Tomato leaves under stress: a comparison of stress response to mild abiotic stress between a cultivated and a wild tomato species.

Tomato is one of the most produced crop plants on earth and growing in the fields and greenhouses all over the world. Breeding with known traits of wild species can enhance stress tolerance of cultivated crops. In this study, we investigated responses of the transcriptome as well as primary and secondary metabolites in leaves of a cultivated and a wild tomato to several abiotic stresses such as nitrogen deficiency, chilling or warmer temperatures, elevated light intensities and combinations thereof. The wild species responded different to varied temperature conditions compared to the cultivated tomato. Nitrogen deficiency caused the strongest responses and induced in particular the secondary metabolism in both species but to much higher extent in the cultivated tomato. Our study supports the potential of a targeted induction of valuable secondary metabolites in green residues of horticultural production, that will otherwise only be composted after fruit harvest. In particular, the cultivated tomato showed a strong induction in the group of mono caffeoylquinic acids in response to nitrogen deficiency. In addition, the observed differences in stress responses between cultivated and wild tomato can lead to new breeding targets for better stress tolerance.

How to make a tumour: cell type specific dissection of Ustilago maydis-induced tumour development in maize leaves.

The biotrophic fungus Ustilago maydis causes smut disease on maize (Zea mays), which is characterized by immense plant tumours. To establish disease and reprogram organ primordia to tumours, U. maydis deploys effector proteins in an organ-specific manner. However, the cellular contribution to leaf tumours remains unknown. We investigated leaf tumour formation at the tissue- and cell type-specific levels. Cytology and metabolite analysis were deployed to understand the cellular basis for tumourigenesis. Laser-capture microdissection was performed to gain a cell type-specific transcriptome of U. maydis during tumour formation. In vivo visualization of plant DNA synthesis identified bundle sheath cells as the origin of hyperplasic tumour cells, while mesophyll cells become hypertrophic tumour cells. Cell type-specific transcriptome profiling of U. maydis revealed tailored expression of fungal effector genes. Moreover, U. maydis See1 was identified as the first cell type-specific fungal effector, being required for induction of cell cycle reactivation in bundle sheath cells. Identification of distinct cellular mechanisms in two different leaf cell types and of See1 as an effector for induction of proliferation of bundle sheath cells are major steps in understanding U. maydis-induced tumour formation. Moreover, the cell type-specific U. maydis transcriptome data are a valuable resource to the scientific community.

Copper amine oxidase 8 regulates arginine-dependent nitric oxide production in Arabidopsis thaliana.

Nitric oxide (NO) is a key signaling molecule in plants, regulating a wide range of physiological processes. However, its origin in plants remains unclear. It can be generated from nitrite through a reductive pathway, notably via the action of the nitrate reductase (NR), and evidence suggests an additional oxidative pathway, involving arginine. From an initial screen of potential Arabidopsis thaliana mutants impaired in NO production, we identified copper amine oxidase 8 (CuAO8). Two cuao8 mutant lines displayed a decreased NO production in seedlings after elicitor treatment and salt stress. The NR-dependent pathway was not responsible for the impaired NO production as no change in NR activity was found in the mutants. However, total arginase activity was strongly increased in cuao8 knockout mutants after salt stress. Moreover, NO production could be restored in the mutants by arginase inhibition or arginine addition. Furthermore, arginine supplementation reversed the root growth phenotype observed in the mutants. These results demonstrate that CuAO8 participates in NO production by influencing arginine availability through the modulation of arginase activity. The influence of CuAO8 on arginine-dependent NO synthesis suggests a new regulatory pathway for NO production in plants.

Leaf wounding or simulated herbivory in young N. attenuata plants reduces carbon delivery to roots and root tips.

MAIN CONCLUSION: In Nicotiana attenuata seedlings, simulated herbivo ry by the specialist Manduca sexta decreases root growth and partitioning of recent photoassimilates to roots in contrast to increased partitioning reported for older plants. Root elongation rate in Nicotiana attenuata has been shown to decrease after leaf herbivory, despite reports of an increased proportion of recently mobilized photoassimilate being delivered towards the root system in many species after similar treatments. To study this apparent contradiction, we measured the distribution of recent photoassimilate within root tissues after wounding or simulated herbivory of N. attenuata leaves. We found no contradiction: herbivory reduced carbon delivery to root tips. However, the speed of phloem transport in both shoot and root, and the delivery of recently assimilated carbon to the entire root system, declined after wounding or simulated herbivory, in contrast with the often-reported increase in root partitioning. We conclude that the herbivory response in N. attenuata seedlings is to favor the shoot and not bunker carbon in the root system.

