S1 basic leucine zipper transcription factors shape plant architecture by controlling C/N partitioning to apical and lateral organs.

Plants tightly control growth of their lateral organs, which led to the concept of apical dominance. However, outgrowth of the dormant lateral primordia is sensitive to the plant's nutritional status, resulting in an immense plasticity in plant architecture. While the impact of hormonal regulation on apical dominance is well characterized, the prime importance of sugar signaling to unleash lateral organ formation has just recently emerged. Here, we aimed to identify transcriptional regulators, which control the trade-off between growth of apical versus lateral organs. Making use of locally inducible gain-of-function as well as single and higher-order loss-of-function approaches of the sugar-responsive S1-basic-leucine-zipper (S1-bZIP) transcription factors, we disclosed their largely redundant function in establishing apical growth dominance. Consistently, comprehensive phenotypical and analytical studies of S1-bZIP mutants show a clear shift of sugar and organic nitrogen (N) allocation from apical to lateral organs, coinciding with strong lateral organ outgrowth. Tissue-specific transcriptomics reveal specific clade III SWEET sugar transporters, crucial for long-distance sugar transport to apical sinks and the glutaminase GLUTAMINE AMIDO-TRANSFERASE 1_2.1, involved in N homeostasis, as direct S1-bZIP targets, linking the architectural and metabolic mutant phenotypes to downstream gene regulation. Based on these results, we propose that S1-bZIPs control carbohydrate (C) partitioning from source leaves to apical organs and tune systemic N supply to restrict lateral organ formation by C/N depletion. Knowledge of the underlying mechanisms controlling plant C/N partitioning is of pivotal importance for breeding strategies to generate plants with desired architectural and nutritional characteristics.

The evolutionarily conserved kinase SnRK1 orchestrates resource mobilization during Arabidopsis seedling establishment.

The onset of plant life is characterized by a major phase transition. During early heterotrophic seedling establishment, seed storage reserves fuel metabolic demands, allowing the plant to switch to autotrophic metabolism. Although metabolic pathways leading to storage compound mobilization are well-described, the regulatory circuits remain largely unresolved. Using an inducible knockdown approach of the evolutionarily conserved energy master regulator Snf1-RELATED-PROTEIN-KINASE1 (SnRK1), phenotypic studies reveal its crucial function in Arabidopsis thaliana seedling establishment. Importantly, glucose feeding largely restores growth defects of the kinase mutant, supporting its major impact in resource mobilization. Detailed metabolite studies reveal sucrose as a primary resource early in seedling establishment, in a SnRK1-independent manner. Later, SnRK1 orchestrates catabolism of triacylglycerols and amino acids. Concurrent transcriptomic studies highlight SnRK1 functions in controlling metabolic hubs fuelling gluconeogenesis, as exemplified by cytosolic PYRUVATE ORTHOPHOSPHATE DIKINASE (cyPPDK). Here, SnRK1 establishes its function via phosphorylation of the transcription factor BASIC LEUCINE ZIPPER63 (bZIP63), which directly targets and activates the cyPPDK promoter. Taken together, our results disclose developmental and catabolic functions of SnRK1 in seed storage mobilization and describe a prototypic gene regulatory mechanism. As seedling establishment is important for plant vigor and crop yield, our findings are of agronomical importance.

High triacylglycerol turnover is required for efficient opening of stomata during heat stress in Arabidopsis.

Heat stress triggers the accumulation of triacylglycerols in Arabidopsis leaves, which increases basal thermotolerance. However, how triacylglycerol synthesis is linked to thermotolerance remains unclear and the mechanisms involved remain to be elucidated. It has been shown that triacylglycerol and starch degradation are required to provide energy for stomatal opening induced by blue light at dawn. To investigate whether triacylglycerol turnover is involved in heat-induced stomatal opening during the day, we performed feeding experiments with labeled fatty acids. Heat stress strongly induced both triacylglycerol synthesis and degradation to channel fatty acids destined for peroxisomal ß-oxidation through the triacylglycerol pool. Analysis of mutants defective in triacylglycerol synthesis or peroxisomal fatty acid uptake revealed that triacylglycerol turnover and fatty acid catabolism are required for heat-induced stomatal opening in illuminated leaves. We show that triacylglycerol turnover is continuous (1.2 mol% per min) in illuminated leaves even at 22°C. The ß-oxidation of triacylglycerol-derived fatty acids generates C2 carbon units that are channeled into the tricarboxylic acid pathway in the light. In addition, carbohydrate catabolism is required to provide oxaloacetate as an acceptor for peroxisomal acetyl-CoA and maintain the tricarboxylic acid pathway for energy and amino acid production during the day.

