Biosynthesis of grana and stroma lamellae in spinach

Park and co-workers (Annu. Rev. Plant Physiol. 22: 395-430) have suggested that stroma lamellae, which perform only photosystem I contain a developing photosystem II which becomes functional upon the folding of these membranes to form grana stacks.     

Carotenoid composition of spinach chloroplast grana and stroma lamellae

Stroma lamellae and grana stacks prepared by French press rupture of spinach (Spinacia oleracea) chloroplasts contain similar amounts of β-carotene on a protein basis. The grana fraction has considerably more xanthophylls than does the stroma fraction. Total carotenoid to chlorophyll ratios are similar for both fractions.     

Chlorophyll,glycerolipid and protein ratios in spinach chloroplast grana and stroma lamellae

The relative molar amounts of glycerolipids are similar in grana and stroma lamellae, as are the ratios of total glycerolipid to weight of membrane protein. However the chlorophyll content relative to protein of grana lamellae is about 40% higher than that of stroma lamellae from the same preparation. Previous reports of chemical composition or enzyme activity based on chlorophyll alone can be highly misleading. The large functional and conformational differences between these two membranes may be related to these differences in pigment content, but are likely to result primarily from qualitative protein differences. The data are in accord with a membrane model in which nonpolar regions of membrane protein bind lipid in fairly constant amounts.     

Analysis of the polypeptide composition of grana and stroma thylakoids by two-dimensional gel electrophoresis. Distribution of photosystem II between grana and stroma lamellae

The polypeptide composition of whole thylakoids and membrane subfragments was studied by using a modified two-dimensional gel electrophoresis technique of O'Farrell [J. Biol. Chem. 250, 4007-4021 (1975)]. The modifications were lithium dodecyl sulphate solubilization instead instead of SDS, reverse isofocusing and sensitive silver staining procedure. This high-resolution technique allowed us to separate and identify about 170 polypeptides of thylakoid membranes. After separating grana and stroma thylakoids it was found that both types of lamellae contained nearly equal amounts of polypeptides, but about 70 polypeptides were different in the two preparations. In grana thylakoids, 54 polypeptides out of 95 were found to be mainly present in grana and 31 of them were only present in grana preparations. In stroma membranes, 43 polypeptides out of 99 were mainly present in stroma lamellae and 38 of these polypeptides were exclusively present in stroma lamellae. In a functional photosystem II preparation, 61 individual polypeptides could be distinguished. Most of these polypeptides were present in both grana and stroma lamellae, but 22 of them were more pronounced in grana than in stroma lamellae. 9 polypeptides of photosystem II were distinctly different in grana and stroma lamellae, and these differences may connect closely with the functional differences of photosystem II in the two types of thylakoids.     

Further evidence for stroma lamellae as a source of Photosystem 1 fractions from spinach chloroplasts

The mechanism of digitonin action on spinach chloroplasts was investigated by thin sectioning. Evidence is presented which shows that digitonin continues to modify membranes for many minutes after the addition of the fixative glutaraldehyde. However, the action of digitonin can be stopped by simultaneous fixation and dilution of the detergent. Such experiments indicate that the initial action of digitonin is to release stroma lamellae which in turn yield a Photosystem 1 fraction. This interpretation is further supported by a significant correlation between the chlorophyll a/chlorophyll b ratio and the ratio of stroma to grana lamellae in spinach chloroplasts.     

The PsbS protein controls the macro-organisation of photosystem II complexes in the grana membranes of higher plant chloroplasts

The PsbS protein is a critical component in the regulation of non-photochemical quenching (NPQ) in higher plant photosynthesis. Electron microscopy and image analysis of grana membrane fragments from wild type and mutant Arabidopsis plants showed that the semi-crystalline domains of photosystem II supercomplexes were identical in the presence and absence of PsbS. However, the frequency of the domains containing crystalline arrays was increased in the absence of PsbS. Conversely, there was a complete absence of such arrays in the membranes of plants containing elevated amounts of this protein. It is proposed that PsbS controls the macro-organisation of the grana membrane, providing an explanation of its role in NPQ.     

