Eudes Aymerick, Mouille Grégory, Thévenin Johanne, Goyallon Arnaud, Minic Zoran, Jouanin Lise
INRA, Centre de Versailles, Institut Jean-Pierre Bourgin, Laboratoire de Biologie Cellulaire, 78026 Versailles cedex, France.
Plant Cell Physiol. 2008 Sep;49(9):1331-41. doi: 10.1093/pcp/pcn108. Epub 2008 Jul 30.
Beta-glucuronidase (GUS) activities have been extensively characterized in bacteria, fungi, and animals, and the bacterial enzyme GUSA from Escherichia coli is commonly used as a reporter for gene expression studies in plants. Although endogenous GUS activity has been observed in plants, the nature and function of the enzymes involved remain elusive. Here we report on tissue-specific localization, partial purification and identification of AtGUS2, a GUS active under acidic conditions from Arabidopsis thaliana. This enzyme belongs to the GH79 family in the Carbohydrate-Active Enzymes database, which also includes mammalian heparanases that degrade the carbohydrate moieties of cell surface proteoglycans, and fungal enzymes active on arabinogalactan proteins (AGPs). We characterized a knockout insertion line (atgus2-1) and transgenic lines overexpressing AtGUS2 (Pro(35S):AtGUS2). Endogenous GUS activity assayed histochemically and biochemically was absent in atgus2-1 tissues and four times higher in Pro(35S):AtGUS2 lines. AGPs purified from atgus2-1 and Pro(35S):AtGUS2 seedlings showed higher and markedly lower glucuronic acid content, respectively. Our results suggest that endogenous GUS activity influences the sugar composition of the complex polysaccharide chains of AGPs. We also show that transgenics display hypocotyl and root growth defects compared to wild-type plants. Hypocotyl and root lengths are increased in Pro(35S):AtGUS2 seedlings, whereas hypocoyl length is reduced in atgus2-1 seedlings. These data are consistent with a role for the carbohydrate moieties of AGPs in cell growth.
β-葡萄糖醛酸酶（GUS）活性已在细菌、真菌和动物中得到广泛表征，大肠杆菌中的细菌酶GUSA常用于植物基因表达研究的报告基因。尽管在植物中已观察到内源性GUS活性，但所涉及酶的性质和功能仍不清楚。在这里，我们报告了拟南芥中一种在酸性条件下具有活性的GUS——AtGUS2的组织特异性定位、部分纯化和鉴定。该酶属于碳水化合物活性酶数据库中的GH79家族，该家族还包括降解细胞表面蛋白聚糖碳水化合物部分的哺乳动物乙酰肝素酶，以及对阿拉伯半乳聚糖蛋白（AGP）有活性的真菌酶。我们对一个敲除插入系（atgus2-1）和过表达AtGUS2的转基因系（Pro(35S):AtGUS2）进行了表征。通过组织化学和生化方法测定，atgus2-1组织中不存在内源性GUS活性，而在Pro(35S):AtGUS2系中则高出四倍。从atgus2-1和Pro(35S):AtGUS2幼苗中纯化的AGP分别显示出较高和明显较低的葡萄糖醛酸含量。我们的结果表明，内源性GUS活性影响AGP复合多糖链的糖组成。我们还表明，与野生型植物相比，转基因植物表现出下胚轴和根生长缺陷。Pro(35S):AtGUS2幼苗的下胚轴和根长度增加，而atgus2-1幼苗的下胚轴长度减少。这些数据与AGP的碳水化合物部分在细胞生长中的作用一致。
