Michał Jasiński

AtOSA1, a Member of the Abc1-Like Family, as a New Factor in Cadmium and Oxidative Stress Response

The analysis of gene expression in Arabidopsis (Arabidopsis thaliana) using cDNA microarrays and reverse transcription-polymerase chain reaction showed that AtOSA1 (A. thaliana oxidative stress-related Abc1-like protein) transcript levels are influenced by Cd2+ treatment. The comparison of protein sequences revealed that AtOSA1 belongs to the family of Abc1 proteins. Up to now, Abc1-like proteins have been identified in prokaryotes and in the mitochondria of eukaryotes. AtOSA1 is the first member of this family to be localized in the chloroplasts. However, despite sharing homology to the mitochondrial ABC1 of Saccharomyces cerevisiae, AtOSA1 was not able to complement yeast strains deleted in the endogenous ABC1 gene, thereby suggesting different function between AtOSA1 and the yeast ABC1. The atosa1-1 and atosa1-2 T-DNA insertion mutants were more affected than wild-type plants by Cd2+ and revealed an increased sensitivity toward oxidative stress (hydrogen peroxide) and high light. The mutants exhibited higher superoxide dismutase activities and differences in the expression of genes involved in the antioxidant pathway. In addition to the conserved Abc1 region in the AtOSA1 protein sequence, putative kinase domains were found. Protein kinase assays in gelo using myelin basic protein as a kinase substrate revealed that chloroplast envelope membrane fractions from the AtOSA1 mutant lacked a 70-kD phosphorylated protein compared to the wild type. Our data suggest that the chloroplast AtOSA1 protein is a new factor playing a role in the balance of oxidative stress.

Changes in the profile of flavonoid accumulation in Medicago truncatula leaves during infection with fungal pathogen Phoma medicaginis.

Medicago truncatula is a model species for the study of the unique secondary metabolism in legumes. LC/MS/MS analysis was used to identify and profile flavonoid glycoconjugates and free aglycones in leaves of M. truncatula (ecotype R108-1) infected with the fungal pathogen Phoma medicaginis. Use of a high resolution analyzer with a collision induced dissociation tandem mass spectrometer (CID MS/MS) permitted structural elucidation of target secondary metabolites and four new acylated flavone glycosides have been identified. Changes in the phytoalexin medicarpin and its isoflavone precursors were quantitatively monitored at various time points after fungal spore application. Application of spores induced disease symptoms in the leaves of infected plants and resulted in an increase in the medicarpin precursors formononetin 7-O-glucoside and malonylated formononetin 7-O-glucoside between one and three days post-infection. Relative concentrations of medicarpin were highest five days post-infection. The rapid increase of these molecules was clearly positively correlated to the infection process as certain of them were absent in uninfected leaves, suggesting that the relative rate of their synthesis is tightly related with the infection process.

A Medicago truncatula ABC transporter belonging to subfamily G modulates the level of isoflavonoids

Full-sized ATP-binding cassette (ABC) transporters of the G subfamily (ABCG) are considered to be essential components of the plant immune system. These proteins have been proposed to be implicated in the active transmembrane transport of various secondary metabolites. Despite the importance of ABCG-based transport for plant-microbe interactions, these proteins are still poorly recognized in legumes. The experiments described here demonstrated that the level of Medicago truncatula ABCG10 (MtABCG10) mRNA was elevated following application of fungal oligosaccharides to plant roots. Spatial expression pattern analysis with a reporter gene revealed that the MtABCG10 promoter was active in various organs, mostly within their vascular tissues. The corresponding protein was located in the plasma membrane. Silencing of MtABCG10 in hairy roots resulted in lower accumulation of the phenylpropanoid pathway-derived medicarpin and its precursors. PCR-based experiments indicated that infection with Fusarium oxysporum, a root-infecting pathogen, progressed faster in MtABCG10-silenced composite plants (consisting of wild-type shoots on transgenic roots) than in the corresponding controls. Based on the presented data, it is proposed that in Medicago, full-sized ABCG transporters might modulate isoflavonoid levels during the defence response associated with de novo synthesis of phytoalexins.

Cytokinin‐induced structural adaptability of a Lupinus luteus PR‐10 protein

Plant pathogenesis‐related (PR) proteins of class 10 are the only group among the 17 PR protein families that are intracellular and cytosolic. Sequence conservation and the wide distribution of PR‐10 proteins throughout the plant kingdom are an indication of an indispensable function in plants, but their true biological role remains obscure. Crystal and solution structures for several homologues have shown a similar overall fold with a vast internal cavity which, together with structural similarities to the steroidogenic acute regulatory protein‐related lipid transfer domain and cytokinin‐specific binding proteins, strongly indicate a ligand‐binding role for the PR‐10 proteins. This article describes the structure of a complex between a classic PR‐10 protein [Lupinus luteus (yellow lupine) PR‐10 protein of subclass 2, LlPR‐10.2B] and N,N′‐diphenylurea, a synthetic cytokinin. Synthetic cytokinins have been shown in various bioassays to exhibit activity similar to that of natural cytokinins. The present 1.95 Å resolution crystallographic model reveals four N,N′‐diphenylurea molecules in the hydrophobic cavity of the protein and a degree of conformational changes accompanying ligand binding. The structural adaptability of LlPR‐10.2B and its ability to bind different cytokinins suggest that this protein, and perhaps other PR‐10 proteins as well, can act as a reservoir of cytokinin molecules in the aqueous environment of a plant cell.

