Molecular dissection of the roles of phytochrome in photoperiodic flowering in rice

Phytochromes mediate the photoperiodic control of flowering in rice (Oryza sativa), a short-day plant. Recent molecular genetics studies have revealed a genetic network that enables the critical daylength response of florigen gene expression. Analyses using a rice phytochrome chromophore-deficient mutant, photoperiod sensitivity5, have so far revealed that within this network, phytochromes are required for expression of Grain number, plant height and heading date7 (Ghd7), a floral repressor gene in rice. There are three phytochrome genes in rice, but the roles of each phytochrome family member in daylength response have not previously been defined. Here, we revealed multiple action points for each phytochrome in the critical daylength response of florigen expression by using single and double phytochrome mutant lines of rice. Our results show that either phyA alone or a genetic combination of phyB and phyC can induce Ghd7 mRNA, whereas phyB alone causes some reduction in levels of Ghd7 mRNA. Moreover, phyB and phyA can affect Ghd7 activity and Early heading date1 (a floral inducer) activity in the network, respectively. Therefore, each phytochrome gene of rice has distinct roles, and all of the phytochrome actions coordinately control the critical daylength response of florigen expression in rice.     

Isolation of rice genes possibly involved in the photoperiodic control of flowering by a fluorescent differential display method

To better understand the molecular mechanisms of the photoperiodic regulation of rice, a short-day plant, we isolated 27 cDNAs that were differentially expressed in the photoperiod-insensitive se5 mutant from approximately 8,400 independent mRNA species by the use of a fluorescent differential display (FDD). For this screening, we isolated mRNAs at five different time points during the night and compared their expression patterns between se5 and the wild type. Of 27 cDNAs isolated, 12 showed diurnal expression patterns often associated with genes involved in the determination of the flowering time. In se5, expression of nine cDNAs was increased. Five of these cDNAs were up-regulated under SD, suggesting that they may promote flowering under SD. They included genes encoding a cDNA containing a putative NAC domain, the fructose-bisphosphate aldolase, and a protease inhibitor. Expression of three cDNAs was decreased in se5 but not photoperiodically regulated. These cDNAs included a rice homolog of Arabidopsis GIGANTEA (GI), lir1, and a gene for myo-inositol 1-phosphate synthase, all of which were previously shown to be under the control of circadian clocks. The expression patterns of the rice homolog of GI, OsGI, were similar to those of the Arabidopsis GI, suggesting the conservation of some mechanisms for the photoperiodic regulation of flowering between these two species.     

A phytochrome-associated protein phosphatase 2A modulates light signals in flowering time control in Arabidopsis

Reversible protein phosphorylation, which is catalyzed by functionally coupled protein kinases and protein phosphatases, is a major signaling mechanism in eukaryotic cellular functions. The red and far-red light-absorbing phytochrome photoreceptors are light-regulated Ser/Thr-specific protein kinases that regulate diverse photomorphogenic processes in plants. Here, we demonstrate that the phytochromes functionally interact with the catalytic subunit of a Ser/Thr-specific protein phosphatase 2A designated FyPP. The interactions were influenced by phosphorylation status and spectral conformation of the phytochromes. Recombinant FyPP efficiently dephosphorylated oat phytochrome A in the presence of Fe(2+) or Zn(2+) in a spectral form-dependent manner. FyPP was expressed predominantly in floral organs. Transgenic Arabidopsis plants with overexpressed or suppressed FyPP levels exhibited delayed or accelerated flowering, respectively, indicating that FyPP modulates phytochrome-mediated light signals in the timing of flowering. Accordingly, expression patterns of the clock genes in the long-day flowering pathway were altered greatly. These results indicate that a self-regulatory phytochrome kinase-phosphatase coupling is a key signaling component in the photoperiodic control of flowering.     

