Characterization and expression analyses of two plastidic enolase genes in rice

To verify the presence of enolase related to the chloroplastic glycolysis in rice, database search was carried out and identified seven putative enolase genes in the rice genome. Among them, <i>OsEno1</i> and <i>OsEno3</i> encode long proteins with N-terminal extensions. GFP protein fusions of these N-terminal extensions were both targeted to plastids of onion epidermal cell. Promoter::<i>GUS</i> analysis showed that <i>OsEno3</i> was highly expressed in young developing leaves, but its expression was drastically decreased during leaf development and greening. On the other hand, the expression of <i>OsEno1</i> was low and detected in limited portions such as leaf sheath at the tiller base. Recombinant OsEno1 protein showed enolase activity with a pH optimum at pH 8.0, whereas OsEno3 did not exhibit detectable activity. Although it remains obscure if OsEno3 encodes a functional enolase <i>in vivo</i>, our results demonstrate that the entire glycolytic pathway does not operate in rice chloroplasts.

为验证水稻中与叶绿体糖酵解相关的烯醇酶（enolase）是否存在，本研究开展数据库检索，在水稻基因组中鉴定出7个推定的烯醇酶基因。其中，OsEno1与OsEno3编码的蛋白序列较长，且带有N端延伸肽段。将这些N端延伸肽段与绿色荧光蛋白（GFP）构建融合蛋白后，二者均定位于洋葱表皮细胞的质体中。启动子::GUS报告基因分析结果显示，OsEno3在幼嫩发育叶片中呈高表达，但在叶片发育及转绿过程中其表达量会急剧下调。与之相反，OsEno1的表达量较低，仅在分蘖基部的叶鞘等有限组织中可被检测到。重组OsEno1蛋白展现出烯醇酶活性，其最适pH为8.0；而OsEno3未检测到可观测的酶活性。尽管目前尚不清楚OsEno3在体内（in vivo）是否编码具有功能的烯醇酶，但本研究结果表明，完整的糖酵解通路并不存在于水稻叶绿体中。