Root Proteomic Analysis of Two Grapevine Rootstock Genotypes Showing Different Susceptibility to Salt Stress.

Keywords: NaCl; Vitis; LC-ESI-MS/MS; carbon and energy metabolism; oxidative stress.

Root proteomic and metabolic analyses reveal specific responses to drought stress in differently tolerant grapevine rootstocks.

BACKGROUND: Roots play a central role in plant response to water stress (WS). They are involved in its perception and signalling to the leaf as well as in allowing the plant to adapt to maintaining an adequate water balance. Only a few studies have investigated the molecular/biochemical responses to WS in roots of perennial plants, such as grapevine. This study compares two grapevine rootstock genotypes (i.e. 101.14 and M4) with different tolerance to WS, evaluating the responses at proteomic and metabolite levels. RESULTS: WS induced changes in the abundance of several proteins in both genotypes (17 and 22% of the detected proteins in 101.14 and M4, respectively). The proteomic analysis revealed changes in many metabolic pathways that fitted well with the metabolite data. M4 showed metabolic responses which were potentially able to counteract the WS effects, such as the drop in cell turgor, increased oxidative stress and loss of cell structure integrity/functionality. However, in 101.14 it was evident that the roots were suffering more severely from these effects. We found that many proteins classified as active in energy metabolism, hormone metabolism, protein, secondary metabolism and stress functional classes showed particular differences between the two rootstocks. CONCLUSION: The proteomic/metabolite comparative analysis carried out provides new information on the possible biochemical and molecular strategies adopted by grapevine roots to counteract WS. Although further work is needed to define in detail the role(s) of the proteins and metabolites that characterize WS response, this study, involving the M4 rootstock genotype, highlights that osmotic responses, modulations of C metabolism, mitochondrial functionality and some specific responses to stress occurring in the roots play a primary role in Vitis spp. tolerance to this type of abiotic stress.

Maize 16-kD γ-zein forms very unusual disulfide-bonded polymers in the endoplasmic reticulum: implications for prolamin evolution.

In the lumen of the endoplasmic reticulum (ER), prolamin storage proteins of cereal seeds form very large, ordered heteropolymers termed protein bodies (PBs), which are insoluble unless treated with alcohol or reducing agents. In maize PBs, 16-kD γ-zein locates at the interface between a core of alcohol-soluble α-zeins and the outermost layer mainly composed of the reduced-soluble 27-kD γ-zein. 16-kD γ-zein originates from 27-kD γ-zein upon whole-genome duplication and is mainly characterized by deletions in the N-terminal domain that eliminate most Pro-rich repeats and part of the Cys residues involved in inter-chain bonds. 27-kD γ-zein also forms insoluble PBs when expressed in transgenic vegetative tissues. We show that in Arabidopsis leaves, 16-kD γ-zein assembles into disulfide-linked polymers that fail to efficiently become insoluble. Instead of forming PBs, these polymers accumulate as very unusual threads that markedly enlarge the ER lumen, resembling amyloid-like fibers. Domain-swapping between the two γ-zeins indicates that the N-terminal region of 16-kD γ-zein has a dominant effect in preventing full insolubilization. Therefore, a newly evolved prolamin has lost the ability to form homotypic PBs, and has acquired a new function in the assembly of natural, heteropolymeric PBs.

Time-Course of Metabolic and Proteomic Responses to Different Nitrate/Ammonium Availabilities in Roots and Leaves of Maize.

The availability of nitrate and ammonium significantly affects plant growth. Co-provision of both nutrients is generally the best nutritional condition, due to metabolic interactions not yet fully elucidated. In this study, maize grown in hydroponics was exposed to different nitrogen (N) availabilities, consisting of nitrate, ammonium and co-provision. Roots and leaves were analyzed after 6, 30, and 54 h by biochemical evaluations and proteomics. The ammonium-fed plants showed the lowest biomass accumulation and the lowest ratio of inorganic to organic N content, suggesting a metabolic need to assimilate ammonium that was not evident in plants grown in co-provision. The N sources differently affected the root proteome, inducing changes in abundance of proteins involved in N and carbon (C) metabolisms, cell water homeostasis, and cell wall metabolism. Notable among these changes was that some root enzymes, such as asparagine synthetase, phosphoenolpyruvate (PEP) carboxylase, and formate dehydrogenase showed a relevant upsurge only under the sole ammonium nutrition. However, the leaf proteome appeared mainly influenced by total N availability, showing changes in the abundance of several proteins involved in photosynthesis and in energy metabolism. Overall, the study provides novel information about the biochemical determinants involved in plant adaptation to different N mineral forms.

