Natural variation in the chickpea metabolome under drought stress.

Chickpea is the world's fourth largest grown legume crop, which significantly contributes to food security by providing calories and dietary protein globally. However, the increased frequency of drought stress has significantly reduced chickpea production in recent years. Here, we have performed a field experiment with 36 diverse chickpea genotypes to evaluate grain yield, photosynthetic activities and molecular traits related to drought stress. For metabolomics analysis, leaf tissue was collected at three time points representing different pod-filling stages. We identified L-threonic acid, fructose and sugar alcohols involved in chickpea adaptive drought response within the mid-pod-filling stage. A stress susceptibility index for each genotype was calculated to identify tolerance capacity under drought, distributing the 36 genotypes into four categories from best to worst performance. To understand how biochemical mechanisms control different traits for genetic improvement, we performed a differential Jacobian analysis, which unveiled the interplay between various metabolic pathways across three time points, including higher flux towards inositol interconversions, glycolysis for high-performing genotypes, fumarate to malate conversion, and carbon and nitrogen metabolism perturbations. Metabolic GWAS (mGWAS) analysis uncovered gene candidates involved in glycolysis and MEP pathway corroborating with the differential biochemical Jacobian results. Accordingly, this proposed data analysis strategy bridges the gap from pure statistical association to causal biochemical relations by exploiting natural variation. Our study offers new perspectives on the genetic and metabolic understanding of drought tolerance-associated diversity in the chickpea metabolome and led to the identification of metabolic control points that can be also tested in other legume crops.

Exo- and endophytic fungi enable rapid transfer of nutrients from ant waste to orchid tissue.

The epiphytic orchid Caularthron bilamellatum sacrifices its water storage tissue for nutrients from the waste of ants lodging inside its hollow pseudobulb. Here, we investigate whether fungi are involved in the rapid translocation of nutrients. Uptake was analysed with a 15 N labelling experiment, subsequent isotope ratio mass spectrometry (IRMS) and secondary ion mass spectrometry (ToF-SIMS and NanoSIMS). We encountered two hyphae types: a thick melanized type assigned to 'black fungi' (Chaetothyriales, Cladosporiales, and Mycosphaerellales) in ant waste, and a thin endophytic type belonging to Hypocreales. In few cell layers, both hyphae types co-occurred. 15 N accumulation in both hyphae types was conspicuous, while for translocation to the vessels only Hypocreales were involved. There is evidence that the occurrence of the two hyphae types results in a synergism in terms of nutrient uptake. Our study provides the first evidence that a pseudobulb (=stem)-born endophytic network of Hypocreales is involved in the rapid translocation of nitrogen from insect-derived waste to the vegetative and reproductive tissue of the host orchid. For C. bilamellatum that has no contact with the soil, ant waste in the hollow pseudobulbs serves as equivalent to soil in terms of nutrient sources.

PANOMICS meets germplasm.

Genotyping-by-sequencing has enabled approaches for genomic selection to improve yield, stress resistance and nutritional value. More and more resource studies are emerging providing 1000 and more genotypes and millions of SNPs for one species covering a hitherto inaccessible intraspecific genetic variation. The larger the databases are growing, the better statistical approaches for genomic selection will be available. However, there are clear limitations on the statistical but also on the biological part. Intraspecific genetic variation is able to explain a high proportion of the phenotypes, but a large part of phenotypic plasticity also stems from environmentally driven transcriptional, post-transcriptional, translational, post-translational, epigenetic and metabolic regulation. Moreover, regulation of the same gene can have different phenotypic outputs in different environments. Consequently, to explain and understand environment-dependent phenotypic plasticity based on the available genotype variation we have to integrate the analysis of further molecular levels reflecting the complete information flow from the gene to metabolism to phenotype. Interestingly, metabolomics platforms are already more cost-effective than NGS platforms and are decisive for the prediction of nutritional value or stress resistance. Here, we propose three fundamental pillars for future breeding strategies in the framework of Green Systems Biology: (i) combining genome selection with environment-dependent PANOMICS analysis and deep learning to improve prediction accuracy for marker-dependent trait performance; (ii) PANOMICS resolution at subtissue, cellular and subcellular level provides information about fundamental functions of selected markers; (iii) combining PANOMICS with genome editing and speed breeding tools to accelerate and enhance large-scale functional validation of trait-specific precision breeding.

System level analysis of cacao seed ripening reveals a sequential interplay of primary and secondary metabolism leading to polyphenol accumulation and preparation of stress resistance.

