[Show abstract][Hide abstract] ABSTRACT: Endogenous gibberellins and brassinosteroids were quantified in 24 axenic microalgae strains from the Chlorophyceae, Trebouxiophyceae, Ulvophyceae and Charophyceae microalgae strains after 4 days in culture. This is the first report of endogenous gibberellins being successfully detected in microalgae. Between 18 and 20 gibberellins were quantified in all strains with concentrations ranging from 342.7 pg mg(-1) DW in Raphidocelis subcapitata MACC 317-4746.1 pg mg(-)(1) DW in Scotiellopsis terrestris MACC 44. Slower growing strains (S. terrestris MACC 44, Gyoerffyana humicola MACC 334, Nautococcus mamillatus MACC 716 and Chlorococcum ellipsoideum MACC 712) exhibited the highest gibberellin contents while lowest levels of gibberellins were found in faster growing strains (R. subcapitata MACC 317 and Coelastrum excentrica MACC 504). In all strains, the active gibberellin detected in the highest concentration was GA6, the predominant intermediates were GA15 and GA53 and the main biosynthetic end products were GA13 and GA51. Gibberellin profiles were similar in all strains except for the presence/absence of GA12 and GA12ald. To date this is the second report of endogenous brassinosteroids in microalgae. Brassinosteroids were detected in all 24 strains with concentrations ranging from 117.3 pg mg(-)(1) DW in R. subcapitata MACC 317-977.8 pg mg(-)(1) DW in Klebsormidium flaccidum MACC 692. Two brassinosteroids, brassinolide and castasterone were determined in all the strains. Generally, brassinolide occurred in higher concentrations than castasterone.
Plant Physiology and Biochemistry 06/2013; 70C:348-353. · 2.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Verticillium longisporum is a vascular pathogen that infects the Brassicaceae host plants Arabidopsis thaliana and Brassica napus. The soil-borne fungus enters the plant via the roots and colonizes the xylem of roots, stems and leaves. During late stages of infections, Verticillium spreads into senescing tissue and switches from biotrophic to a necrotrophic life style. Typical symptoms of Verticillium longisporum-induced disease are stunted growth and leaf chloroses. Expression analyses of the senescence marker genes SENESCENCE ASSOCIATED GENE12, SENESCENCE ASSOCIATED GENE13 and WRKY53 revealed that the observed chloroses are a consequence of premature senescence triggered by Verticillium infection. Our analyses show that concomitant with the development of chloroses levels of trans-zeatin decrease in infected plants. Potentially, induction of cytokinin oxidase expression by Verticillium infection contributes to the observed decreases in cytokinin levels. Stabilisation of Arabidopsis cytokinin levels by both pharmacological and genetic approaches inhibits Verticillium proliferation and coincides with reduced disease symptom development. In summary, our results indicate that Verticillium triggers premature plant senescence for efficient host plant colonization.
[Show abstract][Hide abstract] ABSTRACT: Metastases destroy the function of infested organs and are the main reason of cancer-related mortality. Heteronemin, a natural product derived from a marine sponge, was tested in vitro regarding its properties to prevent tumour cell intravasation through the lymph-endothelial barrier. In three-dimensional (3D) cell cultures consisting of MCF-7 breast cancer cell spheroids that were placed on lymph-endothelial cell (LEC) monolayers, tumour cell spheroids induce "circular chemorepellent-induced defects" (CCIDs) in the LEC monolayer; 12(S)-Hydroxyeicosatetraenoic acid (12(S)-HETE) and NF-κB activity are major factors inducing CCIDs, which are entry gates for tumour emboli intravasating the vasculature. This 3D co-culture is a validated model for the investigation of intravasation mechanisms and of drugs preventing CCID formation and hence lymph node metastasis. Furthermore, Western blot analyses, NF-κB reporter, EROD, SELE, 12(S)-HETE, and adhesion assays were performed to investigate the properties of heteronemin. Five micromolar heteronemin inhibited the directional movement of LECs and, therefore, the formation of CCIDs, which were induced by MCF-7 spheroids. Furthermore, heteronemin reduced the adhesion of MCF-7 cells to LECs and suppressed 12(S)-HETE-induced expression of the EMT marker paxillin, which is a regulator of directional cell migration. The activity of CYP1A1, which contributed to CCID formation, was also inhibited by heteronemin. Hence, heteronemin inhibits important mechanisms contributing to tumour intravasation in vitro and should be tested in vivo.
