Project description:Angiosperms possess a characteristic life cycle with an alternation of sporophyte and gametophyte generations, which happens in plant organs like pistils. Rice pistils contain ovules and receive pollen for successful fertilization to produce grains. The cellular expression profile in rice pistils is largely unknown. Here we show a cell atlas of rice pistils before fertilization by droplet-based single-nucleus RNA sequencing. The Ab initio marker identification validated by in situ hybridization assists the cell-type annotation, revealing cell heterogeneity between ovule and carpel lineages.
Project description:Angiosperms possess a characteristic life cycle with an alternation of sporophyte and gametophyte generations, which happens in plant organs like pistils. Rice pistils contain ovules and receive pollen for successful fertilization to produce grains. The cellular expression profile in rice pistils is largely unknown. Here we show a cell atlas of rice pistils before fertilization by droplet-based single-nucleus RNA sequencing. The Ab initio marker identification validated by in situ hybridization assists the cell-type annotation, revealing cell heterogeneity between ovule and carpel lineages.
Project description:Angiosperms possess a characteristic life cycle with an alternation of sporophyte and gametophyte generations, which happens in plant organs like pistils. Rice pistils contain ovules and receive pollen for successful fertilization to produce grains. The cellular expression profile in rice pistils is largely unknown. Here we show a cell atlas of rice pistils before fertilization by droplet-based single-nucleus RNA sequencing. The Ab initio marker identification validated by in situ hybridization assists the cell-type annotation, revealing cell heterogeneity between ovule and carpel lineages. This SuperSeries is composed of the SubSeries listed below.
Project description:Gene expression throughout the reproductive process in rice (Oryza sativa) beginning with primordia development through pollination/fertilization to zygote formation was analyzed. We analyzed 25 stages/organs of rice reproductive development including early microsporogenesis stages with 57,381 probe sets, and identified around 26,000 expressed probe sets in each stage. Fine dissection of 25 reproductive stages/organs combined with detailed microarray profiling revealed dramatic, coordinated and finely tuned changes in gene expression. Decrease of expressed genes in the pollen maturation process was observed in a similar way with Arabidopsis and maize. An almost equal number of ab initio predicted genes and cloned genes appeared or disappeared coordinated with developmental stage progression. A large number of organ-/stage-specific genes were identified; notably 2,593 probe sets for developing anther, including 932 probe sets corresponding to ab initio predicted genes. Analysis of cell cycle-related genes revealed that several CDKs, cyclins and components of SCF E3 ubiquitin ligase complexes were expressed specifically in reproductive organs. Cell wall biosynthesis or degradation protein genes and transcription factor genes expressed specifically in reproductive stages were also newly identified. Rice genes homologous to reproduction-related genes in other plants showed expression profiles both consistent and inconsistent with their predicted functions. The rice reproductive expression atlas is likely to be the deepest and most comprehensive dataset available, indispensable for unraveling functions of many specific genes in plant reproductive processes that have not yet been thoroughly analyzed. Keywords: developmental stage comparison, tissue comparison, time course Pollinating pistils were collected according to time after pollination. Unpollinatted pistils and pistils 5 min after pollination were separated into stigmas and ovaries. Two or three biological replicates at each stage were analyzed with Affymetrix Rice Genome Array, and total number of samples in this series is 20. Unpollinated samples were references in this series.
Project description:The coordination of pollen tube (PT) growth, guidance and timely growth arrest and rupture mediated by PT-pistil interaction is crucial for the PT to transport sperm cells into ovules for double fertilization. The plasma membrane (PM) represents an important interface for cell–cell interaction, and PM proteins of PTs are pioneers for mediating PT integrity and interaction with pistils. Thus, understanding the mechanisms underlying these events is important for proteomics. Using the efficient aqueous polymer two-phase system and alkali buffer treatment, we prepared high-purity PM from mature and germinated pollen of rice. We used iTRAQ quantitative proteomic methods and identified 1,121 PM-related proteins (PMrPs) (matched to 899 loci); 192 showed differential expression in the two pollen cell types, 119 up- and 73 down-regulated during germination. The PMrP and differentially expressed PMrP sets all showed a functional skew toward signal transduction, transporters, wall remodeling/metabolism and membrane trafficking. Their genomic loci had strong chromosome bias. We found 37 receptor-like kinases (RLKs) from 8 kinase subfamilies and 209 transporters involved in flux of diversified ions and metabolites. In combination with the rice pollen transcriptome data, we revealed that in general, the protein expression of these PMrPs disagreed with their mRNA expression, with inconsistent mRNA expression for 74% of differentially expressed PMrPs. This study, for the first time, identified genome-wide pollen PMrPs, and provided insights into the membrane profile of receptor-like kinases and transporters important for pollen tube growth and interaction with pistils. These pollen PMrPs and their mRNAs showed discordant expression. This work provides novel resource and knowledge to further dissect mechanisms by which pollen or the PT controls PMrP abundance and monitors interactions and ion and metabolite exchanges with female cells in rice.
Project description:DNA methylation is an important epigenetic mark that regulates the expression of genes and transposons. RNA-directed DNA methylation (RdDM) is the main molecular pathway responsible for de novo DNA methylation in plants. Although the mechanism of RdDM has been well understood in Arabidopsis, mutations in RdDM genes cause no visible developmental defects in Arabidopsis. Here, we isolated and cloned Five Elements Mountain 1 (FEM1), which encodes RNA-dependent RNA polymerase 2 in rice. Mutation in OsRDR2 abolished the accumulation of 24-nt small interfering RNAs, and consequently substantially decreased genome-wide CHH methylation levels. Moreover, male and female reproductive development was disturbed, which led to the sterility of osrdr2 mutants. We discovered that OsRDR2-dependent DNA methylation likely regulates the expression of multiple key genes involved in stamen development and meiosis. In wild-type (WT) plants but not in osrdr2 mutants, genome-wide CHH DNA methylation levels were greater in panicles, stamens, and pistils than in seedlings. The global increase of methylation in reproductive organs of the WT was mainly explained by the enhancement of RdDM activity including OsRDR2 activity. Our results, which revealed a global increase in DNA methylation through enhancement of RdDM activity in reproductive organs, demonstrate the crucial role of OsRDR2 in the sexual reproduction of rice.