Sangwan V, Foulds I, Singh J, Dhindsa R S
Department of Biology, McGill University, 1205 Avenue Docteur Penfield, Montreal, Quebec H3A 1B1, Canada.
Plant J. 2001 Jul;27(1):1-12. doi: 10.1046/j.1365-313x.2001.01052.x.
Previous studies on cold-triggered events leading to Ca2+ influx during cold acclimatization have been conducted on either unicellular cyanobacterium Synechocystis or plant cell suspensions, and used transcript levels of cold-induced genes as end-point markers. Whether the results of these studies are valid for intact plants or their organs is not known. Here we examine cold signaling in transgenic Brassica napus seedlings carrying, in addition to the endogenous cold-inducible BN115 gene, the beta-glucuronidase (GUS) gene placed under control of the BN115 promoter. The activity of BN115 promoter was monitored at the transcriptional and translational levels by determining accumulation of BN115 transcripts and by histochemical assay of GUS activity. Cold-activation of BN115 was strongly inhibited by the membrane fluidizer benzyl alcohol, but mimicked at 25 degrees C by the membrane rigidifier dimethylsulfoxide (DMSO). The cold induction of BN115 was also inhibited by stabilizers of microtubules and actin microfilaments, taxol and jasplakinolide, respectively, but was mimicked at 25 degrees C by microtubule destabilizer oryzalin or colchicine, or by microfilament destabilizer latrunculin B. Gd3+ or ruthenium red prevented the cold activation of BN115, but Ca2+ ionophore A23187 or cyclic ADP-ribose activated it at 25 degrees C. Inhibitors of tyrosine kinases, protein kinase C and phosphoinositide kinases prevented the cold activation of BN115, but inhibitors of protein phosphatases (PP) 1 and 2 A activated BN115 at 25 degrees C. Constitutively expressed GUS activity in another transgenic line of the same cultivar of B. napus, was not affected by cold or any of the chemical treatments used in the experimentation. Activation of BN115 at 25 degrees C by DMSO, Ca2+ ionophore, cADPR, and by inhibitors of PP1 and 2A was accompanied by an increased freezing tolerance. It was concluded that the cold-activation of BN115 requires membrane rigidification, cytoskeleton reorganization, Ca2+ influx and action of several types of protein kinases.
先前关于冷驯化过程中导致钙离子内流的冷触发事件的研究，是在单细胞蓝藻集胞藻或植物细胞悬浮液上进行的，并将冷诱导基因的转录水平用作终点标记。这些研究结果对于完整植株或其器官是否有效尚不清楚。在此，我们研究了转基因油菜幼苗中的冷信号传导，这些幼苗除了携带内源性冷诱导基因BN115外，还携带了置于BN115启动子控制下的β-葡萄糖醛酸酶（GUS）基因。通过测定BN115转录本的积累以及通过GUS活性的组织化学分析，在转录和翻译水平上监测BN115启动子的活性。BN115的冷激活受到膜流化剂苄醇的强烈抑制，但在25℃下被膜硬化剂二甲基亚砜（DMSO）模拟。BN115的冷诱导也分别受到微管稳定剂紫杉醇和肌动蛋白微丝稳定剂茉莉酮酸甲酯的抑制，但在25℃下被微管去稳定剂oryzalin或秋水仙碱，或被微丝去稳定剂拉特罗毒素B模拟。钆离子（Gd3 +）或钌红阻止了BN115的冷激活，但钙离子载体A23187或环ADP - 核糖在25℃下激活了它。酪氨酸激酶、蛋白激酶C和磷酸肌醇激酶的抑制剂阻止了BN115的冷激活，但蛋白磷酸酶（PP）1和2A的抑制剂在25℃下激活了BN115。在同一品种的另一个转基因油菜品系中组成型表达的GUS活性，不受冷或实验中使用的任何化学处理的影响。DMSO、钙离子载体、环ADP - 核糖以及PP1和2A的抑制剂在25℃下对BN115的激活伴随着抗冻性的增加。得出的结论是，BN115的冷激活需要膜硬化、细胞骨架重组、钙离子内流以及几种类型蛋白激酶的作用。
