ULTRASTRUCTURAL CYTOLOGY OF CALLUS CULTURES OF STREPTANTHUS TORTUOSUS AS AFFECTED BY TEMPERATURE

Tissue culture cells of Streptanthus tortuosus var. orbiculatus (Cruciferae) which have acquired a spherical viruslike particle located in their nucleoli, designated cell line STV, developed supergranal chloroplasts and lost the ability to differentiate vascular tissues. The effect of temperature on the ultrastructural cytology of one line of the STV tissue, STV-I, was compared with the effect of temperature on the ultrastructural cytology of tissue culture cells lacking the viruslike particles (control cell lines). At 4 C, the cellular and ultrastructural appearance of control tissue culture cells differed from that of tissue grown at 22 C by producing increased amounts of endoplasmic reticulum and dictyosomes and by reduction of chloroplast thylakoids. STV-I cells were generally moribund as a result of 4 C treatment. Chloroplast thylakoids were also reduced in control tissue following growth at 10 C and the apparent quantities of endoplasmic reticulum and dictyosomes were similar to those observed in control cells grown at the control temperature (22 C), but less than those observed in tissue subjected to 4 C. STV-I tissue grown at 10 C demonstrated increased endoplasmic reticulum and dictyosomes and reduction of polysomal configurations. The mitochondrial morphology was variable and the cells contained supergranal chloroplasts and proplastids. At the control temperature (22 C), the fine structural appearance of control tissue culture cells was typical of parenchyma cells, but STV-I cells contained mitochondria of variable morphology and two types of chloroplasts— normal and supergranal. Control tissue grown at 30 C also contained proplastids, but these proplastids contained starch in contrast to the proplastids in control tissue grown at low temperatures. The ultrastructural cytology of STV-I cells grown at elevated temperature (30 C) was characterized by enlarged mitochondria containing massive lipid bodies and the presence of protoplastids with starch and supergranal chloroplasts.     

The Ultrastructure of Chloroplasts in Mineral-deficient Maize Leaves

The ultrastructure of mesophyll chloroplasts in full-nutrient and mineral-deficient maize (Zea mays) leaves was examined by electron microscopy after glutaraldehyde-osmium tetroxide fixation. Nitrogen, calcium, magnesium, phosphorus, potassium, and sulfur deficiencies were induced by growing the plants in nutrient culture. Distinctive chloroplast types were observed with each deficiency. Chloroplasts from nitrogen-deficient plants were reduced in size and had prominent osmiophilic globules and large grana stacks. Magnesium deficiency was characterized by the accumulation of osmiophilic globules and the progressive disruption of the chloroplast membranes. In calcium deficiency, the chloroplast envelope was often ruptured. Chloroplasts from potassium- or phosphorus-deficient plants possessed an extensive system of stroma lamellae. Sulfur deficiency resulted in a pronounced decrease of stroma lamellae, an increase in grana stacking, and the frequent occurrence of long projections extending from the body of the chloroplast. These morphological changes were correlated with functional alterations in the chloroplasts as measured by photosystem I and II activities. In chloroplasts of the nitrogen- and sulfur-deficient plants an increase in grana stacking was associated with an increase in photosystem II activity.     

Development of the Two Heterogeneous Photosystem II Units in Etiolated Bean Leaves

The development of the photosystem II units in relation to the heterogeneity of their photochemical centers was studied in etiolated bean leaves (Phaseolus vulgaris var. red kidney) greened under continuous or intermittent light. The study was done in order to see whether grana are the loci of the units with the efficient photosystem II activity (α units), while the stroma thylakoids are the loci of the units with the less efficient photosystem II activity (β units), as it has been proposed. In addition, the interrelations between α and β centers have been investigated. It was found that the α and the β centers of photosystem II were present in the first photosynthetic membranes irrespective of the mode of greening of the leaves. The magnitude of their respective photochemical rate constants, K′_α and K_β, increased with time in continuous light and it reached the steady-state values of the mature chloroplasts within 16 hours, while in intermittent light it remained smaller. The differentiation of the system II units in α and β centers containing units is more evident under conditions of intermittent illumination, i.e. when the rate of chlorophyll biosynthesis is the limiting step for chloroplast development.     

