Alfredo Simone Negri

Proteomic analysis of somatic embryogenesis in Vitis vinifera

Two dimensional gel electrophoresis coupled to mass spectrometry has been used to study the somatic embryogenesis in Vitis vinifera, by comparing embryogenic and non embryogenic calluses of the Thompson seedless cv. More than 1,000 spots were reproducibly resolved in colloidal Coomassie brilliant blue stained gels over a pI nonlinear range of 3–10 in the first dimension and using homogeneous 12.5% polyacrylamide gels in the second dimension. The expression pattern of 35 spots differed significantly between the two samples. These spots were processed by mass spectrometry analysis and the protein identity was assigned by using both the non-redundant protein and EST databases. Several responsive proteins, some already known to be involved in the somatic embryogenesis process while others, for the first time put into relation with this process, have been described. Moreover, they have been subdivided in functional categories, and their putative role is discussed in terms of their relevance in the somatic embryogenesis process.

Proteome changes in the skin of the grape cultivar Barbera among different stages of ripening

BackgroundGrape ripening represents the third phase of the double sigmoidal curve of berry development and is characterized by deep changes in the organoleptic characteristics. In this process, the skin plays a central role in the synthesis of many compounds of interest (e.g. anthocyanins and aroma volatiles) and represents a fundamental protective barrier against damage by physical injuries and pathogen attacks. In order to improve the knowledge on the role of this tissue during ripening, changes in the protein expression in the skin of the red cultivar Barbera at five different stages from véraison to full maturation were studied by performing a comparative 2-DE analysis.ResultsThe proteomic analysis revealed that 80 spots were differentially expressed throughout berry ripening. Applying a two-way hierarchical clustering analysis to these variations, a clear difference between the first two samplings (up to 14 days after véraison) and the following three (from 28 to 49 days after véraison) emerged, thus suggesting that the most relevant changes in protein expression occurred in the first weeks of ripening. By means of LC-ESI-MS/MS analysis, 69 proteins were characterized. Many of these variations were related to proteins involved in responses to stress (38%), glycolysis and gluconeogenesis (13%), C-compounds and carbohydrate metabolism (13%) and amino acid metabolism (10%).ConclusionThese results give new insights to the skin proteome evolution during ripening, thus underlining some interesting traits of this tissue. In this view, we observed the ripening-related induction of many enzymes involved in primary metabolism, including those of the last five steps of the glycolytic pathway, which had been described as down-regulated in previous studies performed on whole fruit. Moreover, these data emphasize the relevance of this tissue as a physical barrier exerting an important part in berry protection. In fact, the level of many proteins involved in (a)biotic stress responses remarkably changed through the five stages taken into consideration, thus suggesting that their expression may be developmentally regulated.

Evaluation of protein pattern changes in roots and leaves of Zea mays plants in response to nitrate availability by two-dimensional gel electrophoresis analysis

BackgroundNitrogen nutrition is one of the major factors that limit growth and production of crop plants. It affects many processes, such as development, architecture, flowering, senescence and photosynthesis. Although the improvement in technologies for protein study and the widening of gene sequences have made possible the study of the plant proteomes, only limited information on proteome changes occurring in response to nitrogen amount are available up to now. In this work, two-dimensional gel electrophoresis (2-DE) has been used to investigate the protein changes induced by NO3- concentration in both roots and leaves of maize (Zea mays L.) plants. Moreover, in order to better evaluate the proteomic results, some biochemical and physiological parameters were measured.ResultsThrough 2-DE analysis, 20 and 18 spots that significantly changed their amount at least two folds in response to nitrate addition to the growth medium of starved maize plants were found in roots and leaves, respectively. Most of these spots were identified by Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS/MS). In roots, many of these changes were referred to enzymes involved in nitrate assimilation and in metabolic pathways implicated in the balance of the energy and redox status of the cell, among which the pentose phosphate pathway. In leaves, most of the characterized proteins were related to regulation of photosynthesis. Moreover, the up-accumulation of lipoxygenase 10 indicated that the leaf response to a high availability of nitrate may also involve a modification in lipid metabolism.Finally, this proteomic approach suggested that the nutritional status of the plant may affect two different post-translational modifications of phosphoenolpyruvate carboxylase (PEPCase) consisting in monoubiquitination and phosphorylation in roots and leaves, respectively.ConclusionThis work provides a first characterization of the proteome changes that occur in response to nitrate availability in leaves and roots of maize plants. According to previous studies, the work confirms the relationship between nitrogen and carbon metabolisms and it rises some intriguing questions, concerning the possible role of NO and lipoxygenase 10 in roots and leaves, respectively. Although further studies will be necessary, this proteomic analysis underlines the central role of post-translational events in modulating pivotal enzymes, such as PEPCase.

