Enzymatic one-step synthesis of natural 2-pyrones and new-to-nature derivatives from coenzyme A esters.

The 2-pyrone moiety is present in a wide range of structurally diverse natural products with various biological activities. The plant biosynthetic routes towards these compounds mainly depend on the activity of either type III polyketide synthase-like 2-pyrone synthases or hydroxylating 2-oxoglutarate dependent dioxygenases. In the present study, the substrate specificity of these enzymes is investigated by a systematic screening using both natural and artificial substrates with the aims of efficiently forming (new) products and understanding the underlying catalytic mechanisms. In this framework, we focused on the in vitro functional characterization of a 2-pyrone synthase Gh2PS2 from Gerbera x hybrida and two dioxygenases AtF6'H1 and AtF6'H2 from Arabidopsis thaliana using a set of twenty aromatic and aliphatic CoA esters as substrates. UHPLC-ESI-HRMSn based analyses of reaction intermediates and products revealed a broad substrate specificity of the enzymes, enabling the facile "green" synthesis of this important class of natural products and derivatives in a one-step/one-pot reaction in aqueous environment without the need for halogenated or metal reagents and protective groups. Using protein modeling and substrate docking we identified amino acid residues that seem to be important for the observed product scope.

The activation of iron deficiency responses of grapevine rootstocks is dependent to the availability of the nitrogen forms.

BACKGROUND: In viticulture, iron (Fe) chlorosis is a common abiotic stress that impairs plant development and leads to yield and quality losses. Under low availability of the metal, the applied N form (nitrate and ammonium) can play a role in promoting or mitigating Fe deficiency stresses. However, the processes involved are not clear in grapevine. Therefore, the aim of this study was to investigate the response of two grapevine rootstocks to the interaction between N forms and Fe uptake. This process was evaluated in a hydroponic experiment using two ungrafted grapevine rootstocks Fercal (Vitis berlandieri x V. vinifera) tolerant to deficiency induced Fe chlorosis and Couderc 3309 (V. riparia x V. rupestris) susceptible to deficiency induced Fe chlorosis. RESULTS: The results could differentiate Fe deficiency effects, N-forms effects, and rootstock effects. Interveinal chlorosis of young leaves appeared earlier on 3309 C from the second week of treatment with NO3-/NH4+ (1:0)/-Fe, while Fercal leaves showed less severe symptoms after four weeks of treatment, corresponding to decreased chlorophyll concentrations lowered by 75% in 3309 C and 57% in Fercal. Ferric chelate reductase (FCR) activity was by trend enhanced under Fe deficiency in Fercal with both N combinations, whereas 3309 C showed an increase in FCR activity under Fe deficiency only with NO3-/NH4+ (1:1) treatment. With the transcriptome analysis, Gene Ontology (GO) revealed multiple biological processes and molecular functions that were significantly regulated in grapevine rootstocks under Fe-deficient conditions, with more genes regulated in Fercal responses, especially when both forms of N were supplied. Furthermore, the expression of genes involved in the auxin and abscisic acid metabolic pathways was markedly increased by the equal supply of both forms of N under Fe deficiency conditions. In addition, changes in the expression of genes related to Fe uptake, regulation, and transport reflected the different responses of the two grapevine rootstocks to different N forms. CONCLUSIONS: Results show a clear contribution of N forms to the response of the two grapevine rootstocks under Fe deficiency, highlighting the importance of providing both N forms (nitrate and ammonium) in an appropriate ratio in order to ease the rootstock responses to Fe deficiency.

Changing turn-over rates regulate abundance of tryptophan, GS biosynthesis, IAA transport and photosynthesis proteins in Arabidopsis growth defense transitions.

