Import of the barley PSI-F subunit into the thylakoid lumen of isolated chloroplasts

A full-length cDNA clone encoding the PSI-F subunit of barley photosystem I has been isolated and sequenced. The open reading frame encodes a precursor polypeptide with a deduced molecular mass of 24837 Da. The barley PSI-F precursor contains a bipartite presequence with characteristics similar to the presequences of proteins destined to the thylakoid lumen. In vitro import studies demonstrate that an in vitro synthesized precursor is transported across the chloroplast envelope and directed to the thylakoid membrane, where it accumulates in a protease-resistant form. Incubation of the precursor with a chloroplast stromal extract results in processing to a form intermediate in size between the precursor and mature forms. Hydrophobicity analysis of the barley PSI-F protein reveals a hydrophobic region predicted to be a membrane spanning -helix. The hydrophobic nature of PSI-F combined with a bipartite presequence is unusual. We postulate that the second domain in the bipartite presequence of the PSI-F precursor proteins is required to ensure the proper orientation of PSI-F in the thylakoid membrane. The expression of the PsaF gene is light-induced similar to other barley photosystem I genes.Abbreviations 16K 23K and 33K proteins, the 16 kDa, 23 kDa and 33 kDa subunits of the photosystem II oxygen-evolving complex - PSI-N and PSI-F photosystem I subunit N and F - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Knock-out of the chloroplast-encoded PSI-J subunit of photosystem I in Nicotiana tabacum

The plastid-encoded psaJ gene encodes a hydrophobic low-molecular-mass subunit of photosystem I (PSI) containing one transmembrane helix. Homoplastomic transformants with an inactivated psaJ gene were devoid of PSI-J protein. The mutant plants were slightly smaller and paler than wild-type because of a 13% reduction in chlorophyll content per leaf area caused by an approximately 20% reduction in PSI. The amount of the peripheral antenna proteins, Lhca2 and Lhca3, was decreased to the same level as the core subunits, but Lhca1 and Lhca4 were present in relative excess. The functional size of the PSI antenna was not affected, suggesting that PSI-J is not involved in binding of light-harvesting complex I. The specific PSI activity, measured as NADP(+) photoreduction in vitro, revealed a 55% reduction in electron transport through PSI in the mutant. No significant difference in the second-order rate constant for electron transfer from reduced plastocyanin to oxidized P700 was observed in the absence of PSI-J. Instead, a large fraction of PSI was found to be inactive. Immunoblotting analysis revealed a secondary loss of the luminal PSI-N subunit in PSI particles devoid of PSI-J. Presumably PSI-J affects the conformation of PSI-F, which in turn affects the binding of PSI-N. This together renders a fraction of the PSI particles inactive. Thus, PSI-J is an important subunit that, together with PSI-F and PSI-N, is required for formation of the plastocyanin-binding domain of PSI. PSI-J is furthermore important for stability or assembly of the PSI complex.     

Photosystem I reaction centers from maize bundle-sheath and mesophyll chloroplasts lack subunit III

Photosystem I reaction centers were isolated from mesophyll and bundle-sheath chloroplasts of the C4 maize plant. Both preparations were found to be free of chlorophyll b and to have the same spectral properties and chlorophyll/P700 ratio as photosystem I reaction centers isolated from C3 plants. Photosystem I reaction centers from both mesophyll and bundle sheath were found to consist of six subunits with apparent molecular masses of about 70 kDa, 20 kDa, 17 kDa, 16 kDa, 10 kDa and 8 kDa, corresponding to photosystem I reaction center subunits I, II, IV, V, VI and VII of spinach, as tested by their immunological cross-reactivity with antibody raised against the respective spinach subunits. No cross-reactivity was found with antibodies raised against subunit III of spinach, either in whole thylakoids or purified reaction centers of both bundle-sheath and mesophyll chloroplasts. It is concluded that photosystem I reaction centers of bundle-sheath and mesophyll thylakoids of maize are identical and lack the polypeptide corresponding to subunit III present in all C3 plants so far tested.     

