Carla António

JUNGBRUNNEN1, a Reactive Oxygen Species–Responsive NAC Transcription Factor, Regulates Longevity in Arabidopsis

Aging in plants is an intricate process that balances vegetative growth with flowering and reproductive success. This work describes the identification of JUNGBRUNNEN1, a NAC transcription factor that regulates this process in Arabidopsis thaliana and additionally affects abiotic stress tolerance by activating expression of the DREB2A transcription factor. The transition from juvenility through maturation to senescence is a complex process that involves the regulation of longevity. Here, we identify JUNGBRUNNEN1 (JUB1), a hydrogen peroxide (H2O2)-induced NAC transcription factor, as a central longevity regulator in Arabidopsis thaliana. JUB1 overexpression strongly delays senescence, dampens intracellular H2O2 levels, and enhances tolerance to various abiotic stresses, whereas in jub1-1 knockdown plants, precocious senescence and lowered abiotic stress tolerance are observed. A JUB1 binding site containing a RRYGCCGT core sequence is present in the promoter of DREB2A, which plays an important role in abiotic stress responses. JUB1 transactivates DREB2A expression in mesophyll cell protoplasts and transgenic plants and binds directly to the DREB2A promoter. Transcriptome profiling of JUB1 overexpressors revealed elevated expression of several reactive oxygen species–responsive genes, including heat shock protein and glutathione S-transferase genes, whose expression is further induced by H2O2 treatment. Metabolite profiling identified elevated Pro and trehalose levels in JUB1 overexpressors, in accordance with their enhanced abiotic stress tolerance. We suggest that JUB1 constitutes a central regulator of a finely tuned control system that modulates cellular H2O2 level and primes the plants for upcoming stress through a gene regulatory network that involves DREB2A.

Metabolomic applications of HILIC–LC–MS

Hydrophilic interaction liquid chromatography (HILIC), although not a new technique, has enjoyed a recent renaissance with the introduction of robust and reproducible stationary phases. It is consequently finding application in metabolomics studies, which have traditionally relied on the stability of reversed phases (RPs), since the biofluids analyzed are predominantly aqueous and thus contain many polar analytes. HILICs retention of those polar compounds and use of solvents readily compatible with mass spectrometry have seen its increasing adoption in studies of complex aqueous metabolomes. This review describes the stationary phases and their features, surveys HILIC-LC-MSs role in metabolomics experiments, discusses approaches to data extraction and analysis including multivariate analysis, and reviews the literature on HILIC-MS applications in metabolomics.

Molecular mechanisms of desiccation tolerance in the resurrection glacial relic Haberlea rhodopensis

Haberlea rhodopensis is a resurrection plant with remarkable tolerance to desiccation. Haberlea exposed to drought stress, desiccation, and subsequent rehydration showed no signs of damage or severe oxidative stress compared to untreated control plants. Transcriptome analysis by next-generation sequencing revealed a drought-induced reprogramming, which redirected resources from growth towards cell protection. Repression of photosynthetic and growth-related genes during water deficiency was concomitant with induction of transcription factors (members of the NAC, NF-YA, MADS box, HSF, GRAS, and WRKY families) presumably acting as master switches of the genetic reprogramming, as well as with an upregulation of genes related to sugar metabolism, signaling, and genes encoding early light-inducible (ELIP), late embryogenesis abundant (LEA), and heat shock (HSP) proteins. At the same time, genes encoding other LEA, HSP, and stress protective proteins were constitutively expressed at high levels even in unstressed controls. Genes normally involved in tolerance to salinity, chilling, and pathogens were also highly induced, suggesting a possible cross-tolerance against a number of abiotic and biotic stress factors. A notable percentage of the genes highly regulated in dehydration and subsequent rehydration were novel, with no sequence homology to genes from other plant genomes. Additionally, an extensive antioxidant gene network was identified with several gene families possessing a greater number of antioxidant genes than most other species with sequenced genomes. Two of the transcripts most abundant during all conditions encoded catalases and five more catalases were induced in water-deficient samples. Using the pharmacological inhibitor 3-aminotriazole (AT) to compromise catalase activity resulted in increased sensitivity to desiccation. Metabolome analysis by GC or LC–MS revealed accumulation of sucrose, verbascose, spermidine, and γ-aminobutyric acid during drought, as well as particular secondary metabolites accumulating during rehydration. This observation, together with the complex antioxidant system and the constitutive expression of stress protective genes suggests that both constitutive and inducible mechanisms contribute to the extreme desiccation tolerance of H. rhodopensis.

