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1.  On the role of ethylene, auxin and a GOLVEN-like peptide hormone in the regulation of peach ripening 
BMC Plant Biology  2016;16:44.
In 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.
In 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.
1-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.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-016-0730-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4750175  PMID: 26863869
1-methylcyclopropene (1-MCP); Index of absorbance difference (IAD); Microarray; Nectarine; Prunus persica; Hormone peptide; GOLVEN; ROOT GROWTH FACTOR
2.  Time-Resolved Investigation of Molecular Components Involved in the Induction of NO3– High Affinity Transport System in Maize Roots 
The induction, i.e., the rapid increase of nitrate (NO3–) uptake following the exposure of roots to the anion, was studied integrating physiological and molecular levels in maize roots. Responses to NO3– treatment were characterized in terms of changes in NO3– uptake rate and plasma membrane (PM) H+-ATPase activity and related to transcriptional and protein profiles of NRT2, NRT3, and PM H+-ATPase gene families. The behavior of transcripts and proteins of ZmNRT2s and ZmNRT3s suggested that the regulation of the activity of inducible high-affinity transport system (iHATS) is mainly based on the transcriptional/translational modulation of the accessory protein ZmNRT3.1A. Furthermore, ZmNRT2.1 and ZmNRT3.1A appear to be associated in a ∼150 kDa oligomer. The expression trend during the induction of the 11 identified PM H+-ATPase transcripts indicates that those mainly involved in the response to NO3– treatment are ZmHA2 and ZmHA4. Yet, partial correlation between the gene expression, protein levels and enzyme activity suggests an involvement of post-transcriptional and post-translational mechanisms of regulation. A non-denaturing Deriphat-PAGE approach allowed demonstrating for the first time that PM H+-ATPase can occur in vivo as hexameric complex together with the already described monomeric and dimeric forms.
PMCID: PMC5099785  PMID: 27877183
maize; NO-3 induction; ZmNRT2.1; ZmNRT3.1A; PM H+-ATPase; protein complexes
3.  Mineral nitrogen sources differently affect root glutamine synthetase isoforms and amino acid balance among organs in maize 
BMC Plant Biology  2015;15:96.
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.
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.
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.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-015-0482-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4393875  PMID: 25886826
Amino acids; Ammonium; Glutamine synthetase; Maize; Nitrate; Roots
4.  Comparative analysis of fruit aroma patterns in the domesticated wild strawberries “Profumata di Tortona” (F. moschata) and “Regina delle Valli” (F. vesca) 
Strawberry is one of the most valued fruit worldwide. Modern cultivated varieties (Fragaria × ananassa) exhibit large fruits, with intense color and prolonged shell life. Yet, these valuable traits were attained at the cost of the intensity and the variety of the aroma of the berry, two characteristics highly appreciated by consumers. Wild species display smaller fruits and reduced yield compared with cultivated varieties but they accumulate broader and augmented blends of volatile compounds. Because of the large diversity and strength of aromas occurring in natural and domesticated populations, plant breeders regard wild strawberries as important donors of novel scented molecules. Here we report a comprehensive metabolic map of the aroma of the wild strawberry Profumata di Tortona (PdT), an ancient clone of F. moschata, considered as one of the most fragrant strawberry types of all. Comparison with the more renowned woodland strawberry Regina delle Valli (RdV), an aromatic cultivar of F. vesca, revealed a significant enrichment in the total level of esters, alcohols and furanones and a reduction in the content of ketones in in the aroma of PdT berries. Among esters, particularly relevant was the enhanced accumulation of methyl anthranilate, responsible for the intensive sweetish impression of wild strawberries. Interestingly, increased ester accumulation in PdT fruits correlated with enhanced expression of the Strawberry Alcohol Acyltransferase (SAAT) gene, a key regulator of flavor biogenesis in ripening berries. We also detected a remarkable 900-fold increase in the level of mesifurane, the furanone conferring the typical caramel notes to most wild species.