Proteomic characterization of iron deficiency responses in Cucumis sativus L. roots

BackgroundIron deficiency induces in Strategy I plants physiological, biochemical and molecular modifications capable to increase iron uptake from the rhizosphere. This effort needs a reorganization of metabolic pathways to efficiently sustain activities linked to the acquisition of iron; in fact, carbohydrates and the energetic metabolism has been shown to be involved in these responses. The aim of this work was to find both a confirmation of the already expected change in the enzyme concentrations induced in cucumber root tissue in response to iron deficiency as well as to find new insights on the involvement of other pathways.ResultsThe proteome pattern of soluble cytosolic proteins extracted from roots was obtained by 2-DE. Of about two thousand spots found, only those showing at least a two-fold increase or decrease in the concentration were considered for subsequent identification by mass spectrometry. Fifty-seven proteins showed significant changes, and 44 of them were identified. Twenty-one of them were increased in quantity, whereas 23 were decreased in quantity. Most of the increased proteins belong to glycolysis and nitrogen metabolism in agreement with the biochemical evidence. On the other hand, the proteins being decreased belong to the metabolism of sucrose and complex structural carbohydrates and to structural proteins.ConclusionsThe new available techniques allow to cast new light on the mechanisms involved in the changes occurring in plants under iron deficiency. The data obtained from this proteomic study confirm the metabolic changes occurring in cucumber as a response to Fe deficiency. Two main conclusions may be drawn. The first one is the confirmation of the increase in the glycolytic flux and in the anaerobic metabolism to sustain the energetic effort the Fe-deficient plants must undertake. The second conclusion is, on one hand, the decrease in the amount of enzymes linked to the biosynthesis of complex carbohydrates of the cell wall, and, on the other hand, the increase in enzymes linked to the turnover of proteins.

Analysis of grape berry cell wall proteome: A comparative evaluation of extraction methods

Different methods were tested for the extraction of proteins from the cell wall-enriched fraction (CWEf) obtained from a sample formed by skin and seeds of ripe berries of Vitis vinifera L. cv. Cabernet Sauvignon. The CWEf was isolated using a disruptive approach that involves tissue homogenization and precipitation by centrifugation. To extract proteins, the CWEf was treated with CaCl(2) and LiCl in two successive steps or, alternatively, with phenol. The efficiency of the protocols was evaluated by measuring protein yield and by analyzing two-dimensional gel electrophoresis (2-DE) gels for the highest detectable spot number and the greatest spot resolution. The phenol method was also adopted for the extraction of proteins from the cytosolic fraction (CYf). The comparison of 2-DE reference maps of protein extracts from CWEf and CYf indicated the presence of both common traits and unique characteristics. To survey this aspect some spots detected in both fractions or present in only one fraction were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Of the 47 spots identified, some were found to be cell wall proteins, while others were proteins not traditionally considered as localized in the apoplastic space. The data presented here provide initial information regarding the apoplastic proteome of grape berry tissues, but also raise the issue of the technical problems that characterize the isolation of cell wall proteins from these very hardy tissues.

Comprehensive transcript profiling of two grapevine rootstock genotypes contrasting in drought susceptibility links the phenylpropanoid pathway to enhanced tolerance

Highlights Drought tolerance in the M4 grapevine rootstock genotype could be associated with a higher capability to counteract oxidative stresses by enhancing the accumulation of resveratrol in roots.

Proteins involved in biotic and abiotic stress responses as the most significant biomarkers in the ripening of Pinot Noir skins

We propose an integrated approach, obtained by the combination of multivariate statistics and proteomics, useful to isolate candidate biomarkers for the evaluation of grape ripening. We carried out a comparative 2-DE analysis of grape skins collected in three moments of ripening and analyzed the spot volume dataset through the application of principal component analysis followed by forward stepwise-linear discriminant analysis. This technique allowed to discriminate véraison, quite mature and mature samples, and to sort the matched spots according to their significance. We identified 36 spots showing high discriminating coefficients through liquid chromatography - electrospray ionization - tandem mass spectrometry (LC-ESI-MS/MS). Most of them were involved in biotic and abiotic stress responses indicating these enzymes as good candidate markers of berry ripening. These evidences hint at a likely developmental role of these proteins, in addition to their reported activity in stress events. Restricting the same statistical analysis to the samples belonging to the two last stages, it was indicated that this approach can clearly distinguish these close and similar phases of berry development. Taken all together, these results bear out that the employment of the combination of 2-DE and multivariate statistics is a reliable tool in the identification of new protein markers for describing the ripening phases and to assess the overall quality of the fruit.

