| Approximately 30-40% of the world’s arable soils and up to 50% of potentially arable lands are acidic (pH<5.5). Acid soil covers 14 provinces, nearly 21% plough land of Southern China. Aluminum (Al) toxicity is a crucial factor, limiting the crop production in acidic soils. It is now confirmed that the binding of Al3+ by O2·- to form the aluminum superoxide semi reduced radical ion is the primary reason for Al3+ inducing oxidative damage directly and triggering overproduction of reactive oxygen species(ROS), including H2O2, O2-, OH and 1O2, etc. H2O2 is receiving growing attention for not only being a toxic byproduct causing oxidative damage to membrane lipids, protein and DNA at high concentrations, but also involving in signal transduction pathways leading to activate the plant defense against biotic and abiotic stresses at low concentrations. However, the associations of Al tolerance with H2O2 generation, oxidative stress, antioxidant capacity and signal transduction pathways are still unclear. The present study investigated the effects of Al on lipid peroxidation, ROS contents and antioxidant defense systems, the effects of H2O2 pretreatment on Al induced antioxidant responses, the potential source of H2O2 and H2O2 activated mitogen-activated-protein-kinase (MAPK) signal transduction pathway in root tips of Al-sensitive genotype (Yangmai-5) and Al-tolerant genotype (Jian-864) of wheat (Triticum aestivum L.) seedlings under Al stress. The aim of the present study is to elucidate the role of H2O2 signaling in adaptative responses to Al toxicity in wheat plants. The main results are as follows:(1) Hydroponic experiments were carried out to study the effects of a dose-dependent (0,10,20,30,40,50 uM) exposure to 0,10,20,30,40,50μM AlCl3 and a time-dependent (3,6,12,24 h) exposure to 30μM AlCl3 on inhibition of root elongation (IRE), malondialdehyde (MDA) content, ROS (H2O2 and O2·-) contents, Evans blue uptake and antioxidant capacity in root tips of Yangmai-5 and Jian-864. The results suggest that the Al content, IRE, MDA content, H2O2 content, Evans blue uptake increased remarkably and more IRE and oxidative damage were found in Yangmai-5 with the increasing Al concentrations. Exposure to 30μM Al increased levels of MDA, H2O2, O2- and Evans blue uptake in both genotypes, with increases being greater in Yangmai-5 than in Jian-864. In addition, Al treatment increased the activity of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), glutathione reductase (GR) and glutathione peroxidase (GPX), as well as the contents of ascorbate (AsA) and glutathione (GSH) in both the genotypes. The increased activities of the two former enzymes, which are responsible for H2O2 production, were found to be greater in the Al-sensitive genotype than in the Al-tolerant one, whereas the opposite was true for the activities of the latter five enzymes and the contents of AsA and GSH. Consequently, the antioxidant capacity in terms of DPPH (2,2-diphenyl-1-picrylhydrazyl)-radical scavenging activity and FRAP (ferric reducing/antioxidant power) was greater in Jian-864 than in Yangmai-5. These results suggest that Al enhanced the total antioxidant capacity of both non-enzymatic and enzymatic components in wheat seedlings, with the enhancement being greater in the Al-tolerant genotype than in the Al-sensitive genotype. We propose that the total antioxidant capacity is an important factor conferring Al tolerance in plants.(2) Hydroponic experiments were carried out to study the effects of a 24 h exposure to 30μM AlCl3 on root elongation, Al content, MDA content, ROS (H2O2 and O2·-) contents, Evans blue uptake and antioxidant capacity along the roots of Yangmai-5 and Jian-864. Here we found that the Al content, ROS content, MDA content, Evans blue uptake and activity of SOD, CAT, POD, APX and contents of AsA and GSH increased pronouncedly and more IRE and oxidative damage were found in Yangmai-5. The 0-5 mm behind the root tip shows greatest IRE along the roots, where the 0-5 mm segment above the root tip is undergoing higher contents of Al, H2O2, MDA and more Evans blue uptake than each root segments 5-25 mm behind the root tip. Highest activity of SOD, CAT and APX and highest contents of AsA, GSH, DHA and GSSG are also found in 0-5 mm root apical, while the opposite is true for POD. Yangmai-5 exhibited higher SOD and POD activity and lower activity of CAT and APX, lower contents of AsA, GSH, DHA and GSSG than Jian-864. We concluded from these results that 0-5 mm region behind root tip is the most sensitive to Al. The lower H2O2 level and higher antioxidant capacity in 0-5 mm root apical are primarily responsible for the higher Al tolerance of Jian-864.