Theobroma cacao and its popular product, chocolate, are attracting attention due to potential health benefits including antioxidative effects by polyphenols, anti-depressant effects by high serotonin levels, inhibition of platelet aggregation and prevention of obesity-dependent insulin resistance. The development of cacao seeds during fruit ripening is the most crucial process for the accumulation of these compounds. In this study, we analyzed the primary and the secondary metabolome as well as the proteome during Theobroma cacao cv. Forastero seed development by applying an integrative extraction protocol. The combination of multivariate statistics and mathematical modelling revealed a complex consecutive coordination of primary and secondary metabolism and corresponding pathways. Tricarboxylic acid (TCA) cycle and aromatic amino acid metabolism dominated during the early developmental stages (stages 1 and 2; cell division and expansion phase). This was accompanied with a significant shift of proteins from phenylpropanoid metabolism to flavonoid biosynthesis. At stage 3 (reserve accumulation phase), metabolism of sucrose switched from hydrolysis into raffinose synthesis. Lipids as well as proteins involved in lipid metabolism increased whereas amino acids and N-phenylpropenoyl amino acids decreased. Purine alkaloids, polyphenols, and raffinose as well as proteins involved in abiotic and biotic stress accumulated at stage 4 (maturation phase) endowing cacao seeds the characteristic astringent taste and resistance to stress. In summary, metabolic key points of cacao seed development comprise the sequential coordination of primary metabolites, phenylpropanoid, N-phenylpropenoyl amino acid, serotonin, lipid and polyphenol metabolism thereby covering the major compound classes involved in cacao aroma and health benefits.

Metabolism and development - integration of micro computed tomography data and metabolite profiling reveals metabolic reprogramming from floral initiation to silique development.

The interrelationship of morphogenesis and metabolism is a poorly studied phenomenon. The main paradigm is that development is controlled by gene expression. The aim of the present study was to correlate metabolism to early and late stages of flower and fruit development in order to provide the basis for the identification of metabolic adjustment and limitations. A highly detailed picture of morphogenesis is achieved using nondestructive micro computed tomography. This technique was used to quantify morphometric parameters of early and late flower development in an Arabidopsis thaliana mutant with synchronized flower initiation. The synchronized flower phenotype made it possible to sample enough early floral tissue otherwise not accessible for metabolomic analysis. The integration of metabolomic and morphometric data enabled the correlation of metabolic signatures with the process of flower morphogenesis. These signatures changed significantly during development, indicating a pronounced metabolic reprogramming in the tissue. Distinct sets of metabolites involved in these processes were identified and were linked to the findings of previous gene expression studies of flower development. High correlations with basic leucine zipper (bZIP) transcription factors and nitrogen metabolism genes involved in the control of metabolic carbon : nitrogen partitioning were revealed. Based on these observations a model for metabolic adjustment during flower development is proposed.

Influence of the environment on ragweed pollen and their sensitizing capacity in a mouse model of allergic lung inflammation.

Common ragweed (Ambrosia artemisiifolia) is an invasive plant with allergenic pollen. Due to environmental changes, ragweed pollen (RWP) airborne concentrations are predicted to quadruple in Europe by 2050 and more than double allergic sensitization of Europeans by 2060. We developed an experimental RWP model of allergy in BALB/c mice to evaluate how the number of RWP and how RWP collected from different geographical environments influence disease. We administered RWP six times over 3 weeks intranasally to the mice and then evaluated disease parameters 72 h later or allowed the mice to recover for at least 90 days before rechallenging them with RWP to elicit a disease relapse. Doses over 300 pollen grains induced lung eosinophilia. Higher doses of 3,000 and 30,000 pollen grains increased both eosinophils and neutrophils and induced disease relapses. RWP harvested from diverse geographical regions induced a spectrum of allergic lung disease from mild inflammation to moderate eosinophilic and severe mixed eosinophilic-neutrophilic lung infiltrates. After a recovery period, mice rechallenged with pollen developed a robust disease relapse. We found no correlation between Amb a 1 content, the major immunodominant allergen, endotoxin content, or RWP structure with disease severity. These results demonstrate that there is an environmental impact on RWP with clinical consequences that may underlie the increasing sensitization rates and the severity of pollen-induced disease exacerbation in patients. The multitude of diverse environmental factors governing distinctive patterns of disease induced by RWP remains unclear. Further studies are necessary to elucidate how the environment influences the complex interaction between RWP and human health.

Dissecting Metabolism of Leaf Nodules in Ardisia crenata and Psychotria punctata.

