HYPERSENSITIVE TO RED AND BLUE 1 and its C-terminal regulatory function control FLOWERING LOCUS T expression

The red and far-red light-absorbing phytochromes and UV-A/blue light-absorbing cryptochromes regulate seedling de-etiolation and flowering responses. The signaling steps that mediate the photoreceptor regulation on key flowering genes remain largely unknown. We report that a previously identified photomorphogenic mutant, hypersensitive to red and blue 1 (hrb1), flowered late and showed attenuated expression of FLOWERING LOCUS T (FT) over both long days and short days. Transgenic plants that overexpress the full-length HRB1, or its C-terminal half, flowered early and accumulated more FT messages under short-day conditions. The transgenic plants also displayed hyposensitive de-etiolation phenotypes, and the expression of these phenotypes requires the action of PIF4. The double mutant of hrb1/cry2 showed a flowering phenotype and an FT expression pattern similar to hrb1 under long-day conditions, suggesting that HRB1 may function downstream of cry2 under long-day conditions. In contrast, hrb1/phyB-9 showed a flowering phenotype and an FT expression pattern similar to phyB-9 over both long days and short days, indicating a modulatory role of HRB1 in the flowering pathway mediated by phyB. Overexpression of HRB1 did not affect the expression of the central clock oscillators, TOC1 and CCA1. HRB1 therefore represents a signaling step that regulates FT expression downstream of red and blue light perception.     

Characterization of circadian-associated pseudo-response regulators: II. The function of PRR5 and its molecular dissection in Arabidopsis thaliana

Together with PRR1/TOC1, PRR5 belongs to the small family of Pseudo-Response Regulators (PRRs), which function as clock components of Arabidopsis thaliana. We employed a set of transgenic lines, each of which was designed to misexpress a truncated form of the PRR5 molecule, together with the original transgenic line (named PRR5-ox) that misexpresses the entire PRR5 polypeptide. The results of genetic analysis suggested that PRR5-ox seedlings showed a phenotype of hypersensitivity to red light during early photomorphogenesis in a manner dependent on red light photoreceptors (PhyA and PhyB), but independent of PRR1/TOC1. The set of newly constructed transgenic lines (named PRR5-N-ox and PRR5-C-ox) were also characterized in terms of circadian-associated phenotypes. The results suggest that the N-terminal pseudo-receiver domain of the PRR5 molecule seems to be dispensable for the misexpressed PRR5 molecule to bring about the phenotype of red light sensitivity. However, PRR5-N-ox plants, misexpressing only the pseudo-receiver domain, showed a phenotype of long period of free-running circadian rhythms of certain clock-controlled genes. Considering these and other results, we discuss the structure and function of PRR5 in the context of current views of the circadian clock in higher plants.     

Genetic linkages between circadian clock-associated components and phytochrome-dependent red light signal transduction in Arabidopsis thaliana

The current best candidates for Arabidopsis thaliana clock components are CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) and its homolog LHY (LATE ELONGATED HYPOCOTYL). In addition, five members of a small family, PSEUDO-RESPONSE REGULATORS (including PRR1, PRR3, PRR5, PRR7 and PRR9), are believed to be another type of clock component. The originally described member of PRRs is TOC1 (or PRR1) (TIMING OF CAB EXPRESSION 1). Interestingly, seedlings of A. thaliana carrying a certain lesion (i.e. loss-of-function or misexpression) of a given clock-associated gene commonly display a characteristic phenotype of light response during early photomorphogenesis. For instance, cca1 lhy double mutant seedlings show a shorter hypocotyl length than the wild type under a given fluence rate of red light (i.e. hypersensitivity to red light). In contrast, both toc1 single and prr7 prr5 double mutant seedlings with longer hypocotyls are hyposensitive under the same conditions. These phenotypes are indicative of linkage between the circadian clock and red light signal transduction mechanisms. Here this issue was addressed by conducting combinatorial genetic and epistasis analyses with a large number of mutants and transgenic lines carrying lesions in clock-associated genes, including a cca1 lhy toc1 triple mutant and a cca1 lhy prr7 prr5 quadruple mutant. Taking these results together, we propose a genetic model for clock-associated red light signaling, in which CCA1 and LHY function upstream of TOC1 (PRR1) in a negative manner, in turn, TOC1 (PRR1) serves as a positive regulator. PRR7 and PRR5 also act as positive regulators, but independently from TOC1 (PRR1). It is further suggested that these signaling pathways are coordinately integrated into the phytochrome-mediated red light signal transduction pathway, in which PIF3 (PHYTOCHROME-INTERACTING FACTOR 3) functions as a negative regulator immediately downstream of phyB.     

