Comparative Studies on Plastoquinones: V. Changes in Lipophilic Chloroplast Quinones during Development

Changes of lipophilic chloroplast quinones in corn, oats, peas, and Vicia faba are reported after 0, 4, 8, 12, 16, 20, 24, 48, 72, or 96 hours of exposure to light. There is a pronounced increase in plastoquinone A and chlorophyll levels and slight increase, in plastoquinone C_1-6, vitamin K₁, and α-tocopherylquinone content. Coenzyme Q levels, on the other hand, show little change upon exposure to light.     

Comparative Studies on Plastoquinone II. Analysis for Plastoquinones A, B, C, and D

Two different methods for the extraction and assay of plastoquinones A, B, C and D from chloroplasts of green plants have been described. The long procedure involves separation of aqueous and lipid phases of extract in a separatory funnel, column chromatography, purification on thin-layer plates, and spectrophotometric assays for quantitative determination of the various plastoquinones. The short procedure is based on spotting lipid extracts from chloroplasts on thin layer plates and comparing leucomethylene blue spots of unknown quinones with a series of spots produced by known amounts of the 4 standard plastoquinones on the same plate.

Reliability of the 2 procedures is shown by presenting recovery data (82% recovery for PQ A by the long method and 64-100% recovery by the short method). Various solvent systems for quinone purification are described. Separation of plastoquinones B and C into 6 components each is demonstrated for spinach and a tomato mutant, high pigment (hp). Plastoquinone C is shown to be equivalent to C₁-C₄ while D corresponds to PQ C₅ and C₆ according to Griffiths, Wallwork and Pennock's designation. The term PQ D is therefore redundant and should be abandoned in favor of specific designation of PQ C type.

     

Comparative Studies on Plastoquinones. IV. Plastoquinones in Algae

Plastoquinones A and C have been found in all classes of algae, including representatives of greens, yellow-greens, blue-greens, reds, browns and the flagellate, Euglena. Plastoquinone C from red and brown algae can be separated into 6 different types. An additional plastoquinone C has been found in Gigartina and Rhydomela. From chromatographic evidence this may be equivalent to plastoquinone C_o, a C type with a hydroxyl group on the first isoprene unit of the terpenoid sidechain of this substituted benzo-quinone. The ubiquinone, vitamin K and α-tocopherylquinone content of several algae is also reported. The presence of plastoquinone A in all green plants and many algae indicates that it may be a functional element in photosynthesis. Our study shows that plastoquinone C is more regularly present in algae than has been previously shown.     

Ribonuclease activities in bean roots

Vicia faba meristematic and elongating root cells (zones 0–4 and 10–20 mm) contained one nuclease (A₁) and four ribonucleases (A₂, A₃, C₁, C₂). When the overall activity of each enzyme was expressed per cell, the elongating cells contained 4-, 4-, 4-, 10- and 17-fold more activity than meristematic cells for A₁, C₁, C₂, A₂ and A₃, respectively.     

Taxonomic survey for the presence of ribulose-1,5-bisphosphate carboxylase activity in guard cells

Guard cell pairs were dissected from freeze-dried leaves of plants representing 15 families, including monocots, dicots, and pteridophytes. All three major photosynthetic carbon pathways (C₂, C₄, and Crassulacean acid metabolism) were represented. These individual guard cell pairs were assayed quantitatively for ribulose-1,5-bisphosphate carboxylase specific activity. Assay sensitivity averaged 0.08 picomoles of ribulose-P₂ dependent P-glycerate formation (i.e. 100-fold more sensitive than required to detect the activity present in a single Vicia faba mesophyll cell). The calculated specific activities for guard cells and mesophyll cells averaged 4 and 472 millimoles per kilogram dry weight per hour, respectively. For all species surveyed, (a) the enzyme activity calculated for guard cells was below the detection limit of the assay, or (b) the specific activity (weight or cell basis) calculated for guard cells was less than 1% of the specific activity calculated for adjacent mesophyll cells. Based on this survey, the generalization is made that the photosynthetic carbon reduction pathway is absent, or virtually so, in guard cell chloroplasts.     

