Tiago F. Jorge

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.

Mass spectrometry as a quantitative tool in plant metabolomics

Metabolomics is a research field 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 the analysis of a wide range of chemical species with very diverse physico-chemical properties, and therefore powerful analytical tools are required for the separation, characterization and quantification of this vast compound diversity present in plant matrices. In this review, challenges in the use of mass spectrometry (MS) as a quantitative tool in plant metabolomics experiments are discussed, and important criteria for the development and validation of MS-based analytical methods provided. This article is part of the themed issue ‘Quantitative mass spectrometry’.

Cowpea (Vigna unguiculata L. Walp.) Metabolomics: Osmoprotection as a Physiological Strategy for Drought Stress Resistance and Improved Yield

Plants usually tolerate drought by producing organic solutes, which can either act as compatible osmolytes for maintaining turgor, or radical scavengers for protecting cellular functions. However, these two properties of organic solutes are often indistinguishable during stress progression. This study looked at individualizing properties of osmotic adjustment vs. osmoprotection in plants, using cowpea as the model species. Two cultivars were grown in well-watered soil, drought conditions, or drought followed by rewatering through fruit formation. Osmoadaptation was investigated in leaves and roots using photosynthetic traits, water homoeostasis, inorganic ions, and primary and secondary metabolites. Multifactorial analyses indicated allocation of high quantities of amino acids, sugars, and proanthocyanidins into roots, presumably linked to their role in growth and initial stress perception. Physiological and metabolic changes developed in parallel and drought/recovery responses showed a progressive acclimation of the cowpea plant to stress. Of the 88 metabolites studied, proline, galactinol, and a quercetin derivative responded the most to drought as highlighted by multivariate analyses, and their correlations with yield indicated beneficial effects. These metabolites accumulated differently in roots, but similarly in leaves, suggesting a more conservative strategy to cope with drought in the aerial parts. Changes in these compounds roughly reflected energy investment in protective mechanisms, although the ability of plants to adjust osmotically through inorganic ions uptake could not be discounted.

An integrated approach to understand the mechanisms underlying salt stress tolerance in Casuarina glauca and its relation with nitrogen-fixing Frankia Thr

Salinity is one of the most wide spread abiotic stresses affecting agricultural productivity, with an impact on more than 800 million hectares worldwide. A promising solution for the recovery of saline soils encompasses the use of actinorhizal plants, a group of perennial dicotyledonous angiosperms including species highly resilient to extreme environmental conditions. These plants are able to establish root-nodule symbiosis with N2-fixing actinobacteria of the genus Frankia. In this review, we discuss the main physiological and biochemical mechanisms underlying salt tolerance in the model Casuarina glauca supplemented with chemical nitrogen or obtaining it from symbiotic Frankia. In the first part, an overview of the impact of increasing NaCl concentrations in photosynthesis, antioxidative system and membrane integrity is presented. The second part addresses the effect of salt stress in the symbiosis between C. glauca and Frankia strain Thr. Preliminary results from analyses of the branchlets proteome and nodule metabolome are presented as well.

GC-TOF-MS analysis reveals salt stress-responsive primary metabolites in Casuarina glauca tissues

IntroductionCasuarina glauca is a model actinorhizal plant characterized by its ability to establish symbiosis with nitrogen-fixing Frankia bacteria. This species is able to thrive under extreme salinity environments. C. glauca tolerance to high salinity has been previously associated with low tissue dehydration, osmotic adjustments, and high membrane integrity. However, the full characterization of this plant system and the identification of key elements involved in its salt stress tolerance could be significantly improved through a metabolomics approach. To date, very little information is available in the literature about the C. glauca metabolome.ObjectivesThe present study investigates the impact of salt stress on the primary metabolome of nodulated (NOD+) and non-nodulated (KNO3+) C. glauca tissues (nodules, roots, and branchlets) and aims to identify salt-stress responsive metabolites in these two plant groups.MethodsGas chromatography time-of-flight mass spectrometry (GC-TOF-MS) is a powerful tool to measure primary metabolites such as sugars, amino, and organic acids, involved in the regulation of plant developmental processes, and has contributed to better understand how plant metabolism readjusts in response to high salinity.ResultsGC-TOF-MS data reveals major metabolite divergences in amino acid metabolism, which are highly consistent with the previously reported impairment of symbiotic activity, as well as down-regulation of the transcriptional activity of the plant symbiotic genes in the NOD+ plants.ConclusionsAltogether, our results revealed that C. glauca plants can tolerate high levels of salinity through modifications in the levels of some neutral sugars, proline, and ornithine.

Analysis of low abundant trehalose-6-phosphate and related metabolites in Medicago truncatula by hydrophilic interaction liquid chromatography–triple quadrupole mass spectrometry ☆

Trehalose-6-phosphate (T6P) is an important signaling metabolite involved in plant growth control that inhibits the sucrose nonfermenting-1-related protein kinase 1 (SnRK1), a key regulator of energy and carbon metabolism in plants. The quantification of T6P in plant tissues is fundamental to improve our understanding of sugar signaling and the links between plant growth and development in response to stress conditions. However, the almost undetectable levels of T6P together with the complex plant matrix and the presence of T6P isomers such as sucrose-6-phosphate (S6P), makes the detection of this metabolite challenging. This work describes the development and validation of a hydrophilic interaction chromatography (HILIC) method for the on-line coupling with negative ion electrospray (ESI) triple quadrupole tandem mass spectrometry (MS/MS) in the highly sensitive and selective multiple reaction monitoring (MRM) mode for the target analysis of metabolic intermediates of the biosynthesis of trehalose, including glucose-6-phosphate (G6P), uridine 5-diphospho-glucose (UDPG), T6P (and its isomer S6P). Enhanced signal in the MRM mode and improved chromatographic separation for each compound were obtained using piperidine and methylphosphonic acid as additives in the HILIC mobile phase. The optimized HILIC-ESI-QqQ-MS/MS method increases the range of sensitive analytical methodologies for the quantification of key low-abundant metabolites, and was applied to quantify the fluctuations of S6P, T6P and G6P in Medicago truncatula plants in response to environmental stress. The levels of S6P, T6P, and G6P in M. truncatula plant tissues (roots and leaves) exposed to a water deficit and recovery treatment, ranged from 30 to 150pmolg-1 FW, 16-120pmolg-1 FW, and 330-1690pmolg-1 FW, respectively.

