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.

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.

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.

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.

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.

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.

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.

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.

Time-Resolved Investigation of Molecular Components Involved in the Induction of [Formula: see text] High Affinity Transport System in Maize Roots.

The induction, i.e., the rapid increase of nitrate ([Formula: see text]) uptake following the exposure of roots to the anion, was studied integrating physiological and molecular levels in maize roots. Responses to [Formula: see text] treatment were characterized in terms of changes in [Formula: see text] 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 [Formula: see text] 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.

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.

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.

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.

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.

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.

Hyperacidification of vacuoles by the combined action of two different P-ATPases in the tonoplast determines flower color.

The acidification of endomembrane compartments is essential for enzyme activities, sorting, trafficking, and trans-membrane transport of various compounds. Vacuoles are mildly acidic in most plant cells because of the action of V-ATPase and/or pyrophosphatase proton pumps but are hyperacidified in specific cells by mechanisms that remained unclear. Here, we show that the blue petal color of petunia ph mutants is due to a failure to hyperacidify vacuoles. We report that PH1 encodes a P3B-ATPase, hitherto known as Mg2(+) transporters in bacteria only, that resides in the vacuolar membrane (tonoplast). In vivo nuclear magnetic resonance and genetic data show that PH1 is required and, together with the tonoplast H(+) P3A-ATPase PH5, sufficient to hyperacidify vacuoles. PH1 has no H(+) transport activity on its own but can physically interact with PH5 and boost PH5 H(+) transport activity. Hence, the hyperacidification of vacuoles in petals, and possibly other tissues, relies on a heteromeric P-ATPase pump.

Morphological and proteomic responses of Eruca sativa exposed to silver nanoparticles or silver nitrate.

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 L(1) 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.

The proteome of exudates from germinating Lupinus albus seeds is secreted through a selective dual-step process and contains proteins involved in plant defence.

The general knowledge of defence activity during the first steps of seed germination is still largely incomplete. The present study focused on the proteins released in the exudates of germinating white lupin seeds. During the first 24 h, a release of proteins was observed. Initially (i.e. during the first 12 h), the proteins found in exudates reflected the composition of the seed, indicating a passive extrusion of pre-formed proteins. Subsequently, when the rate of protein release was at its highest, the composition of the released proteome changed drastically. This transition occurred in a short time, indicating that more selective and regulated events, such as secretory processes, took place soon after the onset of germination. The present study considered: (a) the characterization of the proteome accumulated in the germinating medium collected after the appearance of the post-extrusion events; (b) the biosynthetic origin and the modalities that are the basis of protein release outside the seeds; and (c) an assessment of antifungal activity of these exudates. The most represented protein in the exudate was chitinase, which was synthesized de novo. The other proteins are involved in the cellular mechanisms responding to stress events, including biotic ones. This exudate was effectively able to inhibit fungal growth. The results of the present study indicate that seed exudation is a dual-step process that leads to the secretion of selected proteins and thus is not a result of passive leakage. The released proteome is involved in protecting the spermosphere environment and thus may act as first defence against pathogens.

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.