Undergraduate Researcher University of Massachusetts, Amherst Eastham, Massachusetts
Body of Abstract: Nitric oxide (NO) is a ubiquitous free-radical signaling molecule involved in a variety of vital cellular pathways related to both normal plant development and stress responses. This research aims to discover mechanisms that control NO levels, and thus NO signaling systems in plants. In Arabidopsis thaliana, as well as other organisms, short-lived NO is integrated into the stable molecule S-nitrosoglutathione (GSNO), the levels of which are primarily regulated by NADH-dependent GSNO reductase (GSNOR). Alternative GSNO reductases remain an unexplored aspect of this dynamic system. Four members of the aldo-keto reductase (AKR) 4C family, AKR4C8, AKR4C9, AKR4C10, and AKR4C11, are highly upregulated in GSNOR-deficient A. thaliana and can catabolize GSNO by using NADPH as a unique and specific source of reducing equivalents. We hypothesize that these AKR4Cs function in GSNOR-deficient A. thaliana as part of a compensatory mechanism to regulate GSNO levels. To understand the function of these AKRs in NO homeostasis, mutant plant lines lacking each of these four genes alone and in combination are being generated using CRISPR-Cas9. Single-knockouts of all four AKR4C genes and two double-knockout lines lacking both AKR4C9 and either AKR4C8 or AKR4C10 have been obtained. Phenotypic analysis of these lines revealed apparent defects in germination in plants lacking AKR4C9 or AKR4C11. We also investigated if other related AKRs can metabolize GSNO, or if this activity is specific to the AKR4C subfamily that is highly upregulated in the absence of GSNOR. Three phylogenetically related AKRs were cloned, expressed, purified and assayed for the ability to reduce GSNO. One of these showed GSNO-reductase activity similar to AKR4C8. In total, our data implicate the AKR4C subfamily, and potentially other AKRs, as NADPH-dependent GSNO reductases that act in addition to NADH-dependent GSNOR in regulating NO homeostasis in plants.