Archives of Toxicology 03/2013; · 5.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Health beneficial effects of xanthohumol have been reported, and basic research provided evidence for anti-cancer effects. Furthermore, xanthohumol was shown to inhibit the migration of endothelial cells. Therefore, this study investigated the anti-metastatic potential of xanthohumol. MCF-7 breast cancer spheroids which are placed on lymphendothelial cells (LECs) induce "circular chemorepellent-induced defects" (CCIDs) in the LEC monolayer resembling gates for intravasating tumour bulks at an early step of lymph node colonisation. NF-κB reporter-, EROD-, SELE-, 12(S)-HETE- and adhesion assays were performed to investigate the anti-metastatic properties of xanthohumol. Western blot analyses were used to elucidate the mechanisms inhibiting CCID formation. Xanthohumol inhibited the activity of CYP, SELE and NF-kB and consequently, the formation of CCIDs at low micromolar concentrations. More specifically, xanthohumol affected ICAM-1 expression and adherence of MCF-7 cells to LECs, which is a prerequisite for CCID formation. Furthermore, markers of epithelial-to-mesenchymal transition (EMT) and of cell mobility such as paxillin, MCL2 and S100A4 were suppressed by xanthohumol. Xanthohumol attenuated tumour cell-mediated defects at the lymphendothelial barrier and inhibited EMT-like effects thereby providing a mechanistic explanation for the anti-intravasative/anti-metastatic properties of xanthohumol.
Archives of Toxicology 03/2013; · 5.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Upward leaf movement, called hyponastic growth, is employed by plants to cope with adverse environmental conditions. Ethylene is a key regulator of this process and, in Arabidopsis thaliana, hyponasty is induced by this phytohormone via promotion of epidermal cell expansion in a proximal zone of the abaxial side of the petiole. ROTUNDIFOLIA3/CYP90C1 encodes an enzyme which was shown to catalyse C-23 hydroxylation of several brassinosteroids (BRs) - phytohormones involved in, for example, organ growth, cell expansion, cell division, and responses to abiotic and biotic stresses. This study tested the interaction between ethylene and BRs in regulating hyponastic growth. A mutant isolated in a forward genetic screen, with reduced hyponastic response to ethylene treatment, was allelic to rot3. The cause of the reduced hyponastic growth in this mutant was examined by studying ethylene-BR interaction during local cell expansion, pharmacological inhibition of BR synthesis and ethylene effects on transcription of BR-related genes. This work demonstrates that rot3 mutants are impaired in local cell expansion driving hyponasty. Moreover, the inhibition of BR biosynthesis reduces ethylene-induced hyponastic growth and ethylene increases sensitivity to BR in promoting cell elongation in Arabidopsis hypocotyls. Together, the results show that ROT3 modulates ethylene-induced petiole movement and that this function is likely BR related.
Journal of Experimental Botany 12/2012; · 5.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In Pinus radiata D. Don, the transition from the juvenile to the mature phase is characterized by a reduction in the tree's organogenic potential, which is usually reverted in breeding programs by reinvigoration procedures to enable vegetative propagation. In this work we have determined the best culture conditions for in vitro reinvigoration of radiata pine buds, tested different cytokinin types (N(6) -benzyladenine, meta-topolin and trans-zeatin) and concentrations (25 and 50 μM), and studied the effect of culture conditions on endogenous cytokinin and indole-3-acetic acid levels at different stages of the organogenic process. To this end, the levels of 43 cytokinins and indole-3-acetic acid were determined in Pinus radiata buds before and during the reinvigoration process. When N(6) -benzyladenine or meta-topolin was applied to the induction medium, we did not observe any significant increase or decrease in endogenous isoprenoid cytokinin content. We also report for the first time the presence of O-glucosides in non-treated Pinus radiata explants from the field and remark the importance of O-glucosides as storage forms.
Physiologia Plantarum 10/2012; · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Jasmonates (JAs) are ubiquitously occurring signaling compounds in plants formed in response to biotic and abiotic stress as well as in development. (+)-7-iso-jasmonoyl isoleucine, the bioactive JA, is involved in most JA-dependent processes mediated by the F-box protein COI1 in a proteasome-dependent manner. However, there is an increasing number of examples, where the precursor of JA biosynthesis, cis-(+)-12-oxophytodienoic acid (OPDA) is active in a JA/COI1-independent manner. Here, we discuss those OPDA-dependent processes, thereby giving emphasis on tomato embryo development. Recent data on seed coat-generated OPDA and its role in embryo development is discussed based on biochemical and genetic evidences.
