Saïd Hamdi

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.

Effect of methyl jasmonate in combination with carbohydrates on gene expression of PR proteins, stilbene and anthocyanin accumulation in grapevine cell cultures

Grapevine (Vitis vinifera L.) is subject to a number of diseases which affect yield and wine quality. After veraison, berries become strongly susceptible to pathogens due to different physiological changes including the accumulation of glucose and fructose, on the one hand, and to the decrease of anti-microbial compounds called stilbenes, on the other. To obtain berry protection, pesticides are excessively used leading to important cost to the grower and to undesirable environmental impact of the residues, especially in grape, soil and water. As a consequence, alternative strategies have to be developed. Exogenously applied biotic elicitors induce defense responses. We studied the effects of methyl jasmonate in combination with sucrose on defense-related gene expression, stilbene and anthocyanin production in grapevine cell suspensions. The methyl jasmonate/sucrose treatment was effective in stimulating phenylalanine ammonia lyase, chalcone synthase, stilbene synthase, UDP-glucose: flavonoid-O-glucosyltransferase, proteinase inhibitor and chitinase gene expression, and triggered accumulation of both piceids and anthocyanins in cells, and trans-resveratrol and piceids in the extracellular medium. Methyl jasmonate treatment might be an efficient natural strategy to protect grapevine berries in vineyard.

1-Deoxy-D-xylulose 5-phosphate synthase from periwinkle: cDNA identification and induced gene expression in terpenoid indole alkaloid-producing cells.

Abstract Terpenoid indole alkaloids (TIAs) arise from the indole and the monoterpene pathways. The latter route derives from isopentenyl diphosphate (IPP). We report on the isolation and characterization of a cDNA ( crdxs ) encoding for 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) in Catharanthus roseus suspension cultures. The enzyme catalyses the formation of the precursor of the non-mevalonate pathway leading to IPP biosynthesis. Expression in Escherichia coli of the truncated DXPS lacking the putative plastid transit peptide revealed that this pseudomature protein was active. crdxs mRNA were detected only in TIA producing-cells and the accumulation of transcripts was found to be associated with TIA production. This result corroborates recent studies obtained with a labelled precursor, which have given evidence that the non-mevalonate pathway is involved in the biosynthesis of the precursor of TIAs secologanin.

Overexpression of VvWRKY2 in tobacco enhances broad resistance to necrotrophic fungal pathogens

WRKY genes encode proteins belonging to a large family of transcription factors that are involved in various developmental and physiological processes and in plant responses to pathogen infections. In the present work, a full-length cDNA from a Vitis vinifera L. cv. Cabernet Sauvignon grape berry library was isolated and characterized. The cDNA, designated VvWRKY2, encodes a polypeptide of 536 amino acids that shows the structural features of group I of WRKY protein family. VvWRKY2 is expressed in the different organs of healthy grapevine plants. In leaves, VvWRKY2 is induced by wounding and after infection with Plasmopara viticola. Constitutive expression of VvWRKY2 in tobacco reduced the susceptibility of transgenic tobacco to three types of fungal pathogens infecting different parts of the plant: Botrytis cinerea (leaves), Pythium spp. (roots) and Alternaria tenuis (seeds). The results indicate that VvWRKY2 may be involved in the resistance of grapevine against the pathogens.

Molecular characterization of a cytokinin-inducible periwinkle protein showing sequence homology with pathogenesis-related proteins and the Bet v 1 allergen family.

Cytokinin treatment of periwinkle callus cultures increased the accumulation of a protein, designated T1, in two-dimensional separated protein extracts. The first 30 NH2-terminal amino acids were determined by Edman degradation and showed significant sequence homology with intracellular pathogenesis-related (IPR) plant proteins and the Bet v 1 allergen family. The deduced amino acid sequence of cDNAs coding for T1, isolated by RT-PCR and 5′ RACE-PCR, exhibited an average sequence identity of 40% with both IPR and Bet v 1-related allergens. T1 and all related proteins contained a p-loop motif typically found in nucleotide-binding proteins as the most conserved sequence feature. Northern blot analysis showed that cytokinin treatment of periwinkle callus induced T1 transcripts, whereas addition of 2,4-dichlorophenoxyacetic acid inhibited this accumulation. Hybridization of genomic periwinkle DNA with the T1 cDNA suggested that the protein is encoded by a single-copy gene. Immunoblot studies with a panel of Bet v 1-specific antibodies and sera from Bet v 1 allergic individuals identified T1 as a protein that is immunologically distinct from the Bet v 1 allergen family and has no allergenic properties.

Effect of cytokinin on alkaloid accumulation in periwinkle callus cultures transformed with a light-inducible ipt gene

The effect of cytokinins on accumulation of indole alkaloids in periwinkle callus cultures was investigated. Firstly, it was found that exogenously-applied cytokinin increased the ajmalicine and serpentine content of untransformed callus culture obtained from cotyledons. Secondly, periwinkle cotyledons were transformed with the isopentenyl transferase (ipt) gene under the control of a light-inducible promoter and two transformed callus lines were used in order to investigate whether endogenously-produced cytokinin could also increase the alkaloid production. We found that the ipt-transgenic tissues accumulated higher levels of isopentenyl transferase transcripts as well as zeatin riboside, even under non-inductive condition, but lower concentration of alkaloids compared to that of untransformed tissues. A 28 kDa polypeptide whose accumulation was previously found to be associated with alkaloid production in a periwinkle cell suspension was also present in the non-transformed tissue and its level was increased in parallel to the CK-enhanced alkaloid production. Neither light induction condition, nor exogenous cytokinin treatment led to the increase of the 28 kDa polypeptide accumulation in the transformed tissues. All these data show that endogenously-produced cytokinin does not mimic the effect of exogenously-applied cytokinin on the alkaloid production in periwinkle calli.

