levels in rve4 6 8 plants (Fig. 7). 523 However, previous studies have demonstrated that modest increases in trough 524 levels of PIF4 and PIF5 transcripts cause increased hypocotyl elongation 525 (Nusinow et al., 2011; Wang et al., 2015). 526 In LL conditions, growth phenotypes in rve4 6 8 cannot be attributed to 527 conflicts of circadian phase with light/dark transitions. However, the same is not 528 true in LD. In this condition, we find that PIF4 and PIF5 expression levels are 529 significantly increased during the dark period (Fig. 7) although not across the 530 entire time series (data not shown). Since PIF proteins are stable in the dark but 531 not the light (Nozue et al., 2007; Lorrain et al., 2008), the night-phased 532 upregulation of these transcripts is likely to lead to a greater accumulation of PIF 533 proteins in rve4 6 8 than in wild type. PIF4 and PIF5 proteins induce expression 534 of genes that positively regulate hypocotyl growth (Franklin et al., 2011; Kunihiro 535 et al., 2011; Nozue et al., 2011; Hornitschek et al., 2012; Sun et al., 2012), 536 suggesting a mechanism to explain enhanced growth in rve4 6 8 plants in LD 537 conditions. Indeed, under this photoperiod, pif4 pif5 is epistatic to rve4 6 8 both 538 at the seedling (Fig. 8) and rosette stage (Fig. 8) of development. Together, 539 these data strongly suggest the growth phenotypes of rve4 6 8 in LD are due to 540 elevated PIF4 and/or PIF5 expression levels at night. 23 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 541 These increased nighttime expression levels of PIF4 and PIF5 in LD may 542 be attributed both to a delay in circadian phase (given the long-period phenotype 543 of rve4 6 8) as well as increased trough levels of the transcripts (Fig. 7). We thus 544 conclude that in LD cycles the rve4 6 8 growth phenotypes are due to alterations 545 in PIF4 and/or PIF5 expression that are caused by both direct RVE control of 546 gene expression and the overall long-period phenotype of this mutant. 547 Recent work has highlighted the importance of both shared and separate 548 molecular pathways controlling growth at different stages of development and in 549 different environmental conditions (Nozue et al., 2015; Seaton et al., 2015). Our 550 current results show that RVE4, RVE6, and RVE8 are partially redundant 551 inhibitors of plant growth throughout plant development, and that both juvenile 552 and adult growth phenotypes of rve mutants require PIF4 and/or PIF5 function 553 (Fig. 8 and Supplemental Fig. 6). These data strongly suggest that these PIFs 554 not only enhance hypocotyl growth but also growth of adult plants. This PIF- 555 dependent promotion of leaf growth at the adult stages of plant development is 556 surprising given previous reports that both loss-of-function and constitutive 557 overexpression of PIF4/PIF5 reduce adult plant size (Fujimori et al., 2004; 558 Lorrain et al., 2008; Keller et al., 2011; Kumar et al., 2012; Nieto et al., 2015; 559 Nozue et al., 2015). The PIF-mediated promotion of adult growth in rve4 6 8 may 560 be due either to the modest increases or the precise time of day at which 561 PIF4/PIF5 expression is increased. 562 Perturbation of clock genes is generally associated with a reduction in 563 adult biomass (Dodd et al., 2005; Graf et al., 2010). Other reported cases in 24 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 564 which mutation of clock genes increases adult plant size can be attributed to a 565 delay in flowering time rather than growth promotion per se. For example, plants 566 overexpressing CCA1 or mutant for PRR5, PRR7, and PRR9 display reduced 567 growth rates as juvenile plants (Ruts et al., 2012) but flower very late (Wang and 568 Tobin, 1998; Nakamichi et al., 2005b) and are thus very large plants at the time 569 of senescence. To our knowledge, the enhanced growth rate we observe in 570 rve8-clade mutants has not previously been reported for a circadian mutant. 