Blue light promotes ascorbate synthesis by deactivating the PAS/LOV photoreceptor that inhibits GDP-L-galactose phosphorylase.

Ascorbate (vitamin C) is an essential antioxidant in fresh fruits and vegetables. To gain insight into the regulation of ascorbate metabolism in plants, we studied mutant tomato plants (Solanum lycopersicum) that produce ascorbate-enriched fruits. The causal mutation, identified by a mapping-by-sequencing strategy, corresponded to a knock-out recessive mutation in a class of photoreceptor named PAS/LOV protein (PLP), which acts as a negative regulator of ascorbate biosynthesis. This trait was confirmed by CRISPR/Cas9 gene editing and further found in all plant organs, including fruit that accumulated 2 to 3 times more ascorbate than in the WT. The functional characterization revealed that PLP interacted with the 2 isoforms of GDP-L-galactose phosphorylase (GGP), known as the controlling step of the L-galactose pathway of ascorbate synthesis. The interaction with GGP occurred in the cytoplasm and the nucleus, but was abolished when PLP was truncated. These results were confirmed by a synthetic approach using an animal cell system, which additionally demonstrated that blue light modulated the PLP-GGP interaction. Assays performed in vitro with heterologously expressed GGP and PLP showed that PLP is a noncompetitive inhibitor of GGP that is inactivated after blue light exposure. This discovery provides a greater understanding of the light-dependent regulation of ascorbate metabolism in plants.

AtALMT5 mediates vacuolar fumarate import and regulates the malate/fumarate balance in Arabidopsis.

Malate and fumarate constitute a significant fraction of the carbon fixed by photosynthesis, and they are at the crossroad of central metabolic pathways. In Arabidopsis thaliana, they are transiently stored in the vacuole to keep cytosolic homeostasis. The malate and fumarate transport systems of the vacuolar membrane are key players in the control of cell metabolism. Notably, the molecular identity of these transport systems remains mostly unresolved. We used a combination of imaging, electrophysiology and molecular physiology to identify an important molecular actor of dicarboxylic acid transport across the tonoplast. Here, we report the function of the A. thaliana Aluminium-Activated Malate Transporter 5 (AtALMT5). We characterised its ionic transport properties, expression pattern, localisation and function in vivo. We show that AtALMT5 is expressed in photosynthetically active tissues and localised in the tonoplast. Patch-clamp and in planta analyses demonstrated that AtALMT5 is an ion channel-mediating fumarate loading of the vacuole. We found in almt5 plants a reduced accumulation of fumarate in the leaves, in parallel with increased malate concentrations. These results identified AtALMT5 as an ion channel-mediating fumarate transport in the vacuoles of mesophyll cells and regulating the malate/fumarate balance in Arabidopsis.

Microbiota responses to mutations affecting NO homeostasis in Arabidopsis thaliana.

Interactions between plants and microorganisms are pivotal for plant growth and productivity. Several plant molecular mechanisms that shape these microbial communities have been identified. However, the importance of nitric oxide (NO) produced by plants for the associated microbiota remains elusive. Using Arabidopsis thaliana isogenic mutants overproducing NO (nox1, NO overexpression) or down-producing NO (i.e. nia1nia2 impaired in the expression of both nitrate reductases NR1/NIA1 and NR2/NIA2; the 35s::GSNOR1 line overexpressing nitrosoglutathione reductase (GSNOR) and 35s::AHB1 line overexpressing haemoglobin 1 (AHB1)), we investigated how altered NO homeostasis affects microbial communities in the rhizosphere and in the roots, soil microbial activity and soil metabolites. We show that the rhizosphere microbiome was affected by the mutant genotypes, with the nox1 and nia1nia2 mutants causing opposite shifts in bacterial and fungal communities compared with the wild-type (WT) Col-0 in the rhizosphere and roots, respectively. These mutants also exhibited distinctive soil metabolite profiles than those from the other genotypes while soil microbial activity did not differ between the mutants and the WT Col-0. Our findings support our hypothesis that changes in NO production by plants can influence the plant microbiome composition with differential effects between fungal and bacterial communities.

