Download The REVEILLE clock genes inhibit growth of

Plant Physiology Preview. Published on March 2, 2017, as DOI:10.1104/pp.17.00109
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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]
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Copyright 2017 by the American Society of Plant Biologists
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ABSTRACT
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and fitness in a constantly changing environment. In Arabidopsis thaliana, the
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clock is comprised of numerous regulatory feedback loops in which REVEILLE8
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(RVE8) and its homologs RVE4 and RVE6 act in a partially redundant manner to
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promote clock pace. Here, we report that the remaining members of the RVE8
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clade, RVE3 and RVE5, play only minor roles in regulation of clock function.
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However, we find that RVE8 clade proteins have unexpected functions in
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modulation of light input to the clock and control of plant growth at multiple
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stages of development. In seedlings, these proteins repress hypocotyl elongation
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in a day-length and sucrose dependent manner. Strikingly, adult rve4 6 8 and
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rve3 4 5 6 8 mutants are much larger than wild type, with both increased leaf
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area and biomass. This size phenotype is associated with a faster growth rate
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and larger cell size and is not simply due to a delay in the transition to flowering.
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Gene expression and epistasis analysis reveal that the growth phenotypes of rve
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mutants are due to misregulation of PHYTOCHROME INTERACTING FACTOR4
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(PIF4) and PIF5 expression. Our results shows that even small changes in PIF
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gene expression caused by perturbation of clock gene function can have large
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effects on the growth of adult plants.
The circadian clock is a complex regulatory network that enhances plant growth
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INTRODUCTION
Circadian rhythms are endogenous, biological rhythms that oscillate with
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an approximately 24-hour period. These rhythms are observed in many
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organisms throughout nature (Dunlap, 1999; Harmer et al., 2001; Harmer, 2009),
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and are particularly vital for plants due to their sessile nature. Numerous studies
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in plants have indicated that an impaired circadian oscillator can contribute to
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diminished growth, impaired defense against herbivores and pathogens, and
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even reduced fitness (Green et al., 2002; Dodd et al., 2005; Yerushalmi et al.,
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2011; Goodspeed et al., 2012; Ruts et al., 2012).
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The molecular network of the Arabidopsis circadian clock is primarily
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comprised of transcription factors that regulate each other’s as well as their own
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expression (Hsu and Harmer, 2014; McClung, 2014). Two homologous single
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MYB-domain transcription factors CIRCADIAN CLOCK ASSOCIATED1 (CCA1)
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and ELONGATED HYPOCOTYL (LHY) (Schaffer et al., 1998; Wang and Tobin,
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1998) have a circadian pattern of transcript peaking at dawn, and loss-of-function
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mutants have a short free-running period in continuous light (Green and Tobin,
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1999; Alabadi et al., 2002; Mizoguchi et al., 2002). CCA1 and LHY comprise a
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negative feedback loop with TIMING OF CAB EXPRESSION (TOC1) in which
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they bind to the evening element (EE) promoter motif of this and other evening-
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phased clock genes to repress expression during the day (Alabadi et al., 2002;
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Harmer and Kay, 2005; Nagel et al., 2015). TOC1 in turn regulates CCA1 and
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LHY by repressing their expression in a time-of-day manner (Gendron et al.,
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2012; Huang et al., 2012; Pokhilko et al., 2012).
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More recent work has characterized the role of the transcription factor
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REVEILLE8 (RVE8), a homolog of CCA1 and LHY, in the circadian network
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(Farinas and Mas, 2011; Rawat et al., 2011). These transcription factors are
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deemed members of the core oscillator, as mutation of these genes results in a
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free-running clock period that is either longer or shorter than wild type.