Light regulation of photosynthetic membrane structure,organization, and function

The light environment during plant growth determines the structural and functional properties of higher plant chloroplasts, thus revealing a dynamically regulated developmental system. Pisum sativum plants growing under intermittent illumination showed chloroplasts with fully functional photosystem (PS) II and PSI reaction centers that lacked the peripheral chlorophyll (Chi) a/b and Chl a light-harvesting complexes (LHC), respectively. The results suggest a light flux differential threshold regulation in the biosynthesis of the photosystem core and peripheral antenna complexes. Sun-adapted species and plants growing under far-red-depleted illumination showed grana stacks composed of few (3–5) thylakoids connected with long intergrana (stroma) thylakoids. They had a PSII/PSI reaction center ratio in the range 1.3–1.9. Shade-adapted species and plants growing under far-red-enrichcd illumination showed large grana stacks composed of several thylakoids, often extending across the entire chloroplast body, and short intergrana stroma thylakoids. They had a higher PSII/PSI reaction center ratio, in the range of 2.2–4.0. Thus, the relative extent of grana and stroma thylakoid formation corresponds with the relative amounts of PSII and PSI in the chloroplast, respectively. The structural and functional adaptation of the photosynthetic membrane system in response to the quality of illumination involves mainly a control on the rate of PSII and PSI complex biosynthesis.     

Chlorophyll a/b-binding proteins, pigment conversions, and early light-induced proteins in a chlorophyll b-less barley mutant.

Monospecific polyclonal antibodies have been raised against synthetic peptides derived from the primary sequences from different plant light-harvesting Chl a/b-binding (LHC) proteins. Together with other monospecific antibodies, these were used to quantify the levels of the 10 different LHC proteins in wild-type and chlorina f2 barley (Hordeum vulgare L.), grown under normal and intermittent light (ImL). Chlorina f2, grown under normal light, lacked Lhcb1 (type I LHC II) and Lhcb6 (CP24) and had reduced amounts of Lhcb2, Lhcb3 (types II and III LHC II), and Lhcb4 (CP 29). Chlorina f2 grown under ImL lacked all LHC proteins, whereas wild-type ImL plants contained Lhcb5 (CP 26) and a small amount of Lhcb2. The chlorina f2 ImL thylakoids were organized in large parallel arrays, but wild-type ImL thylakoids had appressed regions, indicating a possible role for Lhcb5 in grana stacking. Chlorina f2 grown under ImL contained considerable amounts of violaxanthin (2-3/reaction center), representing a pool of phototransformable xanthophyll cycle pigments not associated with LHC proteins. Chlorina f2 and the plants grown under ImL also contained early light-induced proteins (ELIPs) as monitored by western blotting. The levels of both ELIPs and xanthophyll cycle pigments increased during a 1 h of high light treatment, without accumulation of LHC proteins. These data are consistent with the hypothesis that ELIPs are pigment-binding proteins, and we suggest that ELIPs bind photoconvertible xanthophylls and replace "normal" LHC proteins under conditions of light stress.     

Regulation of photosystem stoichiometry,chlorophyll a and chlorophyll b content and relation to chloroplast ultrastructure

The structural-functional organization of higher plant chloroplasts has been investigated in relation to the particular light conditions during plant growth. (1) Light intensity variations during growth caused changes in the $\text{Chl}\text{a}\text{Chl}\text{b}$ ratio, in the light-saturated uncoupled rates of electron transport to a Hill oxidant and in the distribution of the chloroplast volume between the membrane and stroma phases. (2) Light quality differences during growth had an effect on the PS II/PS I reaction center ratio and on the chloroplast membrane phase differentiation into grana and stroma thylakoids. Plants grown under far-red-enriched (680–710 nm) illumination contained higher (20–25%) amounts of PS II and simultaneously lower (20–25%) amounts of PS I reaction centers. They also showed a higher grana density along with thicker grana stacks in their chloroplasts. (3) The size of the light-harvesting antenna pool of PS II centers was estimated from the fluorescence time course of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea-poisoned chloroplasts and was found to be fairly constant (±10%) in spite of the variable PS II/PS I reaction center ratio. The results are compatible with the hypothesis that the structural entities of grana facilitated the centralization and relative concentration increase of a certain group of PS II reaction centers.     

