Effect of the manganese complex on the binding of the extrinsic proteins (17, 23 and 33 kDa) of Photosystem II

Selective extraction-reconstitution experiments with the extrinsic Photosystem II polypeptides (33 kDa, 23 kDa and 17 kDa) have demonstrated that the manganese complex and the 33 kDa polypeptide are both necessary structural elements for the tight binding of the water soluble 17 and 23 kDa species. When the manganese complex is intact the 33 kDa protein interacts strongly with the rest of the photosynthetic complex. Destruction of the Mn-complex has two dramatic effects: i) The binding of the 33 kDa polypeptide is weaker, since it can be removed by exposure of the PS II system to 2 M NaCl, and ii) the 17 and 23 kDa species do not rebind to Mn-depleted Photosystem II membranes that retain the 33 kDa protein.Abbreviations Chl chlorophyll - HQ hydroquinone - MES 2(N-morpholino)ethanesulfonic acid - PS II Photosystem II - Tris 2-amino-2-hydroxymethylpropane-1,3-diol     

Selective extraction of 22 kDa and 10 kDa polypeptides from Photosystem II without removal of 23 kDa and 17 kDa extrinsic proteins

Selective solubilization of Photosystem II membranes with the non-ionic detergent octyl thioglucopyranoside has allowed the isolation of a PS II system which has been depleted of the 22 and 10 kDa polypeptides but retains all three extrinsic proteins (33, 23 and 17 kDa). The PS II membranes which have been depleted of the 22 and 10 kDa species show high rates of oxygen evolution activity, external calcium is not required for activity and the manganese complex is not destroyed by exogenous reductants. When we compared this system to control PS II membranes, we observed a minor modification of the reducing side, and a conversion of the high-potential to the low-potential form of cytochrome b ₅₅₉.Abbreviations Chl- chlorophyll - DCBQ- 2,5-dichloro-p-benzoquinone - DCMU- 3-(3,4-dichlorophenyl)-1,1-dimethylurea - ESR- electron spin resonance - MES- 2-(N-morpholino)ethanesulfonic acid - OTG- octyl--d-thioglucopyranoside - PS II- Photosystem II - PEG- polyethylene glycol, M_r=6000 - Tris- 2-amino-2-hydroxyethylpropane-1,3-diol     

Superoxide radicals are not the main promoters of acceptor-side-induced photoinhibitory damage in spinach thylakoids

Superoxide anion radical formation was studied with isolated spinach thylakoid membranes and oxygen evolving Photosystem II sub-thylakoid preparations using the reaction between superoxide and Tiron (1,2-dihydroxybenzene-3,5-disulphonate) which results in the formation of stable, EPR detectable Tiron radicals.We found that superoxide was produced by illuminated thylakoids but not by Photosystem II preparations. The amount of the radicals was about 70% greater under photoinhibitory conditions than under moderate light intensity. Superoxide production was inhibited by DCMU and enhanced 4–5 times by methyl viologen. These observations suggest that the superoxide in illuminated thylakoids is from the Mehler reaction occurring in Photosystem I, and its formation is not primarily due to electron transport modifications brought about by photoinhibition.Artificial generation of superoxide from riboflavin accelerated slightly the photoinduced degradation of the Photosystem II reaction centre protein D1 but did not accelerate the loss of oxygen evolution supported by a Photosystem II electron acceptor. However, analysis of the protein breakdown products demonstrated that this added superoxide did not increase the amount of fragments brought about by photoinhibition but introduced an additional pathway of damage.On the basis of the above observations we propose that superoxide redicals are not the main promoters of acceptor-side-induced photoinhibition of Photosystem II.Abbreviations DCBQ- 2,5-dichloro-p-benzoquinone - DCMU- 3- (3,4-dichlorophenyl)-1,1-dimethylurea - DMBQ- 2,5-dimethyl-p-benzoquinone - DMPO- 5,5-dimethyl-pyrrolin N-oxide - Hepes- N-(2-hydroxyethyl)-piperazine-N-(2-ethanesulfonic acid) - Mes- 2-(N-morpholino)-ethanesulfonic acid - methyl viologen- 1,1-dimethyl-4,4-bipyridinium dichloride - PS- Photosystem - SOD- Superoxide dismutase (EC 1.15.1.1) - Tiron- 1,2-dihydroxybenzene-3,5-disulphonate - Tris- 2-amino-2-hydroxymethylpropane-1,3-diol     