Characterization of Circadian-associated pseudo-response regulators: I. Comparative studies on a series of transgenic lines misexpressing five distinctive PRR Genes in Arabidopsis thaliana

Every member of a small family of Pseudo-Response Regulator (PRR) genes, including Timing of Cab Expression 1 (TOC1 [or PRR1]), are believed to play roles close to the circadian clock in the model higher plant Arabidopsis thaliana. In this study we established a transgenic line that misexpresses (or overexpresses) the PRR7 gene. As compared with wild-type plants, the resulting PRR7-misexpressing plants (designated PRR7-ox) showed characteristic phenotypes as to hallmarked circadian-associated biological events: (i) early flowering in a manner independent of photoperiodicity, (ii) hypersensitive response to red light during early photomorphogenesis, and (iii) altered free-running rhythms with long period of clock-associated genes. Finally, a series of all transgenic lines (PRR1-ox, PRR3-ox, PRR5-ox, PRR7-ox, and PRR9-ox) were characterized comparatively with regard to their clock-associated roles. The results suggested that the five homologous PRR factors play coordinate roles, distinctively from one another, and closely to the circadian clock in higher plants.     

Characterization of circadian-associated APRR3 pseudo-response regulator belonging to the APRR1/TOC1 quintet in Arabidopsis thaliana

In higher plants, there are wide ranges of biological processes that are controlled through the circadian clock. In this connection, we have been characterizing a small family of proteins, designated as ARABIDOPSIS PSEUDO-RESPONSE REGULATORS (APRR1, APRR3, APRR5, APRR7, and APRR9), among which APRR1 is identical to TOC1 (TIMING OF CAB EXPRESSION1) that is believed to be a component of the central oscillator. Through previous genetic studies, several lines of evidence have already been provided to support the view that, not only APRR1/TOC1, but also other APRR1/TOC1 quintet members are important for a better understanding of the molecular links between circadian rhythm, control of flowering time, and also photomorphogenesis. However, the least characterized one was APRR3 in that no genetic study has been conducted to see if APRR3 also plays an important role in the circadian-associated biological events. Here we show that APRR3-overexpressing transgenic plants (APRR3-ox) exhibited: (i). a phenotype of longer period (and/or delayed phase) of rhythms of certain circadian-controlled genes under continuous white light, (ii). a phenotype of late flowering under long-day photoperiod conditions, (iii). a phenotype of hypo-sensitiveness to red light during early photomorphogenesis of de-etiolated seedlings, supporting the current idea as to the APRR1/TOC1 quintet described above.     

Dual role of TOC1 in the control of circadian and photomorphogenic responses in Arabidopsis

To examine the role of the TOC1 (TIMING OF CAB EXPRESSION1) gene in the Arabidopsis circadian system, we generated a series of transgenic plants expressing a gradation in TOC1 levels. Silencing of the TOC1 gene causes arrhythmia in constant darkness and in various intensities of red light, whereas in blue light, the clock runs faster in silenced plants than in wild-type plants. Increments in TOC1 gene dosage delayed the pace of the clock, whereas TOC1 overexpression abolished rhythmicity in all light conditions tested. Our results show that TOC1 RNA interference and toc1-2 mutant plants displayed an important reduction in sensitivity to red and far-red light in the control of hypocotyl elongation, whereas increments in TOC1 gene dosage clearly enhanced light sensitivity. Furthermore, the red light-mediated induction of CCA1/LHY expression was decreased in TOC1 RNA interference and toc1-2 mutant plants, indicating a role for TOC1 in the phytochrome regulation of circadian gene expression. We conclude that TOC1 is an important component of the circadian clock in Arabidopsis with a crucial function in the integration of light signals to control circadian and morphogenic responses.     

RED1 is necessary for phytochrome B-mediated red light-specific signal transduction in Arabidopsis.

