Biosynthesis of mono- and sesquiterpenes in carrot roots and leaves (Daucus carota L.): metabolic cross talk of cytosolic mevalonate and plastidial methylerythritol phosphate pathways

The biosynthesis of the monoterpenes terpinolene and myrcene and the sesquiterpene beta-caryophyllene in roots and leaves of two carrot varieties (Daucus carota L. cultivars Bolero and Kazan) were investigated by in vivo feeding experiments with [5,5-2H2]-mevalonic acid lactone (d2-MVL) and [5,5-2H2]-1-deoxy-D-xylulose (d2-DOX). The volatiles of the tissues were extracted by stir bar sorptive extraction and analyzed using thermal desorption-multidimensional gas chromatography-mass spectrometry. The experiments demonstrate independent de novo-biosynthesis of terpenoids in carrot roots and in carrot leaves. In both plant tissues monoterpenes are biosynthesized exclusively via the 1-deoxy-D-xylulose/2-C-methyl-D-erythritol-4-phosphate (DOXP/MEP) pathway, whereas sesquiterpenes are generated by the classical mevalonic acid pathway as well as by the DOXP/MEP route. A more detailed investigation of carrot root tissues revealed that the biosynthesis of terpenes is mainly localized in the phloem. Nevertheless, in xylem a de novo-biosynthesis of terpenes was detectable as well, even in the absence of oil ducts in this tissue.     

Metabolism of geraniol in grape berry mesocarp of Vitis vinifera L. cv. Scheurebe: demonstration of stereoselective reduction, E/Z-isomerization, oxidation and glycosylation

The metabolism of deuterium labeled geraniol in grape mesocarp of Vitis vinifera L. cv. Scheurebe was studied by in vivo-feeding experiments. Stereoselective reduction to (S)-citronellol, E/Z-isomerization to nerol, oxidation to neral/geranial and glycosylation of the corresponding monoterpene alcohols could be demonstrated. Time course studies including the determination of conversion rates revealed that the activity of these secondary transformations of monoterpenes is dependent on the ripening stage and can be distinguished from the development of the primary monoterpene synthase activities by the sharp increase at the end of the ripening period. The stereoselective biosynthesis of the potent odorant cis-(2S,4R)-rose oxide from labeled geraniol in grape berry mesocarp is demonstrated as well. Since (S)-citronellol is the precursor of cis-(2S,4R)-rose oxide it can be concluded that especially the last part of the ripening period is important for the generation of this potent odorant. This finding confirms the conclusion that a higher concentration of flavor compounds could be established in the berries by leaving the fruit on the vine for extended periods.     

Localization of stilbene synthase in Vitis vinifera L. during berry development

The localization of stilbene synthase (STS) (EC 2.3.1.95) in grape berry (Vitis vinifera L.) was investigated during fruit development. The berries were collected at 2, 4, 7, 11, and 15 weeks postflowering from the cultivar Nebbiolo during the 2005 and 2006 growing seasons. High-performance liquid chromatography analysis showed that berries accumulated cis- and trans-isomers of resveratrol mainly in the exocarp throughout fruit development. Immunodetection of STS protein was performed on berry extracts and sections with an antibody specifically developed against recombinant grape STS1. In agreement with resveratrol presence, STS was found in berry exocarp tissues during all stages of fruit development. The labeled epidermal cells were few and were randomly distributed, whereas nearly all the outer hypodermis cells were STS-positive. The STS signal decreased gradually from exocarp to mesocarp, where the protein was detected only occasionally. At the subcellular level, STS was found predominantly within vesicles (of varying size), along the plasma membrane and in the cell wall, suggesting protein secretion in the apoplast compartment. Despite the differences in fruit size and structure, the STS localization was the same before and after veraison, the relatively short developmental period during which the firm green berries begin to soften and change color. Nevertheless, the amount of protein detected in both exocarp and mesocarp decreased significantly in ripe berries, in agreement with the lower resveratrol content measured in the same tissues. The location of STS in exocarp cell wall is consistent with its role in synthesizing defense compounds and supports the hypothesis that a differential localization of phenylpropanoid biosynthetic machinery regulates the deposition of specific secondary products at different action sites within cells.     