Thermoluminescence properties of the S2-state in chloride-depleted water oxidizing complexes after reconstituting treatments with various monovalent anions

Under conditions that assured rebinding of the extrinsic 17 and 23 kDa polypeptides, Cl^--depleted Photosystem II membranes isolated from spinach chloroplasts were subjected to reconstituting treatments in media containing NaF, NaCl, NaBr, NaI or NaNO₃, or they were kept in a medium without any added salt other than the buffer. After removing most of the unbound reconstituting anions by washing, the O₂-evolution activities and thermoluminescence properties of the membranes were compared. While the temperature of maximal thermoluminescence emission was lowest for membranes treated with Cl^-, no uniform correlation was evident between the temperature profile of the thermoluminescence emission and the apparent activating effectiveness of the anions in the membranes' water oxidizing machinery. However, the differences between the thermoluminescence features did conform to a trend according to which the emission temperatures were upshifted as the size of the activating anion increased, and its hydration energy decreased, i.e. Cl^-<Br^-<NO₃ ^-<I^-. The inactive F^- anions were not well retained by the membranes. To explain the experimental data it is suggested that the structural environment of the charge accumulating Mn-center is influenced by the ionic conditions encountered by the Photosystem II membranes after Cl^- removal, further enforced by the binding of compatible anions, and then stabilized by the 17 and 23 kDa extrinsic polypeptides. If, as some concepts imply, the anion binding sites are located at or near the functional Mn, only very exceptional characteristics of the water-oxidizing mechanism may account for the observation that the potentially electron-donating I^- anion can serve as activator and that it stabilizes rather than destabilizes the S₂-state.Abbreviations Chl chlorophyll - Hepes 4-(2-hydroxyethyl)-1-piperazine-ethane sulfonic acid - Mes 2-(N-morpholino)ethane sulfonic acid - Pheo the pheophytin a of the Photosystem II reaction center - PS photosystem     

Selective extraction of CP 26 and CP 29 proteins without affecting the binding of the extrinsic proteins (33, 23 and 17 kDa) and the DCMU sensitivity of a Photosystem II core complex

A highly purified oxygen evolving Photosystem II core complex was isolated from PS II membranes solubilized with the non-ionic detergent n-octyl--D-thioglucoside. The three extrinsic proteins (33, 23 and 17 kDa) were functionally bound to the PS II core complex. Selective extraction of the 22, 10 kDa, CP 26 and CP 29 proteins demonstrated that these species are not involved in the binding of the extrinsic proteins (33, 23 and 17 kDa) or the DCMU sensitivity of the Photosystem II complex.Abbreviations Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LHC light-harvesting complex - MES 2-(N-morpholino)ethanesulfonic acid - OGP n-octyl--d-glucoside - OTG n-octyl--d-thioglucoside - PAGE polyacrylamide gel electrophoresis - PS II Photosystem II - SDS sodium dodecyl sulfate     

Use of a monoclonal antibody in structural investigations of the 49-kDa polypeptide of photosystem II

A monoclonal antibody, FAC2, was isolated by immunization of mice with a Photosystem II core preparation followed by splenic fusion and standard monoclonal antibody screening and production techniques. This antibody recognizes the 49-kDa polypeptide of Photosystem II which is the apoprotein of CPal. The antigenic determinant recognized by this antibody lies on a cyanogen bromide fragment which appears as a doublet with an apparent molecular mass of 14.5 kDa. FAC2 was used to follow the effects of trypsin on the 49-kDa polypeptide in a membrane environment. Our results indicate that the extrinsic polypeptides of Photosystem II which are known to be involved in oxygen evolution protect the 49-kDa polypeptide from tryptic attack. Additionally, Photosystem II membranes which are treated with alkaline Tris exhibit a large increase in the ability to bind FAC2. This increase is not observed with membranes treated with calcium chloride or sodium chloride. These results indicate that the 49-kDa polypeptide may be at least structurally associated with the component(s) responsible for oxygen evolution.     

