Root exudation of contrasting drought-stressed pearl millet genotypes conveys varying biological nitrification inhibition (BNI) activity.

Roots secrete a vast array of low molecular weight compounds into the soil broadly referred to as root exudates. It is a key mechanism by which plants and soil microbes interact in the rhizosphere. The effect of drought stress on the exudation process and composition is rarely studied, especially in cereal crops. This study focuses on comparative metabolic profiling of the exudates from sensitive and tolerant genotypes of pearl millet after a period of drought stress. We employed a combined platform of gas and liquid chromatography coupled to mass spectrometry to cover both primary and secondary metabolites. The results obtained demonstrate that both genotype and drought stress have a significant impact on the concentration and composition of root exudates. The complexity and function of these differential root exudates are discussed. To reveal the potential effect of root exudates on the soil microbial community after a period of drought stress, we also tested for biological nitrification inhibition (BNI) activity. The analysis revealed a genotype-dependent enhancement of BNI activity after a defined period of drought stress. In parallel, we observed a genotype-specific relation of elongated root growth and root exudation under drought stress. These data suggest that the drought stress-dependent change in root exudation can manipulate the microbial soil communities to adapt and survive under harsh conditions. Supplementary Information: The online version contains supplementary material available at 10.1007/s00374-021-01578-w.

Adjustment of photosynthetic activity to drought and fluctuating light in wheat.

Drought is a major cause of losses in crop yield. Under field conditions, plants exposed to drought are usually also experiencing rapid changes in light intensity. Accordingly, plants need to acclimate to both, drought and light stress. Two crucial mechanisms in plant acclimation to changes in light conditions comprise thylakoid protein phosphorylation and dissipation of light energy as heat by non-photochemical quenching (NPQ). Here, we analyzed the acclimation efficacy of two different wheat varieties, by applying fluctuating light for analysis of plants, which had been subjected to a slowly developing drought stress as it usually occurs in the field. This novel approach allowed us to distinguish four drought phases, which are critical for grain yield, and to discover acclimatory responses which are independent of photodamage. In short-term, under fluctuating light, the slowdown of NPQ relaxation adjusts the photosynthetic activity to the reduced metabolic capacity. In long-term, the photosynthetic machinery acquires a drought-specific configuration by changing the PSII-LHCII phosphorylation pattern together with protein stoichiometry. Therefore, the fine-tuning of NPQ relaxation and PSII-LHCII phosphorylation pattern represent promising traits for future crop breeding strategies.

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.

mzGroupAnalyzer--predicting pathways and novel chemical structures from untargeted high-throughput metabolomics data.

The metabolome is a highly dynamic entity and the final readout of the genotype x environment x phenotype (GxExP) relationship of an organism. Monitoring metabolite dynamics over time thus theoretically encrypts the whole range of possible chemical and biochemical transformations of small molecules involved in metabolism. The bottleneck is, however, the sheer number of unidentified structures in these samples. This represents the next challenge for metabolomics technology and is comparable with genome sequencing 30 years ago. At the same time it is impossible to handle the amount of data involved in a metabolomics analysis manually. Algorithms are therefore imperative to allow for automated m/z feature extraction and subsequent structure or pathway assignment. Here we provide an automated pathway inference strategy comprising measurements of metabolome time series using LC- MS with high resolution and high mass accuracy. An algorithm was developed, called mzGroupAnalyzer, to automatically explore the metabolome for the detection of metabolite transformations caused by biochemical or chemical modifications. Pathways are extracted directly from the data and putative novel structures can be identified. The detected m/z features can be mapped on a van Krevelen diagram according to their H/C and O/C ratios for pattern recognition and to visualize oxidative processes and biochemical transformations. This method was applied to Arabidopsis thaliana treated simultaneously with cold and high light. Due to a protective antioxidant response the plants turn from green to purple color via the accumulation of flavonoid structures. The detection of potential biochemical pathways resulted in 15 putatively new compounds involved in the flavonoid-pathway. These compounds were further validated by product ion spectra from the same data. The mzGroupAnalyzer is implemented in the graphical user interface (GUI) of the metabolomics toolbox COVAIN (Sun & Weckwerth, 2012, Metabolomics 8: 81-93). The strategy can be extended to any biological system.

Combining microdilution with MicroResp™: microbial substrate utilization, antimicrobial susceptibility and respiration.