Significance of structural variation in thylakoid membranes in maintaining functional photosystems during reproductive growth

Structural variation in the stroma‐grana (SG) arrangement of the thylakoid membranes, such as changes in the thickness of the grana stacks and in the ratio between grana and inter‐grana thylakoid, is often observed. Broadly, such alterations are considered acclimation to changes in growth and the environment. However, the relation of thylakoid morphology to plant growth and photosynthesis remains obscure. Here, we report changes in the thylakoid during leaf development under a fixed light condition. Histological studies on the chloroplasts of fresh green Arabidopsis leaves have shown that characteristically shaped thylakoid membranes lacking the inter‐grana region, referred to hereafter as isolated‐grana (IG), occurred adjacent to highly ordered, large grana layers. This morphology was restored to conventional SG thylakoid membranes with the removal of bolting stems from reproductive plants. Statistical analysis showed a negative correlation between the incidences of IG‐type chloroplasts in mesophyll cells and the rates of leaf growth. Fluorescence parameters calculated from pulse‐amplitude modulated fluorometry measurements and CO₂ assimilation data showed that the IG thylakoids had a photosynthetic ability that was equivalent to that of the SG thylakoids under moderate light. However, clear differences were observed in the chlorophyll a/b ratio. The IG thylakoids were apparently an acclimated phenotype to the internal condition of source leaves. The idea is supported by the fact that the life span of the IG thylakoids increased significantly in the later developing leaves. In conclusion, the heterogeneous state of thylakoid membranes is likely important in maintaining photosynthesis during the reproductive phase of growth.     

Increased zinc content in transplastomic tobacco plants expressing a polyhistidine-tagged Rubisco large subunit

Rubisco is a hexadecameric enzyme composed of two subunits: a small subunit (SSU) encoded by a nuclear gene (rbcS), and a large subunit (LSU) encoded by a plastid gene (rbcL). Due to its high abundance, Rubisco represents an interesting target to express peptides or small proteins as fusion products at high levels. In an attempt to modify the plant metal content, a polyhistidine sequence was fused to Rubisco, the most abundant protein of plants. Plastid transformation was used to express a polyhistidine (6x) fused to the C-terminal extremity of the tobacco LSU. Transplastomic tobacco plants were generated by cotransformation of polyethylene glycol-treated protoplasts using two vectors: one containing the 16SrDNA marker gene, conferring spectinomycin resistance, and the other the polyhistidine-tagged rbcL gene. Homoplasmic plants containing L8-(His)6S8 as a single enzyme species were obtained. These plants contained normal Rubisco amounts and activity and displayed normal photosynthetic properties and growth. Interestingly, transplastomic plants accumulated higher zinc amounts than the wild-type when grown on zinc-enriched media. The highest zinc increase observed exceeded the estimated chelating ability of the polyhistidine sequence, indicating a perturbation in intracellular zinc homeostasis. We discuss the possibility of using Rubisco to express foreign peptides as fusion products and to confer new properties to higher plants.     

The carboxyl-terminal region of the spinach PsaD subunit contains information for its specific assembly into plant thylakoids

The assembly of the multi-subunit membrane-protein Photosystem I (PS I) complex involves incorporation of peripheral proteins into the complex. Here we studied assembly of the PsaD subunit of the cyanobacterial and plant PS I into the thylakoid membranes. We generated partial and chimeric psaD genes from which labeled proteins were synthesized in vitro. Assembly of these proteins into the cyanobacterial or plant thylakoids was assayed. The deletion of leader sequence and N-terminal extension of spinach prePsaD did not inhibit its assembly into spinach or cyanobacterial thylakoids. Addition of these sequences to the cyanobacterial PsaD did not enable it to assemble into plant thylakoids. Moreover, these additions significantly decreased the ability of the chimeric proteins to assemble into cyanobacterial thylakoids. In contrast, when the carboxyl-terminal half of cyanobacterial PsaD was replaced by the corresponding region of the spinach PsaD, the chimeric protein could assemble into both spinach and cyanobacterial thylakoids. Therefore, information in the carboxyl-terminal region of spinach PsaD is crucial for its assembly into plant thylakoids.Abbreviation prePsaD precursor of the PsaD subunit of PS I     

