Function Analysis Of Arabidopsis C2DP1 And Its Homologous Proteins Involved In Flowering Regulation

Flowering is an important process for plants to transition from vegetative growth to reproductive growth.It is an important stage of plant reproduction and is of great significance to plant evolution.At present,much attention has been paid to the mechanism of plant flowering regulation.The regulation mechanism of plant flowering is very complicated,which is regulated by internal factors as well as external environmental factors.At present,from the model plant Arabidopsis thaliana many genes involved in flowering regulation have been identified,mainly involving six regulatory pathways,the photoperiod pathway,vernalization pathway,ambient temperature pathway,gibberellin pathway,autonomous pathway,and age pathway.However,the molecular mechanism of intersection between various signal pathways is not yet fully understood.In order to explore the mechanism of Arabidopsis flowering regulation,in the preliminary work,the laboratory of the author overexpressed some genes in Arabidopsis,and then screened and analyzed the flowering phenotype.It was found that overexpression of a gene encoding a C2-domain protein can cause late flowering in Arabidopsis.Here we temporarily named this gene C2DP1.At the same time,there are two other homologous genes in Arabidopsis genome:C2DP2 and C2DP3.There are few reports on function of these three genes.Since C2DP2 and C2DP3 are homologous genes of C2DP1,it is speculated that overexpression of C2DP2 and C2DP3 can also cause late flowering in Arabidopsis.We overexpressed the two homologous genes C2DP2 and C2DP3 in Arabidopsis thaliana,and found that the overexpression of these two genes can also cause late flowering of Arabidopsis to some extent.Therefore,we guess that these three genes are redundant in function.So we used T-DNA mutant identification technology,CRISPR/Cas9 gene editing technology,and hybrid screening to obtain single mutants and their combination of these genes,c2dp1,c2dp2,and c2dp3,c2dp1/c2dp2,c2dp1/c2dp3,and c2dp2/c2dp3.The double mutants and c2dp1/c2dp2/c2dp3 triple mutants were analyzed for phenotype.The analysis showed that under long day(16 hours of light and 8 hours of darkness)condition,except that the number of leaves at flowering of single mutant of c2dp3 was slightly reduced compared with that of wild type,the number of leaves at flowering of single mutant of c2dp1,c2dp2 and three double mutants were not significantly different from that of wild type,while the number of leaves at flowering of triple mutants was significantly reduced compared with that of wild type.These results indicate that the three genes are redundant in function and can inhibit the early flowering of plants under long day.However,under the normal condition of 22℃ in short day(16 hours dark and 8 hours light),the number of leaves at flowering of single mutant,double mutants and triple mutants was not significantly different from that of wild type,which indicated that these three genes were not involved in flowering regulation of Arabidopsis under short day.In addition,we found that the expression patterns of C2DP1 and C2DP2 in Arabidopsis was very similar,but slightly different through GUS staining.Through subcellular localization analysis,it is found that the proteins of C2DP1 and its homologous genes are mainly distributed in the plasma membrane and cytoplasm.In order to further explore the specific mechanism of the influence of C2DP1 and its homologous genes on flowering regulation,we analyzed the expression level of flowering genes in the mutant materials and found that FLC in the mutants was down-regulated under long-day conditions.It is speculated that C2DP1 and its homologous genes may affect the flowering of Arabidopsis thaliana by regulating the expression of FLC gene.The interaction partner proteins of C2DP1 and C2DP2,VOZ1 and VOZ2 were also screened through the yeast two hybrid screening,and then verified by point-to-point yeast two hybrid method,and bimolecular fluorescence complementation(BiFC)experiments.It was found that the proteins VOZ1 and VOZ2 involved in flowering regulation did interact with C2DP1、C2DP2and C2DP3 proteins respectively,and the interaction mainly occurred in the cell membrane and cytoplasm.Our work provided for the further research with new clues on flowering regulation in Arabidopsis.