In situ fixation of grape berries

Summary Usual immersion protocols in aldehyde solutions fail to fully preserve the fine structure of both exocarp and mesocarp cells of grape berries, especially for theveraison (onset of ripening) and post-veraison stages. In exocarp cells, fixative diffusion is hampered by the thick polysaccharide cell walls. In mesocarp cells, plasma membrane and tonoplast are disrupted before aldehyde crosslinking occurs, owing to the high osmotic pressure and cell wall texture. The fixative was therefore injected under pressure as small droplets in the outer and inner parts of the fruit, with limited changes in the steady-state organization of fruit tissues. Compared to a selective range of immersion protocols, a striking improvement in cell preservation was observed for all berry tissues, allowing new information on various compartments of grape berry cells. The preservation of organ integrity and local concentration of aldehyde molecules are the most critical parameters of improved fixation. This technique may be applicable to a large array of fleshy fruits containing mainly cells comprising a high volumetric proportion of vacuoles accumulating large amounts of organic acids and sugars and bounded by thick-walled exocarp cells.     

Non-mevalonate isoprenoid biosynthesis: enzymes, genes and inhibitors

The essential steps of the novel non-mevalonate pathway of isopentenyl diphosphate and isoprenoid biosynthesis in plants are described. The first five enzymes and genes of this 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway are known. The herbicide fosmidomycin specifically blocks the second enzyme, the DOXP reductoisomerase. The DOXP/MEP pathway is also present in several pathogenic bacteria and the malaria parasite. Hence, all herbicides and inhibitors blocking this novel isoprenoid pathway in plants are also potential drugs against malaria and diseases caused by pathogenic bacteria.     

Cellular expansion and gene expression in the developing grape (Vitis vinifera L.)

Summary. Expression profiles of genes involved in cell wall metabolism and water transport were compared with changes in grape (Vitis vinifera L.) berry growth, basic chemical composition, and the shape, size, and wall thickness of cells within tissues of the berry pericarp. Expression of cell wall-modifying and aquaporin genes in berry pericarp tissues generally followed a bimodal expression profile with high levels of expression coinciding with the two periods of rapid berry growth, stages I and III, and low levels of expression corresponding to the slow-growth period, stage II. Cellular expansion was observed throughout all tissues during stage I, and only mesocarp cellular expansion was observed during stage III. Expansion of only exocarp cells was evident during transition between stages II and III. Cell wall-modifying and aquaporin gene expression profiles followed similar trends in exocarp and mesocarp tissues throughout berry development, with the exception of the up-regulation of pectin methylesterase, pectate lyase, two aquaporin genes (AQ1 and AQ2), and two expansin genes (EXP3 and EXPL) during stage II, which was delayed in the exocarp tissue compared with mesocarp tissue. Exocarp endo-(1→3)-β-glucanase and expansin-like gene expression was concurrent with increases in epidermal and hypodermal cell wall thickness. These results indicate a potential role of the grape berry skin in modulating grape berry growth. Correspondence: P. Bowen, Pacific Agri-Food Research Centre, 4200 Highway 97, Summerland, BC V0H 1Z0, Canada     

Chlorophyta exclusively use the 1-deoxyxylulose 5-phosphate/2-C-methylerythritol 4-phosphate pathway for the biosynthesis of isoprenoids

The biosynthesis of the C5 building block of isoprenoids, isopentenyl diphosphate (IPP), proceeds in higher plants via two basically different pathways; in the cytosolic compartment sterols are formed via mevalonate (MVA), whereas in the plastids the isoprenoids are formed via the 1-deoxyxylulose 5-phosphate/2-C-methylerythritol 4-phosphate pathway (DOXP/MEP pathway). In the present investigation, we found for the Charophyceae, being close relatives to land plants, and in the original green flagellate Mesostignma virilde the same IPP biosynthesis pattern as in higher plants: sterols are formed via MVA, and the phytol-moiety of chlorophylls via the DOXP/MEP pathway. In contrast, representatives of four classes of the Chlorophyta (Chlorophyceae, Ulvophyceae, Trebouxiophyceae, Prasinophyceae) did not incorporate MVA into sterols or phytol. Instead, they incorporated [1-2H1]-1-deoxy-D-xylulose into phytol and sterols. The results indicate that the entire Chlorophyta lineage, which is well separated from the land plant/Charophyceae lineage, is devoid of the acetate/ MVA pathway and uses the DOXP/MEP pathway not only for plastidic, but also for cytosolic isoprenoid formation.     

