Shuji Yokoi

Hd3a Protein Is a Mobile Flowering Signal in Rice

Florigen, the mobile signal that moves from an induced leaf to the shoot apex and causes flowering, has eluded identification since it was first proposed 70 years ago. Understanding the nature of the mobile flowering signal would provide a key insight into the molecular mechanism of floral induction. Recent studies suggest that the Arabidopsis FLOWERING LOCUS T (FT) gene is a candidate for encoding florigen. We show that the protein encoded by Hd3a, a rice ortholog of FT, moves from the leaf to the shoot apical meristem and induces flowering in rice. These results suggest that the Hd3a protein may be the rice florigen.

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.

Hd3a and RFT1 are essential for flowering in rice

RICE FLOWERING LOCUS T 1 (RFT1/FT-L3) is the closest homologue of Heading date 3a (Hd3a), which is thought to encode a mobile flowering signal and promote floral transition under short-day (SD) conditions. RFT1 is located only 11.5 kb from Hd3a on chromosome 6. Although RFT1 RNAi plants flowered normally, double RFT1-Hd3a RNAi plants did not flower up to 300 days after sowing (DAS), indicating that Hd3a and RFT1 are essential for flowering in rice. RFT1 expression was very low in wild-type plants, but there was a marked increase in RFT1 expression by 70 DAS in Hd3a RNAi plants, which flowered 90 DAS. H3K9 acetylation around the transcription initiation site of the RFT1 locus had increased by 70 DAS but not at 35 DAS. In the absence of Hd3a and RFT1 expression, transcription of OsMADS14 and OsMADS15, two rice orthologues of Arabidopsis APETALA1, was strongly reduced, suggesting that they act downstream of Hd3a and RFT1. These results indicate that Hd3a and RFT1 act as floral activators under SD conditions, and that RFT1 expression is partly regulated by chromatin modification.

A gene network for long-day flowering activates RFT1 encoding a mobile flowering signal in rice

Although some genes that encode sensory or regulatory elements for photoperiodic flowering are conserved between the long-day (LD) plant Arabidopsis thaliana and the short-day (SD) plant rice, the gene networks that control rice flowering, and particularly flowering under LD conditions, are not well understood. We show here that RICE FLOWERING LOCUS T 1 (RFT1), the closest homolog to Heading date 3a (Hd3a), is a major floral activator under LD conditions. An RFT1:GFP fusion protein localized in the shoot apical meristem (SAM) under LD conditions, suggesting that RFT1 is a florigen gene in rice. Furthermore, mutants in OsMADS50, a rice ortholog of Arabidopsis SUPPRESOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) did not flower up to 300 days after sowing under LD conditions, indicating that OsMADS50, which acts upstream of RFT1, promotes flowering under LD conditions. We propose that both positive (OsMADS50 and Ehd1) and negative (Hd1, phyB and Ghd7) regulators of RFT1 form a gene network that regulates LD flowering in rice. Among these regulators, Ehd1, a rice-specific floral inducer, integrates multiple pathways to regulate RFT1, leading to flowering under appropriate photoperiod conditions. A rice ortholog of Arabidopsis APETALA1, OsMADS14, was expressed in the floral meristem in wild-type but not in RFT1 RNAi plants, suggesting that OsMADS14 is activated by RFT1 protein in the SAM after the transition to flowering. We have thus exposed a network of genes that regulate LD flowering in rice.

Variations in Hd1 proteins, Hd3a promoters, and Ehd1 expression levels contribute to diversity of flowering time in cultivated rice

Rice is a facultative short-day plant, and molecular genetic studies have identified the major genes involved in short-day flowering. However, the molecular mechanisms promoting the diversity of flowering time in cultivated rice are not known. We used a core collection of 64 rice cultivars that represent the genetic diversity of 332 accessions from around the world and studied the expression levels and polymorphisms of 6 genes in the short-day flowering pathway. The RNA levels of Heading date 3a (Hd3a), encoding a floral activator, are highly correlated with flowering time, and there is a high degree of polymorphism in the Heading date 1 (Hd1) protein, which is a major regulator of Hd3a expression. Functional and nonfunctional alleles of Hd1 are associated with early and late flowering, respectively, suggesting that Hd1 is a major determinant of variation in flowering time of cultivated rice. We also found that the type of Hd3a promoter and the level of Ehd1 expression contribute to the diversity in flowering time and Hd3a expression level. We evaluated the contributions of these 3 factors by a statistical analysis using a simple linear model, and the results supported our experimental observations.