Arrangement of Photosystem II and ATP Synthase in Chloroplast Membranes of Spinach and Pea

We used cryoelectron tomography to reveal the arrangements of photosystem II (PSII) and ATP synthase in vitreous sections of intact chloroplasts and plunge-frozen suspensions of isolated thylakoid membranes. We found that stroma and grana thylakoids are connected at the grana margins by staggered lamellar membrane protrusions. The stacking repeat of grana membranes in frozen-hydrated chloroplasts is 15.7 nm, with a 4.5-nm lumenal space and a 3.2-nm distance between the flat stromal surfaces. The chloroplast ATP synthase is confined to minimally curved regions at the grana end membranes and stroma lamellae, where it covers 20% of the surface area. In total, 85% of the ATP synthases are monomers and the remainder form random assemblies of two or more copies. Supercomplexes of PSII and light-harvesting complex II (LHCII) occasionally form ordered arrays in appressed grana thylakoids, whereas this order is lost in destacked membranes. In the ordered arrays, each membrane on either side of the stromal gap contains a two-dimensional crystal of supercomplexes, with the two lattices arranged such that PSII cores, LHCII trimers, and minor LHCs each face a complex of the same kind in the opposite membrane. Grana formation is likely to result from electrostatic interactions between these complexes across the stromal gap.     

Spatial relationship of photosystem I, photosystem II, and the light- harvesting complex in chloroplast membranes

We have previously demonstrated (Armond, P. A., C. J. Arntzen, J.-M. Briantais, and C. Vernotte. 1976. Arch. Biochem. Biophys. 175:54-63; and Davis, D. J., P. A. Armond, E. L. Gross, and C. J. Arntzen. 1976. Arch. Biochem. Biophys. 175:64-70) that pea seedlings which were exposed to intermittent illumination contained incompletely developed chloroplasts. These plastids were photosynthetically competent, but did not contain grana. We now demonstrate that the incompletely developed plastids have a smaller photosynthetic unit size; this is primarily due to the absence of a major light-harvesting pigment-protein complex which is present in the mature membranes. Upon exposure of intermittent- light seedlings to continuous white light for periods up to 48 h, a ligh-harvesting chlorophyll-protein complex was inserted into the chloroplast membrane with a concomitant appearance of grana stacks and an increase in photosynthetic unit size. Plastid membranes from plants grown under intermediate light were examined by freeze-fracture electron microscopy. The membrane particles on both the outer (PF) and inner (EF) leaflets of the thylakoid membrane were found to be randomly distributed. The particle density of the PF fracture face was approx. four times that of the EF fracture face. While only small changes in particle density were observed during the greening process under continuous light, major changes in particle size were noted, particularly in the EF particles of stacked regions (EFs) of the chloroplast membrane. Both the changes in particle size and an observed aggregation of the EF particles into the newly stacked regions of the membrane were correlated with the insertion of light-harvesting pigment- protein into the membrane. Evidence is presented for identification of the EF particles as the morphological equivalent of a "complete" photosystem II complex, consisting of a phosochemically active "core" complex surrounded by discrete aggregates of the light-harvesting pigment protein. A model demonstrating the spatial relationships of photosystem I, photosystem II, and the light-harvesting complex in the chloroplast membrane is presented.     

