Overproduction of an Arabidopsis aldo-keto reductase increases barley tolerance to oxidative and cadmium stress by an in vivo reactive aldehyde detoxification
Overproduction of an Arabidopsis aldo-keto reductase increases barley tolerance to oxidative and cadmium stress by an in vivo reactive aldehyde detoxification
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DOI：
10.1007/s10725-014-9896-x
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发表时间：
2014-09-01
影响因子：
4.2
通讯作者：
Tamas, Laszlo
中科院分区：
文献类型：
--
作者：
Eva, Csaba;Toth, Gabor;Tamas, Laszlo
在这项工作中，我们在转基因大麦中表达了拟南芥编码蛋白(AKR4C9)，以研究其酶活性，并提高转基因植物的反应醛中和能力(氧化胁迫耐受性的一部分)。从C端或N端His标记的醛酮还原酶(AKR)转基因植株中提取总叶蛋白，用亲和层析进行纯化。拟南芥编码的酶对合成的反应醛和戊二醛表现出中等的活性，对低米氏常数(K-m值)的果糖表现出低的但可检测到的酶活性。C端和N端组氨酸标记的AKRs的活性在相同的范围内。戊二醛也在体内进行了测试，方法是喷洒在植物的叶子上。评价了野生型和转基因植株对反应醛的耐受性，以及这种反应醛处理的一般生理效应。在所有(野生型和转基因)植物中发现生长速度都降低了。高表达AKR的转基因植株表现出较低的呼吸速率，并且表现出较高的鲜重、较高的叶绿素含量和光合作用活性，表明它们具有较高的耐反应醛能力。镉(Cd)处理也被用来验证这一结果。Cd引起强烈的脂质过氧化，但拟南芥酶如预期那样降低了反应醛的含量。本文首次报道了胁迫诱导植物AKR酶还原果糖的动力学参数。此外，还提供了有关反应醛处理对完整植物的影响的数据。
In this work, we expressed an Arabidopsis thaliana-coded protein (AKR4C9) in transgenic barley to study its enzymatic activity and to enhance the reactive aldehyde neutralizing capacity (part of the oxidative stress tolerance) of transgenic plants. Total leaf protein was extracted from transgenic plants expressing either C or N-terminally His-tagged aldo-keto reductase (AKR) enzyme and purified by affinity chromatography. The Arabidopsis-coded enzyme showed moderate activity against the synthetic reactive aldehyde, glutaraldehyde, and low but detectable enzyme activity against fructose with a low Michaelis-Menten constant (K-m value). Activity of the C and the N-terminally His-tagged AKRs were found to be in the same range. Glutaraldehyde was also tested in vivo by spraying onto the leaves of the plants. The reactive aldehyde tolerance of both wild type and transgenic plants, as well as the general physiological effects of this reactive aldehyde treatment were evaluated. The growth rate was found to decrease in all (both wild type and transgenic) plants. The high AKR-expressing transgenic plants showed a lower respiratory rate, and they also showed higher fresh weight, higher chlorophyll content and photosynthetic activity, indicating a higher reactive aldehyde tolerance. Cadmium (Cd) treatment was also performed to validate this result. Cd caused strong lipid peroxidation; however, the Arabidopsis enzyme lowered the reactive aldehyde content as expected. This is the first report in which kinetic parameters of the fructose reduction by the stress inducible plant AKR enzyme are presented. Furthermore, data on the effects of a reactive aldehyde treatment on intact plants are also provided.