Perturbations in plant energy homeostasis prime lateral root initiation via SnRK1-bZIP63-ARF19 signaling.

Plants adjust their energy metabolism to continuous environmental fluctuations, resulting in a tremendous plasticity in their architecture. The regulatory circuits involved, however, remain largely unresolved. In Arabidopsis, moderate perturbations in photosynthetic activity, administered by short-term low light exposure or unexpected darkness, lead to increased lateral root (LR) initiation. Consistent with expression of low-energy markers, these treatments alter energy homeostasis and reduce sugar availability in roots. Here, we demonstrate that the LR response requires the metabolic stress sensor kinase Snf1-RELATED-KINASE1 (SnRK1), which phosphorylates the transcription factor BASIC LEUCINE ZIPPER63 (bZIP63) that directly binds and activates the promoter of AUXIN RESPONSE FACTOR19 (ARF19), a key regulator of LR initiation. Consistently, starvation-induced ARF19 transcription is impaired in bzip63 mutants. This study highlights a positive developmental function of SnRK1. During energy limitation, LRs are initiated and primed for outgrowth upon recovery. Hence, this study provides mechanistic insights into how energy shapes the agronomically important root system.

Tobacco leaf tissue rapidly detoxifies direct salt loads without activation of calcium and SOS signaling.

Salt stress is a major abiotic stress, responsible for declining agricultural productivity. Roots are regarded as hubs for salt detoxification, however, leaf salt concentrations may exceed those of roots. How mature leaves manage acute sodium chloride (NaCl) stress is mostly unknown. To analyze the mechanisms for NaCl redistribution in leaves, salt was infiltrated into intact tobacco leaves. It initiated pronounced osmotically-driven leaf movements. Leaf downward movement caused by hydro-passive turgor loss reached a maximum within 2 h. Salt-driven cellular water release was accompanied by a transient change in membrane depolarization but not an increase in cytosolic calcium ion (Ca2+ ) level. Nonetheless, only half an hour later, the leaves had completely regained turgor. This recovery phase was characterized by an increase in mesophyll cell plasma membrane hydrogen ion (H+ ) pumping, a salt uptake-dependent cytosolic alkalization, and a return of the apoplast osmolality to pre-stress levels. Although, transcript numbers of abscisic acid- and Salt Overly Sensitive pathway elements remained unchanged, salt adaptation depended on the vacuolar H+ /Na+ -exchanger NHX1. Altogether, tobacco leaves can detoxify sodium ions (Na+ ) rapidly even under massive salt loads, based on pre-established posttranslational settings and NHX1 cation/H+ antiport activity. Unlike roots, signaling and processing of salt stress in tobacco leaves does not depend on Ca2+ signaling.

Natural variation of warm temperature-induced raffinose accumulation identifies TREHALOSE-6-PHOSPHATE SYNTHASE 1 as a modulator of thermotolerance.

High-temperature stress limits plant growth and reproduction. Exposure to high temperature, however, also elicits a physiological response, which protects plants from the damage evoked by heat. This response involves a partial reconfiguration of the metabolome including the accumulation of the trisaccharide raffinose. In this study, we explored the intraspecific variation of warm temperature-induced raffinose accumulation as a metabolic marker for temperature responsiveness with the aim to identify genes that contribute to thermotolerance. By combining raffinose measurements in 250 Arabidopsis thaliana accessions following a mild heat treatment with genome-wide association studies, we identified five genomic regions that were associated with the observed trait variation. Subsequent functional analyses confirmed a causal relationship between TREHALOSE-6-PHOSPHATE SYNTHASE 1 (TPS1) and warm temperature-dependent raffinose synthesis. Moreover, complementation of the tps1-1 null mutant with functionally distinct TPS1 isoforms differentially affected carbohydrate metabolism under more severe heat stress. While higher TPS1 activity was associated with reduced endogenous sucrose levels and thermotolerance, disruption of trehalose 6-phosphate signalling resulted in higher accumulation of transitory starch and sucrose and was associated with enhanced heat resistance. Taken together, our findings suggest a role of trehalose 6-phosphate in thermotolerance, most likely through its regulatory function in carbon partitioning and sucrose homoeostasis.

