Carbohydrate Metabolism And Gene Regulation During Anther Development In An Androdioecious Tree,Tapiscia Sinensis

The breeding system of angiosperms is one of the most important research field in Evolutionary Botany,in the long-term evolution process,angiosperms had evolved a variety of breeding systems,such as monoecious,dioecious,gynomonoecy,andromonoecy,gynodioecy and androdioecy.The functional androdioecy breeding system is very rare in the whole flowering plants.Androdioecious might evolved from dioecious or hermaphrodite,but its evolution mechanism was still unclear.At present,most of morphological androdioecy has been proved to be the functional dioecy,and functional androdioecy is in only of 0.005%in angiosperms.Recently,bagging experiments and paternity analysis were used to confirm the functional androdioecy of Tapiscia sinensis,and fruit ripening overlaps with flowering in hermaphroditic individuals.For reproductive assurance,the resource allocation would be partial to the female function,resulting in decreased pollen viability of hermaphroditic flowers.In the process of flower development,carbohydrate is the main nutrients,carbohydrates inputs have larger effect on male and female functions in flowers.In this study,we measured the soluble sugar content of anther of male and hermaphroditic flower,the relative expression of genes related to sugar transport and metabolism,and the expression of temporal changes to gain insight into the mechanisms of the weakening of the male function of hermaphroditic flowers in T,sinensis.The results of this study are as follows:(1)The soluble total sugar and starch content in anther of hermaphroditic flower are lower than male flower anther at vacuolate microspore(VM)and binucleate stage(BN).Compared with the male flower anther,sucrose content of hermaphroditic flowers anther was highly significant reduction at VM and BN stage,and the fructose content at VM stage was extreme significant reduction,and decreased significantly at the BN stage.(2)Compared with the male flowers,SS2,HXK2,AGPase,UGPase and SSasel all showed much higher expression in hermaphroditic flowers than in males at the VM stage.VI was expressed at high levels at the VM and BN stages,and the expression of CWIN and SPS2 were significantly repressed in hermaphroditic flowers at the MMC,VM and BN stages.At BN stage,the CWIN,SS2,HXK2,SPS2,and SSase1 was down regulated in hermaphroditic flowers.The expression of sugar transporter gene ST2 was higher than that of males at the three stages,and the expression of MT was lower than males at the microspore mother cell stage(MMC),but it was higher than that of males at VM and BN stage.During the development of hermaphroditic flowers,expression of the intine synthesis genes Fasciclin-like arabinogalactan(FLA)and UDP-sugar pyrophosphorylase(USP)was lower than the males at the three stages,and the expression was significantly lower than the males at VM stage.(3)In situ hybridization results showed that CWIN was expressed in tapetum and microspores.At tetrads stage(TDS),the expression of CWIN was basically the same in tapetum and microspores of male and hermaphroditic flowers,there were no significant difference.At young microspore(YM)and VM stage of male flowers,CWIN was strongly expressed in the tapetum and microspores,and at mature pollen stage(MP),CWIN is highly expressed in microspores.Compared with male flowers,the expression of CWIN was significantly lower in tapetum and microspores of hermaphroditic flowers at YM and VM,and at MP stage,it almost no expression in pollen grains.Sugar transporter gene ST2 is also expressed in tapetum and microspores,and there were no significant differences between male and hermaphroditic flowers at YM and VM stage.However,at MP stage,ST2 in male microspores with a small amount of expression,and it was no expression in hermaphroditic microspores,but more expression in connective tissue.(4)The gene of cell wall invertase was cloned successfully from T.Sinensis,named as TsCWIN(accession number:KY072915),the full-length cDNA of TsCWIN is 2024 bp,it contains an open reading frame of 1764 bp,a 5' untranslated region of 25 bp and a 3'untranslated region of 235 bp.The TsCWIN gene encoding CWIN protein contains 587 amino acids,and its predicted molecular weight and isoelectric point was 66.35 kDa and 9.05,which belongs to a hydrophobic protein and belongs to the membership of the glycosyl hydrolase family.Phylogenetic tree analysis indicated that TsCWIN protein has the closest genetic relationship with Theobroma cacao CWIN protein.During the development of anther of T.sinensis,there was a severe reproductive burden on hermaphroditic individuals.Reproductive resources was re-allocation in hermaphroditic flowers to ensure the normal development of the fruits and the female function and make the male plant was maintained in hermaphroditic population.Low sugar content was in anther of hermaphroditic flowers,and there was inordinate sugar metabolism and transport,resulting in a resource shortage in hermaphroditic pollen grains,eventually led to a decline of the male function in hermaphrodites.Thus,we thought that hermaphroditic flowers regulated the metabolism and the transport of sugar to realize the resources re-allocation,to maintain the functional androdioecious breeding system in T.sinensis.