In general, plant growth is inhibited under high-density conditions, while it is promoted under low-density conditions. This is known as the "density effect". Growing plants at high densities is often associated with an accelerated flowering time. Three major pathways [the long day (LD), gibberellic acid (GA), and autonomous/vernalization pathways] are known to play important roles in the control of flowering time. Circadian clock genes, namely, LHY, CCA1, GI, and ELF3, regulate the LD pathway. GAI and FCA control flowering via GA and autonomous pathways, respectively. The density effect on plant size is caused by specific factors such as the amount of nutrition obtained from the soil and touch frequency among plants. However, the molecular mechanism underlying the acceleration of flowering time due to density effects remains unclear. Here, we show the density effects on three Brassicaceae plants, namely, Brassica rapa var. nipposinica, Brassica napus, and Brassica chinensis f. honsaitai. They showed shorter stems and leaves when grown at high densities on soil under continuous light (LL). Shorter stems and leaves, as well as accelerated flowering times, were observed when a model plant, Arabidopsis thaliana, was grown under the same conditions. Unexpectedly, ethylene insensitive 2 (ein2) showed no differences in density effects in our experiments. The acceleration of flowering at higher densities was largely suppressed by gai, but not by gi, lhy;cca1, or fca. These results suggest that the promotion of flowering (as a density effect) is likely dependent on the GA pathway, but not the LD or autonomous pathways.
Exposure to salinity causes plants to trigger transcriptional induction of a particular set of genes for initiating salinity-stress responses. Recent transcriptome analyses reveal that expression of a population of salinity-inducible genes also exhibits circadian rhythms. However, since the analyses were performed independently from those with salinity stress, it is unclear whether the observed circadian rhythms simply represent their basal expression levels independently from their induction by salinity, or these rhythms demonstrate the function of the circadian clock to actively limit the timing of occurrence of the salinity induction to particular times in the day. Here, by using tomato, we demonstrate that salt inducibility in expression of particular salinity-stress related genes is temporally controlled in the day. Occurrence of salinity induction in expression of SlSOS2 and P5CS, encoding a sodium/hydrogen antiporter and an enzyme for proline biosynthesis, is limited specifically to the morning, whereas that of SlDREB2, which encodes a transcription factor involved in tomato responses to several abiotic stresses such as salinity and drought, is restricted specifically to the evening. Our findings not only demonstrate potential importance in further investigating the basis and significance of circadian gated salinity stress responses under fluctuating day/night conditions, but also provide the potential to exploit an effective way for improving performance of salinity resistance in tomato.
Seasonal flowering involves responses to changes in day length. In Arabidopsis thaliana, the CONSTANS (CO) transcription factor promotes flowering in the long days of spring and summer. Late flowering in short days is due to instability of CO, which is efficiently ubiquitinated in the dark by the CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) E3 ligase complex. Here we show that CO is also phosphorylated. Phosphorylated and unphosphorylated forms are detected throughout the diurnal cycle but their ratio varies, with the relative abundance of the phosphorylated form being higher in the light and lower in the dark. These changes in relative abundance require COP1, because in the cop1 mutant the phosphorylated form is always more abundant. Inactivation of the PHYTOCHROME A (PHYA), CRYPTOCHROME 1 (CRY1) and CRYPTOCHROME 2 (CRY2) photoreceptors in the phyA cry1 cry2 triple mutant most strongly reduces the amount of the phosphorylated form so that unphosphorylated CO is more abundant. This effect is caused by increased COP1 activity, as it is overcome by introduction of the cop1 mutation in the cop1 phyA cry1 cry2 quadruple mutant. Degradation of CO is also triggered in red light, and as in darkness this increases the relative abundance of unphosphorylated CO. Finally, a fusion protein containing truncated CO protein including only the carboxy-terminal region was phosphorylated in transgenic plants, locating at least one site of phosphorylation in this region. We propose that CO phosphorylation contributes to the photoperiodic flowering response by enhancing the rate of CO turnover via activity of the COP1 ubiquitin ligase.
