Light-induced structural changes of thylakoids in normal and carotenoid deficient chloroplasts of maize

Summary Changes of membrane thickness and loculi were studied after red (650 nm) and far-red (707 nm) light in thylakoids of maize with different stacking and pigment compositions.The most intensive shrinkage of thylakoid membranes occurred in grana and under red light. Membranes of stroma thylakoids responded more to far-red light. Bundle sheath thylakoid membranes did not change in thickness. Loculi decreased in all types of thylakoids under both, red and far-red light. Thylakoids obtained from a -carotenic mutant exhibited a contrasting response: swelling under red light followed by photodestruction. Changes under far-red light were similar to that of normal stroma thylakoids.The data on normal chloroplasts show that the light induced shrinkage of membranes and the decrease of loculi are coupled to a different degree in various kinds of thylakoids; that the thylakoid flattening can be correlated with the Photosystem content of the membranes; and that two kinds of single thylakoids (stroma lamellae and bundle sheath lamellae) are different in molecular structure and function.Data on carotenoid deficient chloroplasts indicate a photooxidative destruction of the thylakoids by Photosystem 2 that occurs in the absence of normal carotenoids.     

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 constant proportion of grana and stroma lamellae in plant chloroplasts

The relative proportion of stroma lamellae and grana end membranes was determined from electron micrographs of 58 chloroplasts from 21 different plant species. The percentage of grana end membranes varied between 1 and 21% of the total thylakoid membrane indicating a large variation in the size of grana stacks. By contrast the stroma lamellae account for 20.3 ± 2.5 ( sd )% of the total thylakoid membrane. A plot of percentage stroma lamellae against percentage of grana end membranes fits a straight line with a slope of zero showing that the proportion of stroma lamellae is independent of the size of the grana stacks. That stroma lamellae account for about 20% of the thylakoid membrane is in agreement with fragmentation and separation analysis (Gadjieva et al . Biochim. Biophys. Acta 144: 92–100, 1999). Chloroplasts from spinach, grown under high or low light, were fragmented by sonication and separated by countercurrent distribution into two vesicle populations originating from grana and stroma lamellae plus end membranes, respectively. The separation diagrams were very similar lending independent support for the notion that the proportion of stroma lamellae is constant. The results are discussed in relation to the composition and function of the chloroplast in plants grown under different environmental conditions, and in relation to a recent quantitative model for the thylakoid (Albertsson, Trends Plant Sci. 6: 349–354, 2001).     

Light-induced De-epoxidation in Lettuce Chloroplasts: VI. De-epoxidation in Grana and in Stoma Lamellae

Grana and stroma lamellae fractions prepared from illuminated chloroplasts (Lactuca sativa L. var. Manoa) by French press treatment contained less violaxanthin and more zeaxanthin than the corresponding fractions from dark controls. In both fractions, only part of the total violaxanthin was de-epoxidized under illumination, and the ratio of de-epoxidized and unchanged violaxanthin was similar. This not only shows that the de-epoxidation system is present in both grana and stroma thylakoids but also that violaxanthin is heterogeneous in both membranes. The presence and similarity of the de-epoxidation system in grana and stroma lamellae suggest that the function of the violaxanthin cycle is linked to photosynthetic activities which are common to both types of membranes.     

Fragmentation and separation analysis of the photosynthetic membrane from spinach

Membrane vesicles, originating from grana, grana core (appressed grana regions), grana margins and stroma lamellae/end membranes, were analysed by counter current distribution (CCD) using aqueous dextran-polyethylene glycol two-phase systems. Each vesicle population gave rise to distinct peaks in the CCD diagram representing different vesicle subpopulations. The grana vesicles and grana core vesicles each separated into 3 different subpopulations having different chlorophyll a/b ratios and PSI/PSII ratios. Two of the grana core subpopulations had a chlorophyll a/b ratio of 2.0 and PSI/PSII ratio of 0.10 and are among the most PSII enriched thylakoid vesicle preparation obtained so far by a non detergent method. The margin vesicles separated into 3 different populations, with about the same chlorophyll a/b ratios, but different fluorescence emission spectra. The stroma lamellae/end membrane vesicles separated into 4 subpopulations. Plastoglobules, connected to membrane vesicles, were highly enriched in 2 of these subpopulations and it is proposed that these 2 subpopulations originate from stroma lamellae while the 2 others originate from end membranes. Fragmentation and separation analysis shows that the margins of grana constitute a distinct domain of the thylakoid and also allows the estimation of the chlorophyll antenna sizes of PSI and PSII in different thylakoid domains.     

