Protein phosphatase 2A (PP2A) regulatory subunit B'γ interacts with cytoplasmic ACONITASE 3 and modulates the abundance of AOX1A and AOX1D in Arabidopsis thaliana.

Organellar reactive oxygen species (ROS) signalling is a key mechanism that promotes the onset of defensive measures in stress-exposed plants. The underlying molecular mechanisms and feedback regulation loops, however, still remain poorly understood. Our previous work has shown that a specific regulatory B'γ subunit of protein phosphatase 2A (PP2A) is required to control organellar ROS signalling and associated metabolic adjustments in Arabidopsis thaliana. Here, we addressed the mechanisms through which PP2A-B'γ impacts on organellar metabolic crosstalk and ROS homeostasis in leaves. Genetic, biochemical and pharmacological approaches, together with a combination of data-dependent acquisition (DDA) and selected reaction monitoring (SRM) MS techniques, were utilized to assess PP2A-B'γ-dependent adjustments in Arabidopsis thaliana. We show that PP2A-B'γ physically interacts with the cytoplasmic form of aconitase, a central metabolic enzyme functionally connected with mitochondrial respiration, oxidative stress responses and regulation of cell death in plants. Furthermore, PP2A-B'γ impacts ROS homeostasis by controlling the abundance of specific alternative oxidase isoforms, AOX1A and AOX1D, in leaf mitochondria. We conclude that PP2A-B'γ-dependent regulatory actions modulate the functional status of metabolic enzymes that essentially contribute to intracellular ROS signalling and metabolic homeostasis in plants.

Regulatory network of secondary metabolism in Brassica rapa: insight into the glucosinolate pathway.

Brassica rapa studies towards metabolic variation have largely been focused on the profiling of the diversity of metabolic compounds in specific crop types or regional varieties, but none aimed to identify genes with regulatory function in metabolite composition. Here we followed a genetical genomics approach to identify regulatory genes for six biosynthetic pathways of health-related phytochemicals, i.e carotenoids, tocopherols, folates, glucosinolates, flavonoids and phenylpropanoids. Leaves from six weeks-old plants of a Brassica rapa doubled haploid population, consisting of 92 genotypes, were profiled for their secondary metabolite composition, using both targeted and LC-MS-based untargeted metabolomics approaches. Furthermore, the same population was profiled for transcript variation using a microarray containing EST sequences mainly derived from three Brassica species: B. napus, B. rapa and B. oleracea. The biochemical pathway analysis was based on the network analyses of both metabolite QTLs (mQTLs) and transcript QTLs (eQTLs). Co-localization of mQTLs and eQTLs lead to the identification of candidate regulatory genes involved in the biosynthesis of carotenoids, tocopherols and glucosinolates. We subsequently focused on the well-characterized glucosinolate pathway and revealed two hotspots of co-localization of eQTLs with mQTLs in linkage groups A03 and A09. Our results indicate that such a large-scale genetical genomics approach combining transcriptomics and metabolomics data can provide new insights into the genetic regulation of metabolite composition of Brassica vegetables.

Changes of phenolic secondary metabolite profiles in the reaction of narrow leaf lupin (Lupinus angustifolius) plants to infections with Colletotrichum lupini fungus or treatment with its toxin.

Plant interactions with environmental factors cause changes in the metabolism and regulation of biochemical and physiological processes. Plant defense against pathogenic microorganisms depends on an innate immunity system that is activated as a result of infection. There are two mechanisms of triggering this system: basal immunity activated as a result of a perception of microbe-associated molecular patterns through pattern recognition receptors situated on the cell surface and effector-triggered immunity (ETI). An induced biosynthesis of bioactive secondary metabolites, in particular phytoalexins, is one of the mechanisms of plant defense to fungal infection. Results of the study on narrow leaf lupin (Lupinus angustifolius L.) plants infected with the anthracnose fungus Colletotrichum lupini and treated with fungal phytotoxic metabolites are described in the paper. The C. lupini phytotoxins were isolated from liquid cultures, purified and partially characterized with physicochemical methods. Accumulation of secondary metabolites on leaf surface and within the tissues of plants either infected, treated with the fungal phytotoxin or submitted to both treatments was studied using GC-MS and LC-MS, respectively. Substantial differences in isoflavone aglycones and glycoconjugate profiles occurred in response to different ways of plant treatment.

Characterization of the natural variation in Arabidopsis thaliana metabolome by the analysis of metabolic distance.

Metabolite fingerprinting is widely used to unravel the chemical characteristics of biological samples. Multivariate data analysis and other statistical tools are subsequently used to analyze and visualize the plasticity of the metabolome and/or the relationship between those samples. However, there are limitations to these approaches for example because of the multi-dimensionality of the data that makes interpretation of the data obtained from untargeted analysis almost impossible for an average human being. These limitations make the biological information that is of prime importance in untargeted studies be partially exploited. Even in the case of full exploitation, current methods for relationship elucidation focus mainly on between groups variation and differences. Therefore, a measure that is capable of exploiting both between- and within-group biological variation would be of great value. Here, we examined the natural variation in the metabolome of nine Arabidopsis thaliana accessions grown under various environmental conditions and established a measure for the metabolic distance between accessions and across environments. This data analysis approach shows that there is just a minor correlation between genetic and metabolic diversity of the nine accessions. On the other hand, it delivers so far in Arabidopsis unexplored chemical information and is shown to be biologically relevant for resistance studies. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0375-3) contains supplementary material, which is available to authorized users.

