Localization of a calcium channel-like protein in the sieve element plasma membrane

Abstract

There is evidence that calcium (Ca²⁺) activity in sieve elements is much higher than in adjacent cells, which raises questions about Ca²⁺ exchange across the sieve element plasma membrane. We looked for the presence of Ca²⁺ channels in tobacco (Nicotiana tabacum L.) and Pistia stratiotes (L.) using a monoclonal antibody to a dihydropyridine (DHP)-type Ca channel (MAB427). Immunolabeling at the light microscope level gave strong signals along the sieve element of both species, and with the transmission electron microscope this label was found associated with the plasma membrane. Western blot analysis using the MAB427 antibody detects a protein between 175 and 220 kDa (approximately the size expected of the α-1 subunit of the Ca²⁺ channel protein in mammals) in blots containing either Pistia or tobacco leaf microsomal membrane protein extracts. As a further test for Ca²⁺ channels, hand-sectioned young Pistia leaves were treated with the fluorescent-tagged Ca²⁺ channel blocker 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-(indacene)propionic acid (DM-BODIPY)-DHP. The phloem, as well as calcium oxalate idioblast cells, exhibited strong fluorescence compared to general parenchyma cells. The binding of DM-BODIPY-DHP was inhibited by pre-treatment of sections with nifedipine, a competitive inhibitor of DHP-type Ca²⁺ channels in animal systems. The results indicate that sieve elements may be enriched with Ca²⁺ channels which might be partly responsible for the high Ca²⁺ activities observed in sieve sap, and also for rapid phloem responses, such as Ca²⁺ activated callose deposition.