A proteomic and biochemical investigation on the effects of sulfadiazine in Arabidopsis thaliana.

Animal manure or bio-solids used as fertilizers are the main routes of antibiotic exposure in the agricultural land, which can have immense detrimental effects on plants. Sulfadiazine (SDZ), belonging to the class of sulfonamides, is one of the most detected antibiotics in the agricultural soil. In this study, the effect of SDZ on the growth, changes in antioxidant metabolite content and enzyme activities related to oxidative stress were analysed. Moreover, the proteome alterations in Arabidopsis thaliana roots in response to SDZ was examined by means of a combined iTRAQ-LC-MS/MS quantitative proteomics approach. A dose-dependent decrease in leaf biomass and root length was evidenced in response to SDZ. Increased malondialdehyde content at higher concentration (2 µM) of SDZ indicated increased lipid peroxidation and suggest the induction of oxidative stress. Glutathione levels were significantly higher compared to control, whereas there was no increase in ascorbate content or the enzyme activities of glutathione metabolism, even at higher concentrations. In total, 48 differentially abundant proteins related to stress/stimuli response followed by transcription and translation, metabolism, transport and other functions were identified. Several proteins related to oxidative, dehydration, salinity and heavy metal stresses were represented. Upregulation of peroxidases was validated with total peroxidase activity. Pathway analysis provided an indication of increased phenylpropanoid biosynthesis. Probable molecular mechanisms altered in response to SDZ are highlighted.

Proteomic analysis of a compatible interaction between sugarcane and Sporisorium scitamineum.

Smut caused by Sporisorium scitamineum is one of the important diseases of sugarcane with global significance. Despite the intriguing nature of sugarcane, S. scitamineum interaction, several pertinent aspects remain unexplored. This study investigates the proteome level alterations occurring in the meristem of a S. scitamineum infected susceptible sugarcane cultivar at whip emergence stage. Differentially abundant proteins were identified by 2DE coupled with MALDI-TOF/TOF-MS. Comprehensively, 53 sugarcane proteins identified were related to defence, stress, metabolism, protein folding, energy, and cell division; in addition, a putative effector of S. scitamineum, chorismate mutase, was identified. Transcript expression vis-à-vis the activity of phenylalanine ammonia lyase was relatively higher in the infected meristem. Abundance of seven candidate proteins in 2D gel profiles was in correlation with its corresponding transcript expression levels as validated by qRT-PCR. Furthermore, this study has opened up new perspectives on the interaction between sugarcane and S. scitamineum.

Comparative proteomics of sugarcane smut fungus - Sporisorium scitamineum unravels dynamic proteomic alterations during the dimorphic transition.

Life cycle of the dimorphic sugarcane smut fungi, Sporisorium scitamineum, involves recognition and mating of compatible saprophytic yeast-like haploid sporidia (MAT-1 and MAT-2) that upon fusion, develop into infective dikaryotic mycelia. Although the dimorphic transition is intrinsically linked with the pathogenicity and virulence of S. scitamineum, it has never been studied using a proteomic approach. In the present study, an iTRAQ-based comparative proteomic analysis of three distinct stages was carried out. The stages were: the dimorphic transition period - haploid sporidial stage (MAT-1 and MAT-2); the transition phase (24 h post co-culturing (hpc)) and the dikaryotic mycelial stage (48 hpc). Functional categorization of differentially abundant proteins showed that the most altered biological processes were energy production, primary metabolism, especially, carbohydrate, amino acid, fatty acid, followed by translation, post-translation and protein turnover. Several differentially abundant proteins (DAPs), especially in the dikaryotic mycelial stage were predicted as effectors. Taken together, key molecular mechanisms underpinning the dimorphic transition in S. scitamineum at the proteome level were highlighted. The catalogue of stage-specific and dimorphic transition-associated-proteins and potential effectors identified herein represents a list of potential candidates for defective mutant screening to elucidate their functional role in the dimorphic transition and pathogenicity in S. scitamineum. BIOLOGICAL SIGNIFICANCE: Being the first comparative proteomics analysis of S. scitamineum, this study comprehensively examined three pivotal life cycle stages of the pathogen: the non-pathogenic haploid phase, the transition phase, and the pathogenic dikaryotic mycelial stage. While previous studies have reported the sugarcane and S. scitamineum interactions, this study endeavored to specifically identify the proteins responsible for pathogenicity. By analyzing the proteomic alterations between the haploid and dikaryotic mycelial phases, the study revealed significant changes in metabolic pathway-associated proteins linked to energy production, notably oxidative phosphorylation, and the citrate cycle. Furthermore, this study successfully identified key metabolic pathways that undergo reprogramming during the transition from the non-pathogenic to the pathogenic stage. The study also deciphered the underlying mechanisms driving the morphological and physiological alterations crucial for the S. scitamineum virulence. By studying its life cycle stages, identifying the key metabolic pathways and stage-specific proteins, it provides unprecedented insights into the pathogenicity and potential avenues for intervention. As proteomics continues to advance, such studies pave the way for a deeper understanding of plant-pathogen interactions and the development of innovative strategies to mitigate the impact of devastating pathogens like S. scitamineum.

