【作者】 王洪政；

【导师】 沈振国；

【作者基本信息】 南京农业大学 ， 植物学， 2006， 博士

【摘要】 Al毒害是酸性土壤上作物生长的主要限制因素。本实验以Al耐性不同的三个小麦品种Atlas 66、Scout 66以及一个江苏省推广品种扬麦9号为实验材料，研究了不同浓度Al处理对小麦的毒害作用。结果显示：0.5mmol·L^-1CaCl₂溶液中，40、10和5μmol·L^-1Al处理24小时分别抑制了Atlas 66、Scout 66和扬麦9号根系伸长的50％左右，表明三个小麦品种Al耐性Atlas 66＞Scout 66＞扬麦9号。铬化青R染色可以原位显示0-80μmol·L^-1Al处理后根尖表面Al的吸附量与根尖伸长受抑制程度具有很好的一致性。5mmol·L^-1 CaCl₂溶液可以有效洗脱10和40μmol·L^-1Al处理24小时后小麦根尖质外体和共质体中可交换态Al。洗脱实验显示，10和40μmol·L^-1Al处理24小时后三个品种小麦根尖交换态Al主要累积在质外体。与对照相比，10μmol·L^-1Al处理24小时显著增加了三个品种根尖质外体以及两个敏感品种根尖共质体交换态Al含量。10和40Alμmol·L^-1Al处理下，Atlas 66根尖质外体和共质体交换态Al含量都显著低于两个敏感品种，因此阻遏Al吸收是Atlas 66Al耐性较高的一个重要机制。与对照相比，10μmol·L^-1Al处理7天后扬麦9号根系形态发生了很大变化，根系伸长受到严重抑制，根尖变粗并且呈棕色，侧根发育受到抑制，在初生根侧形成瘤状突起。通过石蜡组织切片技术我们发现Al处理后根尖分生组织细胞和侧根发生原基外侧都包围了1到几层高度木质化的细胞，这些木质化细胞可能在保护内层具有分化能力的细胞中具有作用，但同时它们也阻止了根系的伸长以及侧根的发育，在Al毒害引起的珊瑚形根系形成中具有重要作用。5μmol·L^-1Al处理24小时即可引起了扬麦9号和Scout 66根尖伸长区细胞活力的降低，而20μmol·L^-1Al处理才可以引起Atlas 66根尖细胞细胞活力的降低。三个品种小麦根尖细胞活力随着处理Al浓度的增加而降低。此外Al处理还引起了三个品种根尖细胞质膜完整性的降低以及根尖细胞ROS的累积，它们可能在Al诱导的小麦根尖死亡中具有重要作用。Atlas 66、Scout 66和扬麦9号分别在40、10和5μmol·L^-1Al胁迫下处理0-12小时没有发现根尖细胞基因组DNA梯形带的出现，因此Al诱导的小麦根尖死亡可能不是功能性的程序化细胞死亡，而是毒害引起的细胞坏死。氧化胁迫是许多生物和非生物因素引起植物伤害的普遍途径之一。我们以Scout66和Atlas 66为材料，比较了两者短期和长期Al处理下抗氧化能力的变化。我们的实验结果显示，Al处理6小时后Al敏感品种Scout 66根系四种抗氧化酶的活力没有显著差异，而相近Al毒害程度下耐性品种Atlas 66根系GR活力显著升高。处理时间延长到12小时，Scout 66根系APX和SOD活力降低，CAT活力显著升高，而Atlas66根系则APX、SOD和GR活力均明显升高。我们推断短期Al毒害下，Atlas 66根系抗氧化酶系统介导的抗氧化能力高于Scout 66，这可能是其Al耐性较高的一个重要原因。5-80μmol·L^-1Al处理24小时显著降低了Scout 66根系APX和sOD的活力，CAT和GR的活力未受影响。与Scout 66不同，Atlas 66根系抗氧化酶活力在较高浓度Al处理下显著高于较低浓度Al处理。表明抗氧化酶系统在较长时间内仍然在Atlas66Al耐性中具有重要作用。5-80μmol·L^-1Al处理48小时，特别是较高浓度Al处理下，Scout 66根系质外体ASA和总抗坏血酸含量显著低于对照，而Atlas 66则显著高于对照。Al处理显著降低了Atlas 66和Scout 66根系共质体ASA和总抗坏血酸的含量，但是Atlas 66的降低幅度显著小于Scout 66。同时我们还发现高浓度Al处理下Atlas 66根系质外体和共质体APX和AO活力没有发生显著变化，而Scout 66则有大幅度降低。