Deep sequencing on genome-wide scale reveals the unique composition and expression patterns of microRNAs in developing pollen of Oryza sativa.

BACKGROUND: Pollen development in flowering plants requires strict control of the gene expression program and genetic information stability by mechanisms possibly including the miRNA pathway. However, our understanding of the miRNA pathway in pollen development remains limited, and the dynamic profile of miRNAs in developing pollen is unknown. RESULTS: Using next-generation sequencing technology, we pyrosequenced small RNA populations from rice uninucleate microspores to tricellular pollen and control sporophytic tissues at the genome-wide level. We identified 292 known miRNAs, including members of all 20 families conserved in plants, and 75 novel miRNAs. Of the 292 known miRNAs, 202 were expressed, with 103 enriched, in developing pollen. More than half of these novel miRNAs displayed pollen-or stage-specific expression. Furthermore, analyzing the 367 miRNAs and their predicted targets indicated that correlation in expression profiles of pollen-enriched known miRNAs and their targets significantly differs from that of sporophyte-enriched known miRNAs and their targets in some functional terms, while novel miRNAs appeared to negatively regulate their targets. Importantly, gene ontology abundance analysis demonstrated chromatin assembly and disassembly was important in the targets of bicellular pollen-expressed novel miRNAs. Principal component analysis revealed pollen of all three stages was discriminated from sporophytes, largely because of the novel and non-conserved known miRNAs. CONCLUSIONS: Our study, for the first time, revealed the differences in composition and expression profiles of miRNAs between developing pollen and sporophytes, with novel and non-conserved known miRNAs the main contributors. Our results suggest the important roles of the miRNA pathway in pollen development.

Integrated proteomic and cytological study of rice endosperms at the storage phase.

The endosperm at the storage phase undergoes a series of coordinated cellular and metabolic events, including starchy endosperm cell death, starch synthesis, and starch granule packaging, which leads to efficient accumulation of starch. However, the mechanism underlying the interconnections remains unknown. We used integrated proteomic and cytological approaches to probe the interconnections in rice (Oryza sativa) endosperm at the storage phase from 12 to 18 days after flowering (DAF). Starch granule packaging was completed first in the inner part of endosperm at 15 DAF and spread to almost the entire endosperm at 18 DAF. Programmed starchy endosperm cell death occurred after the starch granule packaging. Endogenous H(2)O(2) was detectable in the inner part of endosperm at 12 DAF and the region beyond the inner part at 15 DAF, with an H(2)O(2) burst at 15 DAF. Proteomics analysis with 2-D fluorescent difference gel electrophoresis and matrix-assisted laser-desorption ionization time-of-flight/time-of-flight mass spectrometry revealed 317 proteins, including almost all known antioxidants, differentially expressed throughout the 3 stages of the developmental phase. More than two-thirds of the 317 proteins were potential thioredoxin targets, with a preferential skew toward central carbon metabolism, alcoholic fermentation, starch metabolism, amino acid metabolism, and protein synthesis or folding. These proteins implicated in starch synthesis and gluconeogenesis were upregulated, whereas those involved in anabolism of biomacromolecules such as proteins, lipids, and cell wall components were downregulated, with upregulated expression of proteins involved in catabolism of these biomacromolecules, which suggests remobilization of nutrients for starch synthesis. These data suggested important roles of the H(2)O(2)-antioxidant interface in coordinating starch accumulation, programmed cell death of starchy endosperm, and remobilization of nutrients during the cell death.