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.     

The differential scattering of circularly polarized light by chloroplasts and evaluation of their true circular dichroism

Chloroplasts isolated from pea leaves display an intense circular dichroism in the range 600 to 720nm. Circularly polarized light is also differentially scattered by chloroplasts, and this effect can be confused with circular dichroism. By using an instrumental modification it was possible to distinguish, and record separately, the ellipticities of the transmitted light (circular dichroism) and of the scattered light in the same c.d. instrument. By means of a light-scattering apparatus, the intensity of unpolarized light scattered by chloroplasts was measured as a function of wavelength and of angle. This measurement allowed the aforementioned ellipticities to be corrected for mutual interference. At a concentration of 4mug of chlorophyll/ml (the optimum practical concentration of chloroplasts at which there was no significant interaction of scattering and absorption effects) spectra of true circular dichroism (circular differential absorption) and circular differential scattering were obtained. The former showed maxima, positive at 688nm and negative at 676nm, with an intensity Deltatheta=8.3m degrees .litre.(mg of chlorophyll)(-1).cm(-1). The latter had a maximum at 683nm with an intensity of +47m degrees with respect to the solvent baseline; this value is independent of the concentration of chloroplasts in dilute suspensions. It is suggested that the intense circular dichroism of chloroplasts reflects specific chlorophyll-chlorophyll interactions in the light-harvesting pigment. The advantages of this method for determining the c.d. of scattering suspensions over those of other investigators are discussed.     

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.     

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 μs 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.     

The extent to which the spatial separation between photosystems I and II associated with granal formation limits noncyclic electron flow in isolated lettuce chloroplasts

Uncoupled noncyclic electron flow in stacked (granal) chloroplasts with a lateral heterogeneity in the distribution of the two photosystems has been compared with that in unstacked (agranal) chloroplasts with a near-uniform distribution. Chloroplasts were maintained in either structural state in the same assay medium so as to equalize effects of ionic composition which may influence reaction rates. The assay medium, an ion-deficient solution, was capable of supporting high rates of electron flow from water to methyl viologen. At high irradiance, unstacked chloroplasts exhibited an uncoupled rate which was 30% (in chloroplasts isolated from lettuce grown in low light) or 55% (in chloroplasts isolated from lettuce grown in high light) higher than that of stacked chloroplasts; the percentage remained relatively constant in the temperature range 7 to 22 degrees C for both high-light and low-light chloroplasts. At low irradiance, stacked low-light chloroplasts, despite the spatial separation of the two photosystems, gave higher rates of electron flow than did unstacked low-light chloroplasts. The addition of MgCl2 to stacked chloroplasts increased the uncoupled rate of noncyclic electron flow, but only at relatively high irradiances. The differences observed for stacked and unstacked chloroplasts, and for high-light and low-light chloroplasts are discussed. The approach taken in this work should be useful in other comparisons of stacked and unstacked chloroplasts.     

Thylakoid and grana formation during the development of pea chloroplasts,illuminated by white,red, and blue low intensity light

Summary We analyzed the formation of thylakoids and grana during the development of pea chloroplasts, illuminated by white, red and blue low intensity light. The total length of granal and intergranal thylakoids, and the length of granal thylakoids per unit area of plastid section were measured. Initially the greatest increase in length of granal thylakoids and the highest incidence of grana with large thylakoid content occurred in red light. On the other hand, with illumination times of over 12 hours blue light appeared to be more efficient in stimulating grana formation and thylakoid growth.     

Rapid identification of microorganisms by circular-intensity differential scattering

There is a pressing need in clinical medicine for rapid identification of microorganisms. We describe a method that has the potential for such rapid identification: circular-intensity differential scattering, which is based on the differential scattering of left and right circularly polarized light. The scanning time required to obtain the spectral signature of an organism is about 4 min. Using a commercial circular dichrograph modified to measure circular intensity differential scattering at 90 degrees, we obtained significantly different spectra for five different crude influenza viruses. Salmonella typhimurium TA98 and TA100, and Escherichia coli HB101, HB101(pBR322), and HB101(pMB9). Purified supercoiled plasmid pBR322 DNA was readily distinguishable from linear pBR322 DNA; such differences in nucleic acid packaging may be significant factors in the discriminatory power of this technique.     

Rapid identification of microorganisms by circular-intensity differential scattering.

There is a pressing need in clinical medicine for rapid identification of microorganisms. We describe a method that has the potential for such rapid identification: circular-intensity differential scattering, which is based on the differential scattering of left and right circularly polarized light. The scanning time required to obtain the spectral signature of an organism is about 4 min. Using a commercial circular dichrograph modified to measure circular intensity differential scattering at 90 degrees, we obtained significantly different spectra for five different crude influenza viruses. Salmonella typhimurium TA98 and TA100, and Escherichia coli HB101, HB101(pBR322), and HB101(pMB9). Purified supercoiled plasmid pBR322 DNA was readily distinguishable from linear pBR322 DNA; such differences in nucleic acid packaging may be significant factors in the discriminatory power of this technique.     