Mineral nitrogen sources differently affect root glutamine synthetase isoforms and amino acid balance among organs in maize.

BACKGROUND: Glutamine synthetase (GS) catalyzes the first step of nitrogen assimilation in plant cell. The main GS are classified as cytosolic GS1 and plastidial GS2, of which the functionality is variable according to the nitrogen sources, organs and developmental stages. In maize (Zea mays L.) one gene for GS2 and five genes for GS1 subunits are known, but their roles in root metabolism are not yet well defined. In this work, proteomic and biochemical approaches have been used to study root GS enzymes and nitrogen assimilation in maize plants re-supplied with nitrate, ammonium or both. RESULTS: The plant metabolic status highlighted the relevance of root system in maize nitrogen assimilation during both nitrate and ammonium nutrition. The analysis of root proteomes allowed a study to be made of the accumulation and phosphorylation of six GS proteins. Three forms of GS2 were identified, among which only the phosphorylated one showed an accumulation trend consistent with plastidial GS activity. Nitrogen availabilities enabled increments in root total GS synthetase activity, associated with different GS1 isoforms according to the nitrogen sources. Nitrate nutrition induced the specific accumulation of GS1-5 while ammonium led to up-accumulation of both GS1-1 and GS1-5, highlighting co-participation. Moreover, the changes in thermal sensitivity of root GS transferase activity suggested differential rearrangements of the native enzyme. The amino acid accumulation and composition in roots, xylem sap and leaves deeply changed in response to mineral sources. Glutamine showed the prevalent changes in all nitrogen nutritions. Besides, the ammonium nutrition was associated with an accumulation of asparagine and reducing sugars and a drop in glutamic acid level, significantly alleviated by the co-provision with nitrate. CONCLUSION: This work provides new information about the multifaceted regulation of the GS enzyme in maize roots, indicating the involvement of specific isoenzymes/isoforms, post-translational events and biochemical factors. For the first time, the proteomic approach allowed to discriminate the individual contribution of the GS1 isoforms, highlighting the participation of GS1-5 in nitrate metabolism. Moreover, the results give new insights about the influence of amino acid metabolism in plant C/N balance.

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

In light of ongoing climate changes in wine-growing regions, the selection of drought-tolerant rootstocks is becoming a crucial factor for developing a sustainable viticulture. In this study, M4, a new rootstock genotype that shows tolerance to drought, was compared from a genomic and transcriptomic point of view with the less drought-tolerant genotype 101.14. The root and leaf transcriptome of both 101.14 and the M4 rootstock genotype was analysed, following exposure to progressive drought conditions. Multifactorial analyses indicated that stress treatment represents the main factor driving differential gene expression in roots, whereas in leaves the genotype is the prominent factor. Upon stress, M4 roots and leaves showed a higher induction of resveratrol and flavonoid biosynthetic genes, respectively. The higher expression of VvSTS genes in M4, confirmed by the accumulation of higher levels of resveratrol in M4 roots compared with 101.14, was coupled to an up-regulation of several VvWRKY transcription factors. Interestingly, VvSTS promoter analyses performed on both the resequenced genomes highlighted a significantly higher number of W-BOX elements in the tolerant genotype. It is proposed that the elevated synthesis of resveratrol in M4 roots upon water stress could enhance the plant's ability to cope with the oxidative stress usually associated with water deficit.

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

BACKGROUND: Leaf 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. RESULTS: Protein 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. CONCLUSIONS: The 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.

Assembly and sorting of the tonoplast potassium channel AtTPK1 and its turnover by internalization into the vacuole.