Ecological and metabolic implications of the nurse effect of Maihueniopsis camachoi in the Atacama Desert.

Plant-plant positive interactions are key drivers of community structure. Yet, the underlying molecular mechanisms of facilitation processes remain unexplored. We investigated the 'nursing' effect of Maihueniopsis camachoi, a cactus that thrives in the Atacama Desert between c. 2800 and 3800 m above sea level. We hypothesised that an important protective factor is thermal amelioration of less cold-tolerant species with a corresponding impact on molecular phenotypes. To test this hypothesis, we compared plant cover and temperatures within the cactus foliage with open areas and modelled the effect of temperatures on plant distribution. We combined eco-metabolomics and machine learning to test the molecular consequences of this association. Multiple species benefited from the interaction with M. camachoi. A conspicuous example was the extended distribution of Atriplex imbricata to colder elevations in association with M. camachoi (400 m higher as compared to plants in open areas). Metabolomics identified 93 biochemical markers predicting the interaction status of A. imbricata with 79% accuracy, independently of year. These findings place M. camachoi as a key species in Atacama plant communities, driving local biodiversity with an impact on molecular phenotypes of nursed species. Our results support the stress-gradient hypothesis and provide pioneer insights into the metabolic consequences of facilitation.

Multi-regulated GDP-l-galactose phosphorylase calls the tune in ascorbate biosynthesis.

Ascorbate is involved in numerous vital processes, in particular in response to abiotic but also biotic stresses whose frequency and amplitude increase with climate change. Ascorbate levels vary greatly depending on species, tissues, or stages of development, but also in response to stress. Since its discovery, the ascorbate biosynthetic pathway has been intensely studied and it appears that GDP-l-galactose phosphorylase (GGP) is the enzyme with the greatest role in the control of ascorbate biosynthesis. Like other enzymes of this pathway, its expression is induced by various environmental and also developmental factors. Although mRNAs encoding it are among the most abundant in the transcriptome, the protein is only present in very small quantities. In fact, GGP translation is repressed by a negative feedback mechanism involving a small open reading frame located upstream of the coding sequence (uORF). Moreover, its activity is inhibited by a PAS/LOV type photoreceptor, the action of which is counteracted by blue light. Consequently, this multi-level regulation of GGP would allow fine control of ascorbate synthesis. Indeed, experiments varying the expression of GGP have shown that it plays a central role in response to stress. This new understanding will be useful for developing varieties adapted to future environmental conditions.

Deciphering molecular events behind Systemin-induced resistance to Botrytis cinerea in tomato plants.

Plant defence peptides are paramount endogenous danger signals secreted after a challenge, intensifying the plant immune response. The peptidic hormone Systemin (Sys) was shown to participate in resistance in several plant pathosystems, although the mechanisms behind Sys-induced resistance when exogenously applied remain elusive. We performed proteomic, metabolomic, and enzymatic studies to decipher the Sys-induced changes in tomato plants in either the absence or the presence of Botrytis cinerea infection. Sys treatments triggered direct proteomic rearrangement mostly involved in carbon metabolism and photosynthesis. However, the final induction of defence proteins required concurrent challenge, triggering priming of pathogen-targeted proteins. Conversely, at the metabolomic level, Sys-treated plants showed an alternative behaviour following a general priming profile. Of the primed metabolites, the flavonoids rutin and isorhamnetin and two alkaloids correlated with the proteins 4-coumarate-CoA-ligase and chalcone-flavanone-isomerase triggered by Sys treatment. In addition, proteomic and enzymatic analyses revealed that Sys conditioned the primary metabolism towards the production of available sugars that could be fuelling the priming of callose deposition in Sys-treated plants; furthermore, PR1 appeared as a key element in Sys-induced resistance. Collectively, the direct induction of proteins and priming of specific secondary metabolites in Sys-treated plants indicated that post-translational protein regulation is an additional component of priming against necrotrophic fungi.

Phylogenetically diverse wild plant species use common biochemical strategies to thrive in the Atacama Desert.

