The Molecular Genetic And Biochemical Study Of CONSTANS-like Gene OsCOL10 In Photoperiodic Control Of Flowering Time In Rice

Rice （Oryza sativa L.） is one of the most important food crops in the world. The flowering time in rice （also called heading date） is an important agronomic trait closely linked to its productivity. Rice flowering at the appropriate time is not only essential for its success reproduction, but also acts as a key indicator of high and stable yield. In addition, rice heading date determines when and where the people should sow the seeds. Moreover, when the heading date is delayed properly, the rice plants can accumulate more nutrients and in turn increase the yield. Photoperiod is one of the most important and intensively investigated environment factors. Thus, cloning and functional characterization of flowering-associated genes has theoretical and practical significance for the construct of rice flowering network as well as yield increase. CONSTANS （CO） in model plant Arabidopsis plays a central role in the photoperiodic control of flowering time. In rice, the CONSTANS-like （COL） gene family contains at least 16 members, majority of which are not functionally identified. In this study, we first carried out a large-scale screening from dominant gain-of-funciton transcriptional factor （TF） population （pubi::TFs-VP64） and identified a flowering time-associated COL gene, designated OsCOL10; Then we generated various transgenic plants including overexpressing lines as well as RNA interfere nce-mediated knock down plants, investigated the phenotypes of these plants, and compared the expression of previously reported flowering genes in the transgenic plants and wild-type to know the roles of OsCOL10 in the photoperiodic flowering. Further, we characterized the gene in some aspects, including phylogenetic analysis and expression pattern, subcellular localization as well as transcriptional activation activity. Morover, we compared expression of OsCOL10 in various flowering-time NILs or their WTs and corresponding mutants to know the regulatory mechanism of OsCOL10 in the photoperiodic flowering time. Finally, we analyzed various haplotypes of OsCOL10. The main results are as follows:（1）The two independent transgenic lines （OsCOL10-VP64-OX4 and OsCOL10-VP64-OX7） gained from the transcriptional factor gain-of-function population showed delayed flowering compared with wild-type kitaake plants under both short-day （SD） and long-day conditions （LD） conditions. The longer basic vegetative growth phase allowed the mature transgenic lines to grow much taller, produce larger panicles and more seeds than wild-type plants. To investigate whether delayed flowering phenotype in trans genic plants might have been associated with a reduction in growth rate, we compared the leaf emergence rates between the wild-type and transgenic lines. Notably, before the wild-type plants flowering, the leaf emergence rate in transgenic plants was similar to that of the wild-type plants under SD and LD conditions, indicating that flowering delay in transgenic plants was not due to reduction of growth rate but repression of floral induction. Overexpression of OsCOL10 （pubi::OsCOL10） in rice plants delayed flowering similar to pubi:. OsCOL10-VP64 plants. These findings are indicative of OsCOL10 as a constitutive flowering repressor in rice.（2）By comparing the expression of previously reported flowering genes in the transgenic plants and wild-type, we found that OsCOL10 negatively regulated flowering mainly by reducing the expression of integrated gene Ehdl as well as downstream florigen genes Hd3a and RFT1.（3）By using bioinformatic method, we constructed the phylogenetic tree for Arabidopsis and rice CONS TANS-like proteins. We found that these proteins can mainly fall into three subgroups. OsCOL10 belongs to the second subgroup where none has been reported to be involved in flowering regulation. OsCOL10 contains a B-box domain at the N-terminus and a CCT domain at the C-terminus. BLAST searchs of the available genome sequences with the OsCOL10 protein sequence revealed close homo logs in other gramineous plants. Unfortunately, the functions of these homo logs are largely unknown.（4）The expression of OsCOL10 showed circadian oscillation. Under SD, the abundance of OsCOL10 transcript was low at night and increased at 4h before dawn with a peak of expression at the dawn. Under LD, the abundance of OsCOL10 mRNA was low at night, and increased at dawn with a peak of expression after 4h. The peak level of COL10 transcript was higher in plants growing in SD than that growing in LD. Moreover, OsCOL10 accumulated during the vegetative growth from the fourth week and reached the peak at the sixth week under SD, accumulating from the sixth week and peaking at eighth week under LD. We transformed 28-day-old wild-type plants grown under cycles of 12-hour light/12-hour darkness to the continuous light （LL） or continuous darkness （DD）. Remarkably, the rhythmic amplitude of OsCOL10 expression was drastically reduced, but continued to oscillate when released into DD or LL. The results indicate that expression of OsCOL10 is under the control of circadian clock.（5）Expression pattern of OsCOL10 was investigated by quantitative real-time PCR （qRT-PCR） using RNA samples from various tissues and leaves of different developmental stages. The OsCOL10 was preferentially expressed in the leaves, especially in developing young leaves but undetectable in roots. Histochemical staining of transgenic plants carrying the GUS reporter gene driven by the OsCOL10 promoter supported the above expression model and further limited GUS signal at cells outside of vascular tissues by analyzing the slices. In situ hybridization assay by using OsCOL10-specific probes confirmed that OsCOL10 was enriched at the mesophyll cells of leaves.（6）OsCOL10 acts as a transcriptional factor. A transient expression assay in onion epidermis cells was used to assess the subcellular localization of OsCOL10-GFP. In the bombarded cells with the expression of the fusion protein, the signal of GFP fluorescence was localized strictly in the nucleus. The nuclear localization of OsCOL10 was confirmed by the detection of the OsCOL10-GFP fusion protein in transgenic rice root cells. Transcriptional activation assay revealed that full-length OsCOL10 has a weak transcriptional activation activity in yeast cells. Deletion analysis showed that only the middle region was essential for its transcriptional activation activity. These results support the notion that OsCOL10 may play an important role in transcriptional regulation of downstream gene expression.（7）We compared the expression of OsCOL10 in various flowering-time mutants or NILs and corresponding wild-type and found that abundance of OsCOL10 is more in NIL （Ghd7） than that in NIL （ghd7）, suggesting that OsCOL10 is regulated by Ghd7. To verify this data, we introduced a functional Ghd7 driven by its promoter into the Ghd7-deficient cultivar kitaake. The generated transgenic-positive plants showed delayed flowering. The expression of OsCOL10 was significantly increased in transgenic lines. These findings indicate that OsCOL10 is regulated by Ghd7.（8）We selected 100 rice accessions with diverse genetic backgrounds and wide geographic distribution to analyze various haplotypes of OsCOL10. We found that OsCOL10 is highly conserved in rice genus. Hap₃ represents the major haplotypes at the OsCOL10 locus in cultivated rice.