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Specifically, while cca1 and lhy mutants have a short period, the rve8 loss-of-
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function mutant has a long free-running period (Farinas and Mas, 2011; Rawat et
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al., 2011). Although RVE8 binds to similar evening-phased gene targets as
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CCA1 and LHY, this transcription factor activates instead of represses target
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gene expression (Farinas and Mas, 2011; Rawat et al., 2011; Hsu et al., 2013),
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explaining the opposite period phenotypes of these mutants. RVE8 forms an
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additional feedback loop in the clock, in which it positively regulates the evening-
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expressed transcription factor PSEUDO RESPONSE REGULATOR5 (PRR5),
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whose protein in turn represses expression of RVE8 (Rawat et al., 2011).
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RVE8 is one of eleven homologous MYB-like transcription factors in
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Arabidopsis (Rawat et al., 2009). RVE8 acts in a partially redundant manner with
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REVEILLE4 (RVE4) and REVEILLE6 (RVE6), two closely related genes, to
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promote clock pace (Hsu et al., 2013). Two remaining members of the RVE8
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clade, REVEILLE3 (RVE3) and REVEILLE5 (RVE5), were found to associate
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with the EE promoter motif both in vitro and in vivo (Gong et al., 2008; Rawat et
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al., 2011). However, their role in the clock has not been previously characterized.
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Additional key clock proteins include the transcription factors EARLY
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FLOWERING3 (ELF3), EARLY FLOWERING4 (ELF4), and LUX ARRYTHMO
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(LUX), which together comprise the tripartite evening complex (EC) (Nusinow et
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al., 2011). Each component of the EC is necessary for its function, as the lux,
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elf3, and elf4 single mutants are all arrhythmic in continuous light (Hicks et al.,
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2001; Doyle et al., 2002; Hazen et al., 2005). The EC forms in the early evening
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and acts to regulate numerous downstream genes as well as expression of other
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clock components (Dixon et al., 2011; Helfer et al., 2011; Nusinow et al., 2011;
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Mizuno et al., 2014).
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Approximately one-third of the Arabidopsis transcriptome is under
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circadian control (Covington et al., 2008), and consistent with this many aspects
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of plant growth and development are influenced by the circadian clock (Farre,
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2012; Song et al., 2013). Hypocotyl and root elongation and leaf growth undergo
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daily rhythms, and the phases of peak growth rates are shifted but not abolished
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in constant environment conditions (Nozue et al., 2007; Poire et al., 2010; Iijima
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and Matsushita, 2011; Yazdanbakhsh et al., 2011).
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The clock is vital for growth rhythms in multiple organs, as the leaves and
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roots of arrhythmic circadian mutants CCA1-ox and prr9 7 5 exhibit perturbed
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growth (Ruts et al., 2012). The clock also modulates the timing of the transition
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from vegetative to reproductive growth by controlling rhythmic oscillations of key
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transcriptional regulators that promote flowering (Song et al., 2013). Flowering
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time is also regulated by light signaling pathways, which modulate stability of
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clock-regulated proteins to control their activity in a day length-dependent
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manner (Song et al., 2013).
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Many other developmental pathways are regulated not only by the clock
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but also by environmental stimuli. Hypocotyl elongation in Arabidopsis is
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influenced by changes in light and temperature as well as by the clock (Nozue et
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al., 2007; Nomoto et al., 2012). Two key regulators of hypocotyl elongation are
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the basic helix-loop-helix (bHLH) transcription factors PHYTOCHROME
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INTERACTING FACTOR 4 (PIF4) and PIF5 (Leivar et al., 2008; Lorrain et al.,
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2008; Hornitschek et al., 2012; Leivar et al., 2012). The differential patterns of
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hypocotyl growth in constant light and in light-dark cycles are due to the EC-
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mediated circadian regulation of PIF4 and PIF5 expression (Nusinow et al.,
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2011) combined with the post-translational regulation of PIF protein stability by
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red light-activated phytochrome (Nozue et al., 2007; Lorrain et al., 2008). In
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addition, PIF4 and PIF5 influence the rhythmic growth of leaves (Dornbusch et al.,
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2014). PIF proteins have also been shown to act in many other response
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pathways, such as auxin (Franklin et al., 2011; Kunihiro et al., 2011; Nozue et al.,
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2011; Hornitschek et al., 2012; Sun et al., 2012), gibberellin (de Lucas et al.,
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2008), high temperature (Koini et al., 2009; Stavang et al., 2009) and sucrose
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(Liu et al., 2011; Stewart et al., 2011).
