在橄榄（Olea europaea）花粉萌发过程中酯酶的分析和功能分类。
Profiling and functional classification of esterases in olive (Olea europaea) pollen during germination.
机构信息
Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), Granada, Spain.
出版信息
Ann Bot. 2012 Oct;110(5):1035-45. doi: 10.1093/aob/mcs174. Epub 2012 Aug 24.
BACKGROUND AND AIMS
A pollen grain contains a number of esterases, many of which are released upon contact with the stigma surface. However, the identity and function of most of these esterases remain unknown. In this work, esterases from olive pollen during its germination were identifided and functionally characterized.
METHODS
The esterolytic capacity of olive (Olea europaea) pollen was examined using in vitro and in-gel enzymatic assays with different enzyme substrates. The functional analysis of pollen esterases was achieved by inhibition assays by using specific inhibitors. The cellular localization of esterase activities was performed using histochemical methods.
KEY RESULTS
Olive pollen showed high levels of non-specific esterase activity, which remained steady after hydration and germination. Up to 20 esterolytic bands were identified on polyacrylamide gels. All the inhibitors decreased pollen germinability, but only diisopropyl fluorophosphate (DIFP) hampered pollen tube growth. Non-specific esterase activity is localized on the surface of oil bodies (OBs) and small vesicles, in the pollen intine and in the callose layer of the pollen tube wall. Acetylcholinesterase (AChE) activity was mostly observed in the apertures, exine and pollen coat, and attached to the pollen tube wall surface and to small cytoplasmic vesicles.
CONCLUSIONS
In this work, for the first time a systematic functional characterization of esterase enzymes in pollen from a plant species with wet stigma has been carried out. Olive pollen esterases belong to four different functional groups: carboxylesterases, acetylesterases, AChEs and lipases. The cellular localization of esterase activity indicates that the intine is a putative storage site for esterolytic enzymes in olive pollen. Based on inhibition assays and cellular localization of enzymatic activities, it can be concluded that these enzymes are likely to be involved in pollen germination, and pollen tube growth and penetration of the stigma.
背景与目的
花粉粒中含有多种酯酶，其中许多在与柱头表面接触时会被释放出来。然而，这些酯酶中的大多数的身份和功能仍然未知。在这项工作中，鉴定并研究了橄榄花粉在萌发过程中的酯酶。
方法
使用不同酶底物的体外和凝胶内酶促分析检测橄榄（Olea europaea）花粉的酯解能力。通过使用特异性抑制剂进行抑制测定来实现花粉酯酶的功能分析。通过组织化学方法进行酯酶活性的细胞定位。
主要结果
橄榄花粉显示出高水平的非特异性酯酶活性，在水合和萌发后保持稳定。在聚丙烯酰胺凝胶上鉴定出多达 20 个酯解带。所有抑制剂均降低花粉萌发率，但只有二异丙基氟磷酸（DIFP）阻碍花粉管生长。非特异性酯酶活性定位于油体（OB）和小泡、花粉内壁和花粉管细胞壁的胼胝质层表面。乙酰胆碱酯酶（AChE）活性主要观察到在孔、外壁和花粉外壁，附着在花粉管壁表面和小细胞质泡上。
结论
在这项工作中，首次对具有湿柱头的植物物种花粉中的酯酶进行了系统的功能表征。橄榄花粉酯酶属于四个不同的功能组：羧酸酯酶、乙酰酯酶、AChE 和脂肪酶。酯酶活性的细胞定位表明，内壁是橄榄花粉中酯解酶的潜在储存部位。基于抑制测定和酶活性的细胞定位，可以得出结论，这些酶可能参与花粉萌发以及花粉管生长和柱头穿透。
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