Sugar Metabolism Analysis In Rice Flowering Transition Regulated By RID1

Flowering is a crucial process in the life cycle of plants,which represents the transition of shoot apex meristem from vegetative to reproductive stage.Also,flowering transition is the prerequisite for the development of seed and fruit.Therefore,the study of flowering transition is of significance to ensure yield in rice.Previously,we identified a flowering transition gene,RID1(Rice Indeterminate 1),which acts as a master switch for the normal heading in rice.RID1 encodes a Cys-2/His-2-type zinc finger transcription factor.A T-DNA insertion mutant of RID1 shows a "never-flowering" phenotype.Compared to the wild type,the transcript levels of almost sugar metabolic genes were significantly changed in the mature source leaves of rid1.Because Maize ID1 and Arabidopsis thaliana AtIDD8 controlling flowering transition are closely related to sugar metabolism,we further analyzed the sugar metabolism status in rid1.Using qPCR,GC-MS,and 3,5-Dinitrosalicylic acid techniques,we monitor the expression of sugar metabolism-related genes,and the contents of soluble sugar and starch.The result indicated that RID1 might regulate flowering transition closely related with sugar metabolism.At the same time,some genes' mutants related to sugar metabolism were generated employing CRISPR/cas9 system.Those mutants were further examined the heading date phenotype.Our preliminary data indicated that rice flowering transition might be associated with sugar metabolism.The main results are as follows:1.Trehalose metabolism might be involved in flowering transition regulated by RID1: To monitor the expression pattern of trehalose metabolism-related genes in the young leaves and SAM,qRT-PCR result showed that the expression patterns were significantly different in rid1 compared to wild type.Using yeast one-hybrid technique,we found that RID1 can directly bind to the promoter of OsTPS1 and OsTPS8.These results suggest that RID1 regulates the expression of OsTPS1 and OsTPS8,and further affecting the expression of trehalose metabolism-related genes.2.Sucrose and starch metabolism might be involved in flowering transitionregulated by RID1: qRT-PCR result showed that the expression patterns of sucrose and starch metabolism-related genes were significantly different in mature source leaves of rid1 compared to wild type.The gas chromatography-mass spectrometry(GC-MS)was applied to monitor the contents of glucose,fructose and sucrose in the young and mature source leaves.Our results show that the contents of glucose and fructose are decreased.However,sucrose is increased in pre-transition rid1 compared to wild type.We checked the contents of starch in the mature source leaves by 3,5-Dinitrosalicylic acid.The results show that the starch accumulation is significantly higher in rid1 than that in the wild type.These results suggest that rice flowering transition might be associated with sucrose and starch metabolism.3.Generate sugar metabolism mutants using CRISPR/cas9 system: Four and ten mutants which refer to sugar metabolism were obtained by introducing single or double mutant site by CRISPR/cas9 system,respectively.Moreover,we obtained ten sugar metabolism mutants by introducing triple mutant site by CRISPR/cas9 system.Taken together,we obtained 24 sugar metabolism mutants in this study.All these mutants have been confirmed by PCR-RE(PCR restriction enzyme)and sequencing analysis.