【作者】 王华华；

【导师】 毕玉蓉；

【作者基本信息】 兰州大学 ， 植物学， 2009， 博士

【摘要】 本文以野生型拟南芥（WT）和乙烯不敏感突变体etr1-3愈伤组织为材料,研究了拟南芥愈伤组织对盐胁迫的生理响应,并进一步阐明了乙烯（ETH）和一氧化氮（NO）在植物盐适应性过程中的调节作用及其信号转导,同时研究了在盐胁迫下两种拟南芥愈伤组织中交替途径的变化以及H₂O₂与ETH在诱导交替途径中的相互作用关系,并进一步探讨了盐胁迫下交替途径可能的生理功能以及调控机制。主要结果总结如下:1.100 mM NaCl处理下,etr1-3愈伤组织与WT愈伤组织相比表现出了更高水平的离子渗漏和Na⁺/K⁺比以及更低的质膜H⁺-ATPase活性,表明etr1-3愈伤组织对盐更加敏感。盐胁迫下,用乙烯生物合成前体物质1-aminocyclopropane-1-carboxylic acid（ACC）和NO供体硝普钠（sodium nitroprusside,SNP）处理,发现WT愈伤组织中离子渗漏和Na⁺/K⁺比的水平明显的降低,而且H⁺-ATPase活性显著增加;然而,ACC和SNP处理对etr1-3愈伤组织却基本无影响,表明两者均能缓解WT愈伤组织中NaCl诱导的伤害。进一步研究发现,SNP在盐胁迫过程中的作用可以被NO独特的清除剂2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxyde（PTIO）以及乙烯生物合成抑制剂aminooxyacetic acid（AOA）所逆转。在WT愈伤组织中,NO和ETH含量在盐胁迫（100 mM NaCl）初期迅速增加,而且NO的产生显著地刺激了ETH的产生。此外,盐胁迫下ETH诱导了H⁺-ATPase活性和基因表达。恢复试验结果显示,WT愈伤组织中100 mM NaCl诱导的伤害是可逆的,在恢复过程中离子渗漏、Na⁺/K⁺比以及H⁺-ATPase活性均表现出类似的趋势,均恢复到了对照水平。这些结果表明,盐胁迫下,ETH和NO相互协作刺激H⁺-ATPase活性,并进一步调控离子动态平衡,使植物细胞体内的离子平衡重新建立,以此来提高植物对盐分的耐受性,在这一过程中ETH可能是作用于NO下游的一个信号分子。2.100 mM NaCl胁迫处理48 h显著地诱导了WT愈伤组织中交替途径（AP）容量和AP对总呼吸的贡献率,而在etr1-3愈伤组织中,盐胁迫对其AP活性仅有轻微的诱导。盐胁迫下,使用外源ACC处理可进一步增加WT愈伤组织中AP活性,而ACC处理对etr1-3愈伤组织则几乎无影响。另一方面,盐胁迫下,使用乙烯生物合成抑制剂AOA处理WT愈伤组织则抑制了盐胁迫诱导的AP活性,表明ETH是盐胁迫下诱导AP所必须的。盐胁迫下,外源H₂O₂处理增加了WT愈伤组织中ETH的释放,CAT（H₂O₂清除剂）处理则减少了ETH的释放;盐胁迫下,外源ACC处理减少了WT愈伤组织中H₂O₂的产生,AOA处理则增加了H₂O₂的产生,表明盐胁迫下H₂O₂促进了WT愈伤组织中ETH的释放,而ETH减少了H₂O₂的产生。盐胁迫下,AP抑制剂SHAM（salicylhdroxamic acid）处理导致了WT愈伤组织细胞遭受了更严重的伤害,表现在产生了更高水平的H₂O₂,TBARS以及离子渗漏。以上结果表明,盐胁迫下,ETH和H₂O₂均参与了AP的诱导,AP在抗氧化保护过程中起着重要作用,且ETH可能是AP的直接诱导者。更多还原

【Abstract】 In the present study,we used the calli from wild-type Arabidopsis（Arabidopsis thaliana,WT）and ethylene-insensitive mutant etr1-3 to study their physiological responses to salt stress,and further investigated the regulative roles of ethylene and NO under salt stress.In addition,the present study was aimed to investigate the relationship between H₂O₂ and ethylene in the induction of alternative pathway（AP） in Arabidopsis calli from WT and etr1-3 under salt stress.And an effort was also made to demonstrate the possible regulation and physiological function of AP under salt stress.The main results were summarized as follows:1.Results showed that ethylene-insensitive mutant etr1-3 was more sensitive to salt stress than WT.Under 100 mM NaCl,etr1-3 callus displayed a greater electrolyte leakage and Na⁺/K⁺ ratio but a lower plasma membrane（PM）H⁺-ATPase activity than those of WT callus.Application of exogenous 1-aminocyclopropane-1 -carboxylic acid（ACC,an ethylene precursor）or sodium nitroprusside（SNP,a NO donor）alleviated NaCl-induced injury by maintaining a lower Na⁺/K⁺ ratio and an increased PM H⁺-ATPase activity in WT callus but not in etr1-3 callus.The SNP actions in NaCl stress were attenuated by a specific NO scavenger PTIO（2-phenyl-4, 4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxyde）or an ethylene biosynthesis inhibitor AOA（aminooxyacetic acid）in WT callus.Under 100 mM NaCl,the NO accumulation and ethylene emission appeared at early time,and NO production greatly stimulated ethylene emission in WT callus.In addition,ethylene induced the expression of PM H⁺-ATPase genes under salt stress.Furthermore,the recovery experiment showed mat NaCl-induced injury was reversible,as signaled by the similar recovery of Na⁺/K⁺ ratio and PM H⁺-ATPase activity in WT callus.Taken together,the results indicate that ethylene and NO cooperate in stimulating PM H⁺-ATPase activity to modulate ion homeostasis for salt tolerance,and ethylene may be a part of the downstream signal molecular in NO action.2.The capacity of AP and the contribution of AP to the total respiration were significantly induced in the presence of 100 mM NaCl for 48 h in WT Arabidopsis callus but only slightly induced in an ethylene-insensitive mutant,etr1-3.Endogenous ethylene emission was enhanced in WT Arabidopsis callus under salt stress.In addition,application of 1-aminocyclopropane-1-carboxylic acid（ACC,an ethylene precursor）in the presence of NaCl further increased AP capacity in WT callus but had little effects on that of etr1-3 callus,suggesting ethylene is required for the induction of AP.On the other hand,limiting endogenous ethylene production by aminooxyacetic acid（AOA,an ethylene biosynthesis inhibitor）in WT callus eliminated the NaCl-induced increase of ethylene emission and inhibited the induction of AP under salt stress.H₂O₂ enhanced ethylene production while ethylene reduced H₂O₂ generation in WT callus under salt stress.Inhibition of the AP pathway by salicylhydroxamic acid under salt stress resulted in more severe membrane damage as indicated by higher levels of H₂O₂,TBARS and electrolyte leakage in WT callus. Taken together,these results suggest AP can play a role in antioxidant protection under salt stress,and ethylene may be the direct inducer of AP.更多还原