Seedlings of a transgenic Arabidopsis line (ABO) that overexpresses phytochrome B (phyB) display enhanced deetiolation specifically in red light. To identify genetic loci necessary for phytochrome signal transduction in red light, we chemically mutagenized ABO seeds and screened M2 seedlings for revertants of the enhanced deetiolation response. One recessive, red light-specific extragenic revertant, designated red1, was isolated. The mutant phenotype was expressed in the original ABO background as well as in the nontransgenic Nossen (No-0) progenitor background. red1 is also deficient in several other aspects of red light-induced responses known to be mediated by phyB, such as inhibition of petiole elongation and the shade avoidance response. red1 was mapped to the bottom of chromosome 4 at a position distinct from all known photoreceptor loci. Together with complementation analysis, the data show that red1 is a novel photomorphogenic mutant. The evidence suggests that red1 represents a putative phytochrome signal transduction mutant potentially specific to the phyB pathway.     

Phytochrome-regulated PIL1 derepression is developmentally modulated

We define the photoresponsiveness, during seedling de-etiolation,of PHYTOCHROME-INTERACTING FACTOR 3-LIKE 1 (PIL1), initiallyidentified by microarray analysis as an early-response genethat is robustly repressed by first exposure to light. We showthat PIL1 mRNA abundance declines rapidly, with a half-timeof 15 min, to a new steady-state level, 10-fold below the initialdark level, within 45 min of first exposure to red light. Analysisof phy-null mutants indicates that multiple phytochromes, includingphyA and phyB, impose this repression. Conversely, PIL1 expressionis rapidly derepressed by subsequent far-red irradiation ofpreviously red light-exposed seedlings. However, the magnitudeof this derepression is modulated over time, in a biphasic manner,in response to increasing duration of pre-exposure to continuousred light: (i) an early phase (up to about 6 h) of relativelyrapidly increasing effectiveness of far-red reversal of repression,as declining phyA levels relieve initial very low fluence suppressionof this response; and (ii) a second phase (beyond 6 h) of graduallydeclining effectiveness of far-red reversal, to only 20% ofmaximal derepression, within 36 h of continuous red light exposure,with no evidence of circadian modulation of this responsiveness,an observation in striking contrast to a previous report forentrained, green seedlings exposed to vegetative shade. Thesedata, together with analysis of phytochrome signaling mutantsand overexpressors with aberrant de-etiolation phenotypes, suggestthat the second-phase decline in robustness of PIL1 derepressionis an indirect consequence of the global developmental transitionfrom the etiolated to the de-etiolated state, and that circadiancoupling of derepression requires entrainment.     

Physiological interactions of phytochromes A, B1 and B2 in the control of development in tomato

The role of phytochrome B2 (phyB2) in the control of photomorphogenesis in tomato (Solanum lycopersicum L.) has been investigated using recently isolated mutants carrying lesions in the PHYB2 gene. The physiological interactions of phytochrome A (phyA), phytochrome B1 (phyB1) and phyB2 have also been explored, using an isogenic series of all possible mutant combinations and several different phenotypic characteristics. The loss of phyB2 had a negligible effect on the development of white-light-grown wild-type or phyA-deficient plants, but substantially enhanced the elongated pale phenotype of the phyB1 mutant. This redundancy was also seen in the control of de-etiolation under continuous red light (R), where the loss of phyB2 had no detectable effect in the presence of phyB1. Under continuous R, phyA action was largely independent of phyB1 and phyB2 in terms of the control of hypocotyl elongation, but antagonized the effects of phyB1 in the control of anthocyanin synthesis, indicating that photoreceptors may interact differently to control different traits. Irradiance response curves for anthocyanin synthesis revealed that phyB1 and phyB2 together mediate all the detectable response to high-irradiance R, and, surprisingly, that the phyA-dependent low-irradiance component is also strongly reduced in the phyB1 phyB2 double mutant. This is not associated with a reduction in phyA protein content or responsiveness to continuous far-red light (FR), suggesting that phyB1 and phyB2 specifically influence phyA activity under low-irradiance R. Finally, the phyA phyB1 phyB2 triple mutant showed strong residual responsiveness to supplementary daytime FR, indicating that at least one of the two remaining phytochromes plays a significant role in tomato photomorphogenesis.     

Resetting of the circadian clock by phytochromes and cryptochromes in Arabidopsis.