A COMPARISON OF CHLOROPLAST MEMBRANE SURFACES VISUALIZED BY FREEZE-ETCH AND NEGATIVE STAINING TECHNIQUES; AND ULTRASTRUCTURAL CHARACTERIZATION OF MEMBRANE FRACTIONS OBTAINED FROM DIGITONIN-TREATED SPINACH CHLOROPLASTS

Spinach chloroplast lamellae were washed free of negatively staining surface particles (carboxydismutase and coupling factor protein) and the resulting smooth-surfaced lamellae still showed the usual large (175 A) and small (110 A) particles seen by freeze-etching. Therefore, the freeze-fracture plane probably occurs along an internal surface of the chloroplast membrane. Fractions obtained by differential centrifugation of digitonin-treated chloroplast membranes were studied by negative staining, thin sectioning, and freeze-etching techniques for electron microscopy. The material sedimenting between 1,000 g and 10,000 g, enriched in photosystem II activity, was shown to consist of membrane fragments. These freeze-etched membrane fragments were found to have large particles on most of the exposed fracture faces. The large particles had the same size and distribution pattern as the 175 A particles seen in intact chloroplast membranes. The material sedimenting between 50,000 g and 144,000 g, which had only photosystem I activity, was found to consist of particles in various degrees of aggregation. Freeze-etching of this fraction revealed only small particles corresponding to the 110 A particles seen in intact chloroplasts. A model is presented suggesting that chloroplast lamellar membranes have a binary structure, which digitonin splits into two components. The two membrane fragments have different structures, revealed by freeze-etching, and different photochemical and biochemical functions.     

Reversible particle movements associated with unstacking and restacking of chloroplast membranes in vitro

Freeze-fracture and freeze-etch techniques have been employed to study the supramolecular structure of isolated spinach chloroplast membranes and to monitor structural changes associated with in vitro unstacking and restacking of these membranes. High-resolution particle size histograms prepared from the four fracture faces of normal chloroplast membranes reveal the presence of four distinct categories of intramembranous particles that are nonrandomly distributed between grana and stroma membranes. The large surface particles show a one to one relationship with the EF-face particles. Since the distribution of these particles between grana and stroma membranes coincides with the distribution of photosystem II (PS II) activity, it is argued that they could be structural equivalents of PS II complexes. An interpretative model depicting the structural relationship between all categories of particles is presented. Experimental unstacking of chloroplast membranes in low-salt medium for at least 45 min leads to a reorganization of the lamellae and to a concomitant intermixing of the different categories of membrane particles by means of translational movements in the plane of the membrane. In vitro restacking of such experimentally unstacked chloroplast membranes can be achieved by adding 2-20 mM MgCl2 or 100-200 mM NaCl to the membrane suspension. Membranes allowed to restack for at least 1 h at room temperature demonstrate a resegregation of the EF-face particles into the newly formed stacked membrane regions to yield a pattern and a size distribution nearly indistinguishable from the normally stacked controls. Restacking occurs in two steps: a rapid adhesion of adjoining stromal membrane surfaces with little particle movement, and a slower diffusion of additional large intramembranous particles into the stacked regions where they become trapped. Chlorophyll a:chlorophyll b ratios of membrane fraction obtained from normal, unstacked, and restacked membranes show that the particle movements are paralleled by movements of pigment molecules. The directed and reversible movements of membrane particles in isolated chloroplasts are compared with those reported for particles of plasma membranes.     

Chemical modification studies of chloroplast membranes. Water oxidation inhibition by diazonium-benzenesulfonic acid

The water-soluble chemical modifier, diazonium benzene-sulfonic acid, significantly inhibited photosystem II-dependent water oxidation (oxygen evolution) when the compound was reacted with chloroplast membranes in the light but not in the dark. The photochemistry of photosystem II was not affected by the diazonium treatment, shown by complete restoration of photosystem II-dependent electron flow from the alternate electron donor diphenylcarbazide.Paralleling the inhibition of oxygen evolution the illuminated chloroplasts bound significantly more diazonium reagent than did chloroplasts treated in the dark. Both the inhibition of oxygen evolution and the increased binding of the diazonium to the membranes were dependent on photosystem II electron flux, which could not be replaced by photosystem I cyclic electron flow. A dark base to acid or acid to base transition resulted in a similar inhibition of water oxidation and increased diazonium binding.The results suggest a membrane conformational change associated with photosystem II electron flow that exposes otherwise buried diazo reactive groups at the external grana membrane surface.     

