The Redox State Of Ascorbate And The Role Of Exogenous ABA In The Responses Of Rice (Oryza Sativa L.) To High Salinity Or Low Temperature

Analyses of gene transcriptional profiling related to violaxanthin cycle and redox homeostasis in the responses of rice (Oryza sativa L.) to high salinity or low temperature stress were carried out using techniques such as Affymetrix GeneChip microarrays, real-time quantitative RT-PCR (RTqPCR), the chlorophyll a fluorescence technique, gene cloning, gene expression in Escherichia coli, and so on. The main results were as follows:I. Integration of network of ABA-violaxanthin cycle and redox system underlying antioxidant effects in rice responses to high salinity or low temperature.Redox network underlying antioxidant effects was composed of antioxidants such as ascorbate (ASC), glutathione (GSH), tocopherols (Vitamin E), and antioxidant enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and so on. Results from transcriptional profiling using Affymetrix GeneChip microarrays showed that,191genes (or ESTs) were involved in the redox network. The differentially expressed transcripts were identified. There were5/5up-regulation genes in i-93-11/j-NJ-1and2/6down-regulation genes in i-93-11/j-NJ-1induced by chilling, and32/27up-regulation genes in i-93-11/j-NJ1and13/25down-regulation genes in i-93-11/j-NJ1induced by high salinity.Results from transcriptional profiling using Affymetrix GeneChip microarrays showed that,22genes (or ESTs) were involved in ABA-violaxanthin cycle. The differentially expressed transcripts were also identified. There were3/2up-regulation genes in i-93-11/j-NJ-1and no down-regulation genes induced by chilling, and7/6up-regulation genes in i-93-11/j-NJ-1and6/5down-regulation genes in i-93-11/j-NJ-1induced by high salinity. Among these, obviously changes in expression of NCED,8’OX, ZEP and VDE, key genes of ABA biosynthesis, ABA degradation and violaxanthin cycle, respectively, were obtained. It was implyed that ABA-violaxanthin cycle played a key role under stress condition.Futhermore, a network of ABA-violaxanthin cycle and redox system underlying antioxidant effects in rice responses to high salinity or low temperature was integrated by ascorbate with its redox status. This network based on enzymic and nonenzymic biochemistry reactions and involved electron transport chain of both photosynthsis and respiration, and existed in cytoplasm, chloroplast (stroma, thylakoid membrane and lumen), mitochondria (inner membrane and matrix), and peroxisome. As a result, the network, contained191and22genes involved in ROS scavenging and ABA-violaxanthin cycle, respectively, was high complementary and flexibility necessary for land plants, and played a key role in protecting photosynthetic apparatus against oxidative damage. Ⅱ. Protective role of ABA-violaxanthin cycle under high salinity or low temperature.When the third leaf was fully expanded, rice plants were divided into ABA-treatment (+ABA) group and non ABA-treatment (-ABA) group. Both+ABA group and-ABA group plants were further divided into non salt-treatment subgroups (-ABA-aCl,+ABA-NaCl) and salt-treatment subgroups (-ABA+NaCl,+ABA+NaCl), or further divided into normal temperature-treatment subgroups (-ABA-LT,+ABA-T) and low temperature-treatment subgroups (-ABA+LT,+ABA+LT). Changes in actual efficiency of PS Ⅱ photochemistry (φPS Ⅱ), non-photochemical quenching (NPQ), contents of xanthophylls and kinetics of de-epoxidation were studied in ABA-fed and non ABA-fed leaves of rice plants under NaCl stress and low temperature, respectively, and expression levels of genes related to ABA metabolism and violaxanthin cycle were ditermined by real-time fluorescent quantitative RT-PCR (RTqPCR). The predicted promoters of ZEP, NCED and VDE genes were amplified and isolated by using PCR primers as detailed in Table3-2. Abiotic stress-responsive cis-regulatory promoter elements of three genes were studied.Salt stress induced more progressive decrease in actual efficiency of PS Ⅱ photochemistry (φPS Ⅱ), higher reduction state of PS Ⅱ, and a small significant increase of NPQ in NaCl-treatment rice plants as compared with low temperature rice plants, whereas exogenously supplied ABA alleviated more decrease in actual efficiency of PS Ⅱ photochemistry (ΦPS Ⅱ), induced lower reduction state of PS Ⅱ, and caused higher capacity of NPQ in ABA-fed rice plants than in non ABA-fed rice plants. As a result, there were higher activities of photosynthetic electron transport, higher capacity of energy dissipation and lower cumulation of excess light in low temperature treatment rice plants than in NaCl treatment rice plants, and in ABA-fed leaves than in non ABA-fed leaves. Addition of exogenous ABA resulted in enhancing the size of violaxanthin cycle pool, promoting de-epoxidation of violaxanthin cycle components, and raising the level of NPQ by altering kinetics of de-epoxidation of violaxanthin cycle. Protection from photodamage appears to be achieved by coordinated contributions by exogenous ABA and violaxanthin cycle-mediated NPQ. This variety of photoprotective mechanisms may be essential for conferring photodamage-tolerance under NaCl or chilling stress.About7.579-fold increased expression levels of NCED4gene were observed in-ABA+SS rice plants. And about3.777-and3.589-fold increased expression of NCED5and NCED4gene, respectively, were obtained in-ABA+LT rice plants. In addition, there were as high as13.874-and51.253-fold increased expression levels of8’ OX1gene,2.326-and2.956-fold increased expression levels of VDE gene, and2.985-and1.874-fold increased expression levels of ZEP gene under high salinity and low temperature, respectively, as compared with control. NCED,8’OX, VDE and ZEP were the key enzymes of ABA biosynthesis, ABA degradation and violaxanthin