Fragmentation pathways of acylated flavonoid diglucuronides from leaves of Medicago truncatula

INTRODUCTION Flavonoids are important plant compounds occurring in tissues mostly in the form of glycoconjugates. Most frequently the sugar moiety is comprised of mono- or oligosaccharides consisting of common sugars like glucose, rhamnose or galactose. In some plant species the glycosidic moiety contains glucuronic acid and may be acylated by phenylpropenoic acids. METHODOLOGY Flavonoid glyconjugates were extracted from leaves of Medicago truncatula ecotype R108 and submitted to analysis using high-performance liquid chromatography combined with high-resolution tandem (quadrupole-time of flight, QToF) mass spectrometry. RESULTS The studied leaf extracts contained 26 different flavonoid glycosides among which 22 compounds were flavone (apigenin, luteolin, chrysoeriol and tricin) glucuronides and 13 were acylated with aromatic acids (p-coumaric, ferulic or sinapic). The fragmentation pathways observed in positive and negative ion mass spectra differed substantially between each other and from these of flavonoid glycosides which did not contain acidic sugars. The application of high-resolution MS techniques allowed unequivocal differentiation between ions with the same nominal m/z values containing different substituents (e.g. ferulic acid or glucuronic acid). Eleven of the identified flavonoids have not been reported previously in this species. PERSPECTIVES The presented unique fragmentation pathways of flavonoid glucuronates enable detection of these compounds in tissue extracts from different plant species.

Full-Size ABC Transporters from the ABCG Subfamily in Medicago truncatula

Full-size ATP-binding cassette (ABC) transporters belonging to the ABCG subfamily are unique for plants and fungi. There is growing evidence that certain of these proteins play a role in plant defense or signaling systems. As yet, a complete set of full-size ABCG protein genes has been inventoried and classified in only two plants: Arabidopsis thaliana and Oryza sativa. Recently, a domain-based clustering analysis has predicted the presence of at least 12 genes encoding such proteins in the Lotus japonicus genome. Here, we identify and classify 19 genes coding full-size ABCG proteins in Medicago truncatula, a model legume plant. We have found that the majority of these genes are expressed in roots and flowers whereas only a few are expressed in leaves. Expression of several has been induced upon pathogenic infection in both roots and leaves. ABCG messenger RNAs have been detected in root nodules forming during symbiosis of legume plants and nitrogen-fixing bacteria. The data presented provide a scaffold for further studies of the physiological function of Medicago ABCG transporters and their possible role in modulating plant-microorganism interactions.

Medicago truncatula ABCG10 is a transporter of 4-coumarate and liquiritigenin in the medicarpin biosynthetic pathway

A full-size Medicago ABCG protein acts as a distributor of medicarpin early precursors and a modulator of phenylpropanoid pathway activity upon biotic stress.

Defence, Symbiosis and ABCG Transporters

Plant genomes encode more than 100 ATP-binding cassette (ABC) transporters, a number far exceeding those of other organisms. The membrane-bound ABC transporters belonging to the G subfamily (ABCGs) can be categorised by their distinctive topology and taxa distribution. ABCGs form the largest known subfamily of ABC proteins, with 43 and 50 members in Arabidopsis and rice, respectively. Collected experimental data have revealed the great functional diversity of these proteins. The substrates known to be transferred by ABCGs, usually through the plasma membrane, include surface lipids, plant hormones and secondary metabolites. Therefore, ABCGs are recognised as being important for plant development as well as interactions with the environment. Historically, certain members of the ABCG subfamily were considered as proteins that evolved to be involved in pathogenic processes or biotic stress responses. However, recent discoveries have demonstrated that the function of ABCGs in plants extends beyond simply the secretion of anti-microbial molecules. Equally important as defence against invaders are interactions of plants with microorganisms that are beneficial to both partners. Such beneficial interactions include (1) symbiotic associations with fungi of the phylum Glomeromycota, also known as arbuscular mycorrhizae (AM) and (2) legume–rhizobia symbiosis (LRS). We have only just begun to discover that plant ABC transporters are important modulators of symbioses, but how they participate in these processes is unclear. Here, we provide basic information regarding the members of the G subfamily of ABC transporters and position them in the context of the defence reactions and symbiotic associations of plants, with special emphasis on legumes.