Ubiquitin carboxyl-terminal hydrolases are required for period maintenance of the circadian clock at high temperature in Arabidopsis.

Protein ubiquitylation participates in a number of essential cellular processes including signal transduction and transcription, often by initiating the degradation of specific substrates through the 26S proteasome. Within the ubiquitin-proteasome system, deubiquitylating enzymes (DUBs) not only help generate and maintain the supply of free ubiquitin monomers, they also directly control functions and activities of specific target proteins by modulating the pool of ubiquitylated species. Ubiquitin carboxyl-terminal hydrolases (UCHs) belong to an enzymatic subclass of DUBs, and are represented by three members in Arabidopsis, UCH1, UCH2 and UCH3. UCH1 and UCH2 influence auxin-dependent developmental pathways in Arabidopsis through their deubiquitylation activities, whereas biological and enzymatic functions of UCH3 remain unclear. Here, we demonstrate that Arabidopsis UCH3 acts to maintain the period of the circadian clock at high temperatures redundantly with UCH1 and UCH2. Whereas single uch1, uch2 and uch3 mutants have weak circadian phenotypes, the triple uch mutant displays a drastic lengthening of period at high temperatures that is more extreme than the uch1 uch2 double mutant. UCH3 also possesses a broad deubiquitylation activity against a range of substrates that link ubiquitin via peptide and isopeptide linkages. While the protein target(s) of UCH1-3 are not yet known, we propose that these DUBs act on one or more factors that control period length of the circadian clock through removal of their bound ubiquitin moieties, thus ensuring that the clock oscillates with a proper period even at elevated temperatures.

Temporal restriction of salt inducibility in expression of salinity-stress related gene by the circadian clock in Solanum lycopersicum.

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.

PSEUDO RESPONSE REGULATORs stabilize CONSTANS protein to promote flowering in response to day length.

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.

Variation in the flowering gene SELF PRUNING 5G promotes day-neutrality and early yield in tomato.

Plants evolved so that their flowering is triggered by seasonal changes in day length. However, day-length sensitivity in crops limits their geographical range of cultivation, and thus modification of the photoperiod response was critical for their domestication. Here we show that loss of day-length-sensitive flowering in tomato was driven by the florigen paralog and flowering repressor SELF-PRUNING 5G (SP5G). SP5G expression is induced to high levels during long days in wild species, but not in cultivated tomato because of cis-regulatory variation. CRISPR/Cas9-engineered mutations in SP5G cause rapid flowering and enhance the compact determinate growth habit of field tomatoes, resulting in a quick burst of flower production that translates to an early yield. Our findings suggest that pre-existing variation in SP5G facilitated the expansion of cultivated tomato beyond its origin near the equator in South America, and they provide a compelling demonstration of the power of gene editing to rapidly improve yield traits in crop breeding.

Mutations in the N-terminal kinase-like domain of the repressor of photomorphogenesis SPA1 severely impair SPA1 function but not light responsiveness in Arabidopsis.

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.

A loss-of-function mutation in the DWARF4/PETANKO5 gene enhances the late-flowering and semi-dwarf phenotypes of the Arabidopsis clock mutant lhy-12;cca1-101 under continuous light without affecting FLC expression.

The circadian clock plays important roles in the control of photoperiodic flowering in Arabidopsis. Mutations in the LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) genes (lhy;cca1) accelerate flowering under short days, whereas lhy;cca1 delays flowering under continuous light (LL). The lhy;cca1 mutant also exhibits short hypocotyls and petioles under LL. However, the molecular mechanisms underlying the regulation of both flowering time and organ lengths in the LHY/CCA1-dependent pathway are not fully understood. To address these questions, we performed EMS mutagenesis of the lhy-12;cca1-101 line and screened for mutations that enhance the lhy;cca1 phenotypes under LL. In this screen, we identified a novel allele of dwarf4 (dwf4) and named it petanko 5 (pta5). A similar level of enhancement of the delay in flowering was observed in these two dwf4 mutants when combined with the lhy;cca1 mutations. The lhy;cca1 and dwf4 mutations did not significantly affect the expression level of the floral repressor gene FLC under LL. Our results suggest that a defect in brassinosteroid (BR) signaling delayed flowering independent of the FLC expression level, at least in plants with the lhy;cca1 mutation grown under LL. The dwf4/pta5 mutation did not enhance the late-flowering phenotype of plants overexpressing SVP under LL, suggesting that SVP and BR function in a common pathway that controls flowering time. Our results suggest that the lhy;cca1 mutant exhibits delayed flowering due to both the BR signaling-dependent and -independent pathways under LL.

Density effects on late flowering mutants of Arabidopsis thaliana under continuous light.

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.