[Show abstract][Hide abstract] ABSTRACT: Caulogenesis in mature stone pine (Pinus pinea L.) cotyledons is promoted, to varying degrees depending on genotype, by exogenous application of the cytokinin (CK) benzyladenine (BA). In the present study, endogenous CK profiles of cotyledons from open-pollinated plants and two families of stone pine with widely differing organogenic capacities were monitored during caulogenesis and linked to previously characterized BA uptake and induction phases. Changes in levels of free bases, ribosides, ribotides and glucosides of both isoprenoid and aromatic CKs were followed. Before BA application, the pool of endogenous CKs in all sets of cotyledons was dominated by isoprenoid ribotides, but 1h after BA exposure, aromatic CKs (mainly active free bases and ribosides of topolins) accounted for more than 90% of the pool. BA N-glucosides were also observed, levels of which (and topolins) rose from 2d until the end of the (six-day) culture period. The CK profiles of the two selected pine families also differed, although the general trends were similar. During the first 6h, levels of BA and meta-topolin were highest in cotyledons from the family with the strongest caulogenic responses, while levels of ribotides and aromatic glucosides were highest in cotyledons from the other family.
Journal of Plant Physiology 09/2012; · 2.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Polyphenol oxidases (PPOs) are copper-binding enzymes of the plant secondary metabolism that oxidize polyphenols to quinones. Although PPOs are nearly ubiquitous in seed plants, knowledge on their evolution and function in other plant groups is missing. This study reports on the PPO gene family in the moss Physcomitrella patens (Hedw.) B.S.G. asan example for an early divergent plant. The P. patens PPO multigene family comprises 13 paralogues. Phylogenetic analyses suggest that plant PPOs evolved with the colonization of land and that PPO duplications within the monophyletic P. patens paralogue clade occurred after the separation of the moss and seed plant lineages. PPO functionality was demonstrated for recombinant PPO6. P. patens was analysed for phenolic compounds and six substances were detected intracellularly by LC-MS analysis: 4-hydroxybenzoic acid, p-cumaric acid, protocatechuic acid, salicylic acid, caffeic acid, and an ester of caffeic acid. Targeted PPO1 knockout (d|ppo1) plants were generated and plants lacking PPO1 exhibited only ~30% of the wild-type PPO activity in the culture medium, thus suggesting extracellular localization of PPO1, which is in contrast to the mostly plastidic PPO localization in seed plants. Further, d|ppo1 lines formed significantly more gametophores with a reduced areal plant size, which could be related to an increase of endogenously produced cytokinins and indicates an impact of PPO1 on plant development. d|ppo1 plants were less tolerant towards applied 4-methylcatechol compared to the wild type, which suggests a role of extracellular PPO1 in establishing appropriate conditions by the removal of inhibitory extracellular phenolic compounds.
Journal of Experimental Botany 08/2012; 63(14):5121-35. · 5.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Jasmonates are ubiquitously occurring lipid-derived signaling compounds active in plant development and plant responses to biotic and abiotic stresses. Upon environmental stimuli jasmonates are formed and accumulate transiently. During flower and seed development, jasmonic acid (JA) and a remarkable number of different metabolites accumulate organ- and tissue specifically. The accumulation is accompanied with expression of jasmonate-inducible genes. Among these genes there are defense genes and developmentally regulated genes. The profile of jasmonate compounds in flowers and seeds covers active signaling molecules such as JA, its precursor 12-oxophytodienoic acid (OPDA) and amino acid conjugates such as JA-Ile, but also inactive signaling molecules occur such as 12-hydroxy-JA and its sulfated derivative. These latter compounds can occur at several orders of magnitude higher level than JA. Metabolic conversion of JA and JA-Ile to hydroxylated compounds seems to inactivate JA signaling, but also specific functions of jasmonates in flower and seed development were detected. In tomato OPDA is involved in embryo development. Occurrence of jasmonates, expression of JA-inducible genes and JA-dependent processes in flower and seed development will be discussed.
