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.

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.

The circadian clock is required for rhythmic lipid transport in Drosophila in interaction with diet and photic condition.

Modern lifestyle is often at odds with endogenously driven rhythmicity, which can lead to circadian disruption and metabolic syndrome. One signature for circadian disruption is a reduced or altered metabolite cycling in the circulating tissue reflecting the current metabolic status. Drosophila is a well-established model in chronobiology, but day-time dependent variations of transport metabolites in the fly circulation are poorly characterized. Here, we sampled fly hemolymph throughout the day and analyzed diacylglycerols (DGs), phosphoethanolamines (PEs) and phosphocholines (PCs) using LC-MS. In wild-type flies kept on sugar-only medium under a light-dark cycle, all transport lipid species showed a synchronized bimodal oscillation pattern with maxima at the beginning and end of the light phase which were impaired in period01 clock mutants. In wild-type flies under constant dark conditions, the oscillation became monophasic with a maximum in the middle of the subjective day. In strong support of clock-driven oscillations, levels of the targeted lipids peaked once in the middle of the light phase under time-restricted feeding independent of the time of food intake. When wild-type flies were reared on full standard medium, the rhythmic alterations of hemolymph lipid levels were greatly attenuated. Our data suggest that the circadian clock aligns daily oscillations of DGs, PEs, and PCs in the hemolymph to the anabolic siesta phase, with a strong influence of light on phase and modality.

The major trimeric antenna complexes serve as a site for qH-energy dissipation in plants.

Plants and algae are faced with a conundrum: harvesting sufficient light to drive their metabolic needs while dissipating light in excess to prevent photodamage, a process known as nonphotochemical quenching. A slowly relaxing form of energy dissipation, termed qH, is critical for plants' survival under abiotic stress; however, qH location in the photosynthetic membrane is unresolved. Here, we tested whether we could isolate subcomplexes from plants in which qH was induced that would remain in an energy-dissipative state. Interestingly, we found that chlorophyll (Chl) fluorescence lifetimes were decreased by qH in isolated major trimeric antenna complexes, indicating that they serve as a site for qH-energy dissipation and providing a natively quenched complex with physiological relevance to natural conditions. Next, we monitored the changes in thylakoid pigment, protein, and lipid contents of antenna with active or inactive qH but did not detect any evident differences. Finally, we investigated whether specific subunits of the major antenna complexes were required for qH but found that qH was insensitive to trimer composition. Because we previously observed that qH can occur in the absence of specific xanthophylls, and no evident changes in pigments, proteins, or lipids were detected, we tentatively propose that the energy-dissipative state reported here may stem from Chl-Chl excitonic interaction.

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.

Loss of function in the Drosophila clock gene period results in altered intermediary lipid metabolism and increased susceptibility to starvation.

The fruit fly Drosophila is a prime model in circadian research, but still little is known about its circadian regulation of metabolism. Daily rhythmicity in levels of several metabolites has been found, but knowledge about hydrophobic metabolites is limited. We here compared metabolite levels including lipids between period01 (per01) clock mutants and Canton-S wildtype (WTCS) flies in an isogenic and non-isogenic background using LC-MS. In the non-isogenic background, metabolites with differing levels comprised essential amino acids, kynurenines, pterinates, glycero(phospho)lipids, and fatty acid esters. Notably, detectable diacylglycerols (DAG) and acylcarnitines (AC), involved in lipid metabolism, showed lower levels in per01 mutants. Most of these differences disappeared in the isogenic background, yet the level differences for AC as well as DAG were consistent for fly bodies. AC levels were dependent on the time of day in WTCS in phase with food consumption under LD conditions, while DAGs showed weak daily oscillations. Two short-chain ACs continued to cycle even in constant darkness. per01 mutants in LD showed no or very weak diel AC oscillations out of phase with feeding activity. The low levels of DAGs and ACs in per01 did not correlate with lower total food consumption, body mass or weight. Clock mutant flies showed higher sensitivity to starvation independent of their background-dependent activity level. Our results suggest that neither feeding, energy storage nor mobilisation is significantly affected in per01 mutants, but point towards impaired mitochondrial activity, supported by upregulation of the mitochondrial stress marker 4EBP in the clock mutants.

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.

Phospholipid:Diacylglycerol Acyltransferase-Mediated Triacylglyerol Synthesis Augments Basal Thermotolerance.