Plant apocarotenoid metabolism utilizes defense mechanisms against reactive carbonyl species and xenobiotics.

Carotenoid levels in plant tissues depend on the relative rates of synthesis and degradation of the molecules in the pathway. While plant carotenoid biosynthesis has been extensively characterized, research on carotenoid degradation and catabolism into apocarotenoids is a relatively novel field. To identify apocarotenoid metabolic processes, we characterized the transcriptome of transgenic Arabidopsis (Arabidopsis thaliana) roots accumulating high levels of ß-carotene and, consequently, ß-apocarotenoids. Transcriptome analysis revealed feedback regulation on carotenogenic gene transcripts suitable for reducing ß-carotene levels, suggesting involvement of specific apocarotenoid signaling molecules originating directly from ß-carotene degradation or after secondary enzymatic derivatizations. Enzymes implicated in apocarotenoid modification reactions overlapped with detoxification enzymes of xenobiotics and reactive carbonyl species (RCS), while metabolite analysis excluded lipid stress response, a potential secondary effect of carotenoid accumulation. In agreement with structural similarities between RCS and ß-apocarotenoids, RCS detoxification enzymes also converted apocarotenoids derived from ß-carotene and from xanthophylls into apocarotenols and apocarotenoic acids in vitro. Moreover, glycosylation and glutathionylation-related processes and translocators were induced. In view of similarities to mechanisms found in crocin biosynthesis and cellular deposition in saffron (Crocus sativus), our data suggest apocarotenoid metabolization, derivatization and compartmentalization as key processes in (apo)carotenoid metabolism in plants.

Under salt stress guard cells rewire ion transport and abscisic acid signaling.

Soil salinity is an increasingly global problem which hampers plant growth and crop yield. Plant productivity depends on optimal water-use efficiency and photosynthetic capacity balanced by stomatal conductance. Whether and how stomatal behavior contributes to salt sensitivity or tolerance is currently unknown. This work identifies guard cell-specific signaling networks exerted by a salt-sensitive and salt-tolerant plant under ionic and osmotic stress conditions accompanied by increasing NaCl loads. We challenged soil-grown Arabidopsis thaliana and Thellungiella salsuginea plants with short- and long-term salinity stress and monitored genome-wide gene expression and signals of guard cells that determine their function. Arabidopsis plants suffered from both salt regimes and showed reduced stomatal conductance while Thellungiella displayed no obvious stress symptoms. The salt-dependent gene expression changes of guard cells supported the ability of the halophyte to maintain high potassium to sodium ratios and to attenuate the abscisic acid (ABA) signaling pathway which the glycophyte kept activated despite fading ABA concentrations. Our study shows that salinity stress and even the different tolerances are manifested on a single cell level. Halophytic guard cells are less sensitive than glycophytic guard cells, providing opportunities to manipulate stomatal behavior and improve plant productivity.

Elevated Levels of Phosphorylated Sphingobases Do Not Antagonize Sphingobase- or Fumonisin B1-Induced Plant Cell Death.

Long-chain bases (LCBs), also termed sphingobases, are building blocks of sphingolipids, which make up a significant proportion of the cellular membrane system. They are also bioactive molecules regulating intracellular processes. Elevated levels of LCBs like phytosphingosine and dihydrosphingosine can induce cell death in plants and correlate with programmed cell death (PCD) reactions after pathogen recognition. We investigated the previously hypothesized antagonism between phosphorylated and nonphosphorylated LCBs with respect to cell death in Arabidopsis thaliana. Using HPLC-MS/MS, we determined levels of phosphorylated and nonphosphorylated LCBs after cell death induction by LCB application or by Fumonisin B1 (FB1) treatment. We show that previously reported antagonistic effects of phosphorylated LCBs after simultaneous application with nonphosphorylated LCBs are linked to reduced uptake of nonphosphorylated LCBs into the tissue. Furthermore, phosphorylated LCBs did not antagonize PCD induced by avirulence protein recognition. In a functional approach, we used Arabidopsis lines with perturbed levels of phosphorylated LCBs. In these plants, the degree of FB1-induced cell death did not consistently correlate negatively with levels of phosphorylated LCBs, but positively with levels of major nonphosphorylated LCBs phytosphingosine and dihydrosphingosine. As treatment with phosphorylated LCBs did not antagonize cell death, and elevated in vivo levels of these LCB species did not reduce FB1-induced cell death, we conclude that the hypothesized general cell death-antagonizing effect of phosphorylated LCBs in plant cell death reactions should be rejected. Instead, our time-course analysis of LCB levels during cell death reactions showed a positive correlation between levels of nonphosphorylated LCBs and cell death.