The assembly, sorting signals, and turnover of the tonoplast potassium channel AtTPK1 of Arabidopsis (Arabidopsis thaliana) were studied. We used transgenic Arabidopsis expressing a TPK1-green fluorescent protein (GFP) fusion or protoplasts transiently transformed with chimeric constructs based on domain exchange between TPK1 and TPK4, the only TPK family member not located at the tonoplast. The results show that TPK1-GFP is a dimer and that the newly synthesized polypeptides transiently interact with a thus-far unidentified 20-kD polypeptide. A subset of the TPK1-TPK4 chimeras were unable to assemble correctly and these remained located in the endoplasmic reticulum where they interacted with the binding protein chaperone. Therefore, TPK1 must assemble correctly to pass endoplasmic reticulum quality control. Substitution of the cytosolic C terminus of TPK4 with the corresponding domain of TPK1 was sufficient to allow tonoplast delivery, indicating that this domain contains tonoplast sorting information. Pulse-chase labeling indicated that TPK1-GFP has a half-life of at least 24 h. Turnover of the fusion protein involves internalization into the vacuole where the GFP domain is released. This indicates a possible mechanism for the turnover of tonoplast proteins.

The Delay of Raphanus raphanistrum subsp. sativus (L.) Domin Seed Germination Induced by Coumarin Is Mediated by a Lower Ability to Sustain the Energetic Metabolism.

In the present study, the mode of action of coumarin using the germination process as a target was investigated. A dose-response curve, built using a range of concentrations from 0 to 800 µM, allowed us to identify a key concentration (400 µM) inhibiting the germination process, reducing its speed without compromising seed development. Successively, short time-course (0-48 h) experiments were carried out to evaluate the biochemical and metabolic processes involved in coumarin-induced germination delay. The results pointed out that coumarin delayed K+, Ca2+, and Mg2+ reabsorption, suggesting a late membrane reorganisation. Similarly, seed respiration was inhibited during the first 24 h but recovered after 48 h. Those results agreed with ATP levels, which followed the same trend. In addition, the untargeted metabolomic analysis allowed to identify, among the pathways significantly impacted by the treatment, amino acids metabolism, the TCA cycle, and the glyoxylate pathway. The results highlighted that coumarin was able to interact with membranes reorganisation, delaying them and reducing the production of ATP, as also supported by pathway analysis and cell respiration. The in vivo 31P-NMR analysis supported the hypothesis that the concentration chosen was able to affect plant metabolism, maintaining, on the other hand, its viability, which is extremely important for studying natural compounds' mode of action.

Chemical Characterization of a Collagen-Derived Protein Hydrolysate and Biostimulant Activity Assessment of Its Peptidic Components.

Protein hydrolysates (PHs) are plant biostimulants consisting of oligopeptides and free amino acids exploited in agriculture to increase crop productivity. This work aimed to fractionate a commercial collagen-derived protein hydrolysate (CDPH) according to the molecular mass of the peptides and evaluate the bioactivity of different components. First, the CDPH was dialyzed and/or filtrated and analyzed on maize, showing that smaller compounds were particularly active in stimulating lateral root growth. The CDPH was then fractionated through fast protein liquid chromatography and tested on in vitro grown tomatoes proving that all the fractions were bioactive. Furthermore, these fractions were characterized by liquid chromatography-electrospray ionization-tandem mass spectrometry revealing a consensus sequence shared among the identified peptides. Based on this sequence, a synthetic peptide was produced. We assessed its structural similarity with the CDPH, the collagen, and polyproline type II helix by comparing the respective circular dichroism spectra and for the first time, we proved that a signature peptide was as bioactive as the whole CDPH.

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.

Nitrogen Uptake in Plants: The Plasma Membrane Root Transport Systems from a Physiological and Proteomic Perspective.

