Abstract
BackgroundSmall RNA and degradome sequencing have identified a large number of miRNA-target pairs in plant seeds. However, detailed spatial and temporal studies of miRNA-mediated regulation, which can reflect links between seed development and germination are still lacking.ResultsIn this study, we extended our investigation on miRNAs-involved gene regulation by a combined analysis of seed maturation and germination in barley. Through bioinformatics analysis of small RNA sequencing data, a total of 1324 known miRNA families and 448 novel miRNA candidates were identified. Of those, 16 known miRNAs with 40 target genes, and three novel miRNAs with four target genes were confirmed based on degradome sequencing data. Conserved miRNA families such as miR156, miR168, miR166, miR167, and miR894 were highly expressed in embryos of developing and germinating seeds. A barley-specific miRNA, miR5071, which was predicted to target an OsMLA10-like gene, accumulated at a high level, suggesting its involvement in defence response during these two developmental stages. Based on target prediction and Kyoto Encyclopedia of Genes and Genomes analysis of putative targets, nine highly expressed miRNAs were found to be related to phytohormone signalling and hormone cross-talk. Northern blot and qRT-PCR analysis showed that these miRNAs displayed differential expression patterns during seed development and germination, indicating their different roles in hormone signalling pathways. In addition, we showed that miR393 affected seed development through targeting two genes encoding the auxin receptors TIR1/AFBs in barley, as over-expression of miR393 led to an increased length–width ratio of seeds, whereas target mimic (MIM393)-mediated inhibition of its activity decreased the 1000-grain weight of seeds. Furthermore, the expression of auxin-responsive genes, abscisic acid- and gibberellic acid-related genes was altered in miR393 misexpression lines during germination and early seedling growth.ConclusionsOur work indicates that miRNA-target pairs participate in gene expression regulation and hormone interaction in barley embryo and provides evidence that miR393-mediated auxin response regulation affects grain development and influences gibberellic acid and abscisic acid homeostasis during germination.
Highlights
Small RNA and degradome sequencing have identified a large number of miRNA-target pairs in plant seeds
Overview of small RNA (sRNA) expressed in barley embryos during seed maturation and germination To obtain an overview of the miRNA expression profile in embryo during seed maturation and germination, we constructed sRNA libraries using samples at three different developmental stages: one time point in the grain storage phase during seed development (10 DPA), two time points during seed germination including 1 DAG, and 5 DAG (Fig. 1a)
Conserved and barley-specific miRNAs highly expressed in embryo tissues According to bioinformatics analysis, we found that miR156 was the most highly expressed miRNA in embryos of developing and germinating seeds. miR156 is reported to be one of the most abundant miRNAs conserved among most land plants including mosses [36]
Summary
Small RNA and degradome sequencing have identified a large number of miRNA-target pairs in plant seeds. Abscisic acid (ABA) and gibberellic acid (GA), play important roles in determining the physiological state of the seed and regulating the development and germination process. The level of ABA peaks during seed maturation and dormancy, whereas GA level increases during imbibition and remains high during germination and postembryonic growth [1]. It seems to not be the absolute concentration but the balance of ABA and GA that determines the two events. In Arabidopsis, it is reported that auxin plays a role in seed dormancy and germination through its crosstalk with other hormones such as ABA, indicating a coordinating network of auxin and ABA signalling in this important process [9]
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