Root-microbe interaction and its specialized root nodule structures and functions are well studied. In contrast, leaf nodules harboring microbial endophytes in special glandular leaf structures have only recently gained increased interest as plant-microbe phyllosphere interactions. Here, we applied a comprehensive metabolomics platform in combination with natural product isolation and characterization to dissect leaf and leaf nodule metabolism and functions in Ardisia crenata (Primulaceae) and Psychotria punctata (Rubiaceae). The results indicate that abiotic stress resilience plays an important part within the leaf nodule symbiosis of both species. Both species showed metabolic signatures of enhanced nitrogen assimilation/dissimilation pattern and increased polyamine levels in nodules compared to leaf lamina tissue potentially involved in senescence processes and photosynthesis. Multiple links to cytokinin and REDOX-active pathways were found. Our results further demonstrate that secondary metabolite production by endophytes is a key feature of this symbiotic system. Multiple anhydromuropeptides (AhMP) and their derivatives were identified as highly characteristic biomarkers for nodulation within both species. A novel epicatechin derivative was structurally elucidated with NMR and shown to be enriched within the leaf nodules of A. crenata. This enrichment within nodulated tissues was also observed for catechin and other flavonoids indicating that flavonoid metabolism may play an important role for leaf nodule symbiosis of A. crenata. In contrast, pavettamine was only detected in P. punctata and showed no nodule specific enrichment but a developmental effect. Further natural products were detected, including three putative unknown depsipeptide structures in A. crenata leaf nodules. The analysis presents a first metabolomics reference data set for the intimate interaction of microbes and plants in leaf nodules, reveals novel metabolic processes of plant-microbe interaction as well as the potential of natural product discovery in these systems.

Apparent penetration depth in attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy of Allium cepa L. epidermis and cuticle.

Over the past decades, ATR-FTIR has emerged as promising tool for the identification of plants at the genus and (sub-) species level through surface measurements of intact leaves. Theoretical considerations regarding the penetration depth of the evanescent wave into the sample and the thickness of plant leaf cuticles suggest that the structure and composition of the cuticle represent universal taxonomic markers. However, experimental evidence for this hypothesis is scarce. In the current contribution, we present results of a series of simple experiments on epidermal monolayers derived from the bulbs of Allium cepa L. (Amaryllidaceae) as a model system to study the effect of an IR active probe located beyond the theoretical penetration depth of the evanescent wave. We found that this probe had a significant influence on the ATR-FTIR spectra for up to 4 epidermal layers stacked on top of each other corresponding to a total thickness of around 60 µm, exceeding the theoretical penetration depth of the evanescent wave by a factor of around 20. Altogether, our data indicate a major discrepancy between theory and practice in ATR-FTIR spectroscopy in general and provide strong evidence that in general plant leaf spectra cannot be fully explained by the structure and composition of the cuticle alone.

Multiomics approach unravels fertility transition in a pigeonpea line for a two-line hybrid system.

Pigeonpea [Cajanus cajan (L.) Millsp.] is a pulse crop cultivated in the semi-arid regions of Asia and Africa. It is a rich source of protein and capable of alleviating malnutrition, improving soil health and the livelihoods of small-holder farmers. Hybrid breeding has provided remarkable improvements for pigeonpea productivity, but owing to a tedious and costly seed production system, an alternative two-line hybrid technology is being explored. In this regard, an environment-sensitive male sterile line has been characterized as a thermosensitive male sterile line in pigeonpea precisely responding to day temperature. The male sterile and fertile anthers from five developmental stages were studied by integrating transcriptomics, proteomics and metabolomics supported by precise phenotyping and scanning electron microscopic study. Spatio-temporal analysis of anther transcriptome and proteome revealed 17 repressed DEGs/DEPs in sterile anthers that play a critical role in normal cell wall morphogenesis and tapetal cell development. The male fertility to sterility transition was mainly due to a perturbation in auxin homeostasis, leading to impaired cell wall modification and sugar transport. Limited nutrient utilization thus leads to microspore starvation in response to moderately elevated day temperature which could be restored with auxin-treatment in the male sterile line. Our findings outline a molecular mechanism that underpins fertility transition responses thereby providing a process-oriented two-line hybrid breeding framework for pigeonpea.

The Potato Yam Phyllosphere Ectosymbiont Paraburkholderia sp. Msb3 Is a Potent Growth Promotor in Tomato.

The genus Paraburkholderia includes a variety of species with promising features for sustainable biotechnological solutions in agriculture through increasing crop productivity. Here, we present a novel Paraburkholderia isolate, a permanent and predominant member of the Dioscoreae bulbifera (yam family, Dioscoreaceae) phyllosphere, making up to 25% of the microbial community on leaf acumens. The 8.5 Mbp genome of isolate Msb3 encodes an unprecedented combination of features mediating a beneficial plant-associated lifestyle, including biological nitrogen fixation (BNF), plant hormone regulation, detoxification of various xenobiotics, degradation of aromatic compounds and multiple protein secretion systems including both T3SS and T6SS. The isolate exhibits significant growth promotion when applied to agriculturally important plants such as tomato, by increasing the total dry biomass by up to 40%. The open question about the "beneficial" nature of this strain led us to investigate ecological and generic boundaries in Burkholderia sensu lato. In a refined phylogeny including 279 Burkholderia sensu lato isolates strain Msb3 clusters within Clade I Paraburkholderia, which also includes few opportunistic strains that can potentially act as pathogens, as revealed by our ecological meta-data analysis. In fact, we demonstrate that all genera originating from the "plant beneficial and environmental" (PBE) Burkholderia species cluster include opportunists. This indicates that further functional examinations are needed before safe application of these strains in sustainable agricultural settings can be assured.