Shinohara Naoki, Sugiyama Munetaka, Fukuda Hiroo
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo, 113-0033 Tokyo, Japan.
Planta. 2006 Jul;224(2):394-404. doi: 10.1007/s00425-006-0224-1. Epub 2006 Feb 1.
In an optimized liquid medium containing auxin and cytokinin, mesophyll cells isolated from Zinnia elegans L. seedlings can be induced to differentiate into tracheary elements (TEs) at high frequency. However, it is known that buffering the medium at neutral pH severely suppresses TE differentiation. In the process of modifying the medium, we found that excessive administration of auxin restored the suppression. Based on this finding, we physiologically characterized auxin actions involved in TE differentiation by focusing on the influence of extracellular pH. First, dose/response relationships between auxin [1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D)] concentrations and differentiated cell ratios were determined under various extracellular pH conditions. Secondly, intracellular concentrations of free forms and metabolites of auxin species were determined by analyzing extracts from cells cultured with radiolabeled NAA and 2,4-D under different extracellular pH conditions with liquid scintillation counting and thin-layer chromatography autoradiograms. Higher extracellular pH was found to reduce both the auxin potency for inducing TE differentiation and intracellular auxin accumulation. Reduction levels correlatively varied depending on the auxin species. These results suggest that the weakening in auxin potency at higher extracellular pH is ascribed to lower auxin uptake, which leads to decreased intracellular perception of the auxin signal. A model to predict auxin action that considers membrane transport, metabolism, and the perception of auxin is also presented.
在含有生长素和细胞分裂素的优化液体培养基中，从百日草幼苗分离的叶肉细胞可被高频诱导分化为管状分子（TEs）。然而，已知将培养基缓冲至中性pH会严重抑制TE分化。在改良培养基的过程中，我们发现过量施用生长素可恢复这种抑制作用。基于这一发现，我们通过关注细胞外pH的影响，对参与TE分化的生长素作用进行了生理学表征。首先，在各种细胞外pH条件下，测定了生长素[1-萘乙酸（NAA）和2,4-二氯苯氧乙酸（2,4-D）]浓度与分化细胞比例之间的剂量/反应关系。其次，通过液体闪烁计数和薄层色谱放射自显影片分析在不同细胞外pH条件下用放射性标记的NAA和2,4-D培养的细胞提取物，测定生长素种类的游离形式和代谢物的细胞内浓度。发现较高的细胞外pH会降低生长素诱导TE分化的效力以及细胞内生长素的积累。降低水平根据生长素种类而相关变化。这些结果表明，在较高细胞外pH下生长素效力的减弱归因于较低的生长素摄取，这导致细胞内生长素信号感知的降低。还提出了一个预测生长素作用的模型，该模型考虑了膜转运、代谢和生长素的感知。