Ef7 encodes an ELF3-like protein and promotes rice flowering by negatively regulating the floral repressor gene Ghd7 under both short- and long-day conditions

Much progress has been made in our understanding of photoperiodic flowering of rice and the mechanisms underlying short-day (SD) promotion and long-day (LD) repression of floral induction. In this study, we identified and characterized the Ef7 gene, one of the rice orthologs of Arabidopsis EARLY FLOWERING 3 (ELF3). The ef7 mutant HS276, which was induced by γ-irradiation of the japonica rice cultivar 'Gimbozu', flowers late under both SD and LD conditions. Expression analyses of flowering time-related genes demonstrated that Ef7 negatively regulates the expression of Ghd7, which is a repressor of the photoperiodic control of rice flowering, and consequently up-regulates the expression of the downstream Ehd1 and FT-like genes under both SD and LD conditions. Genetic analyses with a non-functional Ghd7 allele provided further evidence that the delayed flowering of ef7 is mediated through the Ghd7 pathway. The analysis of light-induced expression of Ghd7 revealed that the ef7 mutant was more sensitive to red light than the wild-type plant, but the gate of Ghd7 expression was unchanged. Thus, our results show that Ef7 functions as a floral promoter by repressing Ghd7 expression under both SD and LD conditions.     

Bottom-up Assembly of the Phytochrome Network

Plants have developed sophisticated systems to monitor and rapidly acclimate to environmental fluctuations. Light is an essential source of environmental information throughout the plant’s life cycle. The model plant Arabidopsis thaliana possesses five phytochromes (phyA-phyE) with important roles in germination, seedling establishment, shade avoidance, and flowering. However, our understanding of the phytochrome signaling network is incomplete, and little is known about the individual roles of phytochromes and how they function cooperatively to mediate light responses. Here, we used a bottom-up approach to study the phytochrome network. We added each of the five phytochromes to a phytochrome-less background to study their individual roles and then added the phytochromes by pairs to study their interactions. By analyzing the 16 resulting genotypes, we revealed unique roles for each phytochrome and identified novel phytochrome interactions that regulate germination and the onset of flowering. Furthermore, we found that ambient temperature has both phytochrome-dependent and -independent effects, suggesting that multiple pathways integrate temperature and light signaling. Surprisingly, none of the phytochromes alone conferred a photoperiodic response. Although phyE and phyB were the strongest repressors of flowering, both phyB and phyC were needed to confer a flowering response to photoperiod. Thus, a specific combination of phytochromes is required to detect changes in photoperiod, whereas single phytochromes are sufficient to respond to light quality, indicating how phytochromes signal different light cues.     

Phytochrome control of flowering is temperature sensitive and correlates with expression of the floral integrator FT

In Arabidopsis flowering is accelerated by reduced red:far-red (R:FR) ratio which signals the presence of neighbouring vegetation. Hastened flowering is one component of the shade-avoidance syndrome of responses, which alter many aspects of development in response to the threat of potential competition. Of the red/far-red-absorbing photoreceptors it is phyB that plays the most prominent role in shade-avoidance, although other related phytochromes act redundantly with phyB. It is well established that the phyB mutant has a constitutively early flowering phenotype. However, we have shown that the early flowering phenotype of phyB is temperature-dependent. We have established that this temperature-sensitive flowering response defines a pathway that appears to be independent of the autonomous-FLC pathway. Furthermore, we have demonstrated that the phytochromes control the expression of the floral promoter FT. We have also shown that other phyB-controlled responses, including petiole elongation, are not sensitive to the same temperature change. This suggests that discrete pathways control flowering and petiole elongation, components of the shade-avoidance response. This work provides an insight into the phytochrome and temperature interactions that maintain flowering control.     

Changes in photoperiod or temperature alter the functional relationships between phytochromes and reveal roles for phyD and phyE

The phytochromes are one of the means via which plants obtain information about their immediate environment and the changing seasons. Phytochromes have important roles in developmental events such as the switch to flowering, the timing of which can be crucial for the reproductive success of the plant. Analysis of phyB mutants has revealed that phyB plays a major role in this process. We have recently shown, however, that the flowering phenotype of the phyB monogenic mutant is temperature dependent. A modest reduction in temperature to 16 degrees C was sufficient to abolish the phyB mutant early-flowering phenotype present at 22 degrees C. Using mutants null for one or more phytochrome species, we have now shown that phyA, phyD, and phyE, play greater roles with respect to phyB in the control of flowering under cooler conditions. This change in the relative contributions of individual phytochromes appears to be important for maintaining control of flowering in response to modest alterations in ambient temperature. We demonstrate that changes in ambient temperature or photoperiod can alter the hierarchy and/or the functional relationships between phytochrome species. These experiments reveal new roles for phyD and phyE and provide valuable insights into how the phytochromes help to maintain development in the natural environment.     

