Functional Characterization of Genes Coding for Novel ß-D-Glucosidases Involved in the Initial Step of Secoiridoid Glucosides Catabolism in Centaurium erythraea Rafn.

Secoiridoid glucosides (SGs) are monoterpenoids derived from the iridoid cyclopentane-C-pyran skeleton with ß-D glucose linked at C1 position. Coordinated metabolic processes, such as biosynthesis and catabolism of SGs, ensure constitutive presence of these bitter tasting compounds in plant tissues, which plays a decisive role in the defense against pathogens and herbivores. These compounds are susceptible to hydrolysis mediated by enzymes ß-glucosidases, and the resulting aglycones are subsequently directed toward different metabolic pathways in plants. Function of two ß-D-glucosidases (named CeBGlu1 and CeBGlu2) from centaury (Centaurium erythraea Rafn; fam. Gentianaceae), belonging to the glycoside hydrolase 1 (GH1) family, was confirmed using in vitro assays with recombinant proteins, following their heterologous expression in E. coli and His-tag affinity purification. Although they show slightly differential substrate preference, both isoforms display high specificity toward SGs and the organ-specific distribution of transcripts was positively correlated with the content of SGs in diploid and tetraploid C. erythraea plants. Transient overexpression of CeBGlu1 and CeBGlu2 in C. erythraea leaves induced changes in metabolite profiles. The effectiveness of transgene overexpression has been altered by plant ploidy. UHPLC/DAD/(±)HESI - MS2 profiling of leaves of diploid and tetraploid C. erythraea genotypes revealed that the amounts of major SGs; sweroside, swertiamarin, and gentiopicrin was decreased in agroinfiltrated leaves, especially when CeBGlu1 and CeBGlu2 were co-expressed with transgene silencing suppressor p19. The work demonstrates that in planta metabolic engineering adopting transient overexpression of CeBGlu1 and CeBGlu2 is a suitable tool for the modulation of SGs content and glucosides/aglycones ratio, which might have substantial effects on overall phytochemistry of C. erythraea.

In Silico Study of Superoxide Dismutase Gene Family in Potato and Effects of Elevated Temperature and Salicylic Acid on Gene Expression.

Potato (Solanum tuberosum L.) is the most important vegetable crop globally and is very susceptible to high ambient temperatures. Since heat stress causes the accumulation of reactive oxygen species (ROS), investigations regarding major enzymatic components of the antioxidative system are of the essence. Superoxide dismutases (SODs) represent the first line of defense against ROS but detailed in silico analysis and characterization of the potato SOD gene family have not been performed thus far. We have analyzed eight functional SOD genes, three StCuZnSODs, one StMnSOD, and four StFeSODs, annotated in the updated version of potato genome (Spud DB DM v6.1). The StSOD genes and their respective proteins were analyzed in silico to determine the exon-intron organization, splice variants, cis-regulatory promoter elements, conserved domains, signals for subcellular targeting, 3D-structures, and phylogenetic relations. Quantitative PCR analysis revealed higher induction of StCuZnSODs (the major potato SODs) and StFeSOD3 in thermotolerant cultivar Désirée than in thermosensitive Agria and Kennebec during long-term exposure to elevated temperature. StMnSOD was constitutively expressed, while expression of StFeSODs was cultivar-dependent. The effects of salicylic acid (10-5 M) on StSODs expression were minor. Our results provide the basis for further research on StSODs and their regulation in potato, particularly in response to elevated temperatures.

The Arabinogalactan Protein Family of Centaurium erythraea Rafn.

Centaurium erythraea (centaury) is a medicinal plant with exceptional developmental plasticity in vitro and vigorous, often spontaneous, regeneration via shoot organogenesis and somatic embryogenesis, during which arabinogalactan proteins (AGPs) play an important role. AGPs are highly glycosylated proteins belonging to the super family of O-glycosylated plant cell surface hydroxyproline-rich glycoproteins (HRGPs). HRGPs/AGPs are intrinsically disordered and not well conserved, making their homology-based mining ineffective. We have applied a recently developed pipeline for HRGP/AGP mining, ragp, which is based on machine learning prediction of proline hydroxylation, to identify HRGP sequences in centaury transcriptome and to classify them into motif and amino acid bias (MAAB) classes. AGP sequences with low AG glycomotif representation were also identified. Six members of each of the three AGP subclasses, fasciclin-like AGPs, receptor kinase-like AGPs and AG peptides, were selected for phylogenetic and expression analyses. The expression of these 18 genes was recorded over 48 h following leaf mechanical wounding, as well as in 16 tissue samples representing plants from nature, plants cultivated in vitro, and developmental stages during shoot organogenesis and somatic embryogenesis. None of the selected genes were upregulated during both wounding recovery and regeneration. Possible functions of AGPs with the most interesting expression profiles are discussed.

