Deciphering the role of MIR169d:NF-YA2 module under individual as well as combined drought and heat stress in Arabidopsis

Plants are often subjected to a combination of abiotic stresses under natural environmental conditions. The response of plants to combined stresses can be very different from that to the individual stress. Several regulatory mechanisms work in harmony to maintain plant's homeostasis during stress conditions. Among them the roles of microRNAs (miRNAs) in combined stresses are beginning to be unravelled. In this study, we evaluated the MIR169d: NF-YA2 target module in individual as well as combined drought and heat stress (HS) in Arabidopsis. We found that MIR169d is highly HS inducible, however, contrary to the reported downregulation of MIR169a/c forms in drought stress in literature, MIR169d is upregulated in drought. Moreover, while MIR169d expression is upregulated during combined stress, the response is less than individual stresses. Further, Arabidopsis plants overexpressing MIR169d or target nf-ya2 knockout mutant plants are more tolerant to both individual as well as combined heat and drought stress as indicated by the higher expression of stress responsive genes and less Trypan blue staining, while plants overexpressing NF-YA2 or those in which miR169defg isoform is sponged up (MIM169defg) are more prone to individual as well as combined heat and drought stress. The MIR169d promoter harbours both heat and drought stress-responsive cis-elements. Assessment of GUS expression in MIR169d-promoter:GUS and NF-YA2-promoter:GUS transgenic lines shows increased and reduced reporter expression in all the three stress conditions as compared to control, respectively. This suggests a stress-induced transcriptional regulation of the MIR169d:NF-YA2 pair. Thus, the MIR169d:NF-YA2 module can be potentially exploited to engineer crops for resistance to multiple abiotic stresses.