The COP1/SPA complex is an E3 ubiquitin ligase that acts as a key repressor of photomorphogenesis in dark-grown plants. While both COP1 and the four SPA proteins contain coiled-coil and WD-repeat domains, SPA proteins differ from COP1 in carrying an N-terminal kinase-like domain that is not present in COP1. Here, we have analyzed the effects of deletions and missense mutations in the N-terminus of SPA1 when expressed in a spa quadruple mutant background devoid of any other SPA proteins. Deletion of the large N-terminus of SPA1 severely impaired SPA1 activity in transgenic plants with respect to seedling etiolation, leaf expansion and flowering time. This ΔN SPA1 protein showed a strongly reduced affinity for COP1 in vitro and in vivo, indicating that the N-terminus contributes to COP1/SPA complex formation. Deletion of only the highly conserved 95 amino acids of the kinase-like domain did not severely affect SPA1 function nor interactions with COP1 or cryptochromes. In contrast, missense mutations in this part of the kinase-like domain severely abrogated SPA1 function, suggesting an overriding negative effect of these mutations on SPA1 activity. We therefore hypothesize that the sequence of the kinase-like domain has been conserved during evolution because it carries structural information important for the activity of SPA1 in darkness. The N-terminus of SPA1 was not essential for light responsiveness of seedlings, suggesting that photoreceptors can inhibit the COP1/SPA complex in the absence of the SPA1 N-terminal domain. Together, these results uncover an important, but complex role of the SPA1 N-terminus in the suppression of photomorphogenesis.
Fluctuations in the length of the day affect developmental processes and behaviors of many organisms.Mammals and birds reproduce in spring in response to lengthening days and insects pupate in autumn when daylength shortens.These phenomena, called photoperiodism, allow detection of seasonal changes and anticipation of environmental conditions such as low temperatures and desiccation.Photoperiodism was first described in detail by Garner and Allard in 1920 through the demonstration that many plants flower in response to changes in daylength (Garner and Allard, 1920).Subsequently, they showed that some plant species promote flowering when daylength falls below a critical daylength, whereas other plants accelerate flowering in response to daylengths longer than a critical daylength.These plants are called short-day (SD) and long-day (LD) plants, respectively.During the last decade, molecular-genetic approaches were applied to understanding the control of flowering time, mainly in the LD plant Arabidopsis, and notable progress has been made in identifying the molecular mechanisms by which Arabidopsis recognizes daylength and promotes flowering specifically under LDs.Also, recent genetic studies in rice enabled the mechanisms of the daylength response in this SD plant to be compared with those of Arabidopsis.Here we review the recent advances in understanding the regulatory mechanisms for daylength response of flowering in Arabidopsis and compare them with those of rice.
Article7 March 2017Open Access Transparent process PSEUDO RESPONSE REGULATORs stabilize CONSTANS protein to promote flowering in response to day length Ryosuke Hayama Ryosuke Hayama Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Liron Sarid-Krebs Liron Sarid-Krebs Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author René Richter René Richter Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Virginia Fernández Virginia Fernández Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Seonghoe Jang Seonghoe Jang Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author George Coupland Corresponding Author George Coupland [email protected] orcid.org/0000-0001-6988-4172 Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Ryosuke Hayama Ryosuke Hayama Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Liron Sarid-Krebs Liron Sarid-Krebs Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author René Richter René Richter Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Virginia Fernández Virginia Fernández Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Seonghoe