Mass spectrometry‐based plant metabolomics: Metabolite responses to abiotic stress

Metabolomics is one omics approach that can be used to acquire comprehensive information on the composition of a metabolite pool to provide a functional screen of the cellular state. Studies of the plant metabolome include analysis of a wide range of chemical species with diverse physical properties, from ionic inorganic compounds to biochemically derived hydrophilic carbohydrates, organic and amino acids, and a range of hydrophobic lipid-related compounds. This complexitiy brings huge challenges to the analytical technologies employed in current plant metabolomics programs, and powerful analytical tools are required for the separation and characterization of this extremely high compound diversity present in biological sample matrices. The use of mass spectrometry (MS)-based analytical platforms to profile stress-responsive metabolites that allow some plants to adapt to adverse environmental conditions is fundamental in current plant biotechnology research programs for the understanding and development of stress-tolerant plants. In this review, we describe recent applications of metabolomics and emphasize its increasing application to study plant responses to environmental (stress-) factors, including drought, salt, low oxygen caused by waterlogging or flooding of the soil, temperature, light and oxidative stress (or a combination of them). Advances in understanding the global changes occurring in plant metabolism under specific abiotic stress conditions are fundamental to enhance plant fitness and increase stress tolerance.

Hydrophilic interaction chromatography/electrospray mass spectrometry analysis of carbohydrate‐related metabolites from Arabidopsis thaliana leaf tissue

This work describes the development and application of an on-line liquid chromatography/mass spectrometry (LC/MS) method using hydrophilic interaction chromatography (HILIC) coupled to negative ion mode electrospray ionisation ion trap mass spectrometry (ESI-MS) for the analysis of highly polar carbohydrate-related metabolites commonly found in plants, ranging from reducing and non-reducing sugars and sugar alcohols to sugar phosphates. Using this method, separation and detection of a mixture of eight authentic standard compounds containing glucose (Glc), sucrose (Suc), raffinose, verbascose, mannitol, maltitol, glucose-6-phosphate (Glc6P) and trehalose-6-phosphate (Tre6P) were achieved in less than 15 min. The method is rapid, robust, selective, and sensitive, with limits of detection (LODs) ranging from 0.2 microM obtained for neutral sugars, to 1.0 microM obtained for sugar alcohols, and 2.0 microM obtained for negatively charged sugar phosphates. We have studied the negative ion collision-induced dissociation (CID) fragmentation behaviour of the non-reducing raffinose family oligosaccharides (RFOs) raffinose, stachyose, and verbascose. Mainly Bi and Ci glycosidic and Ai cross-ring structurally informative cleavages are observed. We have applied this HILIC/ESI-MS method for the analysis of Arabidopsis thaliana wild-type Columbia-0 (Col-0) and its starchless phosphoglucomutase mutant (pgm1) leaf extracts. The method was used to quantify Glc, Suc, raffinose, and Glc6P in A. thaliana extracts. Data obtained using this HILIC/ESI-MS method were compared with those obtained using a comparable porous graphitic carbon-based LC/ESI-MS method.

Impact of the carbon and nitrogen supply on relationships and connectivity between metabolism and biomass in a broad panel of Arabidopsis accessions

Metabolite profiles support a robust prediction of biomass across a range of conditions and accounts for environmental influences on metabolic networks. Natural genetic diversity provides a powerful tool to study the complex interrelationship between metabolism and growth. Profiling of metabolic traits combined with network-based and statistical analyses allow the comparison of conditions and identification of sets of traits that predict biomass. However, it often remains unclear why a particular set of metabolites is linked with biomass and to what extent the predictive model is applicable beyond a particular growth condition. A panel of 97 genetically diverse Arabidopsis (Arabidopsis thaliana) accessions was grown in near-optimal carbon and nitrogen supply, restricted carbon supply, and restricted nitrogen supply and analyzed for biomass and 54 metabolic traits. Correlation-based metabolic networks were generated from the genotype-dependent variation in each condition to reveal sets of metabolites that show coordinated changes across accessions. The networks were largely specific for a single growth condition. Partial least squares regression from metabolic traits allowed prediction of biomass within and, slightly more weakly, across conditions (cross-validated Pearson correlations in the range of 0.27–0.58 and 0.21–0.51 and P values in the range of <0.001–<0.13 and <0.001–<0.023, respectively). Metabolic traits that correlate with growth or have a high weighting in the partial least squares regression were mainly condition specific and often related to the resource that restricts growth under that condition. Linear mixed-model analysis using the combined metabolic traits from all growth conditions as an input indicated that inclusion of random effects for the conditions improves predictions of biomass. Thus, robust prediction of biomass across a range of conditions requires condition-specific measurement of metabolic traits to take account of environment-dependent changes of the underlying networks.

Analysis of carbohydrates in Lupinus albus stems on imposition of water deficit, using porous graphitic carbon liquid chromatography-electrospray ionization mass spectrometry

This work reports the development and application of a negative ion mode online LC-ESI-MS method for studying the effect of water deficit on the carbohydrate content of Lupinus albus stems, using a porous graphitic carbon (PGC) stationary phase and an ion trap mass spectrometer. Using this method, separation and detection of several water soluble carbohydrates, ranging from mono-, di-, and oligosaccharides (raffinose, stachyose, and verbascose) to sugar alcohols was achieved in approximately 10 min. This on-line PGC-LC-ESI-MS method shows good linearity with correlation coefficients R(2)>0.99, selectivity, short analysis time, and limits of detection (LOD) ranging from 0.4 to 9 pmol for sugars and 4-20 pmol for sugar alcohols. This PGC-LC-ESI-MS method is sensitive and allowed us to detect even small alterations in carbohydrate levels in L. albus stems that resulted from a mild/early water deficit (nmol g(-1)DW). This paper describes details of our method and its application to the quantitative analysis of water soluble underivatised carbohydrates extracted from L. albus stem tissues that have been subjected to early and severe water deficit conditions, followed by a rewatering period.

