Transgenic aequorin monitors cytosolic calcium transients in soybean cells challenged with β-glucan or chitin elicitors
Transgenic aequorin monitors cytosolic calcium transients in soybean cells challenged with β-glucan or chitin elicitors
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DOI：
10.1007/s004250050519
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发表时间：
1999-02-01
期刊：
影响因子：
4.3
通讯作者：
Neuhaus-Url, G
中科院分区：
文献类型：
--
作者：
Mithöfer, A;Ebel, J;Neuhaus-Url, G
转基因大豆（Glycine max L.）表达水母发光蛋白的细胞用于监测响应真菌诱导子处理的胞质Ca 2+浓度的变化。在约60 s的明显滞后期后，几丁质片段和β-葡聚糖激发子均引起胞质Ca浓度的快速增加，其在处理的2-2.5 min内达到峰值。然后Ca 2+浓度降低，在用几丁质片段处理的情况下，在约5分钟后达到基础水平，而在β-葡聚糖处理的情况下，观察到Ca 2+浓度的第二次升高，在约7-8分钟后达到最大值。信号的校准表明，激发子增强了细胞溶质Ca 2+浓度从低至0.1 μ M的静息浓度到约2 μ M的最高水平。剂量-反应实验表明，在50%水平下给予Ca 2+反应的激发子的浓度对于几丁质片段为0.4 nM，对于合成的七-β-葡糖苷和真菌β-葡聚糖部分分别为28 μ M和72 μ M。β-葡聚糖或N，N '，N“，N”'-四乙酰壳四糖（CH_4）诱导的Ca ~（2+）信号被Ca ~（2+）螯合剂1，2-双-（2-氨基苯氧基）乙烷-N，N，N '，N'-四乙酸（BAPTA）和Ca ~（2+）通道抑制剂La ~（3+）抑制。新霉素，其在植物细胞中的靶点尚未明确确定，主要减少β-葡聚糖处理后双相Ca 2+曲线的第二个峰的表达。已知细菌环β-葡聚糖参与大豆细胞的激发子感应机制，并且可能与随后的防御反应的激活有关。
Transgenic soybean (Glycine max L.) cells expressing aequorin were used to monitor changes in cytosolic Ca2+ concentrations in response to treatment with fungal elicitors. After an apparent lag phase of about 60 s, both chitin fragments and beta-glucan elicitors caused a rapid increase in cytosolic Ca concentration, which peaked within 2-2.5 min of treatment. The Ca2+ concentration then decreased and reached the basal level after about 5 min in the case of the treatment with chitin fragments, while a second rise in the Ca2+ concentration with a maximum occurring after about 7-8 min was observed in the case of beta-glucan treatment. Calibration of the signals showed that the elicitors enhanced the cytosolic Ca2+ concentration from resting concentrations as low as 0.1 mu M to highest levels of about 2 mu M. Dose-response experiments showed that the concentration of elicitors giving a Ca2+ response at the 50% level was 0.4 nM for the chitin fragment and 28 mu M and 72 mu M, respectively, for a synthetic hepta-beta-glucoside and a fungal beta-glucan fraction. The beta-glucan- or N,N'.N ",N"'-tetraacetyl chitotetratose (CH4)-induced Ca2+ signals were inhibited by both the Ca2+ chelator 1,2-bis-(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) and by the Ca2+-channel inhibitor La3+. Neomycin, whose target in plant cells has not yet been clearly identified, reduced predominantly the expression of the second peak of the biphasic Ca2+ curve following beta-glucan treatment. Bacterial cyclic beta-glucans known to elicitor-sensing mechanism of soybean cells, and is probably connected with the subsequent activation of defence responses.