Infection Rates and Alkaloid Patterns of Different Grass Species with Systemic Epichloë Endophytes.

Symbiotic Epichloë species are fungal endophytes of cool-season grasses that can produce alkaloids with toxicity to vertebrates and/or invertebrates. Monitoring infections and presence of alkaloids in grasses infected with Epichloë species can provide an estimate of possible intoxication risks for livestock. We sampled 3,046 individuals of 13 different grass species in three regions on 150 study sites in Germany. We determined infection rates and used PCR to identify Epichloë species diversity based on the presence of different alkaloid biosynthesis genes, then confirmed the possible chemotypes with high-performance liquid chromatography (HPLC)/ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) measurements. Infections of Epichloë spp. were found in Festuca pratensis Huds. (81%), Festuca ovina L. aggregate (agg.) (73%), Lolium perenne L. (15%), Festuca rubra L. (15%) and Dactylis glomerata L. (8%). The other eight grass species did not appear to be infected. For the majority of Epichloë-infected L. perenne samples (98%), the alkaloids lolitrem B and peramine were present, but ergovaline was not detected, which was consistent with the genetic evaluation, as dmaW, the gene encoding the first step of the ergot alkaloid biosynthesis pathway, was absent. Epichloë uncinata in F. pratensis produced anti-insect loline compounds. The Epichloë spp. observed in the F. ovina agg. samples showed the greatest level of diversity, and different intermediates of the indole-diterpene pathway could be detected. Epichloë infection rates alone are insufficient to estimate intoxication risks for livestock, as other factors, like the ability of the endophyte to produce the alkaloids, also need to be assessed.IMPORTANCE Severe problems of livestock intoxication from Epichloë-infected forage grasses have been reported from New Zealand, Australia, and the United States, but much less frequently from Europe, and particularly not from Germany. Nevertheless, it is important to monitor infection rates and alkaloids of grasses with Epichloë fungi to estimate possible intoxication risks. Most studies focus on agricultural grass species like Lolium perenne and Festuca arundinacea, but other cool-season grass species can also be infected. We show that in Germany, infection rates and alkaloids differ between grass species and that some of the alkaloids can be toxic to livestock. Changes in grassland management due to changing climate, especially with a shift toward grasslands dominated with Epichloë-infected species such as Lolium perenne, may result in greater numbers of intoxicated livestock in the near future. We therefore suggest regular monitoring of grass species for infections and alkaloids and call for maintaining heterogenous grasslands for livestock.

Staphylococcus aureus Exploits a Non-ribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes.

Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection.

Engineering of new-to-nature halogenated indigo precursors in plants.

Plants are versatile chemists producing a tremendous variety of specialized compounds. Here, we describe the engineering of entirely novel metabolic pathways in planta enabling generation of halogenated indigo precursors as non-natural plant products. Indican (indolyl-ß-D-glucopyranoside) is a secondary metabolite characteristic of a number of dyers plants. Its deglucosylation and subsequent oxidative dimerization leads to the blue dye, indigo. Halogenated indican derivatives are commonly used as detection reagents in histochemical and molecular biology applications; their production, however, relies largely on chemical synthesis. To attain the de novo biosynthesis in a plant-based system devoid of indican, we employed a sequence of enzymes from diverse sources, including three microbial tryptophan halogenases substituting the amino acid at either C5, C6, or C7 of the indole moiety. Subsequent processing of the halotryptophan by bacterial tryptophanase TnaA in concert with a mutant of the human cytochrome P450 monooxygenase 2A6 and glycosylation of the resulting indoxyl derivatives by an endogenous tobacco glucosyltransferase yielded corresponding haloindican variants in transiently transformed Nicotiana benthamiana plants. Accumulation levels were highest when the 5-halogenase PyrH was utilized, reaching 0.93 ±â€¯0.089 mg/g dry weight of 5-chloroindican. The identity of the latter was unambiguously confirmed by NMR analysis. Moreover, our combinatorial approach, facilitated by the modular assembly capabilities of the GoldenBraid cloning system and inspired by the unique compartmentation of plant cells, afforded testing a number of alternative subcellular localizations for pathway design. In consequence, chloroplasts were validated as functional biosynthetic venues for haloindican, with the requisite reducing augmentation of the halogenases as well as the cytochrome P450 monooxygenase fulfilled by catalytic systems native to the organelle. Thus, our study puts forward a viable alternative production platform for halogenated fine chemicals, eschewing reliance on fossil fuel resources and toxic chemicals. We further contend that in planta generation of halogenated indigoid precursors previously unknown to nature offers an extended view on and, indeed, pushes forward the established frontiers of biosynthetic capacity of plants.

