Characterization Of Antioxidant Defense Genes KcCSD And KcTrxf From Kandelia Candel Chloroplast Under NaCl Stress

Mangrove species, thriving in the intertidal zone, have been considered as model trees to elucidate salt resistance in woody plants. Kandelia candel is a major mangrove species along southern China coastlines. We have previously shown that K. candel exhibits a high capacity to restrict salt uptake and subsequent accumulation under increasing salinity. Our microarray analysis has shown that salt stress upregulates the expression of key enzymes in chloroplast antioxidant system (Cu/Zn SOD (CSD), thioredoxin f(Trxf)) in K. candel. However, the link between key enzymes in chloroplast antioxidant system and salinity stress has not yet been established in K. candel. Therefore, in this study, Na+ uptake and transport in K. candel and antioxidative defense were investigated under rising NaCl stress from 100 to 300 mM. Besides, KcCSD and KcTrxf, encoding chloroplast protein in antioxidant system, were cloned and transferred to the functional genetics model tobacco (Nicotiana tabacum L.) to clarify the role in salinity tolerance, respectively.Using ion-selective electrodes, we found that short-term and long-term salinity resulted in Na+ efflux. K. candel roots could maintain a high capacity to extrude Na+ via a PM Na+/H+ antiport system driven by H+-ATPase in the short-term salinity. Salinized K. candel roots had a net Na+ efflux with a declined flux rate during an extended NaCl exposure. Moreover, K. candel plants also exhibit a high capacity to maintain antioxidant enzyme activity under NaCl stress. Under a prolonged stress, K. candel could activate its antioxidant system when roots were unable to effectively limit Na+ uptake and transport in the plant. The build up of Na+ in leaves enhanced H2O2 levels, superoxide dismutase (SOD) activity, and increased transcription of CSD gene encoding a Cu/Zn SOD. What is more, salt treatment enhanced NPTs (non-protein thios) contents and corresponding increased transcription of Trxf in K. candel.Sequence and subcellular localization analyses have revealed that KcCSD is a typical Cu/Zn SOD in chloroplast. Sequence and subcellular localization analyses revealed that KcTrxf is a typical thioredoxin in the chloroplast, which is similar to KcCSD. To clarify the role of chloroplast protein in salinity tolerance, KcCSD and KcTrxf was transferred to the functional genetics model tobacco, respectively. Transgenic lines were more Na+ tolerant than wild-type tobacco in terms of root growth, and survival rate. In the latter,100 mM NaCl led to a remarkable reduction in chlorophyll content and a/b ratio and net photosyntheticrate (Pn). Besides, wild-type tobacco showed higher increase in MDA and H2O2 content under NaCl stress, compared to transgenic lines. The salt damage in WT was presumably resulted from reactive oxygen species (ROS) accumulation, especially H2O2 burst in chloroplast.Controlling ROS production and scavenging in the chloroplast were shown to be essential for tolerance to salinity in transgenic plants. Na+ injury to chloroplast was less pronounced in KcCSD-transgenic plants due to upregulated antioxidant defense. ROS generated due to metabolic imbalances during stress could serve as a stress signal. The high O2- via photosynthetic electron transport chain in salinized transgenic plants was likely due to feed-back activation of O2- production system, thus activating acclimation and defence mechanisms that would in turn counteract stress-associated oxidative stress. KcCSD-transgenic tobacco enhanced SOD activity to converts O2- to O2 and H2O2 by an increment in SOD isoenzymes under 100 mM NaCl stress from 24 h to 7 day. Catalase (CAT) activity rose to scavenge H2O2 in KcCSD overexpressing tobacco plants.KcCSD-transgenic plants better scavenged NaCl-elicited ROS compared to WT ones. KcTrxf-transgenic plants also up-regulated activity of CAT, ascorbate peroxidase (APX) to scavenge ROS. Moreover, KcTrxf transgenic plants were able to increase the activities of monodehydroascorbate reductase (MDAR) and glutathione reductase (GR) in chloroplast AsA-GSH cycle, leading to an increase in the ratio of reduced glutathione (GSH) to oxidized glutathion (GSSG) and NPTs in the leaves.Collectively, the KcCSD-and KcTrxf-transgenic plants could better scavenge the salt-elicited reactive oxygen pecies (ROS) in leaf cells, compared to WT plants. In this way, salt-elicited reduction of chlorophyll content, and chlorophyll a/b ratio of transgenic plants was consequently restricted. As a result, transgenic plants maintain photosynthesis and growth under salinity in the longer term.In conclusion, K. candel effectively excluded Na+ in roots during a short exposure; and increased KcCSD and KcTrxf expression to reduce ROS in chloroplast in a long-term and high saline environment. This benefits the salinized plants to retain the redox balance, and thus reduces the salt-elicited oxidative damage during the prolonged period of salt stress.