Docquier Sarah, Tillemans Vinciane, Deltour Roger, Motte Patrick
Laboratory of Plant Cell Biology, Department of Life Sciences, University of Liège, Bld du Rectorat, 27 Sart Tilman, 4000 Liège, Belgium.
Chromosoma. 2004 Feb;112(5):255-66. doi: 10.1007/s00412-003-0271-3. Epub 2004 Jan 23.
We studied the fine structural organization of nuclear bodies in the root meristem during germination of maize and Arabidopsis thaliana using electron microscopy (EM). Cajal bodies (CBs) were observed in quiescent embryos and germinating cells in both species. The number and distribution of CBs were investigated. To characterize the nuclear splicing domains, immunofluorescence labelling with antibodies against splicing factors (U2B" and m3G-snRNAs) and in situ hybridisation (with U1/U6 antisense probes) were performed combined with confocal microscopy. Antibodies specific to the Arabidopsis SR splicing factor atRSp31 were produced. AtRSp31 was detected in quiescent nuclei and in germinating cells. This study revealed an unexpected speckled nuclear organization of atRSp31 in root epidermal cells where micro-clusters of interchromatin granules were also observed by EM. Therefore, we examined the distribution of green fluorescent protein (GFP)-tagged atRSp31 in living cells after Agrobacterium -mediated transient expression. When expressed transiently, atRSp31-GFP exhibited a speckled distribution in leaf cells. Treatments with alpha-amanitin, okadaic acid, staurosporine or heat shock induced the speckles to reorganize. Furthermore, we generated stable Arabidopsis transgenics expressing atRSp31-GFP. The distribution of the fusion protein was identical to that of endogenous atRSp31. Three-dimensional time-lapse confocal microscopy showed that speckles were highly dynamic domains over time.
我们使用电子显微镜（EM）研究了玉米和拟南芥萌发过程中根分生组织中核体的精细结构组织。在这两个物种的静止胚胎和萌发细胞中均观察到了卡哈尔体（CBs）。我们研究了CBs的数量和分布。为了表征核剪接结构域，我们结合共聚焦显微镜，用针对剪接因子（U2B″和m3G - snRNAs）的抗体进行免疫荧光标记，并进行原位杂交（使用U1/U6反义探针）。制备了针对拟南芥SR剪接因子atRSp31的特异性抗体。在静止细胞核和萌发细胞中检测到了atRSp31。这项研究揭示了根表皮细胞中atRSp31意外的斑点状核组织，在该组织中通过电子显微镜也观察到了染色质间颗粒的微簇。因此，我们在农杆菌介导的瞬时表达后，检测了绿色荧光蛋白（GFP）标记的atRSp31在活细胞中的分布。瞬时表达时，atRSp31 - GFP在叶细胞中呈现斑点状分布。用α - 鹅膏蕈碱、冈田酸、星形孢菌素或热休克处理会诱导这些斑点重新组织。此外，我们生成了表达atRSp31 - GFP的稳定拟南芥转基因植株。融合蛋白的分布与内源性atRSp31的分布相同。三维延时共聚焦显微镜显示，随着时间的推移，斑点是高度动态的结构域。
