Role of Arabidopsis MutSβ in sensing and deploying a saline stress-mediated DNA damage response

FIGUEROA, NICOLÁS; SPAMPINATO, CLAUDIA P.

Mendoza

Congreso; LVIII Reunión Anual SAIB; 2022

In order to maintain the proper integrity of their genetic material, plants have evolved both conserved and idiosyncratic mechanisms to cope with genotoxic stress. One of such mechanisms is the mismatch repair system (MMR) which recognizes and corrects different types of post-replicative errors introduced in the DNA. In addition, the MMR components MutSα (MSH2/MSH6) and MutSγ (MSH2/MSH7) were shown to inhibit genetic recombination and cell cycle progression in the presence of moderate to severe bioticand abiotic stress-derived DNA damage. However, less information is available about the participation of plant MutSβ (MSH2/MSH3) in the DNA damage response pathway upon exposure to environmental stress. In this work, we characterized the responses of three msh3 T-DNA-derived insertional mutant lines to acute salt stress in comparison to their parental wild-type (WT). We chose to work with high salinity, since it is one of the major environmental constraints that affect plant growth and productivity, and it is reported to perturb genome stability by inducing DNA double strand breaks (DSBs). Indeed, formation of DSBs in salt-treated seedlings was confirmed by a neutral comet assay, where differential levels of DNA fragmentation could be observed between msh3 mutants and the WT line. We also performed seed germination assays under saline stress, where we observed an up to 40% increase in the germination rates of all msh3 mutant lines when compared to WT at 150 mM NaCl. This suggests an impaired cell cycle control in the MSH3 defective lines in response to DNA damage. This concurs with our in silico coexpression network analysis, where we observed a coregulation of MSH3 expression with several cell cycle-related genes. Moreover, enrichment analysis of promoter cis-regulatory motifs revealed an overepresentation of E2Fa/b binding motifs in the promoter regions of densely connected coregulated modules, among which MSH3 could be found. This was confirmed by RT-qPCR analysis, which showed a significant increase in relative MSH3 transcripts in both E2Fa - and E2Fb -overexpressing lines. Experiments are underway to demonstrate whether this interaction is direct by both EMSA and ChIP assays, and to assess whether msh3 mutants display alterations in somatic recombination rates under saline stress. Our preliminary data suggests a novel link between the mismatch recognition protein MSH3 and abiotic stress-mediated DNA damage perception and response.