Suppression of the Floral Activator Hd3a Is the Principal Cause of the Night Break Effect in Rice

A short exposure to light in the middle of the night causes inhibition of flowering in short-day plants. This phenomenon is called night break (NB) and has been used extensively as a tool to study the photoperiodic control of flowering for many years. However, at the molecular level, very little is known about this phenomenon. In rice (Oryza sativa), 10 min of light exposure in the middle of a 14-h night caused a clear delay in flowering. A single NB strongly suppressed the mRNA of Hd3a, a homolog of Arabidopsis thaliana FLOWERING LOCUS T (FT), whereas the mRNAs of OsGI and Hd1 were not affected. The NB effect on Hd3a mRNA was maximal in the middle of the 14-h night. The phyB mutation abolished the NB effect on flowering and Hd3a mRNA, indicating that the NB effect was mediated by phytochrome B. Because expression of the other FT-like genes was very low and not appreciably affected by NB, our results strongly suggest that the suppression of Hd3a mRNA is the principal cause of the NB effect on flowering in rice.

Phytochrome B regulates Heading date 1 (Hd1)-mediated expression of rice florigen Hd3a and critical day length in rice

Many plants require circadian clock and light information for the photoperiodic control of flowering. In Arabidopsis, a long-day plant (LDP), flowering is triggered by the circadian clock-controlled expression of CONSTANS (CO) and light stabilization of the CO protein to induce FT (FLOWERING LOCUS T). In rice, a short-day plant (SDP), the CO ortholog Heading date 1 (Hd1) regulates FT ortholog Hd3a, but regulation of Hd3a by Hd1 differs from that in Arabidopsis. Here, we report that phytochrome B (phyB)-mediated suppression of Hd3a is a primary cause of long-day suppression of flowering in rice, based on the three complementary discoveries. First, overexpression of Hd1 causes a delay in flowering under SD conditions and this effect requires phyB, suggesting that light modulates Hd1 control of Hd3a transcription. Second, a single extension of day length decreases Hd3a expression proportionately with the length of daylight. Third, Hd1 protein levels in Hd1-overexpressing plants are not altered in the presence of light. These results also suggest that phyB-mediated suppression of Hd3a expression is a component of the molecular mechanism for critical day length in rice.

Identification of Dynamin as an Interactor of Rice GIGANTEA by Tandem Affinity Purification (TAP)

GIGANTEA (GI), CONSTANS (CO) and FLOWERING LOCUS T (FT) regulate photoperiodic flowering in Arabidopsis. In rice, OsGI, Hd1 and Hd3a were identified as orthologs of GI, CO and FT, respectively, and are also important regulators of flowering. Although GI has roles in both flowering and the circadian clock, our understanding of its biochemical functions is still limited. In this study, we purified novel OsGI-interacting proteins by using the tandem affinity purification (TAP) method. The TAP method has been used effectively in a number of model species to isolate proteins that interact with proteins of interest. However, in plants, the TAP method has been used in only a few studies, and no novel proteins have previously been isolated by this method. We generated transgenic rice plants and cell cultures expressing a TAP-tagged version of OsGI. After a two-step purification procedure, the interacting proteins were analyzed by mass spectrometry. Seven proteins, including dynamin, were identified as OsGI-interacting proteins. The interaction of OsGI with dynamin was verified by co-immunoprecipitation using a myc-tagged version of OsGI. Moreover, an analysis of Arabidopsis dynamin mutants indicated that although the flowering times of the mutants were not different from those of wild-type plants, an aerial rosette phenotype was observed in the mutants. We also found that OsGI is present in both the nucleus and the cytosol by Western blot analysis and by transient assays. These results indicate that the TAP method is effective for the isolation of novel proteins that interact with target proteins in plants.

The Photoperiodic Control of Flowering in Rice, a Short-Day Plant

The photoperiodic sensitivity 5 (se5) mutant of rice, a short-day plant, has a very early flowering phenotype and is completely deficient in photoperiodic response. We have cloned the SE5 gene by candidate cloning and demonstrated that it encodes a putative heme oxygenase. Lack of responses of coleoptile elongation by light pulses and photoreversible phytochromes in crude extracts of se5 indicate that SE5 may function in phytochrome chromophore biosynthesis. Ectopic expression of SE5 cDNA by the CaMV 35S promoter restored the photoperiodic response in the se5 mutant. Our results indicate that phytochromes confer the photoperiodic control of flowering in rice. Comparison of se5 with hy1, a counterpart mutant of Arabidopsis, suggests distinct roles of phytochromes in the photoperiodic control of flowering in these two species.