PMCID: PMC4324068  PMID: 25717332
strawberry; Fragaria moschata; Fragaria vesca; gas chromatography-mass spectrometry; aroma; volatiles
5.  Proteomic and metabolic traits of grape exocarp to explain different anthocyanin concentrations of the cultivars 
The role of grape berry skin as a protective barrier against damage by physical injuries and pathogen attacks requires a metabolism able to sustain biosynthetic activities such as those relating to secondary compounds (i.e., flavonoids). In order to draw the attention on these biochemical processes, a proteomic and metabolomic comparative analysis was performed among Riesling Italico, Pinot Gris, Pinot Noir, and Croatina cultivars, which are known to accumulate anthocyanins to a different extent. The application of multivariate statistics on the dataset pointed out that the cultivars were distinguishable from each other and the order in which they were grouped mainly reflected their relative anthocyanin contents. Sorting the spots according to their significance 100 proteins were characterized by LC-ESI-MS/MS. Through GC-MS, performed in Selected Ion Monitoring (SIM) mode, 57 primary metabolites were analyzed and the differences in abundance of 16 of them resulted statistically significant to ANOVA test. Considering the functional distribution, the identified proteins were involved in many physiological processes such as stress, defense, carbon metabolism, energy conversion and secondary metabolism. The trends of some metabolites were related to those of the protein data. Taken together, the results permitted to highlight the relationships between the secondary compound pathways and the main metabolism (e.g., glycolysis and TCA cycle). Moreover, the trend of accumulation of many proteins involved in stress responses, reinforced the idea that they could play a role in the cultivar specific developmental plan.
PMCID: PMC4523781  PMID: 26300900
anthocyanins; exocarp grape berry; metabolomics; proteomics; stress response; Vitis vinifera
6.  Morphological and Proteomic Responses of Eruca sativa Exposed to Silver Nanoparticles or Silver Nitrate 
PLoS ONE  2013;8(7):e68752.
Silver nanoparticles (AgNPs) are widely used in commercial products, and there are growing concerns about their impact on the environment. Information about the molecular interaction of AgNPs with plants is lacking. To increase our understanding of the mechanisms involved in plant responses to AgNPs and to differentiate between particle specific and ionic silver effects we determined the morphological and proteomic changes induced in Eruca sativa (commonly called rocket) in response to AgNPs or AgNO3. Seedlings were treated for 5 days with different concentrations of AgNPs or AgNO3. A similar increase in root elongation was observed when seedlings were exposed to 10 mg Ag L1 of either PVP-AgNPs or AgNO3. At this concentration we performed electron microscopy investigations and 2-dimensional electrophoresis (2DE) proteomic profiling. The low level of overlap of differentially expressed proteins indicates that AgNPs and AgNO3 cause different plant responses. Both Ag treatments cause changes in proteins involved in the redox regulation and in the sulfur metabolism. These responses could play an important role to maintain cellular homeostasis. Only the AgNP exposure cause the alteration of some proteins related to the endoplasmic reticulum and vacuole indicating these two organelles as targets of the AgNPs action. These data add further evidences that the effects of AgNPs are not simply due to the release of Ag ions.
PMCID: PMC3715538  PMID: 23874747
7.  Proteomic characterization of the Rph15 barley resistance gene-mediated defence responses to leaf rust 
BMC Genomics  2012;13:642.
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.
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.
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.
PMCID: PMC3541957  PMID: 23167439
Barley; Leaf rust; Resistance gene; Rph15; Proteomics; Near isogenic lines
8.  Proteomic characterization of iron deficiency responses in Cucumis sativus L. roots 
BMC Plant Biology  2010;10:268.
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.
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.
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.
PMCID: PMC3016405  PMID: 21122124
9.  Evaluation of protein pattern changes in roots and leaves of Zea mays plants in response to nitrate availability by two-dimensional gel electrophoresis analysis 
BMC Plant Biology  2009;9:113.
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.
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.
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.
PMCID: PMC2744680  PMID: 19698183
10.  Proteome changes in the skin of the grape cultivar Barbera among different stages of ripening 
BMC Genomics  2008;9:378.
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.
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%).
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.
PMCID: PMC2529320  PMID: 18691399

Results 1-10 (10)