Isolation and characterization of a novel peroxisomal choline monooxygenase in barley
Isolation and characterization of a novel peroxisomal choline monooxygenase in barley
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DOI：
10.1007/s00425-011-1478-9
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发表时间：
2011-07
期刊：
影响因子：
4.3
通讯作者：
Shiro Mitsuya;J. Kuwahara;Keiko Ozaki;E. Saeki;T. Fujiwara;T. Takabe
中科院分区：
文献类型：
--
作者：
Shiro Mitsuya;J. Kuwahara;Keiko Ozaki;E. Saeki;T. Fujiwara;T. Takabe
甘氨酸甜菜碱（GB）是许多植物在各种非生物胁迫下积累的相容性溶质。 GB 的合成分两个步骤，胆碱 → 甜菜碱醛 → GB，迄今为止，仅在苋科植物（一种叶绿体铁氧还蛋白依赖性胆碱单加氧酶）中报道了功能性胆碱氧化酶。在这里，我们从大麦 (Hordeum vulgare) 植物中克隆了编码胆碱单加氧酶 (CMO) 的 cDNA，HvCMO。在非胁迫条件下的大麦植株中，GB在所有确定的器官（叶、节间、芒和小花）中积累，其中大部分在叶中。 HvCMO蛋白在叶片中表达较丰富，而甜菜碱醛脱氢酶(BADH)蛋白在芒、小花本体和最幼节间的表达较叶片丰富。高渗透压和低温环境会增加HvCMOmRNA的积累。此外，高 NaCl 的存在会增加 HvCMO 蛋白的表达。 HvCMO蛋白的免疫荧光标记和亚细胞分级分析表明HvCMO蛋白定位于过氧化物酶体。 [14C]胆碱在菠菜（Spinacia oleracea）叶绿体中被氧化为甜菜碱醛和GB，但在大麦中则不然，这表明胆碱氧化酶的亚细胞定位在两种植物物种之间是不同的。我们使用酵母（酿酒酵母）中表达的重组 HvCMO 蛋白研究了胆碱氧化反应。当添加 NADPH 作为辅因子时，表达 HvCMO 的酵母粗提物与重组 BBD2 蛋白结合，将 [14C] 胆碱转化为 GB。这些结果表明，GB 合成中的胆碱氧化是由大麦植物中的过氧化物酶体 NADPH 依赖性胆碱单加氧酶介导的。
Glycine betaine (GB) is a compatible solute accumulated by many plants under various abiotic stresses. GB is synthesized in two steps, choline → betaine aldehyde → GB, where a functional choline-oxidizing enzyme has only been reported in Amaranthaceae (a chloroplastic ferredoxin-dependent choline monooxygenase) thus far. Here, we have cloned a cDNA encoding a choline monooxygenase (CMO) from barley (Hordeum vulgare) plants,HvCMO. In barley plants under non-stress condition, GB had accumulated in all the determined organs (leaves, internodes, awn and floret proper), mostly in the leaves. The expression of HvCMO protein was abundant in the leaves, whereas the expression of betaine aldehyde dehydrogenase (BADH) protein was abundant in the awn, floret proper and the youngest internode than in the leaves. The accumulation ofHvCMOmRNA was increased by high osmotic and low-temperature environments. Also, the expression of HvCMO protein was increased by the presence of high NaCl. Immunofluorescent labeling of HvCMO protein and subcellular fractionation analysis showed that HvCMO protein was localized to peroxisomes. [14C]choline was oxidized to betaine aldehyde and GB in spinach (Spinacia oleracea) chloroplasts but not in barley, which indicates that the subcellular localization of choline-oxidizing enzyme is different between two plant species. We investigated the choline-oxidizing reaction using recombinant HvCMO protein expressed in yeast (Saccharomyces cerevisiae). The crude extract of HvCMO-expressing yeast coupled with recombinant BBD2 protein converted [14C]choline to GB when NADPH was added as a cofactor. These results suggest that choline oxidation in GB synthesis is mediated by a peroxisomal NADPH-dependent choline monooxygenase in barley plants.