Degradome sequencing reveals an integrative miRNA-mediated gene interaction network regulating rice seed vigor

Abstract

Background: It is well known that seed vigor is essential for agricultural production and rice (Oryza sativa L.) is one of the most important crops in the world. Though we previously reported that miR164c regulates rice seed vigor, but whether and how other miRNAs cooperate with miR164c to regulate seed vigor is still unknown.

Results: Based on degradome data of six RNA samples isolated from seeds of the wild-type (WT) indica rice cultivar 'Kasalath' as well as two modified lines in 'Kasalath' background (miR164c-silenced line [MIM164c] and miR164c overexpression line [OE164c]), which were subjected to either no aging treatment or an 8-day artificial aging treatment, 1247 different target transcripts potentially cleaved by 421 miRNAs were identified. The miRNA target genes were functionally annotated via GO and KEGG enrichment analyses. By STRING database assay, a miRNA-mediated gene interaction network regulating seed vigor in rice was revealed, which comprised at least four interconnected pathways: the miR5075-mediated oxidoreductase related pathway, the plant hormone related pathway, the miR164e related pathway, and the previously reported RPS27AA related pathway. Knockout and overexpression of the target gene Os02g0817500 of miR5075 decreased and enhanced seed vigor, respectively. By Y2H assay, the proteins encoded by five seed vigor-related genes, Os08g0295100, Os07g0633100, REFA1, OsPER1 and OsGAPC3, were identified to interact with Os02g0817500.

Conclusions: miRNAs cooperate to regulate seed vigor in rice via an integrative gene interaction network comprising miRNA target genes and other functional genes. The result provided a basis for fully understanding the molecular mechanisms of seed vigor regulation.

Keywords: Degradome; Gene interaction network; Oryza sativa L.; Seed vigor; miRNAs.

Fig. 1

Characterization of unaged and artificially aged seeds of different genotypes of rice. A Seed germination rate; B RT-qPCR analysis of the expression level of miR164c. Data represent mean ± standard deviation (SD; n = 3). Significant differences among samples were determined using Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001). In (B), The expression level of miR164c in unaged WT seeds was set as 1. WT indicates the wild-type rice cultivar ‘Kasalath’; MIM164c and OE164c indicate two modified lines in ‘Kasalath’ background, miR164c-silenced line ‘L13–1–2-1’ and miR164c overexpression line ‘L4–1–3-1’, respectively

Fig. 2

Venn diagram showing the numbers of miRNAs and degradome transcripts in the six seed samples. (A, B) Numbers of miRNAs (A) and degradome transcripts (B) overlapping among the six samples. A1, A2, and A3 indicate unaged WT, MIM164c and OE164c seeds, respectively; B1, B2, and B3 represent artificially aged WT, MIM164c, and OE164c seeds, respectively

Fig. 3

Functional cluster analysis and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of target genes corresponding to the degradome transcripts. (A) Comparison of the abundance of degradome transcripts and TPB value based on the zero-mean normalization analysis of degradome transcripts in unaged and artificially aged WT, MIM164c and OE164c seeds. (B) KEGG enrichments of degradome transcripts unique to unaged and artificially aged WT, MIM164c, and OE164c seeds. (C) GO and (D) KEGG enrichments of target genes common to unaged and artificially aged WT and OE164c seeds but not to unaged and artificially aged MIM164c seeds. In (A) and (B), the greater the intensity of the red color, the higher the abundance of degradome transcripts; and the greater the intensity of the blue color, the lower the abundance of degradome transcripts. A1, A2, and A3 indicate unaged WT, MIM164c and OE164c seeds, respectively; B1, B2, and B3 represent artificially aged WT, MIM164c, and OE164c seeds, respectively

Fig. 4

The miRNA-mediated gene interaction network regulating rice seed vigor. Big and small nodes represent direct and indirect interactions between miR164’s target genes and other genes, respectively

Fig. 5

The simplified gene interaction network regulating rice seed vigor by directly interacting with miR164s’ target genes