Crosstalk between Two bZIP Signaling Pathways Orchestrates Salt-Induced Metabolic Reprogramming in Arabidopsis Roots.

Soil salinity increasingly causes crop losses worldwide. Although roots are the primary targets of salt stress, the signaling networks that facilitate metabolic reprogramming to induce stress tolerance are less understood than those in leaves. Here, a combination of transcriptomic and metabolic approaches was performed in salt-treated Arabidopsis thaliana roots, which revealed that the group S1 basic leucine zipper transcription factors bZIP1 and bZIP53 reprogram primary C- and N-metabolism. In particular, gluconeogenesis and amino acid catabolism are affected by these transcription factors. Importantly, bZIP1 expression reflects cellular stress and energy status in roots. In addition to the well-described abiotic stress response pathway initiated by the hormone abscisic acid (ABA) and executed by SnRK2 (Snf1-RELATED-PROTEIN-KINASE2) and AREB-like bZIP factors, we identify a structurally related ABA-independent signaling module consisting of SnRK1s and S1 bZIPs. Crosstalk between these signaling pathways recruits particular bZIP factor combinations to establish at least four distinct gene expression patterns. Understanding this signaling network provides a framework for securing future crop productivity.

Enzymatic and Non-Enzymatic Mechanisms Contribute to Lipid Oxidation During Seed Aging.

Storage of seeds is accompanied by loss of germination and oxidation of storage and membrane lipids. A lipidomic analysis revealed that during natural and artificial aging of Arabidopsis seeds, levels of several diacylglycerols and free fatty acids, such as linoleic acid and linolenic acid as well as free oxidized fatty acids and oxygenated triacylglycerols, increased. Lipids can be oxidized by enzymatic or non-enzymatic processes. In the enzymatic pathway, lipoxygenases (LOXs) catalyze the first oxygenation step of polyunsaturated fatty acids. Analysis of lipid levels in mutants with defects in the two 9-LOX genes revealed that the strong increase in free 9-hydroxy- and 9-keto-fatty acids is dependent on LOX1 but not LOX5. Fatty acid oxidation correlated with an aging-induced decrease of germination, raising the question of whether these oxylipins negatively regulate germination. However, seeds of the lox1 mutant were only slightly more tolerant to aging, indicating that 9-LOX products contribute to but are not the major cause of loss of germination during aging. In contrast to free oxidized fatty acids, accumulation of oxygenated triacylglycerols upon accelerated aging was mainly based on non-enzymatic oxidation of seed storage lipids.

Enhanced aphid abundance in spring desynchronizes predator-prey and plant-microorganism interactions.

Climate change leads to phenology shifts of many species. However, not all species shift in parallel, which can desynchronize interspecific interactions. Within trophic cascades, herbivores can be top-down controlled by predators or bottom-up controlled by host plant quality and host symbionts, such as plant-associated micro-organisms. Synchronization of trophic levels is required to prevent insect herbivore (pest) outbreaks. In a common garden experiment, we simulated an earlier arrival time (~2 weeks) of the aphid Rhopalosiphum padi on its host grass Lolium perenne by enhancing the aphid abundance during the colonization period. L. perenne was either uninfected or infected with the endophytic fungus Epichloë festucae var. lolii. The plant symbiotic fungus produces insect deterring alkaloids within the host grass. Throughout the season, we tested the effects of enhanced aphid abundance in spring on aphid predators (top-down) and grass-endophyte (bottom-up) responses. Higher aphid population sizes earlier in the season lead to overall higher aphid abundances, as predator occurrence was independent of aphid abundances on the pots. Nonetheless, after predator occurrence, aphids were controlled within 2 weeks on all pots. Possible bottom-up control of aphids by increased endophyte concentrations occurred time delayed after high herbivore abundances. Endophyte-derived alkaloid concentrations were not significantly affected by enhanced aphid abundance but increased throughout the season. We conclude that phenology shifts in an herbivorous species can desynchronize predator-prey and plant-microorganism interactions and might enhance the probability of pest outbreaks with climate change.