Feeding of [5,5-2H(2)]-1-desoxy-D-xylulose and [4,4,6,6,6-2H(5)]-mevalolactone to a geosmin-producing Streptomyces sp. and Fossombronia pusilla

The biosynthesis of the trisnor sesquiterpenoid geosmin (4,8a-dimethyl-octahydro-naphthalen-4a-ol) (1) was investigated by feeding labeled [5,5-2H(2)]-1-desoxy-D-xylulose (11), [4,4,6,6,6-(2)H(5)]-mevalolactone (7) and [2,2-2H(2)]-mevalolactone (9) to Streptomyces sp. JP95 and the liverwort Fossombronia pusilla. The micro-organism produced geosmin via the 1-desoxy-D-xylulose pathway, whereas the liverwort exclusively utilized mevalolactone for terpenoid biosynthesis. Analysis of the labeling pattern in the resulting isotopomers of geosmin (1) by mass spectroscopy (EI/MS) revealed that geosmin is synthesized in both organisms by cyclization of farnesyl diphosphate to a germacradiene-type intermediate 4. Further transformations en route to geosmin (1) involve an oxidative dealkylation of an i-propyl substituent, 1,2-reduction of a resulting conjugated diene, and bicyclization of a germacatriene intermediate 13. The transformations largely resemble the biosynthesis of dehydrogeosmin (2) in cactus flowers but differ with respect to the regioselectivity of the side chain dealkylation and 1,2-reduction     

Isoprenoid biosynthesis via 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway

Higher plants, several algae, bacteria, some strains of Streptomyces and possibly malaria parasite Plasmodium falciparum contain the novel, plastidic DOXP/MEP pathway for isoprenoid biosynthesis. This pathway, alternative with respect to the classical mevalonate pathway, starts with condensation of pyruvate and glyceraldehyde-3-phosphate which yields 1-deoxy-D-xylulose 5-phosphate (DOXP); the latter product can be converted to isopentenyl diphosphate (IPP) and eventually to isoprenoids or thiamine and pyridoxal. Subsequent reactions of this pathway involve transformation of DOXP to 2-C-methyl-D-erythritol 4-phosphate (MEP) which after condensation with CTP forms 4-diphosphocytidyl-2-amethyl-D-erythritol (CDP-ME). Then CDP-ME is phosphorylated to 4-diphosphocytidyl-2-amethyl-D-erythritol 2-phosphate (CDP-ME2P) and to 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (ME-2,4cPP) which is the last known intermediate of the DOXP/MEP pathway. For- mation of IPP and dimethylallyl diphosphate (DMAPP) from ME-2,4cPP still requires clarification. This novel pathway appears to be involved in biosynthesis of carotenoids, phytol (side chain of chlorophylls), isoprene, mono-, di-, tetraterpenes and plastoquinone whereas the mevalonate pathway is responsible for formation of sterols, sesquiterpenes and triterpenes. Several isoprenoids were found to be of mixed origin suggesting that some exchange and/or cooperation exists between these two pathways of different biosynthetic origin. Contradictory results described below could indicate that these two pathways are operating under different physiological conditions of the cell and are dependent on the developmental state of plastids.     

The non-mevalonate isoprenoid biosynthesis of plants as a test system for new herbicides and drugs against pathogenic bacteria and the malaria parasite

Higher plants and several photosynthetic algae contain the plastidic 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate pathway (DOXP/MEP pathway) for isoprenoid biosynthesis. The first four enzymes and their genes are known of this novel pathway. All of the ca. 10 enzymes of this isoprenoid pathway are potential targets for new classes of herbicides. Since the DOXP/MEP pathway also occurs in several pathogenic bacteria, such as Mycobacterium tuberculosis, and in the malaria parasite Plasmodium falciparum, all inhibitors and potential herbicides of the DOXP/MEP pathway in plants are also potential drugs against pathogenic bacteria and the malaria parasite. Plants with their easily to handle DOXP/MEP-pathway are thus very suitable test-systems also for new drugs against pathogenic bacteria and the malaria parasite as no particular security measures are required. In fact, the antibiotic herbicide fosmidomycin specifically inhibited not only the DOXP reductoisomerase in plants, but also that in bacteria and in the parasite P. falciparum, and cures malaria-infected mice. This is the first successful application of a herbicide of the novel isoprenoid pathway as a possible drug against malaria.     

