Hans Kleinig

SC-0051, a 2-benzoyl-cyclohexane-1,3-dione bleaching herbicide, is a potent inhibitor of the enzyme p-hydroxyphenylpyruvate dioxygenase

Growth inhibition of Lemna gibba plantlets by the bleaching herbicide, SC‐0051 (2‐(2‐chloro‐4‐methanesulfonylbenzoyl)‐1,3‐cyclohexanedione)) was alleviated by the addition of homogentisic acid to the growth medium. Homogentisic acid is a key intermediate in the biosynthesis of tyrosine‐derived plant quinones as well as in tyrosine metabolism. The herbicide prevented the incorporation of radioactivity from [14C]tyrosine into lipophilic plant metabolites and, in rat liver extracts, the herbicide inhibited the conversion of tyrosine to homogentisic acid. The enzyme p‐hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27) from both Zea mays seedlings and liver tissues, was found to be subject to strong inhibition by SC‐0051. Inhibition of plant quinone biosynthesis is a new mode of herbicidal action. One of the consequences of quinone depletion in plants in vivo is apparently an indirect inhibition of phytoene desaturation. The enzyme phytoene desaturase itself, however, is not afflicted by the herbicide.

Regulation and activation of phytoene synthase, a key enzyme in carotenoid biosynthesis, during photomorphogenesis

Abstract. During photomorphogenesis in higher plants, a coordinated increase occurs in the chlorophyll and carotenoid contents. The carotenoid level is under phytochrome control, as reflected by the light regulation of the mRNA level of phytoene synthase (PSY), the first enzyme in the carotenoid biosynthetic pathway. We investigated PSY protein levels, enzymatic activity and topological localization during photomorphogenesis. The results revealed that PSY protein levels and enzymatic activity increase during de-etiolation and that the enzyme is localized at thylakoid membranes in mature chloroplasts. However, under certain light conditions (e.g., far-red light) the increases in PSY mRNA and protein levels are not accompanied by an increase in enzymatic activity. Under those conditions, PSY is localized in the prolamellar body fraction in a mostly enzymatically inactive form. Subsequent illumination of dark-grown and/or in far-red light grown seedlings with white light causes the decay of these structures and a topological relocalization of PSY to developing thylakoids which results in its enzymatic activation. This light-dependent mechanism of enzymatic activation of PSY in carotenoid biosynthesis shares common features with the regulation of the NADPH:protochlorophyllide oxidoreductase, the first light-regulated enzyme in chlorophyll biosynthesis. The mechanism of regulation described here may contribute to ensuring a spatially and temporally coordinated increase in both carotenoid and chlorophyll contents.

Inhibition of carotenoid biosynthesis by the herbicide SAN 9789 and its consequences for the action of phytochrome on plastogenesis

Treatment of the mustard (Sinapis alba L.) seedling with the herbicide SAN 9789 inhibits synthesis of colored carotenoids and interferes with the formation of plastid membrane lipids without affecting growth and morphogenesis significantly. In farred light, which is hardly absorbed by chlorophyll, development of plastid ultrastructure, synthesis of ribulosebisphosphate carboxylase and synthesis of chlorophyll are not affected by SAN 9789. It is concluded that normal phytochrome actions on plastid structural development, protein and chlorophyll syntheses are not affected by the absence of carotenoids provided that there is no significant light absorption in chlorophyll. The findings show that the inhibition of synthesis of one set of plastid membrane components (the carotenoids) does not stop synthesis of other components such as chlorophyll and does not halt membrane assembly. Supplementary experiments with the closely related compound SAN 9785, which affects the amount and composition of plastid lipids but not carotenoid and chlorophyll syntheses, suggest that the effect of the herbicide SAN 9789 is due exclusively to its inhibition of synthesis of colored carotenoids. In the presence of SAN 9789 white or red light at high fluence rate causes photodestruction of chlorophyll and ribulosebisphosphate carboxylase and photodecomposition of thylakoids. These effects are interpreted as resulting exclusively from the self-photooxidation and photosensitizing action of chlorophyll once the protection by carotenoids of chlorophyll against self- and sensitized photooxidation is lost.