Drought Stress Tolerance in Plants: Insights from Metabolomics

Global climate change and population growth are two major challenges of the twenty-first century. Crop losses due to variable weather patterns associated with climate change have risen over the past decades, and climate models predict an increased incidence particularly of droughts, floods, and extreme temperatures. This situation together with the growing food demand is anticipated to pose a real threat to global food security. It is therefore imperative to develop strategies to improve food availability substantially in variable environments, and ultimately, transfer this knowledge to farmers in the timeframe needed. Although much progress has been done over the past years in the area of drought tolerance in plants, the mechanisms defining plant resilience to water scarcity are still not completely understood. One approach to improving our knowledge of plant tolerance to drought stress is to reveal the underlying central metabolic pathways that might play an important role in sustaining cellular homeostasis to ensure plant survival in water scarcity conditions. Metabolomics is therefore finding an increasing number of applications to investigate plant metabolite responses to abiotic stress, particularly coupled to mass spectrometry (MS)-dedicated analytical technologies. In this chapter, recent applications of MS-based metabolomics to study plant responses to drought stress are discussed, from sample preparation to the analysis of metabolic responses. Understanding the global drought stress response through changes occurring at the metabolite level (i.e., metabolic reconfiguration and adaptation) is fundamental to improving drought stress tolerance in plants.

Quantification of Low-Abundant Phosphorylated Carbohydrates Using HILIC-QqQ-MS/MS

Phosphorylated carbohydrates are central metabolites involved in key plant metabolic pathways, such as glycolysis and central carbon metabolism. Such pathways influence plant growth, development, and stress responses to environmental changes, and ultimately, reflect the plants energy status. The high polarity of these metabolites, the variety of isomeric structures (e.g., glucose-1-phosphate (G1P)/fructose-6-phosphate (F6P)/mannose-6-phosphate (M6P)/G6P, sucrose-6-phosphate (S6P)/T6P), and rapid metabolic turnover makes their analysis particularly challenging. In this chapter, we describe the use of a set of known phosphorylated carbohydrates to develop and validate a hydrophilic interaction chromatography (HILIC) triple quadrupole (QqQ) tandem mass spectrometry (MS/MS) method in the highly sensitive and selective multiple reaction monitoring (MRM) mode for the target analysis of G1P, F6P, M6P, G6P, S6P, T6P, and the sugar nucleotide uridine 5-diphospho-glucose (UDPG). We present detailed information regarding HILIC column chemistry and practical considerations when coupling it with a QqQ-MS system.

Porous Graphitic Carbon Liquid Chromatography–Mass Spectrometry Analysis of Drought Stress-Responsive Raffinose Family Oligosaccharides in Plant Tissues

Drought is a major limiting factor in agriculture and responsible for dramatic crop yield losses worldwide. The adjustment of the metabolic status via accumulation of drought stress-responsive osmolytes is one of the many strategies that some plants have developed to cope with water deficit conditions. Osmolytes are highly polar compounds, analysis of whcih is difficult with typical reversed-phase chromatography. Porous graphitic carbon (PGC) has shown to be a suitable alternative to reversed-phase stationary phases for the analysis of highly polar compounds typically found in the plant metabolome. In this chapter, we describe the development and validation of a PGC-based liquid chromatography tandem mass spectrometry (LC-MSn) method suitable for the target analysis of water-soluble carbohydrates, such as raffinose family oligosaccharides (RFOs). We present detailed information regarding PGC column equilibration, LC-MSn system operation, data analysis, and important notes to be considered during the steps of method development and validation.

Characterization of the Primary Metabolome of Brachystegia boehmii and Colophospermum mopane under Different Fire Regimes in Miombo and Mopane African Woodlands

Miombo and Mopane are ecological and economic important woodlands from Africa, highly affected by a combination of climate change factors, and anthropogenic fires. Although most species of these ecosystems are fire tolerant, the mechanisms that lead to adaptive responses (metabolic reconfiguration) are unknown. In this context, the aim of this study was to characterize the primary metabolite composition of typical legume trees from these ecosystems, namely, Brachystegia boehmii (Miombo) and Colophospermum mopane (Mopane) subjected to different fire regimes. Fresh leaves from each species were collected in management units and landscapes across varied fire frequencies in the Niassa National Reserve (NNR) and Limpopo National Park (LNP) in Mozambique. Primary metabolites were extracted and analyzed with a well-established gas chromatography time-of-flight mass spectrometry metabolomics platform (GC-TOF-MS). In B. boehmii, 39 primary metabolites were identified from which seven amino acids, two organic acids and two sugars increased significantly, whereas in C. mopane, 41 primary metabolites were identified from which eight amino acids, one sugar and two organic acids significantly increased with increasing fire frequency. The observed changes in the pool of metabolites of C. mopane might be related to high glycolytic and tricarboxylic acid (TCA) rate, which provided increased levels of amino acids and energy yield. In B. boehmii, the high levels of amino acids might be due to inhibition of protein biosynthesis. The osmoprotectant and reactive oxygen species (ROS) scavenging properties of accumulated metabolites in parallel with a high-energy yield might support plants survival under fire stress.