Large-scale histological analysis of leaf mutants using two simple leaf observation methods: identification of novel genetic pathways governing the size and shape of leaves
Large-scale histological analysis of leaf mutants using two simple leaf observation methods: identification of novel genetic pathways governing the size and shape of leaves
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DOI：
10.1111/j.1365-313x.2006.02896.x
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发表时间：
2006-11-01
期刊：
影响因子：
7.2
通讯作者：
Tsukaya, Hirokazu
中科院分区：
文献类型：
--
作者：
Horiguchi, Gorou;Fujikura, Ushio;Tsukaya, Hirokazu
对细胞组织的观察对于理解叶片形态发生的机制是必不可少的。这些观察需要几个准备步骤，如固定和清除器官，这些程序对于大规模分析来说既耗时又费力。因此，我们开发了观察叶表皮和叶肉细胞的简单方法。为了显示表皮，对叶子表面进行了凝胶浇注，然后在光学显微镜下进行观察。为了观察叶片叶肉细胞，将叶片浸泡在含有Triton X-100的溶液中，短暂离心，然后在光学显微镜下观察。这些方法使我们能够通过测量各种参数，包括叶片内细胞的数量、大小和分布，对大量已知的和新分离的拟南芥叶形/大小突变体进行组织表型分析。突变体表现出由细胞数量和/或大小的特定增加或减少引起的叶片大小的变化。此外，还观察到叶片中细胞分布的变化，这是由于沿近、远轴或中轴的细胞数量的增加或减少，或者是沿特定轴的细胞重新聚集而损害了叶片的其他部分。这些结果提供了器官大小控制和叶形模式所涉及的细胞行为的表观视图。
Observations of cellular organization are essential in understanding the mechanisms underlying leaf morphogenesis. These observations require several preparative steps, such as fixation and clearing of organs, and such procedures are time-consuming and labor-intensive for large-scale analyses. Thus, we have developed simple methods for the observation of leaf epidermal and mesophyll cells. To visualize the epidermis, a gel cast was made of the leaf surface, which was then observed under a light microscope. To visualize the leaf mesophyll cells, leaves were immersed in a solution containing Triton X-100, briefly centrifuged, and then viewed under a light microscope. These methods allowed us to conduct a histological phenome analysis for a large number of known and newly isolated leaf-shape/size mutants of Arabidopsis thaliana by measuring various parameters, including cell number, size, and distribution of cells within a leaf blade. Mutants showed changes in leaf size caused by specific increases or decreases in the number and/or size of cells. In addition, altered cell distributions in the leaf blade were observed, resulting from increases or decreases in the number of cells along the proximo-distal or medio-lateral axis, or recruitment of cells along a particular axis at the expense of other leaf parts. These results provide a phenomic view of the cellular behavior involved in organ size control and leaf-shape patterning.