Sakae Katoh

Is the primary cause of thermal inactivation of oxygen evolution in spinach PS II membranes release of the extrinsic 33 kDa protein or of Mn

Abstract Incubation of PS II membranes at 45–50°C for several min resulted in strong inactivation of oxygen evolution, concomitant with release of Mn and the extrinsic proteins of 33, 23 and 17 kDa. No correlation was found between loss of the activity and release of the 33 kDa protein or Mn. However, involvement of the protein release in the mechanism of heat-inactivation was suggested by stabilization of the activity against heat-treatment by immobilization of the 33 kDa protein with a water-soluble carbodiimide. Furthermore, a linear correlation was found between extents of heat-inactivation and amounts of the 33 kDa protein released in the presence of 50 mM CaCl2, which greatly accelerated inactivation of oxygen evolution, release of the 33 kDa protein and aggregation of PS II membranes at high temperatures. Evidence was obtained indicating that the 33 kDa protein released at high temperatures rebinds to its functional site when temperature is lowered but CaCl2 suppresses rebinding of the protein by promoting intensive aggregation of the membranes. Thus, the activity survived in the presence of CaCl2 is proportional to the amounts of the protein remained attached to the membranes during heat-treatment. By contrast, release of Mn was not affected by addition of CaCl2 so that enhanced inactivation of oxygen evolution was not accompanied by corresponding increase in the amount of Mn released. It is concluded, therefore, that the primary cause of heat-inactivation of oxygen evolution is dissociation of the 33 kDa protein but not that of Mn.

Effects of leaf age, nitrogen nutrition and photon flux density on the distribution of nitrogen among leaves of a vine (Ipomoea tricolor Cav.) grown horizontally to avoid mutual shading of leaves

Effects of leaf age, nitrogen nutrition and photon flux density (PFD) on the distribution of nitrogen among leaves were investigated in a vine, Ipomoea tricolor Cav., which had been grown horizontally so as to avoid mutual shading of leaves. The nitrogen content was highest in newly developed young leaves and decreased with age of leaves in plants grown at low nitrate concentrations and with all leaves exposed to full sunlight. Thus, a distinct gradient of leaf nitrogen content was formed along the gradient of leaf age. However, no gradient of leaf nitrogen content was formed in plants grown at a high nitrate concentration. Effects of PFD on the distribution of nitrogen were examined by shading leaves in a manner that simulated changes in the light gradient of an erect herbaceous canopy (i.e., where old leaves were placed under increasingly darker conditions with growth of the canopy). This canopy-type shading steepened the gradient of leaf nitrogen content in plants grown at a low nitrogen supply, and created a gradient in plants grown at high concentrations of nitrate. The steeper the gradient of PFD, the larger the gradient of leaf nitrogen that was formed. When the gradient of shading was inverted, that is, younger leaves were subjected to increasingly heavier shade, while keeping the oldest leaves exposed to full sunlight, an inverted gradient of leaf nitrogen content was formed at high nitrate concentrations. The gradient of leaf nitrogen content generated either by advance of leaf age at low nitrogen availability, or by canopy-type shading, was comparable to those reported for the canopies of erect herbaceous plants. It is concluded that both leaf age and PFD have potential to cause the non-uniform distribution of leaf nitrogen. It is also shown that the contribution of leaf age increases with the decrease in nitrogen nutrition level.

Plastoquinone as a common link between photosynthesis and respiration in a blue-green alga

The role of plastoquinone in a thermophilic blue-green alga, Shynechococcus sp., was studied by measuring reduction kinetics of cytochrome 553 which was oxidized with red flash preferentially exciting photosystem I. Sensitivity of the cytochrome reduction to DBMIBAbbreviations: DCMU = 3-(3,4-dichlorophenyl)-1,1-dimethylurea; DBMIB = 2,5-dib romo-3-methyl-6-isopropyl-p-benzoquinone; HOQNO = 2-n-heptyl-4-hydroxyquinoline-N-oxide indicates that cytochrome 553 accepts electrons from reduced plastoquinone. Plastoquinone is in turn reduced in cells without electrons from photosystem II, since DCMUAbbreviations: DCMU = 3-(3,4-dichlorophenyl)-1,1-dimethylurea; DBMIB = 2,5-dib romo-3-methyl-6-isopropyl-p-benzoquinone; HOQNO = 2-n-heptyl-4-hydroxyquinoline-N-oxide, which inhibited methyl viologen photoreduction more strongly than DBMIB, failed to affect the cytochrome reduction. Participation of cyclic electron transport around photosystem I in cytochrome reduction in the presence of DCMU was excluded, because methyl viologen and antimycin A had no effect on the cytochrome kinetics. On the other hand, electron donation from endogenous substrates to plastoquinone was suggested from decreases in rate of the cytochrome reduction by dark starvation of cells and also from restoration of fast reduction kinetics by the addition of exogenous substrates to or by reillumination of starved cells.KCN, which completely suppressed respiratory O2-uptake, induced a marked acceleration of the cytochrome reduction in starved cells. The poison was less or not effective in stimulating the cytochrome reduction in more extensively starved or reilluminated cells.Results indicate that plastoquinone is functioning not only in the photosynthetic but also in the respiratory electron transport chain, thereby forming a common link between the two energy conservation systems of the blue-green alga.