571 The increased size phenotypes of the rve triple and quintuple mutants 572 might be immediately relevant for agricultural purposes. Phylogenetic analysis 573 shows the RVE8 clade is conserved in both monocot and dicot crop species 574 (Supplemental Fig. 7). Recent studies show that variants in a number of clock 575 gene homologs in crop species, including homologs of genes that are RVE8 576 targets in Arabidopsis, are associated with favorable yield traits (Bendix et al., 577 2015). Perturbations in the circadian clock have been associated with the growth 578 advantages seen in polyploids and in hybrids (Ni et al., 2009). Intriguingly, EE 579 motifs are enriched in the promoters of genes specifically upregulated in 580 polyploids (Ni et al., 2009). Together with our demonstration of enhanced growth 581 rates in rve mutants, this suggests that modulation of RVE8 function may 582 contribute to the hybrid vigor previously associated with alterations in CCA1 and 583 LHY function (Chen, 2010). 584 585 586 25 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 587 588 589 MATERIALS AND METHODS 590 Col; CCR2::LUC wild-type and the rve 4 6 8; CCR2::LUC mutant lines were 591 previously described (Rawat et al., 2011; Hsu et al., 2013) The quintuple rve 592 mutant was generated by introgressing the rve3-1 allele (SALK_001480C) and a 593 rve5-1 mutant allele (SAIL_769_A09) into the previously characterized rve4 6 8 594 triple mutant (Hsu et al., 2013). Genotypic verification of combinatorial mutants 595 was performed via PCR analysis using primers as follows: RVE3 WT; 5’- 596 CAACAACGATACCGGTTTCCATACG-3’ and 5’- 597 TCGCAATGCACTAATCCATGCTTAC-3’; RVE3 TDNA; 5’- 598 CAACAACGATACCGGTTTCCATACG-3’ and 5’-GCGTGGACCGCTTGCTGC 599 AACT-3’. RVE5 WT; 5’-TCTGGGTACTAGAGCAACGAGAC-3’, and 5’- 600 ACTCCCGGTTCCTCTACTATCAC-3’; RVE5 TDNA; 5’- 601 TAGCATCTGAATTTCATAACCAATCTCGATACAC-3’ and 5’ACTCCCGG- 602 TTCCTCTACTATCAC-3’. The primers used to genotype RVE4, RVE6, and 603 RVE8 were previously described (Rawat et al., 2011; Hsu et al., 2013). The 604 qPCR primers used to examine RVE3 expression are 5’- 605 AGCCCTTCATCTATTTGACCGGGA-3’ and 5’- 606 TGTGCGTGGCTTCGTATCTGTATC-3’. The primers used to quantify RVE5 607 expression via semi-quantitative RT-PCR are 5’- 608 ATGGTGTCCGTAAACCCTAGAC-3’ and 5’- 609 CTATTTCAAAGCTTTAGCGCTGTA-3’. The double pif4 5 mutant (pif4-101; 610 GARLIC_114_G06 and pif5-1; SALK_087012) (Fujimori et al., 2004; Lorrain et al., Mutant alleles and genotyping 26 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 611 2008) was crossed to the rve4 6 8 mutant to generate the rve4 6 8 pif4 5 612 quintuple mutant. The primers used to genotype the PIF4 and PIF5 alleles are 613 as follows: PIF4 WT; 5’-CTCGATTTCCGGTTATGG-3’ and 5’- 614 CAGACGGTTGATCATCTG-3’; PIF4 TDNA; 5’-CAGACGGTTGATCATCTG-3’ 615 and 5’-GCATCTGAATTTCATAACCAATC-3’; PIF5 WT; 5’- 616 TCGCTCACTCGCTTACTTAC-3’ and 5’-TCTCTACGAGCTTGGCTTTG-3’; PIF5 617 TDNA; 5’-TCGCTCACTCGCTTACTTAC-3’ and 5’- 618 GGCAATCAGCTGTTGCCCGTCTCACTGGTG-3’. The primers used to amplify 619 the wild-type and T-DNA-specific bands for RVE4, RVE6, and RVE8 have been 620 previously reported (Hsu et al., 2013). 621 622 623 624 Circadian period analysis Seeds were sowed on petri plates containing 1xMS medium (Murashige and 625 Skoog; (Sigma-Aldrich, St. Louis, MO), 3% sucrose, 0.8% agar. Seeds were 626 then stratified for 2-4 days in the dark at 4°C before entrainment in 12 hr light:12 627 hr dark conditions for 6 days at 50 μmol m-2 s-1 white light, 22°C. Seedlings were 628 sprayed with 3 mM D-luciferin (Biosynth) and luciferase activity detected using an 629 ORCA II ER CCD (Hamamatsu Photonics) with illumination provided by light 630 emitting diode SnapLites (Quantum Devices, Inc., Barneveld, WI) emitting 631 continuous monochromatic red or blue light. Neutral density filters (Rosco 632 Laboratories, Sun Valley, CA) were use to generate variable light fluence rates. 633 Bioluminescence was quantified using MetaMorph software (Molecular Devices) 634 and period analysis was performed using Biological Rhythms Analysis Software 635 System (BRASS) http://millar.bio.ed.ac.uk/PEBrown/BRASS/BrassPage.htm). 27 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 636 Hypocotyl analysis 637 Seeds were plated on medium containing 1XMS, 0.8% agar, and concentrations 638 of sucrose ranging from 0-6% (w/v) as indicated. Seeds were stratified and 639 released to the appropriate day-length conditions under cool white fluorescent 640 bulbs at the indicated light intensities, 22°C, for seven days. Neutral density 641 filters were utilized to generate the indicated fluence rates. Individual hypocotyls 642 were transferred to transparencies and scanned following established protocols 643 (http://malooflab.openwetware.org/Hypocotyl_measurement.html). Hypocotyl 644 lengths were measured using ImageJ (http://imagej.nih.gov/ij/). 