Isabelle Fock

Shoot differentiation from protocorm callus cultures of Vanilla planifolia (Orchidaceae): proteomic and metabolic responses at early stage

BackgroundVanilla planifolia is an important Orchid commercially cultivated for the production of natural vanilla flavour. Vanilla plants are conventionally propagated by stem cuttings and thus causing injury to the mother plants. Regeneration and in vitro mass multiplication are proposed as an alternative to minimize damage to mother plants. Because mass production of V. planifolia through indirect shoot differentiation from callus culture is rare and may be a successful use of in vitro techniques for producing somaclonal variants, we have established a novel protocol for the regeneration of vanilla plants and investigated the initial biochemical and molecular mechanisms that trigger shoot organogenesis from embryogenic/organogenic callus.ResultsFor embryogenic callus induction, seeds obtained from 7-month-old green pods of V. planifolia were inoculated on MS basal medium (BM) containing TDZ (0.5 mg l-1). Germination of unorganized mass callus such as protocorm -like structure (PLS) arising from each seed has been observed. The primary embryogenic calli have been formed after transferring on BM containing IAA (0.5 mg l-1) and TDZ (0.5 mg l-1). These calli were maintained by subculturing on BM containing IAA (0.5 mg l-1) and TDZ (0.3 mg l-1) during 6 months and formed embryogenic/organogenic calli. Histological analysis showed that shoot organogenesis was induced between 15 and 20 days after embryogenic/organogenic calli were transferred onto MS basal medium with NAA (0.5 mg l-1). By associating proteomics and metabolomics analyses, the biochemical and molecular markers responsible for shoot induction have been studied in 15-day-old calli at the stage where no differentiating part was visible on organogenic calli. Two-dimensional electrophoresis followed by matrix-assisted laser desorption ionization time-of-flight-tandem mass spectrometry (MALDI-TOF-TOF-MS) analysis revealed that 15 protein spots are significantly expressed (P < 0.05) at earlier stages of shoot differentiation. The majority of these proteins are involved in amino acid-protein metabolism and photosynthetic activity. In accordance with proteomic analysis, metabolic profiling using 1D and 2D NMR techniques showed the importance of numerous compounds related with sugar mobilization and nitrogen metabolism. NMR analysis techniques also allowed the identification of some secondary metabolites such as phenolic compounds whose accumulation was enhanced during shoot differentiation.ConclusionThe subculture of embryogenic/organogenic calli onto shoot differentiation medium triggers the stimulation of cell metabolism principally at three levels namely (i) initiation of photosynthesis, glycolysis and phenolic compounds synthesis; (ii) amino acid - protein synthesis, and protein stabilization; (iii) sugar degradation. These biochemical mechanisms associated with the initiation of shoot formation during protocorm - like body (PLB) organogenesis could be coordinated by the removal of TDZ in callus maintenance medium. These results might contribute to elucidate the complex mechanism that leads to vanilla callus differentiation and subsequent shoot formation into PLB organogenesis. Moreover, our results highlight an early intermediate metabolic event in vanillin biosynthetic pathway with respect to secondary metabolism. Indeed, for the first time in vanilla tissue culture, phenolic compounds such as glucoside A and glucoside B were identified. The degradation of these compounds in specialized tissue (i.e. young green beans) probably contributes to the biosynthesis of glucovanillin, the parent compound of vanillin.

Metabolic changes in different developmental stages of Vanilla planifolia pods.