Temperature Acclimation of Photosynthesis and Related Changes in Photosystem II Electron Transport in Winter Wheat

Winter wheat (Triticum aestivum L. cv Norin No. 61) was grown at 25°C until the third leaves reached about 10 cm in length and then at 15°C, 25°C, or 35°C until full development of the third leaves (about 1 week at 25°C, but 2–3 weeks at 15°C or 35°C). In the leaves developed at 15°C, 25°C, and 35°C, the optimum temperature for CO2-saturated photosynthesis was 15°C to 20°C, 25°C to 30°C, and 35°C, respectively. The photosystem II (PS II) electron transport, determined either polarographically with isolated thylakoids or by measuring the modulated chlorophyll a fluorescence in leaves, also showed the maximum rate near the temperature at which the leaves had developed. Maximum rates of CO2-saturated photosynthesis and PS II electron transport determined at respective optimum temperatures were the highest in the leaves developed at 25°C and lowest in the leaves developed at 35°C. So were the levels of chlorophyll, photosystem I and PS II, whereas the level of Rubisco decreased with increasing temperature at which the leaves had developed. Kinetic analyses of chlorophyll afluorescence changes and P700 reduction showed that the temperature dependence of electron transport at the plastoquinone and water-oxidation sites was modulated by the temperature at which the leaves had developed. These results indicate that the major factor that contributes to thermal acclimation of photosynthesis in winter wheat is the plastic response of PS II electron transport to environmental temperature.

Studies on electron transport associated with Photosystem I. I. Functional site of plastocyanin: Inhibitory effects of HgCl2 on electron transport and plastocyanin in chloroplasts

Abstract 1. Incubation of chloroplasts with HgCl 2 at a molar ratio of HgCl 2 to chlorophyll of about unity, induced a complete inhibition of the methyl viologen Hill reaction, as well as methyl viologen photoreduction with reduced 2,6-dichlorophenolindophenol (DCIP) as electron donor. Photooxidation of cytochrome ƒ was similarly sensitive towards HgCl 2 , whereas photooxidation of P700 was resistant to the poison. Photoreduction of cytochrome ƒ and light-induced increase in fluorescence yield were enhanced by the HgCl 2 treatment of chloroplasts. 2. Photooxidation of reduced yeast cytochrome c catalyzed by Photosystem I particles in the presence of plastocyanin was also suppressed by preincubation with HgCl 2 . The inhibition was recovered by adding more plastocyanin, but not by addition of the particles, to the reaction mixture. 3. Incubation of reduced plastocyanin with HgCl 2 caused a bleaching of the blue color of the protein, whereas the oxidized protein was resistant to HgCl 2 . A similar contrasting difference in sensitivity towards HgCl 2 was found in the Hill reaction in the absence and presence of ferricyanide. 4. Sonic treatment released plastocyanin from the untreated chloroplasts, whereas no plastocyanin was detected in the extract of the HgCl 2 -treated chloroplasts. However, plastocyanin could be reconstructed by dialyzing the extract of the HgCl 2 -treated chloroplasts against cysteine solution and then CuSO 4 solution. 5. The role of plastocyanin in the photosynthetic electron transport is discussed.

Multiple forms of P700-chlorophyll a-protein complexes from Synechococcus sp.: The iron, quinone and carotenoid contents.

The iron, quinone and carotenoid contents of five P700-chlorophyll a-protein complexes having different subunit structures (CP1-a,-b,-c,-d and-e) from the thermophilic cyanobacterium Synechococcus sp. were determined. CP1-a,-b,-c and-d that commonly have four polypeptides of 62,000, 60,000, 14,000 and 10,000 dalton contained 10–14 iron atoms per P700, whereas CP1-e that lacks the two small polypeptides was totally devoid of iron. All CP1 complexes contained vitamin K1 at the molar ratio of vitamin K1 to P700 of about 2 except CP1-e that had only 0.4 vitamin K1 per P700. No plastoquinone was detected in five CP1 complexes. Out of four major carotenoids, β-carotene, zeaxanthin, caloxanthin, and myxoxanthophyll, present in the thylakoid membranes, only β-carotene was found in isolated CP1 complexes; all CP1 complexes contained about 10 β-carotene molecules per P700. The flourescence excitation spectrum showed that β-carotene serves as an efficient antenna of photosystem I. It is concluded that all iron atoms and a larger fraction of vitamin K1 molecules present in the photosystem I reaction center complex are associated with the 14,000 and 10,000 dalton polypeptides, whereas β-carotene exclusively binds to the large polypeptides which carry the functional and antenna chlorophyll a. The possible functions of iron and vitamin K1 as electron carriers and of β-carotene as the accessary pigment and a photoprotectant in the photosystem I complexes are discussed.

