Characterization of plant carbonic anhydrases involvement in nitric oxide production from nitrite and NO-regulated genes during hypersensitive cell death

Since the discovery that Nitric oxide (NO) plays a crucial role in mediating plant defense response in the late nineties, extensive research over the past 20 years revealed that NO is acting as a mediator in plant growth and development, as well as coping with biotic and abiotic stresses. However, both NO biosynthesis and NO downstream signaling during the hypersensitive response triggered by an avirulent pathogen still need further clarification. Two routes for NO production in plants are known, the oxidative pathway and the reductive pathway. To date, the reductive route from nitrite is the most firmly described. Nitrate reductase (NR) can produce NO from nitrite but the physiological relevance of this activity is unclear. Furthermore, exogenous nitrite supply to an NR deficient mutant demonstrates that other routes for NO production from nitrite should exist in plants. Interestingly, it was reported that bovine carbonic anhydrase II, an alpha type carbonic anhydrase (CA), can convert nitrite to NO. Moreover, additional literature reports suggested the involvement of carbonic anhydrases belonging to the beta family of plant CAs in immunity. Therefore, the first aim of this work was to explore the possible involvement of plant carbonic anhydrase enzymes in nitric oxide synthesis during the hypersensitive response (HR). Firstly, we tried to explore the NO producing activity of AtαCA2, an Arabidopsis enzyme belonging to the same family as the bovine CA, which expression was induced by pathogen. We found that this protein requires glycosylation for its activity and localizes to plant thylakoids. Unfortunately, the transient expression in plant system, which yielded a properly glycosylated protein, led to low protein expression not enough to verify its NO production activity. Alternative production system should be eventually considered. Two representatives of β andγtype carbonic anhydrases were also cloned, expressed and purified. As expected, tobacco βCA1 showed high carbonic anhydrase activity, and Arabidopsis γCA2 showed no detectable carbonic anhydrase activity. However, these proteins were not able to catalyze the nitrite conversion to NO. In the second part of this work, we enquired the NO downstream signaling, focusing on transcriptomic changes associated to NO induced cell death. A massive transcriptomic rearrangement was found to be associated to the NO induced plant cell death. The functional class response to stimuli was strongly enriched in the differentially expressed genes modulated by NO. Moreover, we found a large modulation in signaling and transcription factors. Genes encoding for proteins involved in protein degradation or metabolism of nucleic acids were induced, while genes involved in anabolic processes were down-regulated. Importantly, we confirmed that NO treatment leads to a massive metabolic reprogramming, which specially affects lipid metabolism. Finally, among induced genes the enrichment in genes previously found to be involved/associated to cell death confirmed that chosen conditions were adequate to select for genes involved in cell death activation and execution during the HR.