LC/MS profiling of flavonoid glycoconjugates isolated from hairy roots, suspension root cell cultures and seedling roots of Medicago truncatula.

Hairy roots and suspension cell cultures are commonly used in deciphering different problems related to the biochemistry and physiology of plant secondary metabolites. Here, we address about the issue of possible differences in the profiles of flavonoid compounds and their glycoconjugates derived from various plant materials grown in a standard culture media. We compared profiles of flavonoids isolated from seedling roots, hairy roots, and suspension root cell cultures of a model legume plant, Medicago truncatula. The analyses were conducted with plant isolates as well as the media. The LC/MS profiles of target natural products obtained from M. truncatula seedling roots, hairy roots, and suspension root cell cultures differed substantially. The most abundant compounds in seedlings roots were mono- and diglucuronides of isoflavones and/or flavones. This type of glycosylation was not observed in hairy roots or suspension root cell cultures. The only recognized glycoconjugates in the latter samples were glucose derivatives of isoflavones. Application of a high-resolution mass spectrometer helped evaluate the elemental composition of protonated molecules, such as [M + H](+). Comparison of collision-induced dissociation MS/MS spectra registered with a quadrupole time-of-flight analyzer for tissue extracts and standards allowed us to estimate the aglycone structure on the basis of the pseudo-MS(3) experiment. Structures of these natural products were described according to the registered mass spectra and literature data. The analyses conducted represent an overview of flavonoids and their conjugates in different types of plant material representing the model legume, M. truncatula. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0287-2) contains supplementary material, which is available to authorized users.

Differentiation of isomeric malonylated flavonoid glyconjugates in plant extracts with UPLC-ESI/MS/MS.

INTRODUCTION: Glycosylation at different hydroxyl groups of flavonoids and acylation of sugar moieties are ubiquitous modifications observed in plants. These modifications give rise to simultaneous presence of numerous isomeric and isobaric compounds in tissues and extracts thereof. OBJECTIVE: To develop UPLC-MS method capable for resolution of isomeric malonylated glycoconjugates of flavonoids and recognition of structural differences. METHODOLOGY: Flavonoid glycoconjugates were extracted from leaves of blue lupin (Lupinus angustifolius L.) plants with 80% methanol. Extracts were analysed using ultraperformance liquid chromatography (UPLC) combined with tandem (quadrupole-time of flight, QToF) mass spectrometry. RESULTS: Differentiation of malonylated glycosides of isoflavones and flavones is demonstrated in this paper. The use of UPLC-MS/MS enabled 38 flavonoid conjugates to be distinguished, including the discrimination of five different isomers of a single 3'-O-methylluteolin glycoside. Additionally, pseudo MS(3) experiments (CID spectra registered at high cone voltages) enabled confirmation of the aglycone structures by comparison of their spectra with those obtained from aglycone standards. CONCLUSIONS: Application of UPLC-MS/MS allows separation and identification numerous positional isomers of malonylated glycosides of flavonoids and isoflavonoids in plant material. Provided there is strict control of the MS ionisation parameters, this method may be useful for preparation of a flavonoids spectra database, enabling the inter-laboratory comparison of analytical results.

Transcription factor AtDOF4;2 affects phenylpropanoid metabolism in Arabidopsis thaliana.

In a phenotypic screen of plants constitutively overexpressing DOF (DNA-binding-with-one-finger) transcription factors under the control of the Cauliflower mosaic virus 35S promoter, AtDOF4;2 was identified as a gene inducing a bushy plant phenotype and potentially being involved in the regulation of phenylpropanoid metabolism in Arabidopsis. Further molecular and biochemical characterization was performed in parallel using transgenic plants with enhanced and reduced AtDOF4;2 expression. The expression pattern of AtDOF4;2 was determined by quantitative real-time polymerase chain reaction (Q-RTPCR) and through promoter-beta-glucuronidase (GUS) fusions, indicating preferential transcriptional activity in axillary buds of the flower stalk, the hypocotyls periderm and in tapetum cells. Constitutive overexpression and RNAi-mediated silencing of AtDOF4;2 caused reciprocal changes in the expression of flavonoid biosynthetic genes and the accumulation of flavonoids under cold and high-light conditions. Moreover, tapetum-specific overexpression of AtDOF4;2 led to pollen grains devoid of flavonols. In contrast to its negative influence on flavonoid biosynthesis and coincident with high expression in the periderm and tapetum, AtDOF4;2 positively influences the production of hydroxycinnamic acids in the hypocotyl and flower buds, implicating its possible importance for suberin and sporopollenin production. These data provide evidence that AtDOF4;2, influences phenylpropanoid metabolism in an environmental and tissue-specific manner.