ath-miR164c influences plant responses to the combined stress of drought and bacterial infection by regulating proline metabolism

Plants under combined stresses exhibit a prominent shift in molecular responses compared with plants exposed to the same stresses independently. Profiling responses to individual and combined stressors at the gene expression level have identified several genes with intersecting responses to these stressors. However, the upstream regulators at the intersection of plant responses to individual and combined stresses are not known. Here, using the transcriptome of Arabidopsis thaliana under individual and combined drought and Pseudomonas syringae infection, we identified several genes whose expression overlaps between individual and combined stresses. To study the key regulator of such an overlapping gene, we predicted that the expression of 1-Pyrroline-5-carboxylate synthase 1 (AtP5CS1) is regulated by ath-miR164c at post-transcriptional level. Our results from the stem-loop RT-PCR based expression analysis revealed significant downregulation of ath-rniR164c in response to P. syringae infection under both well-irrigated (pathogen only) and drought stress (combined stress) conditions. Furthermore, an Arabidopsis loss-of-function mutant of the miRNA arh-miR164c exhibited resistance to pathogen infection under combined stress, unlike the wild-type plants, implicating the role of ath-miR164c in regulating plant immunity. AtP5CS1 gene expression and proline accumulation were enhanced in the ath-miR164c mutant plants relative to the wild-type plants, demonstrating that ath-rniR164c regulates AtPSCS1 of the proline biosynthesis pathway, which was also validated by 5'RLM-RACE results. This miRNA-mediated modulation of AtP5CS1 gene expression under combined stress fills crucial gaps in identifying the key convergent players in the current understanding of plant stress responses.