2-cysteine peroxiredoxins and thylakoid ascorbate peroxidase create a water-water cycle that is essential to protect the photosynthetic apparatus under high light stress conditions.

Different peroxidases, including 2-cysteine (2-Cys) peroxiredoxins (PRXs) and thylakoid ascorbate peroxidase (tAPX), have been proposed to be involved in the water-water cycle (WWC) and hydrogen peroxide (H2O2)-mediated signaling in plastids. We generated an Arabidopsis (Arabidopsis thaliana) double-mutant line deficient in the two plastid 2-Cys PRXs (2-Cys PRX A and B, 2cpa 2cpb) and a triple mutant deficient in 2-Cys PRXs and tAPX (2cpa 2cpb tapx). In contrast to wild-type and tapx single-knockout plants, 2cpa 2cpb double-knockout plants showed an impairment of photosynthetic efficiency and became photobleached under high light (HL) growth conditions. In addition, double-mutant plants also generated elevated levels of superoxide anion radicals, H2O2, and carbonylated proteins but lacked anthocyanin accumulation under HL stress conditions. Under HL conditions, 2-Cys PRXs seem to be essential in maintaining the WWC, whereas tAPX is dispensable. By comparison, this HL-sensitive phenotype was more severe in 2cpa 2cpb tapx triple-mutant plants, indicating that tAPX partially compensates for the loss of functional 2-Cys PRXs by mutation or inactivation by overoxidation. In response to HL, H2O2- and photooxidative stress-responsive marker genes were found to be dramatically up-regulated in 2cpa 2cpb tapx but not 2cpa 2cpb mutant plants, suggesting that HL-induced plastid to nucleus retrograde photooxidative stress signaling takes place after loss or inactivation of the WWC enzymes 2-Cys PRX A, 2-Cys PRX B, and tAPX.

Two fatty acid desaturases, STEAROYL-ACYL CARRIER PROTEIN Δ9-DESATURASE6 and FATTY ACID DESATURASE3, are involved in drought and hypoxia stress signaling in Arabidopsis crown galls.

Agrobacterium tumefaciens-derived crown galls of Arabidopsis (Arabidopsis thaliana) contain elevated levels of unsaturated fatty acids and strongly express two fatty acid desaturase genes, ω3 FATTY ACID DESATURASE3 (FAD3) and STEAROYL-ACYL CARRIER PROTEIN Δ9-DESATURASE6 (SAD6). The fad3-2 mutant with impaired α-linolenic acid synthesis developed significantly smaller crown galls under normal, but not under high, relative humidity. This strongly suggests that FAD3 plays a role in increasing drought stress tolerance of crown galls. SAD6 is a member of the SAD family of as yet unknown function. Expression of the SAD6 gene is limited to hypoxia, a physiological condition found in crown galls. As no sad6 mutant exists and to link the function of SAD6 with fatty acid desaturation in crown galls, the lipid pattern was analyzed of plants with constitutive SAD6 overexpression (SAD6-OE). SAD6-OE plants contained lower stearic acid and higher oleic acid levels, which upon reduction of SAD6 overexpression by RNA interference (SAD6-OE-RNAi) regained wild-type-like levels. The development of crown galls was not affected either in SAD6-OE or SAD6-OE-RNAi or by RNA interference in crown galls. Since biochemical analysis of SAD6 in yeast (Saccharomyces cerevisiae) and Escherichia coli failed, SAD6 was ectopically expressed in the background of the well-known suppressor of salicylic acid-insensitive2 (ssi2-2) mutant to confirm the desaturase function of SAD6. All known ssi2-2 phenotypes were rescued, including the high stearic acid level. Thus, our findings suggest that SAD6 functions as a Δ9-desaturase, and together with FAD3 it increases the levels of unsaturated fatty acids in crown galls under hypoxia and drought stress conditions.

Lipid profiling of the Arabidopsis hypersensitive response reveals specific lipid peroxidation and fragmentation processes: biogenesis of pimelic and azelaic acid.

