Validating GSNOR Protein Interactors and Their Regulatory Role in Nitric Oxide Signaling in Arabidopsis thaliana

Presenter

Nicole Carol Steinmeyer

Campus

UMass Amherst

Sponsor

Elizabeth Vierling, Department of Biochemistry and Molecular Biology, UMass Amherst

Schedule

Session 4, 2:30 PM - 3:15 PM [Schedule by Time][Poster Grid for Time/Location]

Location

Poster Board A37, Campus Center Auditorium, Row 2 (A21-A40) [Poster Location Map]

Abstract

Nitric Oxide (NO) is a signaling molecule involved in many cellular processes in plants including growth and stress management. NO spontaneously reacts with glutathione (GSH) to form a more stable molecule, S-nitrosoglutathione (GSNO). GSNO levels are regulated by NADH-dependent, S-nitrosoglutathione reductase (GSNOR). We hypothesize that NO homeostasis is controlled in part by the interaction of other proteins with GSNOR. Potential GSNOR interactors in Arabidopsis thaliana were previously identified from literature and proximity labeling. The specific aim of my work is to validate protein-protein interactions with GSNOR using bimolecular fluorescence complementation (BiFC) methodology. In GSNOR BiFC experiments, one-half of YFP is fused to GSNOR, and the other half is fused to a putative interacting protein. These proteins are then expressed in tobacco leaf cells. In previous BiFC experiments YFP fluorescence was observed when the proteins SOD1, TXND9, SERAT3, AHB2, TrxH3, and TrxH5 tagged with N-terminal YFP were expressed with GSNOR tagged with C-terminal YFP. No interaction was detected for NTRA, and CAT3 gave inconsistent results. I observed BiFC interactions when TrxH2, TrxH4, Trx H7, or Trx H9 were expressed with GSNOR, but not with TrxH8. Additional proteins are being cloned for BiFC testing including TrxH1, PAX, UGT, and TrxH2-delta (N-terminal). The ability of thioredoxins to reverse S-nitrosation may play a role in GSNOR regulation. Also, it will be important to rule out that negative results are not due to inadequate protein expression. These experiments will provide a better understanding of how GSNOR, and consequently NO levels, are regulated in A. thaliana.

Keywords

protein-protein Interaction, confocal microscopy, bimolecular fluorescence complementation, thioredoxins

Research Area

Biological Organisms

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