Submitted to: FEBS Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/31/2008
Publication Date: 4/28/2008
Citation: Abat, J.K., Mattoo, A.K., Deswal, R. 2008. S-nitrosylated proteins of a medicinal, cam plant kalanchoe pinnata: ribulose-1, 5-bisphospate carboxylase/oxygenase activity targeted for inhibition. FEBS Journal. 275:2862-2872. Interpretive Summary: Nitrogen is an essential nutrient for plant growth and development. Its utilization, however, results in it being oxidized to toxic forms such as nitric oxides. One such molecule is nitric oxide (NO), which can be both beneficial and toxic to plants and animals. In plants, excessive concentrations of nitric oxides were previously shown to negatively impact photosynthetic apparatus. However, NO is also known to be a bioactive molecule that has been suggested to be a new hormone because it is found to be a part of the plant signaling process in response to abiotic and biotic stresses. Very little is known about the mechanisms by which NO regulates plant processes. In a collaborative work with scientists in India, we have used a proteomics approach and biotin switch assay to identify proteins that are modified by NO, in a process called S-nitrosylation. Nineteen soluble proteins that were modified by NO in a C3 plant, Kalanchoe pinnata, were identified, about nine of which are shown, for the first time, to be nitrosylated. Among these is the carbon fixation enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). For the first time, new insights are provided about potential functions of S-nitrosylation in plants, including the inhibition of Rubisco enzyme activity. Because Rubisco is a key protein involved in carbon fixation, excessive production of NO in plants may negatively impact crop productivity. These findings should be of interest to scientists in the areas of agronomy, physiology, biochemistry, molecular biology, protein science, agriculture and horticulture.
Technical Abstract: Nitric oxide (NO) is a new addition to signaling molecules that affect a myriad of processes in plants. However, the mechanistic details are scanty. NO post-translationally modifies proteins by S-nitrosylation of cysteines. Soluble S-nitrosoproteome of a medicinal, crassulacean acid metabolism (CAM) plant, Kalanchoe pinnata, was purified using the biotin-switch assay. Nineteen targets were identified by MALDI-TOF mass spectrometry, including proteins associated with carbon, nitrogen and sulfur metabolism, cytoskeleton, stress and photosynthesis. Some were similar to those earlier identified in Arabidopsis thaliana but kinesin-like protein, glycolate oxidase, UDP glucose 4-epimerase and DNA topoisomerase II are novel targets for any organism. In vitro and in vivo nitrosylation of ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco), one of the targets, was confirmed by immuno-blotting for validation. Rubisco plays a central role in photosynthesis; therefore effect of S-nitrosylation on its enzymatic activity was determined using NaH14CO3. NO-releasing compound, S-nitrosoglutathione inhibited its activity in a dose dependent manner. Thus, we show for the first time Rubisco inactivation by nitrosylation.