The metabolomic analysis of developing Vanilla planifolia green pods (between 3 and 8 months after pollination) was carried out by nuclear magnetic resonance (NMR) spectroscopy and multivariate data analysis. Multivariate data analysis of the (1)H NMR spectra, such as principal component analysis (PCA) and partial least-squares-discriminant analysis (PLS-DA), showed a trend of separation of those samples based on the metabolites present in the methanol/water (1:1) extract. Older pods had a higher content of glucovanillin, vanillin, p-hydroxybenzaldehyde glucoside, p-hydroxybenzaldehyde, and sucrose, while younger pods had more bis[4-(beta-D-glucopyranosyloxy)-benzyl]-2-isopropyltartrate (glucoside A), bis[4-(beta-D-glucopyranosyloxy)-benzyl]-2-(2-butyl)tartrate (glucoside B), glucose, malic acid, and homocitric acid. A liquid chromatography-mass spectrometry (LC-MS) analysis targeted at phenolic compound content was also performed on the developing pods and confirmed the NMR results. Ratios of aglycones/glucosides were estimated and thus allowed for detection of more minor metabolites in the green vanilla pods. Quantification of compounds based on both LC-MS and NMR analyses showed that free vanillin can reach 24% of the total vanillin content after 8 months of development in the vanilla green pods.

Biological variation of Vanilla planifolia leaf metabolome.

The metabolomic analysis of Vanilla planifolia leaves collected at different developmental stages was carried out using (1)H-nuclear magnetic resonance (NMR) spectroscopy and multivariate data analysis in order to evaluate their variation. Ontogenic changes of the metabolome were considered since leaves of different ages were collected at two different times of the day and in two different seasons. Principal component analysis (PCA) and partial least square modeling discriminate analysis (PLS-DA) of (1)H NMR data provided a clear separation according to leaf age, time of the day and season of collection. Young leaves were found to have higher levels of glucose, bis[4-(beta-D-glucopyranosyloxy)-benzyl]-2-isopropyltartrate (glucoside A) and bis[4-(beta-D-glucopyranosyloxy)-benzyl]-2-(2-butyl)-tartrate (glucoside B), whereas older leaves had more sucrose, acetic acid, homocitric acid and malic acid. Results obtained from PLS-DA analysis showed that leaves collected in March 2008 had higher levels of glucosides A and B as compared to those collected in August 2007. However, the relative standard deviation (RSD) exhibited by the individual values of glucosides A and B showed that those compounds vary more according to their developmental stage (50%) than to the time of day or the season in which they were collected (19%). Although morphological variations of the V. planifolia accessions were observed, no clear separation of the accessions was determined from the analysis of the NMR spectra. The results obtained in this study, show that this method based on the use of (1)H NMR spectroscopy in combination with multivariate analysis has a great potential for further applications in the study of vanilla leaf metabolome.

Somatic hybrids between Solanum melongena and S. sisymbrifolium, as a useful source of resistance against bacterial and fungal wilts

Abstract By using electrofusion of mesophyll protoplasts, 4 clones of somatic hybrids were produced between Solanum melongena cv. Pusa purple cluster and Solanum sisymbrifolium, a sexually incompatible wild relative resistant to bacterial ( Ralstonia solanacearum ) and fungal ( Verticillium dahliae ) wilts. All hybrids were morphologically homogeneous, normal and intermediate between the parents. They set flowers and produced fruits with empty seeds. Their hybrid status was confirmed by examining isoenzymes and random amplified polymorphism DNA analysis. Chloroplast-specific-cleaved amplified polymorphism sequence markers provided evidence that the 4 somatic hybrids possessed chloroplast genome from S. sisymbrifolium . Flow cytometry and chromosome countings revealed that all selected hybrids were tetraploids (2n=4x=48 chromosomes). The parental origin of the hybrid chromosomes was determined by using the techniques of genomic in situ hybridisation. Each hybrid contained one complete set of chromosomes from both parents. Neither spatial separation and recombinant chromosomes nor translocation could be demonstrated during mitotic metaphase. In vitro tests for resistance, using suspensions of two strains of R. solanacearum (race 1 and race 3) and the filtrate of culture medium of one strain of V. dahliae revealed that S. melongena was highly susceptible, whereas S. sisymbrifolium had variable levels of resistance. All somatic hybrids tested showed good levels of resistance, intermediate between those of the parents.