N,N-diethylhydroxylamine: a new electron donor to photosystem II

Diethylhydroxylamine, when added to beet spinach thylakoid membranes in the reaction mixture enhanced both photosystem II mediated dichlorophenolindophenol photoreduction and whole chain electron transport supported by methyl viologen. Diethylhydroxylamine supports dichlorophenolindophenol photoreduction when oxygen evolving complex is inactivated by hydroxylamine washings. All the electron transport assays were found to be highly sensitive to diuron, indicating that diethylhydroxylamine donates electrons to the photosystem II before the herbicide binding site. The stimulation of the photochemical activity by diethylhydroxylamine is not solely due to its action as an uncoupler. It was also observed that the action of diethylhydroxylamine was not altered by preincubations of thylakoids in light in the presence of diethylhydroxylamine. Also, thylakoid membranes did not lose their benzoquinone Hill activity by the pre-incubations with diethylhydroxylamine either in light or in dark. Thus, unlike the photosystem II electron donor, hydroxylamine, diethylhydroxylamine was found to donate electrons without the inactivations of oxygen evolving complex. It is suggested that diethylhydroxylamine is a useful electron donor to the photosystem II.     

Ultrastructural organization of chloroplast membranes in mutants of Pisum sativum L. with impaired activity in the photosystems

The ultrastructural organization and the photosynthesis reactions of chloroplast membranes were studied in three lethal mutants of Pisum sativum, Chl-1, Chl-19 and Chl-5, all lacking the capacity to evolve oxygen. The rates of 2,6-dichloroindophenol reduction, delayed fluorescence and electron-spin-resonance signal 1 indicate that Chl-1 and Chl-19 have an impaired activity in photosystem II (PS II), while in Chl-5 the electron transport is blocked between PS I and the reactions of CO₂ fixation. Ultrathin sectioning demonstrates the presence of giant grana in the chloroplasts of Chl-1 and Chl-19, while the chloroplast structure of the Chl-5 is very similar to that of the wild-type. The grana of the Chl-19 mutant contain large multilamellar regions of tightly packed membranes. When the chloroplast membranes were studied by freeze-fracture, the exoplasmic and protoplasmic fracture faces (EF and PF, respectively) in both stacked and unstacked membranes were found to show large differences in particle concentrations and relative population area (per m²), and also in particle size distribution, between all mutant chloroplast membranes and the wild-type. A close correlation between increasing k_mt (ratio of particle concentrations on PF/EF) and PS II activity was observed. The differences in particle concentrations on both fracture faces in different regions of the intact chloroplast membranes of the wild-type are the consequence of a rearrangement of existing membrane components by lateral particle movements since quantitative measurements demonstrate almost complete conservation of intramembrane particles in number and size during the stacking of stroma thylakoid membranes. The results indicating particle movements strongly support the concept that the chloroplast membranes have a highly dynamic structure.Abbreviations DPIP 2,6-dichloroindophenol - EF and PF exoplasmic and protoplasmic fracture faces, respectively - PS I and PS II photosystems I and II, respectively     

Effect of Evernic Acid on Structure of Spinach Chloroplasts

Detached leaves of Spinacia oleracea were incubated with evernicacid, the main phenolic substance present in Evernia prunastrithalli. This lichen substance produced a decrease in the amountof total chlorophyll and chlorophyll a in treated spinach leaves.Chloroplast structure suffered a decrease in several parameters,i.e. chloroplast area, number of grana, granal width, numberof thylakoids per granum and starch content. The submicroscopicstructure of the chloroplast membranes revealed smaller particlediameters in several of the fracture faces in the evernic acidtreated samples and even a decrease in the density of particlesin the EF, fracture face. The alterations observed may be relatedto changes in photosynthetic activity, probably by modificationof both photosystem I and photosystem II activities. Evernic acid, chloroplast structure, TEM, thylakoidal membrane, freeze-etching, chlorophyll content     

Structural and functional self-organization of Photosystem II in grana thylakoids