Chlorophyll,carotenoid, and lipid content in Triticum sativum L. plastid envelopes,prolamellar bodies,stroma lamellae,and grana

The pigment and lipid content, expressed on a protein basis, is compared in wheat etioplast and chloroplast membrane fractions. Chloroplast envelopes contain less carotenoid and 1/3 more lipid than etioplast envelopes. The minute amount of chlorophyll and carotenoid found in chloroplast envelopes could be due to thylakoid contamination. Prolamellar bodies and grana have nearly the same amount of total lipid and total carotenoid per mg of protein although their respective compositions differ. On a protein basis, the lipid, chlorophyll, and carotenoid contents are lower (2.3, 10, and 20 times, respectively) in stroma lamellae than in grana membranes, but the latter contains a higher proportion of -carotene, chlorophyll a, and sulfolipid.This research represents partial fulfillment of the thesis Doctorat d'Etat ès Sciences requirements of the author     

Differentiation of chloroplast lamellae: Light harvesting efficiency and grana development

Incomplete development of chloroplast lamellae occurred when etiolated pea plants were greened under cycles of 2 min light, 118 min dark. Although the plastids had full photochemical activities, they were nearly agranal. They were also characterized by a high quantum requirement for whole chain electron transport in low light; this is thought to be the result of unequal light absorption by incompletely developed light-harvesting assemblies of photosystem I and II and a lack of regulation of excitation energy distribution between the two photosystems. Continuous illumination induced the final stages of membrane differentiation. These stages were primarily characterized by the appearance of grana stacking and an increase in photosynthetic unit size. A biphasic decrease in quantum requirement for whole chain electron transport correlated directly with the appearance of grana during the final steps of membrane assembly. Structural organization of the membrane may be related to the light-harvesting efficiency of the membrane.     

The grana margins of plant thylakoid membranes

Plant thylakoid membranes contain three structurally distinct domains: the planar appressed membranes of the grana; the planar non-appressed stroma thylakoids; and the highly curved, non-appressed margins of the grana. Evidence is presented to suggest that the grana margins form a significant structural domain, which has hitherto been neglected. If indeed the grana margins contain some of the cytochrome b/f complex, photosystem (PS) I complex and ATP synthase, they form a third functional domain of the laterally heterogeneous continuous thylakoid membrane network. The consequences of grana margins containing complexes are explored with respect to linear electron transport under light-saturating and light-limiting conditions, non-cyclic vs cyclic photophorylation, and the regulation of light energy distribution to both PS I and PS II.     

Relative speed of fixation of glutaraldehyde and osmic acid in plant cells measured by grana appearance in chloroplasts

Summary Brief fixation in a mixture of glutaraldehyde and OsO₄ caused stacked chloroplast grana membranes in leaf cells of wheat, barley, tobacco, maize, cowpea, pigweed or bean plants to distend and vesiculate. Fixation with glutaraldehyde followed by OsO₄ prevented this fixation artifact. In a fixative mixture, OsO₄ apparently reacted with cell contents before glutaraldehyde.     

Movement of a sub-population of the light harvesting complex (LHCII) from grana to stroma lamellae as a consequence of its phosphorylation

Phosphorylation in vitro of the light-harvesting chlorophyll $\text{a}\text{b}$ protein complex associated with Photosystem II (LHC_II) resulted in the lateral migration of a subpopulation of LHC_II from the grana to the stroma lamellae. This movement was characterized by a decrease in the chlorophyll $\text{a}\text{b}$ ratio and an increase in the 77 K fluorescence emission at 681 nm in the stroma lamellae following phosphorylation. Polyacrylamide gel electrophoresis indicated that the principal phosphoproteins under these conditions were polypeptides of 26–27 kDa. These polypeptides increased in relative amount in the stroma lamellae and decreased in the grana during phosphorylation. Pulse/chase experiments confirmed that the polypeptides were labelled in the grana and moved to the stroma lamellae in the subsequent chase period. A fraction at the phospho-LHC_II, however, was unable to move and remained associated with the grana fraction. LHC_II which moved out into the stroma lamellae effectively sensitized Photosystem I (PS I), since the ability to excite fluorescence emission at 735 nm (at 77 K) by chlorophyll b was increased following phosphorylation. These data support the ‘mobile antenna’ hypothesis proposed by Kyle, Staehelin and Arntzen (Arch. Biochem. Biophys. (1983) 222, 527–541) which states that the alterations in the excitation-energy distribution induced by LHC_II phosphorylation are, in part, due to the change in absorptive cross-section of PS II and PS I, resulting specifically from the movement of LHC_II antennae chlorophylls from the PS-II-enriched grana to the PS-I-enriched stroma lamellae.     

