The Mechanisms Of Aluminum Toxicity And Tolerance In Rice (Oryza Sativa L.)

Al toxicity is one of the factors limiting crops production in acid soil. Hydroponic cultures were conducted to investigate the aluminum tolerance and toxicity on the seed germination and growth and on the seedling growth of two varieties, Al-tolerant Azucena and Al-sensitive ER. 64. In addition, aluminum tolerance of rice is discussed.In background of 0.5 mM CaCl₂, 1 mM Al did not significantly inhibited root growth of the two varieties. Root growth of IR 64 were more significantly inhibited by 2 mM Al than that of Azucena. At the stage of germination, considerable increase of hydrogen peroxide production was observed in Al-treated germinated seeds in comparison to control non-treated seeds. And production of H₂O₂ in IR 64 was more than that in Azucena. In the presence of 2 mM Al, addition of 10 mM imidazole (inhibitor of NADPH Oxidase) or 1 mM azide (inhibitor of peroxidase) significantly decreased H₂O₂ production. Compared that in the treatment with Al alone, H₂O₂ production showed decreases of 16% and 43% with imidazole and azide for Azucena, and 21% and 68% for IR 64, respectively. Under Al treatment, the Al-tolerant variety Azucena had significantly higher activities of Catalase, ascorbate peroxidase, dehydroascorbate reducase, glutathione peroxidase and glutathione reductase, and concentrations of reduced glutathione than the Al-sensitive one IR 64. Treatment with buthionine sulfoximine, a specific inhibitor of GSH synthesis, significantly increased H₂O₂ production in the germinating seeds of both varieties in the presence and absence of Al. In contrast, the treatment with GSH significantly decreased the production of H₂O₂ induced by Al stress. Results suggest that cell wall peroxidase and NADPH Oxidase might be implicated in the production of H₂O₂ induced by Al. GSH and antioxidative enzymes may play an important role in scavenging H₂O₂ of germinating rice seeds caused by Al stress.The root apex is the primary target and plays a central role in Al toxicity and tolerance. Al treatment increased Al content of root apices in rice, and Al content of root apices increased when Al concentration of environment increased. The Al content in root apices of Azucena was less than that of IR 64, which suggested that exclusion mechanisms were acting in Azucena. Using the pH indicator (bromocresol purple), it was shown that surface pH of the control (0 Al) root apices was strongly alkalized after 12 h and 24 h. By contrast, the surface pH changed only slightly in the Al-treated root apices. The surface pH of IR 64 was lower than that of Azucena. Moreover, the effects of Al treatments on total phosphate and inorganic phosphate (Pi) content were compared. There was significant difference of the total phosphate content between Azucena and IR 64 in control, and the total phosphate content of root apices in IR 64 was obviously higher than that in Azucena. Al treatments decreased significantly the total phosphate content of root apices in IR 64, but in Azucena. In control, Pi content of root apices in Azucena was evidently higher than that in IR 64. However, Pi content was reduced in Azucena, and was 85% and 79% of control in 20 and 40μM Al stresses. Pi content was increased and was 118% of control by 20μM. Al treatment, then was 80% of control by 40μM Al treatment. Nevertheless, there were not significantly difference between control and Al treatment. Furthermore, Al inhibited the development of lateral root, including the number and length of lateral root. The effect of lateral root in IR 64 was more obvious than that in Azucena. The number and length of lateral root of IR 64 in Al treatment were fewer and lower than those in control. This shows the higher Al tolerance in Azucena.In roots of rice seedlings, we also investigated the role of antioxidative systems under Al stress. The results indicated that the contents of MDA and H₂O₂ in IR 64 roots were higher than those in Azucena roots under 20, 40μM Al stress. Al-induced H₂O₂ accumulation was observed only in apoplast, and cell wall peroxidase and plasma-membrane NADPH Oxidase might be implicated in the production of H₂O₂ induced by Al, using CeCl₃ staining. 