In Silico RNAseq and Biochemical Analyses of Glucose-6-Phosphate Dehydrogenase (G6PDH) from Sweet Pepper Fruits: Involvement of Nitric Oxide (NO) in Ripening and Modulation.

Pepper (Capsicum annuum L.) fruit is a horticultural product consumed worldwide which has great nutritional and economic relevance. Besides the phenotypical changes that pepper fruit undergo during ripening, there are many associated modifications at transcriptomic, proteomic, biochemical, and metabolic levels. Nitric oxide (NO) is a recognized signal molecule that can exert regulatory functions in diverse plant processes including fruit ripening, but the relevance of NADPH as a fingerprinting of the crop physiology including ripening has also been proposed. Glucose-6-phosphate dehydrogenase (G6PDH) is the first and rate-limiting enzyme of the oxidative phase of the pentose phosphate pathway (oxiPPP) with the capacity to generate NADPH. Thus far, the available information on G6PDH and other NADPH-generating enzymatic systems in pepper plants, and their expression during the ripening of sweet pepper fruit, is very scarce. Therefore, an analysis at the transcriptomic, molecular and functional levels of the G6PDH system has been accomplished in this work for the first time. Based on a data-mining approach to the pepper genome and fruit transcriptome (RNA-seq), four G6PDH genes were identified in pepper plants and designated CaG6PDH1 to CaG6PDH4, with all of them also being expressed in fruits. While CaG6PDH1 encodes a cytosolic isozyme, the other genes code for plastid isozymes. The time-course expression analysis of these CaG6PDH genes during different fruit ripening stages, including green immature (G), breaking point (BP), and red ripe (R), showed that they were differentially modulated. Thus, while CaG6PDH2 and CaG6PDH4 were upregulated at ripening, CaG6PDH1 was downregulated, and CaG6PDH3 was slightly affected. Exogenous treatment of fruits with NO gas triggered the downregulation of CaG6PDH2, whereas the other genes were positively regulated. In-gel analysis using non-denaturing PAGE of a 50-75% ammonium-sulfate-enriched protein fraction from pepper fruits allowed for identifying two isozymes designated CaG6PDH I and CaG6PDH II, according to their electrophoretic mobility. In order to test the potential modulation of such pepper G6PDH isozymes, in vitro analyses of green pepper fruit samples in the presence of different compounds including NO donors (S-nitrosoglutathione and nitrosocysteine), peroxynitrite (ONOO-), a hydrogen sulfide (H2S) donor (NaHS, sodium hydrosulfide), and reducing agents such as reduced glutathione (GSH) and L-cysteine (L-Cys) were assayed. While peroxynitrite and the reducing compounds provoked a partial inhibition of one or both isoenzymes, NaHS exerted 100% inhibition of the two CaG6PDHs. Taken together these data provide the first data on the modulation of CaG6PDHs at gene and activity levels which occur in pepper fruit during ripening and after NO post-harvest treatment. As a consequence, this phenomenon may influence the NADPH availability for the redox homeostasis of the fruit and balance its active nitro-oxidative metabolism throughout the ripening process.

Arsenic-induced stress activates sulfur metabolism in different organs of garlic (Allium sativum L.) plants accompanied by a general decline of the NADPH-generating systems in roots.

Arsenic (As) contamination is a major environmental problem which affects most living organisms from plants to animals. This metalloid poses a health risk for humans through its accumulation in crops and water. Using garlic (Allium sativum L.) plants as model crop exposed to 200µM arsenate, a comparative study among their main organs (roots and shoots) was made. The analysis of arsenic, glutathione (GSH), phytochelatins (PCs) and lipid peroxidation contents with the activities of antioxidant enzymes (catalase, superoxide dismutase, ascorbate-glutathione cycle), and the main components of the NADPH-generating system, including glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), NADP-malic enzyme (NADP-ME) and NADP-isocitrate dehydrogenase (NADP-ICDH) was carried out. Data showed a correlation among arsenic accumulation in the different organs, PCs content and the antioxidative response, with a general decline of the NADPH-generating systems in roots. Overall, our results demonstrate that there are clear connections between arsenic uptake, increase of their As-chelating capacity in roots and a decline of antioxidative enzyme activities (catalase and the ascorbate peroxidase) whose alteration provoked As-induced oxidative stress. Thus, the data suggest that roots act as barrier of arsenic mediated by a prominent sulfur metabolism which is characterized by the biosynthesis of high amount of PCs.

Analysis of cytosolic isocitrate dehydrogenase and glutathione reductase 1 in photoperiod-influenced responses to ozone using Arabidopsis knockout mutants.

Oxidative stress caused by ozone (O3 ) affects plant development, but the roles of specific redox-homeostatic enzymes in O3 responses are still unclear. While growth day length may affect oxidative stress outcomes, the potential influence of day length context on equal-time exposures to O3 is not known. In Arabidopsis Col-0, day length affected the outcome of O3 exposure. In short-days (SD), few lesions were elicited by treatments that caused extensive lesions in long days (LD). Lesion formation was not associated with significant perturbation of glutathione, ascorbate, NADP(H) or NAD(H). To investigate roles of two genes potentially underpinning this redox stability, O3 responses of mutants for cytosolic NADP-isocitrate dehydrogenase (icdh) and glutathione reductase 1 (gr1) were analysed. Loss of ICDH function did not affect O3 -induced lesions, but slightly increased glutathione oxidation, induction of other cytosolic NADPH-producing enzymes and pathogenesis-related gene 1 (PR1). In gr1, O3 -triggered lesions, salicylic acid accumulation, and induction of PR1 were all decreased relative to Col-0 despite enhanced accumulation of glutathione. Thus, even at identical irradiance and equal-time exposures, day length strongly influences phenotypes triggered by oxidants of atmospheric origin, while in addition to its antioxidant function, the GR-glutathione system seems to play novel signalling roles during O3 exposure.