High temperatures rapidly induce a genetically programmed heat-shock response (HSR) that is essential to establish short-term acquired thermotolerance. In addition, an immediate HSR-independent metabolic response is triggered, resulting in an accumulation of unsaturated triacylglycerols (TAGs) in the cytosol. The metabolic processes involved in heat-induced TAG formation in plants and their physiological significance remain to be clarified. Lipidomic analyses of Arabidopsis (Arabidopsis thaliana) seedlings indicated that during heat stress, polyunsaturated fatty acids from thylakoid galactolipids are incorporated into cytosolic TAGs. In addition, rapid conversion of plastidic monogalactosyl diacylglycerols (MGDGs) into oligogalactolipids, acylated MGDGs, and diacylglycerols (DAGs), the direct precursor of TAGs, was observed. For TAG synthesis, DAG requires a fatty acid from the acyl-CoA pool or phosphatidylcholine. Since seedlings deficient in PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE1 (PDAT1) were unable to accumulate TAGs after heat stress, phosphatidylcholine appears to be the major fatty acid donor. Results suggest that rapid plastid lipid metabolism drives TAG accumulation during heat stress. PDAT1-mediated TAG accumulation was found to increase heat resistance, since nonacclimated pdat1 mutant seedlings were more sensitive to severe heat stress, as indicated by a more dramatic decline of the maximum efficiency of PSII and lower seedling survival compared to wild-type seedlings. In contrast, nonacclimated trigalactosyldiacylglycerol1 (tgd1) mutants overaccumulating TAGs and oligogalactolipids were more resistant to heat stress. Hence, thylakoid lipid metabolism and TAG formation increases thermotolerance in addition to the genetically encoded HSR.

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.

Lipid-Pro: a computational lipid identification solution for untargeted lipidomics on data-independent acquisition tandem mass spectrometry platforms.

UNLABELLED: A major challenge for mass spectrometric-based lipidomics, aiming at describing all lipid species in a biological sample, lies in the computational and bioinformatic processing of the large amount of data that arises after data acquisition. Lipid-Pro is a software tool that supports the identification of lipids by interpreting large datasets generated by liquid chromatography--tandem mass spectrometry (LC-MS/MS) using the advanced data-independent acquisition mode MS(E). In the MS(E) mode, the instrument fragments all molecular ions generated from a sample and records time-resolved molecular ion data as well as fragment ion data for every detectable molecular ion. Lipid-Pro matches the retention time-aligned mass-to-charge ratio data of molecular- and fragment ions with a lipid database and generates a report on all identified lipid species. For generation of the lipid database, Lipid-Pro provides a module for construction of lipid species and their fragments using a flexible building block approach. Hence, Lipid-Pro is an easy to use analysis tool to interpret complex MS(E) lipidomics data and also offers a module to generate a user-specific lipid database. AVAILABILITY AND IMPLEMENTATION: Lipid-Pro is freely available at: http://www.neurogenetics.biozentrum.uni-wuerzburg.de/en/project/services/lipidpro/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

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.

Reactive electrophilic oxylipins trigger a heat stress-like response through HSFA1 transcription factors.

Abiotic and biotic stresses are often characterized by an induction of reactive electrophile species (RES) such as the jasmonate 12-oxo-phytodienoic acid (OPDA) or the structurally related phytoprostanes. Previously, RES oxylipins have been shown massively to induce heat-shock-response (HSR) genes including HSP101 chaperones. Moreover, jasmonates have been reported to play a role in basal thermotolerance. We show that representative HSR marker genes are strongly induced by RES oxylipins through the four master regulator transcription factors HSFA1a, b, d, and e essential for short-term adaptation to heat stress in Arabidopsis. When compared with Arabidopsis seedlings treated at the optimal acclimation temperature of 37 °C, the exogenous application of RES oxylipins at 20 °C induced a much weaker induction of HSP101 at both the gene and protein expression levels which, however, was not sufficient to confer short-term acquired thermotolerance. Moreover, jasmonate-deficient mutant lines displayed a wild-type-like HSR and were not compromised in acquiring thermotolerance. Hence, the OPDA- and RES oxylipin-induced HSR is not sufficient to protect seedlings from severe heat stress but may help plants to cope better with stresses associated with protein unfolding by inducing a battery of chaperones in the absence of heat.

Light-induced acclimation of the Arabidopsis chlorina1 mutant to singlet oxygen.

Singlet oxygen (¹O2) is a reactive oxygen species that can function as a stress signal in plant leaves leading to programmed cell death. In microalgae, ¹O2-induced transcriptomic changes result in acclimation to ¹O2. Here, using a chlorophyll b-less Arabidopsis thaliana mutant (chlorina1 [ch1]), we show that this phenomenon can also occur in vascular plants. The ch1 mutant is highly photosensitive due to a selective increase in the release of ¹O2 by photosystem II. Under photooxidative stress conditions, the gene expression profile of ch1 mutant leaves very much resembled the gene responses to ¹O2 reported in the Arabidopsis mutant flu. Preexposure of ch1 plants to moderately elevated light intensities eliminated photooxidative damage without suppressing ¹O2 formation, indicating acclimation to ¹O2. Substantial differences in gene expression were observed between acclimation and high-light stress: A number of transcription factors were selectively induced by acclimation, and contrasting effects were observed for the jasmonate pathway. Jasmonate biosynthesis was strongly induced in ch1 mutant plants under high-light stress and was noticeably repressed under acclimation conditions, suggesting the involvement of this hormone in ¹O2-induced cell death. This was confirmed by the decreased tolerance to photooxidative damage of jasmonate-treated ch1 plants and by the increased tolerance of the jasmonate-deficient mutant delayed-dehiscence2.

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.