Molecular Mechanisms Underlying Medium-Chain Free Fatty Acid-Regulated Activity of the Phospholipase PlaF from Pseudomonas aeruginosa.

PlaF is a membrane-bound phospholipase A1 from Pseudomonas aeruginosa that is involved in remodeling membrane glycerophospholipids (GPLs) and modulating virulence-associated signaling and metabolic pathways. Previously, we identified the role of medium-chain free fatty acids (FFAs) in inhibiting PlaF activity and promoting homodimerization, yet the underlying molecular mechanism remained elusive. Here, we used unbiased and biased molecular dynamics simulations and free energy computations to assess how PlaF interacts with FFAs localized in the water milieu surrounding the bilayer or within the bilayer and how these interactions regulate PlaF activity. Medium-chain FFAs localized in the upper bilayer leaflet can stabilize inactive dimeric PlaF, likely through interactions with charged surface residues, as has been experimentally validated. Potential of mean force (PMF) computations indicate that membrane-bound FFAs may facilitate the activation of monomeric PlaF by lowering the activation barrier for changing into a tilted, active configuration. We estimated that the coupled equilibria of PlaF monomerization-dimerization and tilting at the physiological concentration of PlaF lead to the majority of PlaF forming inactive dimers when in a cell membrane loaded with decanoic acid (C10). This is in agreement with a suggested in vivo product feedback loop and gas chromatography-mass spectrometry profiling results, indicating that PlaF catalyzes the release of C10 from P. aeruginosa membranes. Additionally, we found that C10 in the water milieu can access the catalytic site of active monomeric PlaF, contributing to the competitive component of C10-mediated PlaF inhibition. Our study provides mechanistic insights into how medium-chain FFAs may regulate the activity of PlaF, a potential bacterial drug target.

Degradation of sulfadiazine by Microbacterium lacus strain SDZm4, isolated from lysimeters previously manured with slurry from sulfadiazine-medicated pigs.

Sulfadiazine (SDZ)-degrading bacterial cultures were enriched from the topsoil layer of lysimeters that were formerly treated with manure from pigs medicated with (14)C-labeled SDZ. The loss of about 35% of the applied radioactivity after an incubation period of 3 years was attributed to CO2 release due to mineralization processes in the lysimeters. Microcosm experiments with moist soil and soil slurries originating from these lysimeters confirmed the presumed mineralization potential, and an SDZ-degrading bacterium was isolated. It was identified as Microbacterium lacus, denoted strain SDZm4. During degradation studies with M. lacus strain SDZm4 using pyrimidine-ring labeled SDZ, SDZ disappeared completely but no (14)CO2 was released during 10 days of incubation. The entire applied radioactivity (AR) remained in solution and could be assigned to 2-aminopyrimidine. In contrast, for parallel incubations but with phenyl ring-labeled SDZ, 56% of the AR was released as (14)CO2, 16% was linked to biomass, and 21% remained as dissolved, not yet identified (14)C. Thus, it was shown that M. lacus extensively mineralized and partly assimilated the phenyl moiety of the SDZ molecule while forming equimolar amounts of 2-aminopyrimidine. This partial degradation might be an important step in the complete mineralization of SDZ by soil microorganisms.

Implications of Below-Ground Allelopathic Interactions of Camelina sativa and Microorganisms for Phosphate Availability and Habitat Maintenance.

