Functional Analysis Of CRYs And CIB3in Flowering And Senescence Regulation In Soybean (Glycine Max)

Cryptochromes firstly identified in Arabidopsis are blue light receptors found in all evolutionary lineages. Cryptochromes regulate photomorphogenesis in plants and the circadian clock in animals and plants. Although the molecular mechanism of cryptochromes regulating floral initiation has been extensively explored in Arabidopsis, their functions in soybean remain to be elucidated. CIB1, abHLH transcriptional factor, is the first protein identified to interact with CRY2in blue light specific manner. The interaction between CRY2and CIB1in response to blue light signal promotes FT gene expression and triggers floral initiation. Soybean is a typical short-light plant. The study of the photoperiodic flowering mechanism in soybean is critical for the improvement of its agronomic traits. Previously, we studied the functions of GmCRYs, the primary results demonstrate apparently differences between GmCRYs and AtCRYs. In this study we particularly investigated the interaction relationship between GmCRYs and GmCIBs. In addition, we obtained GmCRY and GmCIB3over-expression and RNAi knockdown transgenic soybeans respectively. Furthermore, we tried to find the genetic down-stream genes of GmCRY and GmCIB3through the phenotype analysis and ChIP assay. Those results provided fundamental evidence for the understanding of the Cryptochromes involved photoperiod and senescence mechanisms and useful knowledge for the agronomic trait improvement of soybean cultivar. The main results were listed as follows:(1) Totally twelve GmCIB genes were identified locating on11different chromosomes. We cloned nine GmCIB genes in this study.(2) The transient expression using Arabidopsis protoplast were employed to investigate the subcellular location of the nine GmCIBs. The results indicated that GmCIB6and GmCIB7localize in both nuclear and cytoplasm, and others present majorly in nucleus.(3) The mRNA levels of most GmCIBs peak at the flowering stage, while the expression of GmCIB3is high in both unifoliolate and flowering stage. Furth analysis in long day (LD) or short day (SD) showing that the expression of most GmCIBs is regulated by photeperiod and circadian clock, however no significant correlation were detected between the expression level of GmCIBs and the geographic latitude of different soybean varieties.(4) Phenotype analysis of GmCRY1-OX, GmCRY2-OX, GmCRY2-RNAi and GmCIB3-OX transgenic soybeans under continuous light (LL) showed that GmCRY1or GmCIB3over expression, or GmCRY2knock-down accelerate the senescence progress, otherwise GmCRY2over expression delay senescence phenotype, which indicated that GmCRY1and GmCIB3involved in positively regulation the leaf senescence, while GmCRY2has a negative effect on senescence regulation.(5) Early flowering phenotype of GmCRY1-OX and GmCRY2-RAAi transgenic plants and later flowering phenotype of GmCRY2-OX and GmCIB3-OXtransgenic plants illustrated that those genes also play important role in soybean flowering regulation.(6) Yeast two-hybrid and BiFC assays showed that GmCRY2interacted with GmCIB3in a blue light dependent manner. The PHR domain of GmCRY1or GmCRY2is required for the blue-light-dependent interaction between GmCRY1/2and GmCIB3. In Vivo CoIP or In vitro pull down assays showed that blue light stimulated the formation of the GmCRY2-GmCIB3or GmCRYl-GmCIB3complex.(7) In vitro assays showed that GmCIB3proteins bind to E-box (CANNTG) DNA sequence with the highest affinity. ChIP-PCR showing that GmCIB3interacted with the chromatin E-box regions of the GmFT3,GmFT4or GmWRYK53but not with GmTFL1and GmTFL3, which suggest that GmCIB3may directly suppress GmFT or GmFT4transcription and stimulate GmWRYK53transcription respectively.