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

<p>Abstract</p> <p>Background</p> <p>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 (<it>Zea mays </it>L.) plants. Moreover, in order to better evaluate the proteomic results, some biochemical and physiological parameters were measured.</p> <p>Results</p> <p>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.</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

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

<p>Abstract</p> <p>Background</p> <p>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 (<it>e.g</it>. 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 <it>véraison </it>to full maturation were studied by performing a comparative 2-DE analysis.</p> <p>Results</p> <p>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 <it>véraison</it>) and the following three (from 28 to 49 days after <it>véraison</it>) 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%).</p> <p>Conclusion</p> <p>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.</p

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

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 ([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 3c150 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

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

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

The Delay of <i>Raphanus raphanistrum</i> subsp. <i>sativus</i> (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 Starvation and Nitrate or Ammonium Availability Differently Affect Phenolic Composition in Green and Purple Basil

Basil (Ocimum basilicum L.) comprises green and purple cultivars, worldwide cultivated and appreciated for high contents of rosmarinic acid and anthocyanins, respectively. Although nitrogen (N) fertilization is needed for high yields, in basil it could have detrimental effects on the accumulation of phenolic compounds. In this study, plants of the cultivars &lsquo;Italiano Classico&rsquo; (green) and &lsquo;Red Rubin&rsquo; (purple) were grown in hydroponics and subjected to different nutritional treatments, consisting in N starvation, and nitrate (NO3&minus;) or ammonium (NH4+) nutrition. Plant growth and nutritional status, estimated by the contents of NO3&minus;, NH4+, and amino acids in roots and leaves, were evaluated and put in relation with quality traits of basil leaves, such as chlorophyll content, antioxidant capacity, total phenols, the activity of phenylalanine ammonia-lyase, and the concentrations of individual (poly)phenolic acids and flavonoids. This study reveals that N starvation, as well as the availability of the two inorganic N forms, differently affect the phenolic composition in the two cultivars. Compared to plants grown in NO3&minus; availability, in NH4+ availability, green basil showed a higher content of (poly)phenolic acids, while in purple basil, an increase in the contents of anthocyanins was detected. Overall, the study suggests that the management of NH4+ supply could contribute to enhance crop quality in hydroponics, and provides new knowledge about the relationship between N nutrition and phenolic metabolism in basil