Effects of C60 Fullerene on Thioacetamide-Induced Rat Liver Toxicity and Gut Microbiome Changes.

Thioacetamide (TAA) is widely used to study liver toxicity accompanied by oxidative stress, inflammation, cell necrosis, fibrosis, cholestasis, and hepatocellular carcinoma. As an efficient free radical's scavenger, C60 fullerene is considered a potential liver-protective agent in chemically-induced liver injury. In the present work, we examined the hepatoprotective effects of two C60 doses dissolved in virgin olive oil against TAA-induced hepatotoxicity in rats. We showed that TAA-induced increase in liver oxidative stress, judged by the changes in the activities of SOD, CAT, GPx, GR, GST, the content of GSH and 4-HNE, and expression of HO-1, MnSOD, and CuZnSOD, was more effectively ameliorated with a lower C60 dose. Improvement in liver antioxidative status caused by C60 was accompanied by a decrease in liver HMGB1 expression and an increase in nuclear Nrf2/NF-κB p65 ratio, suggesting a reduction in inflammation, necrosis and fibrosis. These results were in accordance with liver histology analysis, liver comet assay, and changes in serum levels of ALT, AST, and AP. The changes observed in gut microbiome support detrimental effects of TAA and hepatoprotective effects of low C60 dose. Less protective effects of a higher C60 dose could be a consequence of its enhanced aggregation and related pro-oxidant role.

Comparative Metabolite and Gene Expression Analyses in Combination With Gene Characterization Revealed the Patterns of Flavonoid Accumulation During Cistus creticus subsp. creticus Fruit Development.

Cistus creticus L. subsp. creticus (rockrose) is a shrub widespread in Greece and the Mediterranean basin and has been used in traditional medicine as herb tea for colds, for healing and digestive hitches, for the treatment of maladies, as perfumes, and for other purposes. Compounds from its flavonoid fraction have recently drawn attention due to antiviral action against influenza virus and HIV. Although several bioactive metabolites belonging to this group have been chemically characterized in the leaves, the genes involved in their biosynthesis in Cistus remain largely unknown. Flavonoid metabolism during C. creticus fruit development was studied by adopting comparative metabolomic and transcriptomic approaches. The present study highlights the fruit of C. creticus subsp. creticus as a rich source of flavonols, flavan-3-ols, and proanthocyanidins, all of which displayed a decreasing trend during fruit development. The majority of proanthocyanidins recorded in Cistus fruit are B-type procyanidins and prodelphinidins, while gallocatechin and catechin are the dominant flavan-3-ols. The expression patterns of biosynthetic genes and transcription factors were analyzed in flowers and throughout three fruit development stages. Flavonoid biosynthetic genes were developmentally regulated, showing a decrease in transcript levels during fruit maturation. A high degree of positive correlations between the content of targeted metabolites and the expression of biosynthetic genes indicated the transcriptional regulation of flavonoid biosynthesis during C. creticus fruit development. This is further supported by the high degree of significant positive correlations between the expression of biosynthetic genes and transcription factors. The results suggest that leucoanthocyanidin reductase predominates the biosynthetic pathway in the control of flavan-3-ol formation, which results in catechin and gallocatechin as two of the major building blocks for Cistus proanthocyanidins. Additionally, there is a decline in ethylene production rates during non-climacteric Cistus fruit maturation, which coincides with the downregulation of the majority of flavonoid- and ethylene-related biosynthetic genes and corresponding transcription factors as well as with the decline in flavonoid content. Finally, functional characterization of a Cistus flavonoid hydroxylase (F3'5'H) was performed for the first time.

