Physiological Characteristics And Transcriptome Analysis Of Male And Female Flowers In The Mixed Inflorescence Of Castanea Mollissima

Castanea mollissima is an important woody grain tree species in China.It plays an important role in helping poverty alleviation,rural revitalization and maintaining China’s food security.In chestnut production,there was an imbalance in the proportion of male and female,few female flowers and too many male flowers,which restricting the yield of chestnut.Therefore,in this paper,the mixed inflorescences of C.mollissima ’Tanqiao’ were used as materials to clarify the cellular anatomical characteristics of male and female flower differentiation of ’Tanqiao’ mixed inflorescences by means of body microscope and paraffin section technology.The contents of endogenous hormones in male and female flowers of mixed inflorescences at different developmental stages of’Tanqiao’ were analyzed by HPLC and enzyme-linked immunosorbent assay.RNA-seq sequencing technology and bioinformatics were used to screen the related genes regulating the development of male and female flowers.We will provide an important theoretical basis for the morphogenesis and sex regulation of chestnut flowers.The main results are as follows:1.We Clarified the cytological process of female and male flower development of C.mollissima ’Tanqiao’.The external and internal morphology of female and male flowers of C.mollissima ’Tanqiao’ were observed by the microscopy.The results showed that in Changsha City,Hunan Province,the differentiation of mixed inflorescence of ’Tanqiao’ began in mid April and ended after the flowers opened in late May,which lasted about 45 days.The process of female flower development could be divided into eight stages,including flower cluster bract primordium differentiation stage,flower cluster primordium differentiation stage,sepal primordium differentiation stage,stamen primordium differentiation stage,pistil primordium differentiation stage,pistil and pistil development stage,flowering stage and ovary formation stage.The process of male flower development could be divided into 9 stages,including flower cluster bract primordium differentiation stage,flower cluster primordium differentiation stage,sepal primordium differentiation stage,stamen primordium differentiation stage,pistil primordium differentiation stage,stamen elongation stage,anther formation stage,anther development stage and flowering stage.2.We explored the change characteristics of hormone content during the development of male and female flowers of C.mollissima ’Tanqiao’.The contents of endogenous hormones such as gibberellin,auxin,zeatin,abscisic acid,ethylene,brassinolide,salicylic acid and jasmonic acid in different developmental stages of male and female flowers of C.mollissima were determined by high-performance liquid chromatography and enzyme-linked immunosorbent assay.The results showed that there were differences in the content and change trend of endogenous hormones between male and female flowers,and different endogenous hormones had different regulatory effects on the development of male and female flowers.Salicylic acid,gibberellin and abscisic acid in male flowers were significantly higher than those in female flowers at all stages of C.mollissima flower development.These three hormones may be more involved in the process of chestnut male flower development.The contents of Zeatin and jasmonic acid in male flowers were significantly higher than those in male flowers.These two hormones may be more involved in the development of female flowers of chestnut.The contents of Brassinolide and ethylene in female flowers were slightly higher than those in male flowers,and there was yet no significant difference.The content of IAA in both male and female flowers showed an "M" trend in C.mollissima’Tanqiao’.3.We screened the genes related to the differentiation of male and female flowers in the mixed inflorescence of C.mollissima ’Tanqiao’.Transcriptome analysis of male and female flowers of C.mollissima,it was found that 4,331 genes were differentially expressed in female flowers of chestnut at four developmental stages.There were 9,977 differentially expressed genes in the male flower of chestnut at four developmental stages;At the same time,971 genes were differentially expressed in both male and female flowers at four developmental stages.The go and KEGG results of different genes at different developmental stages of female and male flowers and different genes at different developmental stages of male and female flowers of chestnut showed that the biological processes and metabolic pathways closely related to flower development,such as plant hormone synthesis and transduction,starch and sucrose metabolism,were annotated.Twenty differential genes related to plant hormones were excavated.The expression of 9 genes in female flowers was higher than that in male flowers,includingAOS3、YUC4,IAA27、CKX3、UGTK5、UGTK4、TPS28、GA2OX1、CYP724B.The expression of 11 genes in male flowers was higher than that in female flowers,including GA3OX1、GA2OX2、CYP707A1、CYP707A2、CKX1、ARR17、IAA17、IAA7、AUX22、GH3.17、TGA10.At the same time,18 differential genes related to the differentiation of male and female flowers of ’Tanqiao’ were screened.It was found that the expression of some transcription factors decreased with the development of flowers,such as WRKY40,bHLH92,HSP17.7,FD,AHL23,bHLH52,ERF084,VRN1,etc.In contrast,some transcription factors were significantly up-regulated with flower development,such as KUA1,AMS,NAC056,AHL1,MYB26,etc.Through WGCNA analysis,10 genes were excavated,among which IDD7 gene may be the key gene for female flower development of chestnut.These results laid a foundation for further study on the molecular regulation mechanism of mixed inflorescence differentiation of C.mollissima ’Tanqiao’.