cycle, respectively. It was implied that ABA metabolism and violaxanthin cycle could be induced by high salinity or chilling stress, and exogenous ABA could upregulate above genes expression in+ABA+SS and+ABA+LT rice leaves.ZEP gene (LOC_Os04g37619) contains a conserved ABA-responsive cis-acting elements named ABRE (ABA-responsive element, C/GACGTGGC), MYC/MYB, DRE and EP2in its promoter region, and upregulates its expression levels under-ABA+SS,-ABA+LT,+ABA+SS or+ABA+LT conditions. It was implied that expression of ZEP gene (LOC_Os04g37619) might be controlled by ABA-dependent (such as ABRE, MYC/MYB elements) or ABA-independent (such as DRE element) transcriptional regulatory networks.Genes of NCED4, NCED5,8’OX1and VDE contain a conserved ABA-responsive cis-acting element named ABRE in their promoter region that interacts with two ABA activated basic leucine zipper (bZIP) transcription factors AREB and ABF. Their expression were upregulated by high salinity or low temperature, and might be controlled by ABA-receptor pathway.Stress can be converted into a biochemical response and permit plants to acclimate to stressful environmental conditions, by modified enzymic structure shuch as hydrophobic "β-sheet-loop-a-helix" motif at N-terminus of ZEP, an amphipathic α-helix (an α-helix that has both hydrophobic and hydrophilic regions.) at N-terminus of NCED, and the highly conserved four histidines and the highly negatively charged C terminus of VDE.The enzymic activities of ABA metabolism and violaxanthin cycle were controlled by transcription, modification of post transcription, hydrophobic/hydrophilic characteristics of enzymic molecular and pH values.Ⅲ. Redox of ASC and relevant gene/protein expression under high salinity or low temperature stress.To explore the changes in redox status of ASC, the contents of ASC and DHA, the activities of MDHAR, DHAR, GLDH and APX, the expression levels of MDHAR, DHAR, GLDH and APX genes were studied in high salinity-or low temperature-treatment rice, respectively.Salt stress induced more progressive increase in H2O2contents, more decrease in ASC/DHA ratio as compared with control rice plants. The ASC/DHA ratio indicates elevated ROS scavenger capacities.Regeneration of ASC from MDHA and DHA catalized by MDHA reductase (MDHAR) and DHA reductase (DHAR), respectivily. There were2upregulated expression genes (out of5 genes) encoding MDHAR and one upregulated expression gene (out of2genes) encoding DHAR under stress. The activities of MDHAR and DHAR increased and revealed a highly consistent with the upregulated expression genes of MDHAR and DHAR.Biosynthesis of ASC from L-galactono-1,4-lactone was catalized by L-galactono-1,4-lactone dehydrogenase (GLDH), the last step enzyme. GLDH is an integral protein of the inner mitochondrial membrane. The activities of GLDH increased and revealed a highly consistent with the up-regulated expression of GLDH gene under stress conditions.APX, which uses ASC as an electron donor and catalyzes the conversion of H2O2into H2O and O2, is the key enzyme to scavenge ROS and regulate the ratio of ASC/DHA. APX2gene (one of2cytosol isoforms) expression was significantly up-regulated, whereas chloroplast APX gene (stroma and thylakoid isoforms, APX5-8) expression was down-regulated in different degree under stress condition.Three-dimensional structure of APXs was constructed using Deep View Swiss Pdb Viewer. The active site is composed of two substrate-binding sites:the distal histidine site where H2O2binds and the y-heme edge site where the ascorbate binds. Firstly, H170/H164(tAPX/cAPX2) and H41/H43are the proximal and distal histidine, respectively. The proximal H170/H164is an axial ligand. Two residues, R37/R39and H41/H43, in the distal heme pocket have been implicated in acid-base catalysis and cleavage of the peroxide O-O bond during compound I formation, His41/H43as a base acceptor in this process. The distal residue R37/R39enhances the efficiency of the reaction and the binding affinity for ligands. Secondly, in y-heme edge site where the ascorbate binds, some residues forming additional hydrogen bonds with substrate ascorbate. The substrate binding position showed that electron delivery to the heme was through the heme propionate. ASC played an important role in stabilizing three dimentional conformation of APX in addition to a substrate for APX.In addition, there is an additional loop structure composed of residues from P185to S201in APX8(chloroplast isoform), but virtually no structural information comes from homology in APX2(cytosol isoform). The additional loop, which created a neutral and polar microenvirenment near the binding site of ASC, was in favor of biochemical reaction in chloroplast stroma at pH8.0.The deduced molecular weights (MW) of APX8and APX2expressed in Escherichia coli were about28.2KD and44.7KD, respectively. Kinetics analysis showed that APXs catalyze the oxidation of ascorbate and the reduction of H2O2by the ping-pong mechanism. The kmASctSc values for ASC, or the kmH2O2values for H2O2of APX2and APX8were all different. The optimal pH value of APX2and APX8were about6-7and7-8, respectively. The redox status of ASC (or ASC/DHA ratio) were controlled by not only MDHAR, DHAR, GLDH and APX genes at transcritioanal and posttranscriptional levels, but also the redox status of chloroplast stroma and mitochondria matrix.Our results indicated an intricate relationship, at the transcriptional and possibly post-transcriptional levels, between ABA biosynthesis, the violaxanthin cycle, and redox homeostasis mediated ROS-scavenging system, and allowed us to identify candidate genes for follow-up studies to dissect this interaction at the biochemical and molecular level. It is very significant to make some suggestions on breeding new cultivars with antioxidation under stress.