Ezawa Tatsuhiro, Cavagnaro Timothy R, Smith Sally E, Smith F Andrew, Ohtomo Ryo
Graduate School of Bioagricultural Science, Nagoya University, Togo-cho, Aichi 470-0151 Japan.
Soil and Land Systems, School of Earth and Environmental Sciences, The University of Adelaide, SA 5005 Australia.
New Phytol. 2004 Feb;161(2):387-392. doi: 10.1046/j.1469-8137.2003.00966.x. Epub 2003 Dec 12.
The rate of polyphosphate accumulation in extraradical hyphae of an arbuscular mycorrhizal fungus was investigated by conventional histochemistry and a new enzymatic method using a bacterial enzyme, polyphosphate kinase. Marigold (Tagetes patula cv. Bonanza Orange) was inoculated with Archaeospora leptoticha and grown under P-deficient conditions. Extraradical hyphae were harvested at 0, 1, 3 and 24 h after 1 mm P-application. PolyP levels were assessed by both metachromasy of Toluidine blue O and polyphosphate kinase which converted polyP to ATP followed by the ATP-luciferase assay. Percentage of hyphae with metachromatic granules was increased from 25 to 44% from 0 to 1 h, and a maximum of 50% was reach by 3 h. Polyphosphate content was doubled from 1 to 3 h after P-application (4.8-10.0 mol as Pi mg protein) at a rate of 46.4 ± 15.1 nmol min mg . The rate of polyphosphate accumulation in the hyphae was surprisingly rapid as those of polyphosphate-hyper accumulating microorganisms. The enzymatic method employed in the present study allows highly specific and sensitive assessment of polyphosphate in the mycorrhizal system.
通过传统组织化学方法以及一种使用细菌酶——多聚磷酸激酶的新酶法，研究了丛枝菌根真菌根外菌丝中多聚磷酸盐的积累速率。用细凹无梗囊霉接种万寿菊（孔雀草品种Bonanza Orange），并在缺磷条件下培养。在施加1毫米磷后0、1、3和24小时收获根外菌丝。通过甲苯胺蓝O的异染性以及多聚磷酸激酶（其将多聚磷酸转化为ATP，随后进行ATP - 荧光素酶测定）来评估多聚磷酸盐水平。带有异染颗粒的菌丝百分比从0小时的25%增加到1小时的44%，到3小时达到最大值50%。施加磷后1至3小时，多聚磷酸盐含量翻倍（以磷酸根计为4.8 - 10.0微摩尔/毫克蛋白质），速率为46.4±15.1纳摩尔/分钟/毫克。菌丝中多聚磷酸盐的积累速率与多聚磷酸盐超积累微生物的速率一样惊人地快。本研究中采用的酶法能够对菌根系统中的多聚磷酸盐进行高度特异性和灵敏的评估。
