Functional analyses of the flowering time gene OsMADS50, the putative SUPPRESSOR OF OVEREXPRESSION OF CO 1/AGAMOUS-LIKE 20 (SOC1/AGL20) ortholog in rice

A late-flowering mutant was isolated from rice T-DNA-tagging lines. T-DNA had been integrated into the K-box region of Oryza sativa MADS50 (OsMADS50), which shares 50.6% amino acid identity with the Arabidopsis MADS-box gene SUPPRESSOR OF OVEREXPRESSION OF CO 1/AGAMOUS-LIKE 20 (SOC1/AGL20). While overexpression of OsMADS50 caused extremely early flowering at the callus stage, OsMADS50 RNAi plants exhibited phenotypes of late flowering and an increase in the number of elongated internodes. This confirmed that the phenotypes observed in the knockout (KO) plants are because of the mutation in OsMADS50. RT-PCR analyses of the OsMADS50 KO and ubiquitin (ubi):OsMADS50 plants showed that OsMADS50 is an upstream regulator of OsMADS1, OsMADS14, OsMADS15, OsMADS18, and Hd (Heading date)3a, but works either parallel with or downstream of Hd1 and O. sativa GIGANTEA (OsGI). These results suggest that OsMADS50 is an important flowering activator that controls various floral regulators in rice.     

Altered levels of histone deacetylase OsHDT1 affect differential gene expression patterns in hybrid rice

Hybrids between different inbred varieties display novel patterns of gene expression resulted from parental variation in allelic nucleotide sequences. To study the function of chromatin regulators in hybrid gene expression, the histone deacetylase gene OsHDT1 whose expression displayed a circadian rhythm was over-expressed or inactivated by RNAi in an elite rice parent. Increased OsHDT1 expression did not affect plant growth in the parent but led to early flowering in the hybrid. Nonadditive up-regulation of key flowering time genes was found to be related to flowering time of the hybrid. Over-expression of OsHDT1 repressed the nonadditive expression of the key flowering repressors in the hybrid (i.e. OsGI and Hd1) inducing early flowering. Analysis of histone acetylation suggested that OsHDT1 over-expression might promote deacetylation on OsGI and Hd1 chromatin during the peak expression phase. High throughput differential gene expression analysis revealed that altered OsHDT1 levels affected nonadditive expression of many genes in the hybrid. These data demonstrate that nonadditive gene expression was involved in flowering time control in the hybrid rice and that OsHDT1 level was important for nonadditive or differential expression of many genes including the flowering time genes, suggesting that OsHDT1 may be involved in epigenetic control of parental genome interaction for differential gene expression.     

Circadian-associated rice pseudo response regulators (OsPRRs): insight into the control of flowering time

A small family of plant proteins, designated PSEUDO RESPONSE REGULATORS (PRRs), is crucial for a better understanding of the molecular link between circadian rhythm and photoperiodic control of flowering time in the dicotyledonous model plant Arabidopsis thaliana. Recently, we showed that the monocotyledonous model plant Oryza sativa also has homologous members of the OsPRR family (Oryza sativa PRR). In the previous experiments with rice, we mainly characterized a japonica variety (Nipponbare). By employing an indica variety (Kasalath), in this study we further characterized OsPRRs with reference to the photoperiod sensitivity Hd (Heading date) QTL (quantitative trait loci) implicated in the control of flowering time in rice. The circadian-controlled and sequential expression profiles of the five OsPRR genes were observed not only for Nipponbare but also for Kasalath. Then each of these OsPRR genes was mapped on the rice chromosomes. Among these OsPRR genes, OsPRR37 was mapped very closely to Hd2-QTL, which was identified as the major locus that enhances the photoperiod sensitivity of flowering in Nipponbare. Furthermore, we found that Kasalath has a severe mutational lesion in the OsPRR37 coding sequence.     

Combinations of Hd2 and Hd4 genes determine rice adaptability to Heilongjiang Province,northern limit of China

Heading date is a key trait in rice domestication and adaption, and a number of quantitative trait loci (QTLs) have been identified. The rice (Oryza sativa L.) cultivars in the Heilongjiang Province, t...     

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.     

Nonfunctional alleles of long‐day suppressor genes independently regulate flowering time

Due to the remarkable adaptability to various environments, rice varieties with diverse flowering times have been domesticated or improved from Oryza rufipogon. Detailed knowledge of the genetic factors controlling flowering time will facilitate understanding the adaptation mechanism in cultivated rice and enable breeders to design appropriate genotypes for distinct preferences. In this study, four genes (Hd1, DTH8, Ghd7 and OsPRR37) in a rice long‐day suppression pathway were collected and sequenced in 154, 74, 69 and 62 varieties of cultivated rice (Oryza sativa) respectively. Under long‐day conditions, varieties with nonfunctional alleles flowered significantly earlier than those with functional alleles. However, the four genes have different genetic effects in the regulation of flowering time: Hd1 and OsPRR37 are major genes that generally regulate rice flowering time for all varieties, while DTH8 and Ghd7 only regulate regional rice varieties. Geographic analysis and network studies suggested that the nonfunctional alleles of these suppression loci with regional adaptability were derived recently and independently. Alleles with regional adaptability should be taken into consideration for genetic improvement. The rich genetic variations in these four genes, which adapt rice to different environments, provide the flexibility needed for breeding rice varieties with diverse flowering times.     

