Karchi H, Miron D, Ben-Yaacov S, Galili G
Department of Plant Genetics, Weizmann Institute of Science, Rehovot, Israel.
Plant Cell. 1995 Nov;7(11):1963-70. doi: 10.1105/tpc.7.11.1963.
The accumulation of free lysine in tobacco seed triggers the stimulation of lysine-ketoglutarate reductase, an enzyme that acts in lysine catabolism. The mechanism of lysine-ketoglutarate reductase stimulation was studied in two different systems: (1) developing seeds of wild-type plants in which the low basal lysine-ketoglutarate reductase activity can be stimulated by the exogenous addition of lysine; and (2) developing seeds of transgenic tobacco plants expressing a bacterial dihydrodipicolinate synthase in which lysine-ketoglutarate reductase activity is stimulated by endogenous lysine overproduction. In both systems, the stimulation of lysine-ketoglutarate reductase activity was significantly reduced when treated with the Ca2+ chelator EGTA. Moreover, the inhibitory effect of EGTA was overcome by the addition of Ca2+ but not Mg2+, suggesting that the lysine-dependent activation of lysine-ketoglutarate reductase requires Ca2+. This was further confirmed by a significant stimulation of lysine-ketoglutarate reductase activity following the treatment of wild-type seeds with ionomycin (an ionophore that increases Ca2+ flow into the cytoplasm). In addition, treatment of wild-type seeds with the protein phosphatase inhibitor okadaic acid triggered a significant induction in lysine-ketoglutarate reductase activity, whereas treatment of the transgenic seeds with the protein kinase inhibitor K-252a caused a significant reduction in its activity. Thus, we conclude that the stimulation of lysine-ketoglutarate reductase activity by lysine in tobacco seed operates through an intracellular signaling cascade mediated by Ca2+ and protein phosphorylation.
烟草种子中游离赖氨酸的积累会触发赖氨酸-酮戊二酸还原酶的激活，该酶参与赖氨酸的分解代谢。我们在两个不同的系统中研究了赖氨酸-酮戊二酸还原酶激活的机制：（1）野生型植物发育中的种子，其低基础赖氨酸-酮戊二酸还原酶活性可通过外源添加赖氨酸来激活；（2）表达细菌二氢吡啶二羧酸合酶的转基因烟草植物发育中的种子，其中赖氨酸-酮戊二酸还原酶活性因内源性赖氨酸过量产生而被激活。在这两个系统中，用Ca2+螯合剂EGTA处理后，赖氨酸-酮戊二酸还原酶活性的激活显著降低。此外，添加Ca2+可克服EGTA的抑制作用，而添加Mg2+则不能，这表明赖氨酸依赖的赖氨酸-酮戊二酸还原酶激活需要Ca2+。用离子霉素（一种增加Ca2+流入细胞质的离子载体）处理野生型种子后，赖氨酸-酮戊二酸还原酶活性显著激活，进一步证实了这一点。此外，用蛋白磷酸酶抑制剂冈田酸处理野生型种子会引发赖氨酸-酮戊二酸还原酶活性的显著诱导，而用蛋白激酶抑制剂K-252a处理转基因种子则会导致其活性显著降低。因此，我们得出结论，烟草种子中赖氨酸对赖氨酸-酮戊二酸还原酶活性的激活是通过由Ca2+和蛋白磷酸化介导的细胞内信号级联反应来实现的。