Carboxylate groups on the manganese-stabilizing protein are required for efficient binding of the 24 kDa extrinsic protein to photosystem II

The effects of the modification of carboxylate groups on the manganese-stabilizing protein on the binding of the 24 kDa extrinsic protein to Photosystem II were investigated. Carboxylate groups on the manganese-stabilizing protein were modified with glycine methyl ester in a reaction facilitated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The manganese-stabilizing protein which was modified while associated with NaCl-washed membranes could bind to calcium chloride-washed PS II membranes and reconstitute oxygen evolution in a manner similar to that observed for unmodified manganese-stabilizing protein (Frankel, L.K, Cruz, J. C. and Bricker, T. M. (1999) Biochemistry 38, 14271-14278). However, PS II membranes reconstituted with this modified protein were defective in their ability to bind the extrinsic 24 kDa protein of Photosystem II. Mapping of the sites of modification was carried out by trypsin and Staphylococcus V8 protease digestion of the modified protein and analysis by MALDI mass spectrometry. These studies indicated that the domains (1)E-(71)D, (97)D-(144)D, and (180)D-(187)E are labeled when the manganese-stabilizing protein is bound to NaCl-washed Photosystem II membranes. We hypothesize that modified carboxylates, possibly residues (1)E, (32)E, (139)E, and/or (187)E, in these domains are responsible for the altered binding affinity of the 24 kDa protein observed.     

Comparative studies on the polypeptide composition of chloroplast lamellae and lamellar fractions

The polypeptide composition of spinach chloroplast membranes and membrane fractions has been examined by the technique of sodium dodecylsulfate-polyacrylamide gel electrophoresis. Chloroplasts were fragmented into grana (Photosystem II enriched) and stroma lamellae (Photosystem I in character) by the French press technique. The grana lamellae were futher fractionated by the use of digitonin into two fractions, one enriched in Photosystem II and the other enriched in Photosystem I. These membranes are composed of at least 15 polypeptides two of which, with approximate weights of 39 and 50 kdaltons, are observed only in granal fractions. Quantitatively the primarily Photosystem II fractions are enriched in polypeptides in the 30-23 kdalton range whereas the Photosystem I (or Photosystem I-enriched) fractions are enriched in polypeptides in the 60-54 kdalton region. The experiments reported show that contamination by soluble proteins or other membranes is negligible. The results indicate that subtle differences in composition account for the large differences in structure and function within the chloroplast membrane system.     

The inhibitory mechanism of Cu(II) on the Photosystem II electron transport from higher plants

In a previous paper, we reported that Cu(II) inhibited the photosynthetic electron transfer at the level of the pheophytin-Q_A-Fe domain of the Photosystem II reaction center. In this paper we characterize the underlying mechanism of Cu(II) inhibition. Cu(II)-inhibition effect was more sensitive with high pH values. Double-reciprocal plot of the inhibition of oxygen evolution by Cu(II) is shown and its corresponding inhibition constant, K_i, was calculated. Inhibition by Cu(II) was non-competitive with respect to 2,6-dichlorobenzoquinone and 3-(3,4-dichlorophenyl)-1,1-dimethylurea and competitive with respect to protons. The non-competitive inhibition indicates that the Cu(II)-binding site is different from that of the 2,6-dichlorobenzoquinone electron acceptor and 3-(3,4-dichlorophenyl)-1,1-dimethylurea sites, the Q_B niche. On the other hand, the competitive inhibition with respect to protons may indicate that Cu(II) interacts with an essential amino acid group(s) that can be protonated or deprotonated in the inhibitory-binding site.Abbreviations BSA bovine seroalbumin - Chl chlorophyll - DCBQ 2,6-dichlorobenzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - MES 2-(N-morpholino)-ethanesulphonic acid - Pheo pheophytin - Q_A primary quinone acceptor - Q_B secondary quinone acceptor - PS Photosystem - RC reaction center - Tricine N-[Tris(hydroxymethyl)-methyl]-glycine     