Toxic breakdown products of young Camelina sativa (L.) Crantz, glucosinolates can eliminate microorganisms in the soil. Since microorganisms are essential for phosphate cycling, only insensitive microorganisms with phosphate-solubilizing activity can improve C. sativa's phosphate supply. In this study, 33P-labeled phosphate, inductively coupled plasma mass spectrometry and pot experiments unveiled that not only Trichoderma viride and Pseudomonas laurentiana used as phosphate-solubilizing inoculants, but also intrinsic soil microorganisms, including Penicillium aurantiogriseum, and the assemblies of root-colonizing microorganisms solubilized as well phosphate from apatite, trigger off competitive behavior between the organisms. Driving factors in the competitiveness are plant and microbial secondary metabolites, while glucosinolates of Camelina and their breakdown products are regarded as key compounds that inhibit the pathogen P. aurantiogriseum, but also seem to impede root colonization of T. viride. On the other hand, fungal diketopiperazine combined with glucosinolates is fatal to Camelina. The results may contribute to explain the contradictory effects of phosphate-solubilizing microorganisms when used as biofertilizers. Further studies will elucidate impacts of released secondary metabolites on coexisting microorganisms and plants under different environmental conditions.

Combination of long-term 13CO2 labeling and isotopolog profiling allows turnover analysis of photosynthetic pigments in Arabidopsis leaves.

BACKGROUND: Living cells maintain and adjust structural and functional integrity by continual synthesis and degradation of metabolites and macromolecules. The maintenance and adjustment of thylakoid membrane involve turnover of photosynthetic pigments along with subunits of protein complexes. Quantifying their turnover is essential to understand the mechanisms of homeostasis and long-term acclimation of photosynthetic apparatus. Here we report methods combining whole-plant long-term 13CO2 labeling and liquid chromatography - mass spectrometry (LC-MS) analysis to determine the size of non-labeled population (NLP) of carotenoids and chlorophylls (Chl) in leaf pigment extracts of partially 13C-labeled plants. RESULTS: The labeling chamber enabled parallel 13CO2 labeling of up to 15 plants of Arabidopsis thaliana with real-time environmental monitoring ([CO2], light intensity, temperature, relative air humidity and pressure) and recording. No significant difference in growth or photosynthetic pigment composition was found in leaves after 7-d exposure to normal CO2 (~ 400 ppm) or 13CO2 in the labeling chamber, or in ambient air outside the labeling chamber (control). Following chromatographic separation of the pigments and mass peak assignment by high-resolution Fourier-transform ion cyclotron resonance MS, mass spectra of photosynthetic pigments were analyzed by triple quadrupole MS to calculate NLP. The size of NLP remaining after the 7-d 13CO2 labeling was ~ 10.3% and ~ 11.5% for all-trans- and 9-cis-ß-carotene, ~ 21.9% for lutein, ~ 18.8% for Chl a and 33.6% for Chl b, highlighting non-uniform turnover of these pigments in thylakoids. Comparable results were obtained in all replicate plants of the 13CO2 labeling experiment except for three that were showing anthocyanin accumulation and growth impairment due to insufficient water supply (leading to stomatal closure and less 13C incorporation). CONCLUSIONS: Our methods allow 13CO2 labeling and estimation of NLP for photosynthetic pigments with high reproducibility despite potential variations in [13CO2] between the experiments. The results indicate distinct turnover rates of carotenoids and Chls in thylakoid membrane, which can be investigated in the future by time course experiments. Since 13C enrichment can be measured in a range of compounds, long-term 13CO2 labeling chamber, in combination with appropriate MS methods, facilitates turnover analysis of various metabolites and macromolecules in plants on a time scale of hours to days.

Structural, mechanistic, and physiological insights into phospholipase A-mediated membrane phospholipid degradation in Pseudomonas aeruginosa.

Cells steadily adapt their membrane glycerophospholipid (GPL) composition to changing environmental and developmental conditions. While the regulation of membrane homeostasis via GPL synthesis in bacteria has been studied in detail, the mechanisms underlying the controlled degradation of endogenous GPLs remain unknown. Thus far, the function of intracellular phospholipases A (PLAs) in GPL remodeling (Lands cycle) in bacteria is not clearly established. Here, we identified the first cytoplasmic membrane-bound phospholipase A1 (PlaF) from Pseudomonas aeruginosa, which might be involved in the Lands cycle. PlaF is an important virulence factor, as the P. aeruginosa ΔplaF mutant showed strongly attenuated virulence in Galleria mellonella and macrophages. We present a 2.0-Å-resolution crystal structure of PlaF, the first structure that reveals homodimerization of a single-pass transmembrane (TM) full-length protein. PlaF dimerization, mediated solely through the intermolecular interactions of TM and juxtamembrane regions, inhibits its activity. The dimerization site and the catalytic sites are linked by an intricate ligand-mediated interaction network, which might explain the product (fatty acid) feedback inhibition observed with the purified PlaF protein. We used molecular dynamics simulations and configurational free energy computations to suggest a model of PlaF activation through a coupled monomerization and tilting of the monomer in the membrane, which constrains the active site cavity into contact with the GPL substrates. Thus, these data show the importance of the PlaF-mediated GPL remodeling pathway for virulence and could pave the way for the development of novel therapeutics targeting PlaF.