Distribution of plastocyanin in plants, with special reference to its localization in chloroplasts

Abstract 1. 1. Plastocyanin, a copper protein originally discovered in Chlorella ellipsoidea , was found to occur in green leaves of the following plants: spinach, parsley, carrot, turnip, Japanese scallion, and crown daisy. The copper protein was absent from the nonphotosynthetic tissues of the plants. It was not detected in the cells of the photosynthetic purple bacterium, Rhodopseudomonas palustris . 2. 2. Plastocyanin was found to be localized in the chloroplasts of green cells. Spinach chloroplasts contain plastocyanin at a ratio of about 300 chlorophyll molecules per atom of copper of plastocyanin. The chloroplast fragments also contain plastocyanin in concentrations comparable to that of the whole chloroplasts. The plastocyanin in chloroplasts or chloroplast fragments cannot be removed by simple washing with isotonic solution, but a considerable portion can be readily extracted by hypotonic treatment. From these findings, it was inferred that plastocyanin is associated with the chlorophyll-bearing subparticulate components of chloroplasts. 3. 3. The determination of copper has revealed that the copper of the isolated plastocyanin can account for about half the total copper present in the chloroplast.

Isolation and characterization of Photosystem II complexes which lack light-harvesting chlorophyll a/b proteins but retain three extrinsic proteins related to oxygen evolution from spinach

Oxygen-evolving Photosystem II (PS II) complexes, which were largely deprived of major light-harvesting chlorophyll a/b proteins (LHC II) but still associated with the 33 kDa, 23 kDa and 17 kDa extrinsic proteins related to oxygen evolution, were isolated from spinach oxygen-evolving PS II membranes with a non-ionic detergent, n-heptyl thioglucoside. A minor antenna chlorophyll-protein (CP 29) was present but in reducted amounts. The complexes contained all the constituent subunits of PS II reaction center core complexes, the 47 kDa and 43 kDa chlorophyll-carrying proteins, the D1 and D2 proteins and cytochrome b-559. In addition, three hydrophobic proteins of 29 kDa (CP 29 apoprotein), 20 kDa and 10 kDa were present. The antenna size was 80 chlorophyll a per QA, or 76 chlorophyll a per 4 Mn, and the complexes contained about 1 Ca2+ per PS II. With phenyl- or dichloro-p-benzoquinone as electron acceptor, the complexes showed high rates of oxygen evolution in the absence of exogenously added Ca2+. The activity became, however, strongly Ca2+-dependent when the 23 kDa and 17 kDa proteins, but not the bound Ca2+, had been removed with 1.5 M NaCl. The Ca2+ requirement disappeared on reconstitution of the complexes with the two proteins. The complexes were compared with other oxygen-evolving preparations having different polypeptide compositions and functions of several subunit proteins and Ca2+ in PS II electron transport are discussed.

Total immobilization of the extrinsic 33 kDa protein in spinach Photosystem II membrane preparations. Protein stoichiometry and stabilization of oxygen evolution

(1) Treatment of oxygen-evolving Photosystem II membrane fragments (PS II membranes) with a zero-length crosslinker, l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) led to immobilization of all the extrinsic 33 kDa protein molecules without any significant effects on the oxygen-evolving activity and oscillation patterns of flash-induced oxygen evolution and thermoluminescence B band. (2) With increasing concentration of EDC, the chlorophyll-binding 47 kDa protein decreased in parallel with the 33 kDa protein, yielding a crosslinked product consisting of one each of the two proteins. The results, which indicate that the two proteins are present in equimolar amounts in PS II membranes, are consistent with the stoichiometry of one copy of the 33 kDa protein per PS II unit. (3) The total immobilization of the 33 kDa protein stabilized 40 to 60% of the oxygen-evolving activity against urea/NaCl−, CaCl2− and MgCl2-wash, which otherwise solubilize the three extrinsic proteins and strongly inactivate oxygen evolution. The result implies that extraction of the extrinsic proteins may not be the sole cause of the inactivation of oxygen evolution by these treatments. (4) The crosslinking of the 33 kDa protein with EDC had no protecting effect against Tris-, NH2OH- and pH 9.0-treatments. However, the stability of oxygen evolution at alkaline pH levels was slightly but significantly increased by treatment of PS II membranes with dithiobis(suc-cinimidylpropionate), which specifically modifies amino groups.

Crosslinking between the 33 kDa extrinsic protein and the 47 kDa chlorophyll‐carrying protein of the PS II reaction center core complex

Using highly purified oxygen‐evolving photosystem II complexes from spinach, the 33 kDa extrinsic protein was found to crosslink with the 47 kDa chlorophyll‐carrying protein with a cleavable bifunctional crosslinking reagent, dithiobis(succinimidylpropionate).