Laboratory of Crop Science, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601 Japan.
Plant Cell Physiol. 2010 Mar;51(3):341-53. doi: 10.1093/pcp/pcq013. Epub 2010 Jan 22.
In arbuscular mycorrhizal (AM) symbiosis, host plants supply photosynthates to AM fungi and, in return, they receive inorganic nutrients such as phosphate from finely branched fungal arbuscules. Plant cortical cells envelope arbuscules with periarbuscular membranes that are continuous with the plant plasma membranes. We prepared transgenic rice (Oryza sativa) plants that express a fusion of green fluorescent protein with rice AM-inducible phosphate transporter, OsPT11-GFP, and grew them with AM fungi. The fluorescence of the fusion transporter was observed in the arbuscule branch domain, where active nutrient exchange seems to occur. In contrast, a signal was not detected around intracellular hyphal coils on colonization by either Glomus mosseae or Gigaspora rosea, making the difference between Arum- and Paris-type mycorrhizae ambiguous. We also invented a simple device involving glass-bottomed Petri dishes for in planta observation of fluorescent proteins in living AM roots with an inverted fluorescence microscope. The plant bodies remain completely intact, avoiding any stressful procedure such as cutting, staining, etc. Since rice roots exhibit a very low level of autofluorescence, the device enabled clear time-lapse imaging to analyze the formation, function and degeneration of arbuscules. In cortical cells, arbuscules seemed to be functional for only 2-3 d. Suddenly, the arbuscular branches became fragile and they shrank. At this stage, however, the periarbuscular membranes appeared intact. Then, the fluorescence of the transporter disappeared within only 2.5-5.5 h. The collapse of arbuscules occurred in the subsequent several days. Thus, our device has a great advantage for investigation of dynamic features of AM symbiosis.
在丛枝菌根（AM）共生中，宿主植物将光合作用产物供给 AM 真菌，作为回报，真菌从细分支的菌根丛枝中接收无机养分，如磷酸盐。植物皮层细胞用周丛膜将菌根丛枝包裹起来，周丛膜与植物质膜连续。我们制备了表达与水稻 AM 诱导性磷酸盐转运体 OsPT11-GFP 融合的绿色荧光蛋白的转基因水稻（Oryza sativa）植株，并与 AM 真菌一起生长。融合转运体的荧光在活跃的养分交换似乎发生的菌根丛枝分支域中观察到。相比之下，在用 Glomus mosseae 或 Gigaspora rosea 定殖时，在细胞内菌丝线圈周围没有检测到信号，使得 Arum- 和 Paris-型菌根之间的差异变得模糊。我们还发明了一种简单的装置，涉及带有底玻璃的 Petri 盘，用于在倒置荧光显微镜下对活体 AM 根中的荧光蛋白进行植物内观察。植物本体保持完整，避免任何有压力的程序，如切割、染色等。由于水稻根表现出非常低的自发荧光，该装置能够进行清晰的延时成像，以分析菌根丛枝的形成、功能和退化。在皮层细胞中，菌根丛枝似乎只具有 2-3d 的功能。突然，菌根丛枝的分支变得脆弱并收缩。在这个阶段，周丛膜似乎是完整的。然后，在 2.5-5.5 小时内，转运体的荧光消失。菌根丛枝在随后的几天内崩溃。因此，我们的装置在研究 AM 共生的动态特征方面具有很大的优势。
