The Preliminary Study On Cytological And Molecular Mechanism Of Ovule Abortion In Chinese Chestnut

In this study,Chinese chestnut ’huaihuang’ was chosen as experimental material.The fertile ovules that can normally develop into seeds was used as controls,and the causes of high abortion rate of ovules in ovary were investigated.There are some main research methods such as Fluorescein disodium tracer technique,FDA(Fluorescein diacetate)active labeling technique,paraffin technique,electron microscope technique and iTRAQ(Isobaric tag for relative and absolute quantiation)proteomics techniques,and it provides a certain theoretical basis and practical significance for increasing the yield of Chinese chestnut per mu.Main results for this study are as follows:(1)The chestnut anthesis starts mid-June,the rapid growth stage of shell,ovary,fertile ovules occur in 25 days after anthesis(DAA).The analysis of microstructure of ovary suggested that there are 6-9 locules,two ovules per locule in an ovary.But only one of 12-18 ovules could develop nomarlly into seed,the rest aborted.In addition,fluorescent labelling showed that the time of abortion of ovules in ovary was not synchronous and the nutrient transport was hampered at funicle of aborted ovules.All abortive ovules were shriveled and formed seed marks 40 DAA.And the accumulation of callose at the funicle of abortive ovules causes difficulties in the transport of nutrients,which further might lead to the abortion of ovules.(2)The growth and development of chestnut seeds depend on the development of embryo in fertile ovule.It taked 35 to 45 days from anthesis to cotyledon embryo formation,and there was no endosperm in the mature chestnut seeds.The process of embryo development could be divided into four periods,which were globular embryoid period(20 DAA),heart embryoid period(25 DAA),torpedo embryoid period(30 DAA)and cotyledon embryoid period(35 DAA),and the endosperm development went through four stages:free nuclear stage,cellular period,differentiation phase and death phase.The morphology and microstructure of abortive ovules were significantly different from that of fertile ovules at 20 DAA.The results showed that there were no embryo sacs,wide spaces between the integuments,few with nucellus in the abortive ovules.(3)The dynamic changes of ovule cell wall components were observed in different period.The results showed that the development of fertile ovules were accompanied with the increase of cellulose and degradation of callose after initiation of embryogenesis;And,in the cell wall of developing ovules,high esterified and low-esterified homogalacturonan(HG)pectin were abundant,suggesting rapid synthesis and de-methyl-esterification of HG;the arabinan epitopes decreased in developing ovules but were more abundant than galactan epitopes at all stages.In the later development of fertile ovule,the increase of galactan and extensin,the decrease of xylan promote the expansion of cell wall.But the cell shape of abortive ovule was abnormal and the stability and flexibility of cell wall were destroyed,cellulose,callose,HG pectin and arabinan accumulated preferentially in the inner integument.Thus cells in the outer integument may die earlier than that of inner integument;meanwhile,low level of extensin,galactan and xylan suggested the cells of abortive ovules might not be able to synthesize these components.(4)The results of fluorescent labeling of the nucleus showed that the nucleolus were exposed in the cells,the flurescent spots were bright and dispersed;Transmission electron microscopy(TEM)observation showed that cells of abortive ovules had abnormal cell shapes,thickened cell walls,folded cell membranes,condensed cytoplasm,ruptured nuclear membranes,degraded nucleoli;And the presence of TUNEL positive signals in cells indicated that DNA had been fragmented;These results showed that abortion of ovules was caused by programmed cell death(PCD).(5)The results of iTRAQ showed that low level of soluble protein with small molecular weight was found in abortive ovule;The GO enrichment analysis showed that large number of proteins associated with reactive oxygen species metabolic process,response to reactive oxygen species,cell death,cation binding,metal ion binding,calcium ion binding,mitochondrial membrane stystem,respiratory chain and ATP(Adenosine Triphosphate)synthase complex were enriched which might lead to damage of cell compenents and disorder of energy metabolism,and eventually lead to cell senescence and death.