Quinone and pheophytin in the photosynthetic reaction center II from spinach chloroplasts

Prenylquinones and pheophytin a in a preparation of photosynthetic reaction center II from spinach chloroplasts were chemically determined. Each reaction center II had two molecules, each of plastoquinone-9 and pheophytin a, but practically no phylloquinone, α-tocopherylquinone or α-tocopherol.     

Stoichiometry of components in the photosynthetic oxygen evolution system of Photosystem II particles prepared with Triton X-100 from spinach chloroplasts

The stoichiometry of the proteins of the photosynthetic oxygen evolution system and of the electron transport components in Photosystem II particles prepared with Triton X-100 from spinach chloroplasts were determined. Per about 220 chlorophyll molecules, there were one reaction center II, one molecule each of the 33, 24 and 18 kDa proteins, four Mn atoms, two cytochromes b-559 (one high-potential, the other low-potential), and 3.5 plastoquinone-9 molecules, but practically no cytochrome b-563, cytochrome f, phylloquinone, α-tocopherol or α-tocopherylquinone.     

Engineered drought-induced biosynthesis of α-tocopherol alleviates stress-induced leaf damage in tobacco

Tocopherols are members of the vitamin E complex and essential antioxidant compounds synthesized in chloroplasts that protect photosynthetic membranes against oxidative damage triggered by most environmental stresses. Tocopherol deficiency has been shown to affect germination, retard growth and change responses to abiotic stress, suggesting that tocopherols may be involved in a number of diverse physiological processes in plants. Instead of seeking constitutive synthesis of tocopherols to improve stress tolerance, we followed an inducible approach of enhancing α-tocopherol accumulation under dehydration conditions in tobacco. Two uncharacterized stress inducible promoters isolated from Arabidopsis and the VTE2.1 gene from Solanum chilense were used in this work. VTE2.1 encodes the enzyme homogentisate phytyltransferase (HPT), which catalyzes the prenylation step in tocopherol biosynthesis. Transgenic tobacco plants expressing ScVTE2.1 under the control of stress-inducible promoters showed increased levels of α-tocopherol when exposed to drought conditions. The accumulation of α-tocopherol correlated with higher water content and increased photosynthetic performance and less oxidative stress damage as evidenced by reduced lipid peroxidation and delayed leaf senescence. Our results indicate that stress-induced expression of VTE2.1 can be used to increase the vitamin E content and to diminish detrimental effects of environmental stress in plants. The stress-inducible promoters introduced in this work may prove valuable to future biotechnological approaches in improving abiotic stress resistance in plants.     

The formation of homogentisate in the biosynthesis of tocopherol and plastoquinone in spinach chloroplasts

Homogentisate is the precursor in the biosynthesis of -tocopherol and plastoquinone-9 in chloroplasts. It is formed of 4-hydroxyphenylpyruvate of the shikimate pathway by the 4-hydroxyphenylpyruvate dioxygenase. In experiments with spinach the dioxygenase was shown to be localized predominatedly in the chloroplasts. Envelope membranes exhibit the highest specific activity, however, because of the high stromal portion of chloroplasts, 60–80% of the total activity is housed in the stroma. The incorporation of 4-hydroxyphenylpyruvate into 2-methyl-6-phytylquinol as the first intermediate in the tocopherol synthesis by the two-step-reaction: 4-Hydroxyphenylpyruvate Homogentisate 2-Methyl-6-phytylquinol was demonstrated by using envelope membranes. Homogentisate originates directly from 4-hydroxyphenylpyruvate of the shikimate pathway. Additionally, a bypass exists in chloroplasts which forms 4-hydroxyphenylpyruvate from tyrosine by an L-amino-acid oxidase of the thylakoids and in peroxisomes by a transaminase reaction. Former results about the dioxygenase in peroxisomes were verified.     