Effect of the herbicide terbutryn on meiosis,yield and mitotic chromosomes in C2 plants ofVicia faba L.

The herbicide terbutryn induced up to 11.3 % chromosomal abnormalities in cells undergoing meiosis inVicia faba. The mean number of pods/plant and the mean number of seeds/pod were considerably reduced, but the average seed dry mass was not or was only slightly affected by this herbicide. In C₂ plants chromosomal abnormalities were observed in few but significant numbers of cells.     

Cytoplasmic Sterility Factors in VICIA FABA L

Tissues of cytoplasmic male sterile, maintainer, restorer, and restored lines, and sterile plants which reverted to fertility in Vicia faba were examined in ultrathin sections. Cytoplasmic spherical bodies (CSB), ca. 70 nm in diameter, were observed in tissues of all sterile plants but not in tissues of maintainer, restorer or restored sterile plants. No CSB were observed in a reverted fertile branch of a tiller-sterile plant, nor in 5 of 6 reverted fertile plants. One reverted fertile plant contained CSB in ovules. It is proposed that the CSB are the sites of, or possibly, products of, sterility factors in Vicia faba.     

Plastoquinone B

A compound found in spinach and other higher plants previously referred to as R 263 has now been found to be a breakdown product of plastoquinone B. This quinone, PQ B, is found with 8 other quinones in spinach chloroplasts. These 9 quinones are PQ A, PQ B, PQ C, PQ D (7, 8, 15) Vitamin K₁ (10, 12), an unknown naphthoquinone (13) and α-, β- and γ-tocopherylquinones (7, 12). An improved method for purification of plastoquinone B is described. Previous confusion of this compound with other quinoid material on silica gel is described and corrected R_F values are given. The activity of PQ B is similar to the activity of PQ C in restoration studies of the photo-reduction of ferricyanide and indophenol.     

THE TOCOPHEROL,VITAMIN K,AND RELATED ISOPRENOID QUINONE COMPOSITION OF A UNICELLULAR RED ALGA (PORPHYRIDIUM CRUENTUM)1

The total lipids of axenically cultivated cells of Porphyridium cruentum were extracted with aqueous methanol-chloroform mixture and fractionated into neutral and polar lipids by silicic acid column chromatography. Thin-layer and reversed-phase paper chromatographic analyses of the neutral lipid fractions revealed the occurrence of plastoquinones (PQ) A and C, vitamin K₁ (K), ubiquinone-10 (Q₁₀), α-tocopherol (α-T), and α-tocopherolquinone (α-TQ) in the photoautotrophically cultured alga, and the same quinones but no tocopherol in the alga grown photoheterotrophically on glycerol. The plastoquinone A and vitamin K₁ were isolated, identified, and estimated by spectroscopic methods. The results indicated the following decreasing order of concentrations: autotrophic culture, PQ A > K > Q₁₀ > PQ C, α-TQ, α-T; heterotrophic culture, PQ A > Q₁₀ > K > PQ C, α-TQ. Except for the absence of plastoquinone B, the overall quinonoid composition was in general agreement with those previously reported for multicellular members of Rhodophyta, but the concentration level in total lipid was markedly lower.     

Effects of salt stress on nitrogenase activity and growth of four legumes

The effects of salt stress on nitrogenase (N2-ase) activity, growth and nitrogen content ofVicia faba (L.),Medicago sativa (L.) Merrill,Glycine max andVigna sinensis (L.) were investigated. Four levels of salinity were applied and salt treatments were imposed on inoculated and N-fertilized plants.M. sativa tolerated mild levels of salinity but higher salt concentrations depressed N₂-ase activity of this species. The other three legumes were considerably affected by salt treatments, and N₂-ase activity was significantly reduced by salinity. Vicia faba, carrying elongate nodules, could restore a partial N₂-ase activity upon recovery from salt stress whereasG. max andV. sinesis, both with spherical nodules, could not regain significant activity when salinity was removed. Salt stress retarded growth of both inoculated and N-fertilized plants. The nitrogen content of both treatments was also affected by salinity and the effect was more severe for inoculated than N-fertilized plants.     