Antagonistic Interaction between Phosphinothricin and Nepeta rtanjensis Essential Oil Affected Ammonium Metabolism and Antioxidant Defense of Arabidopsis Grown In Vitro.

Phosphinothricin (PPT) is one of the most widely used herbicides. PTT targets glutamine synthetase (GS) activity in plants, and its phytotoxicity is ascribed to ammonium accumulation and reactive oxygen species bursts, which drives rapid lipid peroxidation of cell membranes. In agricultural fields, PPT is extensively sprayed on plant foliage; however, a portion of the herbicide reaches the soil. According to the present study, PPT absorbed via roots can be phytotoxic to Arabidopsis, inducing more adverse effects in roots than in shoots. Alterations in plant physiology caused by 10 days exposure to herbicide via roots are reflected through growth suppression, reduced chlorophyll content, perturbations in the sugar and organic acid metabolism, modifications in the activities and abundances of GS, catalase, peroxidase, and superoxide dismutase. Antagonistic interaction of Nepeta rtanjensis essential oil (NrEO) and PPT, emphasizes the existence of complex control mechanisms at the transcriptional and posttranslational level, which result in the mitigation of PPT-induced ammonium toxicity and in providing more efficient antioxidant defense of plants. Simultaneous application of the two agents in the field cannot be recommended; however, NrEO might be considered as the PPT post-treatment for reducing harmful effects of herbicide residues in the soil on non-target plants.

Secondary Somatic Embryogenesis in Centaurium erythraea Rafn.

Somatic embryogenesis (SE) is a developmental process during which plant somatic cells, under suitable conditions, produce embryogenic cells that develop into somatic embryos (se). SE is the most important method for plant propagation in vitro, having both fundamental and applicative significance. SE can be induced from different tissues and organs, but when se are used as explants, the process is recognized as secondary or cyclic SE. We induced secondary SE in Centaurium erythraea by application of 2,4-dichlorophenoxyacetic acid (2,4-D) and N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU). A medium containing 0.1 mgL-1 2,4-D and 0.25 mgL-1 CPPU was optimal in terms of the number of primary SE explants forming se, the number of well-developed se per explant, and morphological appearance of the obtained se. These concentrations allowed SE to progress through three cycles, whereas at higher concentrations of 0.2 mgL-1 2,4-D and 0.5 mgL-1 CPPU, only two cycles were achieved. Histological analysis revealed that secondary se are formed both directly and indirectly. Secondary SE readily germinated and converted into plantlets. Induction of cyclic SE contributes to the conservation efforts of this endangered medicinal plant and expands the spectrum of in vitro developmental pathways described in centaury-an emerging model in developmental biology.

Transcriptome Profiling of the Potato Exposed to French Marigold Essential Oil with a Special Emphasis on Leaf Starch Metabolism and Defense against Colorado Potato Beetle.

Flower strips of French Marigold are commonly used pest repellents in potato fields. However, the effect of French Marigold volatiles on potato metabolism, physiology and induced defense is unknown. Thus, a microarray transcriptome analysis was performed to study the effects of French Marigold essential oil (EO) on laboratory-grown potato. After 8 h of exposure to EO, with gas chromatography/mass spectrometry (GC/MS)-detected terpinolene and limonene as dominant compounds, 2796 transcripts were differentially expressed with fold change >2 compared to expression in controls. A slightly higher number of transcripts had suppressed expression (1493 down- vs. 1303 up-regulated). Since transcripts, annotated to different photosynthesis-related processes, were mostly down-regulated, we selected a set of 10 genes involved in the leaf starch metabolism pathway, and validated microarray patterns using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Except for decreased synthesis and induced decomposition of starch granule in leaves, 8 h long EO exposure slightly elevated the accumulation of sucrose compared to glucose and fructose in subjected potato plants. An in vitro feeding bioassay with Colorado potato beetle showed that EO-induced alternations on transcriptional level and in the sugars' metabolism caused the enhancement of feeding behavior and overall development of the tested larvae. Results of comprehensive analysis of transcriptional responses in potato exposed to French Marigold EO provide a basis for further elucidation of molecular mechanisms underlying eco-physiological interactions in companion planting cropping systems.

Molecular Characterization and Expression of Four Aquaporin Genes in Impatiens walleriana During Drought Stress and Recovery.