Localization and synthesis of prenylquinones in isolated outer and inner envelope membranes from spinach chloroplasts

The prenylquinone content and biosynthetic capabilities of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts were analyzed. Both envelope membranes contain prenylquinones, and in almost similar amounts (on a protein basis). However, the outer envelope membrane contains more alpha-tocopherol than the inner one although this prenylquinone is the major one in both fractions. On the contrary, plastoquinone-9 is present in higher amounts in the inner envelope membrane than in the outer one. In addition, it has been demonstrated that all the enzymes involved in the last steps of alpha-tocopherol and plastoquinone-9 biosynthesis, i.e., homogentisate decarboxylase polyprenyltransferase, S-adenosyl-methionine:methyl-6-phytylquinol methyltransferase, S-adenosyl-methionine: alpha-tocopherol methyltransferase, homogentisate decarboxylase solanesyltransferase, S-adenosyl-methionine:methyl-6-solanesylquinol methyltransferase, and possibly 2,3-dimethylphytylquinol cyclase, are localized on the inner envelope membrane. These results demonstrate that the inner membrane of the chloroplast envelope plays a key role in chloroplast biogenesis, and especially for the synthesis of the two major plastid prenylquinones.     

Evidence for alpha-Tocopherol Function in the Electron Transport Chain of Chloroplasts

The effect of three different stable radicals-2,2-diphenyl-1-picrylhydrazyl, 1,3,5-triphenyl-verdazyl, and galvinoxyl-was studied in photosystem II of spinach (Spinacia oleracea) chloroplasts. Inhibition by the three was noted on dimethylbenzoquinone reduction in presence of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and on silicomolybdate reduction in presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) in photosystem II and on the H₂O → methylviologen reaction encompassing both photosystems. Inhibition of all photosystem II reactions except silicomolybdate reduction could be partially restored by α-tocopherol or by 9-ethoxy-α-tocopherone but not by other quinones or radical chasers. On this basis, a functional role for α-tocopherol in the electron transport chain of spinach chloroplasts between the DCMU and DBMIB inhibition sites is postulated.     

Incorporation of shikimate and other precursors into aromatic amino acids and prenylquinones of isolated spinach chloroplasts

Under conditions of photosynthesis, shikimate-[1,6-¹⁴C] and D,L-tyrosine-[β-¹⁴C] were incorporated into the aromatic amino acids Phe, Tyr and Trp, and the prenylquinone and α-tocopherol by intact spinach chloroplasts. This might indicate the presence of enzymes of shikimate pathway in chloroplasts.     

Determination of plastoquinone-9 in chloroplasts by high pressure liquid chromatography

A method for the specific determination of nmol quantities of plastoquinone-9 in chloroplasts is described. HPLC of a heptane extract of chloroplasts separated the plastoquinone-9 from other material permitted a final spectrophotometric assay. Levels of plastoquinone-9 plastoquinol-9 were determined in dark adapted and illuminated chloroplasts.     

Adenosine 3′:5′-cyclic monophosphate,adenylate cyclase and a cyclic AMP binding-protein in Phaseolus vulgaris

The validity of using the binding-protein method for determining cyclic AMP in purified and partially purified extracts of Phaseolus tissues has been examined and confirmed. Measurement of cyclic AMP concentration by binding-protein gave similar results to those obtained by direct spectrophotometry of purified extracts. A cyclic AMP binding-protein and adenylate cyclase were demonstrated in Phaseolus extracts. Isolated intact chloroplasts were shown to possess adenylate cyclase activity but persistent cyclic AMP phosphodiesterase activity obviated quantitative assessment.     

The intracellular distribution of tocopherols in Calendula officinalis leaves

From Calendula officinalis leaves, five cellular subtractions (chloroplasts, mitochondria, Golgi membranes, microsomes and cytosol) were obtained and their purity was checked. The contents of α-,γ- and δ-tocopherols were determined in these fractions. There were no tocopherols in Golgi membranes and cytosol. γ-Tocopherol and δ-tocopherol were found in the chloroplasts, mitochondria and microsomes, whereas α-tocopherol was present only in the chloroplasts.     

