铝诱导柱花草和黑麦根系分泌有机酸及其调控机理的研究
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摘要
虽然作为重要的豆科牧草柱花草已广泛种植于酸性土壤地区，但是它对铝的耐性及其抵御机制尚未见报告。本研究发现，5 μmol L~(-1)AlCl_3使紫花苜蓿根长抑制80.0％，铝处理后的根尖被铬天青(ER)明显染色，而柱花草经30 μmol L~(-1)的AlCl_3处理后方观察到根尖明显染色的现象，根的伸长受抑制68.0％，表明柱花草抵御铝毒能力较强。采用高效液相色谱法对根系分泌物的分析发现，铝诱导柱花草根系分泌柠檬酸，并且其分泌量随铝处理浓度(0、10、20、50 μmol L~(-1))和时间(6、12、18、24 h)的增加而增加，表明铝诱导根系分泌柠檬酸是柱花草抵御铝毒害的重要机制。
另一方面，虽然近年来越来越多研究报告在铝胁迫下根系分泌有机酸是一些耐铝植物抵御铝毒的主要机制，但是，至今人们对铝诱导根系分泌有机酸的机制的认识还非常有限。本研究以能够分泌柠檬酸与苹果酸的黑麦为对象，探讨铝诱导根系分泌有机酸的调控因子。在铝胁迫下黑麦根系分泌柠檬酸与苹果酸受阴离子通道抑制剂尼氟灭酸(NA)、苯甲酰甲醛(PG)的明显抑制；5 μmol L~(-1)的水杨酸(SA)显著促进铝诱导的柠檬酸的分泌而抑制苹果酸的分泌；Ca~(2+)通道抑制剂La~(3+)、异搏定(VP)也显著抑制有机酸的分泌，而
广西大学硕士学位论文
r通道抑制剂CsC12、四乙基氮化按(TEACI)对有机酸的分泌无
显著抑制作用;250、500协molL一’的eaZ+专一性鳌合剂EG认显著
抑制柠檬酸的分泌，而对苹果酸分泌无显著影响。这些结果暗示，
阴离子通道参与铝诱导的黑麦根系分泌有机酸，SA和Ca2+可能介
导此分泌过程，而K+对有机酸的分泌无直接调控作用。
As an important forage legume, Stylo (Stylosanthes spp) has been widely planted in acid soil areas. However, there is still no study on aluminum-tolerance of stylo and its tolerance mechanism at present. The present thesis showed that root elongation of alfalfa (Medicago sativa L.) during a 24-h period was inhibited by 80% by the exposure to 5 umol L-1 Al and its roots was obviously stained by eriochrome cyanine R (ER); however, the roots of stylo need to be exposure to 30umol L-1 Al in order to get obviously stained with its root elongation was inhibited by 68%, which shows, for stylo, its ability of tolerance to aluminum is strong. According to analysis of root exudates by HPLC, aluminum induced roots of stylo to secret citrate and its secretion increased with Al concentration treated (0, 10, 20, 50 umol L-1) and the period of exposure to Al (6, 12, 18, 24 h), which indicated that Al-induced secretion of citrate is an important mechanism for stylo to resist the toxicity of aluminum.
On the other hand, although in recent years more and more researches concluded that Al-induced secretion of organic acids from roots is a main mechanism for some Al-tolerance plants, so far in field of study the knowledge of the mechanism responsible for Al-induced secretion of organic acids is still very limited. In present study, taking rye (Secale cereale L.) which can secreted citrate and malate as study object, I explored the regulation of organic acid secretion from roots . Secretion of citrate and malate was obviously inhibited by anion channel inhibitors such as niflumic acid and phenlglyoxal. Five umol L-1 salicylic acid (SA) evidently promoted Al-induced secretion of citrate but inhibited secretion of malate. Ca2+ channel inhibitors (La3+, verapamil), greatly inhibited the secretion, while K+ channel inhibitors (CsCl2, tetraethylammonium chloride) has no obviously inhibit to the secretion. As a special chelator of Ca2+, EGTA of 250, 500umol L-1 inhibited secretion of citrate clearly, while had no d
istinct inhibition to malate secretion. All the above experimental evidence shows that anion channel involve in Al-induced secretion of organic acid from rye roots. Salicylic acid and Ca2+ seem to involve in the process, while K+ has no direct regulation to the secretion of organic acids.