Quality Evaluation of Fair-Trade Cocoa Beans from Different Origins Using Portable Near-Infrared Spectroscopy (NIRS).

Determining cocoa bean quality is crucial for many players in the international supply chain. However, actual methods rely on a cut test protocol, which is limited by its subjective nature, or on time-consuming, expensive and destructive wet-chemistry laboratory procedures. In this context, the application of near infrared (NIR) spectroscopy, particularly with the recent developments of portable NIR spectrometers, may represent a valuable solution for providing a cocoa beans' quality profile, in a rapid, non-destructive, and reliable way. Monitored parameters in this work were dry matter (DM), ash, shell, fat, protein, total polyphenols, fermentation index (FI), titratable acidity (TA) and pH. Different chemometric analyses were performed on the spectral data and calibration models were developed using modified partial least squares regression. Prediction equations were validated using a fivefold cross-validation and a comparison between the different prediction performances for the portable and benchtop NIR spectrometers was provided. The NIRS benchtop instrument provided better performance of quantification considering the whole than the portable device, showing excellent prediction capability in protein and DM quantification. On the other hand, the NIRS portable device, although showing lower but valuable performance of prediction, can represent an appealing alternative to benchtop instruments for food business operators, being applicable in the field.

Quantitative Proteomics of Maize Roots Treated with a Protein Hydrolysate: A Comparative Study with Transcriptomics Highlights the Molecular Mechanisms Responsive to Biostimulants.

Protein hydrolysate (PH)-based biostimulants offer a cost-effective and sustainable approach for the regulation of physiological processes in plants to stimulate growth and improve stress tolerance. Understanding the mode of action of PHs is challenging, but it is indispensable to improve existing candidates and to develop novel molecules with enhanced stimulatory effects. Hence, the present study aimed to understand the proteome level responses in the B73 maize roots treated with APR, a PH biostimulant, at two increasing concentrations and to compare and integrate it with the transcriptomic data obtained previously under identical experimental conditions. Results indicate that APR induced dose-dependent global changes in the transcriptome and proteome of maize roots. APR treatment altered the expression and abundance of several genes and proteins related to redox homeostasis, stress response, glycolysis, tricarboxylic acid cycle, pentose phosphate pathway, and other metabolic pathways of carbohydrates, amino acids, and lipids. Further, metabolic processes of phytohormone, secondary metabolites, especially phenylpropanoids, flavonoids, and terpenoids and transport, and cytoskeletal reorganization associated mechanisms were stimulated. Our results suggest that APR treatment altered the redox homeostasis and thus triggered an oxidative signal. This could be one of the key regulators of the cascade of downstream events involving multiple signaling, hormonal, and metabolic pathways, resulting in an altered physiological and metabolic state which consequently could lead to improved growth and stress adaptation observed in biostimulant-treated plants.

Metabolomic responses triggered by arbuscular mycorrhiza enhance tolerance to water stress in wheat cultivars.