The best ideotypes are under mounting pressure due to increased aridity. Understanding the conserved molecular mechanisms that evolve in wild plants adapted to harsh environments is crucial in developing new strategies for agriculture. Yet our knowledge of such mechanisms in wild species is scant. We performed metabolic pathway reconstruction using transcriptome information from 32 Atacama and phylogenetically related species that do not live in Atacama (sister species). We analyzed reaction enrichment to understand the commonalities and differences of Atacama plants. To gain insights into the mechanisms that ensure survival, we compared expressed gene isoform numbers and gene expression patterns between the annotated biochemical reactions from 32 Atacama and sister species. We found biochemical convergences characterized by reactions enriched in at least 50% of the Atacama species, pointing to potential advantages against drought and nitrogen starvation, for instance. These findings suggest that the adaptation in the Atacama Desert may result in part from shared genetic legacies governing the expression of key metabolic pathways to face harsh conditions. Enriched reactions corresponded to ubiquitous compounds common to extreme and agronomic species and were congruent with our previous metabolomic analyses. Convergent adaptive traits offer promising candidates for improving abiotic stress resilience in crop species.

Different wheat loci are associated to heritable free asparagine content in grain grown under different water and nitrogen availability.

KEY MESSAGE: Different wheat QTLs were associated to the free asparagine content of grain grown in four different conditions. Environmental effects are a key factor when selecting for low acrylamide-forming potential. The amount of free asparagine in grain of a wheat genotype determines its potential to form harmful acrylamide in derivative food products. Here, we explored the variation in the free asparagine, aspartate, glutamine and glutamate contents of 485 accessions reflecting wheat worldwide diversity to define the genetic architecture governing the accumulation of these amino acids in grain. Accessions were grown under high and low nitrogen availability and in water-deficient and well-watered conditions, and plant and grain phenotypes were measured. Free amino acid contents of grain varied from 0.01 to 1.02 mg g-1 among genotypes in a highly heritable way that did not correlate strongly with grain yield, protein content, specific weight, thousand-kernel weight or heading date. Mean free asparagine content was 4% higher under high nitrogen and 3% higher in water-deficient conditions. After genotyping the accessions, single-locus and multi-locus genome-wide association study models were used to identify several QTLs for free asparagine content located on nine chromosomes. Each QTL was associated with a single amino acid and growing environment, and none of the QTLs colocalised with genes known to be involved in the corresponding amino acid metabolism. This suggests that free asparagine content is controlled by several loci with minor effects interacting with the environment. We conclude that breeding for reduced asparagine content is feasible, but should be firmly based on multi-environment field trials. KEY MESSAGE: Different wheat QTLs were associated to the free asparagine content of grain grown in four different conditions. Environmental effects are a key factor when selecting for low acrylamide-forming potential.

Impaired cell growth under ammonium stress explained by modeling the energy cost of vacuole expansion in tomato leaves.

Ammonium (NH4 + )-based fertilization efficiently mitigates the adverse effects of nitrogen fertilization on the environment. However, high concentrations of soil NH4 + provoke growth inhibition, partly caused by the reduction of cell enlargement and associated with modifications of cell composition, such as an increase of sugars and a decrease in organic acids. Cell expansion depends largely on the osmotic-driven enlargement of the vacuole. However, the involvement of subcellular compartmentation in the adaptation of plants to ammonium nutrition has received little attention, until now. To investigate this, tomato (Solanum lycopersicum) plants were cultivated under nitrate and ammonium nutrition and the fourth leaf was harvested at seven developmental stages. The vacuolar expansion was monitored and metabolites and inorganic ion contents, together with intracellular pH, were determined. A data-constrained model was constructed to estimate subcellular concentrations of major metabolites and ions. It was first validated at the three latter developmental stages by comparison with subcellular concentrations obtained experimentally using non-aqueous fractionation. Then, the model was used to estimate the subcellular concentrations at the seven developmental stages and the net vacuolar uptake of solutes along the developmental series. Our results showed ammonium nutrition provokes an acidification of the vacuole and a reduction in the flux of solutes into the vacuoles. Overall, analysis of the subcellular compartmentation reveals a mechanism behind leaf growth inhibition under ammonium stress linked to the higher energy cost of vacuole expansion, as a result of alterations in pH, the inhibition of glycolysis routes and the depletion of organic acids.