An atypical HLH protein OsLF in rice regulates flowering time and interacts with OsPIL13 and OsPIL15

In plants, flowering as a crucial developmental event is highly regulated by both genetic programs and environmental signals. Genetic analysis of flowering time mutants is instrumental in dissecting the regulatory pathways of flowering induction. In this study, we isolated the OsLF gene by its association with the T-DNA insertion in the rice late flowering mutant named A654. The OsLF gene encodes an atypical HLH protein composed of 419 amino acids (aa). Overexpression of the OsLF gene in wild type rice recapitulated the late flowering phenotype of A654, indicating that the OsLF gene negatively regulates flowering. Flowering genes downstream of OsPRR1 such as OsGI and Hd1 were down regulated in the A654 mutant. Yeast two hybrid and colocalization assays revealed that OsLF interacts strongly with OsPIL13 and OsPIL15 that are potentially involved in light signaling. In addition, OsPIL13 and OsPIL15 colocalize with OsPRR1, an ortholog of the Arabidopsis APRR1 gene that controls photoperiodic flowering response through clock function. Together, these results suggest that overexpression of OsLF might repress expression of OsGI and Hd1 by competing with OsPRR1 in interacting with OsPIL13 and OsPIL15 and thus induce late flowering.     

A Set of Three Genes Regulates Photoperiodic Responses of Flowering in Rice (Oryza sativa)

Differentiation in photoperiodic response of flowering has been key to the evolution and wide geographic distribution of rice, an essentially short-day plant. Crosses were made such that the hybrid F1 plants flower later than the late-flowering parents to investigate the genetic basis underlying this differentiation. From initial experiments, three major genes for flowering time were identified from four naturally occurring variants under natural long-day conditions. An F2-derived trigenic mutant line bred-true for a day-neutral response was selected and used as the recipient to synchronize the genetic background for the major genes. Experiments conducted under various daylengths indicated that these genes are responsible for photoperiodic sensitivity and the trihybrid has a critical daylength between 13.5 and 14 h. The three genes regulate photoperiodic responses qualitatively and quantitatively through complementary and other epistatic effects, respectively. The complementation suggests that the three genes act in a linear manner to repress the transition from the vegetative to reproductive phases under long daylengths. This set of genes also provides a model to understand the genetic mechanism underlying the elongated vegetative growth period in the F1 generation, which is usually an obstacle to the use of heterosis, and the selection for early maturation in rice breeding.     

Phytochrome A and Phytochrome B Have Overlapping but Distinct Functions in Arabidopsis Development

Plant responses to red and far-red light are mediated by a family of photoreceptors called phytochromes. In Arabidopsis thaliana, there are genes encoding at least five phytochromes, and it is of interest to learn if the different phytochromes have overlapping or distinct functions. To address this question for two of the phytochromes in Arabidopsis, we have compared light responses of the wild type with those of a phyA null mutant, a phyB null mutant, and a phyA phyB double mutant. We have found that both phyA and phyB mutants have a deficiency in germination, the phyA mutant in far-red light and the phyB mutant in the dark. Furthermore, the germination defect caused by the phyA mutation in far- red light could be suppressed by a phyB mutation, suggesting that phytochrome B (PHYB) can have an inhibitory as well as a stimulatory effect on germination. In red light, the phyA phyB double mutant, but neither single mutant, had poorly developed cotyledons, as well as reduced red-light induction of CAB gene expression and potentiation of chlorophyll induction. The phyA mutant was deficient in sensing a flowering response inductive photoperiod, suggesting that PHYA participates in sensing daylength. In contrast, the phyB mutant flowered earlier than the wild type (and the phyA mutant) under all photoperiods tested, but responded to an inductive photoperiod. Thus, PHYA and PHYB appear to have complementary functions in controlling germination, seedling development, and flowering. We discuss the implications of these results for possible mechanisms of PHYA and PHYB signal transduction.     

