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Genome-wide analysis reveals rapid and dynamic changes in miRNA and siRNA sequence and expression during ovule and fiber development in allotetraploid cotton (Gossypium hirsutum L.).

Pang M, Woodward AW, Agarwal V, Guan X, Ha M, Ramachandran V, Chen X, Triplett BA, Stelly DM, Chen ZJ - Genome Biol. (2009)

Bottom Line: Among 32 miRNA precursors representing 19 unique miRNA families identified, 7 were previously reported, and 25 new miRNA precursors were found in this study.Enrichment of siRNAs in ovules and fibers suggests active small RNA metabolism and chromatin modifications during fiber development, whereas general repression of miRNAs in fibers correlates with upregulation of a dozen validated miRNA targets encoding transcription and phytohormone response factors, including the genes found to be highly expressed in cotton fibers.Rapid and dynamic changes in siRNAs and miRNAs may contribute to ovule and fiber development in allotetraploid cotton.

View Article: PubMed Central - HTML - PubMed

Affiliation: Section of Molecular Cell and Developmental Biology, The University of Texas at Austin, One University Station, A-4800, Austin, TX 78712, USA. davidpang@mail.utexas.edu

ABSTRACT

Background: Cotton fiber development undergoes rapid and dynamic changes in a single cell type, from fiber initiation, elongation, primary and secondary wall biosynthesis, to fiber maturation. Previous studies showed that cotton genes encoding putative MYB transcription factors and phytohormone responsive factors were induced during early stages of ovule and fiber development. Many of these factors are targets of microRNAs (miRNAs) that mediate target gene regulation by mRNA degradation or translational repression.

Results: Here we sequenced and analyzed over 4 million small RNAs derived from fiber and non-fiber tissues in cotton. The 24-nucleotide small interfering RNAs (siRNAs) were more abundant and highly enriched in ovules and fiber-bearing ovules relative to leaves. A total of 31 miRNA families, including 27 conserved, 4 novel miRNA families and a candidate-novel miRNA, were identified in at least one of the cotton tissues examined. Among 32 miRNA precursors representing 19 unique miRNA families identified, 7 were previously reported, and 25 new miRNA precursors were found in this study. Sequencing, miRNA microarray, and small RNA blot analyses showed a trend of repression of miRNAs, including novel miRNAs, during ovule and fiber development, which correlated with upregulation of several target genes tested. Moreover, 223 targets of cotton miRNAs were predicted from the expressed sequence tags derived from cotton tissues, including ovules and fibers. The cotton miRNAs examined triggered cleavage in the predicted sites of the putative cotton targets in ovules and fibers.

Conclusions: Enrichment of siRNAs in ovules and fibers suggests active small RNA metabolism and chromatin modifications during fiber development, whereas general repression of miRNAs in fibers correlates with upregulation of a dozen validated miRNA targets encoding transcription and phytohormone response factors, including the genes found to be highly expressed in cotton fibers. Rapid and dynamic changes in siRNAs and miRNAs may contribute to ovule and fiber development in allotetraploid cotton.

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Quantitative RT-PCR analysis of predicted targets of miRNAs, including three novel miRNAs, in cotton. O (-3), O (0), O (+3), F (+7), and Leaf indicate RNA samples from the ovules at -3 DPA, 0 DPA, and +3 DPA, fiber at +7 DPA, and leaves, respectively. The label above each plot indicates the EST accession number followed by the predicted gene function and the corresponding miRNA. Relative expression levels (REL) were calculated using HISTONE H3 as a control.
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Figure 6: Quantitative RT-PCR analysis of predicted targets of miRNAs, including three novel miRNAs, in cotton. O (-3), O (0), O (+3), F (+7), and Leaf indicate RNA samples from the ovules at -3 DPA, 0 DPA, and +3 DPA, fiber at +7 DPA, and leaves, respectively. The label above each plot indicates the EST accession number followed by the predicted gene function and the corresponding miRNA. Relative expression levels (REL) were calculated using HISTONE H3 as a control.

Mentions: If miRNAs degrade target mRNA transcripts, their expression levels should be negatively correlated. To test this, we compared the expression patterns of predicted miRNA targets in quantitative RT-PCR (qRT-PCR) assays with miRNA accumulation levels in small RNA blot analysis. The expression levels of some miRNA targets are inversely correlated with the accumulation levels of corresponding miRNAs. For example, TC107071 encoding a homolog of Arabidopsis HD-ZIP III protein was cleaved by Gh-miR165/166 (Figure 5) and expressed at relatively high levels in fibers (Figure 6), during which the Gh-miR165/166 levels were relatively low (Figure 4). The expression levels of miR390 and TAS3-like (DW502659) were also negatively correlated. TAS3-like was highly expressed in fibers and leaves (Figure 6), in which miR390 accumulated at low levels (Figure 4). Negative correlation was also observed between Gh-miR164 and its target (TC116985) encoding a NAM-like protein, between Gh-miR2947 and its target (CO105636) encoding a predicted serine/threonine kinase, and between Gh-miR2949 and its target (TC101917) encoding a putative endosomal protein. Interestingly, Gh-miR2949 accumulated at higher levels in ovules (0 DPA) than in fibers (Figure 4), and its target was expressed at lower levels in the ovules than in the fibers (Figure 6). Gh-miR2947 accumulated at lower levels in fibers than in ovules, and its target was expressed at higher levels in fibers than in ovules.


