Water-deficit stress-responsive microRNAs and their targets in four durum wheat genotypes

MicroRNAs (miRNAs) guide regulation at the post-transcriptional level by inducing messenger RNA (mRNA) degradation or translational inhibition of their target protein-coding genes. Durum wheat miRNAs may contribute to the genotypic water-deficit stress response in different durum varieties. Further investigation of the interactive miRNA-target regulatory modules and experimental validation of their response to water stress will contribute to our understanding of the small RNA-mediated molecular networks underlying stress adaptation in durum wheat. In this study, a comprehensive genome-wide in silico analysis using the updated Triticum transcriptome assembly identified 2055 putative targets for 113 conserved durum miRNAs and 131 targets for four novel durum miRNAs that putatively contribute to genotypic stress tolerance. Predicted mRNA targets encode various transcription factors, binding proteins and functional enzymes, which play vital roles in multiple biological pathways such as hormone signalling and metabolic processes. Quantitative PCR profiling further characterised 43 targets and 5 miRNAs with stress-responsive and/or genotype-dependent differential expression in two stress-tolerant and two stress-sensitive durum genotypes subjected to pre-anthesis water-deficit stress. Furthermore, a 5′ RLM-RACE approach validated nine mRNA targets cleaved by water-deficit stress-responsive miRNAs, which, to our knowledge, has not been previously reported in durum wheat. The present study provided experimental evidence of durum miRNAs and target genes in response to water-deficit stress in contrasting durum varieties, providing new insights into the regulatory roles of the miRNA-guided RNAi mechanism underlying stress adaptation in durum wheat.