Antisense suppression of L-galactose dehydrogenase in Arabidopsis thaliana provides evidence for its role in ascorbate synthesis and reveals light modulated L-galactose synthesis
Antisense suppression of L-galactose dehydrogenase in Arabidopsis thaliana provides evidence for its role in ascorbate synthesis and reveals light modulated L-galactose synthesis
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DOI：
10.1046/j.1365-313x.2002.01315.x
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发表时间：
2002-06-01
期刊：
影响因子：
7.2
通讯作者：
Smirnoff, N
中科院分区：
文献类型：
--
作者：
Gatzek, S;Wheeler, GL;Smirnoff, N
L-半乳糖脱氢酶（L-GalDH）是从豌豆幼苗中分离纯化并从拟南芥中克隆的一种新的将L-半乳糖氧化为L-半乳糖内酯（L-GalL）的酶。L-GalL是植物抗坏血酸生物合成的底物，因此通过在烟草中的过表达和在A. thaliana.在烟草中，最高表达株系的可提取活性增加了3.5倍，但这并没有增加叶片抗坏血酸浓度。用反义L-GalDH构建体转化的拟南芥产生了具有30%野生型活性的品系。这些具有较低的叶片抗坏血酸浓度时，在强光条件下生长。L-GalDH活性低的反义转化体中L-Gal池大小增加，并且L-Gal浓度与抗坏血酸呈负相关。这些结果为L-GalDH在抗坏血酸生物合成中的作用提供了直接证据。A中的抗坏血酸池大小。拟南芥的L-GalDH表达量随着光照强度的增加而增加，但L-GalDH表达量不受影响。在适应强光的反义植物中L-Gal积累更高，表明从GDP-甘露糖合成L-Gal的能力增加。由于L-GalL的唯一已知功能是抗坏血酸合成，这些反义植物提供了一个机会，以研究抗坏血酸功能，对碳水化合物代谢的影响最小。其他抗氧化剂的测量显示，抗坏血酸和邻苯三酚依赖的过氧化物酶活性在低抗坏血酸线的增加。由于抗坏血酸是植物中主要的过氧化氢清除抗氧化剂，这可能表明控制过氧化氢浓度的补偿机制。
L-Galactose dehydrogenase (L-GalDH), a novel enzyme that oxidizes L-Gal to L-galactono-1,4-lactone (L-GalL), has been purified from pea seedlings and cloned from Arabidopsis thaliana. L-GalL is a proposed substrate for ascorbate biosynthesis in plants, therefore the function of L-GalDH in ascorbate biosynthesis was investigated by overexpression in tobacco and antisense suppression in A. thaliana. In tobacco the highest expressing lines had a 3.5-fold increase in extractable activity, but this did not increase leaf ascorbate concentration. Arabidopsis thaliana, transformed with an antisense L-GalDH construct, produced lines with 30% of wild-type activity. These had lower leaf ascorbate concentration when grown under high light conditions. L-Gal pool size increased in antisense transformants with low L-GalDH activity, and L-Gal concentration was negatively correlated with ascorbate. The results provide direct evidence for a role of L-GalDH in ascorbate biosynthesis. Ascorbate pool size in A. thaliana is increased by acclimation to high light, but L-GalDH expression was not affected. L-Gal accumulation was higher in antisense plants acclimated to high light, indicating that the capacity to synthesize L-Gal from GDP-mannose is increased. Because the only known function of L-GalL is ascorbate synthesis, these antisense plants provide an opportunity to investigate ascorbate function with minimal effects on carbohydrate metabolism. Measurements of other antioxidants revealed an increase in ascorbate- and pyrogallol-dependent peroxidase activity in low-ascorbate lines. As ascorbate is the major hydrogen peroxide-scavenging antioxidant in plants, this could indicate a compensatory mechanism for controlling hydrogen peroxide concentration.