Effect of Irradiance on the Thermal Stability of Thylakoid Membrane Isolated from Acclimated Wheat Leaves

Thermal stability of thylakoid membranes isolated from acclimated and non-acclimated wheat (Triticum aestivum L. cv. HD 2329) leaves under irradiation was studied. Damage to the photosynthetic electron transport activity was more pronounced in thylakoid membranes isolated from non-acclimated leaves as compared to thylakoid membrane isolated from acclimated wheat leaves at 35 °C. The loss of D1 protein was faster in non-acclimated thylakoid membrane as compared to acclimated thylakoid membranes at 35 °C. However, the effect of elevated temperature on the 33 kDa protein associated with oxygen evolving complex in these two types of thylakoid membranes was minimal. Trypsin digestion of the 33 kDa protein in the thylakoid membranes isolated from control and acclimated seedlings suggested that re-organisation of 33 kDa protein occurs before its release during high temperature treatment.     

Changes of Freeze-Fracture Ultrastructure and Light Harvesting Chlorophyll Protein Complex in Thylakoid Membranes During Ontogeny of Maize

Freeze-fracture electron microscopy enables us to observe and count the freeze-fracture particles which correspond to the different functional components of thylakoid membranes. The present paper reports the observation on freeze-fracture ultrastructure of thylakoid membranes and the analysis of proteins by the SDS-polyacrylamide gel electrophoresis within the membranes from differenly located leaves of maize. In the past, we found that the leaies subtending the ear of maize had a much higher chlorophyll content, a lower chlorophyll a/b ratio and more staking thylakoid membranes and provided the photosynthetic energy used to fill the maize seeds more than that of other leaves. Recently, we have further found that the particle densities of all four faces of thylakoid membranes from the ear leaf were the highest, than those, successively, from the terminal leaf, and the fifth leaf (from the base of the plant). The particle densities on all four fracture faces of thylakoid membranes isolated from the ear leaves of maize were significantly higher than those from the terminal leaves with the increases of 19% in EFs, 28% in PFs and 20% in PFu. Increases in particle densities on the PFs, EFs and PFu faces result in increased densities of LHCP II, PSⅡ and PSI reactions centres, respectively. It is significant that this supramolecular architecture of the ear leaves is consistent with our analytical results of the SDS-polyacrylamide gel electrophoresis within the membranes (a detailed report in another paper). The contents of major polypeptides of 21 kD (LHCP Ⅰ) and 25 kD (LHCP Ⅱ) in thylakoid membranes from the ear leaves were more than those from the terminal leaves. The characteristics of both supramolecular architecture and polypeptide components are in favour of absorbing, transferring, distributing and conversing light energy in the course of photosynthesis of the ear leaves in maize.     

EFFECT OF SHORT-TERM SHADING ON THE CYTOLOGY OF PALISADE TISSUE IN MATURE LEAVES OF THE SUNFLOWER HELIANTHUS ANNUUS

Mature sunflower leaves were exposed to partial shading (35 or 14% of normal sun) or darkness (0% of normal sun) for approximately 8 hr. During this period one-half of each test leaf was shaded; the other half was used as a normal sun control. Palisade cell structure from both halves of each leaf was compared. Shading of leaves had little effect on organelle percent volume values (V_v) with exception of the starch compartment which decreased as shading increased. The surface to volume ratio (S_v) of the chloroplast thylakoids increased while the S_v of the mitochondrial membranes decreased as shading increased. Palisade cell volume did not change in shaded portions of the leaf, except in the fully shaded (dark) tissues where cell volume decreased. Changes in the actual volume of organelle compartments were strongly correlated with changes in cell volume. Thus a general osmotic response may account for some of the volume changes associated with differences in light intensity. Shading increased thylakoid surface areas 10–30% over the full sun controls. The ratio of stromal to granal thylakoid surface area remained constant in both the control and partially shaded samples. However, in darkened samples this ratio decreased as stromal membranes increased more than granal membranes. Changes observed in thylakoid surface areas associated with shading did not support thylakoid models which propose the interconversion of granal membranes to stromal membranes and vice versa.     

