Joel L. Shuman

When Defense Pathways Collide. The Response of Arabidopsis to a Combination of Drought and Heat Stress

Within their natural habitat, plants are subjected to a combination of abiotic conditions that include stresses such as drought and heat. Drought and heat stress have been extensively studied; however, little is known about how their combination impacts plants. The response of Arabidopsis plants to a combination of drought and heat stress was found to be distinct from that of plants subjected to drought or heat stress. Transcriptome analysis of Arabidopsis plants subjected to a combination of drought and heat stress revealed a new pattern of defense response in plants that includes a partial combination of two multigene defense pathways (i.e. drought and heat stress), as well as 454 transcripts that are specifically expressed in plants during a combination of drought and heat stress. Metabolic profiling of plants subjected to drought, heat stress, or a combination of drought and heat stress revealed that plants subject to a combination of drought and heat stress accumulated sucrose and other sugars such as maltose and gulose. In contrast, Pro that accumulated in plants subjected to drought did not accumulate in plants during a combination of drought and heat stress. Heat stress was found to ameliorate the toxicity of Pro to cells, suggesting that during a combination of drought and heat stress sucrose replaces Pro in plants as the major osmoprotectant. Our results highlight the plasticity of the plant genome and demonstrate its ability to respond to complex environmental conditions that occur in the field.

Enhanced Tolerance to Environmental Stress in Transgenic Plants Expressing the Transcriptional Coactivator Multiprotein Bridging Factor 1c

Abiotic stresses cause extensive losses to agricultural production worldwide. Acclimation of plants to abiotic conditions such as drought, salinity, or heat is mediated by a complex network of transcription factors and other regulatory genes that control multiple defense enzymes, proteins, and pathways. Associated with the activity of different transcription factors are transcriptional coactivators that enhance their binding to the basal transcription machinery. Although the importance of stress-response transcription factors was demonstrated in transgenic plants, little is known about the function of transcriptional coactivators associated with abiotic stresses. Here, we report that constitutive expression of the stress-response transcriptional coactivator multiprotein bridging factor 1c (MBF1c) in Arabidopsis (Arabidopsis thaliana) enhances the tolerance of transgenic plants to bacterial infection, heat, and osmotic stress. Moreover, the enhanced tolerance of transgenic plants to osmotic and heat stress was maintained even when these two stresses were combined. The expression of MBF1c in transgenic plants augmented the accumulation of a number of defense transcripts in response to heat stress. Transcriptome profiling and inhibitor studies suggest that MBF1c expression enhances the tolerance of transgenic plants to heat and osmotic stress by partially activating, or perturbing, the ethylene-response signal transduction pathway. Present findings suggest that MBF1 proteins could be used to enhance the tolerance of plants to different abiotic stresses.

The role of neutral lipid nanospheres in Plasmodium falciparum haem crystallization

The intraerythrocytic malaria parasite constructs an intracellular haem crystal, called haemozoin, within an acidic digestive vacuole where haemoglobin is degraded. Haem crystallization is the target of the widely used antimalarial quinoline drugs. The intracellular mechanism of molecular initiation of haem crystallization, whether by proteins, polar membrane lipids or by neutral lipids, has not been fully substantiated. In the present study, we show neutral lipid predominant nanospheres, which envelop haemozoin inside Plasmodium falciparum digestive vacuoles. Subcellular fractionation of parasite-derived haemozoin through a dense 1.7 M sucrose cushion identifies monoacylglycerol and diacylglycerol neutral lipids as well as some polar lipids in close association with the purified haemozoin. Global MS lipidomics detects monopalmitic glycerol and monostearic glycerol, but not mono-oleic glycerol, closely associated with haemozoin. The complex neutral lipid mixture rapidly initiates haem crystallization, with reversible pH-dependent quinoline inhibition associated with quinoline entry into the neutral lipid microenvironment. Neutral lipid nanospheres both enable haem crystallization in the presence of high globin concentrations and protect haem from H2O2 degradation. Conceptually, the present study shifts the intracellular microenvironment of haem crystallization and quinoline inhibition from a polar aqueous location to a non-polar neutral lipid nanosphere able to exclude water for efficient haem crystallization.

