Neutral red as a probe for confocal laser scanning microscopy studies of plant roots
Neutral red as a probe for confocal laser scanning microscopy studies of plant roots
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DOI：
10.1093/aob/mcl045
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发表时间：
2006-06-01
期刊：
影响因子：
4.2
通讯作者：
Menzel, Diedrik
中科院分区：
文献类型：
--
作者：
Dubrovsky, Joseph G.;Guttenberger, Martin;Menzel, Diedrik
中性红（NR）是一种亲脂性的非那嗪染料，作为一种重要的亮场显微镜染色剂，已广泛应用于各种生物系统中。在其未质子化的形式下，它穿透有活力的植物细胞的质膜和质体，然后由于质子化，它被困在酸性隔间中。从植物根系生物学的各个方面探讨了NR在共聚焦激光扫描显微镜（CLSM）研究中的可能应用。方法采用NR作为荧光染料，对野生型和转基因gfp携带系菜豆、韭菜、麻瓜和拟南芥的活根进行荧光检测。用CLSM显示组织。分析了NR对拟南芥细胞骨架完整性和原生根生长速率的影响，以判断该染料的潜在毒性作用。使用NR的主要优点与NR能迅速渗透根组织，与木质素和木质素有亲和力，并能在液泡中积累有关。结果表明，NR是一种合适的探针，可用于观察活根细胞中的原木质部和化木质部元素、内胚层中的Caspafian条带和液泡。短期施用NR后，肌动蛋白细胞骨架和细胞微管系统以及根系生长动态基本保持不变，表明该化学品毒性较低。还发现NR是观察泡状-丛枝菌根根瘤和真菌菌丝内部结构的有效探针。结论NR探针简单、成本低、无需组织处理，是CLSM研究植物根系结构、发育和液泡生物发生的有效探针。
center dot Background and Aims Neutral red (NR), a lipophilic phenazine dye, has been widely used in various biological systems as a vital stain for bright-field microscopy. In its unprotonated form it penetrates the plasma membrane and tonoplast of viable plant cells, then due to protonation it becomes trapped in acidic compartments. The possible applications of NR for confocal laser scanning microscopy (CLSM) studies were examined in various aspects of plant root biology.center dot Methods NR was used as a fluorochrome for living roots of Phaseolus vulgaris, Allium cepa, A. porrum and Arabidopsis thaliana (wild-type and transgenic GFP-carrying lines). The tissues were visualized using CLSM. The effect of NR on the integrity of the cytoskeleton and the growth rate of arabidopsis primary roots was analysed to judge potential toxic effects of the dye.center dot Key Results The main advantages of the use of NR are related to the fact that NR rapidly penetrates root tissues, has affinity to suberin and lignin, and accumulates in the vacuoles. It is shown that NR is a suitable probe for visualization of proto- and metaxylem elements, Caspafian bands in the endodermis, and vacuoles in cells of living roots. The actin cytoskeleton and the microtubule system of the cells, as well as the dynamics of root growth, remain unchanged after short-term application of NR, indicating a relatively low toxicity of this chemical. It was also found that NR is a useful probe for the observation of the internal structures of root nodules and of fungal hyphae in vesicular-arbuscular mycorrhizas.center dot Conclusions Ease, low cost and absence of tissue processing make NR a useful probe for structural, developmental and vacuole-biogenetic studies of plant roots with CLSM.