BACKGROUND: Shifts in dynamic equilibria of the abundance of cellular molecules in plant-pathogen interactions need further exploration. We induced PTI in optimally growing Arabidopsis thaliana seedlings for 16 h, returning them to growth conditions for another 16 h. METHODS: Turn-over and abundance of 99 flg22 responding proteins were measured chronologically using a stable heavy nitrogen isotope partial labeling strategy and targeted liquid chromatography coupled to mass spectrometry (PRM LC-MS). These experiments were complemented by measurements of mRNA and phytohormone levels. RESULTS: Changes in synthesis and degradation rate constants (Ks and Kd) regulated tryptophane and glucosinolate, IAA transport, and photosynthesis-associated protein (PAP) homeostasis in growth/PTI transitions independently of mRNA levels. Ks values increased after elicitation while protein and mRNA levels became uncorrelated. mRNA returned to pre-elicitation levels, yet protein abundance remained at PTI levels even 16 h after media exchange, indicating protein levels were robust and unresponsive to transition back to growth. The abundance of 23 PAPs including FERREDOXIN-NADP( +)-OXIDOREDUCTASE (FNR1) decreased 16 h after PAMP exposure, their depletion was nearly abolished in the myc234 mutant. FNR1 Kd increased as mRNA levels decreased early in PTI, its Ks decreased in prolonged PTI. FNR1 Kd was lower in myc234, mRNA levels decreased as in wild type. CONCLUSIONS: Protein Kd and Ks values change in response to flg22 exposure and constitute an additional layer of protein abundance regulation in growth defense transitions next to changes in mRNA levels. Our results suggest photosystem remodeling in PTI to direct electron flow away from the photosynthetic carbon reaction towards ROS production as an active defense mechanism controlled post-transcriptionally and by MYC2 and homologs. Target proteins accumulated later and PAP and auxin/IAA depletion was repressed in myc234 indicating a positive effect of the transcription factors in the establishment of PTI.

Improved 13C metabolic flux analysis in Escherichia coli metabolism: application of a high-resolution MS (GC-EI-QTOF) for comprehensive assessment of MS/MS fragments.

Gas chromatography-tandem mass spectrometry with electron ionization (GC-EI-MS/MS) provides rich information on stable-isotope labeling for 13C-metabolic flux analysis (13C-MFA). To pave the way for the routine application of tandem MS data for metabolic flux quantification, we aimed to compile a comprehensive library of GC-EI-MS/MS fragments of tert-butyldimethylsilyl (TBDMS) derivatized proteinogenic amino acids. First, we established an analytical workflow that combines high-resolution gas chromatography-quadrupole time-of-flight mass spectrometry and fully 13C-labeled biomass to identify and structurally elucidate tandem MS amino acid fragments. Application of the high-mass accuracy MS procedure resulted into the identification of 129 validated precursor-product ion pairs of 13 amino acids with 30 fragments being accepted for 13C-MFA. The practical benefit of the novel tandem MS data was demonstrated by a proof-of-concept study, which confirmed the importance of the compiled library for high-resolution 13C-MFA. ONE SENTENCE SUMMARY: An analytical workflow that combines high-resolution mass spectrometry (MS) and fully 13C-labeled biomass to identify and structurally elucidate tandem MS amino acid fragments, which provide positional information and therefore offering significant advantages over traditional MS to improve 13C-metabolic flux analysis.

Heterogeneous nutrient supply modulates root exudation and accumulation of medicinally valuable compounds in Artemisia annua and Hypericum perforatum.

Plants have evolved complex mechanisms to adapt to nutrient-deficient environments, including stimulating lateral root proliferation into local soil patches with high nutrient content in response to heterogeneous nutrient distribution. Despite the widespread occurrence of this phenomenon in soil, the effect of heterogeneous nutrient distribution on the accumulation of secondary compounds in plant biomass and their exudation by roots remains largely unknown. This study aims to fill this critical knowledge gap by investigating how deficiency and unequal distributions of nitrogen (N), phosphorus (P), and iron (Fe) affect plant growth and accumulation of the antimalarial drug artemisinin (AN) in leaves and roots of Artemisia annua, as well as AN exudation by roots. Heterogeneous N and P supplies strongly increased root exudation of AN in half of a split-root system exposed to nutrient deficiency. By contrast, exposure to a homogeneous nitrate and phosphate deficiency did not modulate root exudation of AN. This indicates that a combination of local and systemic signals, reflecting low and high nutritional statuses, respectively, were required to enhance AN exudation. This exudation response was independent of the regulation of root hair formation, which was predominantly modulated by the local signal. In contrast to the heterogeneous supply of N and P, heterogeneous Fe supply did not modulate AN root exudation but increased AN accumulation in locally Fe-deficient roots. No modulation of nutrient supply significantly changed the accumulation of AN in A. annua leaves. The impact of a heterogeneous nitrate supply on growth and phytochemical composition was also investigated in Hypericum perforatum plants. Unlike in A. annue, the uneven N supply did not significantly influence the exudation of secondary compounds in the roots of H. perforatum. However, it did enhance the accumulation of several biologically active compounds, such as hypericin, catechin, and rutin isomers, in the leaves of H. perforatum. We propose that the capacity of plants to induce the accumulation and/or differential exudation of secondary compounds under heterogeneous nutrient supply is both species- and compound-specific. The ability to differentially exude AN may contribute to A. annua's adaptation to nutrient disturbances and modulate allelopathic and symbiotic interactions in the rhizosphere.