Fig. 6

Phylogenetic analysis, subcellular localization and yeast two-hybrid (Y2H) assays of Os02g0817500. A Phylogenetic tree. XP_015626381.1, outlined with a red box represents Os02g0817500. B Subcellular localization of Os02g0817500, yellow-green indicated that Os02g0817500 expressed in the cytoplasm. C Nuclear system and (D) membrane system Y2H assays. The interaction partners of Os02g0817500 were identified by growing yeast on SD/−Leu/−Trp/−His/−Ade/X-α-gal and SD/−Leu/−Trp/−His/−Ade/X-gal plates. The pGADT7 and pPR3-N empty vectors were used as negative controls in the nuclear system and membrane system Y2H assays, respectively

Fig. 7

RT-qPCR analysis of the expression of miR164c and target genes, and miR5075-mediated oxidoreductase pathway related genes. (A, B) Expression levels of miR164c and its target genes OsPSK5, Os02g0436400 and OMTN1–6 (A) and miR5075-mediated oxidoreductase pathway related genes including miR1848 and target gene Os02g097300, miR5075 and target gene Os02g0871500, and the interacting genes OsPER1, OsGAPC3, Os08g0295100, REFA1 and Os07g0633100 (B). Data represent mean ± SD (n = 3). Significant differences among different samples were determined using Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001). The expression level of the gene of interest in unaged WT seeds was set as 1

Fig. 8

Artificial aging-induced changes in seed germination rate and RT-qPCR analysis of the gene expression levels in rice cultivars ‘Kasalath’ and ‘Nipponbare’. (A–D) Seed germination rate and seed-specific expression levels of miR164c, miR5075 and the respective target genes OMTN2 and Os02g0817500 in Kasalath (A, C) and Nipponbare (B, D). Data represent mean ± SD (n = 3). Significant differences between the unaged and seeds of each cultivar with different degrees of aging were determined using Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001). In (C, D), the expression level of the gene of interest in unaged WT seeds was set as 1

Fig. 9

Effect of Os02g0817500 knockout on the seed vigor of rice ‘Nipponbare’. A Target sites in the wild type (WT) are marked in red box. Inserted nucleotide is indicated with red uppercase letter and red dashes represent the deleted bases. The original data can be viewed from Additional file 4: Fig. S5. B Analysis of the Os02g0817500 protein sequence in the wild-type (WT) and Os02g0817500-knockout lines. C Photographs of germinated seeds before and after artificial aging of Os02g0817500-knockout lines and ‘Nipponbare’ (WT) after 5 days after germination. (D, E) Comparison of germination rate (D) and simple vigor index (E) between Os02g0817500-knockout lines and ‘Nipponbare’. Bar = 1 cm. Each column presents the means ± standard deviations of three biological replicates. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the WT by Student’s t-test

Fig. 10

Effect of aging on the germination performance of Arabidopsis (Col- WT) and Os02g0817500 ectopic expression lines ‘OE-1’ and ‘OE-2’. A Semi-quantitative RT-PCR analysis of the expression of Os02g0817500 in Arabidopsis leaves. Atactin was used as a positive control. The original data can be viewed from Additional file 5: Fig. S6. B Germination rates of seeds before and after artificial aging. Data represent mean ± SD (n = 3). Significant differences between WT and transgenic samples were determined using Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001). C Phenotypes of 5-day-old seedlings produced from unaged seeds (Left) and 10-day-old seedlings produced from artificially aged seeds (Right). Seedlings were grown on the half-strength Murashige and Skoog (MS) medium

Fig. 11

RT-qPCR analysis of the expression of genes involved in the miR164e-mediated oxidative phosphorylation related pathway. (A) miR164e and the target gene Os10g0571100; (B) miR1846 and the target gene Os10g0576000; (C) miR531b and the target gene Os01g0720300. Data represent mean ± SD (n = 3). Significant differences among different samples were determined using Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001). The expression level of the gene of interest in unaged WT seeds was set as 1