The effect of isoproturon on root growth and ultrastructure of the photosynthetic apparatus of two wheat cultivars and a weed

The effect of isoproturon [N,N-dimethyl N'4-(1-methyl-ethyl) phenyl urea] on the germination, growth of root and ultrastructure of the photosynthetic apparatus was investigated in two wheat cultivars ( Triticum sativum L. cvs Castan and Esquilache) and a weed ( Lolium rigidum Gaud.). No inhibition of germination was apparent in any of the three plants studied. The inhibition of root growth was considerably significant, especially in cv. Esquilache. After 48 h treatment with isoproturon (340 μ M ), the chloroplasts of the two wheat cultivars were found to be similar, showing a higher number of grana with fewer thylakoids per granum (lower stacking degree), broader grana stacks than in the control and an absence of starch. However, after 60 h of treatment, swelling of the thylakoids occurred in the Esquilache cultivar.
The rye grass was the most affected by the herbicide; at low concentration (3.4 μ M ) the number and size of the grana decreased considerably and the stroma thylakoids were destroyed. At higher concentration (17 μ M ), there was a strong accumulation in certain areas of the stroma of a substance with a lipid-like appearance, probably derived from the disintegrating thylakoids. At the same concentrations some chloroplast envelopes were completely desintegrated.
The Hill reaction of the three plants exhibited a similar sensitivity towards the herbicide.     

Chloroplast ultrastructure in leaves of tobacco plants with the introduced gene for the acyl-lipid Δ9-desaturase from <Emphasis Type="Italic">Synechococcus vulcanus</Emphasis> at normal and low temperature

Ultrastructural changes in chloroplasts of tobacco plants (Nicotiana tabacum L.) with the introduced desC gene for the acyl-lipid Δ9-desaturase from the thermophilic cyanobacterium Synechococcus vulcanus were investigated during plant acclimation to cold. Control plants were transformed with an empty pGA482 binary vector. At optimum growth temperature, a decreased number of grana and thylakoids and an increased number of plastoglobules and their larger area were observed in transgenic plants when compared to control ones. In control plants, acclimation to cold (6 days at 10°C) resulted in the larger areas of chloroplasts and grana. These changes indicated starting cold-induced injuries manifested in swelling of the stroma and a slight decrease in the total number of thylakoids in the chloroplast. In contrast, transgenic plants responded to cold by reducing the chloroplast, granal, and plastoglobule areas. Meantime, the number of thylakoids per granum increased noticeably. The total number of thylakoids in the chloroplast increased from 123 to 203. It was concluded that expression of the acyl-lipid Δ9-desaturase gene in tobacco plants provided for the formation of the cell ultrastructure similar to one characteristic of cold-tolerant plants.     

Effect of Doubled-CO2 Concentration on the Ultrastructure of Chloroplasts from Medicago sativa and Setaria italica

