Identification Of In Vitro Induced Flower Reversion And Related Gene Function Study In Switchgrass

Flowering is an essential procedure in the life cycle of angiosperms,it is important to achieve high yield for crops harvesting seeds or fruits.Generally,plants old enough carried out an irreversible one-way procedure of blossom,pollination,fertilized,and seed set.However,differentiated floral meristems of some plant species could re-enter the vegetative phase in some case and cannot flower ordinarily,which termed floral reversion.Switchgrass is a warm-season perennial grass,it was selected as a model energy crop widely in the world since its characteristic of drought-tolerance,barren-tolerance,easy to manage,and high biomass yield.In this study,in vitro incubation of shoot apexes of switchgrass cultivar‘Alamo’at elongation stage 4,which harboring a 1 cm long immature inflorescences,generated flower reversion.It was confirmed by anatomical,histological and cytological analysis subsequently.Next we analyzed the gene expression alteration in different material using RNA-seq and obtained flower reversion related genes.The biological functions of some of the genes were further verified.The main results were showed as follows.1.Shoot apexes incubated on flower reversion medium(MS+3 mg.L^-1 6-BA)generated more than one type of shoots.Anatomy analysis showed that they were different in morphology and distribution.We speculate that the first type of shoots arose from axillary buds on the explants,the second type of shoots arose from callus induced on the nodes,and the third type of shoots arose from florets primordium on the immature inflorescence.Histological analysis showed that floral organs in the third type of shoots degenerated and the apex re-entered vegetative phase.In the adjacent florets,the pistil was replaced by a globoid tissue with neither style nor ovule despite the formation of other whoral of flower organs.Results mentioned above suggesting that the third type of shoot arise from the development pattern alteration of switchgrass floret,which means that they arise from flower reversion instead of adventitious buds.2.We analyzed the transcriptome of flower reversion material from flower reversion induction medium,in vitro developed inflorescence from MS medium and uncultured immature inflorescence.According the development stage and direction,we consider that genes with the same expression level in in vitro developed inflorescence and uncultured immature inflorescence but differently expressed in flower reversion material participated in flower reversion,and through which,517 genes were filtered.Gene annotation by several databases showed that signal transduction,energy metabolism,and transcriptional regulation processes were crucial for switchgrass flower reversion.Comparesion of these 517 genes with flower development realted gene showed that no one of the published flower development related gene was included,which suggested that flower reversion was different from flower development in gene regulation.However,gene expression pattern showed that the flowering promoter AP1 was down regulated and the flowering inhibitor TEM1/TEM2 was up regulated in flower reversion materials despite the change fold less than 2,which indicates that flower reversion was also associated with flower development in some aspects.3.Four genes with relatively high expression level in switchgrass flower reversion material were over-expressed in Arabidopsis.However,no plant showed complete flower reversion in any transgene even.Some of the transgene plants showed expanded leaf area,some of which showed flowering delay and some of which showed abnormal flower organ development.From which we speculated that switchgrass flower reversion is a complex process which including promotion of vegetable growth,suppression of flower formation,and dedifferentiation of flower organs,overexpression of a single gene is insufficient to induce the complete process of flower reversion.4.PvSTK1 gene encodes a serine/threonine protein kinase,the subcellular localization analysis showed that this protein distributed in the whole cell.Over-expression of PvSTK1delayed Arabidopsis inflorescence development and caused sterile florets at the base of the inflorescence.Gene expression pattern analysis showed that flowering promoter genes AP1,AG,FT and LFY were down regulated while flowering inhibitor genes FLC and SVP were up regulated in transgene lines,which suggested that PvSTK1 influenced flower development regulatory network of the photoperiodic pathway,the vernalization pathway and the autonomous pathway,and it function as up-stream of the regulation.Analysis of the infertility floret at the base of the inflorescence showed that,the pollen vitality is normal but the ovules shrink gradually after flowering.Pollination of sterile florets using pollen from wild-type Arabidopsis has not been able to produce silique,which indicating that the PvSTK1 gene influenced Arabidopsis florets fertility by affecting ovule development.In this study,we achieved in vitro floral reversion in switchgrass for the first time,obtained 517 genes that might regulate flower reversion,and verified the biological function of some of the genes.This is an attempt to study plant vegetative growth and reproductive growth from another perspective,which might contribute a fundamental for flower development regulation in switchgrass and other plants.