Interactions of herbicides and azidoquinones at a Photosystem II binding site in the thylakoid membrane

6-Azido-5-decyl-2,3-dimethoxy-p-benzoquinone (6-azido-Q₀C₁₀) was found to replace the native plastoquinone at B (the second stable electron acceptor to Photosystem II (PS II)). The 6-azido-Q₁₀C₁₀ would accept electrons from the primary electron-accepting quinone, Q, thus allowing electron transport through PS II to the plastoquinone pool in thylakoids. The synthetic azidoquinone also competes with the PS II herbicides ioxynil and atrazine for binding. This observation strongly favors the hypothesis that PS II herbicides block electron transport by replacing the native quinone which acts as the second electron carrier on the reducing side of PS II (termed B). Covalent linkage of 6-azido-Q₀C₁₀ to its binding environment by ultraviolet irradiation greatly reduces herbicide-binding affinity but does not lead to a loss in herbicide-binding sites. We take this as evidence that covalent attachment of 6-azido-Q₀C₁₀ allows some freedom of quinone head-group movement such that the herbicides can enter the binding site. This indicates that the protein determinants which regulate quinone and herbicide binding are very closely related, but not identical. A compound somewhat related to 6-azido-Q₀C₁₀ is 2-azido-3-methoxy-5-geranyl-6-methyl-p-benzoquinone (2-azido-Q₂). This compound was found to be an ineffective competitor with respect to herbicide binding. Thus, interactions with protein-binding determinants are highly dependent on the molecular structure of quinones. The 2-azido-Q₂ was an inhibitor of electron flow in the intersystem portion of the chain.     

Surface wax of Andreaea and Pogonatum species

The mosses Andreaea rupestris, Pogonatum aloides and P. urnigerum contain surface waxes in amounts of 0.05–0.12% dry wt. The waxes consisted of esters (C₃₈-C₅₄), primary alcohols (C₂₀-C₃₂), free fatty acids (C₁₆-C₃₀), and alkanes (C₂₁-C₃₁). Additionally, aldehydes (C₂₂-C₃₀) were major constituents in the wax of P. urnigerum. The classes and their chain length distributions in the surface waxes of these mosses are comparable to those of epicuticular waxes of higher plants.     

Expression of human cytomegalovirus pp150 gene in transgenic Vicia faba L. and immunogenicity of pp150 protein in mice

The pp150 gene of human cytomegalovirus (HCMV) was transferred into Vicia faba plants by Agrobacterium tumefaciens-mediated transformation. Three of five hygromycin resistant V. faba plants were identified as positive by PCR and dot-blot hybridization. The ELISA results indicated that pp150 protein from three plants of transformed V. faba leaves and seeds made up 0.005–0.015% of the total soluble protein. The results of detection by immunoblot and inhibition of immunofluorescent assay (IFA) showed that pp150 soluble protein had immunity activity. HCMV pp150-specific antibody (IgG, IgA) and IFN-γ producing T cells were detected in 100% of the mice immunized with pp150 transgenic V. faba seeds by ELISA and intracellular staining and flow cytometry analysis, respectively. The transgenic V. faba plants will provide new material for the development of edible vaccination against HCMV infection.     

In Vitro Biosynthesis of Vicia faba Polyphenoloxidase

Poly A⁺ mRNA was isolated from Vicia faba leaves and translated in vitro using a rabbit reticulocyte translation system. From analysis of the total translation products, the major proteins synthesized in vitro were 32 kilodaltons and 20 kilodaltons. When antibodies to Vicia faba polyphenoloxidase were added, a specific immunoprecipitable protein was observed. This protein's molecular weight was shown to be similar to that of the isolated enzyme (45 kilodaltons). The isolated enzyme successfully competed with the in vitro synthesized product for antipolyphenoloxidase. In addition, the in vitro synthesized product was not immunoprecipitated with antitomato peroxidase and comigrated with isolated and/or iodinated enzyme in sodium dodecylsulfate-polyacrylamide gel electrophoresis. Using in vitro translation and specific immunoprecipitation, a primary translation product corresponding to Vicia faba polyphenoloxidase was identified as a 45 kilodaltons protein.     