It has been reported in quite a number of literatures that doubled CO2 concentration increased the photosynthetic rate and dry matter production of C3 plants, but substantially affected C4 plants little. However, why may CO2 enrichment promote growth and either no change or decrease reproductive allocation of the C3 species, but havinag no effects on growth characteristics of the C4 plants? So far, there has been no satisfactory explanation on that mentioned above, except the differences in their CO2 compensatory points. In the past, although some studies on ultrastructure of the chloroplasts under doubled CO2 concentration were limitedly conducted. Almost all the relevant experimental materials were only from C3 plants not from C4 plants, and even though the results were of inconsistancy. Thereby, it needs to verify whether the differences in photosynthesis of C3 and C4 plants at doubled CO2 level is caused by the difference in their chloroplast deterioration. Experiments to this subject were conducted at the Botanical Garden of Institute of Botany, Academia Sinica in 1993 and 1994. Both experimental materials from C3 plant alfalfa (Medicago sativa) and C4 plant foxtail millet (Setaria italica) were cultivated in the cylindrical open-top chambers (2.2 m in diameter × 2.4 m in height) with aluminum frames covered by polyethylene film. Natural air or air with 350× 10-6 CO2 were blown from the bottom of the chamber space with constant temperature between inside and outside of the chamber 〈0.2℃〉. Electron microscopic observation revealed that the ultrastructure of the chloroplasts from C3 plant Medicago sativa and C4 plant Seteria italica growing under the same doubled CO2 concentration were quite different from each other. The differential characteristics in ultrastructure of chloro plasts displayed mainly in the configuration of thylakoid membrances and the accumulation of starch grains. They were as follows: 1. The most striking feature was the building up of starch grains in the chloroplasts of the bundle sheath cells (BSCs) and the mesophyll cells (MCs) at doubled CO2 concentra tion. The starch grains appeared centrifugally first in the BSCs and then in the chloroplast of the other MCs. It was worthy to note that the starch grains in the chloroplasts of C4 plant Setaria ira/ica were much more than those of the C3 plant Medicago sativa . The decline of photosynthesis in the doubled CO2-grown C4 plants might be caused by an over accumulation of starch grains, that deformed the chloroplast even demaged the stroma thylakoids and grana. There might exsist a correlation between the comformation of thylakoid system and starch grain accumulation, namely conversion and transfer of starch need energy from ATP, and coupling factor (CF) for ATP formation distributed mainly on protoplastic surface (PSu) of stroma thylakoid membranes, as well as end and margin membranes of grana thylakoids. Thereby, these results could provide a conclusive evidence for the reason of non effectiveness on growth characteristics of C4 plant. 2. Under normal condition , the mature chlolroplats of higher plants usually develop complete and regularly arranged photosynthetic membrane systems . Chloroplasts from the C4 plant Setaria italica, however, exerted significant changes on stacking degree, grana width and stroma thylakoid length under doubled CO2 concentration; In these changes, the grana stacks were smaller and more numerous, and the number of thylakoids per granum was greatly increased, and the stroma thylakoid was greatly lengthened as compared to those of the control chloroplasts. But the grana were mutually intertwined by stroma thylakoid. The integrity of some of the grana were damaged due to the augmentation of the intrathylakoid space . Similarly, the stroma thylakoids were also expanded. In case. the plant was seriously effected by doubled CO2 concentration as observed in C4 plant Setaria italica , its chloroplasts contained merely the stroma (matrix) with abundant starch grains, while grana and stroma thylakoid membranes were unrecognizable, or occasionally a few residuous pieces of thylakoid membranes could be visualized, leaving a situation which appeared likely to be chloroplast deterioration. However, under the same condition the C3 plant Medicago sativa possessed normally developed chloroplasts, with intact grana and stroma thylakoid membranes. Its chloroplasts contained grana intertwined with stroma thylakoid membranes, and increased in stacking degree and granum width, in spite of more accumulated starch grains within the chloroplasts. These configuration changes of the thylakoid system were in consistant with the results of the authors another study on chloroplast function, viz. the increased capacity of chloroplasts for light absorption and efficiency of PSⅡ.     

The interaction between plastocyanin and photosystem I is inefficient in transgenic Arabidopsis plants lacking the PSI-N subunit of photosystem I

The PSI-N subunit of photosystem I (PSI) is restricted to higher plants and is the only subunit located entirely in the thylakoid lumen. The role of the PSI-N subunit in the PSI complex was investigated in transgenic Arabidopsis plants which were generated using antisense and co-suppression strategies. Several lines without detectable levels of PSI-N were identified. The plants lacking PSI-N assembled a functional PSI complex and were capable of photoautotrophic growth. When grown on agar media for several weeks the plants became chlorotic and developed significantly more slowly. However, under optimal growth conditions, the plants without PSI-N were visually indistinguishable from the wild-type although several photosynthetic parameters were affected. In the transformants, the second-order rate constant for electron transfer from plastocyanin to P700+, the oxidized reaction centre of PSI, was only 55% of the wild-type value, and steady-state NADP+ reduction was decreased to a similar extent. Quantum yield of oxygen evolution and PSII photochemistry were about 10% lower than in the wild-type at leaf level. Photochemical fluorescence quenching was lowered to a similar extent. Thus, the 40-50% lower activity of PSI at the molecular level was much less significant at the whole-plant level. This was partly explained by a 17% increase in PSI content in the plants lacking PSI-N.     

