Elevated nutrient supply can exert worse effects on Norway spruce than drought, viewed through chemical defence against needle rust.

Abiotic factors such as water and nutrient availability can exert a dominant influence on the susceptibility of plants to various pathogens. Effects of abiotic environmental factors on phenolic compound concentrations in the plant tissue may represent one of the major underlying mechanisms, as these compounds are known to play a substantial role in plant resistance to pests. In particular, this applies to conifer trees, in which a large range of phenolic compounds are produced constitutively and/or induced by pathogen attack. We subjected Norway spruce saplings to water limitation and elevated nutrient supply over 2 years and subsequently controlled infection with the needle rust Chrysomyxa rhododendri (DC.) de Bary and analysed both constitutive and inducible phenolic compound concentrations in the needles as well as the degree of infection. Compared with the control group, both drought and fertilization profoundly modified the constitutive and pathogen-induced profiles of phenolic compounds, but had little impact on the total phenolic content. Fertilization predominantly affected the inducible phenolic response and led to higher infection rates by C. rhododendri. Drought stress, in contrast, mainly shaped the phenolic profiles in healthy plant parts and had no consequences on the plant susceptibility. The results show that specific abiotic effects on individual compounds seem to be decisive for the infection success of C. rhododendri, whereby the impaired induced response in saplings subjected to nutrient supplementation was most critical. Although drought effects were minor, they varied depending on the time and length of water limitation. The results indicate that prolonged drought periods in the future may not significantly alter the foliar defence of Norway spruce against C. rhododendri, but fertilization, often propagated to increase tree growth and forest productivity, can be counterproductive in areas with high pathogen pressure.

Advances in understanding Norway spruce natural resistance to needle bladder rust infection: transcriptional and secondary metabolites profiling.

BACKGROUND: Needle rust caused by the fungus Chrysomyxa rhododendri causes significant growth decline and increased mortality of young Norway spruce trees in subalpine forests. Extremely rare trees with enhanced resistance represent promising candidates for practice-oriented reproduction approaches. They also enable the investigation of tree molecular defence and resistance mechanisms against this fungal disease. Here, we combined RNA-Seq, RT-qPCR and secondary metabolite analyses during a period of 38 days following natural infection to investigate differences in constitutive and infection-induced defence between the resistant genotype PRA-R and three susceptible genotypes. RESULTS: Gene expression and secondary metabolites significantly differed among genotypes from day 7 on and revealed already known, but also novel candidate genes involved in spruce molecular defence against this pathogen. Several key genes related to (here and previously identified) spruce defence pathways to needle rust were differentially expressed in PRA-R compared to susceptible genotypes, both constitutively (in non-symptomatic needles) and infection-induced (in symptomatic needles). These genes encoded both new and well-known antifungal proteins such as endochitinases and chitinases. Specific genetic characteristics concurred with varying phenolic, terpene, and hormone needle contents in the resistant genotype, among them higher accumulation of several flavonoids (mainly kaempferol and taxifolin), stilbenes, geranyl acetone, α-ionone, abscisic acid and salicylic acid. CONCLUSIONS: Combined transcriptional and metabolic profiling of the Norway spruce defence response to infection by C. rhododendri in adult trees under subalpine conditions confirmed the results previously gained on artificially infected young clones in the greenhouse, both regarding timing and development of infection, and providing new insights into genes and metabolic pathways involved. The comparison of genotypes with different degrees of susceptibility proved that several of the identified key genes are differently regulated in PRA-R, and that the resistant genotype combines a strong constitutive defence with an induced response in infected symptomatic needles following fungal invasion. Genetic and metabolic differences between the resistant and susceptible genotypes indicated a more effective hypersensitive response (HR) in needles of PRA-R that prevents penetration and spread of the rust fungus and leads to a lower proportion of symptomatic needles as well as reduced symptom development on the few affected needles.

Does oxygen affect ageing mechanisms of Pinus densiflora seeds? A matter of cytoplasmic physical state.

