Nitric oxide (NO) plays an essential role in a myriad of physiological and pathological processes, but the molecular mechanism of the action and the corresponding direct targets have remained largely unknown. We used cellular, biochemical, and genetic approaches to decipher the potential role of NO in root growth in Arabidopsis thaliana. We specifically demonstrate that exogenous application of NO simulates the phenotype of NO overproducing mutant (noxl), displaying reduced root growth and meristem size. Using root specific cell marker lines, we show that the cell in the cortex layer are more sensitive to NO as they show enhanced size. Examination of total S-nitrosylated proteins showed higher levels in noxl mutant than wild type. Using an in vitro assay we demonstrate that plastidial glyderaldehyde-3-phosphate dehydrogenase (GAPDH) is one of NO direct targets. The function of GAPDH in glycolysis provide a rational for S-nitrosylation of this enzyme and its subsequent reduced activity and ultimately reduced growth in roots. Indeed, the rescue of the root growth phenotype in noxl by exogenous application of glycine and serine, the downstream products of plastidial GAPDH provide unequivocal evidence for mechanism of NO action through S-nitrosylation of key proteins, thereby delicately balancing growth and stress responses. (C) 2017 Elsevier Inc. All rights reserved.