(3) Hydroponic experiments were carried out to study the effects of 2 h of 0.6 mM H2O2 pretreatment on the Al toxicity in the root tips of Yangmai-5 and Jian-864. The results suggest that Al increased levels of H2O2 and O2·- leading to more predominant increase in MDA content, Evans blue uptake and IRE in Yangmai-5 than in Jian-864. However, H2O2 pretreatment alleviated Al-induced deleterious effects in both the genotypes. H2O2 pretreatment increased the activities of SOD, CAT, APX, MDHAR, GR, GPX as well as the levels of AsA and GSH more significantly in Yangmai-5 than in Jian-864. Furthermore, it also increased the total antioxidant capacity evaluated as the DPPH activity and FRAP more significantly in Yangmai-5 than in Jian-864 under Al stress. Therefore, we conclude that H2O2 pretreatment improves wheat Al acclimation during subsequent Al exposure by enhancing the antioxidant capacity, which prevents ROS accumulation, and that the enhancement is greater in the Al-sensitive genotype than in the Al-tolerant genotype.(4) Hydroponic experiments were carried out to study the effects of specific enzyme inhibitors:NADPH oxidase inhibitor diphenyleneiodonium (DPI), SOD inhibitor diethyldithiocarbamic acid (DDC), peroxidase inhibitor NaN3, amine oxidase inhibitor guazatine (GZT) and H2O2 scavenger dimethylthiourea (DMTU) on Al-induced IRE, MDA content, Evans blue uptake, H2O2 content and antioxidant capacity in root tips of Al-sensitive Yangmai-5 and Al-tolerant Jian-864. Here we found that Al increased levels of MDA, H2O2 and Evans blue uptake in both genotypes, with increases being greater in Yangmai-5 than in Jian-864. However, DMTU alleviates IRE and Evans blue uptake, together with decreases the contents of H2O2 and MDA and the activity of SOD, CAT, APX and POD in both wheat genotypes without significant genotypic difference. DPI, DDC, NaN3 and GZT show similar effect as DMTU but with more remarkable decrease in oxidative damage and antioxidant capacity in Yangmai-5. These results suggest that NADPH oxidase is the major source of H2O2 in wheat roots, proxidase and amine oxidase are also contribute to H2O2 generation under Al stress(5) Hydroponic experiments were carried out to study the effects of H2O2 pretreatment, NADPH oxidase inhibitor DPI, SOD inhibitor DDC, H2O2 accelerator (NADH) and scavenger (DMTU) on Al-induced IRE, lipid peroxidation, Evans blue uptake, H2O2 content, NADPH oxidase activity and antioxidant capacity in root tips of Yangmai-5 and Jian-864. Here we found that DMTU, DPI and DDC, together with H2O2 pretreatment decreased the contents of H2O2 and MDA, Evans blue uptake and activities of SOD, CAT, APX and POD and alleviated Al-induced IRE more predominantly in Yangmai-5, while the opposite is true for NADH. Furthermore, DPI and DMTU decreased NADPH oxidase activity to the greater extent in Yangmai-5 while DDC shows little effect on NADPH oxidase activity in either wheat genotype. These results suggest that NADPH oxidase is the major source of H2O2 in wheat roots under Al stress, NADPH oxidase-dependent H2O2 production is the primary cause of oxidative stress and Al-triggered IRE in wheat plants.(6) Hydroponic experiments were carried out to study the effects of MEK1/2 inhibitor PD98059, H2O2 pretreatment, specific enzyme inhibitors (DPI, DDC, NaN3 and GZT), H2O2 scavenger (DMTU), Ca2+ chelator (ethylene glycol tetraacetic acid, EGTA), Ca channel blocker (LaCl3) and CDPK inhibitor (w7) on Al-induced IRE, lipid peroxidation, Evans blue uptake, H2O2 content, antioxidant capacity and the association with MAPK signal transduction in root tips of Al-sensitive Yangmai-5 and Al-tolerant Jian-864 wheat seedlings. Here we found that Al at 30μM increased the levels of MDA, H2O2 and Evans blue uptake and MAPK activity in both genotypes, with increases being greater in Yangmai-5 than in Jian-864. H2O2 pretreatment enhanced MAPK activity in both wheat genotypes under Al stress, while the opposite is true for DMTU, DPI, DDC, GZT and NaN3. However, PD98059 decreased H2O2 content and MAPK activity more remarkably in Yangmai-5 than those of Jian-864. w7, LaCl3 and EGTA alleviate Al-triggered IRE and decrease Evans blue uptake in Yangmai-5 but showed little effect in MDA content in both genotypes. PD98059, LaCl3 and EGTA decreased H2O2 content to the greater extent in Yangmai-5 than in Jian-864 but had shown little effects on MAPK activity. w7 has no obvious effect on either H2O2 content or MAPK activity in both the genotypes. Besides, PD98059 inhibited activity of SOD, CAT and POD in Yangmai-5 and activity of SOD, CAT and APX in Jian-864. EGTA decreased SOD activity in Yangmai-5 under Al stress. w7 and LaCl3 showed little effect on antioxidant capacity in both genotypes. These results suggest that MAPK activation is responsible for the positive regulation of enhancement of endogenous H2O2 level and antioxidant capacity in wheat under Al stress. |
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