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
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 miRNAregulated drought response pathways based on multi-omics approaches remain elusive. Results: We combined sRNA, transcriptome and
... riptome 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 degradomeseq. 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. Background Soybean (Glycine max L.) is the most important legume crop that provides essential vegetable proteins and oil sources for livestock and humans, while its production is severely constrained by global climatic changes, particularly drought stress  . However, the current lack of knowledge 4 regarding the regulatory network underlying soybean responses to drought conditions has restricted an effective management of soybeans productivity in the world. Therefore, characterizing the complex molecular mechanism of drought resistance is a prerequisite for improving soybean yield . In plants, small RNAs play a prominent regulatory role influencing the adaptability to drought stress, especially microRNAs (miRNAs) via degradation or translational inhibition of their target genes . In the past decade, some differentially expressed miRNAs (DEMs) under drought-simulated stress have been filtered by small RNA sequencing (sRNA-seq), mainly in soybean roots [4, 5]. However, only few miRNAs in soybean, e.g. gma-miR394 and gma-miR396, by overexpressing in model plants have been verified to perform an important function in response to drought stress [6, 7]. As known, miR398 participates in stress resistance by regulating the expression of superoxide dismutase (SOD)-related genes in many plants, such as Arabidopsis thaliana , Medicago sativa , potato , and Oryza sativa . Overexpressing miR398 negatively regulates plant stress tolerance in Arabidopsis thaliana and Nicotiana benthamiana [12, 13]. Recently, rice osa-miR398b overexpression plants and mutants in CSD1 and CSD2 were shown to display enhanced basal defenses by altering expression of multiple SODs, indicating that in different plant species miR398 might play different roles in stress resistance [14, 15]. Overall, our understanding of the roles played by miRNAs under drought stress remains limited, especially for seedlings of soybean. Usually, miRNA researchers combined differentially expressed genes (DEGs) to analyze the regulatory mechanisms in which miRNAs might be involved. In terms of genetic control of drought tolerance, generating hypotheses of quite complex data is a difficult mission, and the accurate identification of targets is necessary. Degradome sequencing (degradome-seq) has been important for identification of global targets of miRNAs, which has helped to clarify the miRNA regulatory network . Recently, a few studies on the integrated analysis of miRNA, degradome and mRNA sequencing have been published in plants [17, 18]. However, the miRNA-target pairs that displayed negative correlations during soybean mosaic virus infection were not obtained using the integrated analysis, probably because of the limitations in the number of miRNA targets and DEGs . Overall, gaining an in-depth understanding of the complex molecular mechanisms of soybean miRNAs in the response to drought 5.Zheng Y, Hivrale V, Zhang X, Valliyodan B, Lelandais-Briere C, Farmer AD, May GD, Crespi M, Nguyen HT, Sunkar R: Small RNA profiles in soybean primary root tips under water deficit. BMC systems biology 2016, 10(Suppl 5):126.