Identification of dynamin as an interactor of rice GIGANTEA by tandem affinity purification (TAP)

GIGANTEA (GI), CONSTANS (CO) and FLOWERING LOCUS T (FT) regulatephotoperiodic flowering in Arabidopsis. In rice, OsGI, Hd1 andHd3a were identified as orthologs of GI, CO and FT, respectively,and are also important regulators of flowering. Although GIhas roles in both flowering and the circadian clock, our understandingof its biochemical functions is still limited. In this study,we purified novel OsGI-interacting proteins by using the tandemaffinity purification (TAP) method. The TAP method has beenused effectively in a number of model species to isolate proteinsthat interact with proteins of interest. However, in plants,the TAP method has been used in only a few studies, and no novelproteins have previously been isolated by this method. We generatedtransgenic rice plants and cell cultures expressing a TAP-taggedversion of OsGI. After a two-step purification procedure, theinteracting proteins were analyzed by mass spectrometry. Sevenproteins, including dynamin, were identified as OsGI-interactingproteins. The interaction of OsGI with dynamin was verifiedby co-immunoprecipitation using a myc-tagged version of OsGI.Moreover, an analysis of Arabidopsis dynamin mutants indicatedthat although the flowering times of the mutants were not differentfrom those of wild-type plants, an aerial rosette phenotypewas observed in the mutants. We also found that OsGI is presentin both the nucleus and the cytosol by Western blot analysisand by transient assays. These results indicate that the TAPmethod is effective for the isolation of novel proteins thatinteract with target proteins in plants.     

Characterization of a set of phytochrome-interacting factor-like bHLH proteins in Oryza sativa

The model dicotyledon Arabidopsis thaliana has a characteristic small sub-family of phytochrome-interacting bHLH (basic Helix-Loop-Helix) factors, which are collectively designated the PIL (or PIF) (PHYTOCHROME INTERACTING FACTOR-LIKE) family proteins. In this study, we identified and characterized a set of highly homologous members (designated OsPIL11 to OsPIL16) in the model monocotyledon rice (Oryza sativa). Some of them (OsPIL11, OsPIL12, and OsPIL13) showed the ability to interact with the putative OsPRR1 (PSEUDO-RESPONSE REGULATOR 1) clock component, as far as the results of yeast two-hybrid assays were concerned. It was found that the expression of OsPIL13 is under the control of circadian rhythms (clock), while the expression of OsPIL15 is negatively regulated by light upon the onset to light exposure of etiolated seedlings. When the rice genes (OsPIL11 to OsPIL15) were over-expressed in A. thaliana, the resulting transgenic seedlings displayed anomalous morphologies with very long hypocotyls during early photomorphogenesis. These results suggest the view that the identified OsPILs are functional counterparts (or orthologs) of AtPILs, which are known to play important roles in red light-mediated (phyA and/or phyB-dependent) signal transduction pathways at immediate positions downstream of the photoreceptor in A. thaliana.     

Heading date 1 (Hd1), an ortholog of Arabidopsis CONSTANS, is a possible target of human selection during domestication to diversify flowering times of cultivated rice

During the domestication of rice (Oryza sativa L.), diversification of flowering time was important in expanding the areas of cultivation. Rice is a facultative short day (SD) plant and requires certain periods of dark to induce flowering. Heading date 1 (Hd1), a regulator of the florigen gene Hd3a, is one of the main factors used to generate diversity in flowering. Loss-of-function alleles of Hd1 are common in cultivated rice and cause the diversity of flowering time. However, it is unclear how these functional nucleotide polymorphisms of Hd1 accumulated in the course of evolution. Nucleotide polymorphisms within Hd1 and Hd3a were analyzed in 38 accessions of ancestral wild rice Oryza rufipogon and compared with those of cultivated rice. In contrast to cultivated rice, no nucleotide changes affecting Hd1 function were found in 38 accessions of wild rice ancestors. No functional changes were found in Hd3a in either cultivated or ancestral rice. A phylogenetic analysis indicated that evolution of the Hd1 alleles may have occurred independently in cultivars descended from various accessions of ancestral rice. The non-functional Hd1 alleles found in cultivated rice may be selected during domestication, because they were not found or very rare in wild ancestral rice. In contrast with Hd3a, which has been highly conserved, Hd1 may have undergone human selection to diversify the flowering times of rice during domestication or the early stage of the cultivation period.     

水稻开花光周期调控相关基因研究进展

水稻开花调控是一个极其复杂的生命过程,由自身遗传因素和外界环境共同决定。光周期途径是调控水稻开花的关键途径,在这个途径中成花素基因Hd3a和RTF1处于核心地位,其上游调控途径主要包括Hd1依赖途径、Ehd1依赖途径及不依赖于Hd1和Ehd1的途径。这3条途径在汇集了光信号的各种信息后,将信号在Hd3a和RTF1处整合,并通过成花素形式将信息传递给下游开花基因,调控水稻开花。本文从成花素、光信号感受基因和昼夜节律基因、成花素上游调控基因、互作蛋白和下游调控基因等几方面阐述水稻开花光周期调控相关基因的研究现状,为水稻开花调控的深入研究提供参考。