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Although the roles of the circadian clock and PIFs in regulation of
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hypocotyl elongation in Arabidopsis have been thoroughly examined, clock
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modulation of growth in adult plants is much less understood. Here, we
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demonstrate that loss of RVE function alters PIF gene expression. Surprisingly,
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this perturbation causes greatly enhanced growth at both juvenile and mature
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stages of development, resulting in increased cell size and greater aerial
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biomass in rve mutants than in wild-type controls. These data provide a rare
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example in which alteration of circadian clock function promotes instead of
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inhibits plant growth, potentially opening up novel agricultural applications.
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RESULTS
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The RVEs act in a partially redundant manner to promote the pace of the
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clock
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Previous work has demonstrated that multiple members of the RVE family of
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transcription factors help control the pace of the circadian clock. Whereas
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mutations in the MYB-like transcription factors CCA1 and LHY shorten clock
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pace (Green and Tobin, 1999; Alabadi et al., 2002; Mizoguchi et al., 2002),
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mutations in their homolog RVE8 and two closely related genes, RVE4 and
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RVE6, lengthen circadian period (Farinas and Mas, 2011; Rawat et al., 2011;
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Hsu et al., 2013).
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RVE3 and RVE5 are the remaining members of the RVE8 clade. To
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investigate their role in clock function, we first identified plants with T-DNA
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insertions within these two genes. Neither RVE3 nor RVE5 was detectably
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expressed in the rve3-1 (SALK_001480C) or rve5-1 (SAIL_769_A09) mutants,
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respectively (Supplemental Fig. 1). As neither single mutant has a detectable
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circadian clock phenotype (data not shown), we examined the free-running
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circadian period in rve3-1 rve5-1 (rve3 5) mutants harboring a clock-regulated
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luciferase reporter construct (CCR2::LUC). In contrast to the long-period
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phenotype of the rve4-1 rve6-1 rve8-1 (rve4 6 8) triple mutant (Hsu et al., 2013),
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the rve3 5 double mutant displays a slightly shorter free-running period than wild
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type (Col = 23.95±0.09; rve4 6 8 = 27.83±0.7; rve3 5 = 23.63±0.04) (Fig. 1).
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Although the period of the rve3 5 double mutant is statistically significantly
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different than wild type, the subtle difference in clock pace compared to wild type
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suggests that RVE3 and RVE5 don’t play a major role in clock function.
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We next examined whether RVE3 and RVE5 play a partially redundant
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role in the clock with their homologs RVE4, RVE6, and RVE8. In contrast to the
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slightly shorter period seen in rve3 5 double mutants when compared to wild type,
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plants mutant for all five RVE8-related transcription factors (rve3 4 5 6 8) have a
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slightly longer free-running period than the rve4 6 8 triple mutant (rve4 6 8 =
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27.83±0.7; rve3 4 5 6 8 = 28.33±0.09). The difference in period between the rve
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triple and quintuple mutants is small, although statistically significant (Fig. 1).
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This result further indicates a modest role for RVE3 and RVE5 in the control of
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clock pace.
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We next examined whether the sensitivity of the circadian clock to light is
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altered in rve mutants. For this, we examined the effects of different fluence
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rates of monochromatic red and blue light on free-running circadian period.
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Mutations in light-signaling components have previously been shown to alter the
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relationship between free-running period and fluence rate (Somers et al., 1998;
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Devlin and Kay, 2000). We found that the shortening of period in response to
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high fluence rates of monochromatic red light seen in Col and rve3 5 is less
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pronounced in rve4 6 8 and rve3 4 5 6 8 (Fig. 1), suggesting reduced sensitivity
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of the clock to red light in these mutants. Surprisingly, rve triple and quintuple
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mutants in monochromatic blue light have longer free-running periods at higher
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than at lower fluence rates, the opposite of the trend seen in wild type (Fig. 1).
