Inferring the regulatory network of the miRNA-mediated response to individual and combined heat and drought stress in tomato

Under natural environmental conditions, plants are prone to be challenged simultaneously by combination of stresses like heat and drought stress together, thus affecting their overall growth, development and reproduction. Moreover, future climatic conditions are predicted to be warmer and drier, thus, warranting deep understanding of the stress-responsive regulatory networks for developing stress-management strategies. The role of microRNAs (miRNAs) that are key regulators of different stress signalling cascades in such dual stress conditions using varieties growing in warmer climatic conditions is completely lacking. In this study, we have investigated the effect of drought, heat and the two stresses together (combined stress) on a heat-tolerant tomato (Solanum lycopersicum) variety by evaluating physiological parameters as well as, some stress-responsive miRNA-target modules. Taqman-based qRT-PCR miRNA expression analysis showed enhanced expression of sly-miR482d-3p, sly-miR172d-3p, sly-miR164b-3p, sly-miR398b in individual drought and heat stress with an additive upregulation effect under combined stresses. On the other hand, the expression of sly-miR397-5p and sly-miR396b-3p was less when these two stresses co-occurred than the individual stresses and an antagonistic response was observed for sly-miR166a expression in combined versus single stresses. Several high confidence miRNA targets (101) were identified in-silico using degradome data and were functionally annotated using Gene Ontology enrichment analysis into various stress regulatory networks. The comparative analysis confirmed the inverse expression regulation of the miRNA:target pairs for sly-miR398b:Solyc07g006180, sly-miR164b-3p:Solyc08g061500, sly-miR172d:Solyc04g049800, sly-miR396b-3p:Solyc01g102810 and sly-miR396b-3p:Solyc05g017930 under all the three stress conditions. Since miRNAs are highly conserved across diverse plant species, these miRNAs can be candidates for engineering climate resilient crop plants.