[Show abstract][Hide abstract] ABSTRACT: Receptor-mediated endocytosis is an integral part of signal transduction as it mediates signal attenuation and provides spatial and temporal dimensions to signaling events. One of the best-studied leucine-rich repeat receptor-like kinases in plants, BRASSINOSTEROID INSENSITIVE 1 (BRI1), perceives its ligand, the brassinosteroid (BR) hormone, at the cell surface and is constitutively endocytosed. However, the importance of endocytosis for BR signaling remains unclear. Here we developed a bioactive, fluorescent BR analog, Alexa Fluor 647-castasterone (AFCS), and visualized the endocytosis of BRI1-AFCS complexes in living Arabidopsis thaliana cells. Impairment of endocytosis dependent on clathrin and the guanine nucleotide exchange factor for ARF GTPases (ARF-GEF) GNOM enhanced BR signaling by retaining active BRI1-ligand complexes at the plasma membrane. Increasing the trans-Golgi network/early endosome pool of BRI1-BR complexes did not affect BR signaling. Our findings provide what is to our knowledge the first visualization of receptor-ligand complexes in plants and reveal clathrin- and ARF-GEF-dependent endocytic regulation of BR signaling from the plasma membrane.
Nature Chemical Biology 05/2012; 8(6):583-9. · 12.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: UPLC–ESI–MS/MS method was developed for the quantitative and qualitative analysis of 26 phenolic compounds in plant material, including 15 isoflavonoids (daidzein, genistein, isoformononetin, formononetin, prunetin, biochanin A, daidzin, genistin, ononin, sissotrin, osajin, pomiferin), 5 flavones (apigenin, kaempferol, quercetin, quercetin-3-glucoside, rutin), 4 flavanones (hesperetin, naringenin, naringenin-7-glucoside, naringin), a coumestan (coumestrol) and a coumarine (scopoletin). The analytes were detected in a multiple reaction monitoring mode and quantified using internal standards [13C3]daidzin, [2H6]naringenin and [2H4]genistein. Limits of detection were in the range 0.001–10 pmol injection−1. The precision of the method (relative standard deviation) ranged from 2.6 to 15.1% and the accuracy was –22.1 to 16.6%. Application of the method was demonstrated by the analysis of mung bean (Vigna radiata) sprout extract, in which 14 of the 26 analytes were detected and quantified.
Journal of Food Composition and Analysis 05/2012; 26(1–2):36-42. · 2.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A series of reticulated Arabidopsis thaliana mutants were previously described. All mutants show a reticulate leaf pattern, namely green veins on a pale leaf lamina. They have an aberrant mesophyll structure but an intact layer of bundle sheath cells around the veins. Here, we unravel the function of the previously described reticulated EMS-mutant dov1 (differential development of vascular associated cells 1). By positional cloning, we identified the mutated gene, which encodes glutamine phosphoribosyl pyrophosphate aminotransferase 2 (ATase2), an enzyme catalyzing the first step of purine nucleotide biosynthesis. dov1 is allelic to the previously characterized cia1-2 mutant that was isolated in a screen for mutants with impaired chloroplast protein import. We show that purine-derived total cytokinins are lowered in dov1 and crosses with phytohormone reporter lines revealed differential reporter activity patterns in dov1. Metabolite profiling unraveled that amino acids that are involved in purine biosynthesis are increased in dov1. This study identified the molecular basis of an established mutant line, which has the potential for further investigation of the interaction between metabolism and leaf development.
[Show abstract][Hide abstract] ABSTRACT: Investigating the bioactivity of traditional medical remedies under the controlled conditions of a laboratory is an option to find additional applications, novel formulations or lead structures for the development of new drugs. The present work analysed the anti‑neoplastic activity of increasing polar extracts of the rainforest plant Critonia morifolia (Asteraceae) that has been successfully used as traditional remedy to treat various inflammatory conditions in the long-lasting medical tradition of the Central American Maya, which was here also confirmed in vitro. The apolar petroleum ether extract exhibited the most potent anti‑proliferative and pro‑apoptotic effects in HL‑60 cells and triggered down-regulation of Cdc25C and cyclin D1 within 30 min followed by the inhibition of c-Myc expression and the onset of caspase-3 activation within 2 h. Subsequent to these very rapid molecular responses Chk2 and H2AX became phosphorylated (γ‑H2AX) after 4 h. Analysis of the cell cycle distribution showed an accumulation of cells in the G2-M phase within 8 h and after 24 h in S-phase. This was temporally paralleled by the down-regulation of Cdc25A, Cdc25B, Wee1 and Akt. Therefore, the attenuation of cell cycle progression in the G2-M phase was consistent with the known role of Chk2 for G2-M arrest and with the role of Cdc25B in S-phase progression. These findings suggest the presence of two distinct active principles in the petroleum ether extract of C. moriflia. These facilitated the strong apoptotic response evidenced by the rapid activation of caspase-3 that was later enforced by the inhibition of the survival kinase Akt. Importantly, the efficient down-regulation of Akt, which is successfully tested in current clinical trials, is a unique property of C. morifolia.