In vivo regulation of thylakoid proton motive force in immature leaves

In chloroplast, proton motive force (pmf) is critical for ATP synthesis and photoprotection. To prevent photoinhibition of photosynthetic apparatus, proton gradient (ΔpH) across the thylakoid membranes needs to be built up to minimize the production of reactive oxygen species (ROS) in thylakoid membranes. However, the regulation of thylakoid pmf in immature leaves is little known. In this study, we compared photosynthetic electron sinks, P700 redox state, non-photochemical quenching (NPQ), and electrochromic shift (ECS) signal in immature and mature leaves of a cultivar of Camellia. The immature leaves displayed lower linear electron flow and cyclic electron flow, but higher levels of NPQ and P700 oxidation ratio under high light. Meanwhile, we found that pmf and ΔpH were higher in the immature leaves. Furthermore, the immature leaves showed significantly lower thylakoid proton conductivity than mature leaves. These results strongly indicated that immature leaves can build up enough ΔpH by modulating proton efflux from the lumenal side to the stromal side of thylakoid membranes, which is essential to prevent photoinhibition via thermal energy dissipation and photosynthetic control of electron transfer. This study highlights that the activity of chloroplast ATP synthase is a key safety valve for photoprotection in immature leaves.     

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.     

Freezing damage and frost tolerance of the photosynthetic apparatus studied with isolated mesophyll protoplasts of Valerianella locusta L.

Mesophyll protoplasts were isolated from unhardened and cold-acclimated leaves of Valerianella locusta L. and subjected to freeze-thaw treatment. To evaluate the extent and course of freezing injury, photosynthetic reactions of whole protoplasts and of free thylakoid membranes, liberated from protoplasts by osmotic lysis, were measured. In addition, the integrity of the protoplasts was determined by microscopy. The results reveal an increased frost tolerance of protoplasts isolated from acclimated leaves with respect to all parameters measured. CO₂-dependent O₂ evolution (representing net photosynthetic CO₂ fixation of protoplasts) was the most freezing-sensitive reaction; its inhibition due to freeze-thaw treatment of protoplasts was neither correlated with disintegration of the plasma membrane, nor was it initiated by inactivation of the thylakoid membranes. The frost-induced decline of protoplast integrity was not closely correlated to thylakoid damage either. Freezing injury of the thylakoid membranes was manifested by inhibition of photosynthetic electron transport and photophosphorylation. Both photosystems were affected by freezing and thawing with strongest inhibition occurring in the water-oxidation system or at the oxidizing site of photosystem II. Photophosphorylation responded more sensitively to freezing stress than electron transport, although uncoupling (increased permeability of the thylakoid membranes to protons) was not a conspicuous effect. The data are discussed in relation to freezing injury in leaves and seem to indicate that frost damage in vivo is initiated at multiple sites.Abbreviations Chl chlorphyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCIP 2,6-dichlorophenolindophenol - DPC 1,5-diphenylcarbazide - Hepes 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid - MES 2-(N-morpholino)-ethanesulfonic acid - PS I photosystem I - PS II photosystem II     

Sensitivity of the photosynthetic apparatus to UV-A radiation: role of light-harvesting complex II–photosystem II supercomplex organization

In this work we study the effect of UV-A radiation on the function of the photosynthetic apparatus in thylakoid membranes with different organization of the light-harvesting complex II–photosystem II (LHCII–PSII) supercomplex. Leaves and isolated thylakoid membranes from a number of previously characterized pea species with different LHCII size and organization were subjected to UV-A treatment. A relationship was found between the molecular organization of the LHCII (ratio of the oligomeric to monomeric forms of LHCII) and UV-A-induced changes both in the energy transfer from PSII to PSI and between the chlorophyll–protein complexes within the LHCII–PSII supercomplex. Dependence on the organization of the LHCII was also found with regard to the degree of inhibition of the photosynthetic oxygen evolution. The susceptibility of energy transfer and oxygen evolution to UV-A radiation decreased with increasing LHCII oligomerization when the UV-A treatment was performed on isolated thylakoid membranes, in contrast to the effect observed in thylakoid membranes isolated from pre-irradiated pea leaves. The data suggest that UV-A radiation leads mainly to damage of the PSIIα centers. Comparison of membranes with different organization of their LHCII–PSII supercomplex shows that the oligomeric forms of LHCII play a key role for sensitivity to UV-A radiation of the photosynthetic apparatus. S. G. Taneva is Associated member of the Institute of Biophysics, Bulgarian Academy of Sciences.     

