Functional Analysis Of OsGCD1 In Male Gametophyte Formation,Embryo Pattern Formation And Endosperm Development Of Rice (Oryza Sativa)

In flowering plants(angiosperms),the life history involves alternation between two multicellular generations,a diploid sporophyte and a haploid gametophyte.In a normal life cycle,the two generations are demarcated by meiosis,which produces the spores that will become the gametophyte generation that produces male gametes and female gametes for double fertilization(gamete fusion)and zygote formation,which will become the new sporophyte generation.Pollen formation and male gametogenesis is an intricate process and involves an accurately and tightly controlled set of structural and molecular changes,requiring numerous genes expressed in both gametophytic and sporophytic tissues of anthers.In a certain sense,male gametophyte development can act as a model which offers the superexcellent opportunity to comprehend cell fate specialization,cell signaling and cell polarity.When pollen matures,a sequence of events including pollination,pollen germination and pollen tube growth happen,and the sperm cells are released into the embryo sac and then fuse with two dimorphic female gametes(the egg and the central cell)forming the embryo and endosperm,respectively.In Arabidopsis,the early stages of embryogenesis follow a simple,highly regular and nearly stereotypic pattern,establishing an embryo with apical-basal axis and radial axis.Compared with Arabidopsis,the rice embryo grows by seemingly chaotic,random cell divisions and is not radially symmetrical,which leads to the existence of the third axis,the dorsal-ventral axis.Meanwhile,the syncytial endosperm is formed and subsequently cellularization.The endosperm cells of rice differentiate into the starchy endosperm and the aleurone layer with difference between the ventral and dorsal sides,which is also different from that of Arabidopsis.However,compared to Arabdopsis,the relevant study on rive is less extensive,especially,it remains unclear that realistic significance of the dynamic vacuoles process and procedural nucleus positioning,especially its influence for male gametophyte development and gametogenesis.On the other hand,the regulation mechanisms of embryo dorsal-ventral pattern formation and dorsal-ventral axis differentiation of aleurone layer are essential for rice reproduction,but remain largely unknown.In addition,relatively little is known about the determinant of endosperm free nucleus positioning.To answer above mentioned questions,we employed Osgcd1/+ mutant obtained by CRISPR/Cas9 system as the material in this study.Based on the observation of the Osgcd1/+ male gametophyte development,we revealed that the real role of dynamic central vacuole disappearance and regular nucleus positioning in gametogenesis and vegetative growth of the male gametophyte respectively.At the same time,with the aid of WECLSM,ISH,TUNEL techniques,we analysed the influence of OsGCD1 mutation on embryogenesis,endosperm free nucleus positioning,proper antipodal cell degene-ration and endosperm cellularization,as well as aleurone layer differentiation,with the hope to illuminate in some degree the molecular mechanism for rice embryo pattern formantion and endosperm development.Following are our main research outcomes.1.We screened and obtained the full-length(3349bp)sequence of the OsGCD1(Oryza sativa GAMETE CELL DEFECTIVE 1)gene using bioinformatics analysis and the Institute for Genomic Research(TIGR)database.OsGCD1 and Arabidopsis GCD1 were found to be an orthologous pair,and putative conserved domains were detected.The gene encodes a predicted protein of 345 amino acids localized to mitochondria.The expression of OsGCD1 was evaluated by RT-qPCR(real-time quantitative polymerase chain reaction),which showed that OsGCD1 transcripts were detected in the root,stem,leaf,glume,lemma,palea,and anther,as expected,they were also detected in ovaries at various developmental stages.In addition,RNA in situ hybridization was performed to determine the spatial and temporal patterns of OsGCD1 expression precisely.OsGCDl was found to be expressed in the mature embryo sac,subsequently,it was expressed from 1 to 9 DAP(day after pollination)during embryo development.OsGCD1 was also expressed in the endosperm at both the free-nuclear and cellular endosperm stages.As endosperm development progressed,the signals gradually disappeared from the central part of the endosperm and became concentrated in the aleurone layer.In addition to its expression in ovaries,OsGCD1 was also expressed in anther tapetum and microspores.Our results indicated that OsGCD1 is ubiquitously expressed in the process of plant development,and suggested that it likely plays a key role throughout male gametophyte formation,early embryogenesis and endosperm development of rice.2.To evaluate the function of OsGCDl,the CRISPR/Cas9 system was used to edit three target sites in the first exon of OsGCD1 and successfully create OsGCD1 mutants.All of the mutant plants were heterozygous;no homozygous line was generated.To explain why a homozygous line could not be obtained,reciprocal crosses were carried out,the result suggests that mutated OsGCD1 can be passed to offspring only from the female parent.All of Osgcdl/+ showed normal vegetative and floral organ development,however,the pollen vitality and seed setting rate reduced to a certain degree.The phenotypes of the ovules of the mutants manifested after fertilization.The Osgcdl/+abortive seeds usually carried a partial endosperm and an abnormal embryo,and some had an empty embryo sac.3.The male gametophyte is a pivotal component for rice fertility and sexual reproduction.We found that OsGCD1 plays an indispensable role in the process.In all anther development stages,there are no obvious difference of structure and morphology in anther sporophytic tissues between wild-type and the