The light-harvesting chlorophyll a/b protein acts as a torque aligning chloroplasts in a magnetic field.

Displacement of particles from the purified light-harvesting chlorophyll a/b protein aggregate (LHC) was studied in magnetic fields of various strengths (0 to 1.6 T) by polarized fluorescence measurements. Macromolecular aggregates of LHC have a considerable magnetic susceptibility which enables the particles to rotate and align with their nematic axes parallel with H. As LHC is embedded in a transmembrane direction thylakoids should align perpendicular to H, the mode of alignment experimentally observed in thylakoids. The value of the magnetic susceptibility could be estimated by relating it to the integral susceptibility of the chlorophyll molecules in LHC. The fitting of this value with the field strength dependency of the fluorescence polarization ratio (FP) revealed a relationship between the LHC content of various photosynthetic membranes and their capacity for alignment, which suggested that LHC might be the torque ordering chloroplasts in a magnetic field.

Energization and ultrastructural pattern of thylakoids formed under periodic illumination followed by continuous light.

Bean leaves grown under periodic illumination (56 cycles of 2 min light and 98 min darkness) were subsequently exposed to continuous illumination, and in connection with granum formation and accumulation of the light-harvesting pigment-protein complex thermoluminescence and light-induced shrinkage of thylakoid membranes were studied. Juvenile chloroplasts with large double sheets of thylakoids obtained under periodic light exhibited low temperature spectra of polarized fluorescence yielding fluorescence polarization (FP) values < 1 at 695 nm, characteristic for pheophytin emission. In the course of maturation under continuous light when normal grana appeared and the chlorophyll a/b light-harvesting photosystem II complex was incorporated into the membrane, at 695 nm the relative intensity of fluorescence dropped and FP changed to a value of > 1, suggesting an overlap between the emission of pheophytin and that of the chlorophyll a/b light-harvesting photosystem II complex. Thermoluminescence glow curves recorded with juvenile thylakoids displayed a relatively high proportion of emission at low temperatures (around -10°C) while with mature chloroplasts, more thermoluminescence originated from energetically deeper traps (discharged around 28°C). This means that during thylakoid development the capacity of the membrane to stabilize the separated charges increases, which might be favourable for the ultimate conservation of energy. The more extensive energization of mature thylakoids was also indicated by a light-induced decrease in the thickness of the membranes upon illumination; a change which could not be detected in juvenile thylakoids.

[Polarization fluorescence of chloroplasts oriented in polyacrylamide gel. Experiment and theoretical analysis]. / Poliarizovannaia fluorestsentsiia khloroplastov, orientirovannykh v poliakrilamidnom gele. Eksperiment i teoreticheskii analiz.

Maize mesophyll chloroplasts were oriented in polyacrylamide gel upon squeezing of gel samples. Fluorescence polarization was measured as a function of the gel deformation parameter. Linearly polarized fluorescence spectra were recorded at -140 degrees. The results show that chloroplasts were oriented in polyacrylamide gel as effectively as in magnetic field. Theoretical models are analyzed for orientation of disc-shaped and rod-shaped chloroplasts. Of these two, only the model for disc-shaped chloroplasts can account for the experimental results. Based on experimental data, the approaches are proposed to estimate the order of alignment of thylakoid membranes within chloroplasts, and to calculate the angles made by transition dipoles with the thylakoid membrane plane. Internal structure of chloroplasts (alignment of thylakoid membranes) is shown to be unchanged upon squeezing of the gel samples.

Organization of Chlorophyll a in the Light-Harvesting Chlorophyll a/b Protein Complex as Shown by Circular Dichroism : Liquid Crystal-Like Domains.

The development of thylakoid stacking, accumulation of the light-harvesting chlorophyll a/b protein complex (LHCP), and the changes of circular dichroism (CD) which reflect the organization of chlorophyll molecules in greening thylakoids of bean Phaseolus vulgaris cv Red Kidney leaves were investigated.Chloroplasts formed under intermittent light contained large double sheets of membrane with extensive appression in addition to separate lamellae. Thylakoids of such chloroplasts were devoid of LHCP and exhibited a relatively small CD in the chlorophyll absorption region. Upon continuous illumination, the rearrangement of membranes to characteristic grana and the accumulation of the LHCP was accompanied by the gradual appearance of the very intense CD signal with peaks at 682 to 684 (+) and 665 to 672 nanometers (-). The magnitude of differential absorption was approximately 100 times larger than that of the chlorophyll a in solution. This suggests a superhelical liquid crystal-like organization for LHCP, a texture which can be altered by changes of the electric field in the photosynthetic membranes.

Distribution of characteristic membrane proteins in granum and stroma thylakoids.