On the role of ethylene, auxin and a GOLVEN-like peptide hormone in the regulation of peach ripening

BackgroundIn melting flesh peaches, auxin is necessary for system-2 ethylene synthesis and a cross-talk between ethylene and auxin occurs during the ripening process. To elucidate this interaction at the transition from maturation to ripening and the accompanying switch from system-1 to system-2 ethylene biosynthesis, fruits of melting flesh and stony hard genotypes, the latter unable to produce system-2 ethylene because of insufficient amount of auxin at ripening, were treated with auxin, ethylene and with 1-methylcyclopropene (1-MCP), known to block ethylene receptors. The effects of the treatments on the different genotypes were monitored by hormone quantifications and transcription profiling.ResultsIn melting flesh fruit, 1-MCP responses differed according to the ripening stage. Unexpectedly, 1-MCP induced genes also up-regulated by ripening, ethylene and auxin, as CTG134, similar to GOLVEN (GLV) peptides, and repressed genes also down-regulated by ripening, ethylene and auxin, as CTG85, a calcineurin B-like protein.The nature and transcriptional response of CTG134 led to discover a rise in free auxin in 1-MCP treated fruit. This increase was supported by the induced transcription of CTG475, an IAA-amino acid hydrolase. A melting flesh and a stony hard genotype, differing for their ability to synthetize auxin and ethylene amounts at ripening, were used to study the fine temporal regulation and auxin responsiveness of genes involved in the process. Transcriptional waves showed a tight interdependence between auxin and ethylene actions with the former possibly enhanced by the GLV CTG134. The expression of genes involved in the regulation of ripening, among which are several transcription factors, was similar in the two genotypes or could be rescued by auxin application in the stony hard. Only GLV CTG134 expression could not be rescued by exogenous auxin.Conclusions1-MCP treatment of peach fruit is ineffective in delaying ripening because it stimulates an increase in free auxin. As a consequence, a burst in ethylene production speeding up ripening occurs. Based on a network of gene transcriptional regulations, a model in which appropriate level of CTG134 peptide hormone might be necessary to allow the correct balance between auxin and ethylene for peach ripening to occur is proposed.

Proteomic characterization of the Rph15 barley resistance gene-mediated defence responses to leaf rust

BackgroundLeaf rust, caused by the biotrophic fungal pathogen Puccinia hordei, is one of the most important foliar disease of barley (Hordeum vulgare) and represents a serious threat in many production regions of the world. The leaf rust resistance gene Rph15 is of outstanding interest for resistance breeding because it confers resistance to over 350 Puccinia hordei isolates collected from around the world. Molecular and biochemical mechanisms responsible for the Rph15 effectiveness are currently not investigated. The aim of the present work was to study the Rph15-based defence responses using a proteomic approach.ResultsProtein pattern changes in response to the leaf rust pathogen infection were investigated in two barley near isogenic lines (NILs), Bowman (leaf rust susceptible) and Bowman-Rph15 (leaf rust resistant), differing for the introgression of the leaf rust resistance gene Rph15. Two infection time points, 24 hours and four days post inoculation (dpi), were analysed. No statistically significant differences were identified at the early time point, while at 4 dpi eighteen protein spots were significantly up or down regulated with a fold-change equal or higher than two in response to pathogen infection. Almost all the pathogen-responsive proteins were identified in the Bowman-Rph15 resistant NIL. Protein spots were characterized by LC-MS/MS analysis and found to be involved in photosynthesis and energy metabolism, carbohydrate metabolism, protein degradation and defence. Proteomic data were complemented by transcriptional analysis of the respective genes. The identified proteins can be related to modulation of the photosynthetic apparatus components, re-direction of the metabolism to sustain defence responses and deployment of defence proteins.ConclusionsThe identification of leaf rust infection-modulated defence responses restricted to the resistant NIL support the hypothesis that basal defence responses of Bowman, but not the Rph15 resistance gene-based ones, are suppressed or delayed by pathogen effectors to levels below the detection power of the adopted proteomic approach. Additionally, Rph15-mediated resistance processes identified mainly resides on a modulation of primary metabolism, affecting photosyntesis and carbohydrate pool.

Proteomics of red and white corolla limbs in petunia reveals a novel function of the anthocyanin regulator ANTHOCYANIN1 in determining flower longevity

The Petunia hybrida ANTHOCYANIN1 (AN1) gene encodes a transcription factor that regulates both the expression of genes involved in anthocyanin synthesis and the acidification of the vacuolar lumen in corolla epidermal cells. In this work, the comparison between the red flowers of the R27 line with the white flowers of the isogenic an1 mutant line W225 showed that the AN1 gene has further pleiotropic effects on flavonoid biosynthesis as well as on distant physiological traits. The proteomic profiling showed that the an1 mutation was associated to changes in accumulation of several proteins, affecting both anthocyanin synthesis and primary metabolism. The flavonoid composition study confirmed that the an1 mutation provoked a broad attenuation of the entire flavonoid pathway, probably by indirect biochemical events. Moreover, proteomic changes and variation of biochemical parameters revealed that the an1 mutation induced a delay in the onset of flower senescence in W225, as supported by the enhanced longevity of the W225 flowers in planta and the loss of sensitivity of cut flowers to sugar. This study suggests that AN1 is possibly involved in the perception and/or transduction of ethylene signal during flower senescence.