During desiccation, the cytoplasm of orthodox seeds solidifies into an intracellular glass with highly restricted diffusion and molecular mobility. Temperature and water content govern seed ageing rates, while oxygen (O2) can promote deteriorative reactions. However, whether the cytoplasmic physical state affects involvement of O2 in seed ageing remains unresolved. We aged Pinus densiflora seeds by controlled deterioration (CD) at 45 °C and distinct relative humidity (RH), resulting in cells with a glassy (11% and 30% RH) or fluid (60% and 80% RH) cytoplasm. Hypoxic conditions (0.4% O2) during CD delayed seed deterioration, lipid peroxidation, and decline of antioxidants (glutathione, α-tocopherol, and γ-tocopherol), but only when the cytoplasm was glassy. In contrast, when the cytoplasm was fluid, seeds deteriorated at the same rate regardless of O2 availability, while being associated with limited lipid peroxidation, detoxification of lipid peroxide products, substantial loss of glutathione, and resumption of glutathione synthesis. Changes in metabolite profiles provided evidence of other O2-independent enzymatic reactions in a fluid cytoplasm, including aldo-keto reductase and glutamate decarboxylase activities. Biochemical profiles of seeds stored under seed bank conditions resembled those obtained after CD regimes that maintained a glassy cytoplasm. Overall, O2 contributed more to seed ageing when the cytoplasm was glassy, rather than fluid.

Plant Parasites under Pressure: Effects of Abiotic Stress on the Interactions between Parasitic Plants and Their Hosts.

Parasitic angiosperms, comprising a diverse group of flowering plants, are partially or fully dependent on their hosts to acquire water, mineral nutrients and organic compounds. Some have detrimental effects on agriculturally important crop plants. They are also intriguing model systems to study adaptive mechanisms required for the transition from an autotrophic to a heterotrophic metabolism. No less than any other plant, parasitic plants are affected by abiotic stress factors such as drought and changes in temperature, saline soils or contamination with metals or herbicides. These effects may be attributed to the direct influence of the stress, but also to diminished host availability and suitability. Although several studies on abiotic stress response of parasitic plants are available, still little is known about how abiotic factors affect host preferences, defense mechanisms of both hosts and parasites and the effects of combinations of abiotic and biotic stress experienced by the host plants. The latter effects are of specific interest as parasitic plants pose additional pressure on contemporary agriculture in times of climate change. This review summarizes the existing literature on abiotic stress response of parasitic plants, highlighting knowledge gaps and discussing perspectives for future research and potential agricultural applications.

Redox feedback regulation of ANAC089 signaling alters seed germination and stress response.

The interplay between the phytohormone abscisic acid (ABA) and the gasotransmitter nitric oxide (NO) regulates seed germination and post-germinative seedling growth. We show that GAP1 (germination in ABA and cPTIO 1) encodes the transcription factor ANAC089 with a critical membrane-bound domain and extranuclear localization. ANAC089 mutants lacking the membrane-tethered domain display insensitivity to ABA, salt, and osmotic and cold stresses, revealing a repressor function. Whole-genome transcriptional profiling and DNA-binding specificity reveals that ANAC089 regulates ABA- and redox-related genes. ANAC089 truncated mutants exhibit higher NO and lower ROS and ABA endogenous levels, alongside an altered thiol and disulfide homeostasis. Consistently, translocation of ANAC089 to the nucleus is directed by changes in cellular redox status after treatments with NO scavengers and redox-related compounds. Our results reveal ANAC089 to be a master regulator modulating redox homeostasis and NO levels, able to repress ABA synthesis and signaling during Arabidopsis seed germination and abiotic stress.

Phytohormone release by three isolated lichen mycobionts and the effects of indole-3-acetic acid on their compatible photobionts.

Evidence is emerging that phytohormones represent key inter-kingdom signalling compounds supporting chemical communication between plants, fungi and bacteria. The roles of phytohormones for the lichen symbiosis are poorly understood, particularly in the process of lichenization, i.e. the key events which lead free-living microalgae and fungi to recognize each other, make physical contact and start developing a lichen thallus. Here, we studied cellular and extracellularly released phytohormones in three lichen mycobionts, Cladonia grayi, Xanthoria parietina and Tephromela atra, grown on solid medium, and the effects of indole-3-acetic acid (IAA) on their respective photobionts, Asterochloris glomerata, Trebouxia decolorans, Trebouxia sp. Using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) we found that mycobionts produced IAA, salicylic acid (SA) and jasmonic acid (JA). IAA represented the most abundant phytohormone produced and released by all mycobionts, whereas SA was released by X. parietina and T. atra, and JA was released by C. grayi only. With a half-life of 5.2 days, IAA degraded exponentially in solid BBM in dim light. When IAA was exogenously offered to the mycobionts' compatible photobionts at "physiological" concentrations (as released by their respective mycobionts and accumulated in the medium over seven days), the photobionts' water contents increased up to 4.4%. Treatment with IAA had no effects on the maximum quantum yield of photosystem II, dry mass, and the contents of photosynthetic pigments and α-tocopherol of the photobionts. The data presented may be useful for designing studies aimed at elucidating the roles of phytohormones in lichens.

