Department of Molecular Biology and Radiobiology, Faculty of Agronomy, Mendel University in Brno, Zemědělská 1, CZ-61300 Brno, Czech Republic.
Phytochemistry. 2012 Jul;79:67-77. doi: 10.1016/j.phytochem.2012.03.012. Epub 2012 May 1.
Cytokinin (CK) activity is regulated by the complex interplay of their metabolism, transport, stability and cellular/tissue localization. O-glucosides of zeatin-type CKs are postulated to be storage and/or transport forms. Active CK levels are determined in part by their differential distribution of CK metabolites across different subcellular compartments. We have previously shown that overexpressing chloroplast-localized Zm-p60.1, a maize β-glucosidase capable of releasing active cytokinins from their O- and N3-glucosides, perturbs CK homeostasis in transgenic tobacco. We obtained tobacco (Nicotiana tabacum L., cv Petit Havana SR1) plants overexpressing a recombinant Zm-p60.1 that is targeted to the vacuole. The protein is correctly processed and localized to the vacuole. When grown on medium containing exogenous zeatin, transgenic seedlings rapidly accumulate fresh weight due to ectopic growths at the base of the hypocotyl. The presence of the enzyme in these ectopic structures is shown by histochemical staining. CK quantification reveals that these transgenic seedlings are unable to accumulate zeatin-O-glucoside to levels similar to those observed in the wild type. When crossed with tobacco overexpressing the zeatin-O-glucosyltransferase gene from Phaseolus, the vacuolar variant shows an almost complete reversion in the root elongation assay. This is the first evidence from intact plants that the vacuole is the storage organelle for CK O-glucosides and that they are available to attack by Zm-p60.1. We propose the use of Zm-p60.1 as a robust molecular tool that exploits the reversibility of O-glucosylation and enables delicate manipulations of active CK content at the cellular level.
细胞分裂素 (CK) 的活性受其代谢、运输、稳定性和细胞/组织定位的复杂相互作用调节。玉米素型 CK 的 O-葡糖苷被假定为储存和/或运输形式。细胞分裂素活性水平部分取决于其在不同亚细胞隔室中 CK 代谢物的差异分布。我们之前已经表明，过量表达定位于叶绿体的Zm-p60.1（一种能够从 O-和 N3-葡糖苷中释放活性细胞分裂素的玉米β-葡糖苷酶）会扰乱转基因烟草中的 CK 动态平衡。我们获得了过表达靶向液泡的重组Zm-p60.1 的烟草（Nicotiana tabacum L.，cv Petit Havana SR1）植株。该蛋白被正确加工并定位于液泡。当在含有外源玉米素的培养基上生长时，由于下胚轴基部的异位生长，转基因幼苗会迅速积累鲜重。在这些异位结构中存在该酶，这可以通过组织化学染色来证明。细胞分裂素定量分析表明，这些转基因幼苗无法将玉米素-O-葡糖苷积累到与野生型相似的水平。当与过量表达菜豆 zeatin-O-葡糖基转移酶基因的烟草杂交时，液泡变体在根伸长测定中几乎完全逆转。这是完整植物中第一个证据，表明液泡是 CK O-葡糖苷的储存细胞器，并且它们可被 Zm-p60.1 攻击。我们建议使用 Zm-p60.1 作为一种强大的分子工具，利用 O-葡糖苷化的可逆性，并能够在细胞水平上对活性 CK 含量进行精细操作。