645 646 Adult plant phenotyping and flowering time analysis 647 Arabidopsis seeds were plated on 0.5X MS; 0.8% agar medium and cold 648 stratified for 2-4 days before being transferred to growth chambers illuminated 649 with cool white fluorescent bulbs at 50 μmol m-2 s-1 white light, 22°C, for four days. 650 Upon germination, seedlings were transferred directly to soil and grown under 50 651 μmol m-2 s-1 white light, 22°C, in long-day (16 hr light : 8 hr dark) conditions for 652 the indicated lengths of time. Three-week and six-week old plant leaves were 653 analyzed according to the LeafJ protocol (Maloof et al., 2013). Rosette growth 654 was determined manually by using calipers to measure the distance between the 655 eighth and ninth leaves of every plant. Total aerial biomass was quantified for 656 six-week old plants after they had been stored and dried for three days at 40°C. 657 Flowering time was scored when plants generated a 1 cm bolt in long-day 658 conditions. 28 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 659 Cell size analysis 660 Six-week old plants were dissected and leaves eight and nine were fixed and 661 cleared according to previously described methods (Tsuge et al., 1996; Horiguchi 662 et al., 2006) for two to three hours. Cleared leaves were imaged at 20X 663 magnification using Zeiss Axioscop 2 plus microscope, and mesophyll cell area 664 was determined using ImageJ software (http://imagej.nih.gov/ij/). 665 666 RNA extraction and qRT-PCR analysis 667 Seedlings were grown for six days in 12 hr light:12 hr dark conditions and then 668 released to constant white light for 32 hours before approximately 20-40 669 seedlings were collected per sample for the LL time course. For the LD and SD 670 time courses, seedlings were grown in 16 hr light:8 hr dark or 8 hr light:16 hr dark 671 photocycles for 6 days before sample collection was started. RNA was extracted 672 from whole seedlings using Trizol reagent following the manufacturer’s protocol 673 (Life Technologies, Carlsbad, CA). RNA was subjected to DNAse treatment 674 following the Qiagen RNase-free DNase protocol (Qiagen, Valencia, CA), and 675 cDNA was synthesized using oligo-dT primers and Superscript II Reverse 676 Transcriptase (Life Technologies, Carlsbad, CA). qRT-PCR analysis was 677 performed with reagents as previously described (Martin-Tryon and Harmer, 678 2008) using a BioRad iQ5 thermocycler. Data were analyzed by the delta Ct 679 method using iCycler iQ Optical Systems Software version 3.1 (BioRad 680 Laboratories, Inc., Hercules, CA). Gene expression analysis was conducted 681 using previously described primers (Nusinow et al., 2011; Hsu et al., 2013). 29 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 682 Quantification of gene expression across the LL, LD and SD time series was 683 carried out by computing the area under the curves using natural cubic spline 684 interpolation using the auc function in the MESS package (Ekstrøm, 2016) in the 685 R statistical environment (R Core Team, 2016). Statistical significance was then 686 assessed with linear mixed effect models with genotype as a fixed and replicate 687 as a random effect using the R packages lme4 (Bates et al., 2015) and lmerTest 688 (Kuznetsova et al., 2016). 689 690 Phylogenetic analysis 691 Phylogenetic analysis was performed on the Phylogeny.fr platform (Dereeper et 692 al., 2008). Amino acid sequences for Myb-like factors were aligned with 693 MUSCLE (v3.8.31) configured for highest accuracy (default settings). The 694 phylogenetic tree was reconstructed using the maximum likelihood method 695 implemented in the PhyML program (v3.1/3.0 aLRT). The WAG substitution 696 model was selected assuming an estimated proportion of invariant sites (of 697 0.039) and 4 gamma-distributed rate categories to account for rate heterogeneity 698 across sites. The gamma shape parameter was estimated directly from the data 699 (gamma=1.046). Reliability for internal branch was assessed using the aLRT test 700 (SH-Like). Graphical representation and edition of the phylogenetic tree were 701 performed with TreeDyn (v198.3). UniProtKB/Swiss-Prot