Structural,biochemical and biophysical characterization of four oxygen-evolving Photosystem II preparations from spinach

Four procedures utilizing different detergent and salt conditions were used to isolate oxygen-evolving Photosystem II (PS II) preparations from spinach thylakoid membranes. These PS II preparations have been characterized by freeze-fracture electron microscopy, SDS-polyacrylamide gel electrophoresis, steady-state and pulsed oxygen evolution, 77 K fluorescence, and room-temperature electron paramagnetic resonance. All of the O₂-evolving PS II samples were found to be highly purified grana membrane fractions composed of paired, appressed membrane fragments. The lumenal surfaces of the membranes and thus the O₂-evolving enzyme complex, are directly exposed to the external environment. Biochemical and biophysical analyses indicated that all four preparations are enriched in the chlorophyll $\text{a}\text{b-}\text{light-harvesting}$ complex and Photosystem II, and depleted to varying degrees in the stroma-associated components, Photosystem I and the CF₁-ATPase. The four PS II samples also varied in their cytochrome f content. All preparations showed enhanced stability of oxygen production and oxygen-rate electrode activity compared to control thylakoids, apparently promoted by low concentrations of residual detergent in the PS II preparations. A model is presented which summarizes the effects of the salt and detergent treatments on thylakoid structure and, consequently, on the configuration and composition of the oxygen-evolving PS II samples.     

Polyamines in the photosynthetic apparatus

The three main polyamines putrescine (Put), spermidine (Spd) and spermine (Spm) were characterized by HPLC in intact spinach leaf cells, intact chloroplasts, thylakoid membranes, Photosystem II membranes, the light-harvesting complex and the PS II complex. All contain the three polyamines in various ratios; the HPLC polyamine profiles of highly resolved PS II species (a Photosystem II core and the rection center) suggest an enrichment in the polyamine Spm.Abbreviations Chl chlorophyll - HPLC high performance liquid chromatography - LHC light-harvesting complex - PS II Photosystem II - PS II-RC Photosystem II reaction center - Put putrescine - Spd spermidine - Spm spermine - 10%S-core D₁-D₂-Cyt b559-47 kD-43 kD complex     

On the question of the identity of cytochrome b-560 in thylakoid stromal membranes

Stromal membranes enriched in PS I contain a low potential cytochrome with a reduced -band peak close to 560 nm. The identity of this cytochrome component has been ascribed either to a low potential form of the Photosystem II cytochrome b-559 or to a different cytochrome with a reduced -band of 560 nm. The half-bandwidth of the 560 nm component in stromal membranes is identical to that of purified cytochrome b-559. Western blots show that the stromal membranes contain an amount of PS II cytochrome b-559 -subunit that is more than sufficient to account for the cytochrome b-560 detected spectrophotometrically in these membranes. These immunochemical data and the similarity of (i) the spectral peaks, and (ii) the redox properties of low potential PS II cytochrome b-559 and the b-560 component, suggest that the simplest inference is that the cytochrome b-560 protein in stromal membranes is identical to the PS II cytochrome b-559.Abbreviations: A absorbance - cyt cytochrome - DCBQ 2,5-dichloro-p-benzoquinone - E_mx midpoint potential at pH x - hbw half-bandwidth - LP low potential - MD menadiol - MES 2-(N-morpholino)ethanesulfonic acid - MHQ methylhydroquinone - PS I-PS II photosystems I, II - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis     

Photosystem II in a mutant of Chlamydomonas reinhardtii lacking the 23 kDa psbP protein shows increased sensitivity to photoinhibition in the absence of chloride

