Regulation of symbiotic nitrogen fixation (SNF) during drought stress is complex and not yet fully understood. In the present work, the involvement of nodule C and N metabolism in the regulation of SNF in Medicago truncatula under drought and a subsequent rewatering treatment was analyzed using a combination of metabolomic and proteomic approaches. Drought induced a reduction of SNF rates and major changes in the metabolic profile of nodules, mostly an accumulation of amino acids (Pro, His, and Trp) and carbohydrates (sucrose, galactinol, raffinose, and trehalose). This accumulation was coincidental with a decline in the levels of bacteroid proteins involved in SNF and C metabolism, along with a partial reduction of the levels of plant sucrose synthase 1 (SuSy1). In contrast, the variations in enzymes related to N assimilation were found not to correlate with the reduction in SNF, suggesting that these enzymes do not have a role in the regulation of SNF. Unlike the situation in other legumes such as pea and soybean, the drought-induced inhibition of SNF in M. truncatula appears to be caused by impairment of bacteroid metabolism and N(2)-fixing capacity rather than a limitation of respiratory substrate.

In spite of evidence for positive diversity-productivity relationships increasing plant diversity has highly variable effects on the performance of individual plant species, but the mechanisms behind these differential responses are far from being understood. To gain deeper insights into the physiological responses of individual plant species to increasing plant diversity we performed systematic untargeted metabolite profiling on a number of herbs derived from a grassland biodiversity experiment (Jena Experiment). The Jena Experiment comprises plots of varying species number (1, 2, 4, 8, 16 and 60) and number and composition of functional groups (1 to 4; grasses, legumes, tall herbs, small herbs). In this study the metabolomes of two tall-growing herbs (legume: Medicago x varia; non-legume: Knautia arvensis) and three small-growing herbs (legume: Lotus corniculatus; non-legumes: Bellis perennis, Leontodon autumnalis) in plant communities of increasing diversity were analyzed. For metabolite profiling we combined gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF-MS) and UPLC coupled to FT-ICR-MS (LC-FT-MS) analyses from the same sample. This resulted in several thousands of detected m/z-features. ANOVA and multivariate statistical analysis revealed 139 significantly changed metabolites (30 by GC-TOF-MS and 109 by LC-FT-MS). The small-statured plants L. autumnalis, B. perennis and L. corniculatus showed metabolic response signatures to increasing plant diversity and species richness in contrast to tall-statured plants. Key-metabolites indicated C- and N-limitation for the non-leguminous small-statured species B. perennis and L. autumnalis, while the metabolic signature of the small-statured legume L. corniculatus indicated facilitation by other legumes. Thus, metabolomic analysis provided evidence for negative effects of resource competition on the investigated small-statured herbs that might mechanistically explain their decreasing performance with increasing plant diversity. In contrast, taller species often becoming dominant in mixed plant communities did not show modified metabolite profiles in response to altered resource availability with increasing plant diversity. Taken together, our study demonstrates that metabolite profiling is a strong diagnostic tool to assess individual metabolic phenotypes in response to plant diversity and ecophysiological adjustment.

Non-alcoholic fatty liver disease (NAFLD) results from increased hepatic lipid accumulation and steatosis, and is closely linked to liver one-carbon (C1) metabolism. We assessed in C57BL6/N mice whether NAFLD induced by a high-fat (HF) diet over 8 weeks can be reversed by additional 4 weeks of a dietary methyl-donor supplementation (MDS). MDS in the obese mice failed to reverse NAFLD, but prevented the progression of hepatic steatosis associated with major changes in key hepatic C1-metabolites, e.g. S-adenosyl-methionine and S-adenosyl-homocysteine. Increased phosphorylation of AMPK-α together with enhanced β-HAD activity suggested an increased flux through fatty acid oxidation pathways. This was supported by concomitantly decreased hepatic free fatty acid and acyl-carnitines levels. Although HF diet changed the hepatic phospholipid pattern, MDS did not. Our findings suggest that dietary methyl-donors activate AMPK, a key enzyme in fatty acid β-oxidation control, that mediates increased fatty acid utilization and thereby prevents further hepatic lipid accumulation.

The integral peroxisomal membrane proteins PEX10, PEX2, and PEX12 contain a zinc RING finger close to the C terminus. Loss of function of these peroxins causes embryo lethality at the heart stage in Arabidopsis. Preventing the coordination of Zn 2+ ions by amino acid substitutions in PEX10, PEX2, and PEX12 and overexpressing the resulting conditional sublethal mutations in WT uncovered additional functions of PEX10. Plants overexpressing ΔZn-mutant PEX10 display deformed peroxisomal shapes causing diminished contact with chloroplasts and possibly with mitochondria. These changes correlated with impaired metabolite transfer and, at high CO 2 , recoverable defective photorespiration plus dwarfish phenotype. The N-terminal PEX10 domain is critical for peroxisome biogenesis and plant development. A point mutation in the highly conserved TLGEEY motif results in vermiform peroxisome shape without impairing organelle contact. Addition of an N-terminal T7 tag to WT PEX0 resulted in partially recoverable reduced growth and defective inflorescences persisting under high CO 2 . In contrast, plants overexpressing PEX2-ΔZn-T7 grow like WT in normal atmosphere, contain normal-shaped peroxisomes, but display impaired peroxisomal matrix protein import. PEX12-ΔZn-T7 mutants exhibit unimpaired import of matrix protein and normalshaped peroxisomes when grown in normal atmosphere. During seed germination, glyoxysomes form a reticulum around the lipid bodies for mobilization of storage oil. The formation of this glyoxysomal reticulum seemed to be impaired in PEX10-ΔZn but not in PEX2-ΔZn-T7 or PEX12-ΔZn-T7 plants. Both cytosolic PEX10 domains seem essential for peroxisome structure but differ in metabolic function, suggesting a role for this plant peroxin in addition to the import of matrix protein via ubiquitination of PEX5.Thr Leu Gly Glu Glu Tyr motif in peroxin PEX10 | glyoxysome | metabolomics P eroxisomes perform multiple metabolic processes, including β-oxidation and H 2 O 2 inactivation by catalase. In plants, specialized peroxisomes, the glyoxysomes, contain glyoxylate cycle enzymes for lipid mobilization. Leaf peroxisomes interact with chloroplasts and mitochondria in photorespiration, a metabolic pathway turning two molecules of glycolate in a series of reactions through glyoxylate, glycine (Gly), serine (Ser), and hydroxypyruvate into CO 2 and phosphoglycerate (1-3). When CO 2 is limiting, ribulose-bisphosphate carboxylase/oxygenase functions as an oxygenase and protects the photosynthetic machinery from photodamage by using energy for photorespiration. Mutants lacking enzymes of the photorespiratory cycle are incapable of surviving in ambient air but are able to grow normally in a CO 2 -enriched atmosphere (2). When genes responsible for plant peroxisome biogenesis are impaired, the phenotypes are severe, ranging from sucrose dependency during early seedling development to embryo lethality (4). The biogenesis of peroxisomes relies on a common class of evolutionary conserved genes referred to as "PEX genes" (5, 6).The im...

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