Jang Seonghoe Jang Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author George Coupland Corresponding Author George Coupland [email protected] orcid.org/0000-0001-6988-4172 Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Author Information Ryosuke Hayama1,2, Liron Sarid-Krebs1, René Richter1, Virginia Fernández1, Seonghoe Jang1,3,4 and George Coupland *,1 1Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany 2Present address: International Christian University, Mitaka, Tokyo, Japan 3Present address: Biotechnology Center in Southern Taiwan (BCST), Tainan, Taiwan 4Present address: Agricultural Biotechnology Research Center, Academia Sinica, Nankang, Taipei, Taiwan *Corresponding author. Tel: +49 221 5062 205; Fax: +49 221 5062 207; E-mail: [email protected] The EMBO Journal (2017)36:904-918https://doi.org/10.15252/embj.201693907 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Seasonal reproduction in many organisms requires detection of day length. This is achieved by integrating information on the light environment with an internal photoperiodic time-keeping mechanism. Arabidopsis thaliana promotes flowering in response to long days (LDs), and CONSTANS (CO) transcription factor represents a photoperiodic timer whose stability is higher when plants are exposed to light under LDs. Here, we show that PSEUDO RESPONSE REGULATOR (PRR) proteins directly mediate this stabilization. PRRs interact with and stabilize CO at specific times during the day, thereby mediating its accumulation under LDs. PRR-mediated stabilization increases binding of CO to the promoter of FLOWERING LOCUS T (FT), leading to enhanced FT transcription and early flowering under these conditions. PRRs were previously reported to contribute to timekeeping by regulating CO transcription through their roles in the circadian clock. We propose an additional role for PRRs in which they act upon CO protein to promote flowering, directly coupling information on light exposure to the timekeeper and allowing recognition of LDs. Synopsis To promote flowering of Arabidopsis in response to day length, PSEUDO RESPONSE REGULATOR proteins interact with and stabilize the CONSTANS transcription factor at specific times of day. CONSTANS transcription factor is stabilized in the light by PSEUDO RESPONSE REGULATOR (PRR) proteins. In toc1 prr5 prr7 prr9 quadruple mutants, CO protein levels are reduced independently of the previously reported effects of prr mutations on CO transcription. Reduction of CO levels in toc1 prr5 prr7 prr9 plants requires the ubiquitin ligase COP1. PRR proteins interact directly with CO. In prr mutants, less CO protein is detected directly bound to its target gene FT. Introduction Many organisms recognize seasonal changes in their environment by perceiving day length and utilize this information to control key steps in their life cycle, such as the onset of reproduction or diapause. These responses, referred to as photoperiodism, allow organisms to adapt to high latitude where seasonal climatic fluctuations involving significant temperature changes occur. Photoperiodic responses are generally conferred by a mechanism that allows organisms to measure the length of day or night that fluctuates predictably during the year. This process involves comparison of the light environment against an internal photoperiodic time-keeping mechanism downstream of the circadian clock. In this system, the circadian clock provides an endogenous autonomous rhythm with a period length of ~24 h that regulates the photoperiodic timer. Day length strongly influences the timing of floral initiation in many plants, allowing them to adapt to higher latitude and enabling successful reproduction. Molecular and genetic studies using the model species Arabidopsis thaliana, which flowers specifically in response to photoperiod under long days (LDs) of spring, provided knowledge on the mechanistic framework that couples information on light exposure with the photoperiodic time-keeping mechanism and enables measurement of day length. The CONSTANS (CO) gene was originally isolated as a photoperiodic floral promoter and defined as the integrator of light and timing information. This is achieved through control of its transcription by the circadian clock and regulation of CO protein stability by light exposure (Andres & Coupland, 2012; Song et al, 2014). Accumulation of CO transcripts therefore forms a diurnal rhythm for timekeeping under LDs and SDs (Suarez-Lopez et