Chlorophyll-protein complexes from green algae and higher plants. Heterogeneity of chloroplast complexes from dimorphous tissue of C4 plant leaves

It was demonstrated that the decrease of the activity of photosystem II in agranal chloroplasts of bundle sheath cells of malate C4 plants is due to a loss of the chlorophyll-protein complex of this photosystem. This is paralleled with an increase in the relative amount of the chlorophyll--protein complex of photosystem I and a partial loss of the light-harvesting pigmentprotein complex. These two events may account for the high activity of photosystem I and for the low content of chlorophyll b in the plastids. The cumulative data are indicative of a close interrelationship between the granal state of the chloroplast membranes and the presence of the chlorophyll--protein complex of photosystem II.     

Alignment of biological microparticles by a polarized laser beam

The optical alignment of biological samples is of great relevance to microspectrometry and to the micromanipulation of single particles. Recently, Bayoudh et al. (J. Mod. Opt. 50:1581–1590, 2003) have shown that isolated, disk-shaped chloroplasts can be aligned in a controlled manner using an in-plane-polarized Gaussian beam trap, and suggested that this is due to their nonspherical shape. Here we demonstrate that the orientation of various micrometer-sized isolated biological particles, trapped by optical tweezers, can be altered in a controlled way by changing the plane of linear polarization of the tweezers. In addition to chloroplasts, we show that subchloroplast particles of small size and irregular overall shape, aggregated photosynthetic light-harvesting protein complexes as well as chromosomes can be oriented with the linearly polarized beam of the tweezers. By using a laser scanning confocal microscope equipped with a differential polarization attachment, we also measured the birefringence of magnetically oriented granal chloroplasts, and found that they exhibit strong birefringence with large local variations, which appears to originate from stacked membranes. The size and sign of the birefringence are such that the resulting anisotropic interaction with the linearly polarized laser beam significantly contributes to the torque orienting the chloroplasts.     

Light scattering from suspensions of membrane fragments derived from sonication of beef heart mitochondria.

The intensity of light scattering from suspensions of membrane fragments prepared by sonication of beef heart mitochondria in the presence of EDTA at alkaline pH (ESMP) was determined at 45, 90, and 135 degrees with light of wavelength 546 nm. The dissymmetry ratio Z = I45 degrees c/I135 degrees c, where I45 degrees c and I135 degrees c are the scattering intensities at 45 and 135 degrees extrapolated to zero particle concentration and corrected for reflectance effects, was used to calculate particle size from the Rayleigh-Gans-Debye theory. An average particle diameter D of 184-190 nm was obtained, within the range of particle diameter 50-300 nm determined previously by electron microscopy. This average diameter determined by light scattering is a useful parameter for characterization of ESMP particle size. We propose the term: light scattering average particle diameter, DLS, for this parameter. The refractive index of ESMP was determined to be 1.443 by measurement of scattering intensity in buffer solutions of varying sucrose concentration. The value of Z was independent of sucrose concentration in this determination, showing that the particles are osmotically inactive toward sucrose. The values of average particle diameter DLS and of refractive index fall within the range of validity of the Rayleigh-Gans-Debye theory, for which light scattering changes are attributable solely to dimension change, rather than to change in particle refractive index. Uptake of water accompanying energy-linked salt uptake in ESMP was calculated from light scattering changes to be 0.18 mul of H2O/mg of protein, compared with 0.49 mul of H2O/mg of protein measured by dextran inaccessibility. Measurement of light scattering changes provides a rapid and sensitive method for determining volume changes of ESMP. The magnitude of the volume change observed during energy-linked water and salt uptake and the initial degree of hydration suggests that ESMP are analogous to polyelectrolyte gels with regard to sorption of strong electrolytes and that the Donnan formulation for ion exchange equilibria may be usefully applied to these processes in ESMP.     

Light-induced Changes in the Ultrastructure of Pea Chloroplasts in Vivo: Relationship to Development and Photosynthesis

Light-induced structural changes of chloroplasts and their lamellae were studied in leaves of Pisum sativum L., cv. Blue Bantam, using electron microscopy. Upon illumination of 14-day-old plants with 2000 lux, the chloroplasts decreased in thickness by about 23% with an accompanying increase in electron scattering by the stroma. Concomitantly, the average thickness of granal lamellae (thylakoids) decreased from 195 ± 4 angstroms in the dark to 152 ± 4 angstroms in the light, and this change was half-saturated at only 50 lux. Lamellar flattening at 50 lux and its reversal in the dark both had half-times of a minute or less. The thickness of a partition (a pair of apposed lamellar membranes) was 140 ± 9 angstroms in both the light and the dark, indicating that the observed light-induced change was in the volume enclosed within the thylakoid. The effect of illumination could be inhibited by various uncouplers of photophosphorylation but not by 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea, suggesting that it depended on ATP (or its precursor). In the presence of 0.5 micromolar nigericin, the thickness of the granal lamellae increased in the light to 213 ± 3 angstroms; this may reflect an uptake of K⁺ into an osmotically responding space within the thylakoids.     

