Shimamura Masaki, Brown Roy C, Lemmon Betty E, Akashi Tomohiro, Mizuno Koichi, Nishihara Naohisa, Tomizawa Ken-Ichi, Yoshimoto Katsuhiko, Deguchi Hironori, Hosoya Hiroshi, Horio Tetsuya, Mineyuki Yoshinobu
Department of Biological Science, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.
Plant Cell. 2004 Jan;16(1):45-59. doi: 10.1105/tpc.016501. Epub 2003 Dec 5.
Although seed plants have gamma-tubulin, a ubiquitous component of centrosomes associated with microtubule nucleation in algal and animal cells, they do not have discrete microtubule organizing centers (MTOCs) comparable to animal centrosomes, and the organization of microtubule arrays in plants has remained enigmatic. Spindle development in basal land plants has revealed a surprising variety of MTOCs that may represent milestones in the evolution of the typical diffuse acentrosomal plant spindle. We have isolated and characterized the gamma-tubulin gene from a liverwort, one of the extant basal land plants. Sequence similarity to the gamma-tubulin gene of higher plants suggests that the gamma-tubulin gene is highly conserved in land plants. The G9 antibody to fission yeast gamma-tubulin recognized a single band of 55 kD in immunoblots from bryophytes. Immunohistochemistry with the G9 antibody clearly documented the association of gamma-tubulin with various MTOC sites in basal land plants (e.g., discrete centrosomes with and without centrioles and the plastid surface in monoplastidic meiosis of bryophytes). Changes in the distribution of gamma-tubulin occur in a cell cycle-specific manner during monoplastidic meiosis in the liverwort Dumortiera hirsuta. gamma-Tubulin changes its localization from the plastid surface in prophase I to the spindle, from the spindle to phragmoplasts and the nuclear envelope in telophase I, and back to the plastid surfaces in prophase II. In vitro experiments show that gamma-tubulin is detectable on the surface of isolated plastids and nuclei of D. hirsuta, and microtubules can be repolymerized from the isolated plastids. gamma-Tubulin localization patterns on plastid and nuclear surfaces are not affected by the destruction of microtubules by oryzalin. We conclude that gamma-tubulin is a highly conserved protein associated with microtubule nucleation in basal land plants and that it has a cell cycle-dependent distribution essential for the orderly succession of microtubule arrays.
尽管种子植物含有γ-微管蛋白，这是一种在藻类和动物细胞中与微管成核相关的中心体普遍存在的成分，但它们没有与动物中心体相当的离散微管组织中心（MTOC），植物中微管阵列的组织方式一直是个谜。基部陆地植物中的纺锤体发育揭示了各种各样的MTOC，这些MTOC可能代表了典型的弥散无中心体植物纺锤体进化过程中的里程碑。我们从现存的基部陆地植物之一地钱中分离并鉴定了γ-微管蛋白基因。与高等植物γ-微管蛋白基因的序列相似性表明，γ-微管蛋白基因在陆地植物中高度保守。针对裂殖酵母γ-微管蛋白的G9抗体在苔藓植物的免疫印迹中识别出一条55 kD的单带。用G9抗体进行的免疫组织化学清楚地证明了γ-微管蛋白与基部陆地植物中各种MTOC位点的关联（例如，有和没有中心粒的离散中心体以及苔藓植物单倍体减数分裂中的质体表面）。在多毛地钱的单倍体减数分裂过程中，γ-微管蛋白的分布变化以细胞周期特异性的方式发生。γ-微管蛋白的定位从前期I的质体表面变为纺锤体，从纺锤体变为末期I的成膜体和核膜，然后在前期II又回到质体表面。体外实验表明，在多毛地钱分离的质体和细胞核表面可检测到γ-微管蛋白，并且微管可以从分离的质体中重新聚合。质体和核表面上的γ-微管蛋白定位模式不受oryzalin破坏微管的影响。我们得出结论，γ-微管蛋白是一种与基部陆地植物中微管成核相关的高度保守蛋白，并且它具有细胞周期依赖性分布，这对于微管阵列的有序连续至关重要。
