Cloning And Functional Analysis Of Chloroplast APX In Suaeda Salsa

Suaeda salsa is a leaf succulent euhalophyte that may have evolved the unique salt-tolerant mechanism. The results of most studies suggest that salt tolerance is usually correlated with a more efficient antioxidant system. And there may have distinct difference on the scavenging of reactive oxygen species (ROS) between euhalophyte and nonhalophyte. So cloning those genes that encoding enzymes engaged in scavenging ROS from S. salsa may attribute to get insight into the mechanism of plant salt tolerance.The enhanced production of ROS during salt stress can pose oxidative stress to cells and lead to the oxidative destruction. High NaCl concentrations seem to impair electron transport and lead to the formation of ROS in chloroplasts. Plants have well-developed antioxidant defense systems against ROS. The chief toxicity of O₂·^ˉand H₂O₂ is thought to reside in their ability to initiate cascade reactions that result in the production of the hydroxyl radical and other destruction species such as lipid peroxides. These dangerous cascades are prevented by efficient operation of the cell's antioxidant defenses. SOD converts superoxide radicals (O₂·^ˉ) to hydrogen peroxide (H₂O₂). Hydrogen peroxide acts as a precursor of more cytotoxic or highly reactive oxygen derivatives, such as peroxynitrite or OH·.It is very important to scavenge the excess H₂O₂. CAT and APX are related to H₂O₂-scavenging. In the first part of present study, salt-dilution halophyte S. salsa grown under 200 mmol/L NaCl was used to investigate the activities of O₂·^ˉand H₂O₂-scavenging enzymes and their possible roles in salt adaptations. And the second part was that chloroplast APX of a C3 halophyte Suaeda salsa L. was cloned and its function was analyzed. The main results are as follows.1. Effects of NaCl treatment on growth and net photosynthetic rate of S. salsa seedlings.The growth of S. salsa plants was significantly increased by NaCl treatment. The fresh weight (FW) per plant was increased 93.8% at 200 mM NaCl. Net photosynthetic rate of NaCl-treated plants was significantly higher than that of control plants. These results indicated that S. salsa was a typical halophyte, and no photo-inhibition occurred in S. salsa under 200-mM NaCl treatment. 2. Effects of NaCl treatment on MDA and plasmalemma permeability of Suaeda salsa seedlingsCompared with controls, 200 mmol/L NaCl treatment had no significant effect on the leaf MDA content and leaf plasmalemma permeability suggesting that no significant oxidative damage to biomembranes occurred.3. Effects of NaCl treatment on the activity of antioxidant enzymes of S.salsaSOD,CAT and APX are the basic scavenging-enzyme of O₂·^ˉand H₂O₂ in the leaves of S. salsa. SOD activity was 107.6% of the control. Enzyme (CAT, APX) activities increased significantly under NaCl treatment. The thylakoid-bound and stromal SOD and APX activities were determined in the chloroplast of S. salsa plants grown under NaCl salinity. Both the thylakoid-bound and stromal APX activities were markedly enhanced. At least 50% of chloroplast APX was thylakoid-bound. Stomal APX represented about 74–78% of the total soluble leaf APX activity. The activity of thylakoid-bound SOD constituted the major part of total chloroplast SOD activity. Under salt treatment, similar increase were observed in chloroplast of the thylakoid-bound and stroma SOD and leaf SOD activity of S. salsa.4 cDNA cloning and sequence analyzing of chloroplast APX in halophyte Suaeda salsaBased on sequence homologies to the chloroplast APX genes of Mesembryanthemum crystallinum, Spinacia oleracea, Nicotiana tabacum and Arobidopsis thaliana, we used reverse transcriptase (RT)-PCR amplification and 5'- and 3'- RACE amplification to obtain the full-length transcript sequences of CHLAPX. By alternative processing of the 3'-terminal region as a result of alternative polyadenylation and splicing, two types of mature mRNA variants, one form encoding thylakoid-bound APX (Ss.tAPX) and another encoding stroma APX (Ss.sAPX). Interestingly, the N-terminal 378 amino acids of Ss.sAPX are completely identical with those of Ss.tAPX, whereas the C-terminal 49 amino acids differ. chlAPX isoenzymes have a transit peptide consisting of approximately 64 residues in their N-terminus. Ss.tAPX has one major hydrophobic domain in the C-terminal which is responsible for spanning to the stroma-exposed thylakoid membranes in chloroplasts.5. Expression analysis of chloroplast APX in halophyte Suaeda salsaNorthern blot analysis indicated that the transcription of chloroplast APX was up-regulated by 400 mmol/L NaCl treatment. Amounts of chlAPX transcripts in shoots increased for up to 24 h and then decreased. For 48 h, the mRNA levels of chlAPX in NaCl treatment were still higher than those of controls. In the roots, the transcripts level of chlAPX increased with the treatment time.The level of transcript of chlAPX in the shoots under 48h of 200 mmol/L NaCl treatment was higher than that of 400 mmol/L NaCl treatment. While in the roots the mRNA levels of chlAPX induced by 400 mmol/L NaCl was higher than 200 mmol/L.6. Functional analysis of chlAPXTo determine the function of chlAPX, we over-expressed this gene in the Arabidopsis. No difference was observed in growth between transgenic plants and wild type plants grown under the normal conditions. However, under high light, it is found that the transgenic plants were less oxidative stress than the wild types.Under high light conditions, the MDA content and leaf plasmalemma permeability of transgenic plants were lower than those of wild type. Fv/Fm and chlorophyll contents were higher than those of wile type.We also determined the activity of the antioxidant enzymes such as SOD,CAT,APX. After high light treatment, the enzyme activities were lower than the control. However, the transgenic plants'enzyme activities were significantly higher than those of wild type plants. Insertion of one antioxidant enzyme gene led to a series of antioxidant enzyme activity changes. This suggested that these antioxidant enzymeswere coordinate regulation during the oxidative stress. 7. Functional analysis of cytosolic CAT over-expressed in the chloroplast No difference was observed in growth between transgenic plants and wild type plants grown under the normal conditions. The CAT over-expressed in the chloroplast suppressed the activity of CAT in the leaves. But the activity of SOD,CAT and APX were higher than the wild type's under high light.Summarily, the data in this article indicated that the ability of ROS scavenging system of S. salsa was close correlative to its salt tolerance. The expressions of CHLAPX of S. salsa were significantly up-regulated by salinity at transcription levels. The increase of the antioxidant enzyme activities of S. salsa under salt stress might be one of the reasons for the salt tolerance of S. salsa. They can scavenge the ROS induced by the salinity in time and diminish the oxidative damage. Therefore S. salsa plants can survive in saline soil, even in sea water. Transgenic Arabidopsis plants over-expressed of CHLAPX possess higher antioxidative stress ability than wild type plants, which further indicated the importance of maintaining the ROS production and scavenge equilibrium.