1 h after 40μM Al treatment, ABA level of Azucena root apex increased obviously, and was higher than that of IR 64, then decreased gradually to the level same as the control after 3 h Al treatment. Using CeCl₃ staining, H₂O₂ accumulation at 3 h Al stress in Azucena was obvious, and then was decreased. H₂O₂ content in Azucena at 48 h Al stress was higher than that at control. However, H₂O₂ content in IR 64 was gradually increased, and was more than that in Azucena after 48 h Al treatment. Fluridone, inhibitor of ABA biosynthesis, reduced H₂O₂ accumulation in Azucena root apex caused by Al. The treatment with ABA might decrease H₂O₂ accumulation in root apex as a result of fluridone, and increased H₂O₂ accumulation of root apex in rice. After 3 h Al stress, the activities of APX, GPX and GR in Azucena roots were all increased, were the highest at 6 h Al treatment. But these phenomena were observed in IR 64. Fluridone and tungstate might inhibit antioxidative enzymes' activities caused by Al in Azucena. In condition of Al treatments, roots of Azucena provided with the higher ratios of GSH/GSSG. Using BSO, a specific inhibitor of GSH biosynthesis, we found that GSH had an important role in Al tolerant mechanism. The present data indicated that H₂O₂ was accumulated in apoplast of root apex under Al stress. And H₂O₂ production induced by Al was relative to NADPH Oxidase and cell wall peroxidase. The antioxidative system was rapidly started up by ABA, and then H₂O₂ content was decreased, so oxidative stress was reduced in Al-tolerant variety. These results confirmed with seeds germination under Al stress.The activities of PODs extracted from cell wall are inversely related to root growth. It has been suggested that PODs is likely to result in cell wall stiffening processes related to formation of cross-links among cell wall polymers. The formation of cross-links between cell wall components is mediated by cell wall-associated PODs enzymes. Al treatments increased the activities of POD and FPOD in ionically, covalently bound cell wall of IR 64 roots, but the activities of these POD and FPOD of Azucena roots remained appropriate level. PODs are an essential enzyme during lignin synthesis. Our results indicated that Al treatments induced the increase of lignin content in roots of Al-sensitive variety. Lignifications of cell wall were known to cause the ceasing of cell growth, because lignifications decreased the plasticity of cell wall, which dealt with the inhibition of cell growth. AsA does not only eliminate H₂O₂, but also decrease lignin precursors, which decreases lignin content. In Azucena, there are higher apoplastic AsA contents and APX activity in the presence of Al. Under Al stress, Al-tolerant variety might rapidly start up antioxidative system, avoid excessive H₂O₂ accumulation, and remain definite POD and FPOD activities. Al less caused cross-links among cell wall polymers, and reduce the lignifications of cell wall, so root growth was less inhibited in roots of Al-tolerant variety. However, Al decreased antioxidative system, accumulated excessive H₂O₂, which provided to POD and FPOD, increased POD and FPOD activities, rapidly caused cross-links among cell wall polymers, and increased the lignin synthesis of cell wall, so root growth was more significantly inhibited by Al in root of Al-sensitive variety. Treatment with 0.5 mM H₂O₂ significantly increases H₂O₂ content and lignin content in root of Al-sensitive variety (IR 64). These results suggested that growth inhibition of root caused by Al was associated with H₂O₂ dependent peroxidase-catalyzed formation of cross-linking among cell wall polymers and lignin synthesis. The effects of aluminum on the amount of cell-wall polysaccharides in the roots of Al-resistant (Azucena) and Al-sensitive (IR 64) varieties of rice, as well as the relation between polysaccharides with root elongation was examined. Exposure to 40μM Al for 48 h inhibition root elongation. The amount of pectin in the 10 mm region of root apex was increased by the exposure to Al, especially in IR 64 roots apex. Moreover the amount of hemicellulose was increased by Al treatment in IR 64 roots, but not in Azucena. Using gas chromatography, the increase in the pectin fraction was attributed to increase in the contents of xylose, glucose and galactose. The increase in the content of hemicellulose was due to the increases in xylose, mannose, glucose and galactose. The Al contents in pectin and hemicellulose fractions increased under 40μM Al, and the amounts of Al in the fractions of pectin and hemicellulose of root apex were more accumulated in the Al-sensitive variety than in the Al-resistant one. The results clearly indicated that aluminum reduced root growth by combining with pectin and hemicellulose and altering metabolism of cell-wall polysaccharides.