Abundance and Extracellular Release of Phytohormones in Aero-terrestrial Microalgae (Trebouxiophyceae, Chlorophyta) As a Potential Chemical Signaling Source1.

Phytohormones are pivotal signaling compounds in higher plants, in which they exert their roles intracellularly, but are also released for cell-to-cell communication. In unicellular organisms, extracellularly released phytohormones can be involved in chemical crosstalk with other organisms. However, compared to higher plants, hardly any knowledge is available on the roles of phytohormones in green algae. Here, we studied phytohormone composition and extracellular release in aero-terrestrial Trebouxiophyceae. We investigated (a) which phytohormones are produced and if they are released extracellularly, and if extracellular phytohormone levels are (b) affected by environmental stimuli, and (c) differ between lichen-forming and non-lichen-forming species. Three free-living microalgae (Apatococcus lobatus, Chloroidium ellipsoideum, and Myrmecia bisecta) and three lichen-forming microalgae (Asterochloris glomerata, Trebouxia decolorans, and Trebouxia sp.) were studied. Algae were grown on solid media and the following cellular phytohormones were identified by ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS): indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), abscisic acid (ABA), gibberellin A4 (GA4 ), and zeatin (ZT). Furthermore, IAA, IBA, ABA, jasmonic acid (JA), gibberellin A3 (GA3 ), and GA4 were found to be released extracellularly. IAA and ABA were released by all six species, and IAA was the most concentrated. Phytohormone release was affected by light and water availability, especially IAA in A. glomerata, Trebouxia sp., and C. ellipsoideum. No clear patterns were observed between lichen-forming and non-lichen-forming species. The results are envisaged to contribute valuable baseline information for further studies into the roles of phytohormones in microalgae.

RNA-Seq and secondary metabolite analyses reveal a putative defence-transcriptome in Norway spruce (Picea abies) against needle bladder rust (Chrysomyxa rhododendri) infection.

BACKGROUND: Norway spruce trees in subalpine forests frequently face infections by the needle rust fungus Chrysomyxa rhododendri, which causes significant growth decline and increased mortality of young trees. Yet, it is unknown whether trees actively respond to fungal attack by activating molecular defence responses and/or respective gene expression. RESULTS: Here, we report results from an infection experiment, in which the transcriptomes (via RNA-Seq analysis) and phenolic profiles (via UHPLC-MS) of control and infected trees were compared over a period of 39 days. Gene expression between infected and uninfected ramets significantly differed after 21 days of infection and revealed already known, but also novel candidate genes involved in spruce molecular defence against pathogens. CONCLUSIONS: Combined RNA-Seq and biochemical data suggest that Norway spruce response to infection by C. rhododendri is restricted locally and primarily activated between 9 and 21 days after infestation, involving a potential isolation of the fungus by a hypersensitive response (HR) associated with an activation of phenolic pathways. Identified key regulatory genes represent a solid basis for further specific analyses in spruce varieties with varying susceptibility, to better characterise resistant clones and to elucidate the resistance mechanism.

The non-photochemical quenching protein LHCSR3 prevents oxygen-dependent photoinhibition in Chlamydomonas reinhardtii.