Enzyme-based kinetic modelling of ASC-GSH cycle during tomato fruit development reveals the importance of reducing power and ROS availability.

The ascorbate-glutathione (ASC-GSH) cycle is at the heart of redox metabolism, linking the major redox buffers with central metabolism through the processing of reactive oxygen species (ROS) and pyridine nucleotide metabolism. Tomato fruit development is underpinned by changes in redox buffer contents and their associated enzyme capacities, but interactions between them remain unclear. Based on quantitative data obtained for the core redox metabolism, we built an enzyme-based kinetic model to calculate redox metabolite concentrations with their corresponding fluxes and control coefficients. Dynamic and associated regulations of the ASC-GSH cycle throughout the whole fruit development were analysed and pointed to a sequential metabolic control of redox fluxes by ASC synthesis, NAD(P)H and ROS availability depending on the developmental phase. Furthermore, we highlighted that monodehydroascorbate reductase and the availability of reducing power were found to be the main regulators of the redox state of ASC and GSH during fruit growth under optimal conditions. Our kinetic modelling approach indicated that tomato fruit development displayed growth phase-dependent redox metabolism linked with central metabolism via pyridine nucleotides and H2 O2 availability, while providing a new tool to the scientific community to investigate redox metabolism in fruits.

Predictive metabolomics of multiple Atacama plant species unveils a core set of generic metabolites for extreme climate resilience.

Current crop yield of the best ideotypes is stagnating and threatened by climate change. In this scenario, understanding wild plant adaptations in extreme ecosystems offers an opportunity to learn about new mechanisms for resilience. Previous studies have shown species specificity for metabolites involved in plant adaptation to harsh environments. Here, we combined multispecies ecological metabolomics and machine learning-based generalized linear model predictions to link the metabolome to the plant environment in a set of 24 species belonging to 14 families growing along an altitudinal gradient in the Atacama Desert. Thirty-nine common compounds predicted the plant environment with 79% accuracy, thus establishing the plant metabolome as an excellent integrative predictor of environmental fluctuations. These metabolites were independent of the species and validated both statistically and biologically using an independent dataset from a different sampling year. Thereafter, using multiblock predictive regressions, metabolites were linked to climatic and edaphic stressors such as freezing temperature, water deficit and high solar irradiance. These findings indicate that plants from different evolutionary trajectories use a generic metabolic toolkit to face extreme environments. These core metabolites, also present in agronomic species, provide a unique metabolic goldmine for improving crop performances under abiotic pressure.

PeakForest: a multi-platform digital infrastructure for interoperable metabolite spectral data and metadata management.

INTRODUCTION: Accuracy of feature annotation and metabolite identification in biological samples is a key element in metabolomics research. However, the annotation process is often hampered by the lack of spectral reference data in experimental conditions, as well as logistical difficulties in the spectral data management and exchange of annotations between laboratories. OBJECTIVES: To design an open-source infrastructure allowing hosting both nuclear magnetic resonance (NMR) and mass spectra (MS), with an ergonomic Web interface and Web services to support metabolite annotation and laboratory data management. METHODS: We developed the PeakForest infrastructure, an open-source Java tool with automatic programming interfaces that can be deployed locally to organize spectral data for metabolome annotation in laboratories. Standardized operating procedures and formats were included to ensure data quality and interoperability, in line with international recommendations and FAIR principles. RESULTS: PeakForest is able to capture and store experimental spectral MS and NMR metadata as well as collect and display signal annotations. This modular system provides a structured database with inbuilt tools to curate information, browse and reuse spectral information in data treatment. PeakForest offers data formalization and centralization at the laboratory level, facilitating shared spectral data across laboratories and integration into public databases. CONCLUSION: PeakForest is a comprehensive resource which addresses a technical bottleneck, namely large-scale spectral data annotation and metabolite identification for metabolomics laboratories with multiple instruments. PeakForest databases can be used in conjunction with bespoke data analysis pipelines in the Galaxy environment, offering the opportunity to meet the evolving needs of metabolomics research. Developed and tested by the French metabolomics community, PeakForest is freely-available at https://github.com/peakforest .

