Mechanism Analysis Of Rice Floral Transition Regulated By Rid1 Gene

Rice is one of the most important food crops for half population of the world,the security of rice production is ensure a sustainable development for agriculture and social stability.The heading date of rice directly influences the adaption of region and seasonal changes,thus,regulation of flowering time directly influences successful rice grain production.Our previous investigation identified the Rice Indeterminate 1(RID1)gene acts as the master switch for the transition from the vegetative to reproductive phase and indicated that RID1 activates the expression of florigen genes(Hd3a,RFT1)mainly by regulating the expression of Ehd1.In order to understand the molecular mechanism of rice floral transition regulated by RID1,we cloned the Suppressor of ridl(SID1),and showed RID1-Hd3a/RFT1 autonomous pathway,RID1-OsATG7 metabolism homeostasis pathway,and RID1/DELLA-SCL3 gibberellin pathway cooperatively promoted floral transition.These results were described as follows:1.The cloning of Suppressor of rid1(SID1)generid1 exhibits a never-flowering phenotype.We identify the suppressor of rid1(sid1-D)via the transgenic events of complementation the flowering defects in rid1.The restored flowering of sid1-D results from a dominant mutation of a single gene that is co-segregate with a T-DNA insertion event.The transcript of SID1 which flanked the T-DNA insertion was significantly increased in sidl-D.Genetic analysis verified the gain-of-function of the SID1 was the cause for flowering rescuing in sid1-D.SID1 encodes an INDETERMINATE DOMAIN(IDD)protein with transcriptional activation activity,which was highly expressed in leaves.We generated sidl mutants using the CRISPR-Cas9 system,homozygotes for sidl showed delayed flowering compared to the wild type under natural long-day conditions.In sid1,the transcript levels of Hd3a,RFT1,and Ehdl were partially reduced,suggesting that SID1 might be involved in flowering regulation through modulation of the expression of Hd3a,RFT1,and Ehd1.2.Gain-of-function SID1 directly regulating the expression of Hd3a and RFT1In sidl-D background,the expression patterns of Hd3a,RFT1,and Ehd1 were recovered to normal.A core sequence containing TTTGTC,which was the consensus motif binding by IDDs,was found in the promoter regions of Hd3a and RFT1,respectively.Electrophoretic Mobility Shift Assay(EMSA)verified SID1 could bind to the promoter region of Hd3a and RFT1.Dual Luciferase Reporter(DLR)assay system in ridl protoplasts verified that a considerable amount of SID1 has the transcriptional activation ability to drive the expression of Hd3a and RFT1 when RID1 was abolished.The data suggest that overexpression of SID1 might bind the promoter regions of Hd3a and RFT1,and activate the expression of Hd3a and RFT1 to elicit flowering.3.RID1 directly activates the expression of Hd3a and RFT1 and elicits floweringEMSA and Chromatin Immunoprecipitation assay(ChIP)verified RID1 could bind to the promoter regions of Hd3a and RFT1.Overexpression of Hd3a,but not Ehdl,could restore the flowering of rid1 and reach flowering at the seedling stage.These results indicate that RID1 may initiate the flowering transition through its direct targets Hd3a and RFT1,RID1-Ehdl-Hd3a/RFT1 is not the sole pathway for floral induction mediated by RID1 in rice.4.Physical interaction between SID1 and RID1Yeast two-hybrid assays,bimolecular fluorescence complementation(BiFC)experiments,and pull-down assays verified SID1 interacts with RID1.These results suggest that SID1 and RID1 can form heterodimer,SID1 may act as the coactivater of RID 1 to elicit flowering.5.RID1 promotes the floral transition by regulating starch signaling and amino acid homeostasis via autophagy-related 7To investigate the global binding profiles of RID1,we adopt bioinformatics approach to screen the TTTTGTCC consencus motif in rice genome,and select autophagy-related 7 as the target of RID1.The expression level of OsATG7 in ridl was decreased dramatically,DLR and EMSA assays verified RID1 could bind to the promoter region of OsATG7.Transmission electron microscopy(TEM)assay and western-blot assay verified that the autophagy activity was decrease in rid1,these data indicate that autophagy activity is depressed in rid1.OsATG7 is essential for autophagosome biogenesis and involved in supporting the availability of nutrients and building blocks for cellular function.In ridl,transitory starch to sucrose ratio and free amino acids were decreased in pre-transition mature leaf blades.Complementary test and CRISPR-Cas generated osatg7 transgenic mutant plants verified OsATG7 is a positive regulator of flowering transition.Overall,these results suggest that RID1 promotes the floral transition by regulating starch signaling and amino acid homeostasis via OsATG7.6.DELLA may function as a cofactor of RID 1 in regulation of gibberellin homeostasis and signaling via SCL3The independent transgenic plants overexpressing RID1,in a certain proportion,showed extremely dwarfism and never-flowering phenotypes,which are similar to DELLA mutants.Yeast two hybrid methods and BiFC assays confirmed RID1 directly interacts with DELLA.SCL3,a GA-positive regulator,its expression is induced by DELLA and repressed by GA.Yeast one hybrid assay,DLR and EMSA confirmed that RID1 could directly bind to the promoter region of SCL3.These results suggest that DELLA may function as a cofactor of RID 1 in regulation of gibberellin homeostasis and signaling via SCL3.