The ultrastructure and flexibility of thylakoid membranes in leaves and isolated chloroplasts as revealed by small-angle neutron scattering

We studied the periodicity of the multilamellar membrane system of granal chloroplasts in different isolated plant thylakoid membranes, using different suspension media, as well as on different detached leaves and isolated protoplasts—using small-angle neutron scattering. Freshly isolated thylakoid membranes suspended in isotonic or hypertonic media, containing sorbitol supplemented with cations, displayed Bragg peaks typically between 0.019 and 0.023 Å^− 1, corresponding to spatially and statistically averaged repeat distance values of about 275–330 Å. Similar data obtained earlier led us in previous work to propose an origin from the periodicity of stroma thylakoid membranes. However, detached leaves, of eleven different species, infiltrated with or soaked in D₂O in dim laboratory light or transpired with D₂O prior to measurements, exhibited considerably smaller repeat distances, typically between 210 and 230 Å, ruling out a stromal membrane origin. Similar values were obtained on isolated tobacco and spinach protoplasts. When NaCl was used as osmoticum, the Bragg peaks of isolated thylakoid membranes almost coincided with those in the same batch of leaves and the repeat distances were very close to the electron microscopically determined values in the grana. Although neutron scattering and electron microscopy yield somewhat different values, which is not fully understood, we can conclude that small-angle neutron scattering is a suitable technique to study the periodic organization of granal thylakoid membranes in intact leaves under physiological conditions and with a time resolution of minutes or shorter. We also show here, for the first time on leaves, that the periodicity of thylakoid membranes in situ responds dynamically to moderately strong illumination. This article is part of a Special Issue entitled: Photosynthesis research for sustainability: Keys to produce clean energy.     

Occurrence of a Putative Precursor to the Amyloplast,the “Amyloplast Initial” in Developing Stolons of the Potato

Cells at the apical part of developing stolons of the potato (Solatium tuberosum L. cv. Norin 1) were analyzed for the occurrence of putative precursors to amyloplasts, designated “amyloplast initial.” Ultrastructural studies showed that the cells contained the expected novel organelle. It was about 1 μm in diameter, devoid of thylakoid membranes, and was stained to a similar extent as the stroma of amyloplasts by uranyl acetate and lead citrate. Formation of thylakoid membranes and starch granules takes place at an early stage of development of these initials when they are just a few μm in diameter. At this stage, proliferation of the initials takes places by division at random sites.     

Effects of NaCl on photosynthetic pigments, saccharides, and chloroplast ultrastructure in leaves of tomato cultivars

In leaves of four tomato (Lycopersicon esculentum Mill.) cultivars (Red Cloud, Floradade, Peto 95, and Scorpio) the contents of chlorophyll (Chl) (a+b), Chl a, and -carotene decreased due to 100 mM NaCl treatment as compared with those of controls. The contents of soluble sugars and total saccharides were significantly increased in leaves of NaCl-treated plants, but the starch content was not significantly affected. Transmission electron microscopy indicated that in leaves of NaCl-treated plants, the chloroplasts were aggregated, the cell membranes were distorted and wrinkled, and there was no sign of grana and thylakoid structures in chloroplasts.     

高CO_2浓度对大豆不同叶位叶片叶绿体淀粉粒积累的效应

对高CO_2浓度下生长的大豆(Glycine max(L.)Merr.)不同叶位的叶片进行了电镜观察,揭示出大豆不同叶位叶片的叶绿体对倍增的CO_2浓度反应不一。其显著的超微结构差异特征是:1.叶位居中的叶片叶绿体积累的淀粉粒不仅很大,而且最多,有的叶绿体中的淀粉粒可达20个,几乎充满着叶绿体的基质空间。2.下位叶叶绿体的淀粉粒积累较多,通常为2～5个;3.上位叶叶绿体所含淀粉粒既小又少,虽然有的叶绿体中也积累有3～4个淀粉粒,但大多数叶绿体中所含淀粉粒仅有1～2个。以上结果联系到大豆中位叶的光合作用速率较高及对籽粒产量起作用最大来讨论是很有意义的。     

Association of TMV coat protein with chloroplast membranes in virus-infected leaves