Impact of in vitro phytohormone treatments on the metabolome of the leafy liverwort Radula complanata (L.) Dumort.

INTRODUCTION: Liverworts are a group of non-vascular plants that possess unique metabolism not found in other plants. Many liverwort metabolites have interesting structural and biochemical characteristics, however the fluctuations of these metabolites in response to stressors is largely unknown. OBJECTIVES: To investigate the metabolic stress-response of the leafy liverwort Radula complanata. METHODS: Five phytohormones were applied exogenously to in vitro cultured R. complanata and an untargeted metabolomic analysis was conducted. Compound classification and identification was performed with CANOPUS and SIRIUS while statistical analyses including PCA, ANOVA, and variable selection using BORUTA were conducted to identify metabolic shifts. RESULTS: It was found that R. complanata was predominantly composed of carboxylic acids and derivatives, followed by benzene and substituted derivatives, fatty acyls, organooxygen compounds, prenol lipids, and flavonoids. The PCA revealed that samples grouped based on the type of hormone applied, and the variable selection using BORUTA (Random Forest) revealed 71 identified and/or classified features that fluctuated with phytohormone application. The stress-response treatments largely reduced the production of the selected primary metabolites while the growth treatments resulted in increased production of these compounds. 4-(3-Methyl-2-butenyl)-5-phenethylbenzene-1,3-diol was identified as a biomarker for the growth treatments while GDP-hexose was identified as a biomarker for the stress-response treatments. CONCLUSION: Exogenous phytohormone application caused clear metabolic shifts in Radula complanata that deviate from the responses of vascular plants. Further identification of the selected metabolite features can reveal metabolic biomarkers unique to liverworts and provide more insight into liverwort stress responses.

Host Tree and Geography Induce Metabolic Shifts in the Epiphytic Liverwort Radula complanata.

Bryophytes are prolific producers of unique, specialized metabolites that are not found in other plants. As many of these unique natural products are potentially interesting, for example, pharmacological use, variations in the production regarding ecological or environmental conditions have not often been investigated. Here, we investigate metabolic shifts in the epiphytic Radula complanata L. (Dumort) with regard to different environmental conditions and the type of phorophyte (host tree). Plant material was harvested from three different locations in Sweden, Germany, and Canada and subjected to untargeted liquid chromatography high-resolution mass-spectrometry (UPLC/ESI-QTOF-MS) and data-dependent acquisition (DDA-MS). Using multivariate statistics, variable selection methods, in silico compound identification, and compound classification, a large amount of variation (39%) in the metabolite profiles was attributed to the type of host tree and 25% to differences in environmental conditions. We identified 55 compounds to vary significantly depending on the host tree (36 on the family level) and 23 compounds to characterize R. complanata in different environments. Taken together, we found metabolic shifts mainly in primary metabolites that were associated with the drought response to different humidity levels. The metabolic shifts were highly specific to the host tree, including mostly specialized metabolites suggesting high levels of ecological interaction. As R. complanata is a widely distributed generalist species, we found it to flexibly adapt its metabolome according to different conditions. We found metabolic composition to also mirror the constitution of the habitat, which makes it interesting for conservation measures.

Light exposure of roots in aeroponics enhances the accumulation of phytochemicals in aboveground parts of the medicinal plants Artemisia annua and Hypericum perforatum.

Light acts as a trigger to enhance the accumulation of secondary compounds in the aboveground part of plants; however, whether a similar triggering effect occurs in roots is unclear. Using an aeroponic setup, we investigated the effect of long-term exposure of roots to LED lighting of different wavelengths on the growth and phytochemical composition of two high-value medicinal plants, Artemisia annua and Hypericum perforatum. In A. annua, root exposure to white, blue, and red light enhanced the accumulation of artemisinin in the shoots by 2.3-, 2.5-, and 1.9-fold, respectively. In H. perforatum, root exposure to white, blue, red, and green light enhanced the accumulation of coumaroylquinic acid in leaves by 89, 65, 84, and 74%, respectively. Root lighting also increased flavonol concentrations. In contrast to its effects in the shoots, root illumination did not change phytochemical composition in the roots or root exudates. Thus, root illumination induces a systemic response, resulting in modulation of the phytochemical composition in distal tissues remote from the light exposure site.