Under global climate change forecasts, the pressure of environmental stressors (and in particular drought) on crop productivity is expected to rise and challenge further global food security. The application of beneficial microorganisms may represent an environment friendly tool to secure improved crop performance and yield stability. Accordingly, this current study aimed at elucidating the metabolomic responses triggered by mycorrhizal (Funneliformis mosseae) inoculation of durum (Triticum durum Desf.; cv. 'Mongibello') and bread wheat cultivars (Triticum aestivum L.; cv. 'Chinese Spring') under full irrigation and water deficit regimes. Metabolomics indicated a similar regulation of secondary metabolism in both bread and durum wheat cultivars following water limiting conditions. Nonetheless, a mycorrhizal fungi (AMF) x cultivar interaction could be observed, with the bread wheat cultivar being more affected by arbuscular colonization under water limiting conditions. Discriminant compounds could be mostly related to sugars and lipids, both being positively modulated by AMF colonization under water stress. Moreover, a regulation of metabolites related to oxidative stress and a tuning of crosstalk between phytohormones were also evidenced. Among the latter, the stimulation of the brassinosteroids biosynthetic pathway was particularly evident in inoculated wheat roots, supporting the hypothesis of their involvement in enhancing plant response to water stress and modulation of oxidative stress conditions. This study proposes new insights on the modulation of the tripartite interaction plant-AMF-environmental stress.

Protein Profiling of Arabidopsis Roots Treated With Humic Substances: Insights Into the Metabolic and Interactome Networks.

Background and Aim: Humic substances (HSs) influence the chemical and physical properties of the soil, and are also known to affect plant physiology and nutrient uptake. This study aimed to elucidate plant metabolic pathways and physiological processes influenced by HS activity. Methods: Arabidopsis roots were treated with HS for 8 h. Quantitative mass spectrometry-based proteomics analysis of root proteins was performed using the iTRAQ (Isobaric Tag for Relative and Absolute Quantification) technique. Out of 902 protein families identified and quantified for HS treated vs. untreated roots, 92 proteins had different relative content. Bioinformatic tools such as STRING, KEGG, IIS and Cytoscape were used to interpret the biological function, pathway analysis and visualization of network amongst the identified proteins. Results: From this analysis it was possible to evaluate that all of the identified proteins were functionally classified into several categories, mainly redox homeostasis, response to inorganic substances, energy metabolism, protein synthesis, cell trafficking, and division. Conclusion: In the present study an overview of the metabolic pathways most modified by HS biological activity is provided. Activation of enzymes of the glycolytic pathway and up regulation of ribosomal protein indicated a stimulation in energy metabolism and protein synthesis. Regulation of the enzymes involved in redox homeostasis suggest a pivotal role of reactive oxygen species in the signaling and modulation of HS-induced responses.

Effects of different humic substances concentrations on root anatomy and Cd accumulation in seedlings of Avicennia germinans (black mangrove).

Mangrove areas are among most threatened tropical ecosystems worldwide. Among polluting agents Cadmium is often found in high concentrations in mangrove sediments. Humic substances, complex biomolecules formed in soil and sediments during animal and plant residuals decomposition, have a known biostimulant activity and can be adopted to counteract various plant stresses. This study explores, in controlled conditions, the effect of humic substances on Avicennia germinans seedlings, with or without cadmium contamination. Humic compounds significantly changed plant root architecture, and, when coupled with cadmium, root anatomy and Cortex to Vascular Cylinder diameter ratio. These modifications led to lower Cd uptake by humic substances-treated plants. Humic substances amendment could be effective, depending on their concentrations, on improving plant health in mangrove areas, for forest recuperation and/or dredged sediments phytoremediation purposes.

Possible developments for ex situ phytoremediation of contaminated sediments, in tropical and subtropical regions - Review.

