Aluminum Toxicity In Wheat (Triticum Aestivum L.) Seedlings And The Regulation Of Calcium In Aluminum Toxicity

Al toxicity is one of the factors limiting crops production in acid soil. In present study, we investigated Al toxicity in three wheat cultivars differing in Al resistant. In background of 0.5 mmol·L^-1 CaCl₂, 5-80μmol·L^-1 Al significantly inhibited root growth of the three cultivars. Root growth of Atlas 66, Scout 66 and Yangmai 9 were inhibited by about 50% with 24 h treatment of 40, 10 and 5μmol·L^-1 Al, respectively. It showed that the Al-resistant of the three cultivars follows: Atlas 66 > Scout 66 > Yangmai 9. Using Eriochome cyanine R staining, we found that Al accumulation on surface of root tips was positive related to the inhibition of root growth in treatments of 0 - 80μmol·L^-1 Al for 24 h. Exchangeable Al in apoplast and symplasm of root tips of wheat was effectively desorbed by 5 mmol·L^-1 CaCl₂ at 4℃. The results showed that exchangeable Al was mostly accumulated in apoplast after 10 and 40μmol·L^-1 Al treatment for 24 h. Exchangeable Al in apoplast and symplasm of the two Al-sensitive cultivars was enhanced by treatment of 10μmol·L^-1 Al for 24 h, but only apoplast Al in Atlas 66was enhanced by the same treatment. Additionally, exchangeable Al in both apoplast and symplasm in root tips of the two Al-sensitive cultivars were significantly higher than that of Atlas 66. So we confirmed that exclusion of Al from root tips is an important mechanism of Al resistant in wheat.After treatment of 10μmol·L^-1 Al for 7 days, morphologic changes were founded in roots of Yangmai 9. Root elongation was severely inhibited and root tips were stubbed and brown. Development of lateral roots was arrested and protuberances were founded on the surface of primary roots. Using technology of tissue slice, heavily lignified cells was founded around the cells of merism and anlage of lateral root. The lignified cells maybe play some roles in protection of cells which were differentiating, but simultaneously they also inhibited the root elongation and development of lateral roots. 5μmol·L^-1 Al induced cell death of root tips of Scout 66 and Yangmai 9, but not of Atlas 66. Al increase in cell death was found in treatment of 20μmol·L^-1 Al in root tips of Atlas 66. Cell death in root tips of all the three cultivars were enhanced by the increasing of treatment Al concentration. Loss of integrity of plasma membrane and accumulation of ROS were also founded in root tips exposed to 5-80μmol·L^-1 for 24h. They maybe play some roles in the cell death of root tips. Genomic DNA of root tips was purified during 0-12h of 5, 10 and 40μmol·L^-1 Al treatment for Yangmai 9, Scout 66 and Atlas 66, respectively. DNA ladder was not found in all samples. So cell death induced by Al was not PCD in root tips of all the three wheat cultivars.Oxidative stress is a general mechanism of plant response to many bio- and abio-stress. In present experiments, antioxide capacities in roots of Atlas 66 and Scout 66 were compared in short and long Al treatments. Changes were not found for activities of APX, CAT, GR and SOD in roots of Scout 66 with Al toxicity for 6h, but significantly increasing was found of GR of Atlas 66. When thetreatment time prolonged to 12h, activities of APX and SOD of Scout 66 were decreased. Synchronously, CAT of Scout 66 and APX, SOD and GR of Atlas 66 were increased. We suggested that the rapid increasing anti-oxide ability mediated by antioxide enzymes might play some roles in the Al-resistant of Atlas 66, at least it is the fact in the short time treatment. Treatment of 5-80μmol·L^-1 Al for 24h significantly decreased activities of APX and SOD, and did not effect activities of CAT and GR in roots of Scout 66. Activities of APX, CAT, GR and SOD were decreased by the treatment of low concentration Al, but were increased by high concentration Al. It showed that antioxide enzymes still played roles in Al-resistant of Atlas 66 in the 24h treatment. Apoplastic ASA in roots of Scout 66 was significantly decreased by the treatment of 80μmol·L^-1 Al for 48h, and that of Atlas 66 was increased. 