1,721,088 research outputs found
Optimization of aroma production in grape cell suspension induced by chemical elicitors
No full textIn many functional genomic studies, cell suspension cultures have been very useful experimental tools. Experiments with grape cell suspensions are quick and convenient compared to studies on whole vines and berries, they make it easy to control the environment conditions and to manipulate with accuracy the experiment conditions, and it is possible to carry out experiments all year. Cell suspensions of ‘Cabernet Sauvignon’ have been set up in Gamborgs B5 liquid medium with minimal organics supplemented with 30 gL-1 sucrose, 0.25 gL-1 casein hydrolysate, 0.93 M kinetin and 0.54 M naphthaleneacetic acid by callus cultures established from young berries on the same medium solidified with and 0.8% (w/v) of agar. The cultures were incubated in darkness on an orbital shaker at 100 rpm. The cell suspensions were sub-cultured every 2 weeks using an initial packed cell volume (PCV) of 15%. In a set of preliminary experiments the effect of some elicitors (abscisic acid; epibrassinolide; ethephon; mannitol; jasmonic acid, methyl-jasmonate; polyethylene glycol; salicylic acid; sorbitol; sucrose) on induction of secondary metabolites in cell suspensions were tested. Among those methyl-jasmonate (MeJA) induced the production of at least 25 compounds with sesquiterpene-like mass spectra. Sesquiterpene production was assayed in the cell cultures at 0, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120 hours after the addition of 500 μM MeJA. To study the interaction between MeJA concentration and PCV at the moment of elicitor addition, the production of volatile compounds was assayed in cell suspensions induced with 500 μM of MeJA added at 0, 1, 2, 3, 4, 5 and 6 days after cell suspension subcultures were set up with an initial PCV of 28%. In a subsequent experiment, different concentrations of MeJA (0, 50, 100, 250, 500, 750, 1000, 1500, 2000, 3000 μM) were tested for their effect on volatile production when added to a cell suspension with a PCV of 35%. The production of sequiterpenes began 12 hours after the addition of MeJA, and reached a maximum 72 hours after MeJA addition. The production of sesquiterpenes quickly decreased with an increase of cell suspension PCV at the moment of MeJA addition, suggesting a strong interaction between PCV and MeJA concentration. The most effective concentration of MeJA in stimulating the production of sesquiterpenes, was found to be 500 μM if added when the cell suspensions had a PCV of 35%, and 1000 if added when the cell suspensions had a PCV of 70%. So the optimal concentration of MeJA for induction of sesquiterpenes appeared to be 1.43 μmol per ml of PCV. These results suggest that in the interaction elicitor/density of cell in suspension the real important parameter is the concentration of elicitor per PCV unit and not the concentration of elicitor per total cell suspension volume.
The viability of cells drastically decreased after the addition of MeJA and this decrease depended of concentration of MeJA added. MeJA also induced a significantly greater amount of proanthocyanidins and stilbenes in the cell cultures. The analysis of gene expressions by real-time RT-PCR and microarray analysis revealed that the mechanism by which jasmonates induced the production of secondary metabolites in cultured grape cells varied depending on the pathway [1]
Secondary metabolite biosynthesis in elicited grape cell suspensions
No full textThe aim of this study was to discover genes involved in grape secondary metabolism pathways by stimulating their production in grape cell suspension cultures.
Among numerous elicitors tested, methyl jasmonate (MeJA) induced the production of a wide range of sesquiterpenes in Cabernet Sauvignon cell suspension cultures derived from berries. The efficacy of MeJA in the induction of sesquiterpenes production is strictly dependent upon the density of cells in cell suspension at the moment of elicitor addition. We also found that jasmonic acid (JA) as MeJA activate sesquiterpene synthesis, but when salicylic acid (SA) was added in addition to MeJA, sesquiterpenes were not produced.
Microarray analyses on the induced cultures confirmed the activation of sesquiterpenes biosynthetic pathway by MeJA and JA and the inhibition effect of SA: two terpene synthases on the gene chip were upregulated in the MeJA and JA treated cells.
Microarray data also indicated that genes from tannins and stilbenes pathways are differentially expressed in response to the treatments. Tannins accumulated to higher levels in the MeJA- and JA-treated cell suspension cultures and the expressions of pathway key genes was more than 2-fold higher in the MeJA and JA treatments than the control and MeJA+SA cells. Stilbenes accumulated in all three treatments, but the levels correlated mostly with the expression of stilbene synthase (STS2).