Mechanism of chorismate dehydratase MqnA, the first enzyme of the futalosine pathway, proceeds via substrate-assisted catalysis.

MqnA, the only chorismate dehydratase known so far, catalyzes the initial step in the biosynthesis of menaquinone via the futalosine pathway. Details of the MqnA reaction mechanism remain unclear. Here, we present crystal structures of Streptomyces coelicolor MqnA and its active site mutants in complex with chorismate and the product 3-enolpyruvyl-benzoate, produced during heterologous expression in Escherichia coli. Together with activity studies, our data are in line with dehydration proceeding via substrate assisted catalysis, with the enol pyruvyl group of chorismate acting as catalytic base. Surprisingly, structures of the mutant Asn17Asp with copurified ligand suggest that the enzyme converts to a hydrolase by serendipitous positioning of the carboxyl group. All complex structures presented here exhibit a closed Venus flytrap fold, with the enzyme exploiting the characteristic ligand binding properties of the fold for specific substrate binding and catalysis. The conformational rearrangements that facilitate complete burial of substrate/product, with accompanying topological changes to the enzyme surface, could foster substrate channeling within the biosynthetic pathway.

Bacterial-type ferroxidase tunes iron-dependent phosphate sensing during Arabidopsis root development.

Access to inorganic phosphate (Pi), a principal intermediate of energy and nucleotide metabolism, profoundly affects cellular activities and plant performance. In most soils, antagonistic Pi-metal interactions restrict Pi bioavailability, which guides local root development to maximize Pi interception. Growing root tips scout the essential but immobile mineral nutrient; however, the mechanisms monitoring external Pi status are unknown. Here, we show that Arabidopsis LOW PHOSPHATE ROOT 1 (LPR1), one key determinant of Fe-dependent Pi sensing in root meristems, encodes a novel ferroxidase of high substrate specificity and affinity (apparent KM ∼ 2 µM Fe2+). LPR1 typifies an ancient, Fe-oxidizing multicopper protein family that evolved early upon bacterial land colonization. The ancestor of streptophyte algae and embryophytes (land plants) acquired LPR1-type ferroxidase from soil bacteria via horizontal gene transfer, a hypothesis supported by phylogenomics, homology modeling, and biochemistry. Our molecular and kinetic data on LPR1 regulation indicate that Pi-dependent Fe substrate availability determines LPR1 activity and function. Guided by the metabolic lifestyle of extant sister bacterial genera, we propose that Arabidopsis LPR1 monitors subtle concentration differentials of external Fe availability as a Pi-dependent cue to adjust root meristem maintenance via Fe redox signaling and cell wall modification. We further hypothesize that the acquisition of bacterial LPR1-type ferroxidase by embryophyte progenitors facilitated the evolution of local Pi sensing and acquisition during plant terrestrialization.

A microbiota-root-shoot circuit favours Arabidopsis growth over defence under suboptimal light.

Bidirectional root-shoot signalling is probably key in orchestrating stress responses and ensuring plant survival. Here, we show that Arabidopsis thaliana responses to microbial root commensals and light are interconnected along a microbiota-root-shoot axis. Microbiota and light manipulation experiments in a gnotobiotic plant system reveal that low photosynthetically active radiation perceived by leaves induces long-distance modulation of root bacterial communities but not fungal or oomycete communities. Reciprocally, microbial commensals alleviate plant growth deficiency under low photosynthetically active radiation. This growth rescue was associated with reduced microbiota-induced aboveground defence responses and altered resistance to foliar pathogens compared with the control light condition. Inspection of a set of A. thaliana mutants reveals that this microbiota- and light-dependent growth-defence trade-off is directly explained by belowground bacterial community composition and requires the host transcriptional regulator MYC2. Our work indicates that aboveground stress responses in plants can be modulated by signals from microbial root commensals.

Modulation of Phosphate Deficiency-Induced Metabolic Changes by Iron Availability in Arabidopsis thaliana.