5-80μmol·L^-1 Al enhanced the content of total ascorbate in apoplast of roots of Atlas 66, but it decreased that of Scout 66. ASA and total ascobate in apoplast and symplasm of roots of both Scout 66 and Atlas 66 were decreased by treatment of 5-80μmol·L^-1 Al, but it was more heavily in Scout 66.80μmol.L^-1 Al treatment significantly decreased activities of APX and AO in apoplast and symplasm of roots of Scout 66, and it did not affected that of Atlas 66. Increasing of content of roots apoplastic ASA and maintaining of symplastic ASA and availability of ASA might contribute to the Al-resistant of Atlas 66. Activities of soluble, cell wall ionic and covalent POD in roots of Scout 66 were increased by treatment of 5-80μM Al for 48h. Activity of cell wall covalent POD in roots of Atlas 66 was decreased by the similar treatment, and that of ionic POD was not affected, and that of soluble POD was increased. The increasing of soluble POD of Scout 66 was more heavily than that of Atlas 66. The lower activities of POD in roots of Atlas 66 enhanced the cell wall extensibility under Al stress and that also played some roles in the Al-resistant of Atlas 66.Root growth of the three wheat cultivars exposed to 10 and 40μmol·L^-1 Al was significantly enhanced by application of exogenous CaCl₂. Cell viability and integrity of plasma membrane of root tips exposed to Al was increased, as well as accumulation of Al and ROS was decreased by applicaiton of exogenous CaCl₂. Pretreatment of 2.5-8.5 mmol·L^-1 CaCl₂ significantly increased root growth of Atlas 66 and Scout 66 exposing to 10 or 40μmol·L^-1 Al, but it did not increased that of Yangmai 9. It might be resulted from the different adsorption capacities of Ca and Al in roots of different plants. Exposing only one half of the root system to 2.5 mmol.L^-1 Ca did not reduce the inhibiting effect of Al on the root growth of the exposed half (A1) of the [Al/2.5 mmol·L^-1 Ca plants] compared with that of the [Al/0.5 mmol·L^-1 Ca plants] and the [Al/Al plants], indicated that the amelioration of Al toxicity was due to a direct effect of Ca in the apoplasm. Addition of mmol·L^-1 level MgCl₂ also improved root growth of the three wheat cultivars exposing to 10 or 40μmol·L^-1 Al. But the improvement disappeared when all the Ca²⁺ in treatment solutions was displaced by similar concentration Mg²⁺. It might be caused of the deficiency of Ca required for the root growth. Pretreatment of 8 mmol·L^-1 MgCl₂ significantly increased root growth of Atlas 66 and Yangmai 9 exposing to 40μmol·L^-1 Al. Mg content in roots and shoots of both cultivars treated with 40μmol·L^-1 Al were enhanced by the pretreatment. Effect of MgCl₂ pretreatment on Al toxicity of Yangmai 9 was different to that of CaCl₂ pretreatment. It might be due to the different selective absorption of Ca and Mg in roots of Yangmai 9. Our results confirmed that Ca alleviated Al toxicity of wheat by mechanism of decrease in Al³⁺ on surface of root cells.Increasing of [Ca²⁺]_cyt is an early syndrome of Al toxicity of plant cell. In present study, we investigated the effect of Al stress on variation of [Ca²⁺]_cyt and ROS in cells of intact root tips of an Al-sensitive wheat cultivar, Yangmai 9. Treatment of 40μmol·L^-1 Al for 70 min induced the increasing of [Ca²⁺]_cyt in cells of intact root tips. The increasing was inhibited by the addition of 2 mmol·L^-1 CaCl₂ to the Al treatment solution. It showed that the alleviation of Ca on Al toxicity in Yangmai 9 happened before the induction of variation of [Ca²⁺]_cyt. Pretreatment of 1 mmol·L^-1 Ca²⁺ chelator EGTA and addition of 1 mmol·L^-1 EGTA and inhibitors of Ca²⁺ channels in plasma membrane, La³⁺, verapamil and nifedipine also inhibited the increasing of [Ca²⁺]_cyt in cells treated with Al. Furthermore, we also found that addition of 8 mmol·L^-1 CaCl₂ induced a burst of [Ca²⁺]_cyt in cells in which Al had induce a increasing of [Ca²⁺]_cyt. We suggested that apoplastic Ca²⁺ was one of the most important source for increasing of [Ca²⁺]_cyt induced by Al toxicity in root cells of Yangmai 9. Accumulation of ROS was induced in root cells of Yangmai 9 treated with 40μmol·L^-1 Al for 2 min. Pretreatment of 4mg·mL^-1 CAT inhibited the accumulation of ROS induced by Al stress, as well as the increasing of [Ca²⁺]_cyt. CAT was used to clear up the H₂O₂. It exhibited that H₂O₂ mostly contributed to the accumulation of ROS induced by Al stress in root cells. Accumulation of H₂O₂ upstream regulated the increasing of [Ca²⁺]_cyt in cells of intact root tips stressed with Al.