Non-photochemical quenching (NPQ) helps dissipate surplus light energy, preventing formation of reactive oxygen species (ROS). In Chlamydomonas reinhardtii, the thylakoid membrane protein LHCSR3 is involved in pH-dependent (qE-type) NPQ, lacking in the npq4 mutant. Preventing PSII repair revealed that npq4 lost PSII activity faster than the wild type (WT) in elevated O2, while no difference between strains was observed in O2-depleted conditions. Low Fv/Fm values remained 1.5 h after moving cells out of high light, and this qH-type quenching was independent of LHCSR3 and not accompanied by losses of maximum PSII activity. Culturing cells in historic O2 atmospheres (30-35%) increased the qE of cells, due to increased LHCSR1 and PsbS levels, and LHCSR3 in the WT, showing that atmospheric O2 tensions regulate qE capacity. Colony growth of npq4 was severely restricted at elevated O2, and npq4 accumulated more reactive electrophile species (RES) than the WT, which could damage PSI. Levels of PsaA (PSI) were lower in npq4 grown at 35% O2, while PsbA (PSII) levels remained stable. We conclude that even at high O2 concentrations, the PSII repair cycle is sufficient to maintain net levels of PSII. However, LHCSR3 has an important function in protecting PSI against O2-mediated damage, such as via RES.

Hydrogen Peroxide Metabolism in Interkingdom Interaction Between Bacteria and Wheat Seeds and Seedlings.

In endophytes, the abundance of genes coding for enzymes processing reactive oxygen species (ROS), including hydrogen peroxide (H2O2), argues for a crucial role of ROS metabolism in plant-microbe interaction for plant colonization. Here, we studied H2O2 metabolism of bread wheat (Triticum aestivum L.) seeds and their microbiota during germination and early seedling growth, the most vulnerable stages in the plant life cycle. Treatment with hot steam diminished the seed microbiota, and these seeds produced less extracellular H2O2 than untreated seeds. Using a culture-dependent approach, Pantoea and Pseudomonas genera were the most abundant epiphytes of dry untreated seeds. Incubating intact seedlings from hot steam-treated seeds with Pantoea strains triggered H2O2 production, whereas Pseudomonas strains dampened H2O2 levels, attributable to higher catalase activities. The genus Pantoea was much less represented among seedling endophytes than genus Pseudomonas, with other endophytic genera, including Bacillus and Paenibacillus, also possessing high catalase activities. Overall, our results show that certain bacteria of the seed microbiota are able to modulate the extracellular redox environment during germination and early seedling growth, and high catalase activity is proposed as a key trait of seed endophytes.

Abscisic acid-determined seed vigour differences do not influence redox regulation during ageing.

High seed quality is a key trait to achieve successful crop establishment required for optimum yield and sustainable production. Seed storage conditions greatly impact two key seed quality traits; seed viability (ability to germinate and produce normal seedlings) and vigour (germination performance). Accumulated oxidative damage accompanies the loss of seed vigour and viability during ageing, indicating that redox control is key to longevity. Here, we studied the effects of controlled deterioration at 40°C and 75% relative humidity (RH) ('ageing') under two different O2 concentrations (21 and 78% O2) in Brassica oleracea Two B. oleracea genotypes with allelic differences at two QTLs that result in differences in abscisic acid (ABA) signalling and seed vigour were compared. Ageing led to a similar loss in germination speed in both genotypes that was lost faster under elevated O2 In both genotypes, an equal oxidative shift in the glutathione redox state and a minor loss of α-tocopherol progressively occurred before seed viability was lost. In contrast, ABA levels were not affected by ageing. In conclusion, both ABA signalling and seed ageing impact seed vigour but not necessarily through the same biochemical mechanisms.

Redox poise and metabolite changes in bread wheat seeds are advanced by priming with hot steam.

Fast and uniform germination is key to agricultural production and can be achieved by seed 'priming' techniques. Here, we characterised the responses of bread wheat (Triticum aestivum L.) seeds to a hot steam treatment ('BioFlash'), which accelerated water uptake, resulting in faster germination and seedling growth, typical traits of primed seed. Before the completion of germination, metabolite profiling of seeds revealed advanced accumulation of several amino acids (especially cysteine and serine), sugars (ribose, glucose), and organic acids (glycerate, succinate) in hot steam-treated seeds, whereas sugar alcohols (e.g. arabitol, mannitol) and trehalose decreased in all seeds. Tocochromanols (the 'vitamin E family') rose independently of the hot steam treatment. We further assessed shifts in the half-cell reduction potentials of low-molecular-weight (LMW) thiol-disulfide redox couples [i.e. glutathione disulfide (GSSG)/glutathione (GSH) and cystine/cysteine], alongside the activities of the reactive oxygen species (ROS)-processing enzyme superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase. Upon the first 4 h of imbibition, a rapid conversion of LMW disulfides to thiols occurred. Completion of germination was associated with a re-oxidation of the LMW thiol-disulfide cellular redox environment, before more reducing conditions were re-established during seedling growth, accompanied by an increase in all ROS-processing enzyme activities. Furthermore, changes in the thiol-disulfide cellular redox state were associated to specific stages of wheat seed germination. In conclusion, the priming effect of the hot steam treatment advanced the onset of seed metabolism, including redox shifts associated with germination and seedling growth.

