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Nitric oxide (NO) is a gaseous signaling molecule in plants, transducing information as a result of exposure to low temperatures. However, the underlying molecular mechanism linking NO with chilling stress is not well understood. Here, we functionally characterized the cucumber ( Cucumis sativus L .) nitric oxide synthase-associated gene,  NITRIC OXIDE ASSOCIATED 1  ( CsNOA1 ). Expression analysis of  CsNOA1 , using quantitative real-time PCR,  in situ  hybridization, and a promoter::β-glucuronidase (GUS) reporter assay, revealed that it is expressed mainly in the root and shoot apical meristem (SAM), and that expression is up-regulated by low temperatures. A CsNOA1-GFP fusion protein was found to be localized in the mitochondria, and ectopic expression of  CsNOA1  in the  A. thaliana noa1  mutant partially rescued the normal phenotype. When overexpressing  CsNOA1  in the  Atnoa1  mutant under normal condition, no obvious phenotypic differences was observed between its wild type and transgenic plants. However, the leaves from mutant plant grown under chilling conditions showed hydrophanous spots and wilting. Physiology tolerance markers, chlorophyll fluorescence parameter (Fv/Fm), and electrolyte leakage, were observed to dramatically change, compared mutant to overexpressing lines. Transgenic cucumber plants revealed that the gene is required by seedlings to tolerate chilling stress: constitutive over-expression of  CsNOA1  led to a greater accumulation of soluble sugars, starch, and an up-regulation of Cold-regulatory C-repeat binding factor3 ( CBF3 ) expression as well as a lower chilling damage index (CI). Conversely, suppression of  CsNOA1  expression resulted in the opposite phenotype and a reduced NO content compared to wild type plants. Those results suggest that  CsNOA1  regulates cucumber seedlings chilling tolerance. Additionally, under normal condition, we took several classic inhibitors to perform, and detect endogenous NO levels in wild type cucumber seedling. The results suggest that generation of endogenous NO in cucumber leaves occurs largely independently in the (CsNOA1) and nitrate reductase (NR) pathway.

Cucumber (Cucumis sativus L.) Nitric Oxide Synthase Associated Gene1 (CsNOA1) Plays a Role in Chilling Stress