al, 2001). Under LDs CO transcription is controlled by a clock-controlled blue light photoreceptor FLAVIN-BINDING, KELCH REPEAT, F-BOX1 (FKF1), clock-controlled CYCLING DOF FACTOR (CDFs) transcription factors, and a clock protein GIGANTEA (GI). In this mechanism, a FKF1-GI protein complex temporally promotes transcription of CO by binding to and initiating degradation of CDFs, suppressors of CO transcription, in a light-dependent manner (Imaizumi et al, 2003, 2005; Sawa et al, 2007; Fornara et al, 2009). Through this regulation, transcripts of CO accumulate in the afternoon under LDs when plants are exposed to light. This coincidence between CO mRNA and light exposure allows stabilization of translated CO protein at these times, causing CO to accumulate under LDs and achieving recognition of LDs (Appendix Fig S1; Valverde et al, 2004). CO then directly promotes floral transition through its capacity to activate transcription of the "florigen" gene FLOWERING LOCUS T (FT) specifically under LDs (Andres & Coupland, 2012; Song et al, 2014). By contrast, under SDs, CO transcription occurs only in the dark, and under these conditions, CO protein does not accumulate (Valverde et al, 2004). In contrast to current knowledge of the time-keeping mechanism associated with patterns of CO mRNA accumulation, how information after light exposure is transferred to CO protein is still unclear. The blue light photoreceptors cryptochrome 1 (cry1) and cryptochrome 2 (cry2) and the far-red photoreceptor phytochrome A (phyA) are required to stabilize CO protein (Valverde et al, 2004; Zuo et al, 2011). A protein complex of an E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) and SUPPRESSOR OF PHYA1 (SPA1), which are repressors of A. thaliana photomorphogenesis, mediates between the photoreceptors and CO protein stabilization (Laubinger et al, 2006; Jang et al, 2008; Liu et al, 2008; Zuo et al, 2011; Lau & Deng, 2012; Sarid-Krebs et al, 2015). During the night, the COP1/SPA1 protein complex physically interacts with CO in the nuclei to promote its proteasomal degradation, whereas during the day, the phyA and cry photoreceptors promote COP1 accumulation in the cytoplasm thus allowing CO to accumulate in the nucleus (Osterlund & Deng, 1998; Laubinger et al, 2006; Jang et al, 2008; Liu et al, 2008; Zuo et al, 2011; Sarid-Krebs et al, 2015). Since the COP1/SPA1 complex degrades CO protein during the night, it strongly reduces CO accumulation under SDs (Jang et al, 2008; Liu et al, 2008). However, in contrast to its clear biological function in the perception of SDs, COP1 also interferes with the recognition of LDs where a residual activity in the light further reduces CO protein levels during the day, perhaps because photoreceptor activity under LDs is insufficient to completely suppress COP1 function (Jang et al, 2008). FKF1 might overcome this effect because it has been demonstrated that FKF1 stabilizes CO under LDs (Song et al, 2012). However, activity of FKF1 is strictly limited to the afternoon, whereas CO stabilization occurs at other times during the light period, including the morning (Valverde et al, 2004; Song et al, 2012). Finally, the ubiquitin ligase HOS1 and the APETALA2 (AP2) family protein TARGET OF EAT1 (TOE1), GI, and an E3 ubiquitin ligase ZEITLUPE (ZTL) negatively regulate CO protein abundance, although how their activities are related to light signaling during photoperiodic flowering has not been elucidated (Lazaro et al, 2012, 2015; Song et al, 2014; Zhang et al, 2015). Thus, the mechanisms that ensure CO accumulation in response to light exposure to establish LD recognition and floral transition are not fully understood. In parallel to the studies of day-length measurement in A. thaliana, quantitative trait locus (QTL) analyses of flowering time identified PSEUDO RESPONSE REGULATOR (PRR) genes in a wide range of crop species. Ppd-H1, Ppd-1, BOLTING TIME CONTROL 1 (BvBTC1), PRR37, and SbPRR37 encode PRR proteins in barley, wheat, beet, rice, and sorghum, respectively, and allelic variation at these genes confers natural diversity in flowering time (Turner et al, 2005; Pin et al, 2009; Murphy et al, 2011; Campoli et al, 2012; Shaw et al, 2012; Koo et al, 2013). In A. thaliana, PRR proteins are encoded by a family of five genes and are defined as central components of the circadian clock (TIMING OF CAB EXPRESSION 1 (TOC1), PRR3, PRR5, PRR7, and PRR9) (Strayer et al, 2000; Ito et al, 2003; Yamamoto et al, 2003; Murakami et al, 2004; Para et al, 