Multiple Regression Analysis Reveals MicroRNA Regulatory Networks in Oryza sativa under Drought Stress

Abstract

Drought is a major abiotic stress that reduces rice development and yield. miRNAs (microRNAs) are known to mediate posttranscriptional regulation under drought stress. Although the importance of individual miRNAs has been established, the crosstalks between miRNAs and mRNAs remain unearthed. Here we performed microarray analysis of miRNAs and matched mRNA expression profiles of drought-treated rice cultivar Nipponbare. Drought-responsive miRNA-mRNA regulations were identified by a combination of a partial least square (PLS) regression approach and sequence-based target prediction. A drought-induced network with 13 miRNAs and 58 target mRNAs was constructed, and four miRNA coregulatory modules were revealed. Functional analysis suggested that drought-response miRNA targets are enriched in hormone signaling, lipid and carbohydrate metabolism, and antioxidant defense. 13 candidate miRNAs and target genes were validated by RT-qPCR, hierarchical clustering, and ROC analysis. Two target genes (DWARF-3 and P0651G05.2) of miRNA coregulatory modules were further verified by RLM-5' RACE. Together, our integrative study of miRNA-mRNA interaction provided attractive candidates that will help elucidate the drought-response mechanisms in Oryza sativa.

Figure 1

Flow chart of the general procedure of this study.

Figure 2

The layout of the miRNA-mRNA network and its structural features. (a) miRNA-mRNA network constructed by PLS. (b) Out-degree distribution of the miRNA-mRNA network. (c) In-degree distribution of the miRNA-mRNA network.

Figure 3

Heat map of the 13 DE-miRNAs in drought-stressed and control rice leaves.

Figure 4

(a) Expression level of coregularory miR528-3p/miR812q and their target DWARF-3 by microarray and RT-qPCR. (b) Expression level of coregularory miR395a/miR396g and their target P0651G05.2 by microarray and RT-qPCR. The expression level is expressed as the mean fold changes of 3 replicates. Error bars depict the standard error (n = 3). (c) miR528-3p and miR812q cleavage sites on DWARF-3 identified by RLM-5′ RACE. (d) miR395a and miR396g cleavage sites on P0651G05.2 identified by RLM-5′ RACE. Arrows represent the 5′ termini of mRNA fragments, and the numbers beside them denote the cleavage frequency of cloned PCR products at the exact miRNA cleavage sites. The vertical dashes indicate matched RNA base pairs and circles represent GU mismatch whereas no dashes represent other types of mismatch.

Figure 5

Enriched GO terms of drought-responsive miRNA targets.

Figure 6

Schematic diagram of miRNA-regulated metabolic processes in rice under drought stress. Under drought stress, rice responds with the following physiological changes: (1) activating hormonal signaling pathways, (2) immobilizing fatty acid and downregulating starch and sucrose metabolism, (3) producing antioxidant compounds for detoxification, and (4) inhibiting miRNA biogenesis.