The growing problem of remediation of contaminated sediments dredged from harbor channels needs to be resolved by a cost effective and sustainable technology. Phytoremediation, by ex situ remediation plants, seems to have the potential to replace traditional methods in case of moderately contaminated sediments. On the other side, the need to mix sediments with soil and/or sand to allow an easier establishment of most employed species causes an increase of the volume of the processed substrate up to 30%. Moreover the majority of phytoremediating species are natives of temperate climate belt. Mangroves, with a special focus on the genus Avicennia - a salt secreting species - should represent an effective alternative in terms of adaptation to salty, anoxic sediments and an opportunity to develop ex situ phytoremediation plants in tropical and subtropical regions. The use of humic acid to increase root development, cell antioxidant activity and the potential attenuation of the "heavy metals exclusion strategy" to increase phytoextraction potentials of mangroves will be reviewed.

Proteomic insight into the mitigation of wheat root drought stress by arbuscular mycorrhizae.

Arbuscular mycorrhizal fungi (AMF) are plant growth promoters that ameliorate plant-water relations and the nutrient uptake of wheat. In this work, two cultivars of Triticum spp., a bread and a durum wheat, grown under drought stress and inoculated or not by AMF, are evaluated through a shotgun proteomic approach. The AMF association had beneficial effects as compared to non-mycorrhizal roots, in both bread and durum wheat. The beneficial symbiosis was confirmed by measuring morphological and physiological traits. In our work, we identified 50 statistically differential proteins in the bread wheat cultivar and 66 differential proteins in the durum wheat cultivar. The findings highlighted a modulation of proteins related to sugar metabolism, cell wall rearrangement, cytoskeletal organization and sulphur-containing proteins, as well as proteins related to plant stress responses. Among differentially expressed proteins both cultivars evidenced a decrease in sucrose:fructan 6-fructosyltransferas. In durum wheat oxylipin signalling pathway was involved with two proteins: increased 12-oxo-phytodienoic acid reductase and decreased jasmonate-induced protein, both related to the biosynthesis of jasmonic acid. Interactome analysis highlighted the possible involvement of ubiquitin although not evidenced among differentially expressed proteins. The AMF association helps wheat roots reducing the osmotic stress and maintaining cellular integrity. BIOLOGICAL SIGNIFICANCE: Drought is one of the major constraints that plants must face in some areas of the world, associated to climate change, negatively affecting the worldwide plant productivity. The adoption of innovative agronomic protocols may represent a winning strategy in facing this challenge. The arbuscular mycorrhizal fungi (AMF) inoculation may represent a natural and sustainable way to mitigate the negative effects due to drought in several crop, ameliorating plant growth and development. Studies on the proteomic responses specific to AMF in drought-stressed plants will help clarify how mycorrhization elicits plant growth, nutrient uptake, and stress-tolerance responses. Such studies also offer the potential to find biological markers and genetic targets to be used during breeding for new drought-resistant varieties.

Biostimulant Effects of Seed-Applied Sedaxane Fungicide: Morphological and Physiological Changes in Maize Seedlings.

Most crops are routinely protected against seed-born and soil-borne fungal pathogens through seed-applied fungicides. The recently released succinate dehydrogenase inhibitor (SDHI), sedaxane®, is a broad-spectrum fungicide, used particularly to control Rhizoctonia spp., but also has documented growth-enhancement effects on wheat. This study investigates the potential biostimulant effects of sedaxane and related physiological changes in disease-free maize seedlings (3-leaf stage) at increasing application doses (25, 75 and 150 µg a.i. seed-1) under controlled sterilized conditions. We show sedaxane to have significant auxin-like and gibberellin-like effects, which effect marked morphological and physiological changes according to an approximate saturation dose-response model. Maximum benefits were attained at the intermediate dose, which significantly increased root length (+60%), area (+45%) and forks (+51%), and reduced root diameter as compared to untreated controls. Sedaxane enhanced leaf and root glutamine synthetase (GS) activity resulting in greater protein accumulation, particularly in the above-ground compartment, while glutamate synthase (GOGAT) activity remained almost unchanged. Sedaxane also improved leaf phenylalanine ammonia-lyase (PAL) activity, which may be responsible for the increase in shoot antioxidant activity (phenolic acids), mainly represented by p-coumaric and caffeic acids. We conclude that, in addition to its protective effect, sedaxane can facilitate root establishment and intensify nitrogen and phenylpropanoid metabolism in young maize plants, and may be beneficial in overcoming biotic and abiotic stresses in early growth stages.