2007). The abundance of their transcripts and proteins exhibit circadian rhythms, and under diurnal conditions, they peak in expression sequentially at 2–3 h intervals during the light period in the order PRR9, PRR7, PRR5, PRR3, and TOC1 (Matsushika et al, 2000; Fujiwara et al, 2008). PRR9, PRR7, PRR5, and TOC1 proteins are degraded during the night, so they mainly accumulate during the day when they repress transcription of genes encoding other clock components such as LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) (Mas et al, 2003b; Farré & Kay, 2007; Ito et al, 2007; Kiba et al, 2007; Nakamichi et al, 2010; Huang et al, 2012). Mutations in the PRR genes also delay flowering under LDs (Nakamichi et al, 2005; Ito et al, 2008), but double or triple mutants exhibit much stronger phenotypes suggesting functional redundancy between the genes (Nakamichi et al, 2005, p. 549; Ito et al, 2008). Despite the significance of the PRRs in flowering, how they contribute to this process remains unclear. In A. thaliana, they appear to influence the time-keeping mechanism associated with CO transcription in the photoperiodic flowering pathway indirectly through their role in the circadian clock (Nakamichi et al, 2007; Ito et al, 2008). On the other hand, in crops such as barley and rice, their effects on CO transcription are relatively weak, whereas they strongly affect FT transcription (Turner et al, 2005; Campoli et al, 2012; Koo et al, 2013). Therefore, these PRRs are likely to have a more direct effect on the regulation of the photoperiodic flowering pathway. Here, we identify novel mechanisms contributing to day-length measurement and define unexpectedly direct roles for PRRs in photoperiodic flowering. We show that PRRs convey information on light exposure to the photoperiodic time-keeping mechanism by interacting with and stabilizing CO during the day, thereby allowing CO to accumulate at higher levels under LDs. In A. thaliana, diversity in temporal accumulation patterns among PRRs throughout the day contributes to detection of long-light periods and to CO accumulation in the morning and the evening specifically under LDs. Besides the function of PRRs in controlling the activity of the photoperiodic time-keeping mechanism associated with CO transcription through their role the circadian clock, we find unexpectedly that PRRs also transfer information on light exposure and day length to CO at the post-translational level. This level of regulation allows CO to optimally respond to LDs and accelerates the floral transition under these conditions. Results PRRs induce FT transcription independently of CO transcription In A. thaliana toc1 prr5, prr5 prr7, and prr7 prr9 double mutants, the levels of CO mRNA are slightly reduced, contributing to late flowering (Nakamichi et al, 2007; Ito et al, 2008). However, in these mutants, transcript levels of FLOWERING LOCUS T (FT), which is a direct target of CO and encodes a florigen protein (Tiwari et al, 2010; Andres & Coupland, 2012; Song et al, 2012), are drastically reduced. We confirmed the effects of prr5 prr7 and prr7 prr9 mutations on CO and FT mRNAs under LD. CO mRNA in these mutants was lower than in wild-type (WT) plants but still accumulated at some times, particularly 12 and 16 h after dawn, whereas FT mRNA was strongly reduced throughout the day (Fig EV1A and B). The effects of overexpression of PRR5 and PRR9 on CO and FT mRNA levels under LD were then tested. The abundance of CO mRNA was slightly lower in the overexpression plants compared to WT, especially at 12 and 16 h after dawn, whereas FT mRNA levels were inversely related to CO mRNA and elevated at these times (Fig 1A and B). These results are consistent with a hypothesis that PRRs upregulate FT mRNA level independently from affecting CO mRNA levels. Click here to expand this figure. Figure EV1. Effects of prr mutations on FT expression and flowering time in WT and SUC2::CO backgrounds A, B. Expression pattern of CO (A) and FT (B) mRNA in prr5 prr7 or prr7 prr9 double mutants under LD. Error bars indicate standard error within two biological replicates. In each replicate, two technical replicates were performed. C, D. Effect of single prr mutations on CO (C) and FT (D) mRNA accumulation in SUC2::CO plants under LD. Error bars indicate standard error within two technical replicates. E. Total leaf number and bolting time of prr mutants in the SUC2::CO background grown under LDs. Approximately 40 plants of each genotype were grown under