低温胁迫下拟南芥表皮蜡质的响应机制

doi:10.3864/j.issn.0578-1752.2014.02.005

摘要/Abstract

摘要： 【目的】低温是影响作物产量和品质的主要环境因素之一，而表皮蜡质是植物抵御外界胁迫的第一层保护性屏障。研究低温胁迫对拟南芥表皮蜡质组分含量、结构及相关蜡质基因表达的影响，有助于进一步理解植物表皮蜡质与低温互作机制，从而对后续作物的抗性机制研究起指导作用。【方法】以野生型拟南芥与蜡质突变体cer1、cer3、cer4、cer6、cer10、cer20及kcs1为试验材料，待植株生长至5—6周时进行4℃胁迫处理10 d和18 d。利用扫描电子显微镜观察表皮蜡质晶体结构变化；利用气相色谱-质谱联用仪（GC-MS）分析蜡质组成及含量变化；利用qRT-PCR技术检测蜡质基因CER1、CER3、CER4、KCS1、WIN1的表达。【结果】低温胁迫后，拟南芥蜡质晶体结构在分布密度、形状与大小方面发生了不同程度的变化。野生型拟南芥蜡质晶体熔融成片，大面积覆盖茎秆表面，这可能一定程度上起到了低温阻隔作用，并减少水分散失。cer1突变体表现出松针状晶体显著减少，并以小型树枝状结构为主；cer3与cer10杆状晶体显著减少；而cer6与cer20蜡质晶体在低温胁迫后有所增加；kcs1蜡质晶体在低温胁迫后垂直杆状结构显著减少，水平杆状结构出现，并伴有蜡质晶体熔融现象。低温胁迫对一级醇减少突变体cer4结构无显著影响。GC-MS分析结果表明，拟南芥野生型与各突变体在低温胁迫下表皮蜡质组分含量也发生了不同程度的变化。野生型中醛类与酮类含量显著减少、一级醇类含量显著增加。各突变体则表现出相反的变化，蜡质组分中醛类、酮类含量增加或无显著变化、一级醇含量减少或无显著变化；受低温胁迫较重的cer3﹑cer10表皮蜡质中一级醇含量显著下降。拟南芥表皮蜡质在低温胁迫下显著积累（cer3 无显著变化、cer10 蜡质总量减少），且主要通过增加烷类与次级醇含量来增加蜡质总量。低温胁迫诱导了野生型CER1 基因的强势表达，植株通过上调CER1 的表达促进烷类物质的合成以响应低温胁迫；CER4 的表达上调促进了一级醇的合成。KCS1、CER3与WIN1在低温胁迫下表达下调，暗示了植株蜡质总量的增加主要是通过蜡质合成的下游途径如烷合成途径增加。【结论】低温胁迫可以改变表皮蜡质晶体结构及组分含量，蜡质组分中烷类与次级醇类含量的上升是响应低温胁迫的主要方式，蜡质总量的增加主要来自于烷类的增加。CER1是响应低温胁迫的主要蜡质基因。

关键词: 拟南芥 , 低温胁迫 , 表皮蜡质 , 基因 , 蜡质突变体

Abstract: 【Objective】Cold stress is one of the factors influencing the quality and quantity of crops while the epicuticular wax is believed to be the first protective barrier of the plant to external stresses. In the current study, the effects of cold stress on epicuticular wax morphology and constituents and expression of wax related genes in Arabidopsis stems were examined, aiming to understand the interactive mechanism between plant epicuticular wax and cold stress, and to provide suggestions on researches related to crop resistance to abiotic stresses. 【Method】The materials used in current experiment included wild type Arabidopsis (WT) and wax mutants (cer1, cer3, cer4, cer6, cer10, cer20 and kcs1). The seedlings were grown for 5-6 weeks after germination, and then were placed in a temperature controlled cabinet at 4℃ for ten days and eighteen days, seperately. Scanning electron microscope technology was used to investigate the changes of crystal structure. Gas chromatography and mass spectrometry technology were used to measure the contents of total wax and wax constituents. Real-time Q-PCR was used to analyze the transcripts of wax related genes.【Result】Under cold stress, the density, shape and size of wax crystalloids on stem of mutants and WT changed. Wax crystal structures on WT fused to big horizontal plates, greatly covering the surface of stems, which might help protect plant from cold hardness and decrease water loss. The pine-needle crystals on cer1 reduced significantly and some small dendrites appeared. The rod crystals on cer3 and cer10 reduced significantly while the crystals on cer6 and cer20 increased. The vertical rods on kcs1 surface reduced while the horizontal rods appeared with the fusion of crystals. The cold stress had no effect on the crystal morphology on cer4. The GC-MS analysis showed that the contents of wax constituents and total wax also changed under cold stress. The contents of aldehydes and ketones in Arabidopsis wild type reduced significantly while the content of primary alcohols increased. For the mutants, the contents of acids, aldehydes and ketones increased or changed insignificantly while the contents of primary alcohols reduced or changed insignificantly. For cer3 and cer10 with severe cold harm symptoms, the contents of primary alcohols decreased significantly. The contents of wax accumulated significantly under cold stress except cer3 and cer10, mainly attributing to significant increase in alkanes and secondary alcohols. The content of total wax in cer3 changed insignificantly while that in cer10 reduced. For A. thaliana wild type, the expression of CER1 was induced strongly by the cold stress, suggesting that plant can up-regulate the expression of CER1 to promote the alkane synthesis under cold stress. The up-regulated expression of CER4 promoted the synthesis of primary alcohol. The down-regulated expression of KCS1, CER3 and WIN1 under cold stress indicated that the increase of total wax was due to the promotion of the alkane-synthesizing branch. 【Conclusion】 The crystal structure and constituents of epicuticular wax can be altered by cold stress, and the increase of alkanes and secondary alcohols in epiculticular wax might be the main response way to cold stress. The increase of alkanes contributes to the increase of total wax and cer1 might be the main response wax-related gene to cold stress for A. thaliana.

Key words: Arabidopsis thaliana , cold stress , epicuticular wax , gene , wax mutants

倪郁, 宋超, 王小清. 低温胁迫下拟南芥表皮蜡质的响应机制[J]. 中国农业科学, 2014, 47(2): 252-261.

NI Yu, SONG Chao, WANG Xiao-Qing. Investigation on Response Mechanism of Epicuticular Wax on Arabidopsis thaliana Under Cold Stress[J]. Scientia Agricultura Sinica, 2014, 47(2): 252-261.

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