A phospholipase B from Pseudomonas aeruginosa with activity towards endogenous phospholipids affects biofilm assembly.

Pseudomonas aeruginosa is a severe threat to immunocompromised patients due to its numerous virulence factors and biofilm-mediated multidrug resistance. It produces and secretes various toxins with hydrolytic activities including phospholipases. However, the function of intracellular phospholipases for bacterial virulence has still not been established. Here, we demonstrate that the hypothetical gene pa2927 of P. aeruginosa encodes a novel phospholipase B named PaPlaB. At reaction equilibrium, PaPlaB purified from detergent-solubilized membranes of E. coli released fatty acids (FAs) from sn-1 and sn-2 positions of phospholipids at the molar ratio of 51:49. PaPlaB in vitro hydrolyzed P. aeruginosa phospholipids reconstituted in detergent micelles and phospholipids reconstituted in vesicles. Cellular localization studies indicate that PaPlaB is a cell-bound PLA of P. aeruginosa and that it is peripherally bound to both membranes in E. coli, yet the active form was predominantly associated with the cytoplasmic membrane of E. coli. Decreasing the concentration of purified and detergent-stabilized PaPlaB leads to increased enzymatic activity, and at the same time triggers oligomer dissociation. We showed that the free FA profile, biofilm amount and architecture of the wild type and ΔplaB differ. However, it remains to be established how the PLB activity of PaPlaB is regulated by homooligomerisation and how it relates to the phenotype of the P. aeruginosa ΔplaB. This novel putative virulence factor contributes to our understanding of phospholipid degrading enzymes and might provide a target for new therapeutics against P. aeruginosa biofilms.

Impact of Moderate Cold and Salt Stress on the Accumulation of Antioxidant Flavonoids in the Leaves of Two Capsicum Cultivars.

The horticultural production of bell peppers generates large quantities of residual biomass. Abiotic stress stimulates the production of protective flavonoids, so the deliberate application of stress to the plants after fruit harvest could provide a strategy to valorize horticultural residuals by increasing flavonoid concentrations, facilitating their industrial extraction. Here we exposed two Capsicum cultivars, a chilli and a bell pepper, to cold and salt stress and combinations thereof to determine their valorization potential. Noninvasive image-based phenotyping and multiparametric fluorescence measurements indicated that all stress treatments inhibited plant growth and reduced the leaf chlorophyll fluorescence index, with the chilli cultivar showing greater sensitivity. The fluorescence-based FLAV index allowed the noninvasive assessment of foliar luteolin glycosides. High-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis showed that moderate cold increased the levels of two foliar antioxidant luteolin glycosides in both cultivars, with bell pepper containing the highest amounts (induced to maximum 5.5 mg g-1 DW cynaroside and 37.0 mg g-1 DW graveobioside A) after combined stress treatment. These data confirm the potential of abiotic stress for the valorization of residual leaf biomass to enhance the industrial extraction of antioxidant and bioactive flavonoids.

Carotenoid Isotopolog Profiling in 13C-Labeled Leaf Extracts by LC-MS and LC-FTICR-MS.

Mass spectrometry (MS)-based metabolite analysis combined with stable isotope labeling offers a powerful tool to study dynamic regulation of metabolic pathways and metabolite fluxes in biological systems. Here we describe a method to analyze the composition of carotenoid isotopologs in 13C-labeled leaf extracts by using liquid chromatography (LC)-MS and LC-Fourier transform ion cyclotron resonance (FTICR)-MS. High mass resolution of the latter enables unambiguous assignment of observed mass to a unique chemical formula. Based on peak intensity the relative abundance and the degree of 13C labeling are calculated for individual carotenoid isotopologs.

Direct Analysis of Underivatized Amino Acids in Plant Extracts by LC-MS/MS (Improved Method).

