Project description:DNA methylation is an important epigenetic modification that regulates various plant developmental processes. Rice seed integument determines the seed size. However, the role of DNA methylation in its development remains largely unknown. Here, we report the first dynamic DNA methylomic profiling of rice maternal integument before and after pollination by using a whole-genome bisulfite deep sequencing approach. Analysis of DNA methylation patterns identified 4238 differentially methylated regions underpin 4112 differentially methylated genes, including GW2, DEP1, RGB1 and numerous other regulators participated in maternal integument development. Bisulfite sanger sequencing and qRT-PCR of six differentially methylated genes revealed extensive occurrence of DNA hypomethylation triggered by double fertilization at IAP compared with IBP, suggesting that DNA demethylation might be a key mechanism to activate numerous maternal controlling genes. These results presented here not only greatly expanded the rice methylome dataset, but also shed novel insight into the regulatory roles of DNA methylation in rice seed maternal integument development.
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. This SuperSeries is composed of the following subset Series: GSE13988: Rice expression atlas (1): Anther development GSE14298: Rice expression atlas (2): Pollination - Fertilization GSE14299: Rice expression atlas (3): Early embryogenesis GSE14300: Rice expression atlas (4): Vegetative tissues GSE14301: Rice expression atlas (5): Anther development (Agilent data) Refer to individual Series
Project description:- Pollen tube growth is important process for successful double fertilization, which is critical for grain yield in crop plants. Despite much progress in identification of rapid alkalization factors (RALFs) which serve as ligand for signaling transduction during fertilization in Arabidopsis, there is no functional study of RALF in mono-cotyledon plant. - We functionally characterized two pollen specific RALF in rice (Oryza sativa) using multiple CRISPR/Cas9 induced loss-of-function mutants, peptide treatment, expression analyses, tag reporter lines. - OsRALF17 is specifically expressed in pollen and pollen tube as the strongest level among 41 RALF members in rice. Exogenously applied OsRALF17 inhibits pollen tube germination and elongation at high concentration, but enhances tube elongation at low concentration, indicating the regulation of growth balance. Double mutant of OsRALF17 with OsRALF19 exhibit almost male sterile, with defect on pollen germination and tube elongation. - Our study revealed that functionally-redundant OsRALF17 and 19 peptides binds to the OsMTD2, CrRLK1L family member, and transmits ROS signal for pollen tube germination and integrity maintenance in rice. We provide new insights into the role of RALF and expanding our understanding of the biological role of RALF in regulating rice fertilization.
Project description:The R-loop is a common chromatin feature presented from prokaryotic to eukaryotic genomes and has been revealed to be involved in multiple cellular processes and associated with many human diseases. Here, we take the advantage of our recently developed ssDRIP-seq method to profile genome-wide R-loop levels and provided a first-hand R-loop atlas of Rice (Oryza sativa) at different developmental stages.
Project description:MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) regulate gene expression in eukaryotes. Plant miRNAs modulate their targets mainly via messenger RNA (mRNA) cleavage. Small RNA targets have been extensively investigated in Arabidopsis using computational prediction, experimental validation, and degradome sequencing. However, small RNA targets are largely unknown in rice (Oryza sativa). Here, we report global identification of small RNA targets using high throughput degradome sequencing in the rice indica cultivar 93-11 (Oryza sativa L. ssp. indica). 177 transcripts targeted by total of 87 unique miRNAs were identified. Of targets for the conserved miRNAs between Arabidopsis and rice, transcription factors comprise around 70% (58 in 82), indicating that these miRNAs act as masters of gene regulatory nodes in rice. In contrast, non-conserved miRNAs targeted diverse genes which provide more complex regulatory networks. In addition, 5 AUXIN RESPONSE FACTORS (ARF) cleaved by the TAS3 derived ta-siRNAs were also detected. A total of 40 sRNA targets were further validated via RNA ligase-mediated 5’ rapid amplification of cDNA ends (RLM 5’-RACE). Our degradome results present a detailed sRNA-target interaction atlas, which provides a guide for the study of the roles of sRNAs and their targets in rice.
Project description:Cellularization is a key event during the development of the endosperm. Our understanding of the developmental regulation of cellularization has been limited for plants other than Arabidopsis. We found that the activation of OsbZIP76 coincided with the initiation of cellularization of rice. Either knockdown or knockout of OsbZIP76 led to precocious cellularization. Many genes involved in endosperm development or starch biosynthesis were prematurely activated in the caryopsis at two days after fertilization. The results implied that OsbZIP76 is involved in the regulation of cellularization in rice. As a putative transcription factor, OsbZIP76 alone lacked transcriptional activation activity. However, it was able to interact with OsNF-YB9 and OsNF-YB1, two nuclear factor Y (NF-Y) family transcription factors, both in yeast and in planta. OsbZIP76 and OsNF-YB9 showed similar endosperm-preferential expression patterns and the transiently expressed proteins were colocalized in the epidermal cells of tobacco. As with osnf-yb1 mutants, the osbzip76 mutants showed reduced seed size and reduced apparent amylose content of the seeds. We also confirmed that OsbZIP76 is an imprinted gene in rice, the expression of which depended on the genetic background. Our results suggested that OsbZIP76 is an endosperm-expressed imprinted gene to regulate development of the endosperm in rice.
Project description:We aimed to identify differential expression of microRNAs between superior and inferior spikelets by using a deep sequencing approach developed by Solexa (Illumina). Two small RNA libraries were constructed from superior and inferior spikelets at 18 days after fertilization, and more than nine million small RNA sequence reads were generated for each library. Totals of 351 and 312 known miRNAs were obtained from the superior and inferior spikelets, respectively. Analysis of the expression profiles of these miRNAs showed that 189 miRNAs were differentially expressed between superior spikelets and inferior spikelets. In addition, 43 novel miRNAs were identified mostly by the accumulations of miRNA*s were also expressed differentially. Further analysis shows that these miRNAs may individually participate in regulating hormone metabolism, carbohydrate metabolic pathways, and cell division during rice grain development. These results indicate that slow grain filling and low grain weight of rice inferior spikelets probably relation to the expression and function differences between superior and inferior spikelet miRNAs.