The Function Of TFL1-like Genes RCN And Their Responses To Environmental Factors In Rice

Rice is a typical short-day crop.Heading date is a crucial agronomic trait that determines crop yield,quality and adaptability.Floral transition is an important symbol that rice transforms from vegetative growth to reproductive growth,which is determined by internal factors and external environment.A large number of studies have shown us that Hd3a(Heading date 3a)and RFT1(RICE FLOWERING LOCUS T1)are florigens in rice.Although the molecular regulation network of rice heading has been basically established,the molecular mechanism of floral transition is still unclear.TFL1(TERMINAL FLOWER 1)is a floral inhibitor in Arabidopsis thaliana,which plays an important role in regulating phase transition and inflorescence meristem identity.RICE CENTRORADIALIS 1～4(RCN1～4)genes are homologous genes of TFL1 in rice.Previous studies suggested that RCN genes are functionally redundant.In our study,CRISPR/Cas9 technology was used to create mutants of each RCN gene.We explored their expression patterns,subcellular localizations and interactive genes.Environmental factors were combined to illuminate the functions of RCN family genes.The main findings are as follows:1.Expression patterns and subcellular localization analysis of family genes.The results of GUS staining showed that four RCN genes were expressed in embryos,roots and spikelets.RCN1 and RCN3 were also stained in leaves.RCN genes have a stable basal expression during the whole growth and mainly expressed in vascular tissues.The expression sites were overlapped but still have some differences,suggesting that they may be involved in regulating various developmental processes of rice.We observed that the protein encoded by RCN1 was localized in cell membrane and nucleus through laser confocal microscopy.2.RCN1 interacts with OsFTIP1(Os-FT-INTERACTING PROTEIN 1)in the nucleus.OsFTIPl can transport RFT1 to shoot apical meristem(SAM)and cause floral induction in rice under long-day condition.Although yeast two-hybrid assay didn’t show the interaction between RCN and OsFTIP1,we found that RCN1 interacted with OsFTIP1 in tobacco epidermal cells using bimolecular fluorescence complementation.This indicates that under long-day condition,RCN1 proteins may interact with OsFTIP1 proteins and thus change the localization of the latter proteins,prevent the transport of RFT1 proteins to SAM,which delays floral transition and heading date in rice.3.The effects of RCN1 and RCN3 in regulating rice heading date are more notable,while RCN2 and RCN4 may have function redundancy during floral transition.Heading date of rcnl plants and rcn3 plants were advanced under long-day and short-day condition,respectively.Meanwhile,plant height became lower and total number of leaves decreased.Compared with wildtype,there was no significant difference between rcn2 and rcn4 mutants.In addition,overexpression of RCN1 in rice delayed heading under both photoperiods.Similar phenomenon were observed by dexamethasone-induced RCN1 expression,which further confirm the function of RCN1 to delay floral transition in rice.4.RCN genes affects rice yield formation.The panicle number increased while the spikelet number per panicle decreased in rcnl mutants,and grain filling percentage was significantly decreased without nitrogen application.The spikelet number per panicle decreased in rcn3 mutants.The grain filling percentage of rcn2 and rcn4 were slightly higher than that of wildtype.The other yield components disappeared no significant difference.We also compared the spikelet structure between WT and mutants.We found that the number of branches and spikelets of rcnl and rcn3 mutants decreased,while the number of branches and spikelets of rcn2 and rcn4 mutants slightly increased or had no significant difference.In addition,the plant height of RCN mutants was reduced in various degrees,indicating that this family genes may be involved in the regulation of stem development.5.RCN1 and RCN3 can regulate the differentiation of spikelet in response to nitrogen.Previous research showed that application of nitrogen fertilizer could prolong the differentiation time of stem and spikelet in rice,increase the number of secondary branches and the total number of spikelets under certain conditions.However,after the loss of function of RCN1 or RCN3,the effect of nitrogen on promoting branch and spikelet differentiation on mutants was weaker than that of wildtype.Moreover,this phenomenon was particularly obvious in rcnl/3 double mutant,since there was no significant difference in the number of branches and spikelets under different levels of nitrogen application.6.RCN1 can respond to drought in regulating rice heading date.We found that drought stress both delayed the heading date of rcnl mutant and wildtype,but the extent of the mutant was shorter than that of wildtype.Real-time PCR showed that drought caused the downregulation of flowering-related genes such as Hd3a and RFT1,while RCN1 expression was upregulated,suggesting that RCN1 may be involved in the regulation of drought pathway to delay rice heading.