The Mechanism Of Wheat AGL6 Regulating Stamen Development And Brachypodium Distachyon FUL2 Regulating Flowering Time

MADS-box is a widely-existing transcription factor that plays an important regulatory role during plant growth and development,such as controlling plant flowering time,inflorescence structure,fruit ripening and development,seed development,and development of male and female gametes.Among these transcription factors,two sister subfamilies members,AGL6 and FUL,are analyzed in this study.Previous studies have revealed the functions of rice and maize AGL6 genes Os MADS6 and ZAG3,respectively,in floral development,as well as the important role of the FUL gene in flowering time.Wheat is a main crop worldwide,and the function of the AGL6 gene is unclear.Brachypodium distachyon is an important grass model crop,and the mechanism of FUL gene regulating flowering has not been studied in depth.To better study the function of the MADS-box gene in grasses,this study mainly focused on the following two aspects: on the one hand,the mechanism of wheat AGL6(Ta AGL6)gene regulating stamen development;on the other hand,the mechanism of Brachypodium distachyon FUL2(Bd FUL2)gene regulating flowering time.The main findings of Ta AGL6 regulating stamen development are as follows:1.The full-length c DNA sequence of Ta AGL6 was cloned by 3' RACE and 5' RACE experiments using the c DNA of Chinese Spring wheat inflorescence as template.Results showed that there were three AGL6 homologous genes in wheat,named Ta AGL6-A,Ta AGL6-B,and Ta AGL6-D based on chromosome position.Results of quantitative RT-PCR(q RT-PCR)and in situ hybridization showed that Ta AGL6 genes were mainly expressed in flowers.In florets,these three transcripts of Ta AGL6 gene were mainly expressed in the palea,the lodicule and the pistil.2.To analyze the function of the Ta AGL6 gene,Ta AGL6-A,Ta AGL6-B and Ta AGL6-D were overexpressed in Arabidopsis,respectively.The transgenic plants mainly showed two similar phenotypes: early flowering and dwarf,and increased number of stems or branches.Similarly,a similar phenotype(early flower)also appeared in transgenic wheat overexpressing Ta AGL6-B.3.By analyzing the expression pattern of Ta AGL6 and the phenotype of transgenic Arabidopsis,it is speculated that the function of Ta AGL6 may be redundant.Therefore,RNAi transgenic wheat was obtained by knocking out three wheat AGL6 genes simultaneously.The number and morphology of stamens in RNAi transgenic wheat changed significantly.A normal stamen had four anther cavities,however,in some transgenic stamens,two anther cavities fused together or one stamen only had two anther cavities.At the same time,the vigor of pollen grains was affected in all RNAi stamens,fertility and seed setting rate were significantly reduced compared with the control.Quantitative RT-PCR results showed that the expression levels of Ta AP3 and Ta MGH3,which control stamen development,were significantly down-regulated,and the expression levels of Ta DL,Ta MADS13,and Ta LHS1,which controlled carpel development,were significantly up-regulated.As Ta AGL6 had transcriptional activation activity,the Ta MGH3 and Ta AP3 promoters whose expression was down-regulated were also analyzed.The Ta AGL6 protein was purified and verified by EMSA test.The results showed that Ta AGL6 can bind to Ta AP3 promoter,and Y1 H assay also demonstrated that three copies of the Ta AGL6 gene can bind to Ta AP3.In vivo,it was further verified by D-LUC that Ta AGL6 indeed activated Ta AP3 expression.4.Previous studies in rice have shown that Os MADS6 can interact with class B,C and D genes.Similarly,Y2 H and BIFC had verified that Ta AGL6 transcription factor can interact with the class B-like gene Ta AP3 in wheat,the class C-like gene Ta AG,and the D-like gene Ta MADS13.The interaction between these transcription factors was further verified in vivo by Co-IP.These results indicated that Ta AGL6 had the function of a partial class E transcription factor.The main findings of Bd FUL2 regulating flowering time are as follows:1.Using the c DNA of Brachypodium distachyon Bd21 leaves as the template,we cloned the Bd VRN1(Bd FUL1)and Bd FUL2 genes.q RT-PCR analyses showed that the expression patterns of Bd FUL2 was similar to that of Bd VRN1,and they were expressed in stems,leaves,flowers and seeds.In floret,it was mainly expressed in the glume,the lemma and the palea.Y2 H,Bi FC and Co-IP assays had verified that Bd FUL2 can interact with Bd VRN1.2.It has been previously reported that vernalization induces the expression of Bd VRN1 and Bd FT2.Since the expression pattern of Bd FUL2 is identical to that of Bd VRN1,it is speculated whether vernalization can also induce the expression level of Bd FUL2.Therefore,the expressions of Bd FUL2 in the pre-vernalization,one week of vernalization,two weeks of vernalization and one week after vernalization were detected.It was found that the expression level of Bd FUL2 in vernalization material was significantly higher than that in non-vernalized material.In addition,the expression level of Bd FUL2 was increased gradually with the growth of plant.And Bd VRN1 showed similar expression trend as Bd FUL2.Through the analysis of the concentration of histone H3K4me3 by Chip test,it was found that vernalization indeed induced the expression of Bd FUL2 gene.3.We also obtained Bd FUL2 RNAi transgenic Brachypodium lines.Under long-day conditions,results found that the flowering time of Bd FUL2 RNAi transgenic plants was significantly later than that of the control,regardless of vernalization.As well as the expression levels of Bd FT1 and Bd FT2 were down-regulated.The Bd FUL2 protein was purified and verified by EMSA and Y1 H tests.The results indicated that Bd FUL2 directly regulated the expression of Bd FT1 and Bd FT2.4.Interestingly,we found that the seed germination of Bd FUL2 RNAi was later than control Bd21,consistent with the fact that GA3 content was clearly lower than the control.Therefore,we analyzed the expression levels of related genes in the GA synthesis pathway.Results showed that the expression levels of BRADI2G24980 and BRADI2G19900 were significantly down-regulated in Bd FUL2 RNAi leaves.We further analyzed the CAr G elements in the promoter of BRADI2G24980 and BRADI2G19900,and performed EMSA to test whether the regulation was direct or indirect.Results showed that Bd FUL2 cannot bind to CAr G elements in these two genes.These results indicated that Bd FUL2 indirectly regulated GA synthesis.