Pharmacological studies focus on susceptibility of pathogenic microbes against specific drugs or combinations of them, ecological studies on substrate utilization efficiency of variable microbial communities. The MicroResp™ system was especially developed to study soil microbial communities. It was slightly modified to facilitate exploring of microbial growth efficiency in a concentration-dependent fashion (microdilutions of carbohydrate mixtures or specific toxic chemicals). After turbidimetric growth assessment, colorimetric indicator plates (cresol red agar) were mounted to the assay plates. The substrate utilisation design is illustrated by glucose and a plant carbohydrate mixture, the antimicrobial susceptibility design by the naphthoquinone juglone. Dose-response effects are explored by curve fitting of nonlinear models that especially have been developed to detect hormetic effects that are characterized by stimulation at lower followed by inhibition at higher dosages (U- and inverse U-shaped effects). Multivariate analyses are presented utilizing metavariables that were obtained in the curve fitting process of the measured parameters growth and respiration and the factor growth efficiency.

Hormesis and a Chemical Raison D'être for Secondary Plant Metabolites.

In plants, accumulation in specific compartments and huge structural diversity of secondary metabolites is one trait that is not understood yet. By exploring the diverse abiotic and biotic interactions of plants above- and belowground, we provide examples that are characterized by nonlinear effects of the secondary metabolites. We propose that redox chemistry, specifically the reduction of reactive oxygen species (ROS) and, in their absence, reduction of molecular oxygen by the identical secondary metabolite, is an important component of the hormetic effects caused by these compounds. This is illustrated for selected phenols, terpenoids, and alkaloids. The redox reactions are modulated by the variable availability of transition metals that serve as donors of electrons in a Fenton reaction mode. Low levels of ROS stimulate growth, cell differentiation, and stress resistance; high levels induce programmed cell death. We propose that provision of molecules that can participate in this redox chemistry is the raison d'être for secondary metabolites. In this context, the presence or absence of functional groups in the molecule is more essential than the whole structure. Accordingly, there exist no constraints that limit structural diversity. Redox chemistry is ubiquitous, from the atmosphere to the soil.

Development of a pharmacodynamic screening model with Crithidia fasciculata.

The genus Crithidia is a member of the family Trypanosomatidae and is related to the genera Leishmania and Trypanosoma with which it shares a variety of biochemical mechanisms, such as polyamine synthesis and methionin salvage. In consequence, a screening system for antiparasitic candidate material has been developed with Crithidia fasciculata, a parasite naturally occurring in insects and amphibians, but devoid of pathogenicity for humans. Initially a variety of culture media were evaluated of which TPS was best suited for the maintenance of stock cultures, and E-medium - a newly developed formula - for sensitivity testing. Optimal growth of C. fasciculata was observed under microaerophilic conditions. A system for sensitivity testing was developed and applied to the investigation of extracts from higher tropical plants of the genera Stemona and Aglaia for anticrithidial activity. Extracts with significant anti-crithidial activity were scheduled for chromatographic fractionation and the subsequent isolation, purification and structural identification of individual compounds for further sensitivity testing. Encouraging results were obtained with extracts from Aglaia odorata leaves, A. elaeagnoidea stem bark and A. edulis leaves, with EC(90) values of 1213 ng/ml, 1606 ng/ml, and 1462 ng/ml, respectively.

Dihydrophenanthrenes and other antifungal stilbenoids from Stemona cf. pierrei.

Three new dihydrophenanthrenes, stemanthrenes A-C, along with the new dihydrostilbene stilbostemin G were isolated and identified from the underground parts of Stemona cf. pierrei together with the known pinosylvin, 4'-methylpinosylvin, dihydropinosylvin, stilbostemins B, D, and E as well as the pyrrolo[1,2-a]azepine alkaloids protostemonine and stemonine. The structures of all new stilbenoids, elucidated by NMR analyses, showed a common substitution pattern for aromatic ring A and characteristic C-methylations for ring B. The trivial name racemosol, previously reported for S. collinsae, was renamed to stemanthrene D due to its priority for another compound. Bioautographic tests on TLC plates with Cladosporium herbarum displayed high antifungal activity for compounds with an unsubstituted aromatic ring A, e.g. pinosylvin, but only weak effects for the higher substituted stilbostemin G and stemanthrenes A-C. Similar results were obtained by germ tube inhibition of five microfungi using 2-fold serial broth dilutions determined by a microplate reader. Because of weak inhibition and chemical instability of stemanthrenes, no EC(50) and EC(90) values could be calculated.