LDs. Error bars indicate standard error. Statistical significance between SUC2::CO and each prr mutant, and among multiple prr mutants was calculated using Student's t-test; *P < 0.01; n.s. P > 0.01. Download figure Download PowerPoint Figure 1. PRRs induce FT transcription independently from regulating CO transcription A, B. Expression pattern of CO (A) and FT (B) mRNA in PRR5- or PRR9-overexpressors under LD. The values of CO and FT mRNA levels were normalized to those of PP2a. For all data, error bars indicate standard error within two biological replicates and in each of these two technical replicates were performed. Download figure Download PowerPoint To further analyze the role of PRRs in FT transcription, the effects of prr mutations on FT expression were tested in a transgenic line expressing CO from the SUC2 promoter (An et al, 2004), thereby uncoupling CO transcription from impaired clock function in prr mutants. The SUC2 promoter expresses CO specifically in the phloem companion cells where it acts in WT plants to promote FT transcription (An et al, 2004). In these plants, FT expression was highly induced compared with WT due to increased accumulation of CO mRNA specifically in the phloem cells (Fig 2A and B). The prr mutations were introduced singly and in combination into SUC2::CO plants. Single prr5, prr7, prr9, or toc1 mutations in SUC2::CO background did not dramatically affect CO or FT expression (Fig EV1C and D). By contrast, combining prr mutations in SUC2::CO reduced FT mRNA levels without significantly affecting CO mRNA (Fig 2C and D). In toc1 prr5 SUC2::CO, the levels of FT mRNA were slightly reduced compared to SUC2::CO. In toc1 prr5 prr7 SUC2::CO plants, FT mRNA was further reduced, and this effect was even more enhanced in toc1 prr5 prr7 prr9 SUC2::CO plants (Fig 2C and D). The flowering times of these lines were also measured, and the toc1 prr5 prr7 SUC2::CO as well as the toc1 prr5 prr7 prr9 SUC2::CO line exhibited late-flowering phenotypes compared to SUC2::CO, consistent with their reduced FT mRNA levels (Figs 2E and EV1E). Although FT mRNA levels were reduced in toc1 prr5 SUC2::CO plants compared to SUC2::CO, the mutant line was only mildly later flowering, presumably because FT levels are still so high in toc1 prr5 SUC2::CO that their effect on flowering is close to saturation. These results demonstrate that PRR proteins promote FT transcription independently from CO transcription. Figure 2. PRR genes contribute to SUC2::CO-mediated FT transcription A, B. Comparison of CO (A) and FT (B) mRNA levels between WT and SUC2::CO. C, D. Effect of double, triple, and quadruple prr mutations on CO (C) and FT (D) mRNA in SUC2::CO background under LDs. The values of CO and FT mRNA levels were normalized to those of PP2a. E. Effect of prr mutations on flowering time in SUC2::CO background under LDs. For each line, 16 plants were used for flowering-time measurement. Error bars indicate standard error. Statistical significance between SUC2::CO and each prr mutant, and among multiple prr mutants was calculated using Student's t-test; *P < 0.01; n.s. P > 0.01. Data information: For data in (A–D), error bars indicate standard error within two biological replicates and for each of these two technical replicates were performed. Download figure Download PowerPoint PRRs stabilize CO protein under LD Based on the observation that prr mutations can reduce FT transcript levels independently from CO mRNA, we hypothesized that the prr mutations act in part at the post-transcriptional level to reduce CO protein abundance. To test this idea, the prr mutations were introduced into SUC2::HA:CO, which allowed us to monitor HA:CO protein abundance in the prr mutants. In SUC2::HA:CO under LD, HA:CO protein accumulated during the day, but its levels were rapidly decreased in the night, although CO mRNA abundance remained high at this time (Fig 3A, B and D). This result is consistent with previous data demonstrating that in 35S::CO plants, where CO mRNA is constantly expressed, CO protein accumulates in the day and rapidly disappears in the night, due to light-mediated stabilization of CO protein (Valverde et al, 2004; Song et al, 2012). During the light period under LD, HA:CO levels in toc1 prr5 SUC2::HA:CO were slightly reduced compared to SUC2::HA:CO and a further reduction of HA:CO protein was observed in toc1 prr5 prr7 SUC2::HA:CO and even more dramatically in toc1 prr5 prr7 prr9 SUC2::HA:CO mutants (Fig 3A and B). In these lines, FT mRNA