Membrane fractions obtained by ultrasonication of Vicia faba thylakoids were analyzed by sodium dodecyl sulphate gel electrophoresis. The polypeptide patterns revealed a six-times-higher ratio of the apoprotein (1) of P700 chlorophyll a protein to the apoprotein (2) of one of the chlorophyll a proteins in photosystem II in the light (stroma) fraction as compared with the heavy (grana) fraction indicating different distribution of the two photosystems. Additionally, the light fraction was clearly depleted in chlorophyll a/b apoproteins 2a and 2b of the light-harvesting complex and enriched in CF1 alpha and beta subunits. In contrast to the fairly constant ratio of the CF1 subunits the ratio of chlorophyll a/b apoprotein 2a/2b differed significantly between the light and heavy fraction suggesting a different composition for light-harvesting complex in stroma and grana regions of the thylakoids.

Chiroptical properties of chlorophyll-protein complexes separated on Deriphat/polyacrylamide gel.

Circular dichroism (c.d.) was measured for four chlorophyll-protein complexes, resolved from sodium dodecyl sulphate extracts of chloroplasts by electrophoresis in polyacrylamide gel containing Deriphat 160 (disodium N-dodecyl beta-imidopropionate), a zwitterionic detergent. The slowest-band (1) complex was found to be identical with the complex CP1 as found on electrophoresis in the presence of anion detergent, but it was in a much higher yield (30% of the chlorophyll a). In band-2 and -3 protein complexes a c.d. pattern described for the complex CP2 could be recognized. Another c.d. component of a split-exciton type with extrema at 680 (-) and 669 (+)nm, together with evidence of disorganized chlorophyll, was found in band-2, -3 and -4 complexes. When a barley (Hordeum vulgare) mutant lacking chlorophyll b was examined, only bands 1 and 4 were obtained, and the c.d. of the band-4 complex was much less affected by disorganized chlorophyll. C.D. spectra resembling that of this band-4 complex could be generated by subtracting the c.d. of complex CP1 from the c.d. of photochemically active mutant chloroplast fragments, or by subtracting the c.d. of complexes CP1 and CP2 from pea (Pisum sativum) chloroplast fragments. The Deriphat appears to have preserved at least to some extent a new type of chlorophyll a-protein complex.

Structural and functional stability of isolated intact chloroplasts.

The effect of in vitro ageing on the ultrastructure, electron transport, thermoluminescence and flash-induced 515 nm absorbance change of isolated intact (type A) chloroplasts compared with non-intact (types B and C) chloroplasts was studied.When stored in the dark for 18 h at 5°C, the structural characteristics of intact and non-intact chloroplasts were only slightly altered. The most conspicuous difference between the two was in the coupling of the electron transport which was tighter and more stable in intact chloroplasts. Under dark-storage the activity of PS 2* decreased and the -20°C peak of thermoluminescence increased at the expense of the emission at +25°C. These changes were less pronounced in the intact chloroplasts. PS 1 activity and the flash-induced 515 nm absorbance change were not affected by dark-storage.When kept in the light (80 W m(-2) (400-700 nm) for 1 h at 5°C), the thylakoid system of chloroplasts rapidly became disorganized. Although the initial activity of electron transport was much higher in intact chloroplasts, after a short period of light-storage the linear electron transport and the electron transport around PS 2 decreased in both types of preparations to the same low level. These changes were accompanied by an overall decrease of the intensity of thermoluminescence. PS 1 was not inhibited by light-storage, while the flash-induced 515 nm absorbance change was virtually abolished both in preparations of intact and non-intact chloroplasts.The data show that in stored chloroplast preparations intactness cannot be estimated reliably either by the FeCy test or by inspection under the electron microscope. These tests should be cross-checked on the level and coupling of the electron transport.

Macromolecular organization of chlorophyll a in aggregated chlorophyll a/b protein complex as shown by circular dichroism at room and cryogenic temperatures.

This report concerns the circular dichroic (CD) signal of intact chloroplasts of higher plants. The CD spectra of chloroplasts are compared with the aggregated form of the light-harvesting chlorophyll a/b complex at 25 degrees C and -250 degrees C. The light-harvesting chlorophyll aggregate has a CD of magnitude equal to or greater than chloroplasts, but of opposite sign, and it is not related to the CD of the unaggregated form, and hence its arrangement is an artefact compared to the arrangement in the chloroplast. We suggest that this preparation, which has pseudo-lamellar structure, is a clear example of a large CD signal being generated by macromolecular association. The asymmetry of organization in the chloroplast has an opposite sense to that of the aggregate, but affects only chlorophyll a, not chlorophyll b.

Characteristics of the Flash-induced 515 Nanometer Absorbance Change of Intact Isolated Chloroplasts.