大麦和玉米叶片叶绿体中Rubisco及其活化酶的免疫金标记定位

运用免疫金标记电镜技术研究了禾本科C3植物大麦(Hordeum vulgare L.)和C4植物玉米(Zea mays L.)叶片中Rubisoo及其活化酶(RCA)的细胞定位,结果表明:两种植物叶片解剖结构及叶绿体超微结构差别明显.在大麦叶细胞中,只有一种叶肉细胞叶绿体,Rubisoo和RCA主要分布于叶绿体的间质中.在玉米叶细胞中,存在着维管束鞘细胞和叶肉细胞两种类型叶绿体,Rubisco主要分布于鞘细胞叶绿体的基质中,但在叶肉细胞叶绿体中亦有少量特异性标记;RCA在鞘细胞叶绿体和叶肉细胞叶绿体的基质中都有分布.两种植物叶绿体结构及光合作用关键酶定位的不同,体现了C3植物和C4植物在光合器结构与功能上的差异.     

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.     

ULTRASTRUCTURE OF THE LAMELLAE AND GRANA IN THE CHLOROPLASTS OF ZEA MAYS L

The fine structure of the chloroplasts of maize (Zea mays L.) has been investigated by electron microscopic examination of ultrathin sections of leaves fixed in buffered osmium tetroxide solutions. Both the parenchyma sheath and mesophyll chloroplasts contain a system of densely staining lamellae about 125 A thick immersed in a finely granular matrix material (the stroma), and are bounded by a thin limiting membrane which often appears as a double structure. In the parenchyma sheath chloroplasts, the lamellae usually extend the full width of the disc-shaped plastids, and grana are absent. The mesophyll chloroplasts, however, contain numerous grana of a fairly regular cylindrical form. These consist of highly ordered stacks of dense lamellae, the interlamellar spacing being ca. 125 A. The grana are interlinked by a system of lamellae (intergrana lamellae) which are on the average about one-half as numerous as the lamellae within the grana. In general, this appears to be due to a bifurcation of the lamellae at the periphery of the granum, but more complex interrelationships have been observed. The lamellae of the parenchyma sheath chloroplasts and those of both the grana and intergrana regions of the mesophyll chloroplasts exhibit a compound structure when oriented normally to the plane of the section. A central exceptionally dense line (ca. 35 A thick) designated the P zone is interposed between two less dense layers (the L zones, ca. 45 A thick), the outer borders of which are defined by thin dense lines (the C zones). Within the grana, the C zones, by virtue of their close apposition, give rise to thin dense intermediate lines (I zones) situated midway between adjacent P zones. A model of the lamellar structure is proposed in which mixed lipide layers (L zones) are linked to a protein layer (P zone) by non-polar interaction. Chlorophyll is distributed over the entire lamellar surface and held in the structure by van der Waals interaction of the phytol "tail" with the hydrocarbon moieties of the mixed lipide layers. The evidence in favour of the model is briefly discussed.     

Photosystem II Activity in Agranal Bundle Sheath Chloroplasts from Zea mays

The photochemical activities of chloroplasts isolated from bundle sheath and mesophyll cells of maize (Zea mays var. DS606A) have been measured. Bundle sheath chloroplasts are almost devoid of grana, except in very young leaves, while mesophyll chloroplasts contain grana at all stages of leaf development.     

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.     