The Transcriptional Co-activator MBF1c Is a Key Regulator of Thermotolerance in Arabidopsis thaliana

The ability of an organism to acclimate to its environment is a key determinant in its global distribution and capacity to compete with other organisms. The heat stress response, a highly conserved environmental and developmental program in eukaryotic and prokaryotic organisms, is an important component of the acclimation response of plants. Previous studies have shown that heat shock transcription factors play an important role in thermotolerance in plants and other organisms, controlling the expression of different heat shock proteins and detoxifying enzymes. In contrast, although several other pathways, involving ethylene, salicylic acid (SA), and trehalose, were recently shown to play a central role in thermotolerance in plants, a key regulator of these responses was not identified. Here we report that the highly conserved transcriptional co-activator, MBF1c (multiprotein bridging factor 1c), is a key regulator of thermotolerance in Arabidopsis thaliana. MBF1c protein accumulates rapidly and is localized to nuclei during heat stress. MBF1c is required for thermotolerance and functions upstream to SA, trehalose, ethylene, and pathogenesis-related protein 1 during heat stress. In contrast, MBF1c is not required for the expression of transcripts encoding HSFA2 and different heat shock proteins. Interestingly, MBF1c interacts with TPS5 (trehalose phosphate synthase 5), which is also heat-inducible, and mutants deficient in TPS5 are thermosensitive. Our results provide evidence for the existence of a tightly coordinated heat stress-response network, involving trehalose-, SA-, and ethylene-signaling pathways, that is under the control of MBF1c.

Temporal-Spatial Interaction between Reactive Oxygen Species and Abscisic Acid Regulates Rapid Systemic Acclimation in Plants

An autopropagating wave of reactive oxygen species (the ROS wave) rapidly spreads from a local tissue exposed to stress to the entire plant. In coordination with other systemic signals and abscisic acid, it activates systemic acclimation mechanisms in the entire plant and enhances tolerance to abiotic stress. The enhanced tolerance is specific to the original stress that induces it. Being sessile organisms, plants evolved sophisticated acclimation mechanisms to cope with abiotic challenges in their environment. These are activated at the initial site of exposure to stress, as well as in systemic tissues that have not been subjected to stress (termed systemic acquired acclimation [SAA]). Although SAA is thought to play a key role in plant survival during stress, little is known about the signaling mechanisms underlying it. Here, we report that SAA in plants requires at least two different signals: an autopropagating wave of reactive oxygen species (ROS) that rapidly spreads from the initial site of exposure to the entire plant and a stress-specific signal that conveys abiotic stress specificity. We further demonstrate that SAA is stress specific and that a temporal–spatial interaction between ROS and abscisic acid regulates rapid SAA to heat stress in plants. In addition, we demonstrate that the rapid ROS signal is associated with the propagation of electric signals in Arabidopsis thaliana. Our findings unravel some of the basic signaling mechanisms underlying SAA in plants and reveal that signaling events and transcriptome and metabolome reprogramming of systemic tissues in response to abiotic stress occur at a much faster rate than previously envisioned.

Sensitive and Rapid Method for Amino Acid Quantitation in Malaria Biological Samples Using AccQ•Tag Ultra Performance Liquid Chromatography-Electrospray Ionization-MS/MS with Multiple Reaction Monitoring

An AccQ*Tag ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (AccQ*Tag-UPLC-ESI-MS/MS) method for fast, reproducible, and sensitive amino acid quantitation in biological samples, particularly, the malaria parasite Plasmodium falciparum is presented. The Waters Acquity TQD UPLC/MS system equipped with a photodiode array (PDA) detector was used for amino acid separation and detection. The method was developed and validated using amino acid standard mixtures containing acidic, neutral, and basic amino acids. For MS analysis, the optimum cone voltage implemented, based on direct infusion analysis of a few selected AccQ*Tag amino acids with multiple reaction monitoring, varied from 29 to 39 V, whereas the collision energy varied from 15 to 35 V. Calibration curves were built using both internal and external standardization. Typically, a linear response for all amino acids was observed at concentration ranges of 3 x 10(-3)-25 pmol/muL. For some amino acids, concentration limits of detection were as low as 1.65 fmol. The coefficients of variation for retention times were within the range of 0.08-1.08%. The coefficients of variation for amino acid quantitation, determined from triplicate UPLC-MS/MS runs, were below 8% on the average. The developed AccQ*Tag-UPLC-ESI-MS/MS method revealed good technical and biological reproducibility when applied to P. falciparum and human red blood cells samples. This study should provide a valuable insight into the performance of UPLC-ESI-MS/MS for amino acid quantitation using AccQ*Tag derivatization.

