Annals of Botany

• Background and Aims Pistia stratiotes produces large amounts of calcium (Ca) oxalate crystals in specialized cells called crystal idioblasts. The potential involvement of Ca²⁺ channels in Ca oxalate crystal formation by crystal idioblasts was investigated.

• Methods Anatomical, ultrastructural and physiological analyses were used on plants, fresh or fixed tissues, or protoplasts. Ca²⁺ uptake by protoplasts was measured with ⁴⁵Ca²⁺, and the effect of Ca²⁺ channel blockers studied in intact plants. Labelled Ca²⁺ channel blockers and a channel protein antibody were used to determine if Ca²⁺ channels were associated with crystal idioblasts.

• Key Results ⁴⁵Ca²⁺ 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 µM of the Ca²⁺ channel blockers, isradipine, nifedipine or fluspirilene, showed almost complete inhibition of crystal formation. When fresh tissue sections were treated with the fluorescent dihydropyridine-type Ca²⁺ 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 Ca²⁺ channel 1 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.

• Conclusions The results demonstrate that Ca oxalate crystal idioblasts are enriched, relative to mesophyll cells, in dihydropyridine-type Ca²⁺ channels and that the activity of these channels is important to transport and accumulation of Ca²⁺ required for crystal formation.