Circular intensity differential scattering and chromatin-DNA structure

Circular intensity differential scattering (CIDS) has been proven a powerful method in determining the higher-order structure of large biopolymers, such as chromatin. Theoretical predictions of the expected differential light scattering of circularly polarized light have previously been made for chromatin, either within the Born approximation, treating nucleosomes as noninteracting, oblate ellipsoids, or within a multiple dipole approximation, treating nucleosomes as interacting spheres. In order to conduct a meaningful interpretation of the CIDS signal in terms of given geometric parameters of the chiral structure, we have in this paper combined the two approaches considering the mutual interactions of ellipsoidal nucleosomes. In the process we have also found a confirmation for rthe validity of the Born approximation itself.     

Circular intensity differential scattering and chromatin-DNA structure. A combined theoretical approach

Circular intensity differential scattering (CIDS) has been proven a powerful method in determining the higher-order structure of large biopolymers, such as chromatin. Theoretical predictions of the expected differential light scattering of circularly polarized light have previously been made for chromatin, either within the Born approximation, treating nucleosomes as noninteracting, oblate ellipsoids, or within a multiple dipole approximation, treating nucleosomes as interacting spheres. In order to conduct a meaningful interpretation of the CIDS signal in terms of given geometric parameters of the chiral structure, we have in this paper combined the two approaches considering the mutual interactions of ellipsoidal nucleosomes. In the process we have also found a confirmation for the validity of the Born approximation itself.     

GRASS LEAF ULTRASTRUCTURAL VARIATIONS

The results of an ultrastructural examination of leaf anatomy and cytology of plastids of both non-Kranz and Kranz grasses are presented. Variations of Kranz anatomy examined include agranal and two granal conditions of kranz-cell chloroplasts, details of the two typical parenchyma sheaths of Aristida, and the “distinctive cells” of species of some small panicoid tribes. These conditions are discussed in relation to C₄ photosynthesis, evolution, and classification.     

Green tissue within the haustorium of the dwarf mistletoe Korthalsella (Viscaceae). An ultrastructural comparison between chloroplasts of sucker and aerial stem tissues

Summary Korthalsella (Viscaceae) is a dwarf mistletoe attached to its host branch by a single haustorium. Plants are leafless with flattened or cylindrical stems that function in photosynthesis. When a fresh haustorium is cut the sucker within the host appears bright green. Transmission electron microscopy reveals that this greening is due to chloroplasts, but that their organization differs from those of the aerial stem. The three representatives of Korthalsella endemic to New Zealand were the main species investigated. In the stem, chloroplasts have short stacks of cylindrical grana interconnected by stroma thylakoids typical of normal chloroplasts. Sucker chloroplasts have a more variable organization, with most containing extensive granal stacks and poorly differentiated stroma thylakoids. These granal thylakoids exhibit extensive partitions formed by appression of adjacent membranes. Some sucker plastids also approach etioplasts in having a prominent prolamellar body from which radiate thylakoids with short partitions. Sucker chloroplasts usually contain a few large starch grains, plastoglobuli, and sometimes also a stroma centre. The extensive granal thylakoids in sucker chloroplasts of Korthalsella resemble that found in certain shade plants and tissue grown under low light conditions. Sucker chloroplasts probably have a low level of photosynthesis. This activity might provide a local source of osmotically active material used to assist transport between host and parasite.     

Differential polarization imaging. V. Numerical aperture effects and the contribution of preferential scattering and absorption to the circular dichroism images.

Chiral objects which scatter and absorb preferentially left versus right circularly polarized light give rise to bright-field circular dichroism (CD) images containing contributions from both these two phenomena. These contributions are separated and characterized mathematically, and the effect of the dimensions of the chiral object on their relative magnitude is discussed. CD images of the long-range chiral organization of the thylakoid membranes in chloroplasts are obtained at two different wavelengths to illustrate the diverse wavelength dependence of the preferential absorption and scattering contributions to the images. The bright field CD images not only depend on the magnitude and sign of the preferential scattering and preferential absorption contributions, but also on the numerical aperture of the lens used. This dependence is obtained formally and a method to extract the angle dependent preferential scattering contributions to the images is presented. The validity of this method is confirmed experimentally.     