The biogenesis of the well-ordered macromolecular protein arrangement of photosystem (PS)II and light harvesting complex (LHC)II in grana thylakoid membranes is poorly understood and elusive. In this study we examine the capability of self organization of this arrangement by comparing the PSII distribution and antenna organization in isolated untreated stacked thylakoids with restacked membranes after unstacking. The PS II distribution was deduced from freeze-fracture electron microscopy. Furthermore, changes in the antenna organization and in the oligomerization state of photosystem II were monitored by chlorophyll a fluorescence parameters and size analysis of exoplasmatic fracture face particles. Low-salt induced unstacking leads to a randomization and intermixing of the protein complexes. In contrast, macromolecular PSII arrangement as well as antenna organization in thylakoids after restacking by restoring the original solvent composition is virtually identical to stacked control membranes. This indicates that the supramolecular protein arrangement in grana thylakoids is a self-organized process.     

Immunogold localization of LHC II proteins in chloroplasts with different degrees of grana stacking induced by SAN-9789.

The amount and distribution of proteins of the light-harvesting complex associated with photosystem II (PS II) were investigated using immunogold labelling of chloroplasts of wheat ( Triticum aestivum L. cv. Walde). The seedlings were grown in weak red light (16 mW m⁻²) after imbibition of grains with SAN-9789 (Norflurazon, 0.028 to 28 mg I⁻¹). Chloroplasts of these plants exhibited thylakoids with different degrees of stacking. Thylakoids of untreated plants grown in a greenhouse had most gold particles per unit membrane length in both appressed and non-appressed regions compared to red light grown plants. The ratios of labelling between appressed and non-appressed membranes were fairly constant in red light- and greenhouse-grown plants. The labelling densities were 2.5–3 times higher in the appressed thylakoids compared to the non-appressed thylakoids. However, at a SAN concentration of 2.8 mg I⁻¹ there was a sharp decrease in thylakoid appressions and in labelling density of both appressed and non-appressed membranes. The total amount of particles per chloroplast was also much lower as compared to that at lower SAN concentrations. Plants treated with the highest concentration of SAN (28 mg I⁻¹) contained chloroplasts devoid of normal grana structures. In these plastids, the thylakoids were elongated and single. The labelling density in these membranes was ca 50% of that observed at 2.8 mg I⁻¹. This paper thus supports earlier observations that proteins of the light-harvesting complex of PS II (LHC II) are mainly localized in the appressed regions of the grana membranes, and may be involved in the formation of grana.     

Chloroplast Response to Low Leaf Water Potentials: III. Differing Inhibition of Electron Transport and Photophosphorylation

Cyclic and noncyclic photophosphorylation and electron transport by photosystem 1, photosystem 2, and from water to methyl viologen (“whole chain”) were studied in chloroplasts isolated from sunflower (Helianthus annus L. var Russian Mammoth) leaves that had been desiccated to varying degrees. Electron transport showed considerable inhibition at leaf water potentials of −9 bars when the chloroplasts were exposed to an uncoupler in vitro, and it continued to decline in activity as leaf water potentials decreased. Electron transport by photosystem 2 and coupled electron transport by photosystem 1 and the whole chain were unaffected at leaf water potentials of −10 to −11 bars but became progressively inhibited between leaf water potentials of −11 and −17 bars. A low, stable activity remained at leaf water potentials below −17 bars. In contrast, both types of photophosphorylation were unaffected by leaf water potentials of −10 to −11 bars, but then ultimately became zero at leaf water potentials of −17 bars. Although the chloroplasts isolated from the desiccated leaves were coupled at leaf water potentials of −11 to −12 bars, they became progressively uncoupled as leaf water potentials decreased to −17 bars. Abscisic acid and ribonuclease had no effect on chloroplast photophosphorylation. The results are generally consistent with the idea that chloroplast activity begins to decrease at the same leaf water potentials that cause stomatal closure in sunflower leaves and that chloroplast electron transport begins to limit photosynthesis at leaf water potentials below about −11 bars. However, it suggests that, during severe desiccation, the limitation may shift from electron transport to photophosphorylation.     

Evidence for the role of surface-exposed segments of the light-harvesting complex in cation-mediated control of chloroplast structure and function.