Summary The polypeptide composition of extracts of chloroplasts from tobacco leaves systemically infected with different strains of Tobacco Mosaic Virus (TMV) was analyzed by one- and two-dimensional gel electrophoresis. There were no changes in the protein profiles of chloroplasts from infected leaves when compared to control leaves except for the presence of coat protein (CP) of TMV, identified by immunoblotting. When protease-treated intact chloroplasts isolated on Percoll gradients were osmotically disrupted the CP could be detected in both stroma and membrane fractions. The majority of the CP associated with the thylakoid membranes (about 1–5% of the total thylakoid proteins) was in the form of free molecules while stroma contained aggregated or assembled CP (about 0.1% of the soluble proteins). Thylakoid-associated CP was insensitive to protease digestion unless the membranes were first treated with a detergent, indicating that the CP was embedded inside or otherwise complexed with the thylakoid membranes.Chloroplasts isolated from leaves infected with TMV-PV42, a symptomless strain, contained approximately 10–50 times less CP than did chloroplasts isolated from leaves bearing mosaic symptoms induced by other strains of TMV (U1, PV230 or PV39). A possible role of CP in symptom development is discussed.     

Biological and polymeric self-assembled hybrid systems: Structure and properties of thylakoid/polyelectrolyte complexes

A novel hybrid system composed of biological components and synthetic polymer, thylakoid/polycation complex, has been formed and studied. Effects of complex formation on the structure, electrostatics and functioning of thylakoid membranes have been examined. Thylakoids from bean leaves were used to form complexes with polycation polyallylamine hydrochloride (PAAH) in two systems: (i) thylakoid/polycation complexes formed in an aqueous bulk phase, and (ii) immobilized thylakoid/polycation planar complexes. Immobilized on a solid substrate surface, thylakoid/polycation complexes were prepared using layer-by-layer stepwise alternate adsorption technique, i.e., via the sequential alternate adsorption of thylakoids and polycation molecules. The morphology of built up structures was investigated by scanning electron microscopy. Light-induced electron transport in chloroplasts was studied by the electron paramagnetic resonance (EPR) method. Spin probe technique was employed to study the structural and electrostatic characteristics of thylakoid membranes. We have found that efficiency of light-induced electron transport in thylakoid membranes and membrane structure were not changed noticeably by PAAH binding to thylakoids in a wide range of PAAH concentrations. The data obtained indicate the physiologically-soft character of polycation interactions with thylakoid membranes and demonstrate effectiveness of interfacial self-assembly approach to fabrication of complex planar functional nanostructures from biological components and synthetic polymers.     

Effect of Elevated CO2 Concentration on the Starch Grain Accumulation in Chloroplasts from Soybean Leaves at Different Nodes

Soybean ( Glycine max (L.) Merr. ) plants were grown under ambient and elevated CO2 (plus 350 μL/L) concentration in cylindrical open-top chamber to examine their effects on the ultra- structure of chloroplasts. The upper, lower and mid-node leaves were harvested after 7 days full expansion under different CO2 concentrations and ultrathin section were prepared for transmission electron microscopy. In general, the average content of starch grains and thylakoid membranes in the chloroplasts under the elevated CO2 concentration were always higher than the control. Under higher CO2 concentration, there were smaller and less starch grains in the chloroplasts from upper-node leaves than those from mid-node leaves. The shape of their starch grains changed from elliptical to oval,and their thylakoid membranes and grana remained normal. At lower-node leaves, one or two oval, or three timer starch grains accumulated in the chloroplasts. In the mid-node leaves,however, some chloroplasts under higher CO2 concentration had rather large tim elliptical starch grains which could consequently cause disruption of grana and stroama thylakoids in the chloroplasts, whereas in other chloroplasts, the thylakoid membranes and grana were not deformed as the starch grains were smaller and elliptical. On the other hand, under higher CO2 concentration, the stacking degree of thylakoid membranes and starch grains accumulation in the mid-node leaves were significantly higher than those in the lower-node leaves,and slightly higher than the upper-node leaves. These results, in agreement with the chlorophyll contents and photosynthetic rate which reported by other authors in the past, indicated that the ultrastmcture response of the chloroplasts from different leaf nodes of soybeen under elevated CO2 coneentration were different. The seed yield of soybean at different nodes was decreased gradually from mid-nodes towards both upper- and lower-nodes. The greatest effect of elevated CO2 eoneentrafion on seed yeild was at the mid-node leaves. The variation of seed yields of soybean at different nodes under elevated CO2 concentration was in eoneert with the change in the ultrastmcture of chloroplasts and in turn the change in their photosynthetic rates of leaves at different nodes.     