Aquaporins comprise a large group of transmembrane proteins responsible for water transport, which is crucial for plant survival under stress conditions. Despite the vital role of aquaporins, nothing is known about this protein family in Impatiens walleriana, a commercially important horticultural plant, which is sensitive to drought stress. In the present study, attention is given to the molecular characterization of aquaporins in I. walleriana and their expression during drought stress and recovery. We identified four I. walleriana aquaporins: IwPIP1;4, IwPIP2;2, IwPIP2;7 and IwTIP4;1. All of them had conserved NPA motifs (Asparagine-Proline-Alanine), transmembrane helices (TMh), pore characteristics, stereochemical properties and tetrameric structure of holoprotein. Drought stress and recovery treatment affected the aquaporins expression in I. walleriana leaves, which was up- or downregulated depending on stress intensity. Expression of IwPIP2;7 was the most affected of all analyzed I. walleriana aquaporins. At 15% and 5% soil moisture and recovery from 15% and 5% soil moisture, IwPIP2;7 expression significantly decreased and increased, respectively. Aquaporins IwPIP1;4 and IwTIP4;1 had lower expression in comparison to IwPIP2;7, with moderate expression changes in response to drought and recovery, while IwPIP2;2 expression was of significance only in recovered plants. Insight into the molecular structure of I. walleriana aquaporins expanded knowledge about plant aquaporins, while its expression during drought and recovery contributed to I. walleriana drought tolerance mechanisms and re-acclimation.

Effects of Exogenous Salicylic Acid on Drought Response and Characterization of Dehydrins in Impatiens walleriana.

Impatiens walleriana is a valued ornamental plant sensitive to drought stress. We investigated whether the foliar application of 2mM salicylic acid (SA) can protect potted I. walleriana plants from drought stress. The plants were divided into: watered plants, drought-stressed plants, watered plants treated with SA and drought-stressed plants treated with SA. The number of flowers and flower buds, relative water content (RWC), contents of malondialdehyde (MDA) and proline (Pro) and the activities of superoxide dismutases, catalases and peroxidases were recorded at different time points. Three dehydrin sequences were identified in de novo assembled leaf transcriptome: IwDhn1, IwDhn2.1 and IwDhn2.2. Drought stress caused wilting, floral abortion, reduction of RWC and increased MDA-an indicator of lipid peroxidation. In response to drought, Impatiens accumulated Pro and induced chloroplastic Cu/ZnSOD and two peroxidase isoforms. The most remarkable drought response was strong induction of IwDhn2.1 and IwDhn2.2. Rehydration restored RWC, Pro level, Cu/ZnSOD activity and dehydrins expression in drought-stressed plants approximately to the values of watered plants.SA had ameliorating effects on plants exposed to drought, including prevention of wilting, preservation of RWC, increased Pro accumulation, modulation of antioxidative activities and remarkable decrease of lipid peroxidation, but without effects on flowers' preservation.

Genetic diversity of the Griffon vulture population in Serbia and its importance for conservation efforts in the Balkans.

The Griffon vulture was once a widespread species across the region of Southeast Europe, but it is now endangered and in some parts is completely extinct. In the Balkan Peninsula the largest Griffon vulture inland population inhabits the territory of Serbia. We present, for the first time, the genetic data of this valuable population that could be a source for future reintroduction programs planned in South-eastern Europe. To characterize the genetic structure of this population we used microsatellite markers from ten loci. Blood samples were collected from 57 chicks directly in the nests during the ongoing monitoring program. We performed a comparative analysis of the obtained data with the existing data from three native populations from French Pyrenees, Croatia, and Israel. We have assessed the genetic differentiation between different native populations and determined the existence of two genetic clusters that differentiate the populations from the Balkan and Iberian Peninsulas. Furthermore, we analysed whether the recent bottleneck events influenced the genetic structure of the populations studied, and we found that all native populations experienced a recent bottleneck event, and that the population of Israel was the least affected. Nevertheless, the parameters of genetic diversity suggest that all analysed populations have retained a similar level of genetic diversity and that the Griffon vulture population from Serbia exhibits the highest value for private alleles. The results of this study suggest that the Griffon vulture populations of the Balkan Peninsula are genetically differentiated from the populations of the Iberian Peninsula, which is an important information for future reintroduction strategies.