Chloroplast membranes contain a light-harvesting pigment-protein complex (LHC) which binds chlorophylls a and b. A mild trypsin digestion of intact thylakoid membranes has been utilized to specifically alter the apparent molecular weights of polypeptides of this complex. The modified membrane preparations were analyzed for altered functional and structural properties. Cation-induced changes in room temperature fluorescence intensity and low temperature chlorophyll fluorescence emission spectra, and cation regulation of the quantum yield of photosystem I and II partial reactions at limiting light were lost following the trypsin-induced alteration of the LHC. Electron microscopy revealed that cations can neither maintain nor promote grana stacking in membranes which have been subjected to mild trypsin treatment. Freeze-fracture analysis of these membranes showed no significant differences in particle density or average particle size of membrane subunits on the EF fracture face; structural features of the modified lamellae were comparable to membranes which had been unstacked in a “low salt” buffer. Digitonin digestion of trypsin-treated membranes in the presence of cations followed by differential centrifugation resulted in a subchloroplast fractionation pattern similar to that observed when control chloroplasts were detergent treated in cation-free medium. We conclude that: (a) the initial action of trypsin at the thylakoid membrane surface of pea chloroplasts was the specific alteration of the LHC polypeptides, (b) the segment of the LHC polypeptides which was altered by trypsin is necessary for cation-mediated grana stacking and cation regulation of membrane subunit distribution, and (c) cation regulation of excitation energy distribution between photosystem I and II involves the participation of polypeptide segments of the LHC which are exposed at the membrane surface.     

The role of chlorophyll-protein complexes in the function and structure of chloroplast thylakoids

Summary The photosynthetic pigments of chloroplast thylakoid membranes are complexed with specific intrinsic polypeptides which are included in three supramolecular complexes, photosystem I complex, photosystem II complex and the light-harvesting complex. There is a marked lateral heterogeneity in the distribution of these complexes along the membrane with photosystem II complex and its associated light-harvesting complex being located mainly in the stacked membranes of the grana partitions, while photosystem I complex is found mainly in unstacked thylakoids together with ATP synthetase. In contrast, the intermediate electron transport complex, the cylochrome b-f complex, is rather uniformly distributed in these two membrane regions. The consequences of this lateral heterogeneity in the location of the thylakoid complexes are considered in relation to the function and structure of chloroplasts of higher plants.     

Investigations of the role of the main light-harvesting chlorophyll- protein complex in thylakoid membranes. Reconstitution of depleted membranes from intermittent-light-grown plants with the isolated complex

The functions of the light-harvesting complex of photosystem II (LHC- II) have been studied using thylakoids from intermittent-light-grown (IML) plants, which are deficient in this complex. These chloroplasts have no grana stacks and only limited lamellar appression in situ. In vitro the thylakoids showed limited but significant Mg2+-induced membrane appression and a clear segregation of membrane particles into such regions. This observation, together with the immunological detection of small quantities of LHC-II apoproteins, suggests that the molecular mechanism of appression may be similar to the more extensive thylakoid stacking seen in normal chloroplasts and involve LHC-II polypeptides directly. To study LHC-II function directly, a sonication- freeze-thaw procedure was developed for controlled insertion of purified LHC-II into IML membranes. Incorporation was demonstrated by density gradient centrifugation, antibody agglutination tests, and freeze-fracture electron microscopy. The reconstituted membranes, unlike the parent IML membranes, exhibited both extensive membrane appression and increased room temperature fluorescence in the presence of cations, and a decreased photosystem I activity at low light intensity. These membranes thus mimic normal chloroplasts in this regard, suggesting that the incorporated LHC-II interacts with photosystem II centers in IML membranes and exerts a direct role in the regulation of excitation energy distribution between the two photosystems.     

