Light-dependent absorption and selective scattering changes at 518 nm in chloroplast thylakoid membranes.

Abstract

The light-induced absorbance change at 518 nm of isolated chloroplasts consists of a rapid phase, and a slow phase which is complete in about 20 sec. The slow component of the 518 nm absorbance change correlates with the light-induced change in 900 light scattering at 518 nm. Both show a similar time course, similar pH dependence with a maximum at pH 6.0, and similar sensitivity to inhibitors and to treatment of the chloroplasts with a low concentration of glutaraldehyde. Their light minus dark difference spectra are similar with maxima at about 520 nm. It is concluded that they are manifestations of the same phenomenon, and the slow absorbance increase at 518 nm is due to enhanced scattering. It is proposed that the light-induced changes in scattering at 518 nm reflect alterations in selective ispersion, due to proton uptake and conformational changes in the chloroplast thylakoid membrane.Isolated chloroplasts exhibit reversible light-dependent structural changes, which can be monitored by electron microscopy and by changes in packed volume, transmittance, and 900 light scattering (1-8). Similar changes have also been observed with chloroplasts in idvo (5,(9)(10)(11). Electron microscopy has demonstrated a thinning of the chloroplast thylakoid membrane on illumination (4). The light-induced volume decrease (shrinkage) of chloroplasts and the associated increases in absorbance and 900 light scattering are dependent on photosynthetic electron flow. They are inhibited by uncouplers of photophosphorylation (5, 12). The increase in light scattering parallels the proton uptake which is observed on illumination of chloroplasts (13). It was generally supposed that the increase in light scattering reflected the decrease in chloroplast volume, but further studies (14) showed that this was not the case when chloride was the only anion present in the medium. It was suggested that scattering was influenced by protonation and an increased refractive index of the chloroplast thylakoid system (14), or by changes in the optical properties of the stacked thylakoids (7).In the work described in this paper, we examined lightinduced 90°scattering by isolated chloroplasts in relation to the light-induced absorbance change at 518 nm. It appears that the slow kinetic component of the absorbance increase is caused by an increase in selective scattering which is dependent on the refractive index difference between the chromophore-containing particles of the chloroplast and their environment. MATERIALS AND METHODS Intact chloroplasts were isolated from hydroponically grown spinach plants, essentially by the method of Reeves and Hall (15). Detached leaves (20 g) were preilluminated for 15 min at 40C in white light (16) and ground in a Sorvall Omnimixer for 4 sec at 90% of line voltage (230 V) in a medium containing 0.33 M sorbitol, 10 mM NaCl, 5 MM MgCl2, 1 mM MnCl2, 2 mM EDTA, 2 mM isoascorbate, 0.4% bovine serum albumin, and 50 mM 2-(N-morpholino)ethanesulfonic acid (Mes) buffer, pH 6.5. The brei was filtered through four layer...

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