State of Brassica rapa photosynthetic membranes in microgravity

The structural characteristics of the photosynthetic apparatus of Brassica rapa plants grown on board the space shuttle Columbia (STS-87) for 15 days were examined using the methods of transmission electron microscopy and statistic programme STAT. Maintaining of the same growth conditions for control plants was realized with great accuracy using the Orbiter Environmental simulator in Kennedy Space Center. A grana number per a medial section 1.8 times decreased in microgravity. Considerable changes were also revealed in the grana structure in microgravity in comparison with th ground control, namely: 1/a greater diversity in the thylakoid length with granae and 2/ lateral shifting of the thylakoids lateral shifting of the thylakoids relative one to another. The previous mentioned pheomenon was found for 64% of the invested granae. Shifting of the thylakoids in the granae in microgravity led to increasing of the grana thylakoid surface exposed to a stroma. In addition, the volume of stromal thylakoids increased. The peculiarities in the photosynthetic apparatus structure in microgravity are supposed to be an evidence of decreasing in the light harvesting complex amount of photosystem II (PSII).     

TLP18.3, a novel thylakoid lumen protein regulating photosystem II repair cycle

A proteome analysis of Arabidopsis thaliana thylakoid-associated polysome nascent chain complexes was performed to find novel proteins involved in the biogenesis, maintenance and turnover of thylakoid protein complexes, in particular the PSII (photosystem II) complex, which exhibits a high turnover rate. Four unknown proteins were identified, of which TLP18.3 (thylakoid lumen protein of 18.3 kDa) was selected for further analysis. The Arabidopsis mutants (SALK_109618 and GABI-Kat 459D12) lacking the TLP18.3 protein showed higher susceptibility of PSII to photoinhibition. The increased susceptibility of DeltaTLP18.3 plants to high light probably originates from an inefficient reassembly of PSII monomers into dimers in the grana stacks, as well as from an impaired turnover of the D1 protein in stroma exposed thylakoids. Such dual function of the TLP18.3 protein is in accordance with its even distribution between the grana and stroma thylakoids. Notably, the lack of the TLP18.3 protein does not lead to a severe collapse of the PSII complexes, suggesting a redundancy of proteins assisting these particular repair steps to assure functional PSII. The DeltaTLP18.3 plants showed no clear visual phenotype under standard growth conditions, but when challenged by fluctuating light during growth, the retarded growth of DeltaTLP18.3 plants was evident.     

Three-dimensional architecture of grana and stroma thylakoids of higher plants as determined by electron tomography

We have investigated the three-dimensional (3D) architecture of the thylakoid membranes of Arabidopsis (Arabidopsis thaliana), tobacco (Nicotiana tabacum), and spinach (Spinacia oleracea) with a resolution of approximately 7 nm by electron tomography of high-pressure-frozen/freeze-substituted intact chloroplasts. Higher-plant thylakoids are differentiated into two interconnected and functionally distinct domains, the photosystem II/light-harvesting complex II-enriched stacked grana thylakoids and the photosystem I/ATP synthase-enriched, nonstacked stroma thylakoids. The grana thylakoids are organized in the form of cylindrical stacks and are connected to the stroma thylakoids via tubular junctions. Our data confirm that the stroma thylakoids are wound around the grana stacks in the form of multiple, right-handed helices at an angle of 20° to 25° as postulated by a helical thylakoid model. The junctional connections between the grana and stroma thylakoids all have a slit-like architecture, but their size varies tremendously from approximately 15 × 30 nm to approximately 15 × 435 nm, which is approximately 5 times larger than seen in chemically fixed thylakoids. The variable slit length results in less periodicity in grana/stroma thylakoid organization than proposed in the original helical model. The stroma thylakoids also exhibit considerable architectural variability, which is dependent, in part, on the number and the orientation of adjacent grana stacks to which they are connected. Whereas some stroma thylakoids form solid, sheet-like bridges between adjacent grana, others exhibit a branching geometry with small, more tubular sheet domains also connecting adjacent, parallel stroma thylakoids. We postulate that the tremendous variability in size of the junctional slits may reflect a novel, active role of junctional slits in the regulation of photosynthetic function. In particular, by controlling the size of junctional slits, plants could regulate the flow of ions and membrane molecules between grana and stroma thylakoid membrane domains.     

