A DuPont Company, Agricultural Biotechnology, Wilmington, Delaware 19880, USA.
Plant Physiol. 2012 Jul;159(3):1221-34. doi: 10.1104/pp.112.198309. Epub 2012 May 7.
This study describes a dominant low-seed-oil mutant (lo15571) of Arabidopsis (Arabidopsis thaliana) generated by enhancer tagging. Compositional analysis of developing siliques and mature seeds indicated reduced conversion of photoassimilates to oil. Immunoblot analysis revealed increased levels of At1g01050 protein in developing siliques of lo15571. At1g01050 encodes a soluble, cytosolic pyrophosphatase and is one of five closely related genes that share predicted cytosolic localization and at least 70% amino acid sequence identity. Expression of At1g01050 using a seed-preferred promoter recreated most features of the lo15571 seed phenotype, including low seed oil content and increased levels of transient starch and soluble sugars in developing siliques. Seed-preferred RNA interference-mediated silencing of At1g01050 and At3g53620, a second cytosolic pyrophosphatase gene that shows expression during seed filling, led to a heritable oil increase of 1% to 4%, mostly at the expense of seed storage protein. These results are consistent with a scenario in which the rate of mobilization of sucrose, for precursor supply of seed storage lipid biosynthesis by cytosolic glycolysis, is strongly influenced by the expression of endogenous pyrophosphatase enzymes. This emphasizes the central role of pyrophosphate-dependent reactions supporting cytosolic glycolysis during seed maturation when ATP supply is low, presumably due to hypoxic conditions. This route is the major route providing precursors for seed oil biosynthesis. ATP-dependent reactions at the entry point of glycolysis in the cytosol or plastid cannot fully compensate for the loss of oil content observed in transgenic events with increased expression of cytosolic pyrophosphatase enzyme in the cytosol. These findings shed new light on the dynamic properties of cytosolic pyrophosphate pools in developing seed and their influence on carbon partitioning during seed filling. Finally, our work uniquely demonstrates that genes encoding cytosolic pyrophosphatase enzymes provide novel targets to improve seed composition for plant biotechnology applications.
本研究描述了一种拟南芥（Arabidopsis thaliana）的高油突变体（lo15571），该突变体是通过增强子标记产生的。对发育中的蒴果和成熟种子的成分分析表明，光产物向油脂的转化减少。免疫印迹分析显示，lo15571 发育中的蒴果中 At1g01050 蛋白水平升高。At1g01050 编码一种可溶性胞质焦磷酸酶，是五个密切相关基因之一，它们具有预测的胞质定位和至少 70%的氨基酸序列同一性。使用种子偏好启动子表达 At1g01050 可再现 lo15571 种子表型的大多数特征，包括种子油含量低、发育中的蒴果中瞬时光合产物淀粉和可溶性糖水平升高。种子偏好的 RNAi 介导的 At1g01050 和 At3g53620（另一种在种子填充过程中表达的胞质焦磷酸酶基因）的沉默导致可遗传的油增加 1%至 4%，主要是牺牲了种子贮藏蛋白。这些结果与以下情况一致，即蔗糖的动员速度，即作为种子贮藏脂质生物合成的前体，强烈受内源性焦磷酸酶的表达影响。这强调了在种子成熟时低 ATP 供应的情况下，即由于缺氧条件，依赖焦磷酸的反应在支持胞质糖酵解中发挥核心作用。该途径是为种子油生物合成提供前体的主要途径。在胞质溶胶或质体中糖酵解进入点的 ATP 依赖性反应不能完全补偿由于胞质溶胶中焦磷酸酶的表达增加而导致的转基因事件中观察到的油含量损失。这些发现为发育中的种子中胞质溶胶焦磷酸盐池的动态特性及其对种子填充过程中碳分配的影响提供了新的见解。最后，我们的工作独特地表明，编码胞质溶胶焦磷酸酶的基因是提高植物生物技术应用中种子成分的新靶点。