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Thus clock responsiveness to blue light is fundamentally different in the rve
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mutants than in control plants. Together, these data indicate that the RVEs affect
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both red and blue light signaling to the circadian clock, but in a distinct manner.
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Flowering time is modestly delayed in the rve mutants
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The circadian clock plays an important role in the day-length dependent control
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of the transition from vegetative to reproductive growth (Song et al., 2013). To
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determine whether the RVEs play an important role in the regulation of flowering
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time, we examined both leaf number and days to bolting in Col, rve4 6 8, and
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rve3 4 5 6 8 mutants grown in long-day (LD) (16 hr light:8 hr dark) conditions. In
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our growth conditions, wild-type plants bolt after approximately 24 days, while
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flowering time in the rve4 6 8 triple mutant is delayed by an average of three
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days. The rve3 4 5 6 8 quintuple mutant flowers significantly later than the triple
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mutant, at approximately 29 days (Fig. 2). These slight delays in the timing of
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the transition to flowering are mirrored by changes in leaf number at bolting, with
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the rve4 6 8 mutant generating an average of two more and rve3 4 5 6 8 an
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average of three more rosette leaves than wild type (Fig. 2). Thus the RVE8
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clade genes play a minor role in the control of flowering time.
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Day-length dependence of RVE mutant hypocotyl phenotypes
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The circadian clock drives rhythmic oscillations of hypocotyl elongation in
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Arabidopsis, and mutations in many clock genes results in seedlings with
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perturbed hypocotyl growth patterns (Farre, 2012). Since RVE8 clade genes
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affect clock pace, we investigated the hypocotyl phenotypes of the rve triple and
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rve quintuple mutants at various intensities of white light and under different day
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lengths.
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When grown in short-day (SD) conditions (8 hr light : 16 hr dark), both
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wild-type and rve mutant plants show inhibition of hypocotyl elongation in
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response to increasing fluence rates of light (Fig. 3). However, the hypocotyl
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lengths of the rve triple and rve quintuple mutants are significantly longer than
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wild type at all fluence rates tested, with the maximal difference between the
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mutants and wild type occurring at the highest fluence rate tested (100 μmol m-2
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s-1 white light) (Fig. 3). In LD conditions, hypocotyl lengths of the rve triple and
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quintuple mutants are significantly different from wild type at mid-to-high light
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intensities (7.5-50 μmol m-2 s-1), but not at the lowest (0.6 μmol m-2 s-1) or highest
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(100 μmol m-2 s-1) fluence rates tested (Fig. 3). Similar to LD conditions,
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hypocotyls of the rve4 6 8 and rve3 4 5 6 8 mutants are longer than wild-type at
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mid-range intensities (7.5-25 μmol m-2 s-1) of continuous white light (LL), but are
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no different from the control at very low (0.6 μmol m-2 s-1) or high (50-80 μmol m-2
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s-1) fluence rates (Fig. 3). The hypocotyl lengths of rve4 6 8 and rve3 4 5 6 8 are
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not significantly different from each other under any of the conditions tested.
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Taken together, these data indicate that RVE4, RVE6, and RVE8 contribute to
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regulation of hypocotyl length in a fluence-rate and day-length dependent
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manner, and that RVE3 and RVE5 do not significantly contribute to this
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phenotype in the conditions tested.
