Identification and molecular characterization of propionylcholinesterase, a novel pseudocholinesterase in rice

Cholinesterase is consisting of acetylcholinesterase (AChE) and pseudocholinesterase in vertebrates and invertebrates. AChE gene has been identified in several plant species, while pseudocholinesterase gene has not yet been found in any plant species. In this study, we report that the AChE gene paralog encodes propionylcholinesterase (PChE), a pseudocholinesterase in rice. PChE was found to be located adjacent to AChE (Os07g0586200) on rice chromosome 7 and designated as Os07g0586100. Phylogenetic tree analysis showed a close relationship between rice AChE and PChE. PChE-overexpressing rice had higher hydrolytic activity toward propionylthiocholine than acetylthiocholine and showed extremely low activity against butyrylthiocholine. Therefore, the PChE gene product was characterized as a propionylcholinesterase, a pseudocholinesterase. The rice PChE displayed lower sensitivity to the cholinesterase inhibitor, neostigmine bromide, than electric eel, maize, and rice AChEs. The recombinant PChE functions as a 171 kDa homotetramer. PChE was expressed during the later developmental stage, and it was found be localized in the extracellular spaces of the rice leaf tissue. These results suggest that the rice plant possesses PChE, which functions in the extracellular spaces at a later developmental stage. To the best of our knowledge, this study provides the first direct evidence and molecular characterization of PChE in plants.

胆碱酯酶（Cholinesterase）在脊椎动物与无脊椎动物体内由乙酰胆碱酯酶（acetylcholinesterase，AChE）和假性胆碱酯酶构成。目前已有多种植物物种中鉴定出AChE基因，但尚未在任何植物中发现假性胆碱酯酶基因。本研究发现，水稻中一类AChE基因旁系同源体可编码丙酰胆碱酯酶（propionylcholinesterase，PChE），该酶属于水稻中的假性胆碱酯酶。PChE定位于水稻7号染色体上AChE基因（Os07g0586200）的邻近区域，其基因编号为Os07g0586100。系统发育树分析显示，水稻AChE与PChE之间存在紧密的进化关联。过表达PChE的水稻植株对丙酰硫代胆碱的水解活性高于乙酰硫代胆碱，而对丁酰硫代胆碱的水解活性极低。因此，本研究将PChE的基因产物鉴定为丙酰胆碱酯酶，即一种假性胆碱酯酶。相较于电鳗、玉米及水稻的AChE，水稻PChE对胆碱酯酶抑制剂溴新斯的明（neostigmine bromide）的敏感性更低。重组PChE以171 kDa同源四聚体的形式发挥功能。PChE在水稻发育后期表达，并定位于水稻叶片组织的细胞外间隙。上述结果表明，水稻植株确实存在PChE，该酶在发育后期的细胞外间隙中行使生物学功能。据我们所知，本研究首次为植物中存在PChE提供了直接实验证据，并完成了其分子特征鉴定。