Tissue-specific accumulation and subcellular localization of chalcone isomerase (CHI) in grapevine

Flavonoids are widely distributed secondary metabolic products with many biological functions in plants. Further elucidation of the accumulation and localization patterns of its biosynthesis enzymes will broaden our understanding of flavonoids biosynthesis and regulation. Chalcone isomerase (CHI, EC 5.5.1.6) is an early-step enzyme in the flavonoids biosynthesis pathway. In this study, using an antibody specifically developed against grapevine CHI enzyme, we found that the accumulation of CHI protein exhibited temporal and spatial specificity. In grape berries, CHI was investigated mainly in the outer hypodermis cells of exocarp tissues, in the vascular bundles of mesocarp; and in the integument and the cells around the raphe of seeds. At the subcellular level, CHI was visualized in the cytoplasm, nucleus, and plastids (chloroplasts) of the exocarp cells, while only in the cytoplasm of mesocarp vascular bundle cells. In grapevine vegetable organs, the leaf mesophyll and phloem of leaf veins, the pith ray and primary phloem of stems, the primary phloem and endoderm of roots, and the young leaves, leaf primordium, and the growth point of leaf buds were CHI signal-positive. In these tissue cells, CHI was primarily observed in the cytoplasm, cell wall, and nucleus. The distinct localization patterns of CHI suggested the complexity of flavonoids biosynthesis in grapevine.

     

Biosynthesis of 2-methyl-3-buten-2-ol emitted from needles of Pinus ponderosa via the non-mevalonate DOXP/MEP pathway of isoprenoid formation

The volatile hemiterpene 2-methyl-3-buten-2-ol (MBO) is emitted from the needles of several pine species from the Western United States and contributes to ozone formation in the atmosphere. It is synthesised enzymatically from dimethylallyl diphosphate (DMAPP). We show here that needles of Pinus ponderosa Laws. incorporated [1-2H1]-1-deoxy-D-xylulose (d-DOX) into the emitted MBO, but not D,L-[2-13C]mevalonic acid lactone. Furthermore, MBO emission was inhibited by fosmidomycin, a specific inhibitor of the second enzyme of the mevalonate-independent pathway of isopentenyl diphosphate and DMAPP formation, i.e. the 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway. We thus prove that MBO emitted from needles of P. ponderosa is primarily formed via the DOXP/MEP pathway.     

Expression and In Situ Localization of Two Major PR Proteins of Grapevine Berries during Development and after UV-C Exposition

In grapevine Vitis vinifera L. cv Pinot noir, the Pathogenesis-Related (PR) proteins CHI4D and TL3 are among the most abundant extractable PR proteins of ripe berries and accumulate during berry ripening from véraison until full maturation. Evidence was supplied in favor of the involvement of these two protein families in plant defense mechanisms and plant development. In order to better understand CHI4D and TL3 function in grapevine, we analyzed their temporal and spatial pattern of expression during maturation and after an abiotic stress (UV-C) by in situ hybridization (ISH) and immunohistolocalization. In ripening berries, CHI4D and TL3 genes were mainly expressed in the exocarp and around vascular bundles of the mesocarp. In UV-C exposed berries, CHI4D and TL3 gene expression was strongly induced before véraison. Corresponding proteins localized in the exocarp and, to a lesser extent, around vascular bundles of the mesocarp. The spatial and temporal accumulation of the two PR proteins during berry maturation and after an abiotic stress is discussed in relation to their putative roles in plant defense.     