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The RVEs repress growth in adult plants
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Since we found that the RVEs influence growth at the seedling stage, we
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investigated if this is also true at later stages of development by measuring the
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rosette diameter of six-week old plants grown in LD conditions. We found that
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both rve4 6 8 and rve3 4 5 6 8 are approximately 1.5 fold larger in diameter than
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wild type (Fig. 4). To determine if this rosette size difference is associated with
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increased biomass, we measured the dry weight of aerial tissues of plants
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harvested at six weeks of age. Strikingly, the rve triple and rve quintuple mutants
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exhibit an approximately 30% increase in aerial dry weight compared to wild type
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(Fig. 4). There is no significant difference in aerial biomass between the rve
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triple and rve quintuple mutants, indicating that RVE3 and RVE5 do not
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significantly contribute to growth regulation in LD conditions (Fig. 4).
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To determine whether the extra rosette leaves made by the rve mutants
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before the transition to flowering (Fig. 2) could account for the increased growth
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of these mutants, we assessed rosette diameter by measuring the distance
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between leaves 8 and 9, which were produced by all genotypes. As measured
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this way, the rosette diameter of rve4 6 8 and rve3 4 5 6 8 mutants is
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approximately 25% larger than the wild-type controls (Fig. 4), demonstrating a
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growth phenotype in these plants independent of the flowering time phenotype.
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We next measured blade area, blade length, and petiole length of leaves 8
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and 9 in the rve mutants to determine what aspects of leaf development are
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affected. Blade area of both leaves 8 and 9 is increased approximately 20-30%
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in the triple and quintuple rve mutants compared to wild type (Fig. 4). Average
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blade length of leaf 8 is also increased by about 20%, being ~7mm longer (p-
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value = 6.4x10-4) in the rve triple and ~9mm longer (p-value = 4.8x10-5) in the rve
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quintuple mutant relative to wild type. Likewise, blade length in leaf 9 is
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increased by ~8mm (p-value = 1.6x10-4) and ~10mm (p-value = 5.6x10-6) in the
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rve triple and quintuple mutants, respectively, compared to wild type (Fig. 4).
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The difference in average petiole length is similar between the two mutants and
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wild type, with an increase in petiole length of ~7-8mm (p-value < 5x10-4) in rve4
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6 8 and ~9-10mm (p-value < 5x10-3) in rve3 4 5 6 8 relative to controls (Fig. 4).
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Thus many aspects of leaf growth are enhanced in rve mutants.
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We also measured these growth parameters at three weeks of age, before
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the transition to bolting in both wild-type and rve mutant plants. Similar to later
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stages of growth, rve triple and rve quintuple mutants both display significant
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increases in blade area, blade length, and petiole length compared to wild type
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when grown in LD (Supplemental Fig. 2). In contrast, blade area and length are
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not significantly different between the rve mutants and wild type grown in SD,
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although petiole length in the mutants is greater than in the controls in both SD
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and LD.
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and rve3 4 5 6 8. However, the rve3 4 5 6 8 mutant has a significantly longer
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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
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the roles of RVE3 and RVE5 are influenced by environmental factors. Together,
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our data show that members of the RVE8 clade are inhibitors of plant growth at
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both the seedling and adult stages of development and across different
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environmental conditions.
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Growth rate is accelerated in rve mutants
To better understand why rve mutants are larger than wild type, we
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investigated if the rve leaves are expanding at a faster rate or growing for a
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longer period of time than in control plants. To avoid the confounding effects of
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differences in flowering time, we measured rosette diameter as the distance
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between leaves 8 and 9, which are produced by all genotypes. Significant
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differences in rosette diameter are evident for both the rve triple and quintuple
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mutants throughout all stages of growth, long before the transition to flowering
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(Fig. 5). Both mutant and wild-type plants ceased rosette expansion
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approximately 30 days after germination, indicating that the larger size of rve
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mutants is due to a faster growth rate rather than an extended period of leaf
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growth. These data demonstrate that the larger rosette size in adult rve mutants
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is partly caused by an enhanced growth rate of individual leaves and not just a
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delay in flowering time.