In this chapter we describe a method for quantification of 20 proteinogenic amino acids by liquid chromatography-mass spectrometry which affords neither derivatization nor the use of organic solvents. Analysis of the underivatized amino acids is performed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) in the positive ESI mode. Separation is achieved on a strong cation exchange (SCX) column (Luna 5 µ SCX 100 Å) with 5% acetic acid in water (A) and 75 mM ammonium acetate in water (B). Quantification is accomplished by use of d2-phenylalanine as internal standard achieving limits of detection of 5-50 nM. The method was successfully applied for the determination of proteinogenic amino acids in plant extracts.

Tomato's Green Gold: Bioeconomy Potential of Residual Tomato Leaf Biomass as a Novel Source for the Secondary Metabolite Rutin.

At the end of the annual horticultural production cycle of greenhouse-grown crops, large quantities of residual biomass are discarded. Here, we propose a new value chain to utilize horticultural leaf biomass for the extraction of secondary metabolites. To increase the secondary metabolite content of leaves, greenhouse-grown crop plants were exposed to low-cost abiotic stress treatments after the last fruit harvest. As proof of concept, we evaluated the production of the flavonoid rutin in tomato plants subjected to nitrogen deficiency. In an interdisciplinary approach, we observed the steady accumulation of rutin in young plants under nitrogen deficiency, tested the applicability of nitrogen deficiency in a commercial-like greenhouse, developed a high efficiency extraction for rutin, and evaluated the acceptance of the proposed value chain by its key actors economically. On the basis of the positive interdisciplinary evaluation, we identified opportunities and challenges for the successful establishment of horticultural leaf biomass as a novel source for secondary metabolites.

Analysis of alkaloids in single plant cells by capillary electrophoresis.

In this chapter, capillary electrophoresis (CE) is demonstrated to be a useful technique for the determination of alkaloids in microsamples of single plant cells. A single cell sampling technique with microcapillaries that includes extraction of sample volumes in the pl range from single cells, division into aliquots, addition of internal standard, and injection into the CE capillary is described. The danger of contamination and evaporation of such low sample volumes has been avoided by handling them under an inert protective layer of silicone oil. For the determination of alkaloids in cell samples, CE with direct ultraviolet detection using a high concentration of citric acid as background electrolyte provides sufficient sensitivity.

Analysis of amino acids without derivatization in barley extracts by LC-MS-MS.

A method has been developed for quantification of 20 amino acids as well as 13 (15)N-labeled amino acids in barley plants. The amino acids were extracted from plant tissues using aqueous HCl-ethanol and directly analyzed without further purification. Analysis of the underivatized amino acids was performed by liquid chromatography (LC)-electrospray ionization (ESI) tandem mass spectrometry (MS-MS) in the positive ESI mode. Separation was achieved on a strong cation exchange column (Luna 5micro SCX 100A) with 30 mM ammonium acetate in water (solvent A) and 5% acetic acid in water (solvent B). Quantification was accomplished using d (2)-Phe as an internal standard. Calibration curves were linear over the range 0.5-50 microM, and limits of detection were estimated to be 0.1-3.0 microM. The mass-spectrometric technique was employed to study the regulation of amino acid levels in barley plants grown at 15 degrees C uniform root temperature (RT) and 20-10 degrees C vertical RT gradient (RTG). The LC-MS-MS results demonstrated enhanced concentration of free amino acids in shoots at 20-10 degrees C RTG, while total free amino acid concentration in roots was similarly low for both RT treatments. (15)NO(3) (-) labeling experiments showed lower (15)N/(14)N ratios for Glu, Ser, Ala and Val in plants grown at 20-10 degrees C RTG compared with those grown at 15 degrees C RT.

Column temperature as an active variable in the isocratic, normal-phase high-performance liquid chromatography separation of lipophilic metabolites of nonylphenol ethoxylates.