In intact (type A) chloroplasts isolated from mesophyll protoplasts of maize (Zea mays L. convar. KSC 360) the flash-induced 515 nanometer absorbance change was much higher than in conventionally prepared (types B and C) chloroplasts. The 515 nanometer signal of type A chloroplasts exhibited a biphasic rise: the initial very fast rise (rise time <<1 millisecond) was followed by a slow increase of absorbance (rise time 10 to 20 milliseconds). With decreasing degree of envelope retention the slow phase disappeared. Thus the biphasic rise of the flash-induced 515 nanometer absorbance change can be regarded as an attribute of intact chloroplasts.The 515 nanometer signal of intact chloroplasts was studied at various pH values of the external medium, with various dark intervals between the flashes and at different temperatures. The absorbance change was probed with electron transport inhibitors and ionophores. The data show that the fast phase of the absorbance increase was similar in chloroplasts isolated from protoplasts and in conventional chloroplast preparations. The slow rise, which has not been hitherto recognized in isolated chloroplasts, can be due to a contribution of the proton pump to the electric field which is generated across the thylakoid membranes.

Differential light-scattering of granal and agranal chloroplasts and their fragments.

Intact (class-A) granal and agranal maize chloroplasts and chloroplast fragments were examined for differential scattering of circularly polarized light (measured at 90 degrees) and c.d. (circular dichroism) (measured at 0 degrees) by using a modified spectropolarimeter with a large acceptance angle. Useful c.d. information was obtained by making corrections for scattered light. Chloroplast fragments exhibited a large and characteristic differential scattering of circularly polarized light recognized in the presence of granal chloroplasts. It is confirmed that agranal chloroplasts do not have the intense 682 nm c.d. peak that is assigned to the presence of grana.

Functional characteristics of intact chloroplasts isolated from mesophyll protoplasts and bundle sheath cells of maize.

A procedure was developed to isolate mesophyll and bundle sheath chloroplasts of a high degree of intactness and low cross-contamination. Light-induced (14)CO2 fixation of isolated chloroplasts was similar to that of protoplasts and cells in that it was low and was stimulated by the addition of exogenous substrates. O2 evolution was absent in both bundle sheath chloroplasts and cells. The flash-induced 515 nm absorbance change of intact mesophyll chloroplasts showed a biphasic rise, previously known to be a characteristic only of intact algae. With bundle sheath chloroplasts or cells, no 515 nm signal could be detected. In the presence of 10 µmol l(-1) phenazine methosulphate, bundle sheath chloroplasts exhibited a flash-induced 515 nm signal with a monophasic rise and amplitude comparable to that of the mesophyll chloroplasts. A similar signal was obtained with bundle sheath chloroplasts suspended in an extract prepared from the mesophyll tissue. Both the substrate stimulation of the CO2 fixation and the reconstitution of the 515 nm signal in bundle sheath chloroplasts by the mesophyll extract indicate the requirement of cooperation between the mesophyll and bundle sheath cells of maize leaves.

Flash-induced 515 nm absorbance change in chloroplasts with various granum contents.

The 515 nm absorbance change was studied in mesophyll and bundle sheath chloroplasts of maize, which contain different amounts of grana. The amplitude of the 515 nm signal (induced by 3 micro seconds flashes repeated at 4 s intervals) has shown a correlation with the granum content of the samples. However, upon addition of N-methylphenazonium methosulphate the 515 nm signal became independent of the amount of grana: in agranal thylakoids a large pool of silent Photosystem I was activated and, as a result, the amplitude of the 515 nm signal of agranal chloroplasts increased to the level exhibited by granal chloroplasts. These data show that the 515 nm absorbance change is not limited to small closed vesicles like grana, but in the presence of suitable electron donors single lamellae of bundle sheath chloroplasts can also be active.

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

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.

X-ray microanalytical study of Mn and Fe compartmentation in maize chloroplasts.

X-ray microanalysis of mesophyll and bundle sheath maize chloroplasts showed that bound manganese is located within thylakoids, whereas bound iron is equally present in thylakoids and stroma. A simple correlation between manganese content and oxygen evolution capacity (Photosystem II activity) is unlikely since Mn is also present in bundle sheath thylakoids of low Photosystem II activity. A major part of manganese has probably a distinct function and only a minor part plays a role in the process of oxygen evolution.

Circular dichroism spectra of granal and agranal chloroplasts of maize.

Granum-containing chloroplasts from mesophyll cells of maize (Zea mays L. var. MV 861) leaves exhibited circular dichroism spectra with a large double signal; peaks at 696 nm (+) and 680 nm (-). In the circular dichroism spectra obtained with agranal chloroplasts of bundle sheath cells, this large double signal is absent. Separation of grana lamellae, in a medium of low salt concentration and in KSCN solution, resulted only in a slight decrease of the amplitude, while upon treatment with digitonin the large double signal disappeared. A negative signal of the chlorophyll b peak at 654 nm was observed in the case of both granal and agranal chloroplasts, and it was not affected either by low salt or by digitonin treatment. A positive peak at about 515 nm was higher in granal than in agranal chloroplasts.