Volk Gayle M, Goss Lenora J, Franceschi Vincent R
School of Biological Sciences, Washington State University, Pullman, WA 99164-4236, USA.
Ann Bot. 2004 Jun;93(6):741-53. doi: 10.1093/aob/mch092. Epub 2004 Apr 15.
Pistia stratiotes produces large amounts of calcium (Ca) oxalate crystals in specialized cells called crystal idioblasts. The potential involvement of Ca(2+) channels in Ca oxalate crystal formation by crystal idioblasts was investigated.
Anatomical, ultrastructural and physiological analyses were used on plants, fresh or fixed tissues, or protoplasts. Ca(2+) uptake by protoplasts was measured with (45)Ca(2+), and the effect of Ca(2+) channel blockers studied in intact plants. Labelled Ca(2+) channel blockers and a channel protein antibody were used to determine if Ca(2+) channels were associated with crystal idioblasts.
(45)Ca(2+) uptake was more than two orders of magnitude greater for crystal idioblast protoplasts than mesophyll protoplasts, and idioblast number increased when medium Ca was increased. Plants grown on media containing 1-50 microM of the Ca(2+) channel blockers, isradipine, nifedipine or fluspirilene, showed almost complete inhibition of crystal formation. When fresh tissue sections were treated with the fluorescent dihydropyridine-type Ca(2+) channel blocker, DM-Bodipy-DHP, crystal idioblasts were intensely labelled compared with surrounding mesophyll, and the label appeared to be associated with the plasma membrane and the endoplasmic reticulum, which is shown to be abundant in idioblasts. An antibody to a mammalian Ca(2+) channel alpha1 subunit recognized a single band in a microsomal protein fraction but not soluble protein fraction on western blots, and it selectively and heavily labelled developing crystal idioblasts in tissue sections.
The results demonstrate that Ca oxalate crystal idioblasts are enriched, relative to mesophyll cells, in dihydropyridine-type Ca(2+) channels and that the activity of these channels is important to transport and accumulation of Ca(2+) required for crystal formation.
大薸在称为晶异细胞的特化细胞中产生大量草酸钙晶体。研究了钙离子通道在晶异细胞形成草酸钙晶体过程中的潜在作用。
对植物、新鲜或固定组织或原生质体进行解剖、超微结构和生理学分析。用（45）Ca（2+）测量原生质体对Ca（2+）的摄取，并在完整植物中研究钙离子通道阻滞剂的作用。使用标记的钙离子通道阻滞剂和通道蛋白抗体来确定钙离子通道是否与晶异细胞相关。
晶异细胞原生质体对（45）Ca（2+）的摄取比叶肉原生质体高两个数量级以上，并且当培养基中钙增加时，异细胞数量增加。在含有1-50微摩尔钙离子通道阻滞剂异搏定、硝苯地平或氟司必林的培养基上生长的植物，晶体形成几乎完全受到抑制。当用荧光二氢吡啶型钙离子通道阻滞剂DM-Bodipy-DHP处理新鲜组织切片时，与周围叶肉相比，晶异细胞被强烈标记，并且标记似乎与质膜和内质网相关，内质网在异细胞中丰富。针对哺乳动物钙离子通道α1亚基的抗体在蛋白质印迹中识别微粒体蛋白组分中的一条带，但不识别可溶性蛋白组分中的带，并且它在组织切片中选择性地和强烈地标记发育中的晶异细胞。
结果表明，相对于叶肉细胞，草酸钙晶异细胞富含二氢吡啶型钙离子通道，并且这些通道的活性对于晶体形成所需的钙离子的运输和积累很重要。
