Study Of Arabidopsis RING-type Zinc Finger Protein NCA1on Regulation Of Catalase Activity

Alkaline stress reduces plant growth and crop production severely. Many abiotic stresses including alkaline stress lead to high level of Reactive Oxygen Species (ROS) in plants, which involve in multiple physiological progresses as signal molecules and help plants with general adaption to stresses. However, when stresses continue and the level of ROS is too high, ROS causes damages to cell. Therefore, scavenging of redundant ROS is important to plant stress responses and environment adaption. Catalase (CAT) is an important ROS scavenger which degrades H2O2to H2O and O2. As reported, CAT involves in multiple stress responses including salt, drought and light, and its activity is induced by multiple stresses. However, the mechanism how catalase activity is regulated and is in response to stress stimuli is not very clear.In order to search for key factors in alkaline stress response, we screened T-DNA insertion pool and isolated a high-pH-sensitive mutant phd2(pH homeostasis defective2) in previous work, of which the fresh weight and chlorophyll content were lower than those of Col-0, while the H2O2content in the mutant was higher than that in Col-0. We located the mutated gene to NCA1(No Catalase Activity1) by map-based cloning, which encodes a RING finger protein. The T-DNA insertion introduced a frame shift and a premature stop coden in the fifth exon of NCA1in mutant. We renamed the mutant as ncal-3to keep the consistency. In addition to alkaline stress, ncal-3was hypersensitive to multiple stresses, including salt, low temperature and paraquat. NCA1located in cytosol and expressed in multiple tissues. We demonstrate that NCA1interacted with catalases (CATs) by mass spectrum and the interaction took place in cytosol by Bimolecular Florescence Complementation (BiFC). Besides, the interaction between NCA1and CATs was not affected by stress treatments. NCA1interacted with CATs and increased CATs activity maximally in240kDa complexes in plants. In vitro, NCA1interacted with CAT2in a1:1molar ratio through the NCA1C terminus and activated CAT2through the N terminal RING finger. The zinc ions bonded by RING fingers were required for activating CAT2activity. We isolated peroxisomes and cytosol through percoll-sucrose density gradient centrifugation, and found that NCA1activated catalase activity both in cytosol and in peroxisomes. Subsequently, we found that NCA1has a chaperone holdase activity which helps maintain CATs in a functional folding state.In conclusion, we demonstrate that NCA1interacts with CATs and activates CATs in plant. However, the activation is not affected by alkaline stress, suggesting that NCA1is required for maintaining basal CAT activity. NCA1acts as a chaperone and maintains CATs in a functional folding state, which is required for maximal CAT activity to scavenge stress-induced H2O2and maintain ROS homeostasis to alleviate the stress-induced damages.