Molecular characterization of recombinant T1, a non-allergenic periwinkle (Catharanthus roseus) protein, with sequence similarity to the Bet v 1 plant allergen family.

More than 25% of the population suffer from Type I allergy, an IgE-mediated hypersensitivity disease. Allergens with homology to the major birch ( Betula verrucosa ) pollen allergen, Bet v 1, belong to the most potent elicitors of IgE-mediated allergies. T1, a cytokinin-inducible cytoplasmic periwinkle ( Catharanthus roseus ) protein, with significant sequence similarity to members of the Bet v 1 plant allergen family, was expressed in Escherichia coli. Recombinant T1 (rT1) did not react with IgE antibodies from allergic patients, and failed to induce basophil histamine release and immediate-type skin reactions in Bet v 1-allergic patients. Antibodies raised against purified rT1 could be used for in situ localization of natural T1 by immunogold electron microscopy, but did not cross-react with most of the Bet v 1-related allergens. CD analysis showed significant differences regarding secondary structure and thermal denaturation behaviour between rT1 and recombinant Bet v 1, suggesting that these structural differences are responsible for the different allergenicity of the proteins. T1 represents a non-allergenic member of the Bet v 1 family that may be used to study structural requirements of allergenicity and to engineer hypo-allergenic plants by replacing Bet v 1-related allergens for primary prevention of allergy.

CYTOKININ AND AUXIN-INDUCED REGULATION OF PROTEIN SYNTHESIS AND POLY(A)+ RNA ACCUMULATION IN CATHARANTHUS ROSEUS CELL CULTURES

Summary 2,4-dichlorophenoxyacetic acid (2,4-D)-dependent C. roseus cells were groven for three days in a 2,4-D-free medium, then treated with 4.5 μM 2,4-D, 5 μM zeatin or 4.5 μM 2,4-D + 5 μM zeatin. Hormonetreated cells were labelled in vivo with [ 35 S]-methionine and polypeptides were analyzed by two-dimensional polyacrylamide gel electrophoresis. Translation products of poly (A) + RNAs isolated from cells treated with hormones were also taken as indicators of differential gene expression. Hormone treatments did not achieve dramatic changes in polypeptide patterns and changes in in vitro polypeptide synthesis were fewer than those encountered in vivo . However, accumulation of specific RNAs coding for 18 and 28 kDa polypeptides was demonstrated under conditions of alkaloid production in the cells (i.e., when cells veere grown in 2,4-D-free, zeatin-containing medium). Their molecular masses and isoelectric points are identical to those of two polypeptides whose in vivo synthesis are similarly regulated by 2,4-D and for zeatin. These polypeptides are candidates for a direct or indirect regulatory role in alkaloid synthesis in C. roseus cells, and particularly the polypeptide of 28 kDa whose in vivo and in vitro synthesis are repressed by 2,4D, yet enhanced by zeatin. Another polypeptide of 16 kDa might be derived from the 18 kDa polypeptide in a post-translational process.

The relationship between the accumulation of a 28 kd polypeptide and that of indole alkaloids in Catharanthus roseus cell suspension cultures

Summary We previously found that the accumulation of the indole alkaloid ajmalicine and that of a polypeptide of 28kD were induced by 2,4-dichlorophenoxyacetic acid removal in periwinkle ( Catharanthus roseus ) cell cultures and further increased by zeatin addition to the culture medium. In this work, we compared the effect of various plant growth regulators and stresses on the accumulation patterns of both compounds. In a first series of experiments, the periwinkle cells were subcultured in auxin-free medium for five passages and their contents in ajmalicine and 28 kD polypeptide analysed. The maximal biomass progressively decreased along subcultures; the 28 kD polypeptide and ajmalicine accumulated concomitantly in the cells (treated with or without zeatin) up to the third passage but fully disappeared afterwards. In a second series of experiments, treatment with gibberellic acid deeply inhibited, whereas NaCl treatment (given together with or without zeatin) increased the accumulation of both ajmalicine and the 28 kD polypeptide. Moreover, growing the cells in hypoxia for 2 days inhibited the accumulation of both compounds. These concomitant changes in the accumulation pattern of ajmalicine and the 28 kD polypeptide suggest that the latter can play a (direct or indirect) regulatory role in the indole alkaloid pathway of periwinkle cells.

Changes in the accumulation of cytosolic cyclophilin transcripts in cultured periwinkle cells following hormonal and stress treatments

Summary Cyelophilins (CyPs) are a family of highly conserved proteins that catalyze cis-trans isomerization of peptidylprolyl bonds and also act as chaperones. In spite of the biological importance of these enzymes, the regulation of the CyP genes remains poorly known. We found in periwinkle cell cultures that zeatin, abscisic acid, NaCl, LaCl 3 , high sucrose concentration and cold stress caused an increase in the accumulation of transcripts from a cytosolic CyP gene. By contrast, the expression of the CyP gene was neither heat shock-nor gibberellic acid-responsive, whereas hypoxia or presence of phosphates in the medium led to a rapid and severe decrease in the CyP messages. We also examined the pattern of expression of the CyP gene in young periwinkle plants. CyP transcripts accumulated in all organs studied, but in lower amounts in flower buds than in leaves and roots. From our data, we conclud that: (i) CyP expression is often but not always associated with stress responses; (ii) CyP transcript accumulation is independent of cell mitotic activity; and (iii) there is no correlation between the alkaloid biosynthesis and the content of CyP messages in the periwinkle cells.