Distress and eustress of reactive electrophiles and relevance to light stress acclimation via stimulation of thiol/disulphide-based redox defences.

Photosynthetic organisms suffering from light stress have to cope with an increased formation of reactive short-chain aldehydes. Singlet oxygen generated from highly-charged reaction centres can peroxidise the poly-unsaturated fatty acid (PUFA)-rich thylakoid membranes they are embedded in. Lipid peroxides decay to release α,ß-unsaturated aldehydes that are reactive electrophile species (RES). Acrolein is one of the most abundant and reactive RES produced in chloroplasts. Here, in the model chlorophyte alga Chlamydomonas reinhardtii, a clear concentration-dependent "distress" induced by acrolein intoxication was observed in conjunction with depletion of the glutathione pool. The glutathione redox state (EGSSG/2GSH) strongly correlated (R2 = 0.95) with decreasing Fv/Fm values of chlorophyll fluorescence. However, treatment of C. reinhardtii with sub-toxic acrolein concentrations increased glutathione concentrations and raised the protein levels of a glutathione-S-transferase (GSTS1), mimicking the response to excess light, indicating that at lower concentrations, acrolein may contribute to high light acclimation, which could be interpreted as "eustress". Furthermore, similar patterns of chloroplastic protein carbonylation occurred under light stress and in response to exogenous acrolein. Priming cells by low doses of acrolein increased the alga's resistance to singlet oxygen. A RNA seq. analysis showed a large overlap in gene regulation under singlet oxygen and acrolein stresses. Particularly enriched were transcripts of enzymes involved in thiol/disulphide exchanges. Some of the genes are regulated by the SOR1 transcription factor, but acrolein treatment still induced an increase in glutathione contents and enhanced singlet oxygen tolerance of the sor1 mutant. The results support a role for RES in chloroplast-to-nucleus retrograde signalling during high light acclimation, with involvement of SOR1 and other pathways.

Foliar Phenolic Compounds in Norway Spruce with Varying Susceptibility to Chrysomyxa rhododendri: Analyses of Seasonal and Infection-Induced Accumulation Patterns.

Secondary phenolic metabolites are involved in plant responses to various biotic stress factors, and are apparently important for the defense against fungal pathogens. In this study, we investigated their role in defense against the rust Chrysomyxa rhododendri in Norway spruce. The fungal pathogen undergoes a seasonal lifecycle with host shift; after overwintering in rhododendron shrubs, it attacks the sprouting current-year spruce needles and causes needle fall in autumn. Repeated infections lead to reduced timber yield and severe problems with rejuvenation in subalpine Norway spruce forests. Trees with varying susceptibility to infection by C. rhododendri were selected and foliar phenolic composition was assessed using UHPLC-MS. We report on seasonal accumulation patterns and infection-related changes in the concentrations of 16 metabolites, including flavonoids, stilbenes, simple phenylpropanoids and the precursor shikimic acid, and their correlation with the infection degree of the tree. We found significant variation in the phenolic profiles during needle development: flavonoids were predominant in the first weeks after sprouting, whereas stilbenes, picein and shikimic acid increased during the first year. Following infection, several flavonoids and resveratrol increased up to 1.8 fold in concentration, whereas picein and shikimic acid were reduced by about 70 and 60%, respectively. The constitutive and early stage infection-induced concentrations of kaempferol, quercetin and taxifolin as well as the late stage infection-induced concentrations of stilbenes and picein were negatively correlated with infection degree. We conclude that a combination of constitutive and inducible accumulation of phenolic compounds is associated with the lower susceptibility of individual trees to C. rhododendri. The potentially fungicidal flavonoid aglycones may limit hyphal growth and prevent development of infection symptoms, and high levels of stilbenes may impede the infection of older needles. The presented results underline a highly compound-specific seasonal accumulation and defense response of Norway spruce and may facilitate the selection of promising trees for breeding programs.