Membranes of Tetrahymena: III. The effect of temperature on membrane core structures and fatty acid composition of Tetrahymena cells

Abstract Membrane core structures as revealed by the freeze-etch electron microscopy and the fatty acid composition measured by gas-liquid chromatography have been analyzed in Tetrahymena cells exposed to low temperature for varying periods. When cells were grown to mid-log phase at the optimal growth temperature of 28 °C and then chilled to 10 °C, cell division was inhibited. However, within 16 h the cells adapted to the low temperature. Chilling effected drastic structural alterations in the cores of different membrane types (membranes of the pellicula, the alveolar sacs, the endoplasmic reticulum and the nuclei). In all cases, there was a segregation of smooth faces from particle-rich faces in the fracture planes. However, the native membrane state, i.e. like that of cells grown at 28 °C, reappeared when the cells adapted to the low temperature. The total lipids of Tetrahymena cells contained primarily even-numbered fatty acids ranging from C 12 to C 18 , but we also detected appreciable amounts of C 20 acids; this has not been reported before. During the initial phase of chilling, when cell division is inhibited, about 50% of the saturated fatty acids were replaced by unsaturated fatty acids, primarily monoenoic, dienoic and trienoic acids. We conclude that the structural recovery of the membranes in chilled Tetrahymena cells is accomplished by a desaturation of membrane fatty acids. This is discussed with respect to membrane “fluidity”.

Protein filaments-structural components of the phloem exudate : I. Observations with Cucurbita and Nicotiana.

SummaryFine structure and chemical composition of the phloem exudate of Cucurbita maxima and Nicotaiana glauca x suaveolens are investigated. Filamentous structures, several microns in length, are identified as structural components of the exudate by means of negative staining and electron microscopy. Two types of filaments are described: one form measures nearly 40 Å in diameter and shows a beaded appearance with regular spacings of about 50 Å; it is termed “elementary filament”. The second form has a diameter of about 90 Å and presumably consists of two helically arranged 40 Å subunits.The proteinaceous nature of the filaments is indicated by chemical analysis. The main macromolecular component of the exudate is demonstrated to be protein. Only traces of nucleic acids are detectable, lipids and polysaccharides cannot be found. The identity of the protein filaments with the filamentous structures (“slime”, “P-protein”), as revealed in thin sections of mature sieve tubes, is discussed.

The site of carotenogenic enzymes in chromoplasts from Narcissus pseudonarcissus L.

The membranes from the chromoplasts of Narcissus pseudonarcissus L. which are derived from the inner envelope membrane are the site of β-carotene synthesis from [1-14C]isopentenyl diphosphate. The enzymes involved are partly peripheral membrane proteins (prenyltransferase, phytoene synthase) and partly integral membrane proteins (cis-trans isomerase, dehydrogenase(s), cyclase(s)). Metabolic channeling is suggested.

The pigments of Flexibacter elegans: Novel and chemosystematically useful compounds

Abstract1.Flexibacter elegans Fx e1 contains 3 pigments; their quantitative proportion is 2.5 : 2.5 : 95%. The absolute pigment content is 0.6–0.8 μg per mg dry cells.2.Contrary to statements in the literature, these pigments are not carotenoids. This can be deduced from their electronic spectra which lack fine structure and are pH-dependent; from their resistance to photooxidation; from their failure to incorporate 14C-mevalonate; and from their mass spectra which do not show the M-92 and M-106 fragments typical for carotenoids.3.The main pigment is characterized in detail; it has been named flexirubin.4.The new pigments are widely distributed among members of the Cytophaga-Flexibacter group and seem to be valuable chemosystematic markers.5.Specific pigment content of the bacteria does neither change along the growth curve, nor under illumination. Low pH or high phosphate concentrations depress specific pigment content.6.The taxonomical position of experimental strain Fx e1 is discussed: The organism shows a cyclic change in cell shape. In young cultures there are long and very agile thread cells which fragment later in culture development, until finally only very short rods remain. A redefinition of the genus Flexibacter based on this shape change is proposed.

β-Carotene synthesis in isolated chromoplasts from Narcissus pseudonarcissus

A system has been established from isolated intact chromoplasts of Narcissus pseudonarcissus flowers that synthesizes geranylgeraniol, an unknown polyprenoid alcohol, phytoene, and β-carotene from [1-14C]isopentenyl pyrophosphate in a good yeild. Long chain pyrophosphates are not accumulated. San 6706 inhibits the dehydrogenation of phytoene, whereas nicotine does not lead to an accumulation of lycopene. Separation and identification of polyprenoid lipids was performed by HPLC. The properties and advantages of the chromoplast system are discussed.