FORMATION OF THE PROLAMELLAR BODY IN 8-DAY,DARK-GROWN SEEDLINGS

The development of the prolamellar body in etioplasts of dark-grown seedlings of Phaseolus vulgaris is followed through the 8th day. From 2 to 6 days there is an increase in plastid size and starch content and synthesis of a system of porous lamellae which appear to arise, as such, from the inner component of the plastid envelope. From 6 to 8 days much of the starch disappears accompanied by rapid membrane synthesis resulting in an extensive prolamellar body. A model of the prolamellar body is discussed in which the basic structural unit is a six-pointed star with four tubules joining at each node. Observation of face views of the porous peripheral lamellae at their juncture with the prolamellar body suggests the origin of the prolamellar body by the continued contraction of the porous lamellae and the formation of interconnecting tubules between adjacent lamellae. The pores of the peripheral lamellae appear to correspond to the areas of stroma within each star module. Short lengths of membranes of individual peripheral lamellae fuse, forming short overlaps which resemble small, two-compartmented grana. It is postulated that this is the initial step in grana formation.     

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.     

Localization of photophosphorylation and proton transport activities in various regions of the chloroplast lamellae

Membrane fragments released by French pressure cell treatment of whole chloroplasts and isolated by differential centrifugation have been characterized structurally and with respect to phosophorylating and proton transport activities. In agreement with results of other workers, the heavy fraction released by pressure treatment was found by electron microscopy studies to be made up of mostly intact grana stacks while the light fraction was comprised of vesicles derived from the stromal lamellae. Both fractions were found to carry out rapid rates of cyclic photophosphorylation catalyzed by phenazine methosulfate (PMS). However, only the grana membranes demonstrated active proton accumulation in the presence of PMS. No light induced H⁺ uptake could be detected in the stromal lamellae fraction; and as expected, proton gradient dissipating agents such as NH₄Cl, nigericin in the presence of K⁺, and gramicidin were only slightly inhibitory to phosphorylation at concentrations which were very inhibitory in the grana membrane fraction.

Further evidence that stromal lamellae do not have active proton transport in the intact chloroplast was obtained by comparing various chloroplasts having different amounts of stromal and grana membranes. Comparative studies on young and old chloroplasts from lettuce, mesophyll and bundle sheath cell plastids from sorghum, and greening plastids from etiolated corn seedlings revealed a direct correlation between the extent of grana formation and the amount of proton transport activity. Samples which had larger amounts of stromal lamellae had high rates of ATP formation but a reduced capacity for H⁺ accumulation.     

Immunocytochemical and Cytochemical Localization of Photosystems I and II

Cytochemical and immunocytochemical methods were used to localize photosystems I and II in barley (Hordeum vulgare L. cv Himalaya) chloroplasts. PSI activity, monitored by diaminobenzidine oxidation, was associated with the lumen side of the thylakoids of both grana and stroma lamellae. The P₇₀₀ chlorophyll a protein, the reaction center of PSI, was localized on thin sections of barley chloroplasts using monospecific antibodies to this protein and the peroxidase-antiperoxidase procedure. Results obtained by immunocytochemistry were similar to those of the diaminobenzidine oxidation: both grana and stroma lamellae contained immunocytochemically reactive material. Both the grana and stroma lamellae were also labeled when isolated thylakoids were reacted with the P₇₀₀ chlorophyll a protein antiserum and then processed by the peroxidase-antiperoxidase procedure. PSII activity was localized cytochemically by monitoring the photoreduction of thiocarbamyl nitroblue tetrazolium, a reaction sensitive to the PSII inhibitor, DCMU. PSII reactions occurred primarily on the grana lamellae, with weaker reactions on the stroma lamellae.     

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.     

Entwicklung und Struktur der Proplastiden

In this study the proplastid development in embryonic cells is described for the apical meristem of Elodea canadensis, embryo sacs from Lilies, and Begonia leaf buds. The formation of these cell organelles originates with submicroscopical particles which consist of a homogeneous stroma with a surrounding double membrane. When these proplastids reach an average size of 1 µ, the inner layer of the membrane begins to invaginate into the stroma. This process is comparable to tubuli formation in mitochondria. Under growth conditions with sufficient exposure to light, the development of the grana and stroma lamellae proceeds without interruption. If the plants are kept in the dark, small vesicles are formed which accumulate in the prolamellar body of the proplastids. After illumination these elementary vesicles merge to form membranes which evolve into grana and stroma lamellae. The structural similarity of the early proplastid stages with the mitochondria seems to indicate that there exists some phylogenetic relationship between the two cell organelles.     