Biological and polymeric self-assembled hybrid systems: structure and properties of thylakoid/polyelectrolyte complexes

A novel hybrid system composed of biological components and synthetic polymer, thylakoid/polycation complex, has been formed and studied. Effects of complex formation on the structure, electrostatics and functioning of thylakoid membranes have been examined. Thylakoids from bean leaves were used to form complexes with polycation polyallylamine hydrochloride (PAAH) in two systems: (i) thylakoid/polycation complexes formed in an aqueous bulk phase, and (ii) immobilized thylakoid/polycation planar complexes. Immobilized on a solid substrate surface, thylakoid/polycation complexes were prepared using layer-by-layer stepwise alternate adsorption technique, i.e., via the sequential alternate adsorption of thylakoids and polycation molecules. The morphology of built up structures was investigated by scanning electron microscopy. Light-induced electron transport in chloroplasts was studied by the electron paramagnetic resonance (EPR) method. Spin probe technique was employed to study the structural and electrostatic characteristics of thylakoid membranes. We have found that efficiency of light-induced electron transport in thylakoid membranes and membrane structure were not changed noticeably by PAAH binding to thylakoids in a wide range of PAAH concentrations. The data obtained indicate the physiologically-soft character of polycation interactions with thylakoid membranes and demonstrate effectiveness of interfacial self-assembly approach to fabrication of complex planar functional nanostructures from biological components and synthetic polymers.     

Thylakoid membrane stability to heat stress studied by flash spectroscopic measurements of the electrochromic shift in intact potato leaves: influence of the xanthophyll content

A flash-induced transthylakoid electric field was measured at 515 nm as an electrochromic absorbance shift in intact potato leaves using a double flash differential spectrophotometer. The decay rate of the electrochromic shift in dark-adapted samples was used to examine the conductance to ions of thylakoid membranes. Heat stress (39.5 °C for 15 min) was found to accelerate drastically the electric field decay, with the half decay time falling from more than 200 ms to less than 45 ms. Heat-induced acceleration of the electric field breakdown was insensitive to the PSII electron donor Hydroxylamine and to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD), thus indicating that it reflects an increase in thylakoid membrane permeability after heat stress. This phenomenon did not involve peroxidative damage of membrane lipids. Acceleration of the electric field relaxation exhibited the same temperature dependence as that of PSII deactivation, suggesting that the ionic permeability of thylakoid membranes is one of the most heat-sensitive components of the photosynthetic apparatus. When potato leaves were infiltrated with 100 mol m^?3 ascorbate (in a buffer of pH 5), there was massive conversion of the carotenoid violaxanthin to zeaxanthin. This change in carotenoid composition protected thylakoid membranes against heat-induced changes in permeability, as revealed by the maintenance of a slow decay of the 515 nm absorbance change after heat stress. No such effect was observed after treatments which did not induce the vio-laxanthin-to-zeaxanthin conversion: leaf infiltration with 0 mol m^?3 ascorbate (at pH 5 or 8), 100 mol m^?3 ascorbate at pH 8 or 100 mol m^?3 ascorbate +5 mol m^?3 dithiothreitol at pH 5. Increased stability of the permeability properties of thylakoid membranes was also observed after a mild heat treatment (2 h at 35 °C). The data presented suggest that de-epoxidized xanthophylls in vivo stabilize thylakoid membranes and protect thylakoids against heat-induced disorganization.     

Effect of polyamines on stabilization of molecular complexes in thylakoid membranes of osmotically stressed oat leaves