Effect of growth temperature on chloroplast structure and activity in barley

Seedlings of barley (Hordeum vulgare L. cv. Abyssinian) were grown at constant temperature and light intensity and the properties and structure of chloroplasts in the primary leaf were examined. Seventeen growth temperatures ranging from 2 to 37 C were employed. Three major effects of the growth temperature were seen. (a) At very low and high growth temperatures chloroplast biogenesis was inhibited. This occurred in plants grown at temperatures above 32 C while growth at 2 C resulted in a mixed population of pale yellow, pale green, and green plants. (b) Chloroplasts were produced at all other temperatures tested but growth temperatures within a few degrees of those inhibitory to chloroplast development resulted in chloroplasts with abnormal properties and structure. Chloroplasts in the green plants grown at 2 and 5 C showed a number of structural peculiarities, including a characteristic crimping of granal thylakoids. Photoreductive activity, measured using ferricyanide as the Hill oxidant in the presence of gramicidin D, was high, but this activity in chloroplasts isolated from plants grown at 2 C showed thermal inactivation at temperatures 5 degrees lower than was the case with plants grown at higher temperatures. High growth temperatures (30 to 32 C) yielded chloroplasts with reduced photoreductive activity and a tendency toward the formation of large grana and disorientation of the lamellar systems with respect to one another. Chloroplasts of the most affected plants (grown at 32 C) frequently contained a very large elongated granum, with narrow intrathylakoid spaces. (c) Photoreductive activity was not constant at intermediate growth temperatures but steadily declined with decreasing growth temperatures between 27 and 11 C. Some alterations in chloroplast structure were also observed.

The changes in chloroplast activity and structure indicate that acclimation to temperature takes place over the entire temperature range in which chloroplast development is permitted.

     

Studies of the Ndh complex and photosystem II from mesophyll and bundle sheath chloroplasts of the C4-type plant Zea mays

In C(4) plants, granal mesophyll (MS) chloroplasts contain higher photosystem (PS) II and lower PS I activity than agranal bundle sheath (BS) chloroplasts. The maize NAD(P)H dehydrogenase or NAD(P)H-plastoquinone oxidoreductase (also named Ndh complex) from MS and BS chloroplasts, contains at least 11 subunits (NdhA-K) and is homologous to NADH dehydrogenase or Complex I from mitochondria and bacteria. The amount of Ndh complex is higher in BS compared with MS chloroplasts. However, there is little information about the interdependence of the PS II and Ndh complex in chlororespiration and linear and cyclic electron transport in C(4) plants. To characterize the expression of the PS II and Ndh complex in maize plastids, we used cytochrome b559 (cyt b559) antibodies and Ndh immunoglobulins (IgG) to analyze the Ndh complex and PS II in both MS and BS chloroplasts from maize leaves by Western blotting and immunolabeling. In Western blot experiments, it was found that the amount of cyt b559 (a marker for PS II) is 7-8 times higher in MS than BS chloroplasts. Conversely, the NdhH, -J, -K and -E content is 2.5-3 times higher in BS than MS chloroplasts. Similar results were obtained in immunolabeling experiments using Ndh IgGs and cyt b559 antibodies in MS and BS chloroplasts. These data suggest that in BS chloroplasts, ATP could be produced mainly by cyclic electron transport around PS I and Ndh complexes. Conversely, the linear electron transport in BS chloroplasts via PS II could have a lower production of ATP. These results also suggest that the contribution of the Ndh complex in the production of ATP in MS chloroplasts is minimal and that instead, this complex could have a chlororespiratory role.     

Laser-flash-activated electron paramagnetic resonance studies of primary photochemical reactions in chloroplasts.

Electron paramagnetic resonance studies of the primary reactants of Photosystems I and II have been conducted at cryogenic temperatures after laser-flash activation with monochromatic light.P-700 photooxidation occurs irreversibly in chloroplasts and in Photosystem I fragments after activation with a 730 nm laser flash at a temperature of 35 degrees K. Flash activation of chloroplasts or Photosystem II chloroplast fragments with 660 nm light results in the production of a free-radical signal (g = 2.002, linewidth approximately 8 gauss) which decays with a half-time of 5.0 ms at 35 degrees K. The half-time of decay is independent of temperature in the range of 10-77 degrees K. This reversible signal can be eliminated by preillumination of the sample at 35 degrees K with 660 nm light (but not by 730 nm light), by preillumination with 660 nm light at room temperature in the presence of 3-(3',4'-dichlorophenyl)-1,1'-dimethylurea (DCMU) plus hydroxylamine, or by adjustment of the oxidation-reduction potential of the chloroplasts to - 150 mV prior to freezing. In the presence of ferricyanide (20-50 mM), two free-radical signals are photoinduced during a 660 nm flash at 35 degrees K. One signal decays with a half-time of 5 ms, whereas the second signal is formed irreversibly. These results are discussed in terms of a current model for the Photosystem II primary reaction at low temperature which postulates a back-reaction between P-680+ and the primary electron acceptor.