Osgcd1/+,suggesting that sterile pollen is due to abnormal microspore development.These pollen grains linger-ingly developed and could not germinate in the medium,indicating that OsGCD1 mutation results in the defect of pollen,and thus,male mutated OsGCD1 can't be passed to offspring.In Osgcd1/+ plants the big central vacuole could be kept to the mature pollen stage and never disappeared.The generative nucleus was thus still peripheral located due to the special occupation of the central vacuole.In addition,abnormal vacuole may contribute to the block of vegetative cell fully development and starch accumulation,which seems necessary for the pollen germination,so the vegetative growth of the male gametophyte showed abnormalities with thinner intine layer,lower level of cytoplasmic contents and few starch grains.Interestingly,the gametogenesis was not disturbed and two sperm cells were still well produced.Our observations suggest that gametogenesis is a comparably independent process from the vegetative development of the male gametophyte,which is not influenced by the abnormal existence of the central vacuole and altered position of the generative nucleus caused by OsGCD1 mutation.4.The maturity and morphology of egg cell was not really affected in Osgcd1/+ plants,and egg cell participated in the fertilization,and the zygote was capable of initiating cleavage in Osgcd1/+ ovules.Embryogenesis was initiated,but embryo pattern formation was disrupted,in Osgcdl/+ mutants.The embryo developed slowly or even stagnated in the early stages.Subsequently pattern formation,particularly the dorsal-ventral pattern and symmetry establishment,of embryos was disturbed.Thus,the embryos showed various morphological and structural dysplasias.The transcript levels of genes involved in early embryogenesis including GE,OSH1,HAZI,PNH,OsMPK6,RAmy1A,ROC1 and SCR were determined.The result showed that OsGCD1 mutation influenced on the differentiation and formation of basic axes,particularly the dorsal-ventral axis,at the molecular level.The spatial distribution of GE and OSH1 expression in whole embryos was evaluated using RNA in situ hybridization and further evaluated the effect of the OsGCD1 mutation on the expression of genes related to the dorsal-ventral axis.Irregular GE expression in Osgcdl/+ abnormal embryos indicated that OsGCD1 mutation disturbed scutellum development and made differentiation between the embryonic dorsal and ventral regions difficult.Moreover,OSH1 was expressed in a smaller area of the abnormal embryo,suggesting that OsGCD1 mutation influenced not only the dorsal-ventral but also the apical-basal pattern formation,and SAM development as well.5.The syncytial endosperm of Osgcdl/+ mutant seeds was distributed only at the bottom part of the embryo sac,indicating that the directed positioning of the free nucleus was disturbed by OsGCD1 mutation.Following,the disorganized proliferation of the endosperm nuclei and irregular formation of the endosperm cell walls were observed in Osgcd1/+.Starchy endosperm cells of the wild type initiated PCD when endosperm cellularization was complete,and the profile of the starchy endosperm became gradually blurred.However,PCD was not triggered in the Osgcdl1+ mutant and the endosperm cells maintained discernible cell morphology,indicating that OsGCD1 mutation results in failed PCD,which may lead to failure of endosperm cell death and starch synthesis.In addition,the phenotypic analysis and relative expression levels analysis of the rice aleurone layer-specific genes proved that dysfunction of OsGCD1 disturbed the number of cell layer the dorsal-ventral differentiation of the aleurone layer.6.In rice,antipodal cells continue to proliferate and form the highly distinct antipodal cell cluster in the mature embryo sac.Our results showed that in wild-type antipodal cell cluster persisted after fertilization until the initiation of endosperm cellularization and formation of the first cellular endosperm layer at the periphery of embryo sacs.However,antipodal cell degradation was delayed in Osgcd1/+ mutant seeds as the endosperm cellularization postponed.In addition,the free endosperm nuclei of Osgcd1/+ mutant seed were concentrated at the micropylar end and absent around antipodal cells.The above-mentioned event indicates that the fate of antipodal cells may be controlled by particular period of endosperm development,whereas antipodal cells may influence endosperm free nucleus positioning.In summary,OsGCDl encoding a mitochondrial protein ubiquitously expressed in the process of plant reproduction development.No mutant plants were homozygous,and mutated OsGCD1 can't be passed to offspring from the male parent although maternal heritability also dropped.OsGCDl plays a pivotal role in the process of male gametophyte development by regulating their vegetative growth,the organized dynamics process of vacuole and generative nucleus positioning.The maturity and morphology of egg cell was not really affected in Osgcd1/+ plants and embryogenesis can be initiated,but pattern formation,particularly the dorsal-ventral pattern and symmetry establish-ment of embryo was disrupted.In addition,OsGCDl works on the directed positioning of the free nucleus of the syncytial endosperm and the dorsal-ventral differentiation of the aleurone layer.Meanwhile,rice antipodal cells lifespan seems to be related to accurate early endosperm development.Thus,we demonstrate that the regular dynamics process of vacuole is critical for the formation of functional male gametophyte,but the male gametogenesis seems a self-programmed process and not really influenced by the variation of vacuole dynamic and generative cell positioning once the cell is created.We also provide an illustration for the molecular mechanism involving in embryo dorsal-ventral axis establishment,endosperm free nucleus positioning and subsequent aleurone differentiation.