Alterations in nepetalactone metabolism during polyethylene glycol (PEG)-induced dehydration stress in two Nepeta species.

A number of Nepeta species (fam. Lamiaceae) are interesting medicinal crops for arid and semi-arid areas, due to their ability to maintain essential developmental and physiological processes and to rationalize their specialized metabolism under water deficit growth conditions. The present research is, to our knowledge, the first attempt to investigate the molecular background of the dehydration-induced changes in specialized metabolism of Nepeta species, which will help to understand relations between dehydration stress on one hand and biomass production and yield of nepetalactone (NL) on the other. During the 6 days exposure of Nepeta rtanjensis Diklic & Milojevic and Nepeta argolica Bory & Chaub. ssp. argolica plants to PEG-induced dehydration stress under experimental in vitro conditions, decrease in transcript levels of the majority of 10 NL biosynthetic genes, and some of the 5 transcription factors (TFs) were recorded, simultaneously with the initial reduction in NL content. The two model species evidently employ similar strategies in response to severe dehydration stress; however N. rtanjensis is highlighted as the species more efficient in maintaining NL amounts in tissues. The results suggest trichome-specific and co-ordinately regulated NL biosynthesis at the level of gene expression, with trichome enriched MYC2 and YABBY5 TFs being the potential positive regulators. Manipulation of such TFs can be effective for engineering the NL biosynthetic pathway, and for the increased production of cis,trans-NL in N. argolica ssp. argolica and trans,cis-NL in N. rtanjensis.

Silencing of germacrene A synthase genes reduces guaianolide oxalate content in Cichorium intybus L.

Chicory (Cichorium intybus L.) is a medicinal and industrial plant from the Asteraceae family that produces a variety of sesquiterpene lactones (STLs), most importantly bitter guaianolides: lactucin, lactucopicrin and 8-deoxylactucin as well as their modified forms such as oxalates. These compounds have medicinal properties; however, they also hamper the extraction of inulin - a very important food industry product from chicory roots. The first step in guaianolide biosynthesis is catalyzed by germacrene A synthase (GAS) which in chicory exists in two isoforms - GAS long (encoded by CiGASlo) and GAS short (encoded by CiGASsh). AmiRNA silencing was used to obtain plants with reduced GAS gene expression and level of downstream metabolites, guaianolide-15-oxalates, as the major STLs in chicory. This approach could be beneficial for engineering new chicory varieties with varying STL content, and especially varieties with reduced bitter compounds more suitable for inulin production.

Secoiridoids Metabolism Response to Wounding in Common Centaury (Centaurium erythraea Rafn) Leaves.

Centaurium erythraea Rafn produces and accumulates various biologically active specialized metabolites, including secoiridoid glucosides (SGs), which help plants to cope with unfavorable environmental conditions. Specialized metabolism is commonly modulated in a way to increase the level of protective metabolites, such as SGs. Here, we report the molecular background of the wounding-induced changes in SGs metabolism for the first time. The mechanical wounding of leaves leads to a coordinated up-regulation of SGs biosynthetic genes and corresponding JA-related transcription factors (TFs) after 24 h, which results in the increase of metabolic flux through the biosynthetic pathway and, finally, leads to the elevated accumulation of SGs 96 h upon injury. The most pronounced increase in relative expression was detected for secologanin synthase (CeSLS), highlighting this enzyme as an important point for the regulation of biosynthetic flux through the SG pathway. A similar expression pattern was observed for CeBIS1, imposing itself as the TF that is prominently involved in wound-induced regulation of SGs biosynthesis genes. The high degree of positive correlations between and among the biosynthetic genes and targeted TFs expressions indicate the transcriptional regulation of SGs biosynthesis in response to wounding with a significant role of CeBIS1, which is a known component of the jasmonic acid (JA) signaling pathway.

ragp: Pipeline for mining of plant hydroxyproline-rich glycoproteins with implementation in R.