另一方面，虽然近年来越来越多研究报告在铝胁迫下根系分泌有机酸是一些耐铝植物抵御铝毒的主要机制，但是，至今人们对铝诱导根系分泌有机酸的机制的认识还非常有限。本研究以能够分泌柠檬酸与苹果酸的黑麦为对象，探讨铝诱导根系分泌有机酸的调控因子。在铝胁迫下黑麦根系分泌柠檬酸与苹果酸受阴离子通道抑制剂尼氟灭酸(NA)、苯甲酰甲醛(PG)的明显抑制；5 μmol L~(-1)的水杨酸(SA)显著促进铝诱导的柠檬酸的分泌而抑制苹果酸的分泌；Ca~(2+)通道抑制剂La~(3+)、异搏定(VP)也显著抑制有机酸的分泌，而
广西大学硕士学位论文
r通道抑制剂CsC12、四乙基氮化按(TEACI)对有机酸的分泌无
显著抑制作用;250、500协molL一’的eaZ+专一性鳌合剂EG认显著
抑制柠檬酸的分泌，而对苹果酸分泌无显著影响。这些结果暗示，
阴离子通道参与铝诱导的黑麦根系分泌有机酸，SA和Ca2+可能介
导此分泌过程，而K+对有机酸的分泌无直接调控作用。
As an important forage legume, Stylo (Stylosanthes spp) has been widely planted in acid soil areas. However, there is still no study on aluminum-tolerance of stylo and its tolerance mechanism at present. The present thesis showed that root elongation of alfalfa (Medicago sativa L.) during a 24-h period was inhibited by 80% by the exposure to 5 umol L-1 Al and its roots was obviously stained by eriochrome cyanine R (ER); however, the roots of stylo need to be exposure to 30umol L-1 Al in order to get obviously stained with its root elongation was inhibited by 68%, which shows, for stylo, its ability of tolerance to aluminum is strong. According to analysis of root exudates by HPLC, aluminum induced roots of stylo to secret citrate and its secretion increased with Al concentration treated (0, 10, 20, 50 umol L-1) and the period of exposure to Al (6, 12, 18, 24 h), which indicated that Al-induced secretion of citrate is an important mechanism for stylo to resist the toxicity of aluminum.
On the other hand, although in recent years more and more researches concluded that Al-induced secretion of organic acids from roots is a main mechanism for some Al-tolerance plants, so far in field of study the knowledge of the mechanism responsible for Al-induced secretion of organic acids is still very limited. In present study, taking rye (Secale cereale L.) which can secreted citrate and malate as study object, I explored the regulation of organic acid secretion from roots . Secretion of citrate and malate was obviously inhibited by anion channel inhibitors such as niflumic acid and phenlglyoxal. Five umol L-1 salicylic acid (SA) evidently promoted Al-induced secretion of citrate but inhibited secretion of malate. Ca2+ channel inhibitors (La3+, verapamil), greatly inhibited the secretion, while K+ channel inhibitors (CsCl2, tetraethylammonium chloride) has no obviously inhibit to the secretion. As a special chelator of Ca2+, EGTA of 250, 500umol L-1 inhibited secretion of citrate clearly, while had no d
istinct inhibition to malate secretion. All the above experimental evidence shows that anion channel involve in Al-induced secretion of organic acid from rye roots. Salicylic acid and Ca2+ seem to involve in the process, while K+ has no direct regulation to the secretion of organic acids.
引文
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[2]Hideaki Matsumoto. Cell biology of aluminum toxicity and tolerance in higher plants. International Review of Cytology, 2000, 200:1～46.
[3]Kochian LV. Cellular mechanisms of aluminum toxicity and resistance in plants. Ann. Rev. Plant Physiol. Plant Mol. Biol,1995,46:237～260.
[4]Ma JF. Role of organic acid in detoxification of aluminum in higher plants. Plant Cell Physiol, 2000, 41: 383～390.
[5]Delhaize E, Ryan PR. Aluminum toxicity and tolerance in plants. Plant Pysiol, 1995, 107:315～332.
[6]Foy CD. The physiology of plant adaptation to mineral stress. Iowa State J.Res., 1983, 57: 355～392.
[7]Wallace SU, Henning SJ, Anderson IC. Elongation, Al concentration and hematoxylin staining of aluminum-treated wheat roots. Iowa State J.Res.,1981,57:97～106.
[8]Bennet RJ, Breen CM. The aluminum signal: new dimensions to mechanisms of aluminum tolerance. Plant Siol, 1991, 134:153～166.
[9]Ryan PR, Ditomaso JM, Kochian LV. Aluminum toxicity in roots: an investigation of spatial sensitivity and to role of the root cap. J. Experimental Botany, 1993, 44: 437～446.
[10]Sung Ju Ahn, Sivaguru M., Osawa H., et al., Aluminum inhibits the H~+-ATPase activity by permanently altering the plasma membrane surface potentials in squash roots. Plant Physiol, 2001,126:1381～1390.
[11]Taylor GJ. Current views of the aluminum stress response: the physiological basis of tolerance. Curr. Top. Plant Biochem. Physiol., 1991,10:57～93.
[12]Hue NV, Craddock GR,Adams F. Effect of organic acids on aluminum toxicity in subsoils. Soil Sci Am J., 1986,50:28～34.
[13]Ma JF, Hiradate S, Nomoto K, et al. Internal detoxification mechanism of Al in hydrangea (identification of Al form in the leaves). Plant Physiol, 1997, 113:
1033～1039.
[14]Li XF, Ma JF, Matsumoto H. Aluminum-induced secretion of both citrate and malate in rye. Plant and Soil, 2002, 242: 235～243.
[15]Ma JF, Zheng SJ, Hiradate S, et al. Detoxifying aluminum with buckwheat. Nature, 1997, 39: 569～570.
[16]Ma JF, Hiradate S, Matsumoto H. High Aluminum Resistance in Buckwheat. Ⅱ. Oxalic Acid Detoxifies Aluminum Internally. Plant Physiol, 1998,117:753～759.
[17]Zheng SJ, Ma JF, Matsumoto H. High aluminum resistance in buckwheat: Ⅰ. Al-indueed specific secretion of oxalic acid from root tips. Plant Physiol, 1998, 117:745～751.
[18]Foy CD, Chancy RL, White MC. The physiology of metal toxicity in plants. Annu. Rev Plant Physiol, 1978,29:511～566.
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