Biosynthesis,accumulation and emission of carotenoids, α-tocopherol,plastoquinone, and isoprene in leaves under high photosynthetic irradiance

The localization of isoprenoid lipids in chloroplasts, the accumulation of particular isoprenoids under high irradiance conditions, and channelling of photosynthetically fixed carbon into plastidic thylakoid isoprenoids, volatile isoprenoids, and cytosolic sterols are reviewed. During leaf and chloroplast development in spring plastidic isoprenoid biosynthesis provides primarily thylakoid carotenoids, the phytyl side-chain of chlorophylls and the electron carriers phylloquinone K1, alpha-tocoquinone and alpha-tocopherol, as well as the nona-prenyl side-chain of plastoquinone-9. Under high irradiance, plants develop sun leaves and high light (HL) leaves with sun-type chloroplasts that possess, besides higher photosynthetic CO2 assimilation rates, different quantitative levels of pigments and prenylquinones as compared to shade leaves and low light (LL) leaves. After completion of chloroplast thylakoid synthesis plastidic isoprenoid biosynthesis continues at high irradiance conditions, constantly accumulating alpha-tocopherol (alpha-T) and the reduced form of plastoquinone-9 (PQ-9H2) deposited in the steadily enlarging osmiophilic plastoglobuli, the lipid reservoir of the chloroplast stroma. In sun leaves of beech (Fagus) and in 3-year-old sunlit Ficus leaves the level of alpha-T and PQ-9 can exceed that of chlorophyll b. Most plants respond to HL conditions (sun leaves, leaves suddenly lit by the sun) with a 1.4-2-fold increase of xanthophyll cycle carotenoids (violaxanthin, zeaxanthin, neoxanthin), an enhanced operation of the xanthophyll cycle and an increase of beta-carotene levels. This is documented by significantly lower values for the weight ratio chlorophylls to carotenoids (range: 3.6-4.6) as compared to shade and LL leaves (range: 4.8-7.0). Many plant leaves emit under HL and high temperature conditions at high rates the volatile compounds isoprene (broadleaf trees) or methylbutenol (American ponderosa pines), both of which are formed via the plastidic 1-deoxy-D: -xylulose-phosphate/2-C-methylerythritol 5-phosphate (DOXP/MEP) pathway. Other plants by contrast, accumulate particular mono- and diterpenes. Under adequate photosynthetic conditions the chloroplastidic DOXP/MEP isoprenoid pathway essentially contributes, with its C5 isoprenoid precusors, to cytosolic sterol biosynthesis. The possible cross-talk between the two cellular isoprenoid pathways, the acetate/MVA and the DOXP/MEP pathways, that preferentially proceeds in a plastid-to-cytosol direction, is shortly discussed.     

An NMR microscopic study of grape (Vitis vinifera L.)

Summary Mature healthy grape berries and berries wound-inoculated with the fungusBotrytis cinerea were examined by¹H NMR microimaging using 2D and 3D spin echo and gradient echo procedures. These NMR images were compared with representations obtained by conventional histology, where possible using the same specimens. 3D imaging datasets from excised seeds were reconstructed by surface rendering and maximum intensity projection to allow interpretation of their internal structure. T₂-weighted spin echo images revealed the major features of the pericarp, septum and loculi of whole berries. T₁-weighted images were less discriminatory of parenchyma tissues in the fruit but revealed the endosperm in seeds as a chemically shifted feature. A non-invasive study by T₁-weighted spin echo NMR imaging of infection byB. cinerea over a 6-day period showed that the disease spread throughout the exocarp but failed to spread in the mesocarp, a result confirmed by histological examination of the same specimen. Surface rendering of 3D datasets of excised seeds revealed the two ruminations of the endosperm and the distal location of the chalaza. The position of the embryonic axis was revealed in T₂-weighted maximum intensity projections. This noninvasive study revealed the need to apply a range of imaging techniques and parameters to visualise the structural features of the different parts of the grape berry.Abbrevations BF bright field - FDA fluorescein diacetate - FI field inhomogeneity - FOV field of view - NMR nuclear magnetic resonance - RF radiofrequency - T₁ spin-lattice relaxation time - T₂ spin-spin relaxation time - TE echo time - TMS tetramethylsilane - TR repeat time