Normal-phase separation of technical grade nonylphenol (t-NP, about 90% 4-nonylphenol), 4-nonylphenol mono-ethoxylate (4-NP1EO) and 4-nonylphenol di-ethoxylate (4-NP2EO) was assessed, with the inclusion of column temperature as an active variable. The compound 2,4,6-trimethylphenol was evaluated for use as internal standard. Isocratic elution with 2-propanol/hexanes mixtures from an amino-silica column and spectrometric UV detection at 277 nm were employed. Technical nonylphenol presented a significant contribution from unknown substances that eluted with retention times similar to that of 4-NP1EO. GC-MS analysis of the unknowns allowed to identify them as isomers of 2-NP. The response of the system to joint variations in flow rate, eluent composition and column temperature was investigated by means of Doehlert statistical experimental design. A model for retention of the analytes as a function of the experimental variables was proposed, and separation selectivity was studied. Selection of the optimal working zone was made through desirability function (D) calculations. Potential co-elution of 2-NP isomers with 4-NP1EO was considered when optimizing the separation. The occurrence of a restricted region of the experimental space where baseline resolution of analytes, associated impurities and internal standard results feasible (D not equal to 0) is apparent.

OrganoCat pretreatment of perennial plants: Synergies between a biogenic fractionation and valuable feedstocks.

A successful biorefinery needs to align suitable pretreatment with sustainable production of biomasses. Herein, four perennial plants, (Sida, Silphium, Miscanthus and Szarvasi) regarded as promising feedstocks for biorefineries were subjected to the OrganoCat pretreatment. The technology was successfully applied to the different perennial plants revealing that pretreatment of grasses was more efficient than of non-grasses. Thorough analyses of the lignocellulose - before and after fractionation - enabled a detailed description of the fate of cellulosic, non-cellulosic polysaccharides and lignin during the pretreatment. Especially Szarvasi pulp displayed outstanding results in terms of fractionation efficiency and enzymatic digestibility, though in all cases successful lignocellulose fractionation was observed. These insights into the structural composition of different perennial plant species and the impact of the OrganoCat pretreatment on the plant material leads to useful information to strategically adapt such processes to the individual lignocellulosic material aiming for a full valorisation.

Separation of nonylphenol ethoxylates and nonylphenol by non-aqueous capillary electrophoresis.

Capillary electrophoresis based on non-aqueous solvent background electrolytes was employed, with single and multiple wavelength UV detection, to evaluate discrimination among oligomer components of mixtures of non-ionic, long chain nonylphenol ethoxylates (NPnEO, with n = number of ethoxy units) and their lipophilic degradation products. The tested organic solvents included acetonitrile, methanol, ethanol, 1- and 2-propanol, 1-butanol and tetrahydrofurane in the presence of sodium acetate. A rational variation of composition of background electrolyte solvent mixtures allowed to modify the mobility of electroosmotic flow and the type and degree of interactions between the ionic additive (sodium acetate) and the components of the analyte mixtures. The physicochemical properties of the solvents, such as dielectric constant, viscosity and electron donor-acceptor ability regarding the additive, were considered to improve the resolution of lipophilic compounds with less than three ethoxy groups and the discrimination attainable for longer chain oligomers. The studied methodologies also allowed discerning between surfactants of similar (nominal) ethoxy chain lengths. This was demonstrated by the different peak distribution patterns observed for NPnEO compounds with n = 7.5 and 10, respectively.

An efficient laboratory workflow for environmental risk assessment of organic chemicals.

In this study, we demonstrate a fast and efficient workflow to investigate the transformation mechanism of organic chemicals and evaluate the toxicity of their transformation products (TPs) in laboratory scale. The transformation process of organic chemicals was first simulated by electrochemistry coupled online to mass spectrometry (EC-MS). The simulated reactions were scaled up in a batch EC reactor to receive larger amounts of a reaction mixture. The mixture sample was purified and concentrated by solid phase extraction (SPE) for the further ecotoxicological testing. The combined toxicity of the reaction mixture was evaluated in fish egg test (FET) (Danio rerio) compared to the parent compound. The workflow was verified with carbamazepine (CBZ). By using EC-MS seven primary TPs of CBZ were identified; the degradation mechanism was elucidated and confirmed by comparison to literature. The reaction mixture and one primary product (acridine) showed higher ecotoxicity in fish egg assay with 96 h EC50 values of 1.6 and 1.0 mg L(-1) than CBZ with the value of 60.8 mg L(-1). The results highlight the importance of transformation mechanism study and toxicological effect evaluation for organic chemicals brought into the environment since transformation of them may increase the toxicity. The developed process contributes a fast and efficient laboratory method for the risk assessment of organic chemicals and their TPs.