A new solid-state microelectrode for measuring intra-cellular chloride activities

Solid-state microelectrodes for measuring intracellular Cl^? activity (aⁱCl) were made by sealing the tips of tapered glass capillaries (tip diameter 0.3 μm), coating them under vacuum with a 0.2–0.3 μm thick layer of spectroscopic grade silver, and sealing them (except for the terminal 2–5 μm of the tip) inside tapered glass shields. 106 microelectrodes had an average slope of 55.0 ± 0.6 mV (S.E.) per decade change in α_Cl. Tip resistance was (77.1 ± 3.1 × 10⁹ω (n=30). Electrode response was rapid (10–20 s), was unaffected by HCO₃^?, H₂PO₄^2? or protein, and remained essentially unchanged over a 24-h period. αⁱ_Cl in frog sartorius muscle fibers and epithelial cells of bullfrog small intestine was measured in vitro. In both tissues, αⁱ_Cl significantly exceeded the value corresponding to equilibrium distribution of Cl^? across the cell membrane.     

Functional Modeling Identifies Paralogous Solanesyl-diphosphate Synthases That Assemble the Side Chain of Plastoquinone-9 in Plastids

It is a little known fact that plastoquinone-9, a vital redox cofactor of photosynthesis, doubles as a precursor for the biosynthesis of a vitamin E analog called plastochromanol-8, the physiological significance of which has remained elusive. Gene network reconstruction, GFP fusion experiments, and targeted metabolite profiling of insertion mutants indicated that Arabidopsis possesses two paralogous solanesyl-diphosphate synthases, AtSPS1 (At1g78510) and AtSPS2 (At1g17050), that assemble the side chain of plastoquinone-9 in plastids. Similar paralogous pairs were detected throughout terrestrial plant lineages but were not distinguished in the literature and genomic databases from mitochondrial homologs involved in the biosynthesis of ubiquinone. The leaves of the atsps2 knock-out were devoid of plastochromanol-8 and displayed severe losses of both non-photoactive and photoactive plastoquinone-9, resulting in near complete photoinhibition at high light intensity. Such a photoinhibition was paralleled by significant damage to photosystem II but not to photosystem I. In contrast, in the atsps1 knock-out, a small loss of plastoquinone-9, restricted to the non-photoactive pool, was sufficient to eliminate half of the plastochromanol-8 content of the leaves. Taken together, these results demonstrate that plastochromanol-8 originates from a subfraction of the non-photoactive pool of plastoquinone-9. In contrast to other plastochromanol-8 biosynthetic mutants, neither the single atsps knock-outs nor the atsps1 atsps2 double knock-out displayed any defects in tocopherols accumulation or germination.     

Photosynthetic apparatus of chilling-sensitive plants IX. The involvement of α-tocopherol in the electron transport chain and the anti-oxidizing system in chloroplasts of tomato leaves

1. The role of tocopherols in tomato chloroplasts from fresh, cold and dark-stored as well as stored and illuminated leaves was studied.2. The cold and dark storage of leaves results in a loss of chloroplast α- and γ-tocopherols of about 30–40% accompanied by an increase in chloroplast δ-tocopherol of about 40%. On illumination of stored leaves, an elevation of α- and γ-tocopherol level to about 110 and 95% of the control, respectively, occurs, whilst δ-tocopherol content is not affected.3. Experiments performed with 2,2-diphenyl-1-picrylhydrazyl-treated chloroplasts show that only about 70% of total α-tocopherol is functionally active in the electron transport of Photosystem II between the diphenyl-carbazide (DPC) donation site and the inhibition site of DBMIB.4. A small amount of α-tocopherol quinone (about 10% of α-tocopherol content) is found in chloroplasts from fresh, fresh and illuminated as well as cold and dark-stored tomato leaves, whereas the illumination of the latter increases the chloroplast α-tocopherol quinone content 3-fold. Moreover, following the illumination of chloroplasts from cold and dark-stored as well as stored and illuminated leaves, the oxidation of exogenous α-tocopherol to α-tocopherol quinone is 2-fold faster then in chloroplasts from fresh leaves.5. The primary product (‘α-tocopheroxide’) formed during the α-tocopherol oxidation by illuminated chloroplasts was identified as 8a-hydroxy-α-tocopheron.6. Exogenous α-tocopherol inhibits the lipid photoperoxidation by about 40–50% in chloroplasts from all three kinds of tomato leaf.7. The results seem to suggest that chloroplast α-tocopherol is involved in both electron transport of PS II and antioxidizing system of chloroplasts.     