Overproduction of ascorbic acid impairs pollen fertility in tomato.

Ascorbate is a major antioxidant buffer in plants. Several approaches have been used to increase the ascorbate content of fruits and vegetables. Here, we combined forward genetics with mapping-by-sequencing approaches using an ethyl methanesulfonate (EMS)-mutagenized Micro-Tom population to identify putative regulators underlying a high-ascorbate phenotype in tomato fruits. Among the ascorbate-enriched mutants, the family with the highest fruit ascorbate level (P17C5, up to 5-fold wild-type level) had strongly impaired flower development and produced seedless fruit. Genetic characterization was performed by outcrossing P17C5 with cv. M82. We identified the mutation responsible for the ascorbate-enriched trait in a cis-acting upstream open reading frame (uORF) involved in the downstream regulation of GDP-l-galactose phosphorylase (GGP). Using a specific CRISPR strategy, we generated uORF-GGP1 mutants and confirmed the ascorbate-enriched phenotype. We further investigated the impact of the ascorbate-enriched trait in tomato plants by phenotyping the original P17C5 EMS mutant, the population of outcrossed P17C5 × M82 plants, and the CRISPR-mutated line. These studies revealed that high ascorbate content is linked to impaired floral organ architecture, particularly anther and pollen development, leading to male sterility. RNA-seq analysis suggested that uORF-GGP1 acts as a regulator of ascorbate synthesis that maintains redox homeostasis to allow appropriate plant development.

Modeling Protein Destiny in Developing Fruit.

Protein synthesis and degradation are essential processes that regulate cell status. Because labeling in bulky organs, such as fruits, is difficult, we developed a modeling approach to study protein turnover at the global scale in developing tomato (Solanum lycopersicum) fruit. Quantitative data were collected for transcripts and proteins during fruit development. Clustering analysis showed smaller changes in protein abundance compared to mRNA abundance. Furthermore, protein and transcript abundance were poorly correlated, and the coefficient of correlation decreased during fruit development and ripening, with transcript levels decreasing more than protein levels. A mathematical model with one ordinary differential equation was used to estimate translation (kt ) and degradation (kd ) rate constants for almost 2,400 detected transcript-protein pairs and was satisfactorily fitted for >1,000 pairs. The model predicted median values of ∼2 min for the translation of a protein, and a protein lifetime of ∼11 d. The constants were validated and inspected for biological relevance. Proteins involved in protein synthesis had higher kt and kd values, indicating that the protein machinery is particularly flexible. Our model also predicts that protein concentration is more strongly affected by the rate of translation than that of degradation.

Molecular underpinnings of methyl jasmonate-induced resistance in Norway spruce.

In response to various stimuli, plants acquire resistance against pests and/or pathogens. Such acquired or induced resistance allows plants to rapidly adapt to their environment. Spraying the bark of mature Norway spruce (Picea abies) trees with the phytohormone methyl jasmonate (MeJA) enhances resistance to tree-killing bark beetles and their associated phytopathogenic fungi. Analysis of spruce chemical defenses and beetle colonization success suggests that MeJA treatment both directly induces immune responses and primes inducible defenses for a faster and stronger response to subsequent beetle attack. We used metabolite and transcriptome profiling to explore the mechanisms underlying MeJA-induced resistance in Norway spruce. We demonstrated that MeJA treatment caused substantial changes in the bark transcriptional response to a triggering stress (mechanical wounding). Profiling of mRNA expression showed a suite of spruce inducible defenses are primed following MeJA treatment. Although monoterpenes and diterpene resin acids increased more rapidly after wounding in MeJA-treated than control bark, expression of their biosynthesis genes did not. We suggest that priming of inducible defenses is part of a complex mixture of defense responses that underpins the increased resistance against bark beetle colonization observed in Norway spruce. This study provides the most detailed insights yet into the mechanisms underlying induced resistance in a long-lived gymnosperm.