OsELF3-1, an Ortholog of Arabidopsis EARLY FLOWERING 3, Regulates Rice Circadian Rhythm and Photoperiodic Flowering

Arabidopsis thaliana EARLY FLOWERING 3 (ELF3) as a zeitnehmer (time taker) is responsible for generation of circadian rhythm and regulation of photoperiodic flowering. There are two orthologs (OsELF3-1 and OsELF3-2) of ELF3 in rice (Oryza sativa), but their roles have not yet been fully identified. Here, we performed a functional characterization of OsELF3-1 and revealed it plays a more predominant role than OsELF3-2 in rice heading. Our results suggest OsELF3-1 can affect rice circadian systems via positive regulation of OsLHY expression and negative regulation of OsPRR1, OsPRR37, OsPRR73 and OsPRR95 expression. In addition, OsELF3-1 is involved in blue light signaling by activating EARLY HEADING DATE 1 (Ehd1) expression to promote rice flowering under short-day (SD) conditions. Moreover, OsELF3-1 suppresses a flowering repressor GRAIN NUMBER, PLANT HEIGHT AND HEADING DATE 7 (Ghd7) to indirectly accelerate flowering under long-day (LD) conditions. Taken together, our results indicate OsELF3-1 is essential for circadian regulation and photoperiodic flowering in rice.     

Integration of light signaling with photoperiodic flowering and circadian rhythm

PLANT DE-ETIOLATION IS TRIGGERED BY LIGHT SIGNALS Light is arguably the most important resource for plants, and plants have evolved an array of photosensory pig- ments enabling them to develop optimally in a broad range of ambient light conditions. The ph…     

Mutation in TERMINAL FLOWER1 reverses the photoperiodic requirement for flowering in the wild strawberry Fragaria vesca

Photoperiodic flowering has been extensively studied in the annual short-day and long-day plants rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), whereas less is known about the control of flowering in perennials. In the perennial wild strawberry, Fragaria vesca (Rosaceae), short-day and perpetual flowering long-day accessions occur. Genetic analyses showed that differences in their flowering responses are caused by a single gene, SEASONAL FLOWERING LOCUS, which may encode the F. vesca homolog of TERMINAL FLOWER1 (FvTFL1). We show through high-resolution mapping and transgenic approaches that FvTFL1 is the basis of this change in flowering behavior and demonstrate that FvTFL1 acts as a photoperiodically regulated repressor. In short-day F. vesca, long photoperiods activate FvTFL1 mRNA expression and short days suppress it, promoting flower induction. These seasonal cycles in FvTFL1 mRNA level confer seasonal cycling of vegetative and reproductive development. Mutations in FvTFL1 prevent long-day suppression of flowering, and the early flowering that then occurs under long days is dependent on the F. vesca homolog of FLOWERING LOCUS T. This photoperiodic response mechanism differs from those described in model annual plants. We suggest that this mechanism controls flowering within the perennial growth cycle in F. vesca and demonstrate that a change in a single gene reverses the photoperiodic requirements for flowering.     

Phytochromes A1 and B1 have distinct functions in the photoperiodic control of flowering in the obligate long-day plant Nicotiana sylvestris

The obligate long-day plant Nicotiana sylvestris with a nominal critical day length of 12 h was used to dissect the roles of two major phytochromes (phyA1 and phyB1) in the photoperiodic control of flowering using transgenic plants under-expressing PHYA1 (SUA2), over-expressing PHYB1 (SOB36), or cosuppressing the PHYB1 gene (SCB35). When tungsten filament lamps were used to extend an 8 h main photoperiod, SCB35 and SOB36 flowered earlier and later, respectively, than wild-type plants, while flowering was greatly delayed in SUA2. These results are consistent with those obtained with other long-day plants in that phyB has a negative role in the control of flowering, while phyA is required for sensing day-length extensions. However, evidence was obtained for a positive role for PHYB1 in the control of flowering. Firstly, transgenic plants under-expressing both PHYA1 and PHYB1 exhibited extreme insensitivity to day-length extensions. Secondly, flowering in SCB35 was completely repressed under 8 h extensions with far-red-deficient light from fluorescent lamps. This indicates that the dual requirement for both far-red and red for maximum floral induction is mediated by an interaction between phyA1 and phyB1. In addition, a diurnal periodicity to the sensitivity of both negative and positive light signals was observed. This is consistent with existing models in which photoperiodic time measurement is not based on the actual measurement of the duration of either the light or dark period, but rather the coincidence of endogenous rhythms of sensitivity - both positive and negative - and the presence of light cues.