Lipid peroxidation (LPO) is induced by a variety of abiotic and biotic stresses. Although LPO is involved in diverse signaling processes, little is known about the oxidation mechanisms and major lipid targets. A systematic lipidomics analysis of LPO in the interaction of Arabidopsis (Arabidopsis thaliana) with Pseudomonas syringae revealed that LPO is predominantly confined to plastid lipids comprising galactolipid and triacylglyceride species and precedes programmed cell death. Singlet oxygen was identified as the major cause of lipid oxidation under basal conditions, while a 13-lipoxygenase (LOX2) and free radical-catalyzed lipid oxidation substantially contribute to the increase upon pathogen infection. Analysis of lox2 mutants revealed that LOX2 is essential for enzymatic membrane peroxidation but not for the pathogen-induced free jasmonate production. Despite massive oxidative modification of plastid lipids, levels of nonoxidized lipids dramatically increased after infection. Pathogen infection also induced an accumulation of fragmented lipids. Analysis of mutants defective in 9-lipoxygenases and LOX2 showed that galactolipid fragmentation is independent of LOXs. We provide strong in vivo evidence for a free radical-catalyzed galactolipid fragmentation mechanism responsible for the formation of the essential biotin precursor pimelic acid as well as of azelaic acid, which was previously postulated to prime the immune response of Arabidopsis. Our results suggest that azelaic acid is a general marker for LPO rather than a general immune signal. The proposed fragmentation mechanism rationalizes the pathogen-induced radical amplification and formation of electrophile signals such as phytoprostanes, malondialdehyde, and hexenal in plastids.

Peramine and lolitrem B from endophyte-grass associations cascade up the food chain.

Endophytic fungi in cool-season grass species produce herbivore-toxic alkaloids, which are assumed to harm higher trophic levels along food chains. Previous studies have shown fitness disadvantages for higher trophic levels that feed on aphids that were exclusively reared on perennial ryegrass (Lolium perenne) infected with the endophytic fungus Neotyphodium lolii. However, it is unknown whether the alkaloids produced by the fungus-grass association can be assimilated by plant sap-sucking insects like aphids. Using an ultra high performance liquid chromatography method combined with mass spectrometry, we provide the first evidence that the alkaloids peramine and lolitrem B are present in aphids (Rhopalosiphum padi) and in aphid predators when the aphids are reared on endophyte-infected grass. We conclude that alkaloids can enter the plant sap of the grass and are responsible for longer pupal stages of the ladybird Harmonia axyridis and for fitness disadvantages of aphids and their predators as shown in previous studies.

Design of Glycoengineered IL-4 Antagonists Employing Chemical and Biosynthetic Glycosylation.

Interleukin-4 (IL-4) plays a key role in atopic diseases. It coordinates T-helper cell differentiation to subtype 2, thereby directing defense toward humoral immunity. Together with Interleukin-13, IL-4 further induces immunoglobulin class switch to IgE. Antibodies of this type activate mast cells and basophilic and eosinophilic granulocytes, which release pro-inflammatory mediators accounting for the typical symptoms of atopic diseases. IL-4 and IL-13 are thus major targets for pharmaceutical intervention strategies to treat atopic diseases. Besides neutralizing antibodies against IL-4, IL-13, or its receptors, IL-4 antagonists can present valuable alternatives. Pitrakinra, an Escherichia coli-derived IL-4 antagonist, has been evaluated in clinical trials for asthma treatment in the past; however, deficits such as short serum lifetime and potential immunogenicity among others stopped further development. To overcome such deficits, PEGylation of therapeutically important proteins has been used to increase the lifetime and proteolytic stability. As an alternative, glycoengineering is an emerging strategy used to improve pharmacokinetics of protein therapeutics. In this study, we have established different strategies to attach glycan moieties to defined positions in IL-4. Different chemical attachment strategies employing thiol chemistry were used to attach a glucose molecule at amino acid position 121, thereby converting IL-4 into a highly effective antagonist. To enhance the proteolytic stability of this IL-4 antagonist, additional glycan structures were introduced by glycoengineering utilizing eucaryotic expression. IL-4 antagonists with a combination of chemical and biosynthetic glycoengineering could be useful as therapeutic alternatives to IL-4 neutralizing antibodies already used to treat atopic diseases.