Concurrent suboptimal supply of several nutrients requires the coordination of nutrient-specific transcriptional, phenotypic, and metabolic changes in plants in order to optimize growth and development in most agricultural and natural ecosystems. Phosphate (Pi) and iron (Fe) deficiency induce overlapping but mostly opposing transcriptional and root growth responses in Arabidopsis thaliana. On the metabolite level, Pi deficiency negatively modulates Fe deficiency-induced coumarin accumulation, which is controlled by Fe as well as Pi deficiency response regulators. Here, we report the impact of Fe availability on seedling growth under Pi limiting conditions and on Pi deficiency-induced accumulation of amino acids and organic acids, which play important roles in Pi use efficiency. Fe deficiency in Pi replete conditions hardly changed growth and metabolite profiles in roots and shoots of Arabidopsis thaliana, but partially rescued growth under conditions of Pi starvation and severely modulated Pi deficiency-induced metabolic adjustments. Analysis of T-DNA insertion lines revealed the concerted coordination of metabolic profiles by regulators of Fe (FIT, bHLH104, BRUTUS, PYE) as well as of Pi (SPX1, PHR1, PHL1, bHLH32) starvation responses. The results show the interdependency of Pi and Fe availability and the interplay between Pi and Fe starvation signaling on the generation of plant metabolite profiles.

Hypoglycin A in Cow's Milk-A Pilot Study.

Hypoglycin A (HGA) originating from soapberry fruits (litchi, and ackee) seeds or seedlings from the sycamore maple (SM) tree (related to Sapindaceae) may cause Jamaican vomiting sickness in humans and atypical myopathy in horses and ruminants. A possible transfer into dairy cow's milk cannot be ruled out since the literature has revealed HGA in the milk of mares and in the offal of captured deer following HGA intoxication. From a study, carried out for another purpose, bulk raw milk samples from four randomly selected dairy farms were available. The cows were pastured in the daytime. A sycamore maple tree was found on the pasture of farm No. 1 only. Bulk milk from the individual tank or milk filling station was sampled in parallels and analyzed for HGA by LC-ESI-MS/MS. Measurable concentrations of HGA occurred only in milk from farm No. 1 and amounted to 120 and 489 nmol/L. Despite low and very variable HGA concentrations, the results indicate that the ingested toxin, once eaten, is transferred into the milk. However, it is unknown how much HGA the individual cow ingested during grazing and what amount was transferred into the bulk milk samples. As a prerequisite for a possible future safety assessment, carry-over studies are needed. Furthermore, the toxins' stability during milk processing should also be investigated as well.

Free amino acid contents of selected Ethiopian plant and fungi species: a search for alternative natural free amino acid sources for cosmeceutical applications.

Free amino acids (FAAs), the major constituents of the natural moisturizing factor (NMF), are very important for maintaining the moisture balance of human skin and their deficiency results in dry skin conditions. There is a great interest in the identification and use of nature-based sources of these molecules for such cosmeceutical applications. The objective of the present study was, therefore, to investigate the FAA contents of selected Ethiopian plant and fungi species; and select the best sources so as to use them for the stated purpose. About 59 different plant species and oyster mushroom were included in the study and the concentrations of 27 FAAs were analyzed. Each sample was collected, lyophilized, extracted using aqueous solvent, derivatized with Fluorenylmethoxycarbonyl chloride (Fmoc-Cl) prior to solid-phase extraction and quantified using Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC-ESI-MS/MS) system. All the 27 FAAs were detected in most of the samples. The dominant FAAs that are part of the NMF were found at sufficiently high concentration in the mushroom and some of the plants. This indicates that FAAs that could be included in the preparations for the management of dry skin condition can be obtained from a single natural resource and the use of these resources for the specified purpose have both economic and therapeutic advantage in addition to fulfilling customer needs.

Reshaping of the Arabidopsis thaliana Proteome Landscape and Co-regulation of Proteins in Development and Immunity.