The psbP gene product, the so called 23 kDa extrinsic protein, is involved in water oxidation carried out by Photosystem II. However, the protein is not absolutely required for water oxidation. Here we have studied Photosystem II mediated electron transfer in a mutant of Chlamydomonas reinhardtii, the FUD 39 mutant, that lacks the psbP protein. When grown in dim light the Photosystem II content in thylakoid membranes of FUD 39 is approximately similar to that in the wild-type. The oxygen evolution is dependent on the presence of chloride as a cofactor, which activates the water oxidation with a dissociation constant of about 4 mM. In the mutant, the oxygen evolution is very sensitive to photoinhibition when assayed at low chloride concentrations while chloride protects against photoinhibition with a dissociation constant of about 5 mM. The photoinhibition is irreversible as oxygen evolution cannot be restored by the addition of chloride to inhibited samples. In addition the inhibition seems to be targeted primarily to the Mn-cluster in Photosystem II as the electron transfer through the remaining part of Photosystem II is photoinhibited with slower kinetics. Thus, this mutant provides an experimental system in which effects of photoinhibition induced by lesions at the donor side of Photosystem II can be studied in vivo.Abbreviations Chl chlorophyll - DCIP 2,6-dichlorophenolindophenol - DPC 2,2-diphenylcarbonic dihydrazide - HEPES 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid - P₆₈₀ the primary electron donor to PS II - PpBQ phenyl-p-benzoquinone - PS II Photosystem II - Q_A the first quinone acceptor of PS II - Q_B the second quinone acceptor of PS II - SDS sodium dodecyl sulfate - Tris tris(hydroxymethyl)aminomethane - Tyr_D accessory electron donor on the D₂-protein - Tyr_Z tyrosine residue, acting as electron carrier between P₆₈₀ and the water oxidizing system     

Photosynthetic electron transport in thylakoid preparations from two marine red algae (Rhodophyta).

Thylakoid membrane preparations active in photosynthetic electron transport have been obtained from two marine red algae, Griffithsia monilis and Anotrichium tenue. High concentrations (0.5-1.0 M) of salts such as phosphate, citrate, succinate and tartrate stabilized functional binding of phycobilisomes to the membrane and also stabilized Photosystem II-catalysed electron-transport activity. High concentrations (1.0 M) of chloride and nitrate, or 30 mM-Tricine/NaOH buffer (pH 7.2) in the absence of salts, detached phycobilisomes and inhibited electron transport through Photosystem II. The O2-evolving system was identified as the electron-transport chain component that was inhibited under these conditions. Washing membranes with buffers containing 1.0-1.5 M-sorbitol and 5-50 mM concentrations of various salts removed the outer part of the phycobilisome but retained 30-70% of the allophycocyanin 'core' of the phycobilisome. These preparations were 30-70% active in O2 evolution compared with unwashed membranes. In the sensitivity of their O2-evolving apparatus to the composition of the medium in vitro, the red algae resembled blue-green algae and differed from other eukaryotic algae and higher plants. It is suggested that an environment of structured water may be essential for the functional integrity of Photosystem II in biliprotein-containing algae.     

Replacement of tyrosine D with phenylalanine affects the normal proton transfer pathways for the reduction of P680+ in oxygen-evolving photosystem II particles from Chlamydomonas

We have probed the electrostatics of P680(+) reduction in oxygenic photosynthesis using histidine-tagged and histidine-tagged Y(D)-less Photosystem II cores. We make two main observations: (i) that His-tagged Chlamydomonas cores show kinetics which are essentially identical to those of Photosystem II enriched thylakoid membranes from spinach; (ii) that the microsecond kinetics, previously shown to be proton/hydrogen transfer limited [Schilstra et al. (1998) Biochemistry 37, 3974-3981], are significantly different in Y(D)-less Chlamydomonas particles when compared with both the His-tagged Chlamydomonas particles and the spinach membranes. The oscillatory nature of the kinetics in both Chlamydomonas samples is normal, indicating that S-state cycling is unaffected by either the histidine-tagging or the replacement of tyrosine D with phenylalanine. We propose that the effects on the proton-coupled electron transfers of P680(+) reduction in the absence of Y(D) are likely to be due to pK shifts of residues in a hydrogen-bonded network of amino acids in the vicinity of Y(Z). Tyrosine D is 35 A from Y(Z) and yet has a significant influence on proton-coupled electron transfer events in the vicinity of Y(Z). This finding emphasizes the delicacy of the proton balance that Photosystem II has to achieve during the water splitting process.     

