铝诱导柱花草根系分泌柠檬酸及其调控机制的研究
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摘要
铝毒是酸性土壤上限制植物生长的最重要的限制因子之一。有机酸分泌被认为是一些耐铝植物抵御铝毒害的重要机制，但人们对有关铝胁迫下，植物根系分泌有机酸的调控机制的认识还比较有限。本研究以柱花草为材料，通过水培试验，比较柱花草热研2号和热研7号的耐铝性差异及与其根系分泌有机酸，以明确柱花草品种间耐铝性差异的机制。研究还以耐铝性较强的热研2号品种为材料，在研究了柱花草根系柠檬酸分泌特点的基础上，进一步从阴离子通道、蛋白质合成及修饰、有机酸代谢与运输以及细胞信号转导等方面探讨铝诱导柱花草根系分泌柠檬酸的调控机制。研究结果表明：
柱花草品种间存在耐铝性差异，10、20、30μmol·L~(-1)Al处理24h后，热研2号相对根伸长率均大于热研7号的伸长率，而根尖被铬天青R染色较浅，说明热研2号耐铝性较强。在铝胁迫下热研2号根系分泌柠檬酸的速率显著大于铝较敏感的热研7号品种，且柠檬酸分泌受阴离子通道抑制剂、蛋白合成抑制剂、呼吸抑制剂抑制时，根尖的铝含量显著增加，说明了铝诱导根系分泌柠檬酸是柱花草品种间耐铝性差异的重要机制。
铝胁迫下距根尖0.5-1.0cm的根段分泌的柠檬酸数量显著高于0-0.5、1.0-1.5、1.5-2.0cm根段，说明0.5-1.0cm的根段是柠檬酸的主要分泌位点。
缺磷也能诱导柱花草根系分泌柠檬酸，在处理后的第6天开始缺磷处理根系分泌的柠檬酸显著高于对照处理的柠檬酸，并在缺磷处理后的第8d至第10d时达最大并在此后减少，暗示根系分泌柠檬酸并非柱花草对铝胁迫的专一性反应。
铝诱导柱花草根系柠檬酸的分泌有一个明显的滞缓期，在开始铝处理的0.5、2、4h根系分泌的柠檬酸很少，而处理8h后分泌量明显增加，说明柱花草以第二类模式分泌有机酸。
10μmol L~(-1)阴离子通道抑制剂(PG、NIF、DIDS、A-9-C)处理均抑制铝诱导的柠檬酸分泌。其次，在铝溶液中加入的蛋白合成抑制剂(CHM)、铝处理前CHM的预处理及铝处理后加入的CHM均抑制柠檬酸分泌。此外，呼吸抑制剂(NaN_3)、柠檬酸载体抑制剂(PITC)也显著抑制铝诱导的柠檬酸分泌。另一方面，铝处理溶液中的异三聚G蛋白激活剂(CTX)使根尖细胞内的游离钙信号加强并促进柠檬酸的分泌，而加入G蛋白抑制剂(PTX)削弱钙信号强度并抑制柠檬酸的分泌。这些结果说明：(1)阴离子通道介导铝诱导柱花草根系柠檬酸分泌，且柠檬酸的分泌需新合成蛋白的参与；(2)在铝胁迫下根尖的呼吸作用及柠檬酸在线粒体膜上的跨膜运输影响着根系柠檬酸的分泌；(3)异三聚体G蛋白介导铝胁迫的信号转导，并参与铝诱导柱花草根系分泌柠檬酸的调控过程。
Aluminum (Al) is one of the most important factors limiting plant growth in acid soils. Although it has been considered that Al-induced secretion of organic acids from roots is a main mechanism for some Al-tolerance plants, so far the knowledge about the mechanisms responsible for Al-induced secretion of organic acids is still very limited. In present studies, two cultivars of stylo (Stylosanthes Spp), Reyan No.2 and Reyan No. 7, were objected to compare the difference of Al-tolerance between cultivars and exudation of organic acid from the roots to elucidate the mechanism responsible for Al-tolerant diversity in stylos. Furthermore, on the base of study on the characteristics of Al-induced secretion of citrate in stylo, the factors regulated citrate secretion under Al stress, in terms of anion channels, protein synthesis, metabolizion and transportion of organic acids and cell signal transduction, were investigated in Reyan No.2. The results were shown as follows:
There was great genetic variation in Al tolerance between cultivars of stylo. More inhibition of root elongation and stronger staining on the surfaces of roots with eriochrome cyanine R were found in Reyan No.2 contrasting to that of Reyan No.7 after exposure of roots to Al~(3+) (10, 20, 30μmol·L~(-1)) solution, which indicated that Reyan No.2 was an Al-tolerance cultivars. Aluminum-induced secretion of citrate from roots was more significantly in Reyan No.2. In other way, Al content increased significantly when citrate exudation was inhibited by the inhibitors of anion channels, protein synthesis and respiration. These results demonstrated that Al-reduced secretion of citrate from roots was an important mechanism for the difference in Al-tolerance between cultivars of stylo.