Feinbau und Carotinoide von Tribonema (Xanthophyceae)

SummaryThe fine structure and the carotenoids of three species of the genus Tribonema were investigated: Tr. viride, Tr. aequale and Tr. minus. The cells of the latter two species (cultured material) contained one nucleus, whereas the cells of the former (collected near Freiburg/Black Forest) possessed numerous nuclei. This multinucleate material is supposed to represent stages in the formation of zoospores as is also indicated by the occurrence of flagella.The ultrastructure of the H-shaped walls which are characteristic for these Heterotrichales is described in detail. The general appearance of the cell interior of Tribonema resembles closely that of the related siphoneous genus Botrydium (Falk, 1967). This is true for mitochondria, centrioles, the parabasal position of the dictyosomes, the association of the forming face of the Golgi apparatus with the nuclear envelope, cisternae of the ER-system surrounding the plastids and for the electron-transparent DNA-containing areas at the ends of the plastids. With respect to certain other structural features, however, differences between these two Xanthophycean algae are apparent:a)In Tribonema, only one distinct dictyosome per nucleus can be recognized.b)The plastids lack a pyrenoid area traversed by parallel bands of thylakoids. However, some of the plastids bulge somewhat into the cytoplasm and form outpocketings, free of thylakoids. If these outpocketings do represent pyrenoids, then they belong to the “bulging type” found in some brown algae, Euglenophyceae and Haptophyceae. c) In Tribonema plastids the three thylakoids forming a band are appressed and are not separated by an interthylakoidal space. These structural differences are discussed with particular reference to their taxonomic value in assessing the position of the Xanthophyceae and other related groups of the Chromophyta.Furthermore, evidence is presented that a limited area in the space between the plastid envelope and the surrounding ER-cisterna is occupied by a network of smooth ER-tubules. This network seems to be continuous with the periplastidal ER-cisterna. Similar tubular structures are reported for other groups of the Chromophyta. This suggests that the periplastidal network is of widespread occurrence among the Chromophyta.Analysis of the carotenoid pigments of Tribonema has demonstrated that besides the carotenoids reported to be common in Xanthophyceae three triesters of a recently described new carotenoid, vaucheriaxanthin also are present. The esterified vaucheriaxanthin amounts up to 40% of the total carotenoid content. Since these triesters so far have been found only in Vaucheria, Botrydium and Tribonema they may provide additional taxonomic characterization for the Xanthophyceae.ZusammenfassungZell-Feinbau und Carotinoidausstattung wurde bei drei Arten der Xanthophyceengattung Tribonema untersucht: Tr. viride (Freilandmaterial), Tr. aequale und Tr. minus (Kulturmaterial). Während die beiden letzteren nur einen Kern je Zelle enthielten, traten bei Tr. viride bis zu sechs Kerne je Zelle auf. Wahrscheinlich handelte es sich dabei um Fäden, die im Begriff waren, Zoosporen zu bilden, was auch durch das vereinzelte Auftreten von Geißeln nahegelegt wird.Der Zellfeinbau von Tribonema ist dem von Botrydium sehr ähnlich. Einige bemerkenswerte Abweichungen hiervon (Dictyosomenzahl, Assoziationstyp der Thylakoide, Pyrenoid) werden unter dem Gesichtspunkt der Verwertbarkeit von Feinstrukturdaten für die Algensystematik ebenso diskutiert wie Übereinstimmungen mit anderen Chromophytengruppen (unter anderem periplastidäre ER-Cisterne, parabasale Lage des Golgi-Apparates, Lagebeziehung Kernhülle — Dictyosom). Bei den bereits für verschiedene Chromophytengruppen beschriebenen “tubulären Strukturen” im Raum zwischen Periplastidärcisterne und Plastidenhülle handelt es sich um ein flächiges Maschenwerk aus nicht mit Ribosomen besetzten, untereinander kommunizierenden Tubuli (“Periplastidär-Reticulum”), das mit der periplastidären Cisterne des ER in Verbindung steht.Die Analyse der Carotinoide ergab, daß bei Tribonema außer den üblichen Xanthophyceen-Carotinoiden drei Triester des erst kürzlich in Vaucheria und Botrydium gefundenen, bisher unbekannten Carotinoids Vaucheriaxanthin vorkommen. Es wird angenommen, daß Vaucheriaxanthin ein Pigment von chemotaxonomischem Wert zur Charakterisierung der Xanthophyceen ist.

On the compartmentation of isopentenyl diphosphate synthesis and utilization in plant cells

Purified spinach chloroplast and daffodil chromoplast preparations do not use mevalonate, phosphomevalonate, and diphosphomevalonate for the synthesis of isopentenyl diphosphate. Isopentenyl diphosphate, on the other hand, is incorporated into plastidal polyprenoids in large amounts. In the presence of a cytoplasmic supernatant, however, mevalonate and the phosphomevalonates were incorporated into the plastidal polyprenoids in equally large amounts, which demonstrates that the enzymes mevalonate kinase (EC 2.7.1.36), phosphomevalonate kinase (EC 2.7.4.2), and diphosphomevalonate decarboxylase (EC 4.1.1.33) are soluble cytoplasmic enzymes and that they apparently do not occur as isoenzymes within the plastids. The concept is developed that isopentenyl diphosphate is a central intermediate in plant polyprenoid formation which is channeled into several compartment for different biosynthetic pathways.