Further investigations on structural and catalytic properties of O2 evolving preparations from tobacco and two chlorophyll deficient tobacco mutants

Previous investigations (Specht, S., Pistorius, E.K. and Schmid, G.H.: Photosynthesis Res. 13, 47–56, 1987) of Photosystem II membranes from tobacco (Nicotiana tabacum L. cv. John William's Broadleaf) which contain normally stacked thylakoid membranes and from two chlorophyll deficient tobacco mutants (Su/su and Su/su var. Aurea) which have low stacked or essentially unstacked thylakoids with occasional membrane doublings, have been extended by using monospecific antisera raised against the three extrinsic polypeptides of 33,21 and 16 kDa. The results show that all three peptides are synthesized as well in wild type tobacco as in the two mutants to about the same level and that they are present in thylakoid membranes of all three plants. However, in the mutants the 16 and 21 kDa peptides (but not the 33 kDa peptide) are easily lost during solubilization of Photosystem II membranes. In the absence of the 16 and 21 kDa peptide Photosystem II membranes from the mutants have a higher O₂ evolving activity without addition of CaCl₂ than the wild type Photosystem II membranes. On the other hand, after removal of the 33 kDa peptide no significant differences in the binding of Mn could be detected among the three plants. The results also show that reaction center complexes from wild type tobacco and the mutant Su/su are almost identical to the Triton-solubilized Photosystem II membranes from the mutant Su/su var. Aurea.Abbreviations PS photosystem - chl chlorophyll - LHCP light harvesting chlorophyll a/b protein complex - WT wild type - OEE1, OEE2 and OEE3 oxygen evolution enhancing complex of 29–36 kDa, 21–24 kDa and 16–18 kDa, respectively     

The unusually strong stabilizing effects of glycine betaine on the structure and function of the oxygen-evolving Photosystem II complex

Natural osmoregulatory substances (osmolytes) allow a wide variety of organisms to adjust to environments with high salt and/or low water content. In addition to their role in osmoregulation, some osmolytes protect proteins from denaturation and deactivation by, for example, elevated temperature and chaotropic compounds. A ubiquitous protein-stabilizing osmolyte is glycine betaine (N-trimethyl glycine). Its presence has been reported in bacteria, in particular cyanobacteria, in animals and in plants from higher plants to algae. In the present review we describe the experimental evidence related to the ability of glycine betaine to enhance and stabilize the oxygen-evolving activity of the Photosystem II protein complexes of higher plants and cyanobacteria. The osmolyte protects the Photosystem II complex against dissociation of the regulatory extrinsic proteins (the 18 kD, 23 kD and 33 kD proteins of higher plants and the 9 kD protein of cyanobacteria) from the intrinsic components of the Photosystem II complex, and it also stabilizes the coordination of the Mn cluster to the protein cleft. By contrast, glycine betaine has no stabilizing effect on partial photosynthetic processes that do not involve the oxygen-evolving site of the Photosystem II complex. It is suggested that glycine betaine might act, in part, as a solute that is excluded from charged surface domains of proteins and also as a contact solute at hydrophobic surface domains.     

Salicylic acid may indirectly influence the photosynthetic electron transport

Salicylic acid (SA) is a phenolic phytohormone with important roles in plant development, transpiration, endogenous signaling and defense against pathogens. One of the pathways of SA biosynthesis is located in the chloroplasts. The aim of the present work was to investigate the possible regulatory effects of SA on photosynthetic electron transport processes. Here we show that SA also affects leaf photosynthesis, via inducing stomatal closure and also by slowing down Photosystem II (PS II) electron transport. Photosynthetic CO? incorporation and stomatal conductivity (measured with an infrared gas analyzer) were much lower in SA-infiltrated tobacco leaves than in untreated or water-infiltrated controls. PS II electron transport (calculated from PAM chlorophyll fluorescence data) was more sensitive to SA than Photosystem I (PS I) (measured with far red absorption). Direct probing of PS II charge separation and stabilization (measured with thermoluminescence), however, showed that these events were less affected in isolated thylakoid membranes than in leaves, suggesting that the effect of SA on PS II is indirect and different from similar effects of phenolic herbicides.     