reference sequences 702 are as follows: Arabidopsis thaliana: At_CCA1 (P92973), At_LHY (Q6R0H1), 703 At_RVE1 (F4KGY6), At_RVE2 (F4K5X6), At_RVE3 (Q6R0H0), At_RVE4 30 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 704 (Q6R0G4), At_RVE5 (C0SVG5), At_RVE6 (Q8H0W3), At_RVE7 (B3H5A8), 705 At_RVE7-like (F4J2J6), and At_RVE8 (Q8RWU3); Oryza sativa: 706 Os01g06320 (Q5ZCD7), Os_02g45670 (B9F1T8), Os_02g46030 (Q6ZHD2), 707 Os_04g49450 (A3AWS7), Os_05g07010 (C7J2V5), Os_06g01670 (Q5VS69), 708 Os_06g45840 (B9FQF0), Os_06g51260 (B5AEI9), and Os_08g06110 709 (Q0J7W9); Solanum lycopersicum: Sl_01g095030 (K4AZP3), Sl_02g036370 710 (K4B5T0), Sl_03g098320 (K4BJP7), Sl_06g036300 (K4C4Z7), Sl_10g005080 711 (K4CX41), and Sl_10g084370 (K4D3M3). 712 31 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 713 FIGURE LEGENDS 714 Figure 1. Free-running circadian period is lengthened in rve triple and quintuple 715 mutants. Plants were entrained for 6d in 12 hr light:12 hr dark cycles at 22°C 716 before release to constant light (LL) at the indicated fluence rates at 22°C. A, 717 CCR2::LUC expression was assayed in constant red plus blue light (86 μmol m-2 718 s-1 total fluence, equal contributions red and blue light) (n=35-45). B, Fluence rate 719 response curves for CCR2::LUC period in constant red light (n=20-30). C, 720 Fluence rate response curves for CCR2::LUC period in constant blue light (n=20- 721 30). Error bars represent SEM. ** indicates p < 0.01, *** indicates p < 0.001, 722 Student’s t test. Data shown are illustrative of at least two independent 723 experiments. 724 725 Figure 2. Flowering time is modestly delayed in rve mutants. Plants were grown 726 in 16 hr light:8 hr dark cycles; bolting date was defined as the day at which the 727 inflorescence reached 1 cm. A, Days to bolting (n=25-36); B, Rosette leaf 728 number at bolting (n=25-36). Error bars represent SEM. *** indicates p < 0.001, 729 Student’s t test. Data shown are illustrative of at least two independent 730 experiments. 731 732 Figure 3. Hypocotyl lengths of Col and rve mutants grown in different 733 photoperiods. Seedlings were grown at 22°C in white light at the indicated 734 fluence rates for seven days. A, Hypocotyl lengths of Col and rve mutants in 8 hr 735 light:16 hr dark (short day, SD) conditions (n=15-40). B, Hypocotyl lengths in 16 32 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 736 hr light:8 hr dark (long day, LD) conditions (n=23-35). C, Hypocotyl lengths in 737 constant light (LL) conditions (n=20-40). Error bars represent SEM. ** indicates p 738 < 0.01, *** indicates p < 0.001, Student’s t test. Data shown are illustrative of at 739 least two independent experiments. 740 741 Figure 4. Aerial growth phenotypes of 6-wk-old wild type and rve mutants. 742 Plants were grown in LD cycles (55 μmol m-2 s-1 white light) for six weeks. A, Col 743 and rve3 4 5 6 8 plants. B, Rosette diameter measured at the greatest distance 744 between leaves of Col, rve4 6 8, and rve3 4 5 6 8 plants (n=28-33). C, Biomass 745 measured as total aerial dry weight (n=16-23). D, Rosette diameter measured as 746 the distance between leaves 8 and 9 (n=28-33). E, Blade areas of leaves 8 and 9 747 (n=12). F, Blade lengths of leaves 8 and 9 (n=12). G, Petiole lengths of leaves 8 748 and 9 (n=12). Error bars represent SEM. * indicates p < 0.05, ** indicates p < 749 0.01, *** indicates p < 0.001, Student’s t test. Data shown are illustrative of at 750 least two independent experiments. 751 752 Figure 5. The RVEs reduce rosette growth rate and cell size. Col, rve4 6 8, and 753 rve3 4 5 6 8 plants were grown in LD cycles (55 μmol m-2 s-1 white light). A, 754 Rosette diameter measured as the distance between leaves 8 and 9 (n=28-33). 755 Asterisks indicate significant difference between the mutants and Col. *** 756 indicates p < 0.001, Student’s t test. B, Size of leaf blade mesophyll cells in six- 757 week-old Col and rve4 6 8 plants (n=12 leaves per genotype; n=20-40 cells per 758 leaf). Images were taken at 20X magnification with Zeiss Axioscop 2 plus 33 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 759 microscope. Scale bar represents 100 μm. C, Quantification of mesophyll cell 760 area. Error bars represent SEM. *** indicates p < 0.001, Student’s t test; N.S., 761 not significant. Data shown are illustrative of at least two independent 762 experiments. 