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.

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.

Orientation of emitting dipoles of chlorophyll A in thylakoids: considerations on the orientation factor in vivo.

Orientation angles of five emitting dipoles of chlorophyll a in thylakoids were estimated from low temperature fluorescence polarization ratio spectra of magnetically oriented chloroplasts. A simple expression is given also for the evaluation of data from linear dichroism measurements. It is shown that the Qy dipoles of chlorophylls lie more in the plane of the membranes and span a larger angular interval than was previously thought. Values for the orientation factor are calculated using various models corresponding to different degrees of local order of the Qy dipoles of chlorophylls in the thylakoid. We show that the characteristic orientation pattern of the Qy dipoles of chlorophylls in the membrane, i.e., increasing dichroism toward longer wavelengths, may favour energy transfer between the antenna chlorophylls as well as funnel the excitation energy into the reaction centers.

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.

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.

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.