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We examined whether this increased growth rate is due to larger average
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cell size or more cells per organ in the mutant compared to control. We found
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that the average area of mesophyll cells in the leaves of six-week old rve4 6 8
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mutants is increased by approximately 30% compared to wild type (Col =
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7.01x103 μm2; rve4 6 8 = 9.27x103 μm2; p-value = 5.3x10-36) (Fig. 5). This
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increase in cell size is similar to the increase in total blade area in the mutant (Fig.
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4), suggesting that larger average cell size is primarily responsible for the
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increased leaf size in rve mutants.
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rve mutants display an enhanced sensitivity to sucrose
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Sugars are well-known regulators of plant growth (Lastdrager et al., 2014). We
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therefore investigated whether rve mutants have an altered response to sucrose.
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We grew rve4 6 8, rve3 4 5 6 8, and Col seedlings on various concentrations of
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sucrose in 12 hr light:12 hr dark conditions and measured hypocotyl lengths.
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The maximal enhancement of hypocotyl elongation for all genotypes occurred at
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2% sucrose, and diminished at higher concentrations (Fig. 6). We next examined
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responsiveness to sucrose by comparing the degree of sucrose-mediated growth
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enhancement relative to the no-sucrose controls for each genotype (Fig. 6).
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Notably, both rve triple and quintuple mutants displayed an enhanced response
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to sucrose compared to Col at concentration of sucrose between 0.5 and 3% (Fig.
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6). The quintuple rve mutant demonstrated an even greater response to sucrose
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than the rve triple at 2% and 6% sucrose (Fig. 6), suggesting that RVE3, RVE5,
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or both act in a partially redundant manner with RVE4, RVE6, and RVE8 to
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repress sucrose growth responses.
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Expression of PIF transcription factors is altered in rve mutants
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Previous studies have revealed a role for the clock-regulated transcription factors
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PIF4 and PIF5 in the promotion of hypocotyl elongation in response to sucrose
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(Liu et al., 2011; Stewart et al., 2011). Since the rve mutants are hyper-
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responsive to sucrose (Fig. 6), we investigated whether PIF expression patterns
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are altered in rve mutants in either constant light and/or light/dark cycles. First,
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wild-type and mutant seedlings were grown in light-dark cycles and then
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transferred to constant light and temperature conditions at dawn (Zeitgeber Time
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0; ZT 0). After 32 hours in constant conditions, plants were harvested at three-
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hour intervals and gene expression was determined using qRT-PCR. As
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expected given the long period phenotype of rve4 6 8 mutants (Fig. 1), the
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circadian patterns of expression of both PIF4 and PIF5 show obvious phase
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delays (Fig. 7 and Supplemental Fig. 3). Notably, the maximal abundance of
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PIF4 and PIF5 is not appreciably different from wild type in these conditions.
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However, the waveforms of PIF gene expression are altered in rve4 6 8 such that
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trough levels of PIF gene expression are increased during the subjective night
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(ZT 36 – 48) and the peaks of gene expression are broader (Fig. 7). This
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suggests that overall PIF expression levels may be higher in the mutant than in
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wild type. Indeed, quantification of PIF4 and PIF5 expression across the entire
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LL time series, determined by computing the area under the curves using natural
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cubic spline interpolation, reveals significantly greater expression levels in the rve
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mutant compared to wild type (Fig. 7). These data suggest that the growth
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phenotypes of rve mutants maintained in constant light (Fig. 3) might be due to
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increased overall levels of PIF gene expression.
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We next examined PIF expression in seedlings grown in LD and SD
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cycles (Fig. 7 and Supplemental Fig. 3). Similar to the LL data, we saw no
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difference in peak levels of PIF4 and PIF5 expression but did see an increase in
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trough levels. Quantification of PIF gene expression across the entire LD and
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SD time courses did not reveal significant differences between the rve mutant
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and wild type (data not shown). However, since PIF4 and PIF5 proteins are
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destabilized in the light (Nozue et al., 2007; Lorrain et al., 2008), differences in
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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
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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
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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
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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
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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
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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.
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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.
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***
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
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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.
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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.
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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.
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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.
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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.
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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.
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