Probing lectin binding sites on isolated viable generative and sperm cells of tobacco

Generative cells and sperm cells of tobacco were isolated and labeled by fluorescein isothiocyanate conjugated concanavalin agglutinin (FITC-Con A), wheat germ agglutinin (FITC-WGA) and soybean agglutinin (FITC-SBA) to compare lectin binding site (LBS) distributive patterns between generative cells and sperm cells, and between a pair of sperm cells from the same pollen tube. With the help of cooled charge couple device (CCD) and confocal microscope, Con A binding sites were found on the surface of 96% generative cells and 91% sperm cells. WGA binding sites were also detected on plasma membrane of generative cells and inner plasma membrane of vegetative cells and the crosswall of male germ unit (MGU), but it was hardly found on most of sperm cell surface. No soybean agglutinin (SBA) binding sites were detected on the plasma membrane of both generative cells and sperm cells. The experiment revealed that LBSs on the membrane surface of germ cell were reorganized or modified during the development from generative cell to sperm cell, indicating sperm formation might couple with its function specialization. The possible dimorphism on surface glycoprotein distribution of sperm cells was not confirmed in tobacco in this experiment since sperm cells derived from the same generative cell could be both labeled or not labeled at all by FITC-Con A. Based on the finding in present work and our former reports, we proposed that there might be functional difference of sperm in the population of living sperms of higher plants.