Molecular Mechanisms For Rice Interploidy Crosses Resulting In Abnormal Seed Development

In angiosperm plants,seed is formed by a process known as "double fertilization":the embryo is formed after one sperm fertilizes the egg to form a zygote,and the other sperm fertilizes two central cell nuclei to form an endosperm cell.It contains 1 maternal and 1 paternal genome(1 maternal genome to 1 paternal genome,1m:1p)in the embryo;and 2 maternal and 1 paternal genome(2m:1p)in the endosperm.When the 2m:1p ratio is disrupted in endosperm,the seed development is impaired.The molecular mechanism that how parental genomic dosage imbalance effects the seed development is unknown.In this study,diploid(2n=2x=24)rice(Oryza sativa L.susp.japonica cv.02428)and its isogenic autotetraploid(2n=4x=48)were used as parents,to get the reciprocal interploidy crosses.Pollinating the diploid plant's pistil with tetraploid plant's pollen could get paternal genome excess cross(2X4);pollinating the tetraploid plant's pistil with diploid plant's pollen could get maternal genome excess cross(4X2).Genome-wide mRNA-Seq and sRNA-Seq data were used to analyze the expression level of genes and small RNAs in the endosperm of interploidy crosses,and discussed the molecular mechanism that the effect of parental genomic dosage imbalance on seed development in rice.In this study,the main results include the following points:1.Interploidy crosses disrupted the normal process of seed development in rice.Seeds from both interploidy crosses were smaller than those in the diploid or tetraploid parent at 6 day after pollination(DAP),seeds were significantly smaller in the maternal-excess cross than other crosses;seeds from both interploidy crosses seemed to stop develop at 9 and 13 DAP,and aborted finally.2.The most abundant small RNAs were 24-nt small interfering RNAs(siRNAs)(52.5-58.6%)in rice endosperm.In the paternal-excess cross,24-nt siRNAs derived from protein-coding genes have the highest expression level;in the maternal-excess cross,the expression level of 24-nt siRNAs inherited the level of its diploid and tetraploid parents.3.Parental genomic dosage imbalance induced a mass of nonadditively expressed genes in rice endosperm.Many downregulated genes were associated with starch and storage proteins accumulation in the interploidy crosses.Downregulated expression of these genes reduced the accumulation of storage matter in the endosperm,resulting in abnormal seed development.However,many stress-responsive genes were upregulated in the interploidy crosses,may response to parental genomic dosage imbalance.4.Imprinted genes play an important role in the endosperm development process,we analyzed the effect of parental genomic dosage imbalance on imprinted genes expression in this study.63-72%maternal expressed genes and 42-58%paternal expressed genes were nonadditively expressed in the paternal-excess and maternal-excess crosses,respectively.In addition,24-nt siRNAs level were negative correlation with imprinted genes expression level.24%nonadditively expressed imprinted genes were associated with nonadditive 24-nt siRNAs in the flanking sequence of genes.5.The timing of endosperm celluarization is essential for normal seeds development,Polycomb Repressive Complex2(PRC2)is required for this process.Interploidy crosses disrupted some PRC2 genes expression level.In addition,OsMET1b and OsCMT3a which are required for maintenance CG and CHG methylation(H=A,T or C)in rice,respectively,were upregulated in the paternal-excess cross,whereas downregulated in the maternal-excess cross.Differentially expressed PRC2 genes and DNA methylation genes may induce the precocious or delayed timing of endosperm celluarization,leading to the abnormal seed development in the interploidy crosses.6.OsWOX2L(WUSCHEL homeobox2-like)gene was downregulated expression in the interploidy crosses,and is an endosperm high-expressed gene.In this study,we used the CRISPR/Cas9 system to edit OsWOX2L gene,lost of function of OsWOX2L gene resulted in dramatically reduced seed set,a lot of seeds could not accumulate starch during filling stage.In conclusion,our research showed that the rice interploidy crosses disrupt sRNA and gene expression,leading to abnomal seeds development;maintaining the 2m:1p genome ratio in the endosperm is essential for normal grain development in rice and other cereal crops.