Nitrogen nutrition in plants is a key determinant in crop productivity. The availability of nitrogen nutrients in the soil, both inorganic (nitrate and ammonium) and organic (urea and free amino acids), highly differs and influences plant physiology, growth, metabolism, and root morphology. Deciphering this multifaceted scenario is mandatory to improve the agricultural sustainability. In root cells, specific proteins located at the plasma membrane play key roles in the transport and sensing of nitrogen forms. This review outlines the current knowledge regarding the biochemical and physiological aspects behind the uptake of the individual nitrogen forms, their reciprocal interactions, the influences on root system architecture, and the relations with other proteins sustaining fundamental plasma membrane functionalities, such as aquaporins and H+-ATPase. This topic is explored starting from the information achieved in the model plant Arabidopsis and moving to crops in agricultural soils. Moreover, the main contributions provided by proteomics are described in order to highlight the goals and pitfalls of this approach and to get new hints for future studies.

Biochemical and Proteomic Changes in the Roots of M4 Grapevine Rootstock in Response to Nitrate Availability.

In agricultural soils, nitrate (NO3-) is the major nitrogen (N) nutrient for plants, but few studies have analyzed molecular and biochemical responses involved in its acquisition by grapevine roots. In viticulture, considering grafting, NO3- acquisition is strictly dependent on rootstock. To improve the knowledge about N nutrition in grapevine, this study analyzed biochemical and proteomic changes induced by, NO3- availability, in a hydroponic system, in the roots of M4, a recently selected grapevine rootstock. The evaluation of biochemical parameters, such as NO3-, sugar and amino acid contents in roots, and the abundance of nitrate reductase, allowed us to define the time course of the metabolic adaptations to NO3- supply. On the basis of these results, the proteomic analysis was conducted by comparing the root profiles in N-starved plants and after 30 h of NO3- resupply. The analysis quantified 461 proteins, 26% of which differed in abundance between conditions. Overall, this approach highlighted, together with an increased N assimilatory metabolism, a concomitant rise in the oxidative pentose phosphate pathway and glycolysis, needed to fulfill the redox power and carbon skeleton demands, respectively. Moreover, a wide modulation of protein and amino acid metabolisms and changes of proteins involved in root development were observed. Finally, some results open new questions about the importance of redox-related post-translational modifications and of NO3- availability in modulating the dialog between root and rhizosphere.

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

BACKGROUND: Iron 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. RESULTS: The 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. CONCLUSIONS: The 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.

Oxidations in white grape (Vitis vinifera L.) skins: Comparison between ripening process and photooxidative sunburn symptoms.

Oxidations in grape berries are gaining major interest as they affect grape characteristics and quality. Considering berries, Reactive Oxygen Species are involved in the responses to both ripening process and stresses, including photooxidative sunburn. Redox metabolism involves a multitude of chemical and enzymatic reactions. In this study, four white grape cultivars were examined for natural ripening and photooxidative sunburn effects (obtained in artificial conditions) on berry pigmentation, chemical composition and enzymatic activity. The measured parameters included reflectance spectra, pigmentation (including berry browning), content of photosynthetic pigments, organic acid profiles, antioxidant activity, concentrations of antioxidants (total phenolics, ascorbic acid and reduced glutathione), enzymatic activities (guaiacol peroxidases, ascorbate peroxidase and catalase). The effects of the treatment (natural ripening and artificial photooxidative sunburn) on each considered parameter are described in the paper. Photooxidative sunburn strongly affected the contents of antioxidants and chlorophylls, increased the browning index and modulated the enzymatic activities investigated. Samples clearly clustered depending on the oxidation status. Furthermore, the PCA highlighted the similarities and differences in the responses to oxidative stress during ripening and photooxidative sunburn. PCA produced five functions with eigenvalues higher than 1, representing 87.03% of the total variability. In particular, the scores of the function 1 discriminated the samples based on the oxidation status, while the function 2 separated the samples based on the sampling date, representing the physiological responses characteristic of ripening. Our work sheds light on this topic, and will allow a more conscious vineyard management, thus supporting the agricultural adaptation to climate changes.

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

BACKGROUND: Nitrogen 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. RESULTS: Through 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. CONCLUSION: This 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.

Insight into Composition of Bioactive Phenolic Compounds in Leaves and Flowers of Green and Purple Basil.