Proteome remodeling is a fundamental adaptive response, and proteins in complexes and functionally related proteins are often co-expressed. Using a deep sampling strategy we define core proteomes of Arabidopsis thaliana tissues with around 10 000 proteins per tissue, and absolutely quantify (copy numbers per cell) nearly 16 000 proteins throughout the plant lifecycle. A proteome-wide survey of global post-translational modification revealed amino acid exchanges pointing to potential conservation of translational infidelity in eukaryotes. Correlation analysis of protein abundance uncovered potentially new tissue- and age-specific roles of entire signaling modules regulating transcription in photosynthesis, seed development, and senescence and abscission. Among others, the data suggest a potential function of RD26 and other NAC transcription factors in seed development related to desiccation tolerance as well as a possible function of cysteine-rich receptor-like kinases (CRKs) as ROS sensors in senescence. All of the components of ribosome biogenesis factor (RBF) complexes were found to be co-expressed in a tissue- and age-specific manner, indicating functional promiscuity in the assembly of these less-studied protein complexes in Arabidopsis.Furthermore, we characterized detailed proteome remodeling in basal immunity by treating Arabidopsis seeldings with flg22. Through simultaneously monitoring phytohormone and transcript changes upon flg22 treatment, we obtained strong evidence of suppression of jasmonate (JA) and JA-isoleucine (JA-Ile) levels by deconjugation and hydroxylation by IAA-ALA RESISTANT3 (IAR3) and JASMONATE-INDUCED OXYGENASE 2 (JOX2), respectively, under the control of JASMONATE INSENSITIVE 1 (MYC2), suggesting an unrecognized role of a new JA regulatory switch in pattern-triggered immunity. Taken together, the datasets generated in this study present extensive coverage of the Arabidopsis proteome in various biological scenarios, providing a rich resource available to the whole plant science community.

An 8 week open-label interventional multicenter study to explore the lung clearance index as endpoint for clinical trials in cystic fibrosis patients ≥8 years of age, chronically infected with Pseudomonas aeruginosa.

BACKGROUND: Forced expiratory volume in 1 second (FEV1) is the only parameter currently recognized as a surrogate endpoint in cystic fibrosis (CF) trials. However, FEV1 is relatively insensitive to changes in the small airways of patients with milder lung disease. This pilot study aimed to explore the lung clearance index (LCI) as a marker for use in efficacy trials with inhaled antibiotics in CF. METHODS: This open-label, single-arm study enrolled CF patients with Pseudomonas aeruginosa infection, who were treated with tobramycin (28-day on/off regime). FEV1, LCI and bacterial load in sputum (CFU) were assessed at baseline, after 1, 4 and 8 weeks of treatment. RESULTS: All patients (n = 17) showed elevated LCI of > 11 despite 3 patients having normal FEV1 (> 90% predicted) at baseline. Overall, LCI improved in 8 (47%) patients and FEV1 in 9 (53%) patients. At week 4, LCI improved by 0.88, FEV1 increased by 0.52%, and P. aeruginosa reduced by 30,481.3 CFU/mL. These changes were however statistically non-significant. Six adverse events occurred in 5/17 (29.4%) patients, most of which were mild-to-moderate in severity. CONCLUSIONS: Due to the low evaluable sample size, no specific trend was observed related to the changes between LCI, FEV1 and CFU. Based on the individual data from this study and from recently published literature, LCI has been shown to be a more sensitive parameter than FEV1 for lung function. LCI can be hypothesized to be an appropriate endpoint for efficacy trials in CF patients if the heterogeneity in lung function is limited by enrolling younger patients or patients with more milder lung disease and thus, limiting the ventilation inhomogeneities. TRIAL REGISTRATION: The study is registered with ClinicalTrials.gov, identifier: NCT02248922.

Sensitive and Selective Amino Acid Profiling of Minute Tissue Amounts by HPLC/Electrospray Negative Tandem Mass Spectrometry Using 9-Fluorenylmethoxycarbonyl (Fmoc-Cl) Derivatization.

A method for selective and sensitive quantification of amino acids is described. The combination of established derivatization procedures of secondary and primary amino groups with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) and subsequent detection of derivatized amino acids by LC-ESI-MS/MS using multiple reaction monitoring provides high selectivity. The attachment of an apolar moiety enables purification of derivatized amino acids from matrix by a single solid-phase extraction step, which increases sensitivity by reduced ion suppression during LC-ESI-MS/MS detection. Additionally, chromatography of all amino acids can be performed on reversed-phase HPLC columns using eluents without additives, which are known to cause significant decreases in signal to noise ratios. The method has been routinely applied for amino acid profiling of low amounts of liquids and tissues of various origins with a sample throughput of about 50-100 samples a day. In addition to a detailed description of the method, some representative examples are presented.

Quantitative Analysis of Free Amino Acids and Urea Derived from Isolated Corneocytes of Healthy Young, Healthy Aged, and Diseased Skin.