Metabolite profiling of non-sterile rhizosphere soil.

Rhizosphere chemistry is the sum of root exudation chemicals, their breakdown products and the microbial products of soil-derived chemicals. To date, most studies about root exudation chemistry are based on sterile cultivation systems, which limits the discovery of microbial breakdown products that act as semiochemicals and shape microbial rhizosphere communities. Here, we present a method for untargeted metabolic profiling of non-sterile rhizosphere soil. We have developed an experimental growth system that enables the collection and analysis of rhizosphere chemicals from different plant species. High-throughput sequencing of 16SrRNA genes demonstrated that plants in the growth system support a microbial rhizosphere effect. To collect a range of (a)polar chemicals from the system, we developed extraction methods that do not cause detectable damage to root cells or soil-inhabiting microbes, thus preventing contamination with cellular metabolites. Untargeted metabolite profiling by UPLC-Q-TOF mass spectrometry, followed by uni- and multivariate statistical analyses, identified a wide range of secondary metabolites that are enriched in plant-containing soil, compared with control soil without roots. We show that the method is suitable for profiling the rhizosphere chemistry of Zea mays (maize) in agricultural soil, thereby demonstrating the applicability to different plant-soil combinations. Our study provides a robust method for the comprehensive metabolite profiling of non-sterile rhizosphere soil, which represents a technical advance towards the establishment of causal relationships between the chemistry and microbial composition of the rhizosphere.

Mechanisms of glacial-to-future atmospheric CO2 effects on plant immunity.

The impacts of rising atmospheric CO2 concentrations on plant disease have received increasing attention, but with little consensus emerging on the direct mechanisms by which CO2 shapes plant immunity. Furthermore, the impact of sub-ambient CO2 concentrations, which plants have experienced repeatedly over the past 800 000 yr, has been largely overlooked. A combination of gene expression analysis, phenotypic characterisation of mutants and mass spectrometry-based metabolic profiling was used to determine development-independent effects of sub-ambient CO2 (saCO2 ) and elevated CO2 (eCO2 ) on Arabidopsis immunity. Resistance to the necrotrophic Plectosphaerella cucumerina (Pc) was repressed at saCO2 and enhanced at eCO2 . This CO2 -dependent resistance was associated with priming of jasmonic acid (JA)-dependent gene expression and required intact JA biosynthesis and signalling. Resistance to the biotrophic oomycete Hyaloperonospora arabidopsidis (Hpa) increased at both eCO2 and saCO2 . Although eCO2 primed salicylic acid (SA)-dependent gene expression, mutations affecting SA signalling only partially suppressed Hpa resistance at eCO2 , suggesting additional mechanisms are involved. Induced production of intracellular reactive oxygen species (ROS) at saCO2 corresponded to a loss of resistance in glycolate oxidase mutants and increased transcription of the peroxisomal catalase gene CAT2, unveiling a mechanism by which photorespiration-derived ROS determined Hpa resistance at saCO2 . By separating indirect developmental impacts from direct immunological effects, we uncover distinct mechanisms by which CO2 shapes plant immunity and discuss their evolutionary significance.

Chemical priming of immunity without costs to plant growth.

ß-Aminobutyric acid (BABA) induces broad-spectrum disease resistance, but also represses plant growth, which has limited its exploitation in crop protection. BABA perception relies on binding to the aspartyl-tRNA synthetase (AspRS) IBI1, which primes the enzyme for secondary defense activity. This study aimed to identify structural BABA analogues that induce resistance without stunting plant growth. Using site-directed mutagenesis, we demonstrate that the (l)-aspartic acid-binding domain of IBI1 is critical for BABA perception. Based on interaction models of this domain, we screened a small library of structural BABA analogues for growth repression and induced resistance against biotrophic Hyaloperonospora arabidopsidis (Hpa). A range of resistance-inducing compounds were identified, of which (R)-ß-homoserine (RBH) was the most effective. Surprisingly, RBH acted through different pathways than BABA. RBH-induced resistance (RBH-IR) against Hpa functioned independently of salicylic acid, partially relied on camalexin, and was associated with augmented cell wall defense. RBH-IR against necrotrophic Plectosphaerella cucumerina acted via priming of ethylene and jasmonic acid defenses. RBH-IR was also effective in tomato against Botrytis cinerea. Metabolic profiling revealed that RBH, unlike BABA, does not majorly affect plant metabolism. RBH primes distinct defense pathways against biotrophic and necrotrophic pathogens without stunting plant growth, signifying strong potential for exploitation in crop protection.