Metabolomics as a Hypothesis-Generating Functional Genomics Tool for the Annotation of Arabidopsis thaliana Genes of “Unknown Function”

Metabolomics is the methodology that identifies and measures global pools of small molecules (of less than about 1,000 Da) of a biological sample, which are collectively called the metabolome. Metabolomics can therefore reveal the metabolic outcome of a genetic or environmental perturbation of a metabolic regulatory network, and thus provide insights into the structure and regulation of that network. Because of the chemical complexity of the metabolome and limitations associated with individual analytical platforms for determining the metabolome, it is currently difficult to capture the complete metabolome of an organism or tissue, which is in contrast to genomics and transcriptomics. This paper describes the analysis of Arabidopsis metabolomics data sets acquired by a consortium that includes five analytical laboratories, bioinformaticists, and biostatisticians, which aims to develop and validate metabolomics as a hypothesis-generating functional genomics tool. The consortium is determining the metabolomes of Arabidopsis T-DNA mutant stocks, grown in standardized controlled environment optimized to minimize environmental impacts on the metabolomes. Metabolomics data were generated with seven analytical platforms, and the combined data is being provided to the research community to formulate initial hypotheses about genes of unknown function (GUFs). A public database (www.PlantMetabolomics.org) has been developed to provide the scientific community with access to the data along with tools to allow for its interactive analysis. Exemplary datasets are discussed to validate the approach, which illustrate how initial hypotheses can be generated from the consortium-produced metabolomics data, integrated with prior knowledge to provide a testable hypothesis concerning the functionality of GUFs.

Plant Metabolomics by GC-MS and Differential Analysis

Metabolomics is a new genomics approach that aims at measuring all or a subset of metabolites in the cell. Several approaches to plant metabolomics are currently used in plant research. These include targeted analysis, metabolite profiling, and metabolic fingerprinting. Metabolic fingerprinting, unlike metabolite profiling, does not aim at separating or identifying all the metabolites present in the sample, but rather generates a fingerprint that characterizes a specific metabolic state of the plant system under investigation. This chapter describes the implementation of metabolic fingerprinting approach using gas chromatography coupled to mass spectrometry (GC-MS) and discriminant function analysis combined with genetic algorithm (GA-DFA). This approach enables the identification of specific metabolites that are biologically relevant, and which may go undetected if direct infusion-based fingerprinting approaches were used due to the sample complexity and matrix suppression effects.

Wound-healing properties of nut oil from Pouteria lucuma.

Background  Cell migration, angiogenesis, inflammation, and extracellular matrix remodeling are key events in wound healing. Natural products, including fatty acids (FAs), can accelerate wound healing by modulating the aforementioned events.

Quantitative characterization of hemozoin in Plasmodium berghei and vivax

The incidence and global distribution of chloroquine resistant (CR) Plasmodium vivax infection has increased since emerging in 1989. The mechanism of resistance in CR P. vivax has not been defined. The resistance likely relates to the formation and disposition of hemozoin as chloroquines primary mechanism of action involves disruption of hemozoin formation. CR P. berghei strains, like CR P. vivax strains, are confined to reticulocyte host cells and reportedly they do not accumulate appreciable intraerythrocytic hemozoin. Reports comparing hemozoin production between P. vivax strains and CR to chloroquine sensitive (CS) P. berghei are absent. Here we compare in vivo patterns of hemozoin formation and distribution in blood, spleen and liver tissue of male Swiss mice infected with CS or CR P. berghei not treated with chloroquine and CR P. berghei also treated with chloroquine. Light microscopy, laser desorption mass spectrometry and a colorimetric hemozoin assay detect trace hemozoin in the blood of CR P. berghei infected mice but significant hemozoin accumulation in liver and spleen tissue. Field emission in lens scanning electron microscopy reveals CR P. berghei hemozoin crystals are morphologically smaller but similar to those formed by CS parasites. CR P. berghei produces approximately five-fold less total hemozoin than CS strain. Lipid analysis of CS and CR P. berghei sucrose gradient purified bloodstage hemozoin indicates a similar lipid environment around the isolated hemozoin, predominately monopalmitic glycerol and monostearic glycerol. In contrast to CR and CS P. berghei, colorimetric hemozoin analysis of P. vivax strains indicates similar amounts of hemozoin are produced despite differing chloroquine sensitivities. These results suggest CR P. berghei forms significant hemozoin which accumulates in liver and spleen tissues and that the P. vivax chloroquine resistance mechanism differs from P. berghei.