Changes in low-molecular-weight thiol-disulphide redox couples are part of bread wheat seed germination and early seedling growth.

The tripeptide antioxidant glutathione (γ-l-glutamyl-l-cysteinyl-glycine; GSH) essentially contributes to thiol-disulphide conversions, which are involved in the control of seed development, germination, and seedling establishment. However, the relative contribution of GSH metabolism in different seed structures is not fully understood. We studied the GSH/glutathione disulphide (GSSG) redox couple and associated low-molecular-weight (LMW) thiols and disulphides related to GSH metabolism in bread wheat (Triticum aestivum L.) seeds, focussing on redox changes in the embryo and endosperm during germination. In dry seeds, GSH was the predominant LMW thiol and, 15 h after the onset of imbibition, embryos of non-germinated seeds contained 12 times more LMW thiols than the endosperm. In germinated seeds, the embryo contained 17 and 11 times more LMW thiols than the endosperm after 15 and 48 h, respectively. This resulted in the embryo having significantly more reducing half-cell reduction potentials of GSH/GSSG and cysteine (Cys)/cystine (CySS) redox couples (EGSSG/2GSH and ECySS/2Cys, respectively). Upon seed germination and early seedling growth, Cys and CySS concentrations significantly increased in both embryo and endosperm, progressively contributing to the cellular LMW thiol-disulphide redox environment (Ethiol-disulphide). The changes in ECySS/2Cys could be related to the mobilisation of storage proteins in the endosperm during early seedling growth. We suggest that EGSSG/2GSH and ECySS/2Cys can be used as markers of the physiological and developmental stage of embryo and endosperm. We also present a model of interaction between LMW thiols and disulphides with hydrogen peroxide (H2O2) in redox regulation of bread wheat germination and early seedling growth.

Chlamydomonas reinhardtii responding to high light: a role for 2-propenal (acrolein).

High light causes photosystem II to generate singlet oxygen (1 O2 ), a reactive oxygen species (ROS) that can react with membrane lipids, releasing reactive electrophile species (RES), such as acrolein. To investigate how RES may contribute to light stress responses, Chlamydomonas reinhardtii was high light-treated in photoautotrophic and mixotrophic conditions and also in an oxygen-enriched atmosphere to elevate ROS production. The responses were compared to exogenous acrolein. Non-photochemical quenching (NPQ) was higher in photoautotrophic cells, as a consequence of a more de-epoxidized state of the xanthophyll cycle pool and more LHCSR3 protein, showing that photosynthesis was under more pressure than in mixotrophic cells. Photoautotrophic cells had lowered α-tocopherol and ß-carotene contents and a higher level of protein carbonylation, indicators of elevated 1 O2 production. Levels of glutathione, glutathione peroxidase (GPX5) and glutathione-S-transferase (GST1), important antioxidants against RES, were also increased in photoautotrophic cells. In parallel to the wild-type, the LHCSR3-deficient npq4 mutant was high light-treated, which in photoautotrophic conditions exhibited particular sensitivity under elevated oxygen, the treatment that induced the highest RES levels, including acrolein. The npq4 mutant had more GPX5 and GST1 alongside higher levels of carbonylated protein and a more oxidized glutathione redox state. In wild-type cells glutathione contents doubled after 4 h treatment, either with high light under elevated oxygen or with a non-critical dose (600 ppm) of acrolein. Exogenous acrolein also increased GST1 levels, but not GPX5. Overall, RES-associated oxidative damage and glutathione metabolism are prominently associated with light stress and potentially in signaling responses of C. reinhardtii.

Association genetics of phenolic needle compounds in Norway spruce with variable susceptibility to needle bladder rust.

KEY MESSAGE: Accumulation of phenolic needle metabolites in Norway spruce is regulated by many genes with small and additive effects and is correlated with the susceptibility against fungal attack. Norway spruce accumulates high foliar concentrations of secondary phenolic metabolites, with important functions for pathogen defence responses. However, the molecular genetic basis underlying the quantitative variation of phenolic compounds and their role in enhanced resistance of spruce to infection by needle bladder rust are unknown. To address these questions, a set of 1035 genome-wide single nucleotide polymorphisms (SNPs) was associated to the quantitative variation of four simple phenylpropanoids, eight stilbenes, nine flavonoids, six related arithmetic parameters and the susceptibility to infection by Chrysomyxa rhododendri in an unstructured natural population of Norway spruce. Thirty-one significant genetic associations for the flavonoids gallocatechin, kaempferol 3-glucoside and quercetin 3-glucoside and the stilbenes resveratrol, piceatannol, astringin and isorhapontin were discovered, explaining 22-59% of phenotypic variation, and indicating a regulation of phenolic accumulation by many genes with small and additive effects. The phenolics profile differed between trees with high and low susceptibility to the fungus, underlining the importance of phenolic compounds in the defence mechanisms of Norway spruce to C. rhododendri. Results highlight the utility of association studies in non-model tree species and may enable marker-assisted selection of Norway spruce adapted to severe pathogen attack.