763 764 Figure 6. The RVEs repress growth responses to sucrose. A, Hypocotyl lengths 765 of 6d-old seedlings grown in 12 hr light:12 hr dark cycles (55 μmol m-2 s-1 white 766 light) at 22°C (n=28-47) with the indicated concentrations of sucrose in the media. 767 B, As above, but hypocotyl lengths were normalized within each genotype so that 768 the 0% sucrose control = 100%. *** indicates p < 0.001, N.S., not significant, 769 Student’s t test. 770 771 Figure 7. PIF4 and PIF5 expression is elevated in rve4 6 8 mutants. A – F, Time 772 course of PIF4 and PIF5 expression. Seedlings were grown in (A, B) 12 hr 773 light:12 hr dark cycles at 25 μmol m-2 s-1 white light for 6d before transfer to LL at 774 the same fluence rate, (C, D) LD, or (E, F) SD cycles under 25 μmol m-2 s-1 white 775 light. A, C, E, PIF4 and B, D, F, PIF5 expression normalized relative to PP2a. 776 Times represent hours since the last transition from dark to light; error bars 777 represent SEM of two independent biological replicates. G, H, Expression of 778 PIF4 (G) or PIF5 (H) integrated across the entire LL time series (left y-axes) or 779 during the dark portions of the LD and SD time series (right y-axes) computed as 780 the area under the curve. Error bars represent range of values across two 781 independent time course experiments. *** indicates p < 0.001, ** indicates p < 34 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 782 0.005, and * indicates p < 0.05, linear mixed effect model with genotype as a 783 fixed and replicate as a random effect; N.S., not significant. 784 785 Figure 8. The PIFs are epistatic to the RVEs at both seedling and adult stages of 786 development. A, Seedlings were grown in LL at 7.5 μmol m-2 s-1 (white light) for 787 6d at 22°C. B, Quantification of hypocotyl length of plants grown as in (A) (n=68- 788 80). C –D, Seedlings were grown in LD (C) or SD (D) at 25 μmol m-2 s-1 (white 789 light) on media containing 3% sucrose for 6d at 22°C and hypocotyl length 790 measured (n= 72-92). E-H, Col, rve4 6 8, pif4 5, and rve4 6 8; pif4 5 plants were 791 grown in LD cycles (55 μmol m-2 s-1) for 3 weeks. E, 3-wk-old plants. F, Leaf 5 792 blade area, G, blade length, and H, petiole length of indicated genotypes of 3-wk- 793 old plants (n=12). Lines within the box-and-whisker plots represent the medians; 794 whiskers and outliers plotted with the Tukey method. Significance is indicated as 795 determined by linear mixed effect model with genotype as a fixed and replicate 796 as a random effect. Error bars represent SEM. Data shown are illustrative of at 797 least two independent experiments. 798 799 SUPPLEMENTAL DATA 800 Supplementary Figure 1. Gene expression in rve3 and rve5 mutants. 801 Supplementary Figure 2. Aerial growth phenotypes of 3-wk-old wild type and 802 rve mutants grown in long or short days. 803 Supplementary Figure 3. PIF4 and PIF5 expression is elevated in rve4 6 8 804 mutants. 35 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 805 Supplementary Figure 4. PRR5, TOC1, and LUX expression is altered in rve4 806 6 8 mutants. 807 Supplementary Figure 5. PRR5, TOC1, and LUX expression is altered in rve4 808 6 8 mutants. 809 Supplementary Figure 6. The PIFs are epistatic to the RVEs when grown on 810 media lacking sucrose. 811 Supplementary Figure 7. The RVE8 clade is conserved in monocot and dicot 812 species. 813 814 Supplementary Figure 1. Gene expression in rve3 and rve5 mutants. A, RVE3 815 expression in Col and rve3-1 plants harvested two hours after subjective dawn 816 (CT 2) as determined by qRT-PCR (RVE3/PP2a). B, Semi-quantitative RT-PCR 817 detects RVE5 signal in cDNA generated from Col, but not rve5-1 plants, after 30 818 and 35 cycles of amplification. Primers against RVE4 were used as a positive 819 control. 820 821 Supplementary Figure 2. Aerial growth phenotypes of 3-wk-old wild type and 822 rve mutants grown in long or short days. Plants were grown in (A – C) LD cycles 823 (55 μmol m-2 s-1 white light) or (D – F) SD cycles (120 μmol m-2 s-1 white light) for 824 three weeks. A, D, Blade areas of leaf 5 (n=12). B, E, Blade lengths of leaf 5 825 (n=12). C, F, Petiole lengths of leaf 5 (n=12). The trend of larger leaf 826 dimensions in the rve mutants was similar for four different leaves tested 827 amongst respective light conditions. Error bars represent SEM. * indicates p < 36 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 828 0.05, ** indicates p < 0.01, *** indicates p < 0.001, Student’s t test. Data shown 829 are illustrative of at least two independent experiments. 