levels were reduced and late flowering was also observed, consistent with the reduced HA:CO protein levels (Figs 3C and EV2F). CO mRNA levels were not significantly affected in these lines (Fig 3D). Taken together, these data indicate that the PRRs act redundantly to enhance CO protein accumulation under LDs. Figure 3. PRR genes contribute to CO protein accumulation under LD A, B. Effect of double, triple, and quadruple prr mutations on HA:CO protein accumulation in SUC2::HA:CO under LD. C, D. FT (C) and CO (D) mRNA expression under the same conditions as in (A, B). E, F. Expression pattern of HA:CO protein in pCO::HA:CO under LD and SD. G, H. FT (G) and HA:CO (H) mRNA expression in pCO::HA:CO under LD and SD. I–L. Effect of prr9 and toc1 prr5 prr7 mutations on HA:CO protein accumulation in SUC2::HA:CO background under LD. Samples collected early in the day were harvested at Zeitgeber time (ZT) 1 for (I and J) and at ZT 0.5 for (K and L). Data information: The values of HA:CO levels were normalized to those of histone 3a. The values of CO and FT mRNA levels were normalized to those of PP2a. For all data except for (B), error bars indicate standard error within two biological replicates. For (B), error bars indicate standard error within three biological replicates. For the RNA analyses, two technical replicates were performed for each biological replicate. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Stabilization and accumulation of HA:CO through PRR-mediated inhibition of its proteasomal degradation, and its effect on flowering time A. Effect of MG132 treatment on recovery of HA:CO protein accumulation in toc1 prr5 prr7 prr9 SUC2::HA:CO. 10-day-old SUC2::HA:CO and toc1 prr5 prr7 prr9 SUC2::HA:CO were treated with either MG132 or DMSO at ZT5 and harvested at ZT8 under LD. The levels of HA:CO were normalized to those of H3a. The ratio of the values after MG132 treatment to those after DMSO treatment was calculated in each line, and fold change in HA:CO accumulation by the MG132 treatment was measured. Error bars indicate standard error within two biological replicates. In each replicate, two technical replicates were performed. B, C. Accumulation of HA:CO protein in pCO::HA:CO in the morning under LD and SD. D. Accumulation of HA:CO mRNA in pCO::HA:CO in the morning under LD and SD. Error bars indicate standard error within two technical replicates. E. Accumulation of FT mRNA in pCO::HA:CO in the morning under LD and SD, and FT mRNA accumulation in the morning requires CO. Error bars indicate standard error within two technical replicates. F. Total leaf number of prr mutants in the SUC2::HA:CO background under LDs. 16 plants of each genotype were grown under LDs. Error bars indicate standard error. Statistical significance between SUC2::HA:CO and each prr mutant, and among multiple prr mutants was calculated using Student's t-test; *P < 0.01; n.s. P > 0.01. Download figure Download PowerPoint When CO mRNA is expressed constitutively from the 35S promoter under LDs, CO protein peaks in abundance early in the morning and again in the afternoon (Valverde et al, 2004; Song et al, 2012). We tested this pattern in pCO::HA:CO lines in which HA:CO is expressed from the endogenous CO promoter. As in the 35S lines, the levels of HA:CO protein increased early in the morning and in the afternoon, and these peaks appeared more strongly under LD than SD (Figs 3E, F and H, and EV2B–D). The diurnal accumulation of FT mRNA was also similar to that of HA:CO protein (Figs 3G and EV2E), suggesting that both the morning and the evening peak of CO contribute to LD-induction of flowering. In a co mutant induction of FT in the morning was impaired, indicating that FT transcription in the morning depends on CO activity (Fig EV2E; Kim et al, 2005). Next, we analyzed the temporal HA:CO protein accumulation in single and higher order prr mutants at different times during the day and compared those to the WT. In agreement with previous reports on PRR9 function in the morning, HA:CO accumulation was specifically reduced in the morning in prr9 SUC2::HA:CO plants (Figs 3I–L and EV3A), whereas the toc1 prr5 prr7 mutations strongly reduced the evening peak under LD (Fig 3I–L; Fujiwara et al, 2008). These data indicate that diversity in the timing of expression and activity of PRRs cause CO to accumulate at specific times during the day to generate the typical LD-specific accumulation pattern. Previously, FKF1 was reported to stabilize CO protein specifically in the evening