Hydroxyproline-rich glycoproteins (HRGPs) are one of the most complex families of macromolecules found in plants, due to the diversity of glycans decorating the protein backbone, as well as the heterogeneity of the protein backbones. While this diversity is responsible for a wide array of physiological functions associated with HRGPs, it hinders attempts for homology based identification. Current approaches, based on identifying sequences with characteristic motifs and biased amino acid composition, are limited to prototypical sequences. Ragp is an R package for mining and analysis of HRGPs, with emphasis on arabinogalactan proteins. The ragp filtering pipeline exploits one of the HRGPs key features, the presence of hydroxyprolines which represent glycosylation sites. Main package features include prediction of proline hydroxylation sites, amino acid motif and bias analyses, efficient communication with web servers for prediction of N-terminal signal peptides, glycosylphosphatidylinositol modification sites and disordered regions and the ability to annotate sequences through hmmscan and subsequent GO enrichment, based on predicted Pfam domains. As such, ragp extends R's rich ecosystem for high-throughput sequence data analyses. The ragp R package is available under the MIT Open Source license and is freely available to download from GitHub at: https://github.com/missuse/ragp.

Phenotypic and Genetic Variation of an Interspecific Centaurium Hybrid (Gentianaceae) and Its Parental Species.

Interspecific hybridization is one of the major actuators of evolutionary changes in plants. As the result of allopolyploid hybridization, offspring may gain different ploidy levels in comparison to parental species, which can provide them instant reproductive isolation. Two tetraploid sister species, Centaurium erythraea and C. littorale, readily cross-fertilize, resulting in hybrids of various ploidy. In northern Serbia, two stable populations of a hexaploid taxon C. pannonicum have been documented. It has been proposed previously that this taxon emerged after an interspecific hybridization event between two tetraploid sister-species: C. erythraea and C. littorale subsp. compressum. The existing populations of the hybridogenic taxon, as well as neighboring populations of the two parental taxa were here characterized by both morphometrics and molecular markers (EST-SSR and trnL-F). Three leaf and two flower characteristics were found to be informative in delimitation of the parental taxa and in their discernment from hybrid individuals, the latter having intermediate values. Eight microsatellite markers were found to have good ability to distinguish studied taxa, placing C. pannonicum in closer relationship with C. erythraea. Conversely, trnL-F plastid marker nominated C. littorale subsp. compressum to be the donor of the C. pannonicum plastid DNA. Reproductive isolation of the hexaploid hybrid individuals from the parental species should be examined as the next logical step in describing the new species.

Nepetalactone-rich essential oil mitigates phosphinothricin-induced ammonium toxicity in Arabidopsis thaliana (L.) Heynh.

Active ingredient of the commercial herbicide BASTA (B), phosphinothricin, acts as an inhibitor of glutamine synthetase (GS), a key enzyme in ammonium assimilation. The treatment with BASTA leads to an elevation of ammonium levels in plants and further to various physiological alterations, ammonium toxicity and lethality. Results of the present study emphasize the complexity underlying control mechanisms that determine BASTA interaction with essential oil (EO) from Nepeta rtanjensis (NrEO), bioherbicide inducing oxidative stress in target plants. Simultaneous application of NrEO and BASTA, two agents showing differential mode of action, suspends BASTA-induced ammonium toxicity in Arabidopsis thaliana plants. This is achieved through maintaining GS activity, which sustains a sub-toxic and/or sub-lethal ammonium concentration in tissues. As revealed by the present study, regulation of GS activity, as influenced by BASTA and NrEO, occurs at transcriptional, posttranscriptional, and/or posttranslational levels. Two genes encoding cytosolic GS, GLN1;1 and GLN1;3, are highlighted as the main isozymes in Arabidopsis shoots contributing to NrEO-induced overcoming of BASTA-generated ammonium toxicity. The effects of NrEO might be ascribed to its major component nepetalactone, but the contribution of minor EO components should not be neglected. Although of fundamental significance, the results of the present study suggest possible low efficiency of BASTA in plantations of medicinal/aromatic plants such as Nepeta species. Furthermore, these results highlight the possibility of using NrEO as a bioherbicide in BASTA-treated crop fields to mitigate the effect of BASTA residues in contaminated soils.

Chemodiversity of two closely related tetraploid Centaurium species and their hexaploid hybrid: Metabolomic search for high-resolution taxonomic classifiers.

