Will Casuarina glauca Stress Resilience Be Maintained in the Face of Climate Change?

Actinorhizal plants have been regarded as promising species in the current climate change context due to their high tolerance to a multitude of abiotic stresses. While combined salt-heat stress effects have been studied in crop species, their impact on the model actinorhizal plant, Casuarina glauca, has not yet been fully addressed. The effect of single salt (400 mM NaCl) and heat (control at 26/22 °C, supra optimal temperatures at 35/22 °C and 45/22 °C day/night) conditions on C. glauca branchlets was characterised at the physiological level, and stress-induced metabolite changes were characterised by mass spectrometry-based metabolomics. C. glauca could withstand single salt and heat conditions. However, the harshest stress condition (400 mM NaCl, 45 °C) revealed photosynthetic impairments due to mesophyll and membrane permeability limitations as well as major stress-specific differential responses in C and N metabolism. The increased activity of enzymatic ROS scavengers was, however, revealed to be sufficient to control the plant oxidative status. Although C. glauca could tolerate single salt and heat stresses, their negative interaction enhanced the effects of salt stress. Results demonstrated that C. glauca responses to combined salt-heat stress could be explained as a sum of the responses from each single applied stress.

Antitumour and Toxicity Evaluation of a Ru(II)-Cyclopentadienyl Complex in a Prostate Cancer Model by Imaging Tools.

BACKGROUND: Ruthenium complexes have been extensively investigated for their prospective value as alternatives to cisplatin. Recently, we reported the in vitro anticancer properties of a family of organometallic ruthenium( II)-cyclopentadienyl complexes and have explored their mechanism of action. OBJECTIVE: The purpose of this study was to evaluate the in vivo antitumour efficacy and toxicity of one of these Ru(II) compounds, [RuCp(mTPPMSNa)(2,2'-bipy)][CF3SO2] (TM85) which displayed an interesting spectrum of activity against several cancer cells. METHODS: Studies to assess the antitumour activity and toxicity were performed in a metastatic prostate (PC3) mice model using ICP-MS, nuclear microscopy, elemental analysis and Transmission Electron Microscopy (TEM). RESULTS: TM85 showed low systemic toxicity but no significant tumour reduction, when administered at tolerated dose (20mg/kg) over 10 days. Ru was mainly retained in the liver and less in kidneys, with low accumulation in tumour. Increased bilirubin levels, anomalous Ca and Fe concentrations in liver and mitochondria alterations were indicative of liver injury. The hepatotoxicity observed was less severe than that of cisplatin and no nephrotoxicity was found. CONCLUSION: Under the experimental conditions of this study, TM85 is less toxic than cisplatin, induces similar tumour reduction and avoids the formation of metastatic foci. No renal toxicity was observed by the analysis of creatinine levels and the effective renal plasma flow by 99mTc-MAG3 clearance. Hence, it can be considered a valuable compound for further studies in the field of Ru-based anticancer drugs.

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 plant's 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.

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.

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.

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'.

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. © 2015 Wiley Periodicals, Inc. Mass Spec Rev 35:620-649, 2016.

New water-soluble ruthenium(II) cytotoxic complex: biological activity and cellular distribution.

A novel water soluble organometallic compound, [RuCp(mTPPMSNa)(2,2'-bipy)][CF3SO3] (TM85, where Cp=η(5)-cyclopentadienyl, mTPPMS=diphenylphosphane-benzene-3-sulfonate and 2,2'-bipy=2,2'-bipyridine) is presented herein. Studies of interactions with relevant proteins were performed to understand the behavior and mode of action of this complex in the biological environment. Electrochemical and fluorescence studies showed that TM85 strongly binds to albumin. Studies carried out to study the formation of TM85 which adducts with ubiquitin and cytochrome c were performed by electrospray ionization mass spectrometry (ESI-MS). Antitumor activity was evaluated against a variety of human cancer cell lines, namely A2780, A2780cisR, MCF7, MDAMB231, HT29, PC3 and V79 non-tumorigenic cells and compared with the reference drug cisplatin. TM85 cytotoxic effect was reduced in the presence of endocytosis modulators at low temperatures, suggesting an energy-dependent mechanism consistent with endocytosis. Ultrastructural analysis by transmission electron microscopy (TEM) revealed that TM85 targets the endomembranar system disrupting the Golgi and also affects the mitochondria. Disruption of plasma membrane observed by flow cytometry could lead to cellular damage and cell death. On the whole, the biological activity evaluated herein combined with the water solubility property suggests that complex TM85 could be a promising anticancer agent.