Genome-wide analysis reveals rapid and dynamic changes in miRNA and siRNA sequence and expression during ovule and fiber development in allotetraploid cotton (Gossypium hirsutum L.).

Pang M, Woodward AW, Agarwal V, Guan X, Ha M, Ramachandran V, Chen X, Triplett BA, Stelly DM, Chen ZJ - Genome Biol. (2009)

Quantitative RT-PCR analysis of predicted targets of miRNAs, including three novel miRNAs, in cotton. O (-3), O (0), O (+3), F (+7), and Leaf indicate RNA samples from the ovules at -3 DPA, 0 DPA, and +3 DPA, fiber at +7 DPA, and leaves, respectively. The label above each plot indicates the EST accession number followed by the predicted gene function and the corresponding miRNA. Relative expression levels (REL) were calculated using HISTONE H3 as a control.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3091316&req=5

Figure 6: Quantitative RT-PCR analysis of predicted targets of miRNAs, including three novel miRNAs, in cotton. O (-3), O (0), O (+3), F (+7), and Leaf indicate RNA samples from the ovules at -3 DPA, 0 DPA, and +3 DPA, fiber at +7 DPA, and leaves, respectively. The label above each plot indicates the EST accession number followed by the predicted gene function and the corresponding miRNA. Relative expression levels (REL) were calculated using HISTONE H3 as a control.
Mentions: If miRNAs degrade target mRNA transcripts, their expression levels should be negatively correlated. To test this, we compared the expression patterns of predicted miRNA targets in quantitative RT-PCR (qRT-PCR) assays with miRNA accumulation levels in small RNA blot analysis. The expression levels of some miRNA targets are inversely correlated with the accumulation levels of corresponding miRNAs. For example, TC107071 encoding a homolog of Arabidopsis HD-ZIP III protein was cleaved by Gh-miR165/166 (Figure 5) and expressed at relatively high levels in fibers (Figure 6), during which the Gh-miR165/166 levels were relatively low (Figure 4). The expression levels of miR390 and TAS3-like (DW502659) were also negatively correlated. TAS3-like was highly expressed in fibers and leaves (Figure 6), in which miR390 accumulated at low levels (Figure 4). Negative correlation was also observed between Gh-miR164 and its target (TC116985) encoding a NAM-like protein, between Gh-miR2947 and its target (CO105636) encoding a predicted serine/threonine kinase, and between Gh-miR2949 and its target (TC101917) encoding a putative endosomal protein. Interestingly, Gh-miR2949 accumulated at higher levels in ovules (0 DPA) than in fibers (Figure 4), and its target was expressed at lower levels in the ovules than in the fibers (Figure 6). Gh-miR2947 accumulated at lower levels in fibers than in ovules, and its target was expressed at higher levels in fibers than in ovules.

Bottom Line: Among 32 miRNA precursors representing 19 unique miRNA families identified, 7 were previously reported, and 25 new miRNA precursors were found in this study.Enrichment of siRNAs in ovules and fibers suggests active small RNA metabolism and chromatin modifications during fiber development, whereas general repression of miRNAs in fibers correlates with upregulation of a dozen validated miRNA targets encoding transcription and phytohormone response factors, including the genes found to be highly expressed in cotton fibers.Rapid and dynamic changes in siRNAs and miRNAs may contribute to ovule and fiber development in allotetraploid cotton.

View Article: PubMed Central - HTML - PubMed

Affiliation: Section of Molecular Cell and Developmental Biology, The University of Texas at Austin, One University Station, A-4800, Austin, TX 78712, USA. davidpang@mail.utexas.edu

ABSTRACT

Background: Cotton fiber development undergoes rapid and dynamic changes in a single cell type, from fiber initiation, elongation, primary and secondary wall biosynthesis, to fiber maturation. Previous studies showed that cotton genes encoding putative MYB transcription factors and phytohormone responsive factors were induced during early stages of ovule and fiber development. Many of these factors are targets of microRNAs (miRNAs) that mediate target gene regulation by mRNA degradation or translational repression.

Results: Here we sequenced and analyzed over 4 million small RNAs derived from fiber and non-fiber tissues in cotton. The 24-nucleotide small interfering RNAs (siRNAs) were more abundant and highly enriched in ovules and fiber-bearing ovules relative to leaves. A total of 31 miRNA families, including 27 conserved, 4 novel miRNA families and a candidate-novel miRNA, were identified in at least one of the cotton tissues examined. Among 32 miRNA precursors representing 19 unique miRNA families identified, 7 were previously reported, and 25 new miRNA precursors were found in this study. Sequencing, miRNA microarray, and small RNA blot analyses showed a trend of repression of miRNAs, including novel miRNAs, during ovule and fiber development, which correlated with upregulation of several target genes tested. Moreover, 223 targets of cotton miRNAs were predicted from the expressed sequence tags derived from cotton tissues, including ovules and fibers. The cotton miRNAs examined triggered cleavage in the predicted sites of the putative cotton targets in ovules and fibers.

Conclusions: Enrichment of siRNAs in ovules and fibers suggests active small RNA metabolism and chromatin modifications during fiber development, whereas general repression of miRNAs in fibers correlates with upregulation of a dozen validated miRNA targets encoding transcription and phytohormone response factors, including the genes found to be highly expressed in cotton fibers. Rapid and dynamic changes in siRNAs and miRNAs may contribute to ovule and fiber development in allotetraploid cotton.

Show MeSH
Related in: MedlinePlus