Flow Cytometry of Spinach Chloroplasts

Abstract

Intact spinach (Spinacia oleracea) chloroplasts, thylakoid membranes, and inside-out or right-side-out thylakoid vesicles have been characterized by flow cytometry with respect to forward angle light scatter, right angle light scatter, and chlorophyll fluorescence. Analysis of intact chloroplasts with respect to forward light scatter and the chlorophyll fluorescence parameter revealed the presence of truly "intact" and "disrupted" chloroplasts. The forward light scatter parameter, normally considered to reflect object size, was instead found to reflect the particle density. One essential advantage of flow cytometry is that additional parameters such as Ricinus communis agglutinin (linked to fluorescein isothiocyanate) fluorescence can be determined through logical conditions placed on bit-maps, amounting to an analytical purification procedure. In the present case, chloroplast subpopulations with fully preserved envelopes, thylakoid membrane, and inside-out or right-side-out thylakoid membranes vesicles can be distinguished. Flow cytometry is also a useful tool to address the question of availability of glycosyl moities on the membrane surfaces if one keeps in mind that organelle-to-organelle interactions could be partially mediated through a recognition process. A high specific binding of R. communis agglutinin and peanut lectin to the chloroplast envelope was detected. This showed that galactose residues were exposed and accessible to specific lectins on the chloroplast surface. No exposed glucose, fucose, or mannose residues could be detected by the appropriate lectins. Ricin binding to the intact chloroplasts caused a strong aggregation. Disruption of these aggregates by resuspension or during passage in the flow cytometer induced partial breakage of the chloroplasts. Only minor binding of R. communis agglutinin and peanut lectin to the purified thylakoid membranes was detected; the binding was found to be low for both inside-out and right-side-out vesicles of the thylakoid membranes.Among the tools available to characterize cellular compartments by morphological and functional criteria, flow cytometry occupies an important place. Advantages of this 1 W.P.S. was supported by the Federation of European Biochemical Societies and the Swedish Institute.

Purification and functional characterization of protoplasts and intact vacuoles from grape cells

“…These findings regarding vacuole characterization by microscopy and flow cytometry analysis were previously summarized in the book chapter by Papadakis et al [ 5 ]. So far, only spinach chloroplasts [ 11 ] and rice protoplasts [ 12 ] have been studied by flow cytometry. The present results open new perspectives for future work concerning the study of calcium transport across the tonoplast with calcium sensitive fluorescent probes and flow cytometry analysis.…”

The carbon reactions of photosynthesis: role of lectins and glycoregulation

“…Thylakoid membranes from several plants have been demonstrated to possess lectins and, in at least one case, they have been shown to interact with Rubisco in a galacturonic acid-dependent manner (Schröder and Petit 1992, Hincha et al 1993, Aleksidze et al 2002. More recently, it was shown that some lectins in stroma provide ligands to Rubisco (Kovalchuk et al 2012).…”

Cytometry and plant sciences: A personal retrospective

“…ISAC member Bob Ashcroft did some of the first work (81) in the flow analysis of chloroplasts and was able to characterize intact chloroplasts and thylakoids prepared from spinach and maize by using forward and 90°light scatter and fluorescence emission signals. ISAC member Patrice Petit took this work forward by using forward angle and 90°light scatter and chlorophyll autofluorescence measurements to characterize the integrity of isolated spinach chloroplasts and, in combination with labeling with fluorescein isothiocyanate-linked lectins, to distinguish intact chloroplasts from thylakoid membranes and from various chloroplast membrane subfractions (82). ISAC member Julie Auger was involved in the further characterization of the fluorescence signals produced by chloroplasts (83).…”