Integration of sRNA, degradome, transcriptome analysis and functional investigation reveals gma-miR398c negatively regulates drought tolerance via GmCSDs and GmCCS in transgenic Arabidopsis and soybean

Abstract

Background: Drought conditions adversely affect soybean growth, resulting in severe yield losses worldwide. Increasing experimental evidence indicates miRNAs are important post-transcriptional regulators of gene expression. However, the drought-responsive molecular mechanism underlying miRNA–mRNA interactions remains largely uncharacterized in soybean. Meanwhile, the miRNA-regulated drought response pathways based on multi-omics approaches remain elusive. Results: We combined sRNA, transcriptome and degradome sequencing to elucidate the complex regulatory mechanism mediating soybean drought resistance. One-thousand transcripts from 384 target genes of 365 miRNAs, which were enriched in the peroxisome, were validated by degradome-seq. An integrated analysis showed 42 miRNA–target pairs exhibited inversely related expression profiles. Among these pairs, a strong induction of gma-miR398c as a hub gene negatively regulates multiple peroxisome-related genes ( GmCSD 1a/b, GmCSD 2a/b/c and GmCCS ). Meanwhile, we detected that alternative splicing of GmCSD1a/b might affect soybean drought tolerance by bypassing gma-miR398c regulation. Overexpressing gma-miR398c in Arabidopsis thaliana L. resulted in decreased percentage germination, increased leaf water loss, and reduced survival under water deficiency, which displayed sensitivity to drought during seed germination and seedling growth. Furthermore, overexpressing gma-miR398c in soybean decreased GmCSD 1a/b, GmCSD 2a/b/c and GmCCS expression, which weakened the ability to scavenge O 2 .− , resulting in increased relative electrolyte leakage and stomatal opening compared with knockout miR398c and wild-type soybean under drought conditions. Conclusion: The study indicates that gma-miR398c negatively regulates soybean drought tolerance, and provides novel insights useful for breeding programs to improve drought resistance by CRISPR technology.