Reorganization of the chloroplast thylakoid system during decay of acquired thermotolerance of photosynthesis and aging of wheat leaves

A 3 hours heating at 39 degrees C of 14-day old wheat plants increases the termotolerance of photosynthesis, and also the length and number of thylakoids in chloroplast in mature leaves. The acquired termotolerance disappears within 10 days. Simultaneously the intensity of photosynthesis and the length of thylakoids decrease. Reduction of photosynthesis ability and of thylakoid membranes occurs in the first leaves of non-hardened plants during 14-29 days after sowing. The intensity of photosynthesis in plants of both variants positively correlates with the length of grana membranes and with the total length of membranes of all thylakoids. Besides, a positive correlation was detected between the intensity of photosynthesis and the share of small (2-7 thylakoids) grana and the length of their membranes in non-hardened plants. The level of thermotolerance of photosynthesis in leaves in heat hardened plants correlates positively with the length of grana membranes and with the total length of all thylakoid membranes and the share of small grana.     

Chloroplast ultrastructure,photosynthetic apparatus activities and production of steviol glycosides in <Emphasis Type="Italic">Stevia rebaudiana in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

The accumulation of steviol glycosides (SGs) in cells of Stevia rebaudiana Bertoni both in vivo and in vitro was related to the extent of the development of the membrane system of chloroplasts and the content of photosynthetic pigments. Chloroplasts of the in vitro plants, unlike those of the intact plants, had poorly developed membrane system. The callus cells grown in the light contained proplastids of almost round shape and their thylakoid system was represented by short thylakoids sometimes forming a little number of grana consisting of 2–3 thylakoids. In cells of the etiolated in vitro regenerants and the callus culture grown in the dark, only proplastids practically lacking the membrane system were observed. All the chloroplasts having developed thylakoids and forming at least a little number of grana were equipped with photochemically active reaction centers of photosystems 1 and 2. Leaves of in vivo plants accumulated greater amount of the pigments than leaves of the in vitro plants. In both the callus culture grown in the light and the etiolated in vitro regenerants, the content of the pigments was one order of magnitude lower than that in leaves of the intact plants. The callus tissue grown in the dark contained merely trace amounts of the pigments. Leaves of the intact and the in vitro plants did not exhibit any significant differences in photosynthetic O₂ evolution rate. However, photosynthetic O₂ evolution rate in the callus cells was much lower than that in the differentiated plant cells. The in vitro cell cultures containing merely proplastids did not practically produce SGs. However, after transferring these cultures in the light, both the formation of chloroplasts and the production of SGs in them were detected.     

Segregation of the photosystems in higher plant thylakoids and short- and long-term regulation by a mesoscopic approach

In this paper we consider the relationship between the lateral segregation of photosystems I and II in the grana and characteristics of the short- and long-term regulation in thylakoids following the mesoscopic approach. Our study is thermodynamic; it is based on the Flory-Huggins theory for binary mixtures and the McMillan-Mayer theory of heterogeneous solutions. We demonstrate that state transitions promote rearrangement of photosystems by either favoring their mixing after LHCII phosphorylation or enhancing their segregation after LHCII dephosphorylation. This regulation influences the entire system properties locally. We also demonstrate that the variations of the photosystem ratio promote rearrangement of the photosystems preserving their segregation. This regulation influences the entire system properties globally. The studies presented are another indication of the importance of the segregation of the photosystems in the grana thylakoids of higher plants. Segregation of PSIs and PSIIs is a signature of the spinodal decomposition, which is a fine regulatory mechanism, related to both the short- and long-term adaptations of the photosynthetic apparatus in higher plant thylakoids.     

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.