Photosynthesis of Grass Species Differing in Carbon Dioxide Fixation Pathways : VIII. Ultrastructural Characteristics of Panicum Species in the Laxa Group

Ultrastructural studies of leaves of seven Panicum species in or closely related to the Laxa group and classified as C₃, C₄ or C₃-C₄ intermediate were undertaken to examine features associated with C₃ and C₄ photosynthesis. The C₃ species Panicum rivulare Trin. had few organelles in bundle sheath cell profiles (2 chloroplasts, 1.1 mitochondria, and 0.3 peroxisomes per cell section) compared to an average of 10.6 chloroplasts, 17.7 mitochondria, and 3.2 peroxisomes per bundle sheath cell profile for three C₃-C₄ species, Panicum milioides Nees ex Trin., Panicum decipiens Nees ex Trin. and Panicum schenckii Hack. However, two other C₃ species, Panicum laxum Sw. and Panicum hylaeicum Mez, contained about 0.7, 0.5, and 0.3 as many chloroplasts, mitochondria, and peroxisomes, respectively, as in bundle sheath cell profiles of the C₃-C₄ species. Chloroplasts and mitochondria in bundle sheath cells were larger than those in mesophyll cells for the C₄ species Panicum prionitis Griseb. and the C₃-C₄ species, but in C₃ species the organelles were similar in size or were smaller in the bundle sheath cells. The C₃-C₄ species and P. laxum and P. hylaeicum exhibited an unusually close association of organelles in bundle sheath cells with mitochondria frequently surrounded in profile by chloroplasts. The high concentrations in bundle sheath cells of somewhat larger organelles than in mesophyll cells correlates with the reduced photorespiration of the C₃-C₄ species.     

Sesquiterpenoid aldehyde quinones and derivatives in pigment glands of Gossypium

The resistance of Gossypium species to insects is enhanced by compounds in their lysigenous pigment glands. In cultivated cottons, glands in achlorophyllous plant parts contained predominately the terpenoid aldehyde gossypol in G. hirsutum, and gossypol and its methyl and dimethyl ethers in G. barbadense. Glands in young green tissues, however, contained hemigossypolone as the predominant terpenoid aldehyde in G. hirsutum, and a new quinone, hemigossypolone-7-methyl ether, in G. barbadense. As glands aged in green tissues, the sesquiterpenoid quinones were replaced by several C₂₅-terpenoids formed by the Diels-Alder reaction of the quinones with myrcene or trans-β-ocimene. Two C₂₅-terpenoids isolated from G. barbadense, but not G. hirsutum, were the methyl ethers of heliocides H₁ and H₄ and were designated heliocides B₁ and B₄, respectively. A dark red pigment, gossyrubilone, from glands of young leaves of both species is the isopentylimine of hemigossypolone. Similar red imines, formed from sesquiterpenoid quinones and amino acids, resembled the red coloration of the envelope cells surrounding the gland sac. The terpenoid quinones of Gossypium had physical characteristics different from quinones in Bombax which apparently were incorrectly identified as being the same. A survey of the terpenoid quinones and their heliocide derivates in wild Gossypium species and related genera in the Gossypieae showed considerable diversity which may be used for establishing biochemical and phylogenetic relationships.     

Chryseobacterium yeoncheonense sp. nov., with ginsenoside converting activity isolated from soil of a ginseng field

A Gram-staining negative, aerobic, non-motile, non-flagellate, yellow-pigmented, rod-shaped bacterial strain, designated strain DCY67^T, was isolated from ginseng field in Republic of Korea. Strain DCY67^T contained β-glucosidase activity which converts ginsenoside Rb1 to compound K. Optimum growth of DCY67^T occurred at 30 °C and pH 6.0–6.5. Analysis of the 16S rRNA gene sequences revealed that strain DCY67^T belonged to the family Flavobacteriaceae and was most closely related to Chryseobacterium ginsenosidimutans THG 15^T (97.5 %). The genomic DNA G+C content was 36.1 mol%. The predominant quinones were MK-6 (90.9 %) and MK-7 (9.15 %). The major fatty acids were iso-C_15:0, summed feature 3 (containing C_16:1 ω7c and/or C_16:1 ω6c) and iso-C_17:0 3-OH. On the basis of these phenotypic, genotypic and chemotaxonomic studies, strain DCY67^T represents a novel species of the genus Chryseobacterium, for which, name Chryseobacterium yeoncheonense sp. nov. proposed the type strain is DCY67^T (=KCTC 32090^T = JCM 18516^T).     

