Studies On The Characteristics Of Female Flower Development And Its Hormone Regulation Mechanism In Tung Tree

Tung tree(Vernicia fordii),native to China,belongs to the Euphorbiaceae family of woody angiosperms.Tung tree is considered as one of the four major woody oil trees in China.The oil from tung seeds was called for tung oil.Tung oil contains special trivalent conjugated double bond fatty acid(eleostearic acid),so it has active chemical properties.In recent years,tung oil has been attracted world-wide attention due to production security,environmental concerns,and negative effect of synthetic chemical coatings on human health.As a monoecious plant species,tung tree produces a low ratio of female to male flowers(approximately 1:27)and functionally abnormal female flowers,which results in low fruit yield in tung orchards.Toward this end,this study conducted integrated analyses of morphological and cytological observations,endogenous phytohormone assay and RNA-seq of flower buds and male and female flowers.Based on these analyses,this study proposed a possible mechanism for female flowers differentiation and development in tung tree.This study not only provides an important theoretical significance in high-yield cultivation techniques of tung tree,but also has an important practical value for tung tree.The main results are as follows:1.The regulation of flower buds differentiation and male and female flowers development in tung tree was identified.The differentiation of flower buds and development of male and female flowers in multi-female flower tung tree and male flower tung tree were researched using the sectioning technology system.The result showed the differentiation of flower buds in tung tree were divided into four stages:leaf primordial stage,inflorescence primordial stage,inflorescence axis differentiation stage,and floral organ differentiation stage.The male flower buds differentiation were earlier than multi-female flower buds.The male flower buds differentiation began at the middle of June and the key period of sex differentiation was at the late of June,while the multi-female flower buds differentiation began at the early of July and the key period of sex differentiation was at the late of July.The organ morphology and the cytological process of the diferentiation and development in male and female were researched using a 3D super depth digital microscope and the sectioning technology system.The result found the development of male and female flowers needed ten months which began at June or July.The development of female flowers in tung tree were divided into six stages:floral primordium differentiation stage,pistil development stage,dormancy stage,carpellar union(stamen degeneration)stage,stigma elongation stage,and flowering stage.The development of male flowers in tung tree were divided into six stages:floral primordium differentiation stage,Stamen development stage,dormant stage,pollen formation stage,filament elongation stage,flowering stage.The cytological process of female and male gametogenesis were researched using the sectioning technology system.The result showed the development of female and male gametogenesis began at March of the following year and the duration time was approximately one month.The key period of female gametogenesis development was at the late of March and the mature embryo sac had seven cells with eight nuclei,while the key period of male gametogenesis development was at the middle of March and the mature pollen had two-cell.2.Exogenous hormones of female flowers differentiation were selected.Before the key period of sex differentiation,the male flower buds were treated by different concentration of indole-3-acetic acid(IAA),ethephon(CEPA),gibberellin(GA),and N-(Phenylmethyl)-9H-purin-6-amine(6-BA).The result found IAA and CEPA had not effect for the differentiation of the male flower bud.The GA3 could slightly increase the ratio of female flowers in male flower buds.The 6-BA could significantly increase the ratio of female flowers in male flower buds,and with the concentration increasing,the ratio of female flowers was increased.The male flower buds had the most female flowers at the concentration of 160 mg/L 6-BA,and the male flower buds transferred to female flower buds at the concentration of 640 mg/L 6-BA.Besides,the induced female flowers in male flower buds could produce tung fruits and the characteristic of the tung fruits was similar to normal tung fruits.Therefore,the 6BA was the important exogenous hormone to promote the differentiation of the female flower in male flower buds.3.The regulating mechanism of 6-BA of the differentiation of the female flower in male flower buds was described.The male flower buds which were treated by 640 mg/L 6-BA after 0-h,12-h,and 36-h,were researched using the HPLC-MSn technology,real-time qPCR technology,transcriptome sequencing technology,and bioinformatics technology.The result showed levels of cytokinin were negatively correlated with auxin.The level of jasmonic acid increased at 12-h,while the level of GAs decreased at 12-h.Besides,based on the analysis of the expression pattern of floral meristem-,stamen primordium-,and flowering time-related genes,the research found 6-BA could promote the expression of floral meristem-related genes,and suppress the expression of stamen primordium-and flowering time-related genes.Finally,the male flower buds transferred to female flower buds in tung tree.4.The key reason leading to stamen abortion in female flowers was identified.The organ morphology and the cytological process of 35 days before flowering(DBF),30 DBF,25 DBF,20 DBF,15 DBF,and 10 DBF in male and female were researched using a 3D super depth digital microscope and the sectioning technology system.The result found female flowers possessed obvious stamens at 35 DBF and 30 DBF which initially degenerated from 25 DBF and completely disappeared until 10 DBF,whereas no pistil was observed and only intact stamen developed in male flowers across all the development stages.The stamens in female flowers(SFF)developed into sporogenous cell stage at 35 DBF,while the stamens in male flowers(SMF)developed into early microspore mother cell(MMC)stage.When the SFF developed into early MMC stage at 30 DBF,the SMF developed into later MMC stage.Interestingly,the SFF stopped growth at early MMC stage with tapetum cells degeneration and large vacuoles in MMC at 25 DBF,while the SMF developed into tetrads stage.From 20 to 10 DBF,the SFF shrank at carpel base,and the MMC was mostly occupied by vacuoles with nucleuses in MMC disappearing,while the SMF developed into binuclear pollen stage.Taken together,the abortion of SFF in tung tree belongs to the type of PCD.The tapetum cell degeneration should contribute to the abortion of SFF,finally leading to PCD in MMC.5.The regulating mechanism of salicylic acid of the development of the female flower in tung tree was described.Based on the analyses of the HPLC-MSn analysis,real-time qPCR analysis,transcriptome sequencing analysis,and bioinformatics analysis in male and female flowers of 30 DBF,20 DBF,10 DBF,we found 17 key genes in anther and pollen development were suppressed in 20 DBF and 10 DBF,and 37 PCD-related genes were identified based on their expression patterns which included 26 positive genes and 11 negative genes.Most of the positive genes of PCD-related genes exhibited high expression levels in 20 DBF and 10 DBF,while negative genes showed low expression levels in 20 DBF and 10 DBF and high expression in 30 DBF.Further analyses of phytohormone levels and co-expression networks suggested that salicylic acid(SA)accumulation could trigger PCD and inhibit the development of SFF in tung tree.Therefore,PCD-related genes are activated and anther and pollen development genes are suppressed which result in the abortion of SFF.Consequently,the normal female flower develops in tung tree.