Title : Unraveling the role of mirnas in combined heat and drought stress in tomato
Abstract:
lant microRNAs (miRNAs) control entangled gene regulatory networks and have been implicated in important developmental switches as well as in different stress responses. Adverse climatic conditions like high temperature, water deficit, osmotic imbalance, etc., negatively affect agricultural food production world-wide. In nature, a number of different stresses can occur simultaneously and studies have revealed that the response of plants to a combination of different stresses is unique and cannot be directly deduced by analyzing the response of plant to each of the different stresses applied individually. The effect of combined stresses may in fact be either additive or retrograde in nature. Combination of heat and drought stresses has adverse effect on the plant’s physiological status, growth and productivity and represents an excellent example of simultaneous occurrence of two different abiotic stresses in nature. While differential mRNA transcript profiles in response to combined heat and drought stresses have been evaluated in some plants, studies on involvement of miRNAs in combined stress is completely lacking. In the present study, we identified the core miRNome involved in combined heat and drought stress in tomato (Solanum lycopersicum L.) leaf tissue. We identified miRNAs that showed up- regulation at both mature and precursor levels in individual heat, drought and combined stress like miR172b. We thus chose to characterize MIR172 family in-depth. In-house developed bioinformatics screening pipeline revealed five new putative loci for miR172 apart from the four loci reported at miRBase for tomato. Out of above five loci, four loci have been validated by cloning and sequencing. All these miR172 isoforms show different degree of stress- inducibility in individual and/or combined stresses at mature as well as precursor level pointing to specific stress-responsive roles. Transient assays in Nicotiana benthamiana revealed that different miR172 precursors exhibit different processivity to yield mature miRNA. Moreover, the same mature form exhibits variable target cleavage efficiencies when different targets were assessed. The main targets of miR172 are genes that encode AP2- like transcription factors. With the help of degradome data we identified HSF18 as a novel target of miR172. We find that upon HS, miRNA172 is up-regulated which in turn decreases HSF18 transcripts. Functional characterization of HSF18 by knocking-down its expression using virus induced gene silencing (VIGS) in tomato established its role as a negative regulator of HS-tolerance. Interestingly, miR172 promoter analysis identifies cis-regulatory elements for binding of AP2s as well as HSFs. Yeast one hybrid screen confirms HSF-mediated transcriptional control of miR172. Experiments to establish a possible feedback regulatory loop between AP2s, HSFs and miRNA are underway. Using short tandem target mimic (STTM) constructs against different isoforms of miR172 and miR-resistant version of HSF18, will help elucidate the role of miRNA172 in the regulatory networks involved in individual and combined stress response.