Interaction of exogenous quinones with membranes of higher plant chloroplasts: modulation of quinone capacities as photochemical and non-photochemical quenchers of energy in Photosystem II during light-dark transitions

Light modulation of the ability of three artificial quinones, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), 2,6-dichloro-p-benzoquinone (DCBQ), and tetramethyl-p-benzoquinone (duroquinone), to quench chlorophyll (Chl) fluorescence photochemically or non-photochemically was studied to simulate the functions of endogenous plastoquinones during the thermal phase of fast Chl fluorescence induction kinetics. DBMIB was found to suppress by severalfold the basal level of Chl fluorescence (F_o) and to markedly retard the light-induced rise of variable fluorescence (F_v). After irradiation with actinic light, Chl fluorescence rapidly dropped down to the level corresponding to F_o level in untreated thylakoids and then slowly declined to the initial level. DBMIB was found to be an efficient photochemical quencher of energy in Photosystem II (PSII) in the dark, but not after prolonged irradiation. Those events were owing to DBMIB reduction under light and its oxidation in the dark. At high concentrations, DCBQ exhibited quenching behaviours similar to those of DBMIB. In contrast, duroquinone demonstrated the ability to quench F_v at low concentration, while F_o was declined only at high concentrations of this artificial quinone. Unlike for DBMIB and DCBQ, quenched F_o level was attained rapidly after actinic light had been turned off in the presence of high duroquinone concentrations. That finding evidenced that the capacity of duroquinone to non-photochemically quench excitation energy in PSII was maintained during irradiation, which is likely owing to the rapid electron transfer from duroquinol to Photosystem I (PSI). It was suggested that DBMIB and DCBQ at high concentration, on the one hand, and duroquinone, on the other hand, mimic the properties of plastoquinones as photochemical and non-photochemical quenchers of energy in PSII under different conditions. The first model corresponds to the conditions under which the plastoquinone pool can be largely reduced (weak electron release from PSII to PSI compared to PSII-driven electron flow from water under strong light and weak PSI photochemical capacity because of inactive electron transport on its reducing side), while the second one mimics the behaviour of the plastoquinone pool when it cannot be filled up with electrons (weak or moderate light and high photochemical competence of PSI).     

STRANDEDNESS OF VICIA FABA CHROMOSOMES AS REVEALED BY ENZYME DIGESTION STUDIES

Chromosomes and nuclei isolated from neutral formalin-fixed Vicia faba lateral roots were treated with trypsin, pepsin, RNase, or DNase. Only trypsin affected the morphology of the chromosomes and nuclei. The appearance of the chromosomes after trypsin digestion indicated that each chromatid contained four strands that could be seen with an ordinary light microscope. The experiments are interpreted as indicating that mitotic chromosomes of Vicia faba are multistranded and that the linear continuity of the chromosome is dependent on protein.     

Comparative electrophoretical analysis of plasmid-like mitochondrial DNAs in Vicia faba and in some other legumes

Minicircular DNAs found in mitochondria of 6-d-old etiolated seedlings of Vicia faba L. were also present in mitochondria isolated from cell suspension cultures and from green leaves of this plant. These results support the suggestion that the plasmid-like molecules found in mitochondria of V. faba are an essential component of the mitochondrial genome. The minicircular DNAs were, apparently, peculiar for the species V. faba since they were found in all three cultivars of this species which were studied. The distribution pattern of plasmid-like DNAs in Vicia villosa L. was completely different and mitochondria from Medicago sativa L. also contained specific minicircular DNAs. Thus, minicircular DNAs are typical for the mitochondrial genomes of several legumes and different plant species have their specific mitochondrial plasmid-like DNAs