830 831 Supplementary Figure 3. PIF4 and PIF5 expression is elevated in rve4 6 8 832 mutants. Seedlings were harvested in LL (A, B), LD (C,D), or SD (E,F) 833 conditions; these are the same data shown in Figure 7 but are plotted on a linear 834 rather than log scale on the y-axis. A, C, E, PIF4 expression relative to PP2a. B, 835 D, F, PIF5 expression relative to PP2a. n=10-30 for all genotypes tested. Error 836 bars represent SEM for two independent biological replicates. 837 838 Supplementary Figure 4. PRR5, TOC1, and LUX expression is altered in rve4 839 6 8 mutants. Seedlings were grown as described in the legend for Figure 7 and 840 were harvested in LL (A-C), LD (D-F), or SD (G-H) conditions. A, D, G, PRR5 841 expression relative to PP2a. B, E, H, TOC1 expression relative to PP2a. C, F, I, 842 LUX expression relative to PP2a. n=10-30 for all genotypes tested. Error bars 843 represent SEM for two independent biological replicates. J – L, Expression of 844 PRR5 (J), TOC1 (K), or LUX (L) (integrated across the entire LL, LD, and SD 845 time series) computed as the area under the curve. Error bars represent range 846 of values across two independent time course experiments. *** indicates p < 847 0.001, and * indicates p < 0.05, linear mixed effect model with genotype as a 848 fixed and replicate as a random effect; N.S., not significant. 849 37 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 850 Supplementary Figure 5. PRR5, TOC1, and LUX expression is altered in rve4 851 6 8 mutants. Seedlings were harvested in LL (A-C), LD (D-F), or SD (G-H) 852 conditions; these are the same data shown in Supplemental Figure 4 but are 853 plotted on a log rather than linear scale on the y-axis. A, D, G, PRR5 expression 854 relative to PP2a. B, E, H, TOC1 expression relative to PP2a control. C, F, I, LUX 855 expression relative to PP2a control. n=10-30 for all genotypes tested. Error bars 856 represent SEM for two independent biological replicates. 857 858 Supplementary Figure 6. The PIFs are epistatic to the RVEs when grown on 859 media lacking sucrose. Seedlings were grown for 6d in 12 hr light:12 hr dark 860 cycles at 50 μmol m-2 s-1 (white light) at 22°C on media A, containing 3% sucrose 861 (n=32-45), or B, lacking sucrose (n=15-24). Error bars represent SEM. * 862 indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, Student’s t test. 863 Data shown are illustrative of at least two independent experiments. 864 865 Supplementary Figure 7. The RVE8 clade is conserved in monocot and dicot 866 species. Phylogenetic tree of single Myb-like proteins from Arabidopsis thaliana 867 (At), Oryza sativa (Os), and Solanum lycopersicum (Sl). Values for branches 868 represent proportion of bootstrap data sets that support the indicated topography. 869 38 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 870 ACKNOWLEDGEMENTS 871 We thank the Arabidopsis Biological Resource Center and Kazunari Nozue for 872 providing seeds. We also thank members of the Harmer lab, Kazunari Nozue, 873 and Julin Maloof for helpful discussions. 874 875 AUTHOR CONTRIBUTIONS 876 JAG, ASK, PYH, and SLH designed the research; JAG, ASK, DNC, and PYH 877 performed the research; JAG, ASK, PYH, and SLH analyzed data; and JAG and 878 SLH wrote the paper. 879 880 39 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 LITERATURE CITED Alabadi D, Yanovsky MJ, Mas P, Harmer SL, Kay SA (2002) Critical role for CCA1 and LHY in maintaining circadian rhythmicity in Arabidopsis. Curr Biol 12: 757761 Aschoff J (1960) Exogenous and endogenous components in circadian rhythms. 