The results of this work confirm that an inducible cell culture system is a powerful tool for functional genomics studies as we can associate specific genes from biosynthesis pathways with changes in metabolite production. We are now working towards the functional characterisation of terpene synthases, modifying enzymes and transport proteins associated with volatile production in grapes. We have also identified several transcription factors that have expression patterns matching the induction patterns seen for the three pathways described above. These are also targets of future work as they may help us understand the regulation of these secondary metabolite pathways in grape berries
Analysis of grape cell suspensions reveals changes in secondary metabolism in response to various elicitors
No full textSecondary metabolism is irnportant tbr the procluction of many grape-derived compounds that contribute to the quality of r,vine. The airn of this study was to discover genes involved in grape secondary rnetabolism pathways by stirnulating their production in grape cell suspension cultures.
Among numerous elicitors tested, methyl jasmonate (MeJA) induced the production of a wide range of sesquitelpenes in Cabernet Sauvignon cell suspension cultures derived from berries. To enable significant microarray comparisons, the interactions of other elicitors alone and in combination with
with MeJA were explored. We found that MeJA and jasmonic acid (JA) activate sesquitelpene synthesis, but when salicylic acid (SA) was added in addition to MeJA, sesquiterpenes were not produced. Microaray analyses on the induced cultures confirmed the results of the volatile analyses, and also indicated that genes from other secondary metabolite pathways are differentially expressed in response to the treatments. Our fìndings suggest that such inducible cell culture systems are powerful tools for discovering genes associated with secondary metabolite production
Flavour Biosynthesis Pathways in Grape Cell Cultures: Sesquiterpenes Biosynthesis
No full textThe aim of this study was to discover genes involved in grape flavour pathways by elicitation of grape cell suspension. Among numerous elicitors tested, methyl jasmonate (MeJA) and jasmonic acid (JA) induce a wide range of sesquiterpenes in ‘Cabernet Sauvignon’ and Riesling berry cell suspension cultures. Sesquiterpenes, the largest subclass of natural terpenes compounds, may be synthesised by two different pathways: the mevalonate pathways (MVA) localised in the cytosol and the methylerytritol phoshate pathway (DOXP/MEP) localised in the plastids. Using specific inhibitors we found that in our cell suspension MeJA induced sequiterpenes are synthesised by the MVA pathway. A set of experiments performed to optimize sesquiterpenes production, indicated that the efficacy of MeJA concentration is strictly dependent upon the packed cell volume (PCV) of cell suspension at the moment of elicitor addition. In time course studies it was found that the peak in sesquiterpene accumulation was recorded 72 hours after MeJA addition. For the time-course experiment, we analysed the expression of 10 terpene synthase homologues found in the grape EST database by real time PCR. The expression of at least 4 of these putative sesquiterpenes synthase genes are induced by MeJA, with maximum expression at 24 hours, therefore 48 hours before the peak of sesquiterpene accumulation
L’analisi dell'espressione genica per mezzo dei microarrays in colture cellulari di vite (Vitis vinifera) trattate con elicitori
No full textAnalisi del contenuto dei metaboliti secondari che determinano la qualità delle uve e l’analisi dei microarrays sono state condotte su sospensioni cellulari di acino di “Cabernet Sauvignon” trattate con metil-jasmonato, acido jasmonico, acido salicilico e la combinazione metil-jasmonato+acido salicilico. Il metil-jasmonato e l’acido jasmonico hanno indotto la produzione di sesquiterpeni, stilbeni e tannini, e l’analisi dei microarrays ha evidenziato l’espressione dei geni delle relative vie biosintetiche. L’acido salicilico non ha mostrato le suddette attività e inoltre ha inibito l’azione del metil-jasmonato. Le analisi con la PCR real-time hanno confermato l’espressione di alcuni geni rilevata con l’analisi dei microarrays
Molecular characterization of aroma genes in Vitis vinifera var. ‘Moscato bianco’
No full textMOLECULAR CHARACTERIZATION OF AROMA GENES IN VITIS VINIFERA var. MOSCATO BIANCO
D’Onofrio C.*, De Lorenzis G.*, Boss P.K. **
*) Department of Fruit Science and Plant Protection of Woody Species ’G. Scaramuzzi‘, Fruit Science Section, University of Pisa, Via del Borghetto 80, I-56124 Pisa, Italy
**) CSIRO Plant Industry, PO Box 350 Glen Osmond SA 5064 Australia
Corresponding author: D’Onofrio C. ([email protected])
Running Head
Muscat Grape Aroma Genes Characterization
Key words
gene expression, grape flavour, monoterpenes, muscat, terpenoids
Background and Aims
Grape-derived flavour compounds and some flavour precursors modified during fermentation, wine evolution and ageing, are fundamental in determining the organoleptic parameters used to define wine quality as the aromatic quality, persistency and complexity are wine characteristics that may influence consumer choice. Although hundreds of secondary metabolites that potentially contribute to wine flavour have been detected in grapes, the knowledge of the biosynthesis of many of these compounds is not well understood and only a few genes involved in flavour pathways have been discovered and characterised. The flavour and aroma of certain Vitis vinifera grape varieties is dominated by volatile terpenes and small volatile aldehydes. Typical monoterpenol components of Muscat cultivars, a group of Vitis vinifera aromatic varieties, are linalool, geraniol, nerol, citronellol, and alpha-terpineol. The aim of this research is the discovery and characterization of genes involved in terpenoid biosynthesis of “Moscato Bianco”, one of the most important Italian Muscat varieties.