Monocotyledonous leaves subjected to osmotica used for protoplast isolation accumulate a massive amount of putrescine (Put), lose chlorophyll and senesce rapidly. Treatment with spermidine (Spd) or spermine (Spm) prevents the loss of chlorophyll, indicating preservation of the thylakoid membranes at the site of the chlorophyll-protein complexes. Using several recently produced antibody probes, the effects on the stabilization of thylakoid membranes of applying either difluoromethylarginine (DFMA), a specific inhibitor of putrescine synthesis via arginine decarboxylase, or the polyamines Spd, Spm, or diaminopropane (Dap) to osmotically shocked oat leaves (Avena sativa L.) have been investigated. High protein levels were maintained in thylakoid membranes of leaf tissue incubated in the dark in the presence of 0.6 M sorbitol when pretreated with DFMA. After 48 h incubation, the level of the thylakoid protein D1, at the core of photosystem II, was higher in the DFMA-pretreated leaves as was the stromal protein ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco; as indicated by the level of large subunits). Applications of Spd, Spm or Dap were effective in retarding the loss of D1, D2 and cytochrome f from the thylakoid membranes as well as Rubisco large subunits and chlorophyll from the leaf tissue. The effects of polyamine applications may be mediated through Dap since most of the added Spd or Spm was converted to Dap within 6 h. The possible mechanisms of action of polyamine applications and DFMA-pretreatment on stabilizing the composition of the thylakoid membrane are also discussed.Abbreviations Cyt cytochrome - Dap diaminopropane - DFMA DL--difluoromethylarginine - LSU large subunit (of Rubisco) - Put putrescine - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - Spd spermidine - Spm spermine - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis This research was supported by the Agricultural and Food Research Council and by the British-Spanish joint research programme Acción Integrade HB-079 (R.T.B. and A.F.T.), British Council SPN/BAR/991 (R.T.B.) and Comision Interministerial de Cienica y Tecnologia 90-130 (A.F.T.). We thank Merrell Dow Research Center (Cincinnati, Ohio) for the gift of DFMA and Teresa Capell and Xavier Figueras (Univ. Barcelona) for help and suggestions.     

Activation of latent phenolase during spinach leaf senescence

The observed increase of phenolase activity and of its rate of activation during spinach leaf senescence is due to reduced binding of latent phenolase to the thylakoid membranes and not to de novo synthesis. The same amount of phenolase which is active in isolated thylakoid membranes from senescent leaves can be found in the membranes of non-senescent leaves after activation of latent enzyme. Tracer experiments give evidence that one multiple form which is responsible for the bulk activity in senescent leaves, is synthesized before, but not after the onset of senescence, indicating that pre-existing latent phenolase is converted to easily activating forms.     

Bicarbonate effects on photo-inhibition. Including an explanation for the sensitivity to photo-inhibition under anaerobic conditions

Isolated thylakoid membranes were found to be efficiently protected against photo-inhibition by sodium bicarbonate (20 mM NaHCO3) under both anaerobic and aerobic conditions. Furthermore, data are presented which indicate that the pronounced sensitivity to photo-inhibition under anaerobic compared to aerobic conditions is due to the removal of protecting bicarbonate, rather than oxygen, from the medium. A second type of bicarbonate effect on photo-inhibition, in apparent contradiction to the protective effect of added NaHCO3, is that thylakoid membranes that were depleted in their endogenous bicarbonate by treatment with formate were found to be less susceptible to photo-inhibition than thylakoids in the normal non-depleted state.     

Dithiol oxidant and disulfide reductant dynamically regulate the phosphorylation of light-harvesting complex II proteins in thylakoid membranes

Light-induced phosphorylation of light-harvesting chlorophyll a/b complex II (LHCII) proteins in plant thylakoid membranes requires an activation of the LHCII kinase via binding of plastoquinol to cytochrome b(6)f complex. However, a gradual down-regulation of LHCII protein phosphorylation occurs in higher plant leaves in vivo with increasing light intensity. This inhibition is likely to be mediated by increasing concentration of thiol reductants in the chloroplast. Here, we have determined the components involved in thiol redox regulation of the LHCII kinase by studying the restoration of LHCII protein phosphorylation in thylakoid membranes isolated from high-light-illuminated leaves of pumpkin (Cucurbita pepo), spinach (Spinacia oleracea), and Arabidopsis. We demonstrate an experimental separation of two dynamic activities associated with isolated thylakoid membranes and involved in thiol regulation of the LHCII kinase. First, a thioredoxin-like compound, responsible for inhibition of the LHCII kinase, became tightly associated and/or activated within thylakoid membranes upon illumination of leaves at high light intensities. This reducing activity was completely missing from membranes isolated from leaves with active LHCII protein phosphorylation, such as dark-treated and low-light-illuminated leaves. Second, hydrogen peroxide was shown to serve as an oxidant that restored the catalytic activity of the LHCII kinase in thylakoids isolated from leaves with inhibited LHCII kinase. We propose a dynamic mechanism by which counteracting oxidizing and reducing activities exert a stimulatory and inhibitory effect, respectively, on the phosphorylation of LHCII proteins in vivo via a novel membrane-bound thiol component, which itself is controlled by the thiol redox potential in chloroplast stroma.