in 35S::CO plants (Song et al, 2012). However, in contrast to the dramatic reduction of HA:CO levels in toc1 prr5 prr7 prr9 SUC2::HA:CO, the fkf1 mutation did not strongly affect HA:CO levels in the same SUC2::HA:CO line (Fig EV3C–E). This result suggests that PRRs are more heavily involved than FKF1 in increasing CO protein levels when it is expressed from the SUC2 promoter in the phloem companion cells, the tissue in which CO promotes floral induction. Click here to expand this figure. Figure EV3. Effect of prr9 and fkf1 mutations on HA:CO accumulation A, B. Accumulation of HA:CO (A) and FT mRNA (B) in SUC2::HA:CO and prr9 SUC2::HA:CO in the morning. Each genotype was grown in four independent agar plates and harvested at ZT1 under LD. The levels of HA:CO and FT mRNA were analyzed in these samples. Error bars indicate standard error within these samples. Statistical significance was calculated using Student's t-test, *P < 0.01. As the control, HA:CO levels among those lines grown in one plate for each and harvested at ZT12 is shown. C–E. Effect of the fkf1-2 mutation on HA:CO accumulation in SUC2::HA:CO under LD. In (D), each genotype was grown on two independent agar plates and analyzed. Download figure Download PowerPoint PRRs contribute to light-mediated accumulation of CO PRR proteins accumulate during the day and are degraded during the night (Mas et al, 2003b; Farré & Kay, 2007; Ito et al, 2007; Kiba et al, 2007; Fujiwara et al, 2008), so they could directly contribute to CO protein stabilization in light. CO is also known to be stabilized in plants exposed to blue light (BL) and far-red light (FR) (Valverde et al, 2004). To understand whether CO protein abundance under different light regimes is dependent on PRR function, HA:CO protein accumulation was followed under BL and FR in SUC2::HA:CO plants and after introduction of prr mutations. In SUC2::HA:CO plants, the levels of HA:CO protein were low in the dark, but increased strongly in BL or FR independently of CO mRNA abundance (Fig 4A and B). The toc1 prr5 prr7 SUC2::HA:CO and toc1 prr5 prr7 prr9 SUC2::HA:CO lines showed strongly reduced HA:CO protein levels in both BL and FR when compared to SUC2::HA:CO (Fig 4A and B). Moreover, FT mRNA levels under both conditions correlated with the abundance of HA:CO protein, but not with CO mRNA (Fig 4C and D). Together with the observations that PRR proteins accumulate during the day, these results support the hypothesis that the PRR proteins contribute to light-mediated accumulation of CO. Figure 4. PRR genes are required for BL- and FR-mediated CO protein accumulation A, B. Effect of double, triple, and quadruple prr mutations on HA:CO protein accumulation in blue light (BL) and far-red light (FR). Plants were grown under LDs and then transferred to darkness for 24 h. The plants were then transferred to continuous BL or FR at ZT 0. A population of plants was kept in darkness (D) as control. HA:CO protein was analyzed in each genotype at the illustrated times under BL, FR, or control D treatment. C. FT mRNA accumulation under the same conditions as in (A, B). D. CO mRNA accumulation under the same conditions as in (A, B). E–G. PRRs suppress ability of COP1 to degrade CO. (E, F) Effect of quadruple prr, cop1, and quintuple prr/cop1 mutations on CO protein abundance in SUC2::HA:CO background through a time course under LDs. (G) CO mRNA levels in genotypes used in (F). H, I. Abundance of HY5 protein in prr quadruple mutant in continuous light (LL) and continuous dark (DD). Plants were grown under LDs for 7 days, transferred to LL or DD at ZT 0 and kept for 40 h before harvesting. Data information: The values of HA:CO and HY5 levels were normalized to those of histone 3a. Student's t-test revealed no significant difference between genotypes. The values of CO and FT mRNA levels were normalized by those of PP2a. For all data except for (B), error bars indicate standard error within two biological replicates. For (B), error bars indicate standard error within three biological replicates. For the RNA analyses, two technical replicates were performed in each biological replicate. Download figure Download PowerPoint In A. thaliana light-signaling pathways, COP1 acts as a negative regulator to suppress photomorphogenesis by targeting proteins such as the transcription factor ELONGATED HYPOCOTYL 5 (HY5) for proteasomal degradation during the dark (Osterlund et al, 2000). In the light, phys and crys suppress COP1 function, allowing HY5 to accumulate