GISH confirmation of somatic hybrids between Solanum melongena and S. torvum: assessment of resistance to both fungal and bacterial wilts

Interspecific somatic hybrids between Solanum melongena L. (2n = 2x = 24) and two accessions of Solanum torvum Sw. (2n = 2x = 24) were produced in view of transferring resistance to two soil-born pathogens, Ralstonia solanacearum and Verticillium dahliae, from the wild species into the cultivated eggplant. All somatic hybrids were phenotypically homogenous and intermediate between the parents. Their hybrid nature was confirmed by analysis of isozymes and RAPDs. They showed reduced pollen viability, and all but one possessed the chloroplasts from either one or the other parent. As S. melongena and S. torvum chromosomes were morphologically indistinguishable, genomic in situ hybridisation (GISH) was applied to recognise the chromosomes from each parent in the hybrids. As expected, the selected tetraploid plants contained one complete set of chromosomes from each fusion partner. On spread preparations, the two parental genomes were not spatially separated at any time of the cell cycle. Translocation or recombinant chromosomes could not be demonstrated in the mitotic metaphase. Tests for resistance performed in vitro by using suspensions of two strains of R. solanacearum (race 1 and 3) and filtrate of culture medium of one strain of V. dahliae, revealed that S. melongena was susceptible, whereas both accessions of S. torvum had high levels of resistance. Except for two hybrid clones, which were found susceptible to race 3, as was S. melongena, all somatic hybrids tested showed good levels of bacterial and fungal resistance, either intermediate or as high as that of the wild parent.

Ralstonia solanacearum induces soluble amine-oxidase activity in Solanum torvum stem calli.

Solanum torvum is reported to carry resistance to bacterial wilt caused by Ralstonia solanacearum. So, this wild species is used as rootskock for eggplants or tomatoes in naturally infected soil. This study aimed to investigate the involvement of the polyamine metabolism pathway in the resistance mechanisms of this species. Calli induced from Solanum torvum stem explants were inoculated with the bacteria under partial vacuum. All calli showed a hypersensitive response after infiltration. Furthermore, amine oxidase activity with aldehyde and H(2)O(2) production was detected in soluble protein extracts of calli infiltrated by the bacteria. Due to its preferential affinity for aliphatic amines, this enzyme was supposed to have amine oxidase-like (AO-like) activity. Moreover, the length of aliphatic chain cycle altered the oxidative deamination kinetics of potential substrates. The AO-like catalytic activity was significantly inhibited by chelator agents such as ethylene-diamine-tretraacetic (EDTA), and also by semi-carbazide as aminoguanidine. These results suggested that (i) the prosthetic group of the AO-like enzyme could be a tyrosine-derived 6-hydroxytopaquinone structure, copper containing; (ii) this enzyme could be a semi-carbazide sensitive amine oxidase (SSAO).

Evidence of parietal amine oxidase activity in Solanum torvum Sw. stem calli after Ralstonia solanacearum inoculation.

Calli induced from Solanum torvum stem explants were inoculated with Ralstonia solanacearum under partial vacuum. All calli showed a hypersensitive response after infiltration. Furthermore, amine oxidase activity with aldehyde and H(2)O(2) production was detected in semi-purified cell walls of calli infiltrated by the bacteria. Due to its preferential affinity for monoamines, this enzyme is supposed to have monoamine oxidase-like (MAO-like) activity. Moreover, the presence of hydroxyl radicals in the aromatic cycle alters the oxidative deamination kinetics of potential substrates. Indeed, the oxidation of dopamine (+2, OH) was shown to be faster than that of tyramine (+1, OH), which in turn was faster than that of phenylethylamine (0, OH). The MAO-like catalytic activity was significantly inhibited by some reducing agents such as sodium bisulphite and cysteine, and also by tryptamine under anaerobiosis. This latter result suggested that the prosthetic group of the MAO-like enzyme could be a tyrosine-derived 6-hydroxytopaquinone structure. Finally, the sigmoid kinetics of the MAO-like enzyme in semi-purified cell walls did not correspond to that expected for a purified MAO, suggesting that the kinetics were affected by some factors present in cell walls.