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Mol Ecol 20: 1155-1165 45 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. A 29 *** Period (hrs) 28 *** *** 27 26 ** 25 24 23 22 Col rve 35 B Col rve3 5 rve 468 rve4 6 8 rve 34568 rve3 4 5 6 8 30 Period (hrs) 28 26 24 1 10 100 fluence rate (µmol/m2/sec) 1000 1 10 100 fluence rate (µmol/m2/sec) 1000 C 30 Period (hrs) 28 26 24 Figure 1. Free-running circadian period is lengthened in rve triple and quintuple mutants. Plants were entrained for 6d in 12 hr light:12 hr dark cycles at 22°C before release to constant light (LL) at the indicated fluence rates at 22°C. A, CCR2::LUC expression was assayed in constant red plus blue light (86 μmol m-2 s-1 total fluence, equal contributions red and blue light) (n=35-45). B, Fluence rate response curves for CCR2::LUC period in constant red light (n=20-30). C, Fluence rate response curves for CCR2::LUC period in constant blue light (n=20-30). Error bars represent SEM. ** indicates p < 0.01, *** indicates p < 0.001, Student’s t test. Data shown are illustrative of at least two independent experiments. Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. *** A 35 *** days to bolting 30 *** 25 20 15 10 5 0 Col rve 468 *** B leaf number at bolting 15 *** rve 34568 *** 10 5 0 Col rve 468 rve 34568 Figure 2. Flowering time is modestly delayed in rve mutants. Plants were grown in 16 hr light:8 hr dark cycles; bolting date was defined as the day at which the inflorescence reached 1 cm. A, Days to bolting (n=2536); B, Rosette leaf number at bolting (n=25-36). Error bars represent SEM. *** indicates p < 0.001, Student’s t test. Data shown are illustrative of at least two independent experiments Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. hypocotyl length (mm) A 12 *** 4 hypocotyl length (mm) 1 10 100 12 8 *** 4 *** *** long days 0 hypocotyl length (mm) 0 *** Col rve4 6 8 rve3 4 5 6 8 short days C *** 8 0 B *** 0 1 10 100 12 *** ** 8 *** 4 constant light 0 0 1 10 fluence rate (µmol/m2/sec) 100 Figure 3. Hypocotyl lengths of Col and rve mutants grown in different photoperiods. Seedlings were grown at 22°C in white light at the indicated fluence rates for seven days. A, Hypocotyl lengths of Col and rve mutants in 8 hr light:16 hr dark (short day, SD) conditions (n=15-40). B, Hypocotyl lengths in 16 hr light:8 hr dark (long day, LD) conditions (n=23-35). C, Hypocotyl lengths in constant light (LL) conditions (n=20-40). Error bars represent SEM. ** indicates p < 0.01, *** indicates p < 0.001, Student’s t test. Data shown are illustrative of at least two independent experiments. Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. rve3 4 5 6 8 *** C * E ** ** * 80 40 0 600 500 400 300 200 leaf 8 Col rve4 6 8 leaf 9 rve3 4 5 6 8 Col F 45 blade length (mm) blade area (mm 2) 700 * dry weight (g) 120 rve rve 468 34568 *** *** 0.2 0.1 0 Col *** *** 35 30 25 leaf 8 rve4 6 8 Col *** 120 0.3 40 20 D * rve rve 468 34568 leaf 9 rve3 4 5 6 8 rosette diameter (leaves 8 & 9) (mm) Col *** G *** 80 40 0 Col ** *** 25 petiole length (mm) B rosette diameter (mm) A rve rve 468 34568 *** *** 20 15 10 5 leaf 8 rve4 6 8 Col leaf 9 rve3 4 5 6 8 Figure 4. Aerial growth phenotypes of 6-wk-old wild type and rve mutants. Plants were grown in LD cycles (55 μmol m-2 s-1 white light) for six weeks. A, Col and rve3 4 5 6 8 plants. B, Rosette diameter measured at the greatest distance between leaves of Col, rve4 6 8, and rve3 4 5 6 8 plants (n=28-33). C, Biomass measured as total aerial dry weight (n=16-23). D, Rosette diameter measured as the distance between leaves 8 and 9 (n=28-33). E, Blade areas of leaves 8 and 9 (n=12). F, Blade lengths of leaves 8 and 9 (n=12). G, Petiole lengths of leaves 8 and 9 (n=12). Error bars represent SEM. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, Student’s t test. Data shown are illustrative of at least two independent experiments. Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. rossette diameter (leaves 8 & 9) (mm) A 125 100 *** *** *** 50 0 *** *** *** 10 Col 20 Day rve4 6 8 30 40 C 10000 cell area (μm2) B *** 75 25 *** *** Col rve4 6 8 rve3 4 5 6 8 *** 7500 5000 2500 0 Col rve 468 Figure 5. The RVEs reduce rosette growth rate and cell size. Col, rve4 6 8, and rve3 4 5 6 8 plants were grown in LD cycles (55 μmol m-2 s-1 white light). A, Rosette diameter measured as the distance between leaves 8 and 9 (n=28-33). Asterisks indicate significant difference between the mutants and Col. *** indicates p < 0.001, Student’s t test. B, Size of leaf blade mesophyll cells in six-week-old Col and rve4 6 8 plants (n=12 leaves per genotype; n=20-40 cells per leaf). Images were taken at 20X magnification with Zeiss Axioscop 2 plus microscope. Scale bar represents 100 μm. C, Quantification of mesophyll cell area. Error bars represent SEM. *** indicates p < 0.001, Student’s t test; N.S., not significant. Data shown are illustrative of at least two independent experiments. Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. hypocotyl length (mm) A 5 4 3 2 1 % growth compared to no sucrose B Col rve4 6 8 rve3 4 5 6 8 0 2 4 sucrose concentration (%) 200 *** ** *** Col rve4 6 8 rve3 4 5 6 8 180 N.S. 160 N.S. 140 *** *** *** *** 6 N.S. *** *** *** *** N.S. 120 100 0.5 1 2 % sucrose (w/v) 3 6 Figure 6. The RVEs repress growth responses to sucrose. A, Hypocotyl lengths of 6d-old seedlings grown in 12 hr light:12 hr dark cycles (55 μmol m-2 s-1 white light) at 22°C (n=28-47) with the indicated concentrations of sucrose in the media. B, As above, but hypocotyl lengths were normalized within each genotype so that the 0% sucrose control = 100%. *** indicates p < 0.001, N.S., not significant, Student’s t test. Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 4.0 Col rve468 B LL PIF5 expression PIF4 expression A 1.0 0.2 4.0 2.0 1.0 0.5 0.2 0.1 32 36 40 44 48 52 56 60 64 68 4.0 D LD 1.0 PIF5 expression PIF4 expression C 32 36 40 44 48 52 56 60 64 68 0.2 0.1 4.0 2.0 1.0 0.5 0.2 0.1 12 16 20 15 45 10 40 35 * 5 30 0 D -S -S rv e4 68 D -L D 25 ol -S D D -S rv e4 68 D ol -L 68 e4 rv C L -L C ol -L -L 68 rv e4 ol D 0 L 25 N.S. ** C 30 50 D 5 *** 12 16 20 68 35 8 e4 40 4 rv 10 0 PIF5 expression, night * ** 45 C Time (hours) H 12 16 20 0.2 0.1 15 50 8 0.5 L 12 16 20 1.0 -L 8 2.0 ol 4 4.0 L 0 12 16 20 -L 12 16 20 8 -L 0.2 4 C 0.5 0 68 PIF5 expression 1.0 12 16 20 e4 F SD 8 rv 8 PIF4 expression, night PIF4 expression, constant light 4 2.0 8 G 0 ol 12 16 20 PIF5 expression, constant light PIF4 expression E 8 C 0.0 Figure 7. PIF4 and PIF5 expression is elevated in rve4 6 8 mutants. A – F, Time course of PIF4 and PIF5 expression. Seedlings were grown in (A, B) 12 hr light:12 hr dark cycles at 25 μmol m-2 s-1 white light for 6d before transfer to LL at the same fluence rate, (C, D) LD, or (E, F) SD cycles under 25 μmol m-2 s-1 white light. A, C, E, PIF4 and B, D, F, PIF5 expression normalized relative to PP2a. Times represent hours since the last transition from dark to light; error bars represent SEM of two independent biological replicates. G, H, Expression of PIF4 (G) or PIF5 (H) integrated across the entire LL time series (left y-axes) or during the dark portions of the LD and SD time series (right y-axes) computed as the area under the curve. Error bars represent range of values across two independent time course experiments. *** indicates p < 0.001, ** indicates p < 0.005, and * indicates p < 0.05, linear mixed effect model with genotype as a fixed and replicate as a random effect; N.S., not significant. Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. B 8 rve468 pif45 pif45 rve468 constant light E Col p < 2e-16 6 p = 0.03 4 2 rve468 pif45 pif45 rve468 p = 8e-6 F blade area (mm2) Col hypocotyl length (mm) A 200 p = 0.4 150 100 0 8 long days p < 2e-16 6 p = 0.002 4 p = 5e-5 G 22 blade length (mm) hypocotyl length (mm) C 2 p < 2e-16 short days p < 2e-16 10 5 0 Col rve468 pif45 pif45 rve468 H 18 16 14 20 petiole length (mm) hypocotyl length (mm) 15 p = 0.2 12 0 D 20 p = 1e-9 15 p = 0.07 10 5 Col rve468 pif45 pif45 rve468 Figure 8. The PIFs are epistatic to the RVEs at both seedling and adult stages of development. A, Seedlings were grown in LL at 7.5 μmol m-2 s-1 (white light) for 6d at 22°C. B, Quantification of hypocotyl length of plants grown as in (A) (n=68-80). C –D, Seedlings were grown in LD (C) or SD (D) at 25 μmol m-2 s-1 (white light) on media containing 3% sucrose for 6d at 22°C and hypocotyl length measured (n= 72-92). E-H, Col, rve4 6 8, pif4 5, and rve4 6 8; pif4 5 plants were grown in LD cycles (55 μmol m-2 s-1) for 3 weeks. E, 3-wk-old plants. F, Leaf 5 blade area, G, blade length, and H, petiole length of indicated genotypes of 3-wk-old plants (n=12). Lines within the box-and-whisker plots represent the medians; whiskers and outliers plotted with the Tukey method. Significance is indicated as determined by linear mixed effect model with genotype as a fixed and replicate as a random effect. Error bars represent SEM. Data shown are illustrative of at least two independent experiments. Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved. 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