Jean-Pierre Carde

Characterization of a Grapevine R2R3-MYB Transcription Factor That Regulates the Phenylpropanoid Pathway

The ripening of grape (Vitis vinifera) berry is characterized by dramatic changes in gene expression, enzymatic activities, and metabolism that lead to the production of compounds essential for berry quality. The phenylpropanoid metabolic pathway is one of the components involved in these changes. In this study, we describe the cloning and functional characterization of VvMYB5a, a cDNA isolated from a grape L. cv Cabernet Sauvignon berry library. VvMYB5a encodes a protein belonging to a small subfamily of R2R3-MYB transcription factors. Expression studies in grapevine indicate that the VvMYB5a gene is mainly expressed during the early steps of berry development in skin, flesh, and seeds. Overexpression of VvMYB5a in tobacco (Nicotiana tabacum) affects the expression of structural genes controlling the synthesis of phenylpropanoid and impacts on the metabolism of anthocyanins, flavonols, tannins, and lignins. Overexpressing VvMYB5a induces a strong accumulation of several phenolic compounds, including keracyanin (cyanidin-3-rhamnoglucoside) and quercetin-3-rhamnoglucoside, which are the main anthocyanin and flavonol compounds in tobacco. In addition, VvMYB5a overexpression increases the biosynthesis of condensed tannins and alters lignin metabolism. These findings suggest that VvMYB5a may be involved in the control of different branches of the phenylpropanoid pathway in grapevine.

Purification of plant mitochondria by isopycnic centrifugation in density gradients of Percoll.

Abstract Mitochondria from potato tubers have been separated from contaminating organelles and membrane vesicles on self-generated Percoll gradients and in a relatively short time. The Percoll-purified mitochondria devoid of carotenoids and galactolipids showed no contamination with intact plastids, microbodies, or vacuolar enzymes. Percoll-purified mitochondria exhibited intact membranes and a dense matrix. The intactness of purified mitochondrial preparations was ascertained by the measurement of KCN-sensitive ascorbate cyt c-dependent O2 uptake. When compared with washed mitochondria, Percoll-purified mitochondria showed improved rates of substrate oxidation, respiratory control, and ADP:O ratios. The recovery of the cyt oxidase was 70–90% and on a cyt oxidase basis the rate of succinate oxidation by unpurified mitochondria was equal to that recorded for Percoll-purified mitochondria. The great flexibility of purification procedure involving silica sols was extended from mitochondria to the isolation of intact peroxisomes.

Characterization of Lipid Rafts from Medicago truncatula Root Plasma Membranes: A Proteomic Study Reveals the Presence of a Raft-Associated Redox System

Several studies have provided new insights into the role of sphingolipid/sterol-rich domains so-called lipid rafts of the plasma membrane (PM) from mammalian cells, and more recently from leaves, cell cultures, and seedlings of higher plants. Here we show that lipid raft domains, defined as Triton X-100-insoluble membranes, can also be prepared from Medicago truncatula root PMs. These domains have been extensively characterized by ultrastructural studies as well as by analysis of their content in lipids and proteins. M. truncatula lipid domains are shown to be enriched in sphingolipids and Δ7-sterols, with spinasterol as the major compound, but also in steryl glycosides and acyl-steryl glycosides. A large number of proteins (i.e. 270) have been identified. Among them, receptor kinases and proteins related to signaling, cellular trafficking, and cell wall functioning were well represented whereas those involved in transport and metabolism were poorly represented. Evidence is also given for the presence of a complete PM redox system in the lipid rafts.

Cell Expansion and Endoreduplication Show a Large Genetic Variability in Pericarp and Contribute Strongly to Tomato Fruit Growth

Postanthesis growth of tomato (Solanum lycopersicon) as of many types of fruit relies on cell division and cell expansion, so that some of the largest cells to be found in plants occur in fleshy fruit. Endoreduplication is known to occur in such materials, which suggests its involvement in cell expansion, although no data have demonstrated this hypothesis as yet. We have analyzed pattern formation, cell size, and ploidy in tomato fruit pericarp. A first set of data was collected in one cherry tomato line throughout fruit development. A second set of data was obtained from 20 tomato lines displaying a large weight range in fruit, which were compared as ovaries at anthesis and as fully grown fruit at breaker stage. A remarkable conservation of pericarp pattern, including cell layer number and cell size, is observed in all of the 20 tomato lines at anthesis, whereas large variations of growth occur afterward. A strong, positive correlation, combining development and genetic diversity, is demonstrated between mean cell size and ploidy, which holds for mean cell diameters from 10 to 350 μm (i.e. a 32,000-times volume variation) and for mean ploidy levels from 3 to 80 C. Fruit weight appears also significantly correlated with cell size and ploidy. These data provide a framework of pericarp patterning and growth. They strongly suggest the quantitative importance of polyploidy-associated cell expansion as a determinant of fruit weight in tomato.

A Ubiquitous Plant Housekeeping Gene, PAP, Encodes a Major Protein Component of Bell Pepper Chromoplasts

We have isolated a cDNA (PAP) corresponding to a single nuclear gene that encodes an approximately 30-kD major protein of bell pepper (Capsicum annuum L.) fruit chromoplasts. RNA and protein analyses revealed that, although at a low level, this gene is also expressed in every organ of the plant, the amount of the corresponding transcript and protein dramatically increasing in the latter stages of fruit development. Western-blot and immunocytochemical analyses of purified chloroplasts from leaves and fruits and of chromoplasts from red fruits showed that the encoded protein is the major component of plastoglobules and fibrils and is localized on the outer surface of these lipid structures. Analyses of PAP in plants belonging to different taxa revealed that it is expressed and highly conserved in both monocotyledonous and dicotyledonous plants. The presence of the protein in plastids not differentiating into chromoplasts indicates that PAP is expressed irrespective of the ontogeny of various plastid lines. In light of our results and since the encoded protein, identical to that previously named ChrB or fibrillin, is present in plastoglobules from several species and accumulates in the fibrils of bell pepper chromoplast, we propose to designate it as a plastid-lipid-associated protein.

