Nguyen Suong T T, McCurdy David W
Centre for Plant Science, School of Environmental and Life Sciences, The University of Newcastle, Newcastle, NSW, 2308, Australia.
BMC Plant Biol. 2015 Apr 23;15:109. doi: 10.1186/s12870-015-0483-8.
Transfer cells (TCs) are trans-differentiated versions of existing cell types designed to facilitate enhanced membrane transport of nutrients at symplasmic/apoplasmic interfaces. This transport capacity is conferred by intricate wall ingrowths deposited secondarily on the inner face of the primary cell wall, hence promoting the potential trans-membrane flux of solutes and consequently assigning TCs as having key roles in plant growth and productivity. However, TCs are typically positioned deep within tissues and have been studied mostly by electron microscopy. Recent advances in fluorophore labelling of plant cell walls using a modified pseudo-Schiff-propidium iodide (mPS-PI) staining procedure in combination with high-resolution confocal microscopy have allowed visualization of cellular details of individual tissue layers in whole mounts, hence enabling study of tissue and cellular architecture without the need for tissue sectioning. Here we apply a simplified version of the mPS-PI procedure for confocal imaging of cellulose-enriched wall ingrowths in vascular TCs at the whole tissue level.
The simplified mPS-PI staining procedure produced high-resolution three-dimensional images of individual cell types in vascular bundles and, importantly, wall ingrowths in phloem parenchyma (PP) TCs in minor veins of Arabidopsis leaves and companion cell TCs in pea. More efficient staining of tissues was obtained by replacing complex clearing procedures with a simple post-fixation bleaching step. We used this modified procedure to survey the presence of PP TCs in other tissues of Arabidopsis including cotyledons, cauline leaves and sepals. This high-resolution imaging enabled us to classify different stages of wall ingrowth development in Arabidopsis leaves, hence enabling semi-quantitative assessment of the extent of wall ingrowth deposition in PP TCs at the whole leaf level. Finally, we conducted a defoliation experiment as an example of using this approach to statistically analyze responses of PP TC development to leaf ablation.
Use of a modified mPS-PI staining technique resulted in high-resolution confocal imaging of polarized wall ingrowth deposition in TCs. This technique can be used in place of conventional electron microscopy and opens new possibilities to study mechanisms determining polarized deposition of wall ingrowths and use reverse genetics to identify regulatory genes controlling TC trans-differentiation.
传递细胞（TCs）是现有细胞类型经转分化形成的，旨在促进共质体/质外体界面处营养物质的增强膜转运。这种转运能力由次生沉积在初生细胞壁内表面的复杂壁内突赋予，从而促进溶质的潜在跨膜通量，因此赋予传递细胞在植物生长和生产力方面的关键作用。然而，传递细胞通常位于组织深处，并且大多通过电子显微镜进行研究。最近，使用改良的假席夫 - 碘化丙啶（mPS - PI）染色程序结合高分辨率共聚焦显微镜对植物细胞壁进行荧光团标记的进展，使得能够在整装片中可视化单个组织层的细胞细节，从而无需组织切片就能研究组织和细胞结构。在这里，我们应用mPS - PI程序的简化版本，对整个组织水平上维管束中富含纤维素的壁内突进行共聚焦成像，以观察传递细胞。
简化的mPS - PI染色程序产生了维管束中单个细胞类型的高分辨率三维图像，重要的是，还产生了拟南芥叶片小叶脉韧皮薄壁细胞（PP）传递细胞和豌豆伴胞传递细胞中的壁内突图像。通过用简单的固定后漂白步骤取代复杂的清除程序，获得了更有效的组织染色。我们使用这种改良程序来调查拟南芥其他组织（包括子叶、茎生叶和萼片）中PP传递细胞的存在情况。这种高分辨率成像使我们能够对拟南芥叶片壁内突发育的不同阶段进行分类，从而能够在整个叶片水平上对PP传递细胞中壁内突沉积程度进行半定量评估。最后，我们进行了去叶实验，作为使用这种方法统计分析PP传递细胞发育对叶片切除反应的一个例子。
使用改良的mPS - PI染色技术实现了对传递细胞壁内突极化沉积的高分辨率共聚焦成像。该技术可用于替代传统电子显微镜，并为研究决定壁内突极化沉积的机制以及利用反向遗传学鉴定控制传递细胞转分化的调控基因开辟了新的可能性。
