High-performance liquid chromatography with fluorescence detection for mycophenolic acid determination in saliva samples.

BACKGROUND: For therapeutic drug monitoring (TDM) of mycophenolic acid (MPA), which is frequently proposed, saliva might be a suitable and easy-to-obtain biological matrix. The study aimed to validate an HPLC method with fluorescence detection for determining mycophenolic acid in saliva (sMPA) in children with nephrotic syndrome. METHODS: The mobile phase was composed of methanol and tetrabutylammonium bromide with disodium hydrogen phosphate (pH 8.5) at a 48:52 ratio. To prepare the saliva samples, 100 µL of saliva, 50 µL of calibration standards, and 50 µL of levofloxacin (used as an internal standard) were mixed and evaporated to dryness at 45 °C for 2 h. The resulting dry extract was reconstituted in the mobile phase and injected into the HPLC system after centrifugation. Saliva samples from study participants were collected using Salivette® devices. RESULTS: The method was linear within the range of 5-2000 ng/mL, was selective with no carry-over effect and met the acceptance criteria for within-run and between-run accuracy and precision. Saliva samples can be stored for up to 2 h at room temperature, for up to 4 h at 4 °C, and for up to 6 months at - 80 °C. MPA was stable in saliva after three freeze-thaw cycles, in dry extract for 20 h at 4 °C, and for 4 h in the autosampler at room temperature. MPA recovery from Salivette® cotton swabs was within the range of 94-105%. The sMPA concentrations in the two children with nephrotic syndrome who were treated with mycophenolate mofetil were within 5-112 ng/mL. CONCLUSIONS: The sMPA determination method is specific, selective, and meets the validation requirements for analytic methods. It may be used in children with nephrotic syndrome; however further studies are required to investigate focusing on sMPA and the correlation between sMPA and total MPA and its possible contribution to MPA TDM is required.

Restriction of access to the central cavity is a major contributor to substrate selectivity in plant ABCG transporters.

ABCG46 of the legume Medicago truncatula is an ABC-type transporter responsible for highly selective translocation of the phenylpropanoids, 4-coumarate, and liquiritigenin, over the plasma membrane. To investigate molecular determinants of the observed substrate selectivity, we applied a combination of phylogenetic and biochemical analyses, AlphaFold2 structure prediction, molecular dynamics simulations, and mutagenesis. We discovered an unusually narrow transient access path to the central cavity of MtABCG46 that constitutes an initial filter responsible for the selective translocation of phenylpropanoids through a lipid bilayer. Furthermore, we identified remote residue F562 as pivotal for maintaining the stability of this filter. The determination of individual amino acids that impact the selective transport of specialized metabolites may provide new opportunities associated with ABCGs being of interest, in many biological scenarios.

The plant immune system ­ zig-zag model / System odpornosciowy roslin ­ model zygzakowy.

Unlike vertebrates, plants lack specialized mobile immune cells and antibodies. However, it doesn't mean that plants are not able to effectively defend themselves against pathogens. In the case of plants, almost every cell appears to be able of triggering an effective defense response thanks to the action of the immune system. The latter is well described by zig-zag model proposed by JD Jones and JL Dangle, which encompasses two branches of the plant immune system. Importantly, the zig-zag model, for the first time, has clearly presented the complex interplay between plants and their pathogens, moreover, it is extremely useful from a didactic point of view.

ATP-binding cassette transporters in nonmodel plants.

Knowledge about plant ATP-binding cassette (ABC) proteins is of great value for sustainable agriculture, economic yield, and the generation of high-quality products, especially under unfavorable growth conditions. We have learned much about ABC proteins in model organisms, notably Arabidopsis thaliana; however, the importance of research dedicated to these transporters extends far beyond Arabidopsis biology. Recent progress in genomic and transcriptomic approaches for nonmodel and noncanonical model plants allows us to look at ABC transporters from a wider perspective and consider chemodiversity and functionally driven adaptation as distinctive mechanisms during their evolution. Here, by considering several representatives from agriculturally important families and recent progress in functional characterization of nonArabidopsis ABC proteins, we aim to bring attention to understanding the evolutionary background, distribution among lineages and possible mechanisms underlying the adaptation of this versatile transport system for plant needs. Increasing the knowledge of ABC proteins in nonmodel plants will facilitate breeding and development of new varieties based on, for example, genetic variations of endogenous genes and/or genome editing, representing an alternative to transgenic approaches.

A roadmap of plant membrane transporters in arbuscular mycorrhizal and legume-rhizobium symbioses.

Most land plants live in close contact with beneficial soil microbes: the majority of land plant species establish symbiosis with arbuscular mycorrhizal fungi, while most legumes, the third largest plant family, can form a symbiosis with nitrogen-fixing rhizobia. These microbes contribute to plant nutrition via endosymbiotic processes that require modulating the expression and function of plant transporter systems. The efficient contribution of these symbionts involves precisely controlled integration of transport, which is enabled by the adaptability and plasticity of their transporters. Advances in our understanding of these systems, driven by functional genomics research, are rapidly filling the gap in knowledge about plant membrane transport involved in these plant-microbe interactions. In this review, we synthesize recent findings associated with different stages of these symbioses, from the pre-symbiotic stage to nutrient exchange, and describe the role of host transport systems in both mycorrhizal and legume-rhizobia symbioses.

Early stages of legume-rhizobia symbiosis are controlled by ABCG-mediated transport of active cytokinins.