Inhibition of photosystem II activity by Cu++ ion. Choice of buffer and reagent is critical

Cupric ion (Cu⁺⁺) inhibits the rate of photosystem II electron transport and the intensity of the variable part of chl a fluorescence in isolated chloroplast thylakoids. The inhibition is markedly dependent on the nature of the buffer used in the assay medium. In MES and HEPES buffers, complete inhibition of photosystem II occurs at 30 M of Cu⁺⁺, while in Tricine no inhibition occurred even at 200 M Cu⁺⁺. In other buffers used (TES, Phosphate, Tris), the efficacy of Cu⁺⁺ inhibition is intermediate. The calculated binding constants are found to correspond to the observed I₅₀ values for the six buffers used. It is concluded that the previous reports on copper inhibition, where buffers have been used indiscriminately should be reconsidered. Certain reagents such as hydroxylamine, ascorbate and diphenyl carbazide, which react with Cu⁺⁺, should be avoided.Abbreviations Chl chlorophyll - DCIP 2,6-dichlorophenol indophenol - DCMU 3-(3,4 dichlorophenyl)-1,1-dimethyl urea - DAD diaminodurene - DPC diphenyl carbazide - Fv variable chl fluorescence - HEPES N-2-hydroxyethyl piperazine sulfonic acid - I ₃₀ inhibitor concentration causing 30% inhibition of Fv - MES 2-(N-morpholino) ethane sulfonic acid - MV Methyl viologen - PS II Photosystem II - PS I Photosystem I - TES N-tris(hydroxymethyl)-methyl-2-amino sulfonic acid - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - Tricine N-tris(hydroxymethyl) ethylglycine - Tris N-tris(hydroxymethyl)amino ethane     

Effects of natural shade on soybean thylakoid membrane composition

The effect of natural shade on chloroplast thylakoid membrane activity and composition was examined for soybean (Glycine Max. cv. Young) grown under field conditions. Plots with high (10 plants m^–1 row) or low (1 plant m^–1 row) plant density were established. Expanding leaves were tagged at 50, 58 and 65 days after planting (DAP). At 92 DAP, tagged leaves were used as reference points to characterize canopy light environments and isolate thylakoid membranes. Light environments ranged from a photosynthetic photon flux density (PPFD) of 87% of full sun to a PPFD of 10% of full sun. The decline in PPFD was accompanied by an increase in the far-red/red (735 nm/645 nm) ratio from 0.9 to approximately six. The major effects of shade on chloroplast thylakoid membranes were a reduction in chloroplast coupling factor and a shift in light-harvesting capacity from Photosystem I to Photosystem II. Photosynthetic electron transport capacity was not affected by differences in PPFD, but was 20 to 30% higher in the 1 plant m^–1 row treatment. The plant density effect on electron transport was associated with differences in plastocyanin concentration, suggesting that plastocyanin is a limiting factor in soybean. Shade did not have a significant effect on the concentration of Photosystem II, Cyt b₆f, or Photosystem I complexes.Abbreviations CF₁ chloroplast coupling factor - DAP days after planting - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCIP 2,6-dichlorophenolindophenol - FR/R far-red/red - PBS 10 mM sodium phosphate (pH 7.0), 150 mM NaCl - PPFD photosynthetic photon flux density - PS I Photosystem I - PS II Photosystem II - P700 reaction center of Photosystem I - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - TBS 20 mM Tris-HCl (pH 7.5), 500 mM NaCl - TTBS 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 0.05% (w/v) polyoxyethylenesorbitan monolaurate (Tween-20) The US Government right to retain a non-exclusive, royalty-free licence in and to any copyright is acknowledged.The US Government right to retain a non-exclusive, royalty-free licence in and to any copyright is acknowledged.