Methods and Results
Samples of flowers and berry at different developmental stages, from fruit set to technological ripening, have been analysed for their aroma content and the expression of both previously characterised genes and some that are predicted to be involved in grape flavour pathways. The aroma compounds have been extracted by SPE and SPME procedures and analysed by GS-MS, while the expression of characterised grape aroma genes and some candidate genes have been analysed by real-time RT-PCR.
Among the genes we analysed, some showed a peak of expression at berry set, while others are expressed at veraison or during the berry ripening. In particular, some candidate genes showed a peak of expression in flowers (3 putative geranyl or geranylgeranyl diphosphate synthase, alpha-terpineol synthase, valencene and germacrene D synthase, and 3 other additional putative terpene synthase genes), another during fruit set (HMG-CoA reductase), one at veraison (carotenoid cleavage dioxygenase 1) and others during ripening (1 putative geranyl or geranylgeranyl diphosphate synthase, 3 putative terpene synthases).
Conclusions
The analysis of correlations between aroma accumulation and gene expression has allowed the identification of candidate genes potentially involved in grape aroma compound biosynthesis and these will be functionally characterised.
Significance of Study
The discover and characterization of genes that encode the enzymes from grapevine flavour pathways and analysis of their activity during berry development would support decisions on management of genotype, environment and viticultural practices for improving grape flavour and aroma potential
Molecular characterization of terpenoid genes in Vitis vinifera var. Muscat
No full textThe flavour and aroma of certain Vitis vinifera grape varieties is dominated by volatile terpenes and small volatile aldehydes. Monoterpenes contribute to the final grape and wine aroma and flavour in form of free volatiles and as glycoside conjugates of monoterpene alcohols. Typical monoterpenol components of the cultivar Muscat and other aroma-rich grape varieties are linalool, geraniol, nerol, citronellol, and alpha-terpineol. These compounds are widely recognized by humans as important fragrance, flavour and aroma compounds and can present desirable quality traits for plant breeding in viticulture.
The aim of this project is the molecular characterization of genes of terpenoid formation in Muscat, an aromatic Vitis vinifera varieties. In this work, we describe the expression of different monoterpene and sesquiterpene synthases, and other genes involved flavour biosynthesis pathway in flower buds, open flowers, and during berry ripening and we compare transcriptional profiles with aroma profiles obtained by solid phase extraction analysis.
The experiments reveal that monoterpenes synthases are almost synthesized during berry setting and the sesquiterpenes synthases are almost synthesized in flower buds, open flowers, during berry setting and in harvest stage. The aroma profiles show almost terpenoids production in flower buds and in harvest stage
Induction of secondary metabolism in grape cell cultures by jasmonates
No full textThe use of a genetic approach to study the biosynthetic pathways leading to the production of secondary metabolites in grapes is difficult given the long generation times and difficulty in transforming this species. In the present study, GC/MS and microarray experiments were used to identify compounds produced in grape cell cultures in response to jasmonates and to examine the expression of genes from pathways that produce these secondary metabolites. Methyl jasmonate (MeJA) and jasmonic acid (JA) treatments resulted in the production of at least 25 compounds with sesquiterpenelike mass spectra in the cell cultures.Asignificantly greater amount of proanthocyanidins was produced in the MeJA-treated
cell cultures compared with controls and stilbene biosynthesis was induced in both MeJA- and JA-treated cells. Salicylic acid (SA) suppressed the MeJA-associated increase in sesquiterpenes and proanthocyanidins, butSAdid not suppress the stilbene production induced by MeJA treatment. The mechanism by which jasmonates induced secondary metabolite production in cultured grape cells varied depending on the pathway. The increased production of proanthocyanidins and stilbenes was associated with the induction of all of the genes in associated biosynthesis pathways, including those involved in the production of phenylalanine, whereas increased sesquiterpene synthesis was linked to the induction of certain genes from relevant biosynthesis pathways
Functional characterization of terpene synthases of 'aromatic' and 'non-aromatic' grapevine cultivars
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