Regulation of Primary Metabolism in Response to Low Oxygen Availability as Revealed by Carbon and Nitrogen Isotope Redistribution

During low-oxygen stress, activation of Ala and GABA metabolism and bifurcation of the tricarboxylic acid cycle explains the down-regulation of respiratory oxygen consumption. Based on enzyme activity assays and metabolic responses to waterlogging of the legume Lotus japonicus, it was previously suggested that, during hypoxia, the tricarboxylic acid cycle switches to a noncyclic operation mode. Hypotheses were postulated to explain the alternative metabolic pathways involved, but as yet, a direct analysis of the relative redistribution of label through the corresponding pathways was not made. Here, we describe the use of stable isotope-labeling experiments for studying metabolism under hypoxia using wild-type roots of the crop legume soybean (Glycine max). [13C]Pyruvate labeling was performed to compare metabolism through the tricarboxylic acid cycle, fermentation, alanine metabolism, and the γ-aminobutyric acid shunt, while [13C]glutamate and [15N]ammonium labeling were performed to address the metabolism via glutamate to succinate. Following these labelings, the time course for the redistribution of the 13C/15N label throughout the metabolic network was evaluated with gas chromatography-time of flight-mass spectrometry. Our combined labeling data suggest the inhibition of the tricarboxylic acid cycle enzyme succinate dehydrogenase, also known as complex II of the mitochondrial electron transport chain, providing support for the bifurcation of the cycle and the down-regulation of the rate of respiration measured during hypoxic stress. Moreover, up-regulation of the γ-aminobutyric acid shunt and alanine metabolism explained the accumulation of succinate and alanine during hypoxia.

Initial water deficit effects on Lupinus albus photosynthetic performance, carbon metabolism, and hormonal balance: metabolic reorganization prior to early stress responses

The early (2-4 d) effects of slowly imposed soil water deficit on Lupinus albus photosynthetic performance, carbon metabolism, and hormonal balance in different organs (leaf blade, stem stele, stem cortex, and root) were evaluated on 23-d-old plants (growth chamber assay). Our work shows that several metabolic adjustments occurred prior to alteration of the plant water status, implying that water deficit is perceived before the change in plant water status. The slow, progressive decline in soil water content started to be visible 3 d after withholding water (3 DAW). The earliest plant changes were associated with organ-specific metabolic responses (particularly in the leaves) and with leaf conductance and only later with plant water status and photosynthetic rate (4 DAW) or photosynthetic capacity (according to the Farquhar model; 6 DAW). Principal component analysis (PCA) of the physiological parameters, the carbohydrate and the hormone levels and their relative values, as well as leaf water-soluble metabolites full scan data (LC-MS/MS), showed separation of the different sampling dates. At 6 DAW classically described stress responses are observed, with plant water status, ABA level, and root hormonal balance contributing to the separation of these samples. Discrimination of earlier stress stages (3 and 4 DAW) is only achieved when the relative levels of indole-3-acetic acid (IAA), cytokinins (Cks), and carbon metabolism (glucose, sucrose, raffinose, and starch levels) are taken into account. Our working hypothesis is that, in addition to single responses (e.g. ABA increase), the combined alterations in hormone and carbohydrate levels play an important role in the stress response mechanism. Response to more advanced stress appears to be associated with a combination of cumulative changes, occurring in several plant organs. The carbohydrate and hormonal balance in the leaf (IAA to bioactive-Cks; soluble sugars to IAA and starch to IAA; relative abundances of the different soluble sugars) flag the initial responses to the slight decrease in soil water availability (10-15% decrease). Further alterations in sucrose to ABA and in raffinose to ABA relative values (in all organs) indicate that soil water availability continues to decrease. Such alterations when associated with changes in the root hormone balance indicate that the stress response is initiated. It is concluded that metabolic balance (e.g. IAA/bioactive Cks, carbohydrates/IAA, sucrose/ABA, raffinose/ABA, ABA/IAA) is relevant in triggering adjustment mechanisms.

Metabolic efficiency underpins performance trade-offs in growth of Arabidopsis thaliana

Growth often involves a trade-off between the performance of contending tasks; metabolic plasticity can play an important role. Here we grow 97 Arabidopsis thaliana accessions in three conditions with a differing supply of carbon and nitrogen and identify a trade-off between two tasks required for rosette growth: increasing the physical size and increasing the protein concentration. We employ the Pareto performance frontier concept to rank accessions based on their multitask performance; only a few accessions achieve a good trade-off under all three growth conditions. We determine metabolic efficiency in each accession and condition by using metabolite levels and activities of enzymes involved in growth and protein synthesis. We demonstrate that accessions with high metabolic efficiency lie closer to the performance frontier and show increased metabolic plasticity. We illustrate how public domain data can be used to search for additional contending tasks, which may underlie the sub-optimality in some accessions.