IC4R002-miRNA-2011-21679406

Project Title

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

The Background of This Project

[[File:|700px|thumb|right|Figure 1. GWA Analysis of Al Tolerance within and across Rice Subpopulations.]]

• 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.
• MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are two types of small non-coding RNAs (20 to 24 nucleotides in length) identified in nearly all eukaryotes. The pool of small RNAs in plants is highly complex, consisting primarily of many low-abundant siRNAs and a small number of highly expressed 21-nucleotide sequences; most of the latter are miRNAs. Most miRNA loci are encoded by independent transcriptional units in intergenic regions that are transcribed by RNA polymerase II. In plants, miRNAs are processed from stem-loop regions of long primary transcripts by a Dicer-like enzyme and are loaded into silencing complexes, where they generally direct cleavage of complementary mRNAs. Although miRNAs were identified in plants just recently, studies have revealed that miRNAs play crucial roles in each major stage of plant development, often targeting the transcription factors that mediate transition from one developmen-tal stage to the next.
• Haploid pollen (also called the gametophyte) is a key regulator of sexual reproduction in flowering plants and is produced from diploid pollen mother cells via meiosis. In contrast to animals, in which products of meiosis directly develop into sperm cells, in plants, the product of meiosis undergoes a unique postmeiotic pollen development process, finally giving rise to sperm cells. During this process, haploid uninucleate microspores (UNMs) generated from meiosis first undergo asymmetric mitosis to generate bicellular pollen (BCP) consisting of a large vegetative cell and a small generative cell enclosed in the vegetative cell. The two types of cells have different fates: the vegetative cell exits the cell cycle and can develop into a polarly growing pollen tube, whereas the generative cell under goes further mitosis to produce two sperm cells. This postmeiotic pollen development is orderly and precisely regulated; however, the mechanism underlying the main developmental events remains largely unknown.

Plant Culture & Treatment

• Rice cultivar Zhonghua 10 (O. sativa L. ssp. japonica)was planted in a climate chamber under a 12-hour light/12-hour dark cycle at 28°C for 2 weeks, then the roots and leaves were collected. One-month-old callus cells were generated from rice embryos on N6 solid medium containing 2, 4-Dichlorophenoxyacetic acid (2mg/l) in the dark at 25°C. Pollen at UNM, BCP and TCP stages was obtained as described previously.

Research Findings

• Of the millions of high-quality small RNAs from the individual libraries, 72.4% were 20 to 24 nucleotides in length, which is the typical size range for Dicer-derived products. The major component of small RNAs in UNMs was 24 nucleotides long. Throughout pollen development, the proportion of 24-nucleotide small RNAs decreased and the 21-nucleotide population increased in BCP, while TCP contained mostly 21-nucleotide small RNAs (Figure 1). In the sporophytic samples, although two peaks occurred in all three libraries, 24-nucleotide small RNAs were the most abundant in callus cells and roots, and 21-nucleotide small RNAs were the most abundant in leaves (Figure 1).

[[File:|700px|thumb|right|Figure 2. Haplotype analysis of the Nrat1 gene region.]]

Labs working on this Project

• Research Center for Molecular and Developmental Biology, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, 20 Nanxincun, Xiangshan, Beijing 100093, China
• National Center for Plant Gene Research, 20 Nanxincun Xiangshan Haidianqu, Beijing 100093, China. 3 Graduate School of Chinese Academy of Sciences, 19A Yuquanlu Shijingshanqu, Beijing 100049, China

Corresponding Author

• Tai Wang(twang@ibcas.ac.cn)