Phytochrome-mediated control of grana and stroma thylakoid formation in plastids of mustard cotyledons

The etioplast»chloroplast transition in the cotyledons of mustard seedlings (Sinapis alba L.) has been studied by electron microscopy. It was found that the active form of phytochrome, established by a red light pulse pretreatment, increases the initial rate and eliminates the lag of grana and stroma thylakoid formation after the onset of white light 60 h after sowing. The effect of a pretreatment with 15 s red light pulses is fully reversible by 756 nm light pulses. This reversibility is lost within 5 min. Evidence is presented which suggests that the time course of grana and stroma thylakoid formation is not correlated with the time course of the dispersal of the prolamellar body. The different functions of phytochrome and chlorophyll in controlling thylakoid formation are discussed.     

Lack of the light-harvesting complex CP24 affects the structure and function of the grana membranes of higher plant chloroplasts

The photosystem II (PSII) light-harvesting antenna in higher plants contains a number of highly conserved gene products whose function is unknown. Arabidopsis thaliana plants depleted of one of these, the CP24 light-harvesting complex, have been analyzed. CP24-deficient plants showed a decrease in light-limited photosynthetic rate and growth, but the pigment and protein content of the thylakoid membranes were otherwise almost unchanged. However, there was a major change in the macroorganization of PSII within these membranes; electron microscopy and image analysis revealed the complete absence of the C(2)S(2)M(2) light-harvesting complex II (LHCII)/PSII supercomplex predominant in wild-type plants. Instead, only C(2)S(2) supercomplexes, which are deficient in the LHCIIb M-trimers, were found. Spectroscopic analysis confirmed the disruption of the wild-type macroorganization of PSII. It was found that the functions of the PSII antenna were disturbed: connectivity between PSII centers was reduced, and maximum photochemical yield was lowered; rapidly reversible nonphotochemical quenching was inhibited; and the state transitions were altered kinetically. CP24 is therefore an important factor in determining the structure and function of the PSII light-harvesting antenna, providing the linker for association of the M-trimer into the PSII complex, allowing a specific macroorganization that is necessary both for maximum quantum efficiency and for photoprotective dissipation of excess excitation energy.     

Endopeptidases in the stroma and thylakoids of pea chloroplasts

Three endopeptidases (EP2, EP3, and EP4) were identified after fractionation of pea (Pisum sativum, var Feltham First) chloroplast stromal extracts. All three were identified by their ability to cleave in vitro-synthesized preplastocyanin to lower molecular weight forms. EP2 is inhibited by phenylmethylsulfonylfluoride, and both EP2 and EP3 are inhibited by the heavy metal chelators 1,10-phenanthroline and EDTA. A further endopeptidase, EP5, was identified in Triton X-100 extracts of thylakoid membranes. Experiments involving contrifugation through a sucrose pad indicate that EP5 either has a high molecular weight or is associated with a thylakoid protein complex. EP5 is effectively inhibited by phenylmethylsulfonylfluoride, iodoacetate, and 1,10-phenanthroline, but not by EGTA. The implications of these results for the analysis of chloroplast protein maturation are discussed, and an improved protocol for the purification of the stromal processing peptidase is described which ensures the removal of EP2, the most active of the stromal peptidases analysed in this study.     

Circularly polarized chlorophyll luminescence reflects the macro-organization of grana in pea chloroplasts

Circular polarization of luminescence (CPL; Steinberg IZ (1978) Annu Rev Biophys Bioeng 7: 113–137) was applied to study pea chloroplasts in different structural states. The structural changes of chloroplasts were induced by variation of osmotic pressure, concentration of magnesium-ions or photoinhibition. Both large CPL and psi-type circular dichroism (psi, polymerization and salt induced) signals appeared in the presence of granal macrostructure and were sensitive to structural changes of the grana. The relation was studied between the amount of CPL expressed as an emission anisotropy factor g _em and amplitudes of the red psi-type CD bands. The positive psi-type CD band was not directly correlated with g _em possibly due to a large contribution of circular intensity differential scattering to the measured CD spectra. However, a linear correlation between the amplitude of the negative psi-type CD band and g _em was found. The CPL signal of pea chloroplasts was attributed to a psi-type origin, which is observed in macroaggregates with densely packed chromophores with a long-range chiral order, and directly depends on the level of macroorganization. With the use of CPL-based microscopy, the long-range packing of LHC II particles can be studied in individual chloroplasts in future. In addition, the CPL method in general allows the study of the macro-organization of grana in green leaves, where conventional light-transmission methods fail. This revised version was published online in June 2006 with corrections to the Cover Date.