BACKGROUND/AIMS: Free amino acids (FAAs) and urea, present inside the corneocytes, can be important indicators of skin condition. However, due to the lack of a standard extraction protocol for FAAs from corneocytes, conflicting research results have been reported. Therefore, the purpose of this study was (1) to standardize the extraction protocol and (2) to investigate FAA profiles in healthy young and healthy old volunteers, as well as in psoriasis and atopic dermatitis patients. METHODS: Skin samples were collected from four groups (healthy young, healthy old, and psoriasis and atopic dermatitis patients) with 5 volunteers per group. Corneocytes were isolated and examined microscopically. FAAs and urea were extracted from the isolated corneocytes, and their amounts were quantified using LC-ESI/MS/MS (after derivatization with Fmoc-Cl) and colorimetric methods, respectively. RESULTS: The micrographs of the corneocytes showed no morphological features attributable to age or disease conditions. The highest and lowest concentrations of total FAAs and urea were observed in the healthy old group and the healthy young group, respectively. Unlike the other FAAs and urea, citrulline was found at a higher level in the healthy young group than in the disease groups. CONCLUSION: This study suggests that the levels of FAAs and urea in the skin are affected by age and skin conditions (healthy/diseased). However, further studies are needed to show the effects of different skin conditions on the levels of FAAs and urea.

Iron and Phosphate Deficiency Regulators Concertedly Control Coumarin Profiles in Arabidopsis thaliana Roots During Iron, Phosphate, and Combined Deficiencies.

Plants face varying nutrient conditions, to which they have to adapt to. Adaptive responses are nutrient-specific and strategies to ensure supply and homeostasis for one nutrient might be opposite to another one, as shown for phosphate (Pi) and iron (Fe) deficiency responses, where many genes are regulated in an opposing manner. This was also observed on the metabolite levels. Whereas root and exudate levels of catechol-type coumarins, phenylpropanoid-derived 2-benzopyranones, which facilitate Fe acquisition, are elevated after Fe deficiency, they are decreased after Pi deficiency. Exposing plants to combined Pi and Fe deficiency showed that the generation of coumarin profiles in Arabidopsis thaliana roots by Pi deficiency considerably depends on the availability of Fe. Similarly, the effect of Fe deficiency on coumarin profiles is different at low compared to high Pi availability. These findings suggest a fine-tuning of coumarin profiles, which depends on Fe and Pi availability. T-DNA insertion lines exhibiting aberrant expression of genes involved in the regulation of Pi starvation responses (PHO1, PHR1, bHLH32, PHL1, SPX1) and Fe starvation responses (BRUTUS, PYE, bHLH104, FIT) were used to analyze the regulation of the generation of coumarin profiles in Arabidopsis thaliana roots by Pi, Fe, and combined Pi and Fe deficiency. The analysis revealed a role of several Fe-deficiency response regulators in the regulation of Fe and of Pi deficiency-induced coumarin profiles as well as for Pi deficiency response regulators in the regulation of Pi and of Fe deficiency-induced coumarin profiles. Additionally, the regulation of Fe deficiency-induced coumarin profiles by Fe deficiency response regulators is influenced by Pi availability. Conversely, regulation of Pi deficiency-induced coumarin profiles by Pi deficiency response regulators is modified by Fe availability.

Balancing trade-offs between biotic and abiotic stress responses through leaf age-dependent variation in stress hormone cross-talk.

In nature, plants must respond to multiple stresses simultaneously, which likely demands cross-talk between stress-response pathways to minimize fitness costs. Here we provide genetic evidence that biotic and abiotic stress responses are differentially prioritized in Arabidopsis thaliana leaves of different ages to maintain growth and reproduction under combined biotic and abiotic stresses. Abiotic stresses, such as high salinity and drought, blunted immune responses in older rosette leaves through the phytohormone abscisic acid signaling, whereas this antagonistic effect was blocked in younger rosette leaves by PBS3, a signaling component of the defense phytohormone salicylic acid. Plants lacking PBS3 exhibited enhanced abiotic stress tolerance at the cost of decreased fitness under combined biotic and abiotic stresses. Together with this role, PBS3 is also indispensable for the establishment of salt stress- and leaf age-dependent phyllosphere bacterial communities. Collectively, our work reveals a mechanism that balances trade-offs upon conflicting stresses at the organism level and identifies a genetic intersection among plant immunity, leaf microbiota, and abiotic stress tolerance.