The Molecular Mechanisms In The Regulation Of Heading Time And Development By TaTOC1s Genes In Wheat

Heading time is an important agronomic trait of common wheat and determines the adaptability to different ecological environments.Both the photoperiodic flowering pathway mediated by day-length and the vernalization pathway mediated by low temperature play important roles in determination of wheat heading time.In Arabidopsis,TIMING OF CAB EXPRESSION 1(TOC1),a member of PSEUDO-RESPONSE REGULATOR(PRR)family,is an important transcriptional factor and essential for some biology process,for example,the maintaince of circandian clock,flowering time and so on.Wheat,an staple food crop,is a long-day monocotyledon.There are many significant differences in developmental traits between wheat and Arabidopsis.It is an interesting scientific question to explore the function and mechanism of TOC1 in wheat.In this study,we knocked down the expression of TaTOC1 s in wheat using biotechnology and studied systematically the function of TaTOC1 s involved in wheat development.The main results are as follows:(1)Three TaTOC1 s were isolated from hexaploid wheat Lunxuan987.Their evolutionary relationship with others PRR family members in multiple species was analysed.The result showed that three Ta TOC1 proteins were closely related to barley HvTOC1.In situ hybridization experiments were peformed and showed that TaTOC1 s were predominantly expressed during the spikelets development process,suggesting that these genes may play important roles in wheat flowering regulation.(2)We obtained a number of TaTOC1 s silenced transgenic plants(termed as TaTOC1-RNAi)by RNAi strategy.The TaTOC1-RNAi transgenic lines flowered earlier about 7 days than controls(Lunxuan987)under both chamber and field growth conditions.Moreover,we found the development of shoot apical meristem(SAM)of transgenic plants was accelerated,but the duration of each developmental stage changed indistinctively.(3)Besides heading time,some other important agronomic traits of TaTOC1-RNAi transgenic lines were also changed,for example,the plant height become lower,the flag leaf become larger and the angle of flag leaf were smaller.The chlorophyll content and the net photosynthetic rate also changed.Then,we analyzed the wheat spike traits and yields.The spike number and spike length had no obvious change.The thousand kernel weight decreased but the grain number per spike and kernels per spikelet increased significantly compared with those in control.Finally the single plant weight and plot yield changed a little.In summary,decreased TaTOC1 s caused the reduction of plant height and eary heading time,but the yield remained stable,implying the application potential of TaTOC1 s in wheat genetic improvement.(4)To explain the early flowering phenotype of TaTOC1-RNAi transgenic lines,we detected the expression level of flowering-related genes and found the early flowering phenotype of TaTOC1-RNAi transgenic lines was correlated with the up-regulation of TaGI and TaFT and down-regulation of TaCO.(5)By comparative analysis of the RNA-seq data at different development stages of flag leaf in TaTOC1-RNAi transgenic lines and controls,we obtained 40081 different expression genes(DEGs)(20288 genes up-regulated and 19793 genes down-regulated).Some of them were related to cell wall organization,cell cycles and response to auxin.The GO and KEGG pathway analysis indicated some of DEGs were focused on the plant circadian rhythms and photosynthesis pathways,which provided some important information for further study on the functions of TaTOC1 s in wheat.(6)With more detailed evidences,we further confirmed tae-mi R408 to target TaTOC1 s.The expression level of several flowering-related genes in tae-miR408 overexpressing transgenic lines were determinated.The results suggested the early flowering phenotype of tae-mi R408 transgenic lines caused by reduced TaTOC1 s,upregulated TaGI and TaFT,as well as downregulated the expression of TaCO.(7)By comparative analysis of the RNA-seq data in leaves and SAMs at differently vernalized TaTOC1-RNAi transgenic lines and controls,a total of 33493 gene up-regulated and 30881 genes down-regulated were obtained.GO term analysis showed 11 genes involved in response to temperature stimulus and flowering regulation.Moreover,a number of TOC1 binding sites were identified in the promoters of these genes.These results provided important data for studying the reduced vernalization requirements in TaTOC1-RNAi transgenic lines.In summary,we studied the photoperiod-mediated flowering regulation in wheat,analyzed the molecular mechanism of phenotype variations and reduced vernalization requirements in TaTOC1-RNAi transgenic lines by RNA-seq technologies,these results provide important information for studying the molecular mechanism of TaTOC1 s in wheat flowering and development regulation.