A new type of subchloroplast fragments isolated from pea chloroplasts in the presence of digitonin

Abstract

Heavy fragments were isolated from pea chloroplasts using digitonin treatment and differential centrifugation. The particles were characterized by a significantly lowered chlorophyll a/b ratio, contents of photosystem I (PS I) proteins and ATPase, as well as of amount of P700. The content of photosystem II (PS II) proteins decreased insignificantly, whereas that of proteins of the light-harvesting complex II did not change. The absorption and low-temperature fluorescence spectra were indicative of a decreased content of PS I. Electron microscopy of ultrathin sections of heavy fragment preparations identified them as grana with reduced content of thylakoids. The diameter of these particles was practically the same as within chloroplasts. Comparison of various characteristics of the fragments and chloroplasts from which the fragments were isolated allowed us to define a high degree of preservation of marginal regions in thylakoids present in the heavy fragment particles. Analysis of the results shows that the procedure of fragmentation produces grana with high extent of thylakoid integrity. The phenomenon of reduction of the thylakoid content in grana, occurring as our heavy fragments, is considered in the frame of our previous hypothesis concerning the peculiarities of grana organization in the transversal direction.

Short-term heating causes thylakoid restructuring in pea chloroplasts and modifies spectral properties of pigment-protein complexes

“…The isolated chloroplasts were suspended in the medium containing 10 mM Tricine buffer (pH 7.5), 0.4 M sucrose, 10 mM NaCl, and 5 mM MgCl 2 . Subchloroplast fragments were obtained by solubilization of the chloroplasts with 0.3 % digitonin, and the subsequent differential centrifugation according to the previously published procedure [13]. The particles sedimented at 1000 g and 40000 g after 20000 g were used for the measurements.…”

“…The particles sedimented at 1000 g and 40000 g after 20000 g were used for the measurements. Chlorophyll concentration (Chl) was estimated spectrophotometrically in 80 % acetone extracts [13] and the yield of the fractions was calculated as the ratio of the total Chl in the particles to the total Chl in chloroplast suspension taken for fragmentation. Chl a/b ratio was calculated as described [13].…”

“…The films were prepared as described earlier [14] and placed into an optical cryostat filled with vapours of liquid nitrogen. The fluorescence spectra were measured as described earlier [13] using modified lab-made setup. The films were highly transparent and their low temperature fluorescence spectra proved to be identical to those of corresponding to frozen buffer-glycerol suspension [13].…”

“…The fluorescence spectra were measured as described earlier [13] using modified lab-made setup. The films were highly transparent and their low temperature fluorescence spectra proved to be identical to those of corresponding to frozen buffer-glycerol suspension [13]. Light absorption by the samples in a peak of the red band did not exceed 15 %.…”

“…Excitation spectra of fluorescence were obtained at the detection wavelength of 735 nm for the same samples as for the measurement of the fluorescence spectra as described earlier [13]. The samples were illuminated with the monochromatic light in the interval 630-720 nm using an excitation monochromator with the band-pass of 2 nm and a sampling step of 0.5 nm.…”