Growing evidence has highlighted the essential role of plant hormones, notably, cytokinins (CKs), in nitrogen-fixing symbiosis, both at early and late nodulation stages1,2. Despite numerous studies showing the central role of CK in nodulation, the importance of CK transport in the symbiosis is unknown. Here, we show the role of ABCG56, a full-size ATP-binding cassette (ABC) transporter in the early stages of the nodulation. MtABCG56 is expressed in roots and nodules and its messenger RNA levels increase upon treatment with symbiotic bacteria, isolated Nod factor and CKs, accumulating within the epidermis and root cortex. MtABCG56 exports bioactive CKs in an ATP-dependent manner over the plasma membrane and its disruption results in an impairment of nodulation. Our data indicate that ABCG-mediated cytokinin transport is important for proper establishment of N-fixing nodules.

The Full-Size ABCG Transporter of Medicago truncatula Is Involved in Strigolactone Secretion, Affecting Arbuscular Mycorrhiza.

Strigolactones (SLs) are plant-derived signaling molecules that stimulate the hyphal branching of arbuscular mycorrhizal fungi (AMF), and consequently promote symbiotic interaction between the fungus and the plant. Currently, our knowledge on the molecular mechanism of SL transport is restricted to the Solanaceae family. In the Solanaceae family, SL translocation toward the rhizosphere occurs through the exodermis via hypodermal passage cells and involves a member of the G subfamily, of the ATP-binding cassette (ABC) membrane transporters. Most Fabaceae species, including those that are agriculturally important, have a different root anatomy compared to most angiosperm plants (i.e., lacking an exodermis). Thus, we have investigated how SL transport occurs in the model legume Medicago truncatula. Here, we show that overexpression of a SL transporter from petunia (PaPDR1) enhances AMF colonization rates in M. truncatula. This result demonstrates the importance of ABCG proteins for the translocation of orobanchol-type molecules to facilitate arbuscular mycorrhiza, regardless of root anatomy and phylogenetic relationships. Moreover, our research has led to the identification of Medicago ABCG59, a close homologue of Petunia PDR1, that exhibits root specific expression and is up-regulated by phosphate starvation as well as in the presence of rac-GR24, a synthetic SL. Its promoter is active in cortical cells, root tips, and the meristematic zone of nodules. The mtabcg59 loss-of-function mutant displayed a reduced level of mycorrhization compared to the WT plants but had no impact on the number of nodules after Sinorhizobium meliloti inoculation. The reduced mycorrhization indicates that less SLs are secreted by the mutant plants, which is in line with the observation that mtabcg59 exudates exhibit a reduced stimulatory effect on the germination of the parasitic plant Phelipanche ramosa compared to the corresponding wild type.

MtABCG20 is an ABA exporter influencing root morphology and seed germination of Medicago truncatula.

Abscisic acid (ABA) integrates internal and external signals to coordinate plant development, growth and architecture. It plays a central role in stomatal closure, and prevents germination of freshly produced seeds and germination of non-dormant seeds under unfavorable circumstances. Here, we describe a Medicago truncatula ATP-binding cassette (ABC) transporter, MtABCG20, as an ABA exporter present in roots and germinating seeds. In seeds, MtABCG20 was found in the hypocotyl-radicle transition zone of the embryonic axis. Seeds of mtabcg20 plants were more sensitive to ABA upon germination, due to the fact that ABA translocation within mtabcg20 embryos was impaired. Additionally, the mtabcg20 produced fewer lateral roots and formed more nodules compared with wild-type plants in conditions mimicking drought stress. Heterologous expression in Arabidopsis thaliana provided evidence that MtABCG20 is a plasma membrane protein that is likely to form homodimers. Moreover, export of ABA from Nicotiana tabacum BY2 cells expressing MtABCG20 was faster than in the BY2 without MtABCG20. Our results have implications both in legume crop research and determination of the fundamental molecular processes involved in drought response and germination.

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

The ABCG10 protein of the model legume Medicago truncatula is required for efficient de novo production of the phenylpropanoid-derived phytoalexin medicarpin. Silencing the expression of MtABCG10 results, inter alia, in a lower accumulation of medicarpin and its precursors. In this study, we demonstrate that the impairment of medicarpin biosynthesis can be partially averted by the exogenous application of 4-coumarate, an early precursor of the core phenylpropanoid pathway, and the deoxyisoflavonoid formononetin. Experiments conducted using HPLC/MS in a heterologous system as well as in vitro transport assays with labelled substrate revealed that MtABCG10 is responsible for the membrane translocation of 4-coumarate and liquiritigenin, molecules representing key branching points in the phenylpropanoid pathway. The identification of transporters participating in the distribution of precursors is an important step in understanding phenylpropanoid biosynthesis.

Abscisic acid - an overlooked player in plant-microbe symbioses formation?

Abscisic acid (ABA) is an ubiquitous plant hormone and one of the foremost signalling molecules, controlling plants' growth and development, as well as their response to environmental stresses. To date, the function of ABA has been extensively investigated as an abiotic stress molecule which regulates the plants' water status. However, in the context of symbiotic associations, ABA is less recognized. In contrast to well-described auxin/cytokinin and gibberellin/strigolactone involvement in symbioses, ABA has long been underestimated. Interestingly, ABA emerges as an important player in arbuscular mycorrhiza and legume-rhizobium symbiosis. The plant's use of stress hormones like ABA in regulation of those interactions directly links the efficiency of these processes to the environmental status of the plant, notably during drought stress. Here we provide an overview of ABA interplay in beneficial associations of plants with microorganisms and propose ABA as a potential factor determining whether the investment in establishing the interaction is higher than the profit coming from it.

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.

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.

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.