The amount of citrate secreted from the segment of 0.5-1.0cm from root apex was greater than other segment(0-0.5, 1.0-1.5, 1.5-2.0cm). It was illuminated that citrate was secreted mainly from 0.5-1.0cm root segment in stylo under Al stress.
Phosphor (P) absence could also induce secretion of citrate from roots in stylo, and the amount came to the most from the 8~(th) and 10~(th) day of P deficiency treatment. The result showed that secretion of citrate from roots in stylo did not the special response to Al stress.
Another, there was obvious a lag between Al-induced secretion of citrate and the initial to Al treatment in stylo. The secretion of citrate was limited after the treatment with Al for 0.5, 2, 4 hours, while the secretion enhanced significantly from the 8~(th) hour of Al treatment. It indicated that citrate in stylo was secreted from roots under Al stress in pattern II.
The anion channels inhibitors including PG, NIF, DIDS and A-9-C in solution (10μmol·L~(-1)) could all inhibit Al-induced secretion of citrate. Secondly, the inhibition of citrate secretion was found after addition of protein synthesis inhibitor (CHM, 25 or 50μmol·L~(-1)) in Al solution as well as pre-treatment of CHM before Al treatment and the treatment with CHM after Al treatment. Furthermore, respiration inhibitor (NaN_3, 10μmol·L~(-1)) or the inhibitor (PITC, 10μmol·L~(-1)) of citrate carrier on mitochondorial membrane all inhibited the exudation. On the other hand, the treatment with heterotrimeric G protein activator (CTX) enhanced [Ca~(2+)]cyt signal in root tip surface cells and the exudation of citrate during the course of Al-induced secretion of citrate, while the inhibitor (PTX) of heterotrimeric G protein depressed effectively Ca~(2+) signal transduction and the secretion of citrate. All the results demonstrated that: (1) Anion channels involved in Al-induced secretion of citrate in stylo, de novo synthesis and activation of an anion channel proteins might be required for the secretion; (2) Both the respiration of root tips and citrate transportation crossing mitochondorial membrane impacted the secretion of citrate under Al stress. (3) Heterotrimeric G protein would intervene in the signal transduction of Al stress and participated in the regulation of Al-induced secretion of citrate in stylo.
柱花草品种间存在耐铝性差异，10、20、30μmol·L~(-1)Al处理24h后，热研2号相对根伸长率均大于热研7号的伸长率，而根尖被铬天青R染色较浅，说明热研2号耐铝性较强。在铝胁迫下热研2号根系分泌柠檬酸的速率显著大于铝较敏感的热研7号品种，且柠檬酸分泌受阴离子通道抑制剂、蛋白合成抑制剂、呼吸抑制剂抑制时，根尖的铝含量显著增加，说明了铝诱导根系分泌柠檬酸是柱花草品种间耐铝性差异的重要机制。
铝胁迫下距根尖0.5-1.0cm的根段分泌的柠檬酸数量显著高于0-0.5、1.0-1.5、1.5-2.0cm根段，说明0.5-1.0cm的根段是柠檬酸的主要分泌位点。
缺磷也能诱导柱花草根系分泌柠檬酸，在处理后的第6天开始缺磷处理根系分泌的柠檬酸显著高于对照处理的柠檬酸，并在缺磷处理后的第8d至第10d时达最大并在此后减少，暗示根系分泌柠檬酸并非柱花草对铝胁迫的专一性反应。
铝诱导柱花草根系柠檬酸的分泌有一个明显的滞缓期，在开始铝处理的0.5、2、4h根系分泌的柠檬酸很少，而处理8h后分泌量明显增加，说明柱花草以第二类模式分泌有机酸。
10μmol L~(-1)阴离子通道抑制剂(PG、NIF、DIDS、A-9-C)处理均抑制铝诱导的柠檬酸分泌。其次，在铝溶液中加入的蛋白合成抑制剂(CHM)、铝处理前CHM的预处理及铝处理后加入的CHM均抑制柠檬酸分泌。此外，呼吸抑制剂(NaN_3)、柠檬酸载体抑制剂(PITC)也显著抑制铝诱导的柠檬酸分泌。另一方面，铝处理溶液中的异三聚G蛋白激活剂(CTX)使根尖细胞内的游离钙信号加强并促进柠檬酸的分泌，而加入G蛋白抑制剂(PTX)削弱钙信号强度并抑制柠檬酸的分泌。这些结果说明：(1)阴离子通道介导铝诱导柱花草根系柠檬酸分泌，且柠檬酸的分泌需新合成蛋白的参与；(2)在铝胁迫下根尖的呼吸作用及柠檬酸在线粒体膜上的跨膜运输影响着根系柠檬酸的分泌；(3)异三聚体G蛋白介导铝胁迫的信号转导，并参与铝诱导柱花草根系分泌柠檬酸的调控过程。
Aluminum (Al) is one of the most important factors limiting plant growth in acid soils. Although it has been considered that Al-induced secretion of organic acids from roots is a main mechanism for some Al-tolerance plants, so far the knowledge about the mechanisms responsible for Al-induced secretion of organic acids is still very limited. In present studies, two cultivars of stylo (Stylosanthes Spp), Reyan No.2 and Reyan No. 7, were objected to compare the difference of Al-tolerance between cultivars and exudation of organic acid from the roots to elucidate the mechanism responsible for Al-tolerant diversity in stylos. Furthermore, on the base of study on the characteristics of Al-induced secretion of citrate in stylo, the factors regulated citrate secretion under Al stress, in terms of anion channels, protein synthesis, metabolizion and transportion of organic acids and cell signal transduction, were investigated in Reyan No.2. The results were shown as follows:
There was great genetic variation in Al tolerance between cultivars of stylo. More inhibition of root elongation and stronger staining on the surfaces of roots with eriochrome cyanine R were found in Reyan No.2 contrasting to that of Reyan No.7 after exposure of roots to Al~(3+) (10, 20, 30μmol·L~(-1)) solution, which indicated that Reyan No.2 was an Al-tolerance cultivars. Aluminum-induced secretion of citrate from roots was more significantly in Reyan No.2. In other way, Al content increased significantly when citrate exudation was inhibited by the inhibitors of anion channels, protein synthesis and respiration. These results demonstrated that Al-reduced secretion of citrate from roots was an important mechanism for the difference in Al-tolerance between cultivars of stylo.
The amount of citrate secreted from the segment of 0.5-1.0cm from root apex was greater than other segment(0-0.5, 1.0-1.5, 1.5-2.0cm). It was illuminated that citrate was secreted mainly from 0.5-1.0cm root segment in stylo under Al stress.
Phosphor (P) absence could also induce secretion of citrate from roots in stylo, and the amount came to the most from the 8~(th) and 10~(th) day of P deficiency treatment. The result showed that secretion of citrate from roots in stylo did not the special response to Al stress.
Another, there was obvious a lag between Al-induced secretion of citrate and the initial to Al treatment in stylo. The secretion of citrate was limited after the treatment with Al for 0.5, 2, 4 hours, while the secretion enhanced significantly from the 8~(th) hour of Al treatment. It indicated that citrate in stylo was secreted from roots under Al stress in pattern II.
The anion channels inhibitors including PG, NIF, DIDS and A-9-C in solution (10μmol·L~(-1)) could all inhibit Al-induced secretion of citrate. Secondly, the inhibition of citrate secretion was found after addition of protein synthesis inhibitor (CHM, 25 or 50μmol·L~(-1)) in Al solution as well as pre-treatment of CHM before Al treatment and the treatment with CHM after Al treatment. Furthermore, respiration inhibitor (NaN_3, 10μmol·L~(-1)) or the inhibitor (PITC, 10μmol·L~(-1)) of citrate carrier on mitochondorial membrane all inhibited the exudation. On the other hand, the treatment with heterotrimeric G protein activator (CTX) enhanced [Ca~(2+)]cyt signal in root tip surface cells and the exudation of citrate during the course of Al-induced secretion of citrate, while the inhibitor (PTX) of heterotrimeric G protein depressed effectively Ca~(2+) signal transduction and the secretion of citrate. All the results demonstrated that: (1) Anion channels involved in Al-induced secretion of citrate in stylo, de novo synthesis and activation of an anion channel proteins might be required for the secretion; (2) Both the respiration of root tips and citrate transportation crossing mitochondorial membrane impacted the secretion of citrate under Al stress. (3) Heterotrimeric G protein would intervene in the signal transduction of Al stress and participated in the regulation of Al-induced secretion of citrate in stylo.
引文
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