Formation of lipid bodies and changes in fatty acid composition upon pre-akinete formation in Arctic and Antarctic Zygnema (Zygnematophyceae, Streptophyta) strains.

Filamentous green algae of the genus Zygnema (Zygnematophyceae, Streptophyta) are key components of polar hydro-terrestrial mats where they face various stressors including UV irradiation, freezing, desiccation and osmotic stress. Their vegetative cells can develop into pre-akinetes, i.e. reserve-rich, mature cells. We investigated lipid accumulation and fatty acid (FA) composition upon pre-akinete formation in an Arctic and an Antarctic Zygnema strain using transmission electron microscopy and gas chromatography coupled with mass spectrometry. Pre-akinetes formed after 9 weeks of cultivation in nitrogen-free medium, which was accompanied by massive accumulation of lipid bodies. The composition of FAs was similar in both strains, and α-linolenic acid (C18:3) dominated in young vegetative cells. Pre-akinete formation coincided with a significant change in FA composition. Oleic (C18:1) and linoleic (C18:2) acid increased the most (up to 17- and 8-fold, respectively). Small amounts of long-chain polyunsaturated FAs were also detected, e.g. arachidonic (C20:4) and eicosapentaenoic (C20:5) acid. Pre-akinetes exposed to desiccation at 86% relative humidity were able to recover maximum quantum yield of photosystem II, but desiccation had no major effect on FA composition. The results are discussed with regard to the capability of Zygnema spp. to thrive in extreme conditions.

A rapid HPLC-MS/MS method for the simultaneous quantification of cyclosporine A, tacrolimus, sirolimus and everolimus in human blood samples.

In the past few years, high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) has matured to a true alternative to antibody-based immunoassays in routine therapeutic drug monitoring. In transplantation medicine, mass spectrometry-based assessment of immunosuppressant drug levels is considered a gold standard diagnostic procedure. We describe a fast state-of-the-art routine online solid-phase extraction (SPE) HPLC-MS/MS analysis platform that allows monitoring of cyclosporine A, tacrolimus, sirolimus and everolimus from 50-microl aliquots of EDTA whole blood specimens within 3.4 min total analysis time. Sample purification is done by offline protein precipitation followed by two automated chromatographic separation steps. Mass spectrometry-based analyte quantification relies on selected reaction monitoring experiments. The assay underwent complete validation and performance evaluation studies and performs very well in several international proficiency testing schemes. In daily routine, it allows reporting of about 75 patient sample results per work shift with a typical total individual sample turnaround time of less than 3 h.

Capillary electrochromatography of biologically relevant flavonoids.

Flavonoids were separated utilizing CEC technique. Baseline separation of biologically relevant flavonoids was obtained using a 100 microm ID fused-silica capillary filled with 3 microm Silica-C18 material and an optimized mobile phase comprising of 20 mM Tris-HCl (pH 6.5), ACN and water at a ratio of 10/40/50 v/v/v. Separations were carried out at 25 kV and a column temperature of 25 degrees C. The influence of relevant parameters for the CEC separation, such as buffer concentration, pH, separation voltage, and ACN concentration, was investigated and optimized. Dependencies of the electroendoosmotic flow (EOF) on these parameters and effects on the resolution of the analytes were studied. During analyses the solvents used for dissolving the samples turned out to have significant effects on the separation of flavonoids. The optimized system was then successfully used for the separation of the flavonoids epicatechin, myricetin, quercetin, naringenin, and hesperetin. CEC turned out to be a useful complementary tool for the economic analysis of flavonoids in addition to common HPLC, muHPLC, and CE methodologies. This method can be used for real applications in phytomics.