Hill Reaction, Hydrogen Peroxide Scavenging, and Ascorbate Peroxidase Activity of Mesophyll and Bundle Sheath Chloroplasts of NADP-Malic Enzyme Type C(4) Species

Intact mesophyll and bundle sheath chloroplasts wee isolated from the NADP-malic enzyme type C₄ plants maize, sorghum (monocots), and Flaveria trinervia (dicot) using enzymic digestion and mechanical isolation techniques. Bundle sheath chloroplasts of this C₄ subgroup tend to be agranal and were previously reported to be deficient in photosystem II activity. However, following injection of intact bundle sheath chloroplasts into hypotonic medium, thylakoids had high Hill reaction activity, similar to that of mesophyll chloroplasts with the Hill oxidants dichlorophenolindophenol, p-benzoquinone, and ferricyanide (approximately 200 to 300 micromoles O₂ evolved per mg chlorophyll per hour). In comparison to that of mesophyll chloroplasts, the Hill reaction activity of bundle sheath chloroplasts of maize and sorghum was labile and lost activity during assay. Bundle sheath chloroplasts of maize also exhibited some capacity for 3-phosphoglycerate dependent O₂ evolution (29 to 58 micromoles O₂ evolved per milligram chlorophyll per hour). Both the mesophyll and bundle sheath chloroplasts were equally effective in light dependent scavenging of hydrogen peroxide. The results suggest that both chloroplast types have noncyclic electron transport and the enzymology to reduce hydrogen peroxide to water. The activities of ascorbate peroxidase from these chloroplast types was consistent with their capacity to scavenge hydrogen peroxide.     

A new type of subchloroplast fragments isolated from pea chloroplasts in the presence of digitonin

Heavy fragments were isolated from pea chloroplasts using digitonin treatment and differential centrifugation. The particles were characterized by a significantly lowered chlorophyll a/b ratio, contents of photosystem I (PS I) proteins and ATPase, as well as of amount of P700. The content of photosystem II (PS II) proteins decreased insignificantly, whereas that of proteins of the light-harvesting complex II did not change. The absorption and low-temperature fluorescence spectra were indicative of a decreased content of PS I. Electron microscopy of ultrathin sections of heavy fragment preparations identified them as grana with reduced content of thylakoids. The diameter of these particles was practically the same as within chloroplasts. Comparison of various characteristics of the fragments and chloroplasts from which the fragments were isolated allowed us to define a high degree of preservation of marginal regions in thylakoids present in the heavy fragment particles. Analysis of the results shows that the procedure of fragmentation produces grana with high extent of thylakoid integrity. The phenomenon of reduction of the thylakoid content in grana, occurring as our heavy fragments, is considered in the frame of our previous hypothesis concerning the peculiarities of grana organization in the transversal direction.     

Photochemical Apparatus Organization in the Chloroplasts of Two Beta vulgaris Genotypes

The composition and structural organization of thylakoid membranes of a low chlorophyll mutant of Beta vulgaris was investigated using spectroscopic, kinetic and electrophoretic techniques. The data obtained were compared with those of a standard F1 hybrid of the same species. The mutant was depleted in chlorophyll b relative to the hybrid and it had a higher photosystem II/photosystem I reaction center (Q/P₇₀₀) ratio and a smaller functional chlorophyll antenna size. Analysis of thylakoid membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the mutant lacked a portion of the chlorophyll a/b light-harvesting complex but was enriched in the photosystem II reaction center chlorophyll protein complex. Comparison of functional antenna sizes and of photosystem stoichiometries determined electrophoretically were in good agreement with those determined spectroscopically. Both approaches indicated that about 30% of the total chlorophyll was associated with photosystem I and about 70% with photosystem II. A greater proportion of photosystem II_β was detected in the mutant. The results suggest that a higher photosystem II to photosystem I ratio in the sugar beet mutant has apparently compensated for the smaller photosystem II chlorophyll light-harvesting antenna in its chloroplasts. Moreover, a lack of chlorophyll a/b light-harvesting complex correlates with the abundance of photosystem II_β. It is proposed that a developmental relationship exists between the two types of photosystem II where photosystem II_β is a precursor form of photosystem II_α occurring prior to the addition of the chlorophyll a/b light-harvesting complex and grana formation.