The authors sought to investigate the role of phytochromes A and B (phyA and phyB) and cryptochromes 1 and 2 (cryl and cry2) in the synchronization of the leaf position rhythm in Arabidopsis thaliana. The seedlings were transferred from white light-dark cycles to free-running conditions with or without exposure to a light treatment during the final hours of the last dark period. The phase advance caused by a far-red light treatment was absent in the phyA mutant, deficient in the fhy1 and fhy3 mutants involved in phyA signaling, and normal in the cryl and cryl cry2 mutants. The phase shift caused by blue light was normal in the cry2 mutant; reduced in the phyA, cryl, phyA cry1, and cry1 cry2 mutants; and abolished in the phyA cryl cry2 triple mutant. The phase shift caused by red light was partially retained by the phyA phyB double mutant. The authors conclude that cryl and cry2 participate as photoreceptors in the blue light input to the clock but are not required for the phyA-mediated effects on the phase of the circadian rhythm of leaf position. The signaling proteins FHY1 and FHY3 are shared by phyA-mediated photomorphogenesis and phyA input to the clock.     

Light- and temperature-entrainable circadian clock in soybean development

In plants, the spatiotemporal expression of circadian oscillators provides adaptive advantages in diverse species. However, the molecular basis of circadian clock in soybean is not known. In this study, we used soybean hairy roots expression system to monitor endogenous circadian rhythms and the sensitivity of circadian clock to environmental stimuli. We discovered in experiments with constant light and temperature conditions that the promoters of clock genes GmLCLb2 and GmPRR9b1 drive a self-sustained, robust oscillation of about 24-h in soybean hairy roots. Moreover, we demonstrate that circadian clock is entrainable by ambient light/dark or temperature cycles. Specifically, we show that light and cold temperature pulses can induce phase shifts of circadian rhythm, and we found that the magnitude and direction of phase responses depends on the specific time of these two zeitgeber stimuli. We obtained a quadruple mutant lacking the soybean gene GmLCLa1, LCLa2, LCLb1, and LCLb2 using CRISPR, and found that loss-of-function of these four GmLCL orthologs leads to an extreme short-period circadian rhythm and late-flowering phenotype in transgenic soybean. Our study establishes that the morning-phased GmLCLs genes act constitutively to maintain circadian rhythmicity and demonstrates that their absence delays the transition from vegetative growth to reproductive development.     

Interaction of phytochromes A and B in the control of de-etiolation and flowering in pea

The interactions of phytochrome A (phyA) and phytochrome B (phyB) in the photocontrol of vegetative and reproductive development in pea have been investigated using null mutants for each phytochrome. White-light-grown phyA phyB double mutant plants show severely impaired de-etiolation both at the seedling stage and later in development, with a reduced rate of leaf production and swollen, twisted internodes, and enlarged cells in all stem tissues. PhyA and phyB act in a highly redundant manner to control de-etiolation under continuous, high-irradiance red light. The phyA phyB double mutant shows no significant residual phytochrome responses for either de-etiolation or shade-avoidance, but undergoes partial de-etiolation in blue light. PhyB is shown to inhibit flowering under both long and short photoperiods and this inhibition is required for expression of the promotive effect of phyA. PhyA is solely responsible for the promotion of flowering by night-breaks with white light, whereas phyB appears to play a major role in detection of light quality in end-of-day light treatments, night breaks and day extensions. Finally, the inhibitory effect of phyB is not graft-transmissible, suggesting that phyB acts in a different manner and after phyA in the control of flower induction.     

Arabidopsis response regulators ARR3 and ARR4 play cytokinin-independent roles in the control of circadian period

Light and temperature are potent environmental signals used to synchronize the circadian oscillator with external time and photoperiod. Phytochrome and cryptochrome photoreceptors integrate light quantity and quality to modulate the pace and phase of the clock. PHYTOCHROME B (phyB) controls period length in red light as well as the phase of the clock in white light. phyB interacts with ARABIDOPSIS RESPONSE REGULATOR4 (ARR4) in a light-dependent manner. Accordingly, we tested ARR4 and other members of the type-A ARR family for roles in clock function and show that ARR4 and its closest relative, ARR3, act redundantly in the Arabidopsis thaliana circadian system. Loss of ARR3 and ARR4 lengthens the period of the clock even in the absence of light, demonstrating that they do so independently of active phyB. In addition, in white light, arr3,4 mutants show a leading phase similar to phyB mutants, suggesting that circadian light input is modulated by the interaction of phyB with ARR4. Although type-A ARRs are involved in cytokinin signaling, the circadian defects appear to be independent of cytokinin, as exogenous cytokinin affects the phase but not the period of the clock. Therefore, ARR3 and ARR4 are critical for proper circadian period and define an additional level of regulation of the circadian clock in Arabidopsis.