Photoperiod Affects the Phenotype of Mitochondrial Complex I Mutants.

Plant mutants for genes encoding subunits of mitochondrial complex I (CI; NADH:ubiquinone oxidoreductase), the first enzyme of the respiratory chain, display various phenotypes depending on growth conditions. Here, we examined the impact of photoperiod, a major environmental factor controlling plant development, on two Arabidopsis (Arabidopsis thaliana) CI mutants: a new insertion mutant interrupted in both ndufs8.1 and ndufs8.2 genes encoding the NDUFS8 subunit and the previously characterized ndufs4 CI mutant. In the long day (LD) condition, both ndufs8.1 and ndufs8.2 single mutants were indistinguishable from Columbia-0 at phenotypic and biochemical levels, whereas the ndufs8.1 ndufs8.2 double mutant was devoid of detectable holo-CI assembly/activity, showed higher alternative oxidase content/activity, and displayed a growth retardation phenotype similar to that of the ndufs4 mutant. Although growth was more affected in ndufs4 than in ndufs8.1 ndufs8.2 under the short day (SD) condition, both mutants displayed a similar impairment of growth acceleration after transfer to LD compared with the wild type. Untargeted and targeted metabolomics showed that overall metabolism was less responsive to the SD-to-LD transition in mutants than in the wild type. The typical LD acclimation of carbon and nitrogen assimilation as well as redox-related parameters was not observed in ndufs8.1 ndufs8 Similarly, NAD(H) content, which was higher in the SD condition in both mutants than in Columbia-0, did not adjust under LD We propose that altered redox homeostasis and NAD(H) content/redox state control the phenotype of CI mutants and photoperiod acclimation in Arabidopsis.

Spore Density Determines Infection Strategy by the Plant Pathogenic Fungus Plectosphaerella cucumerina.

Necrotrophic and biotrophic pathogens are resisted by different plant defenses. While necrotrophic pathogens are sensitive to jasmonic acid (JA)-dependent resistance, biotrophic pathogens are resisted by salicylic acid (SA)- and reactive oxygen species (ROS)-dependent resistance. Although many pathogens switch from biotrophy to necrotrophy during infection, little is known about the signals triggering this transition. This study is based on the observation that the early colonization pattern and symptom development by the ascomycete pathogen Plectosphaerella cucumerina (P. cucumerina) vary between inoculation methods. Using the Arabidopsis (Arabidopsis thaliana) defense response as a proxy for infection strategy, we examined whether P. cucumerina alternates between hemibiotrophic and necrotrophic lifestyles, depending on initial spore density and distribution on the leaf surface. Untargeted metabolome analysis revealed profound differences in metabolic defense signatures upon different inoculation methods. Quantification of JA and SA, marker gene expression, and cell death confirmed that infection from high spore densities activates JA-dependent defenses with excessive cell death, while infection from low spore densities induces SA-dependent defenses with lower levels of cell death. Phenotyping of Arabidopsis mutants in JA, SA, and ROS signaling confirmed that P. cucumerina is differentially resisted by JA- and SA/ROS-dependent defenses, depending on initial spore density and distribution on the leaf. Furthermore, in situ staining for early callose deposition at the infection sites revealed that necrotrophy by P. cucumerina is associated with elevated host defense. We conclude that P. cucumerina adapts to early-acting plant defenses by switching from a hemibiotrophic to a necrotrophic infection program, thereby gaining an advantage of immunity-related cell death in the host.