Gibbs J, Morrell S, Valdez A, Setter T L, Greenway H
Faculty of Agriculture (Plant Sciences), University of Western Australia, Nedlands, Australia.
J Exp Bot. 2000 Apr;51(345):785-96.
To investigate regulation of anaerobic carbohydrate catabolism in anoxia-tolerant plant tissue, rate of alcoholic fermentation and maximum catalytic activities of four key enzymes were assessed in coleoptiles of two rice cultivars that differ in tolerance to anoxia. The enzymes were ATP-dependent phosphofructokinase (PFK), pyrophosphate-dependent phosphofructokinase (PFP), pyruvate decarboxylase (PDC), and alcohol dehydrogenase (ADH). During anoxia, rates of coleoptile elongation and ethanol synthesis were faster in the more tolerant variety Calrose than in IR22. Calrose coleoptiles, in contrast to IR22, also showed a sustained Pasteur effect, with the estimated rate of glycolysis during anoxia being 1.4-1.7-fold faster than that of aerobic coleoptiles. In Calrose after 5 d anoxia, maximum catalytic activities of crude enzyme extracts were (in mumol substrate g-1 fresh weight min.-1) 170-240 for ADH, 4-6 for PDC and PFP and 0.4-0.7 for PFK. During anoxia, activity per coleoptile of all four enzymes increased 3-5.5-fold, suggesting that PFK, and PFP, like PDC and ADH, are synthesised in anoxic rice coleoptiles. Enzyme activities, on a fresh weight basis, were lower in IR22 than in Calrose. In vivo activities of PDC and PFK in anoxic coleoptiles from both cultivars were calculated using in vitro activities, estimated substrate levels, cytoplasmic pH, and S0.5 (the substrate level at which 0.5Vmax is reached, without inferring Michaelis-Menten kinetics). Data indicated that potential carbon flux through PFK, rather than through PDC, more closely approximated rates of alcoholic fermentation. That PFK is an important site of regulation was supported further for Calrose coleoptiles by a decrease in the concentration of its substrate pool (F-6-P + G-6-P) following the onset of anoxia. By contrast, in IR22, there was little evidence for control by PFK, consistent with recent evidence that suggests substrate supply limits alcoholic fermentation in this cultivar.
为了研究耐缺氧植物组织中无氧碳水化合物分解代谢的调控机制，我们评估了两种耐缺氧能力不同的水稻品种胚芽鞘中酒精发酵速率和四种关键酶的最大催化活性。这四种酶分别是ATP依赖的磷酸果糖激酶（PFK）、焦磷酸依赖的磷酸果糖激酶（PFP）、丙酮酸脱羧酶（PDC）和乙醇脱氢酶（ADH）。在缺氧期间，耐缺氧能力更强的品种Calrose胚芽鞘的伸长速率和乙醇合成速率比IR22更快。与IR22不同，Calrose胚芽鞘还表现出持续的巴斯德效应，缺氧期间估计的糖酵解速率比需氧胚芽鞘快1.4 - 1.7倍。在缺氧5天后的Calrose中，粗酶提取物的最大催化活性（以μmol底物g-1鲜重min.-1计），ADH为170 - 240，PDC和PFP为4 - 6，PFK为0.4 - 0.7。在缺氧期间，所有四种酶的每个胚芽鞘活性增加了3 - 5.5倍，这表明PFK和PFP与PDC和ADH一样，是在缺氧水稻胚芽鞘中合成的。以鲜重为基础，IR22中的酶活性低于Calrose。使用体外活性、估计的底物水平、细胞质pH值和S0.5（达到0.5Vmax时的底物水平，无需推断米氏动力学）计算了两个品种缺氧胚芽鞘中PDC和PFK的体内活性。数据表明，通过PFK而非PDC的潜在碳通量更接近酒精发酵速率。缺氧开始后，Calrose胚芽鞘底物池（F - 6 - P + G - 6 - P）浓度降低，进一步支持了PFK是一个重要调控位点的观点。相比之下，在IR22中，几乎没有证据表明PFK起到调控作用，这与最近的证据一致，即该品种中底物供应限制了酒精发酵。
