Debnam Phillip M, Fernie Alisdair R, Leisse Andrea, Golding Alison, Bowsher Caroline G, Grimshaw Caroline, Knight Jacqueline S, Emes Michael J
College of Biological Science, University of Guelph, Gordon Street, Guelph, Ont., Canada N1G 2W1.
Plant J. 2004 Apr;38(1):49-59. doi: 10.1111/j.1365-313X.2004.02017.x.
Expression of one specific isoform of plastidic glucose 6-phosphate dehydrogenase (G6PDH) was manipulated in transgenic tobacco. Antisense and sense constructs of the endogenous P2 form of G6PDH were used to transform plants under the control of the cauliflower mosaic virus (CaMV) 35S promotor. Recombinant plants with altered expression were taken through to homozygosity by selective screening. Northern analyses revealed substantial changes in the expression of the P2 form of G6PDH, with no apparent impact on the activity of the cytosolic isoenzyme. Analysis of G6PDH activity in chloroplasts showed that despite the large changes in expression of P2-G6PDH, the range of enzyme activity varied only from approximately 50 to 200% of the wild type, reflecting the presence of a second G6PDH chloroplastic isoform (P1). Although none of the transgenic plants showed any visible phenotype, there were marked differences in metabolism of both sense and antisense lines when compared with wild-type/control lines. Sucrose, glucose and fructose contents of leaves were higher in antisense lines, whereas in overexpressing lines, the soluble sugar content was reduced below that of control plants. Even more striking was the observation that contents of glucose 6-phosphate (Glc6P) and 6-phosphogluconate (6PG) changed, such that the ratio of Glc6P:6PG was some 2.5-fold greater in the most severe antisense lines, compared with those with the highest levels of overexpression. Because of the distinctive biochemical properties of P2-G6PDH, we investigated the impact of altered expression on the contents of antioxidants and the response of plants to oxidative stress induced by methyl viologen (MV). Plants with decreased expression of P2-G6PDH showed increased content of reduced glutathione (GSH) compared to other lines. They also possessed elevated contents of ascorbate and exhibited a much higher ratio of reduced:oxidised ascorbate. When exposed to MV, leaf discs of wild-type and overexpressing lines demonstrated increased oxidative damage as measured by lipid peroxidation. Remarkably, leaf discs from plants with decreased P2-G6PDH did not show any change in lipid peroxidation in response to increasing concentrations of up to 15 micro m MV. The results are discussed from the perspective of the role of G6PDH in carbohydrate metabolism and oxidative stress. It is suggested that the activity of P2-G6PDH may be crucial in balancing the redox poise in chloroplasts.
在转基因烟草中对质体葡萄糖-6-磷酸脱氢酶（G6PDH）的一种特定同工型的表达进行了调控。利用G6PDH内源性P2形式的反义构建体和正义构建体，在花椰菜花叶病毒（CaMV）35S启动子的控制下转化植物。通过选择性筛选使表达改变的重组植物达到纯合状态。Northern分析显示G6PDH的P2形式的表达有显著变化，而对胞质同工酶的活性没有明显影响。叶绿体中G6PDH活性分析表明，尽管P2-G6PDH的表达有很大变化，但酶活性范围仅在野生型的约50%至200%之间变化，这反映了存在第二种叶绿体G6PDH同工型（P1）。尽管没有转基因植物表现出任何可见表型，但与野生型/对照品系相比，正义和反义品系在代谢方面存在明显差异。反义品系叶片中的蔗糖、葡萄糖和果糖含量较高，而在过表达品系中，可溶性糖含量低于对照植株。更引人注目的是观察到葡萄糖-6-磷酸（Glc6P）和6-磷酸葡萄糖酸（6PG）的含量发生了变化，与过表达水平最高的品系相比，在最严重的反义品系中Glc6P:6PG的比值大约高2.5倍。由于P2-G6PDH具有独特的生化特性，我们研究了表达改变对抗氧化剂含量以及植物对甲基紫精（MV）诱导的氧化胁迫的响应的影响。与其他品系相比，P2-G6PDH表达降低的植物中还原型谷胱甘肽（GSH）含量增加。它们还具有较高的抗坏血酸含量，并且还原型:氧化型抗坏血酸的比值更高。当暴露于MV时，野生型和过表达品系的叶盘通过脂质过氧化测量显示氧化损伤增加。值得注意的是，P2-G6PDH降低的植物的叶盘在高达15微摩尔MV浓度增加时，脂质过氧化没有任何变化。从G6PDH在碳水化合物代谢和氧化胁迫中的作用角度对结果进行了讨论。有人提出P2-G6PDH的活性可能对平衡叶绿体中的氧化还原平衡至关重要。
