Department of Molecular Genetics and Biotechnology Center, The Ohio State University, 1060 Carmack Road, Columbus, Ohio 43210-1002.
Plant Physiol. 1992 Aug;99(4):1642-9. doi: 10.1104/pp.99.4.1642.
The expression of Delta(1)-pyrroline-5-carboxylate reductase (P5CR) gene was found to be higher in soybean root nodules than in leaves and roots, and its expression in roots appeared to be osmoregulated (AJ Delauney, DPS Verma [1990] Mol Gen Genet 221: 299-305). P5CR was purified to homogeneity as a monomeric protein of 29 kilodaltons by overexpression of a soybean P5CR cDNA clone in Escherichia coli. The pH optimum of the purified P5CR was altered by increasing the salt concentration, and maximum enzyme activity was attainable at a lower pH under high salt (0.2-1 molar NaCl). Kinetic studies of the purified enzyme suggested that nicotinamide adenine dinucleotide phosphate(+) inhibited P5CR activity, whereas nicotinamide adenine dinucleotide(+) did not. Subcellular fractionation and antibodies raised against purified soybean P5CR were used to investigate location of the enzyme in different parts of soybean as well as in leaves of transgenic tobacco plants synthesizing soybean P5CR. P5CR activity was present in cytoplasm of soybean roots and nodules as well as in leaves, but in leaves, about 15% of the activity was detected in the plastid fraction. The location of P5CR was further confirmed by western blot assay of the proteins from cytosol and plastid fractions of different parts of the plant. Expression of soybean nodule cytosolic P5CR in transgenic tobacco under the control of cauliflower mosaic virus 35S promoter led to the accumulation of this protein exclusively in the cytoplasm, suggesting that the chloroplastic activity may be due to the presence of a plastid form of the enzyme. The different locations of P5CR in root and leaf suggested that proline may be synthesized in different subcellular compartments in root and leaf. Proline concentration was not significantly increased in transgenic plants exhibiting high level P5CR activity, indicating that reduction of P5C is not a rate-limiting step in proline production.
Delta(1)-吡咯啉-5-羧酸还原酶（P5CR）基因的表达在大豆根瘤中高于叶片和根，其在根中的表达似乎受到渗透调节（AJ Delauney， DPS Verma [1990] Mol Gen Genet 221: 299-305）。通过在大肠杆菌中过表达大豆 P5CR cDNA 克隆，将 P5CR 纯化至单体蛋白 29 千道尔顿的均一性。纯化的 P5CR 的 pH 最适值通过增加盐浓度而改变，并且在高盐（0.2-1 摩尔 NaCl）下可以在较低的 pH 下达到最大酶活性。纯化酶的动力学研究表明，烟酰胺腺嘌呤二核苷酸磷酸（+）抑制 P5CR 活性，而烟酰胺腺嘌呤二核苷酸（+）没有。亚细胞分级分离和针对纯化的大豆 P5CR 产生的抗体用于研究该酶在大豆不同部位以及合成大豆 P5CR 的转基因烟草叶片中的位置。P5CR 活性存在于大豆根和根瘤以及叶片的细胞质中，但在叶片中，约 15%的活性存在于质体部分中。P5CR 的位置通过对来自植物不同部位的细胞质和质体部分的蛋白质进行 western blot 分析进一步得到证实。在花椰菜花叶病毒 35S 启动子的控制下，在转基因烟草中表达大豆根瘤细胞质 P5CR 导致该蛋白仅在细胞质中积累，这表明质体活性可能是由于存在该酶的质体形式。P5CR 在根和叶中的不同位置表明脯氨酸可能在根和叶的不同亚细胞隔室中合成。在表现出高 P5CR 活性的转基因植物中，脯氨酸浓度没有显著增加，这表明 P5C 的还原不是脯氨酸产生的限速步骤。
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