| Endosperm is one product of double fertilization in angiosperms,acting as a source of nourishment for embryo development and germination.Endosperm development follows an initial syncytial phase of free nuclear divisions without cytokinesis and then cellularization.Cellularization is one of the key developmental processes that determines the viability and final size of seeds.Previous research has shown that loss of function of FIS class genes causes over proliferation of uncellularized endosperm in the fertilized fis class mutant seeds and autonomous endosperm formation in absence of fertilization.FIS1 is a member of the Polycomb Repressive Complex 2(PRC2),which catalyzes the trimethylation of histone H3 at lysine 27(H3K27me3).In this study,fisl homozygous seeds were mutagenized with EMS,isolating four independent mutant lines showing increased viable seed formation.These suppressor mutations mapped to a CHD3 chromatin remodeling family member gene PICKLE RELATED 2(PKR2),a PEG specifically expressed in syncytial endosperm.We further showed that the suppressor mutation also suppressed seed defect in.a paternal excess interploidy cross,supporting the notion that imprinted genes are involved in establishing postzygotic hybridization barriers and adding new evidence of the antagonism between CHD3 and PRC2 in other tissues.1.The fisl homozygote was used in reciprocal crosses with a suppressor line,which each cross giving rise to approximately 10%plump seeds,suggesting that the suppressor mutations are recessive.Crosses between the four suppressor lines gave approximately 50%wild type-like seeds,suggesting that the mutations in each line are allelic to each other.Whole genome sequencing of one suppressor line revealed a mutation in the CHD3 chromatin remodeling family member gene PKR2.Sanger sequencing of the other three suppressor lines revealed that each line had a mutation in PKR2 but at different locations,confirming that mutations in PKR2 suppress seed abortion of fis1 in the suppressor lines.2.Seed development of Ler,pkr2-2,fis1 and fis1 pkr2-2,was determined by sectioning seeds harvested at 5,8 and 10 day after pollination,and found that the restoration of cellularization in fis1 pkr2-2 allows embryo development to maturation.Mutation in PKR2 was also shown to suppress the seed defects in fis2,suggesting that suppression is not specific to the fisl mutation.Comparing the frequencies of autonomous seed development between fis1 and fis1 pkr2-2,suggested that autonomous seed formation in fis1 is only slightly suppressed by the pkr2 mutation.3.To further understand PKR2 function in endosperm development,the suppressor line fis1 pkr2-2 was transformed with a complementary vector PKR2::GFP,generating 11 plants which showed reduced formation of viable seeds,indicating that the PKR2::GFP restored PKR2 function and complemented the phenotype of suppressor line.By crossing one representative GFP line to the pkr2-2 single mutant,we isolated homozygous GFP lines with or without fis1 for detailed GFP characterization.PKR2::GFP lines showed syncytial endosperm expression,overlapping with that shown in FIS1::GFP plants.GFP expression in the ovules only occurred when PKR2::GFPline was used as pollen donors to cross with Ler,suggesting that PKR2 is a paternally expressed imprinted gene(PEG).Conversely,GFP expression was shown in reciprocal crosses of fisl pkr2 PKR2::GFP and Ler,suggesting the PKR2 maternal copy is repressed by FIS1.4.Compared with the fisl in Ler,an allelic mutant mea was also found in Col.pkr2-1 mea double mutant produced increased plump seeds,and most of which could germinate and develop into viable seedlings,similar to that in the fisl pkr2-2 suppressor line in Ler.It has been shown that mutation in another PEG,ADM could also suppress seed abortion in mea.Mutations in ADM and PKR2 were found to additively suppress seed abortion.pkr2 mutation also slightly suppressed autonomous seed formation in mea.We also noticed that the adm single mutant has a slight reduction of seed weight but there was no further seed weight reduction in the adm pkr2-1 double mutant.5.The developmental defects observed in fis class seed are similar to those of aborted seeds in 2n X 4n paternal excess interploidy crosses.To determine whether PKR2 could repress aborted seeds in 2n X 4n crosses,we converted diploid(2n)pkr2-1,adm and adm pkr2-1 into tetraploid(4n).pkr2-1 2n pollinated by pkr2-1 4n resulted in 40%plump seeds,suggesting the pkr2 mutation promotes seed viability in paternal genome excess interploidy crosses.We also observed additive suppression of seed abortion when adm pkr2 2n was pollinated by the 4n double mutant.6.Transcriptome data were generated from RNA libraries of WT Ler,pkr2-2,fis1 and fis1 pkr2-2 seed at 3 and 6 DAP,with three biological repeats.A large number of genes deregulated in fis1 mutant are normalized in the fis1 pkr2-2 mutant(normalized genes).However normalized genes poorly overlapped between these two time points,suggesting that different genes are involved in different developmental stages.We analyzed the GO function of normalized genes and found that genes related to motor and microtubule motor activity were enriched among the down-normalized genes in the suppressor line.Genes related to hydrolase activity and specifically in glycosyl hydrolyzing activity,were enriched among the up-normalized genes in the suppressor line.Microtubule motor activity is essential for the construction of a new cell wall in the phragmoplast,whilst glycosyl hydrolyzing enzymes can degrade pectin,which is assumed to be a key step in the deconstruction of plant cell walls.Increased expression of genes that are potentially required to build the phragmoplast together with reduced expression of genes that degrade cell walls correlates with the observed cellularization in fis1 pkr2-2 seeds,indicating PKR2 promotes fisl mutant endosperm cellularization to allow viable seed formation.Further support that PRC2 and CHD3 antagonistically regulate endosperm cellularization. |
PDF Full Text Request