Core protein phosphorylation facilitates the repair of photodamaged photosystem II at high light

Phosphorylation of photosystem II (PSII) reaction center protein D1 has been hypothesised to function as a signal for the migration of photodamaged PSII core complex from grana membranes to stroma lamellae for concerted degradation and replacement of the photodamaged D1 protein. Here, by using the mutants with impaired capacity (stn8) or complete lack (stn7 stn8) in phosphorylation of PSII core proteins, the role of phosphorylation in PSII photodamage and repair was investigated. We show that the lack of PSII core protein phosphorylation disturbs the disassembly of PSII supercomplexes at high light, which is a prerequisite for efficient migration of damaged PSII complexes from grana to stroma lamellae for repair. This results in accumulation of photodamaged PSII complexes, which in turn results, upon prolonged exposure to high light (HL), in general oxidative damage of photosynthetic proteins in the thylakoid membrane.     

Lipid composition of envelopes,prolamellar bodies and other plastid membranes in etiolated,green and greening wheat leaves

Summary Comparative studies of lipid composition were made on prolamellar bodies, envelopes and other plastid membranes separately extracted from etiolated, green or greening (intermittent or continuous light) wheat (Triticum sativum L.) leaves. The different membrane fractions were examined by electron microscopy.The major lipid was digalactosyldiglyceride in the envelopes and prolamellar bodies and monogalactosyldiglyceride in stroma lamellae and grana. Phosphatidylcholine represented 60% of total phospholipids in the envelopes, 30% in prolamellar bodies and 14% in grana. All types of envelopes had the same lipid proportions.For all lipids the lowest fatty acid unsaturation was always found in the envelope membranes. The relative amount of {ie193-1} acid in the phosphatidylglycerol of envelopes increased from 4% (etioplasts) to an average of 15% (etiochloroplasts and chloroplasts).Abbreviations DGDG digalactosyldiglyceride - MGDG monogalactosyldiglyceride - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - PI phosphatidylinositol - PS phosphatidylserine - SL sulfolipid     

THE ULTRASTRUCTURAL DEVELOPMENT OF THE DIMORPHIC PLASTIDS OF ZEA MAYS L.

The development of the dimorphic chloroplasts of Zea mays L. in adult foliage leaves is described, and a method of correlating ultrastructural stages by means of leaf chlorophyll is presented. In addition, the developmental changes in chlorophyll a/b ratio are discussed. Both the mesophyll and the bundle sheath plastids contain small grana at the earliest stages of plastid development. As the plastids enlarge, the mesophyll grana stacks increase in both length of the appressed membrane and in the number of thylakoids per granum. Initially, the grana stacks in the bundle sheath plastids also enlarge, but as the plastids approach full size, most of the membrane appression is lost. However, the remaining areas of appression in the bundle sheath plastids show an increase in the number of thylakoids in each small granum.     

In situ localization of the sites of paraquat action

Abstract. Effects of paraquat were monitored by electron microscopic, cytochemical, and in vivo spectrophotometric procedures. Electron microscopy of paraquat-treated pea leaf discs indicates that the chloroplasts are affected primarily with both thylakoid dilation and apparent lamellar fusions, resulting in a 'honeycomb' of lamellae. No ultra-structural effects were noted when leaf discs were incubated in both DCMU and paraquat. Paraquat-induced peroxide was located cytochemically along the stroma lamellae, on the ends of the grana stack and in the dilated thylakoids. Less destruction was noted in those samples where peroxide was precipitated by cerium chloride, indicating that peroxide or one of its products is one of the major causes of paraquat toxicily. In C₄ plants mesophyll plastids exhibited much more peroxide deposition than was detected in bundle sheath plastids. No plastid peroxide depositions were noted in a photobleaching mutant that is resistant to paraquat. In situ cytochrome f oxidation-reduction was followed during paraquat treatments and resulted in a hastening of the dark re-reduction of cytochrome f after only 1 h of treatment. This effect was prevented by DCMU or CeCI₃. Electron transport, as measured by cytochrome f oxidation-reduction, was completely obliterated by a 24 h paraquat treatment. These in situ studies on paraquat confirm previous studies that the primary effect in the light is on the plastid and involves peroxide or further products generated by peroxide.