Isolation and Functional Analysis of Homogentisate Phytyltransferase from Synechocystis sp. PCC 6803 and Arabidopsis

Tocopherols, collectively known as vitamin E, are lipid-soluble antioxidants synthesized exclusively by photosynthetic organisms and are required components of mammalian diets. The committed step in tocopherol biosynthesis involves condensation of homogentisic acid and phytyl diphosphate (PDP) catalyzed by a membrane-bound homogentisate phytyltransferase (HPT). HPTs were identified from Synechocystis sp. PCC 6803 and Arabidopsis based on their sequence similarity to chlorophyll synthases, which utilize PDP in a similar prenylation reaction. HPTs from both organisms used homogentisic acid and PDP as their preferred substrates in vitro but only Synechocystis sp. PCC 6803 HPT was active with geranylgeranyl diphosphate as a substrate. Neither enzyme could utilize solanesyl diphosphate, the prenyl substrate for plastoquinone-9 synthesis. In addition, disruption of Synechocystis sp. PCC 6803 HPT function causes an absence of tocopherols without affecting plastoquinone-9 levels, indicating that separate polyprenyltransferases exist for tocopherol and plastoquinone synthesis in Synechocystis sp. PCC 6803. It is surprising that the absence of tocopherols in this mutant had no discernible effect on cell growth and photosynthesis.     

Biosynthesis of tocopherols: A re-examination of the biosynthesis and metabolism of 2-methyl-6-phytyl-1,4-benzoquinol

A number of previous studies of the involvement of 2-methyl-6-phytyl-1,4-benzoquinol in the biosynthesis of α-tocopherol have failed to take account of the fact that this quinol and its quinone have very similar chromatographic properties to those of 2-methyl-3-phytyl-1,4-benzoquinol and 2-methyl-3-phytyl-1,4-benzoquinone respectively. It has now been shown that the two quinones can be separated from each other either by multidevelopment TLC or by HPLC and that the claims made earlier with regard to the biosynthesis and metabolism of 2-methyl-6-phytyl-1,4-benzoquinol in chloroplasts are correct. In particular, it has been established that this quinol is the only methyl phytylbenzoquinol formed from homogentisate and phytyl pyrophosphate in chloroplast preparations. It has also been shown for the first time that lettuce chloroplasts are able to synthesize ³H-labelled α- and γ-tocopherols from [methylene-³H] homogentisate.     

Differences in phytochrome action on the formation of carotenoids and quinones during chloroplast development in seedlings of barley (Hordeum vulgare)

The influence of phytochrome on the light induced formation of carotenoids and quinones was investigated using etiolated seedlings of barley ( Hordeum vulgare L. cv. Villa). The biosynthesis of both the quinones and the carotenoids was enhanced by active phytochrome, but the formation of the individual carotenoids and quinones was influenced by quite different thresholds. In both younger and older plants the biosynthesis of β-carotene, lutein and violaxanthin was promoted by a low P_fr threshold. The formation of plastoquinone-9, plastohydroquinone-9, α-tocoquinone and phylloquinone was also influenced by a low P_fr threshold. The biosynthesis of zeaxanthin and neoxanthin required a much higher amount of P_fr. Only antheraxanthin-likeα-tocopherol, desmethylphylloquinone and, in older leaves, α-tocoquinone exhibited a complete reversibility of the phytochrome action in their biosynthesis. The effect of phytochrome on the biosynthesis of carotenoids and quinones was different in seedlings of different age.     