Basil (Ocimum basilicum L.) is a culinary, medicinal, and ornamental plant appreciated for its antioxidant properties, mainly attributed to high content of rosmarinic acid. This species also includes purple varieties, characterized by the accumulation of anthocyanins in leaves and flowers. In this work, we compared the main morphological characteristics, the antioxidant capacity and the chemical composition in leaves, flowers, and corollas of green ('Italiano Classico') and purple ('Red Rubin' and 'Dark Opal') basil varieties. The LC-ESI-MS/MS analysis of individual compounds allowed quantifying 17 (poly)phenolic acids and 18 flavonoids, differently accumulated in leaves and flowers of the three varieties. The study revealed that in addition to rosmarinic acid, basil contains several members of the salvianolic acid family, only scarcely descripted in this species, as well as, especially in flowers, simple phenolic acids, such as 4-hydroxybenzoic acid and salvianic acid A. Moreover, the study revealed that purple leaves mainly contain highly acylated anthocyanins, while purple flowers accumulate anthocyanins with low degree of decoration. Overall, this study provides new biochemical information about the presence of not yet characterized bioactive compounds in basil that could contribute to boosting the use of this crop and to gaining new knowledge about the roles of these compounds in plant physiology.

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

BACKGROUND: Grape 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. RESULTS: The 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%). CONCLUSION: These 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.

Combined 2D electrophoretic approaches for the study of white lupin mature seed storage proteome.

Seed proteome analysis by 2D IEF/SDS-PAGE techniques is challenging for the intrinsic difficulties related to quantitative disparity of the seed proteins, i.e. storage and non-storage proteins, their polymorphic nature, the extensive post-translational modifications and the paucity of deposited primary structures available. Conversely, 2D maps of seed proteomes can be extremely useful for a number of fundamental and applied investigations. In this work, we have used a combination of two experimental approaches to identify the main protein components of an emerging protein-rich legume seed, that is white lupin seed (Lupinus albus, L.). One is the canonical proteomic approach including 2D electrophoretic separation and mass spectrometry of selected trypsin-digested polypeptides; the other approach is a group comparative 2D electrophoretic analysis of cotyledonary protein families. To this second purpose, the three main families of lupin seed proteins, namely alpha-conglutins, the 11S globulin fraction, beta-conglutins, the 7S globulin fraction, and gamma-conglutin, a basic 7S protein, were isolated by conventional biochemical techniques and their 2D reference maps were compared with the total protein map. With the first approach 37 out of 40 spots, making up about 35% of total spot volumes in the 2D map, were found to belong to the main seed protein families. Thanks to cDNA-deduced lupin storage protein sequences, determined on purpose and deposited, most of the identification statistical parameters were very good. Moreover, it was possible to identify several endogenously proteolysed subunits in the map. The second comparative approach, beside confirming these attributions, allowed to allocate 124 polypeptides within the three main lupin protein families. These two approaches proved to be mutually validating and their combined use was effective for the establishment of a seed proteome map even in the case of sequence and protein post-translational processing lack of information. The results obtained also extend our knowledge of the seed storage protein polymorphism of white lupin.

Proteomic Comparison of Fruit Ripening between 'Hedelfinger' Sweet Cherry (Prunus avium L.) and Its Somaclonal Variant 'HS'.

The somaclonal variant HS, from sweet cherry (Prunus avium L.) 'Hedelfinger' (H), was previously selected for reduced tree vegetative vigor and lesser canopy density. In this work, we compared H and HS fruits at early unripe (green) and full ripe (dark red) stages by biochemical and proteomic approaches. The main biochemical parameters showed that fruit quality was not affected by somaclonal variation. The proteomic analysis identified 39 proteins differentially accumulated between H and HS fruits at the two ripening stages, embracing enzymes involved in several pathways, such as carbon metabolism, cell wall modification, stress response, and secondary metabolism. The evaluation of fruit phenolic composition by mass spectrometry showed that HS sweet cherries have higher levels of procyanidin, flavonol, and anthocyanin compounds. This work provides the first proteomic characterization of fruit ripening in sweet cherry, revealing new positive traits of the HS somaclonal variant.