PRESENCE OF LEUCOPLASTS IN SECRETORY CELLS AND OF MONOTERPENES IN THE ESSENTIAL OIL: A CORRELATIVE STUDY

ABSTRACT Analytical and structural studies were performed on 45 species of higher plants containing specialized secretory structures and/or producing essential oils or resins. Significant amounts of volatile compounds, mainly monoterpenes and sesquiterpenes, were recovered from these species. The specialized structures included glandular hairs, resin ducts, secretory cavities and idioblasts. During the ultrastructural investigation, special attention was paid to the plastidome. A number of secretory cells contain true leucoplasts, devoid of thylakoids and ribosomes. The comparison between analytical and structural data showed a very close correlation between the presence of leucoplasts in secretory cells and the significant quantities of monoterpenes (hydrocarbons or oxygenated compounds) in the volatile extract. Moreover, a morphometric estimation of leucoplast development in the cells suggested a quantitative relationship between the expansion of this plastidome and the ratio of monoterpenes in the oil....

Monoterpene hydrocarbon biosynthesis by isolated leucoplasts of Citrofortunella mitis.

A plastid vesicle preparation isolated from exocarpium of young Citrofortunella mitis (calamondin) fruits was able to synthesise monoterpene hydrocarbons when incubated with isopentenyl pyrophosphate. The electron-microscope comparison between this organelle fraction and the various plastid classes present in the peel tissues has shown the structural identity between these plastid vesicles and the leucoplasts of the epithelial cells lining the secretory pockets. The monoterpene biosynthesis required the presence of dimethylallyl pyrophosphate, Mn2+ or Mg2+ and was increased by addition of 2-mercaptoethanol. Evidence is provided that the leucoplast vesicles act as a complete system in which occur all the successive steps involved in monoterpene hydrocarbon elaboration from isopentenyl pyrophosphate.

The thylakoid membrane protein ALB3 associates with the cpSecY-translocase in Arabidopsis thaliana

The integration of light-harvesting chlorophyll proteins (LHCPs) into the thylakoid membrane requires the integral thylakoid membrane protein ALB3, a homologue of the bacterial cytoplasmic membrane protein YidC. In bacteria, YidC is associated with the SecY-translocase and facilitates the integration of Sec-dependent proteins into the plasma membrane. In addition, it is also involved in the insertion of Sec-independent proteins. In the present study we demonstrate, in Arabidopsis thaliana, that most ALB3 is a constituent of an oligomeric complex of approx. 180 kDa. In addition, we detected ALB3 in several higher-molecular-mass complexes (up to 700 kDa). Furthermore, we show that most ALB3 co-fractionates with cpSecY during gel-filtration analysis and blue native gel electrophoresis, suggesting an association of ALB3 with the cpSecY complex. A direct interaction of ALB3 with the cpSecY complex was demonstrated by co-immunoprecipitation experiments using digitonin-solubilized thylakoid membrane proteins and anti-cpSecY or anti-ALB3 antibodies. This result was further confirmed by electron microscopic co-immunolocalization of ALB3 and cpSecY. In addition, an association of ALB3 with the cpSecY complex was demonstrated directly by cross-linking experiments using the chemical cross-linker disuccinimidyl suberate.

In vivo formation of sesquiterpene hydrocarbons in the endoplasmic reticulum of pine

Abstract Memebrane fractions were isolated from an homogenate of primary leaves of maritime pine (Pinus pinaster Ait.) seedlings, labelled in vivo by [1-14C]acetate. The various membranes from the 38 000 × g pellet were isolated by discontinuous density gradient, characterized with enzymic markers and identified by electron microscopy. It is shown that two fractions containing labelled sesquiterpene hydrocarbons were enriched in endoplasmic reticulum. A fraction devoid of radioactivity located between these fractions is very probably enriched in tonoplast.

Inhibition of the sterol pathway in leek seedlings impairs phosphatidylserine and glucosylceramide synthesis but triggers an accumulation of triacylglycerols

Like most higher plants, leek seedlings (Allium porrum L.) contain a mixture of Delta(5)-sterols in which sitosterol largely predominates. As previously reported (Plant Physiol., 117 (1998) 931), these compounds, which are synthesized at the endoplasmic reticulum level, were shown to be actively transported to the plasma membrane via a membrane-mediated process, together with phosphatidylserine (PS). In the present work, leek seedlings were allowed to germinate for 7 days in the presence of fenpropimorph, a sterol biosynthesis inhibitor. Such a treatment was found to trigger an almost complete replacement of the usual sterols by 9beta,19-cyclopropylsterols (mainly cycloeucalenol and 29-norcycloartenol). Extensive lipid analyses and labeling experiments with sodium [14C]acetate were performed to examine potential changes in the content and the rate of synthesis of the other lipid molecular species. The results indicate that the inhibition of the sterol pathway was accompanied by a severe decrease in PS and glucosylceramide synthesis as well as by a redirection of fatty acids toward the storage triacylglycerol pathway. Triacyglycerols are shown to accumulate concomitantly with a significant increase in intracellular lipid droplets in both aerial parts and roots of leek seedlings. Taken together, the present data emphasize that a coordinated regulation of the biosynthetic pathways of sterols and some specific lipid molecular species could take place during plant membrane biogenesis.