In vitro secretomic analysis identifies putative pathogenicity-related proteins of Sporisorium scitamineum - The sugarcane smut fungus.

Sporisorium scitamineum, the sugarcane smut pathogen, relies predominantly on its secretome to successfully colonise its host, in accordance with other related smut fungi. Considering the significance of deciphering its secretome, we have examined alterations in the in vitro secretome of S. scitamineum in response to synthetic and sugarcane meristem tissue-amended growth media, so as to identify host signal responsive secretory proteins. Secretory proteins that were differentially abundant and exclusively secreted in response to host extract media were identified by two-dimensional gel electrophoresis coupled with MALDI-TOF/TOF MS. Of the 16 differentially abundant and exclusively secreted proteins, nine proteins were identified. Among which, six were related to cell wall modification, morphogenesis, polysaccharide degradation, and carbohydrate metabolism. In planta gene expression profiling indicated that five in vitro secreted proteins were expressed in distinct patterns by S. scitamineum during different stages of infection with relatively higher expression at 1 day after inoculation, suggesting that these proteins could be aiding S. scitamineum at early time points in penetration and colonisation of sugarcane cells. The present study has provided insights into the alterations occurring in the secretome of S. scitamineum at in vitro conditions and has resulted in the identification of secretory proteins that are possibly associated with pathogenicity of the sugarcane smut fungus.

Interceptive therapy for maxillary constriction: a cast-metal-type splint expander.

A new cast-metal-type splint expander was used to treat severe maxillary constriction. This new appliance has the same advantages of a rapid expander on acrylic splints, compared to a Haas or to a banded expander. Furthermore, it is characterized by greater respect for the gingival tissue, both during the therapy and immediately after the removal of the expander. It is also more comfortable for the patient, so that it is possible to maintain the expander in the mouth for several months without gingival suffering. This appliance is very versatile and well indicated for permanent, mixed and deciduous dentitions.

Assisted phytoremediation of mixed metal(loid)-polluted pyrite waste: effects of foliar and substrate IBA application on fodder radish.

Exogenous application of plant-growth promoting substances may potentially improve phytoremediation of metal-polluted substrates by increasing shoot and root growth. In a pot-based study, fodder radish (Raphanus sativus L. var. oleiformis Pers.) was grown in As-Zn-Cu-Co-Pb-contaminated pyrite waste, and treated with indolebutyric acid (IBA) either by foliar spraying (10 mgL(-1)), or by direct application of IBA to the substrate (0.1 and 1 mgkg(-1)) in association, or not, with foliar spraying. With the exception of foliar spraying, IBA reduced above-ground biomass, whilst direct application of IBA to the substrate surface reduced root biomass (-59%). Trace element concentrations were generally increased, but removals (mg per plant) greatly reduced with IBA application, together with greater metal leaching from the substrate. It is concluded that, in our case, IBA had a negative effect on plant growth and phytoextraction of trace elements, possibly due to unsuitable root indoleacetic acid concentration following soil IBA application, the direct chelating effect of IBA and the low microbial activity in the pyrite waste affecting its breakdown.

Protein expression changes in maize roots in response to humic substances.

Humic substances are known to affect plant metabolism at different levels. We characterized humic substances extracted from earthworm feces by diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy and used them to treat corn, Zea mays L., seedlings to investigate changes in patterns of root protein expression. After root plasma membrane extraction and purification, proteins were separated by two-dimensional gel electrophoresis, and differential spot intensities were evaluated by image analysis. Finally, 42 differentially expressed proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The majority of them were downregulated by the treatment with humic substances. The proteins identified included malate dehydrogenase, ATPases, cytoskeleton proteins, and different enzymes belonging to the glycolytic/gluconeogenic pathways and sucrose metabolism. The identification of factors involved in plant responses to humic substances may improve our understanding of plant-soil cross-talk, and enable a better management of soil resources.