利用锌特异性探针HL~1示踪植物细胞外Zn~(2+)的分布

以拟南芥(Arabidopsis thaliana)和谷子(Setaria italic)为研究材料,利用锌特异性探针HL1,使用荧光分光光度仪、等温滴定热量测定仪(ITC200)和倒置荧光显微镜等仪器探究了该化学探针的特性以及植物细胞外游离Zn~(2+)的分布。结果表明,当HL1与不同元素溶液混合时,只与Zn~(2+)特异性结合,在紫外光(UV)激发下,发射出波长为500 nm的蓝色荧光;生成物的平衡解离常数KD=7.02×10–4 mol·L–1,具有很好的稳定性。拟南芥叶片中的Zn~(2+)分布于细胞间隙及叶表皮毛的外周和表层,且叶表皮毛的荧光强度具有明显的浓度依赖性;谷子叶片中的Zn~(2+)分布在细胞间隙以及维管组织。拟南芥根中的Zn~(2+)分布于根的伸长区,且荧光强度也明显地表现出与浓度相关。由此推断,根伸长区与Zn~(2+)运输有关,叶的维管组织是植物细胞外运输Zn~(2+)的主要途径,细胞间隙和叶表皮毛是植物储存Zn~(2+)的主要区域。HL1适用于检测细胞外Zn~(2+)的分布。

Fluorescence Imaging of the Extracellular Zinc Distribution in Plants by Using a Highly Specific Fluorescent Probe

We chose Arabidopsis thaliana and Setaria italic as material to study the character of HL¹ that can specifically combine with zinc, and the distribution of extracellular Zn²⁺ by using fluorescence spectrophotometry, isothermal titration calorimetry (ITC200) and inverted fluorescence microscopy. Fluorescence intensity for HL¹ was greatly enhanced with the addition of Zn²⁺ but not other ions. The dissociation constant (K_D=7.02×10^-4 mol·L^-1) exhibited product stability with the combined reaction of HL¹ and Zn²⁺. In A. thaliana, the distribution of extracellular free Zn²⁺ was mainly located in leaf intercellular space and surface of the trichome where fluorescence intensity was corresponding to the concentration of Zn²⁺. The distribution of Zn²⁺ were located in intercellular space and fibrovascular tissue in the leaf of S. italic. The root of elongation zone was existing the blue fluorescence corresponded to the presence and concentration of Zn²⁺. The root elongation zone relates to Zn²⁺ transportation, and the leaf intercellular space and trichome surface are related to Zn²⁺ storage. In conclusion, Investigation of extracellular free Zn²⁺ by using HL¹ is efficient.