Synthesis and Secretion of Acid Phosphatase in Halotolerant Zygosaccharomyces rouxii

Changes in the contents of defensive substances against the active oxygen in water-stressed spinach plants were examined. The contents of ascorbate peroxidase (AP; EC 1.11.1.7), glutathione reductase (GR; EC 1.6.4.2) and α-tocopherol increased remarkably in water-stressed spinach leaves, while those of Superoxide dismutase (SOD; EC 1.15.1.1), dehydroascorbate reductase (EC 1.8.5.1), ascorbate and glutathione changed little. The content of α-tocopherol in chloroplast thylakoid membranes isolated from water-stressed leaves was higher than that from normal leaves. It is, therefore, conceivable that GR, AP and α-tocopherol might be related to the tolerance of plants to water deficiency.     

Plastoquinol as a singlet oxygen scavenger in photosystem II

It has been found that in Chlamydomonas reinhardtii cells, under high-light stress, the level of reduced plastoquinone considerably increases while in the presence of pyrazolate, an inhibitor of plastoquinone and tocopherol biosynthesis, the content of reduced plastoquinone quickly decreases, similarly to α-tocopherol. In relation to chlorophyll, after 18 h of growth under low light with the inhibitor, the content of α-tocopherol was 22.2 mol/1000 mol chlorophyll and that of total plastoquinone (oxidized and reduced) was 19 mol/1000 mol chlorophyll, while after 2 h of high-light stress the corresponding amounts dropped to 6.4 and 6.2 mol/1000 mol chlorophyll for α-tocopherol and total plastoquinone, respectively. The degradation of both prenyllipids was partially reversed by diphenylamine, a singlet oxygen scavenger. It was concluded that plastoquinol, as well as α-tocopherol is decomposed under high-light stress as a result of a scavenging reaction of singlet oxygen generated in photosystem II. The levels of both α-tocopherol and of the reduced plastoquinone are not affected significantly in the absence of the inhibitor due to a high turnover rate of both prenyllipids, i.e., their degradation is compensated by fast biosynthesis. The calculated turnover rates under high-light conditions were twofold higher for total plastoquinone (0.23 nmol/h/ml of cell culture) than for α-tocopherol (0.11 nmol/h/ml). We have also found that the level of α-tocopherolquinone, an oxidation product of α-tocopherol, increases as the α-tocopherol is consumed. The same correlation was also observed for γ-tocopherol and its quinone form. Moreover, in the presence of pyrazolate under low-light growth conditions, the synthesis of plastoquinone-C, a hydroxylated plastoquinone derivative, was stimulated in contrast to plastoquinone, indicating for the first time a functional role for plastoquinone-C. The presented data also suggest that the two plastoquinones may have different biosynthetic pathways in C. reinhardtii.     

The subcellular distribution and biosynthesis of castaprenols and plastoquinone in the leaves of Aesculus hippocastanum

Intact chloroplasts and cell walls were prepared from horse-chestnut leaves that had previously metabolized [2-(14)C]mevalonate. The bulk of the castaprenols and plastoquinone-9 was found within the chloroplasts. The remaining portion of the castaprenols was associated with the cell-wall preparation whereas that of the plastoquinone-9 was probably localized in the soluble fraction of the plant cell. The (14)C content of these compounds of different cell fractions indicated the presence of polyisoprenoid-synthesizing activity both inside and outside the chloroplasts. This was confirmed by the relative incorporation of (14)C when ultrasonically treated and intact chloroplasts were incubated with [2-(14)C]mevalonate. As the leaves aged (on the tree) an increase in extraplastidic castaprenols and plastoquinone-9, together with associated synthesizing activities, was observed.