Differential S-nitrosylation and characterization of purified S-nitrosoglutathione reductase (GSNOR) from Brassica juncea shows multiple forms of the enzyme.

S-nitrosoglutathione reductase (GSNOR). a master regulator of NO homeostasis, is a single-copy gene in most plants. In Lotus japonicus, two GSNOR isoforms were identified exhibiting similar kinetic properties but differential tissue-specific expressions. Previously, a genome-wide identification in Brassica juncea revealed four copies of GSNOR, each encoding proteins that vary in subunit molecular weights and pI. Here, we report multiple forms of GSNOR using 2D immunoblot which showed 4 immunopositive spots of 41.5 kDa (pl 5.79 and 6.78) and 43 kDa (pl 6.16 and 6.23). To confirm, purification of GSNOR using anion-exchange chromatography yielded 2 distinct pools (GSNOR-A & GSNOR-B) with GSNOR activities. Subsequently, affinity-based purification resulted in 1 polypeptide from GSNOR-A and 2 polypeptides from GSNOR-B. Size exclusion-HPLC confirmed 3 GSNORs with molecular weight of 87.48 ± 2.74 KDa (GSNOR-A); 87.36 ± 3.25 and 82.74 ± 2.75 kDa (GSNOR-B). Kinetic analysis showed Km of 118 ± 11 µM and Vmax of 287 ± 22 nkat/mg for GSNOR-A, whereas Km of 96.4 ± 8 µM and Vmax of 349 ± 15 nkat/mg for GSNOR-B. S-nitrosylation and inhibition by NO showed redox regulation of all BjGSNORs. Both purified GSNORs exhibited variable denitrosylation efficiency as depicted by Biotin Switch assay. To the best of our knowledge, this is the first report confirming multiple isoforms of GSNOR in B. juncea.

Nitric oxide (NO) modulates low temperature-stress signaling via S-nitrosation, a NO PTM, inducing ethylene biosynthesis inhibition leading to enhanced post-harvest shelf-life of agricultural produce.

Low temperature (cold) stress is one of the major abiotic stress conditions affecting crop productivity worldwide. Nitric oxide (NO) is a dynamic signaling molecule that interacts with various stress regulators and provides abiotic stress tolerance. Stress enhanced NO contributes to S-nitrosothiol accumulation which causes oxidation of the -SH group in proteins leading to S-nitrosation, a post-translational modification. Cold stress induced in vivo S-nitrosation of > 240 proteins majorly belonging to stress/signaling/redox (myrosinase, SOD, GST, CS, DHAR), photosynthesis (RuBisCO, PRK), metabolism (FBA, GAPDH, TPI, SBPase), and cell wall modification (Beta-xylosidases, alpha-l-arabinogalactan) in different crop plants indicated role of NO in these important cellular and metabolic pathways. NO mediated regulation of a transcription factor CBF (C-repeat Binding Factor, a transcription factor) at transcriptional and post-translational level was shown in Solanum lycopersicum seedlings. NO donor priming enhances seed germination, breaks dormancy and provides tolerance to stress in crops. Its role in averting stress, promoting seed germination, and delaying senescence paved the way for use of NO and NO releasing compounds to prevent crop loss and increase the shelf-life of fruits and vegetables. An alternative to energy consuming and expensive cold storage led to development of a storage device called "shelf-life enhancer" that delays senescence and increases shelf-life at ambient temperature (25-27 °C) using NO donor. The present review summarizes NO research in plants and exploration of NO for its translational potential to improve agricultural yield and post-harvest crop loss. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01371-z.