IC4R002-miRNA-2011-21679406
Contents
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
- 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).
- miRNAs are derived from hairpin-like precursors, originating from a single-stranded RNA transcript through sequential processing by Dicer or Dicer-like (DCL) proteins. miRNA precursors have a characteristic fold-back structure, which is the primary criterion to annotate nov-miRs. Therefore, we predicted nov-miRs as follows. First, by folding the flanking genome sequence of the above un-annotated small RNAs, followed by analysis of structural features, we excluded small RNAs that cannot form the characteristic fold-back structure. Second, recently evolved/evolving miRNAs have a single locus in the genome, so small RNAs with multiple loci in the rice genome were excluded. Third, to minimize noise, we also eliminated small RNAs of low abundance (with total number of reads fewer than five) and those originating from both strands, which would generate siRNA-like small RNAs. Fourth, all the remaining un-annotated small RNA sequences were subjected to ‘MIREAP’, which recovers most kn-miRs with only a few exceptions whose structures cannot satisfy the common features of an miRNA gene. Finally, to distinguish miRNAs from miniature inverted repeat transposable elements (MITEs), we blasted the precursor and mature sequences of the small RNAs with characteristic fold-back structure against the Oryza Repeat Database, and the homologs of repetitive sequences were discarded. From the above analyses, our predicated nov-miRs satisfied the following criteria: precursors had a characteristic fold-back structure, contained no repetitive sequences, and matched the genome only once, and most were located in the intergenic region; lengths of mature miRNA ranged from 20 to 24 nucleotides, and the number of reads was greater than five; the mature sequences could be sequenced in two or more libraries, or the miRNA* sequence could be identified in at least one library; and the targets of predicted miRNAs could be predicted using an upgraded version of miRU.
- To directly compare the expression patterns of these miRNAs in the developing pollen and in sporophytes, we normalized the counts to 1 million, and the abundance of each miRNA was expressed as transcripts per million(TPM). Using Z-score transformation, with ratio > 2.0 and Z-score > 2.0 cutoffs, we identified 103 kn-miRs expressed preferentially in developing pollen and 122 preferentially in sporophytic tissues (Additional file 1). Clustering analysis revealed a high proportion of kn-miRs expressed constitutively in all samples (clusters 4, 5 and 9 in Figure 2a) or preferentially in sporophytic tissues (clusters 1 and 10 in Figure 2a), and most of the conserved knmiRs were in these clusters (Additional file 1). However, this analysis also showed some kn-miRs accumulated to a large extent in developing rice pollen or at individual stages of pollen development. For example, the members of clusters 8, 3 and 13 (Figure 2a) displayed UNM-, BCP and TCP-enriched expression, respectively; and those in cluster 18 (Figure 2a) accumulated to a greater extent in pollen than in sporophytic tissues. Moreover, some conserved kn-miRs, such as osa-miR160e, osa-miR162b, osa-miR169e and n/o, osa-miR171a, osa-miR396a/b and osa-miR399h, were also present in pollen-enriched clusters.
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)