因此Al胁迫下Atlas 66根系质外体抗坏血酸含量的升高和共质体抗坏血酸含量的维持以及严重Al毒害下抗坏血酸利用率的保持可能是其Al耐性的一个重要机制。5-80μmol·L^-1Al处理48小时Scout 66根系可溶性、细胞壁离子和共价结合POD活力都显著升高。同样处理下Atlas 66根系细胞壁离子结合POD活力没有发生显著变化，共价结合POD活力则显著低于对照，可溶性POD活力虽然高于对照，但是升高幅度显著小于Scout 66。结果表明Al处理下Atlas 66根系低POD活力降低了Al引起的根系细胞壁硬化，从而保证Atlas 66根系保持较高的伸长生长速率。添加高浓度外源CaCl₂显著降低了10和40μmol·L^-1Al处理下三个品种小麦根系伸长生长的受抑制程度。高浓度外源CaCl₂提高了Al处理下三个品种小麦根尖的细胞活力，降低了Al毒害引起的根尖细胞质膜受破坏程度以及根尖细胞ROS的累积。2.5-8.5 mmol·L^-1 CaCl₂预处理显著改善了Atlas 66和Scout 66随后Al处理中的根系伸长生长，而预处理对扬麦9号没有显著影响。这可能与不同植物根系对Ca、Al的相对吸附能力不同有关。在分根实验中，三个品种小麦根系一侧的高浓度CaCl₂供应并没有显著改善另一侧Al毒害下根系的伸长生长，因此小麦幼苗不能通过提高Ca吸收而缓解Al毒害，表明Ca没有参与小麦幼苗的体内解毒机制。添加2和4 mmol·L^-1 MgCl₂也可以显著改善三个品种小麦幼苗10和40μmol·L^-1Al处理下的根系伸长生长，并且在0.5 mmol·L^-1 CaCl₂存在时具有与添加相同浓度CaCl₂同近的缓解效果，但是在将0.5 mmol·L^-1CaCl₂等摩尔替换为MgCl₂时，MgCl₂的缓解效果消失，这可能与根系伸长对培养液Ca²⁺的绝对需求有关。8 mmol·L^-1 MgCl₂预处理显著提高了Atlas 66和扬麦9号40μmol·L^-1Al处理中根系的伸长生长，同时经过预处理的小麦幼苗在Al处理后保持了较高的根系和地上部Mg含量，说明两个品种小麦幼苗在Mg处理过程中吸附吸收了较多的Mg，从而缓解了随后的Al毒害。扬麦9号在Ca预处理和Mg预处理后对Al毒害的反应不同，说明扬麦9号对Ca和Mg的选择性吸附吸收能力不同。通过上面的实验进一步确定了Ca对小麦Al毒害的缓解作用是通过降低根系表面Al³⁺活度，从而降低根尖对Al的吸附和吸收实现的。Ca²⁺信号紊乱是植物细胞Al毒害的早期现象。本实验以扬麦9号为材料，研究了Al诱导的完整小麦根尖细胞[Ca²⁺]_cyt变化。结果发现40μmol·L^-1Al处理在70分钟内诱导了扬麦9号根尖表皮细胞[Ca²⁺]_cyt的持续升高，而处理液中同时含有2.5 mmol·L^-1CaCl₂时，[Ca²⁺]_cyt在70分钟内则没有发生显著变化。1 mmol·L^-1 Ca²⁺螯合剂EGTA预处理30分钟以及处理液中添加1 mmol·L^-1 EGTA或者质膜Ca²⁺通道抑制剂LAGl₃、verapamil和nifedipine也抑制了Al诱导的根尖细胞[Ca²⁺]_cyt升高。同时我们还发现在Al诱导根尖细胞[Ca²⁺]_cyt升高出现后处理液中添加8 mmol·L^-1 CaCl₂会引发[Ca²⁺]_cyt的急剧升高。因此我们推断质外体Ca²⁺是Al毒害诱导的扬麦9号根尖细胞[Ca²⁺]_cyt升高的主要Ca²⁺来源之一，而外源CaCl₂对小麦Al毒害的缓解作用发生在Al诱导的细胞[Ca²⁺]_cyt升高之前。40μmol·L^-1Al处理30分钟内诱导了扬麦9号根尖细胞ROS含量的快速增加。4mg mL^-1 CAT预处理30分钟抑制了Al诱导的ROS升高以及[Ca²⁺]_cyt升高。CAT在植物细胞中的主要作用是清除H₂O₂，因此可以推断Al处理引起的细胞ROS累积主要是H₂O₂的累积，而此H₂O₂累积可能上游调控了[Ca²⁺]_cyt的升高。更多还原

【Abstract】 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.更多还原