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 'Italiano Classico' (green) and 'Red Rubin' (purple) were grown in hydroponics and subjected to different nutritional treatments, consisting in N starvation, and nitrate (NO3 12) or ammonium (NH4+) nutrition. Plant growth and nutritional status, estimated by the contents of NO3 12, 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 12 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

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

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 (\u2018Italiano Classico\u2019) and purple (\u2018Red Rubin\u2019 and \u2018Dark Opal\u2019) 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

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

Experimental approaches on in vitro grafting in Prunus armeniaca and P. spinosa

An in vitro grafting protocol was established with micropropagated shoots of the apricot cultivars \u2018Caldesi\u2019 and \u2018Alb 3\u2019, (apricot selection from \u2018Cricot\u2019 x \u2018NAJ 53\u2019 cross) and the P. spinosa seedling \u2018PS123\u2019(open pollination from a wild tree). Compatible combinations (homografts) were ascertained by considering vascular continuity between scion and stock, as determined by fast toluidine blue staining procedure, and by restoration of phloem connections as determined by application of 5(6)-carboxyfluorescein (CF) to the scion. 15 days after graft establishment, the differentiation of new xylematic tissue resulting in unification of the vascular elements between stock and scion was observed in all the genotypes which, moreover, showed restoration of active phloem. High correlation between the differentiation of new xylem and the differentiation of new phloem at the graft point was found. Graft-take, however, did not correlate with the scion-stock compatibility, in that heterograft-take was high but did not reflect xylematic continuity or phloematic transport, which were not established in any of the tested scion-stock combination. The use of rooted shoots as rootstock resulted in unsuccessful graft technique

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

Triosephosphate Isomerases in Italian Ryegrass (Lolium multiflorum) : Characterization and Susceptibility to Herbicides

The effect of treatments with four herbicides and a safener on the activity of triosephosphate isomerase (TPI) extracted from shoots of Italian ryegrass was investigated. It was found that atrazine and fluorodifen, herbicides which interfere with photosynthesis, caused a decrease in measured enzyme activity. In addition, the in vitro effect of oxidized glutathione (GSSG), a compound produced in situations of oxidative stress, on TPI activity was investigated. It was shown that GSSG was a strong inhibitor of enzyme activity, at low concentrations in a dose-timedependent manner. The enzyme extracts were submitted to chromatographic purifications and to two-dimensional electrophoresis. Some spots had molecular masses ranging between 20 and 30 kDa and were characterized and identified by LC-ESI-MS/MS as TPIs. The mass spectrometry also made it possible to identify the presence of cysteine residues that could be subjected to S-glutathionylation, which regulate the enzyme activity

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 stepwiselinear discriminant analysis. This technique allowed to discriminate v\ue9raison, 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

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

New insights into the biodegradation of thiodiglycol, the hydrolysis product of Yperite (sulfur mustard gas)

Aims: To isolate thiodiglycol (TDG)-degrading bacteria, the mustard gas hydrolysis product, and to characterize the metabolites formed and the enzymes involved in the degradation. Methods and Results: Two strains, identified as Achromobacter xylosoxydans G5 and Paracoccus denitrificans E4, isolated from a petroleum-contaminated soil, utilized TDG as sole carbon and sulfur source. During the degradation of TDG by strain E4 [(2-hydroxyethyl)thio] acetic acid (HETA), thiodiglycolic acid (TDGA) and bis-(2-hydroxyethyl)disulfide (BHEDS) were identified by gas chromatography\u2013mass spectrometry analysis, while HETA and TDGA were identified for strain G5. Two-dimensional isoelectric focussing-gel electrophoresis (2-D IEF/SDS\u2013PAGE) maps of protein extracts of P. denitrificans E4 grown on TDG showed a spot identified as a methanol dehydrogenase. Increased expression of a putative iscS gene, involved in sulfur assimilation, was observed in TDG-grown cells of A. xylosoxydans G5. Conclusions: TDG degradation by P. denitrificans E4 occurred through two pathways: one involved cleavage of the C\u2013S bond of HETA, yielding BHEDS and the other, oxidation of the alcoholic groups of TDG, yielding TDGA. The cleavage of the C\u2013S bond of TDGA gave mercaptoacetic acid, further oxidized to acetate and sulfate. Significance and Impact of the Study: Increased knowledge of TDG-degrading bacteria and the possibility of using them in a tailored-two-stage mustard gas destruction process