Density effects on semi-dwarf and early flowering mutants of Arabidopsis thaliana under continuous light.

Plant growth promotion and inhibition under low- and high-density conditions (referred to as the density effect) has been studied extensively. Here, we show that such density effects were unaffected by the position of wild-type (WT) and gibberellic acid insensitive (gai) strains of Arabidopsis thaliana (Arabidopsis) within pots. Additionally, petanko 1 (pta1) and pta5 were newly discovered alleles of the ROTUNDIFOLIA 3 (ROT3) and DWARF 4 (DWF4) genes that are involved in brassinosteroid biosynthesis. Unlike gai, the semi-dwarf mutants of pta1 and pta5 exhibited normal flowering times and a shortening of rosette leaves at high densities. Moreover, the pta1 and pta5 variants suppressed flowering stem shortening at high densities. pta5, but not pta1 suppressed the reduction in silique number at intermediate densities. SPINDLY (SPY) is a negative regulator of GA signaling, while PHYTOCHROME B (PHYB) is a red-light photoreceptor. High-density growth did not reduce the flowering time of phyB mutants, but did affect that of spy mutants. Neither spy nor phyB suppressed the shortening of rosette leaves at high densities; however, spy suppressed flowering stem shortening. Moreover, spy suppressed the reduction of silique number at high densities, while and phyB promoted the decrease. These data suggest that GA, BR, and light signaling pathways play important roles in the density effect.

Phosphorylation of CONSTANS and its COP1-dependent degradation during photoperiodic flowering of Arabidopsis.

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.

A circadian rhythm set by dusk determines the expression of FT homologs and the short-day photoperiodic flowering response in Pharbitis.

Seasonal control of flowering through responsiveness to daylength shows extreme variation. Different species flower in response to long days or short days (SDs), and this difference evolved several times. The molecular mechanisms conferring these responses have been compared in detail only in Arabidopsis thaliana and rice (Oryza sativa) and suggest that a conserved pathway confers daylength responses through regulation of FLOWERING LOCUS T (FT) transcription by CONSTANS (CO). We studied Pharbitis (Ipomoea nil; formerly, Pharbitis nil), a widely used SD model species and a member of the Convolvulaceae, and showed using transgenic plants together with detailed expression analysis that two putative orthologs of FT (Pn FT1 and Pn FT2) promote flowering specifically under SDs. These genes are expressed only under SDs, and light flashes given during the night reduce their expression and prevent flowering. We demonstrate that in Pharbitis a circadian rhythm set by the light-to-dark transition at dusk regulates Pn FT expression, which rises only when the night is longer than 11 h. Furthermore, Pharbitis accessions that differ in their critical night-length responses express Pn FT at different times after dusk, demonstrating that natural genetic variation influencing the clock regulating Pn FT expression alters the flowering response. In these assays, Pn FT mRNA abundance was not related to Pn CO expression, suggesting that Pn FT may be regulated by a different transcription factor in Pharbitis. We conclude that SD response in Pharbitis is controlled by a dedicated light sensitive clock, set by dusk, that activates Pn FT transcription in darkness, a different mechanism for measuring daylength than described for Arabidopsis and rice.

Adaptation of photoperiodic control pathways produces short-day flowering in rice.

The photoperiodic control of flowering is one of the important developmental processes of plants because it is directly related to successful reproduction. Although the molecular genetic analysis of Arabidopsis thaliana, a long-day (LD) plant, has provided models to explain the control of flowering time in this species, very little is known about its molecular mechanisms for short-day (SD) plants. Here we show how the photoperiodic control of flowering is regulated in rice, a SD plant. Overexpression of OsGI, an orthologue of the Arabidopsis GIGANTEA (GI) gene in transgenic rice, caused late flowering under both SD and LD conditions. Expression of the rice orthologue of the Arabidopsis CONSTANS (CO) gene was increased in the transgenic rice, whereas expression of the rice orthologue of FLOWERING LOCUS T (FT) was suppressed. Our results indicate that three key regulatory genes for the photoperiodic control of flowering are conserved between Arabidopsis, a LD plant, and rice, a SD plant, but regulation of the FT gene by CO was reversed, resulting in the suppression of flowering in rice under LD conditions.

Shedding light on the circadian clock and the photoperiodic control of flowering.

Recently, notable progress has been made towards understanding the genetic interactions that underlie the function of the circadian clock in plants, and how these functions are related to the seasonal control of flowering time. The LHY/CCA1 and TOC1 genes have been proposed to participate in a negative feedback loop that is part of the central oscillator of the circadian clock. Furthermore, analysis of a flowering-time pathway has suggested how transcriptional regulation by the circadian clock, combined with post-transcriptional regulation by light, could activate proteins that control flowering time in response to appropriate daylengths.