Species within the genus Centaurium readily hybridize and polyploid complexes are often seen in natural populations. We describe phytochemical profiles of newly discovered allohexaploid hybrid, here named Centaurium pannonicum, and its parental tetraploid species C. erythraea and rare C. littorale ssp. compressum. Our aim was to examine chemodiversity of these taxa in the area of Vojvodina (North Serbia) and to perform metabolomics search for chemical classifiers which would provide high resolution discrimination of parental and hybrid individuals. In sum, UHPLC-MS/MS Orbitrap metabolomics fingerprinting revealed seventy compounds in methanol extracts. Despite the lack of qualitative chemical novelty in hybrid plants, UHPLC-qqqMS targeted metabolomics approach, aimed at three secoiridoid compounds and seventeen phenolics, pointed to considerable differences in quantitative composition of these dominant compounds among the plant taxa studied. In addition to the difference in the ploidy levels, the hybrid taxon was well distinguished from both parental species based on metabolite profiles, and, for most individuals, positioned intermediately to the parental taxa in both PCA and hierarchical clustering. After optimizing and comparing several statistical learning methods, it was possible to narrow the number of taxonomic classifiers to five (three xanthones, one secoiridoid glycoside, and one phenolic acid), while increasing the differentiation resolution. The presented metabolomics approach will certainly, along with morphometrics and molecular genetics studies, have high impact on further elucidation of complex relationships among taxa within the genus Centaurium.

Knockout mutants as a tool to identify the subunit composition of Arabidopsis glutamine synthetase isoforms.

Glutamine synthetase (GS) is a key enzyme in nitrogen assimilation, which catalyzes the formation of glutamine from ammonia and glutamate. Plant GS isoforms are multimeric enzymes, recently shown to be decamers. The Arabidopsis genome encodes five cytosolic (GS1) proteins labeled as GLN1;1 through GLN1;5 and one chloroplastic (GS2) isoform, GLN2;0. However, as many as 11 GS activity bands were resolved from different Arabidopsis tissues by Native PAGE and activity staining. Western analysis showed that all 11 isoforms are composed exclusively of 40 kDa GS1 subunits. Of five GS1 genes, only GLN1;1, GLN1;2 and GLN1;3 transcripts accumulated to significant levels in vegetative tissues, indicating that only subunits encoded by these three genes produce the 11-band zymogram. Even though the GS2 gene also had significant expression, the corresponding activity was not detected, probably due to inactivation. To resolve the subunit composition of 11 active GS1 isoforms, homozygous knockout mutants deficient in the expression of different GS1 genes were selected from the progeny of T-DNA insertional SALK and SAIL lines. Comparison of GS isoenzyme patterns of the selected GS1 knockout mutants indicated that all of the detected isoforms consist of varying proportions of GLN1;1, GLN1;2 and GLN1;3 subunits, and that GLN1;1 and GLN1;3, as well as GLN1;2 and GLN1;3 and possibly GLN1;1 and GLN1;2 proteins combine in all proportions to form active homo- and heterodecamers.

Sugars and acid invertase mediate the physiological response of Schenkia spicata root cultures to salt stress.

A heterotrophic model system was established in our studies in order to differentiate the effect of high salt concentrations in external medium on growth and sugar metabolism in roots from the effect of reduced sugar availability resulting from decreased photosynthesis under salinity. Soluble sugar content and the activity of acid invertase in root cultures of salt-tolerant (ST) and salt-sensitive (SS) Schenkia spicata (L.) Mansion genotypes were investigated during exposure to different NaCl concentrations (0-200 mM). Their response to severe salinity was characterized by a metabolic adjustment that led to the accumulation of sucrose (Suc) in root tissues. There was clear evidence that cell wall invertase (CW-Inv) is the major contributor to the Suc/hexose ratio in roots during exposure to elevated salinity. The results of CW-Inv activity and immunodetection assays in our study suggest that the regulation of CW-Inv expression is most likely achieved in a salt stress dependent manner. Also, NaCl modulated soluble acid invertase (SA-Inv) expression differentially in SS and ST genotypes of S. spicata. Regardless of the salt treatment, genotype, or the amount of enzyme, SA-Inv activity was generally low, indicating regulation at the posttranslational level. The results suggest no direct role of SA-Inv in the regulation of the root tissue carbohydrate pool and therefore in the control of the availability of glucose and fructose for the primary metabolism and/or osmotic adjustment in the present heterotrophic model system.