Selective scattering spectra as an approach to internal structure of granal and agranal chloroplasts.

Abstract

Selective scattering spectra of granal and agranal chloroplasts were measured in the red spectral region and compared with calculations based on the Mie theory. The spectra were influenced considerably by the intactness and ultrastructural pattern of the chloroplasts. It was demonstrated that the spectra consist of two components: one attributable to grana and the other, to single lamellae. The dependence of the selective scattering spectra on the ultrastructural characteristics offers a convenient method for monitoring the quality of chloroplast preparations by a procedure much faster than electron microscopy. Selective scattering spectra of algal cells such as Chlorella and Anacystis, as well as of spinach chloroplasts and fragments, have been reported in the literature (1)(2)(3)(4)(5)(6)(7)(8). In these studies the photosynthetic lamellar system was considered as a whole with no regard to the ultrastructural pattern characteristic of the chloroplasts. As shown under the electron microscope, chloroplasts consist of two types of lamellae: single lamellae that appear never to fuse with each other, and stacked lamellae that build up the highly ordered piles called "grana." The individual contributions of single and stacked lamellae in the selective scattering spectra have not been distinguished in previous reports.The aim of the present work was to study the selective scattering spectra of chloroplasts containing various amounts of grana and to compare intact class A and class B chloroplasts (9) in order to obtain information on the relationship between ultrastructure and selective scattering. Selective spectra of chloroplasts were shown to be dependent on their intactness and ultrastructure. This phenomenon offers a convenient method for checking the quality of chloroplast preparations. MATERIALS AND METHODSChloroplast preparations with high and low granum contents were obtained from maize leaves (Zea mays L. var. MV SC 660). The plants were grown in a greenhouse for 9-l1 days.Class A chloroplasts were isolated from a suspension of mesophyll protoplasts (10) or bundle sheath cells. Mesophyll protoplasts were prepared in a digestion medium containing 0.6 M d-sorbitol, 0.1 M potassium phosphate, 0.5% (wt/vol) Macerozyme R-10, 2.0% (wt/vol) cellulase Onozuka R-10, and 0.5% (wt/vol) potassium dextran sulfate, pH 5.8. Bundle sheath cells were separated in a maceration medium containing 0.4 M d-sorbitol, 0.1 M potassium phosphate, 0.5% Macerozyme R-10, 1.0% cellulase Onozuka R-10, and 1.0% Helicase, pH 5.8. Macerozyme and cellulase preparations were purchased from Kinki Yakult MFG Co. Ltd., Nishinomiya, Japan; the Helicase was obtained from Industrie Biologique Frangaise, Gennevilliers, France; and potassium dextran sulfate was from Meito Sangyo Co. Ltd., Nagoya, Japan. The incubation was for 90 min at 37°. Protoplasts and cells were washed three times in 0.6 M d-sorbitol and transferred to the medium of Anderson and Boardman (11) containing 0.3 M sucrose, 0.05 M potassium phosphate, and 0.01 M potassium chlor...

Optical Property Analyses of Plant Cells for Adaptive Optics Microscopy

“…In addition, chloroplasts refract and scatter light. [30,31] The chloroplast stroma (the internal fluid of the chloroplast) has a refractive index of 1.42, [25,32] (stacks of photosynthetic membranes surrounded by the stroma) has higher pigment and protein contents, indicating that it likely has an even higher refractive index (n > 1.5). [28,29] These data suggest that chloroplasts likely also cause optical disturbances in imaging of live plant cells.…”

Reconciliation of theory and experiment on 90° selective scattering spectra as a measure of intact or broken granal or agranal chloroplasts

“…This could not be reconciled [4] by making reasonable changes to the particle parameters. They explained the frequency shift between intact and broken chloroplasts as being due to the decrease in chloroplast refractive index caused by dilution with the surrounding medium when the outer envelope was broken.…”

“…However, only Mie theory gives the light intensity scattered at 90'; the experimental quantity measured in [4].…”

“…To calculate the scattering spectrum for intact granal chloroplasts, we use in angular measure: w. = 27.802 X lOi s-' y = 1.583 x 1o14 s-l and from the absorption spectra [4] we find Co = 0.85 and Cr = 0.0022. Using a particle radius of 2000 nm, which is typical of chloroplast size, the scattering spectrum was calculated for the angles 90" _+ 3", corresponding to the experimental situation in [4]. This is compared with the theoretical broken chloroplast spectrum in fig.2.…”

“…The experimental spectrum of 90" scattered light in the 650-750 nm region was later measured [4], for chloroplasts containing different degrees of grana, with the outer envelopes either intact or broken. They found that the scattering spectrum for intact granal chloroplasts had a peak at 703 nm, and non-intact chloroplasts gave a peak at 690 nm.…”