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.     

Diversified mechanisms for regulating flowering time in a short-day plant rice

Flowering in rice is influenced by not only endogenous factors that comprise an autonomous pathway, but also environmental effects, such as photoperiod, water availability, and temperature just before floral initiation. Recent molecular genetics studies have elucidated the functional roles of genes involved in the photoperiod pathway, e.g., photoreceptors, circadian clock components, and short-day (SD) promotion factors. Although these molecular players are well conserved between rice andArabidopsis, their actual genetic functions are distinct. This is exemplified byHd1 (aCO counterpart) and phytochromes, in particular, ricePHYA. Hd1 has a dual role in regulating flowering time and the expression ofHd3a (anFT counterpart) repression under long-day (LD) conditions while promotion under SDs. Models have been proposed to explain these photoperiod-dependent antagonistic activities. Some regulatory factors are present in only one of the model systems, e.g.,FLC inArabidopsis orEhd1 in rice. Furthermore, epistatic relationships vary among such flowering regulators asHd3a (FT), OsMADS50 (SOCT), andOsMADS14 (AP1). Further experiments to probe these differences will be essential to enlarging our understanding of the diversified flowering regulation mechanisms in rice.     

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.     

Rice SVP-group MADS-box proteins, OsMADS22 and OsMADS55, are negative regulators of brassinosteroid responses

Most short vegetative phase (SVP)-group MADS-box genes control meristem identity and flowering time. Among the three SVP-group genes in rice, OsMADS47 has been reported as a negative regulator of brassinosteroid (BR) responses. Here, we investigated the functional roles of two close homologs, OsMADS22 and OsMADS55, by generating single, double and triple RNAi lines and overexpression lines. Analyses of the plants showed that their roles in regulating meristem identity are well conserved; however, the involvement of these genes in determining flowering time has diversified. Most importantly, OsMADS55 works as a major negative regulator of BR responses, and OsMADS22 functions to support OsMADS55. Whereas single OsMADS55 RNAi plants display weak BR responses in the lamina joint (LJ), OsMADS22 - OsMADS55 double and OsMADS22 - OsMADS47 - OsMADS55 triple RNAi plants manifest dramatic BR responses with regard to LJ inclination, coleoptile elongation and senescence. Stem elongation is also notably reduced in the double and triple RNAi plants, probably because of BR oversensitivity. Expression analyses indicate the diversified roles in age-dependent BR responses. Altogether, our study demonstrates that all three rice SVP-group genes work as negative regulators of BR responses, but that their spatial and temporal roles are diversified.     

Alteration of floral organ identity in rice through ectopic expression of<Emphasis Type="Italic"> OsMADS16</Emphasis>

We used a transgenic approach and yeast two-hybrid experiments to study the role of the rice ( Oryza sativa L.) B-function MADS-box gene, OsMADS16. Transgenic rice plants were generated that ectopically expressed OsMADS16 under the control of the maize ( Zea mays L.) ubiquitin1 promoter. Microscopic observations revealed that the innermost-whorl carpels had been replaced by stamen-like organs, which resembled the flowers of the previously described Arabidopsis thaliana (L.) Heynh. mutation superman as well as those ectopically expressing the AP3 gene. These results indicate that expression of OsMADS16 in the innermost whorl induces stamen development. Occasionally, carpels had completely disappeared. In addition, ectopic expression of OsMADS16 enhanced expression of OsMADS4, another B-function gene, causing superman phenotypes. In the yeast two-hybrid system, OsMADS16 did not form a homodimer but, rather, the protein interacted with OsMADS4. OsMADS16 also interacted with OsMADS6 and OSMADS8, both of which are homologous to SEPALLATA proteins required for the proper function of class-B and class-C genes in Arabidopsis. Based on the gene expression pattern and our yeast two-hybrid data, we discuss a quartet model of MADS-domain protein interactions in the lodicule and stamen whorls of rice florets.