International Journal of Oncology 03/2012; 40(6):2131-9. · 2.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the distribution of IAA (indole-3-acetic acid) and the IAA synthetic cells in maize coleoptiles, we established immunohistochemistry of IAA using an anti-IAA-C-monoclonal antibody. We first confirmed the specificity of the antibody by comparing the amounts of endogenous free and conjugated IAA to the IAA signal obtained from the IAA antibody. Depletion of endogenous IAA showed a corresponding decrease in immuno-signal intensity and negligible cross-reactivity against IAA-related compounds, including tryptophan, indole-3-acetamide, and conjugated-IAA was observed. Immunolocalization showed that the IAA signal was intense in the approximately 1 mm region and the outer epidermis at the approximately 0.5 mm region from the top of coleoptiles treated with 1-N-naphthylphthalamic acid. By contrast, the IAA immuno-signal in the outer epidermis almost disappeared after 5-methyl-tryptophan treatment. Immunogold labeling of IAA with an anti-IAA-N-polyclonal antibody in the outer-epidermal cells showed cytoplasmic localization of free-IAA, but none in cell walls or vacuoles. These findings indicated that IAA is synthesized in the 0–2.0 mm region of maize coleoptile tips from Trp, in which the outer-epidermal cells of the 0.5 mm tip are the most active IAA synthetic cells.
[Show abstract][Hide abstract] ABSTRACT: Cytokinins are phytohormones that are involved in various regulatory processes throughout plant development, but they are also produced by pathogens and known to modulate plant immunity. A novel transgenic approach enabling autoregulated cytokinin synthesis in response to pathogen infection showed that cytokinins mediate enhanced resistance against the virulent hemibiotrophic pathogen Pseudomonas syringae pv tabaci. This was confirmed by two additional independent transgenic approaches to increase endogenous cytokinin production and by exogenous supply of adenine- and phenylurea-derived cytokinins. The cytokinin-mediated resistance strongly correlated with an increased level of bactericidal activities and up-regulated synthesis of the two major antimicrobial phytoalexins in tobacco (Nicotiana tabacum), scopoletin and capsidiol. The key role of these phytoalexins in the underlying mechanism was functionally proven by the finding that scopoletin and capsidiol substitute in planta for the cytokinin signal: phytoalexin pretreatment increased resistance against P. syringae. In contrast to a cytokinin defense mechanism in Arabidopsis (Arabidopsis thaliana) based on salicylic acid-dependent transcriptional control, the cytokinin-mediated resistance in tobacco is essentially independent from salicylic acid and differs in pathogen specificity. It is also independent of jasmonate levels, reactive oxygen species, and high sugar resistance. The novel function of cytokinins in the primary defense response of solanaceous plant species is rather mediated through a high phytoalexin-pathogen ratio in the early phase of infection, which efficiently restricts pathogen growth. The implications of this mechanism for the coevolution of host plants and cytokinin-producing pathogens and the practical application in agriculture are discussed.
[Show abstract][Hide abstract] ABSTRACT: Hormone pathway interactions are crucial in shaping plant development, such as synergism between the auxin and brassinosteroid pathways in cell elongation. Both hormone pathways have been characterized in detail, revealing several feedback loops. The complexity of this network, combined with a shortage of kinetic data, renders its quantitative analysis virtually impossible at present.
As a first step towards overcoming these obstacles, we analyzed the network using a Boolean logic approach to build models of auxin and brassinosteroid signaling, and their interaction. To compare these discrete dynamic models across conditions, we transformed them into qualitative continuous systems, which predict network component states more accurately and can accommodate kinetic data as they become available. To this end, we developed an extension for the SQUAD software, allowing semi-quantitative analysis of network states. Contrasting the developmental output depending on cell type-specific modulators enabled us to identify a most parsimonious model, which explains initially paradoxical mutant phenotypes and revealed a novel physiological feature.
The package SQUADD is freely available via the Bioconductor repository at http://www.bioconductor.org/help/bioc-views/release/bioc/html/SQUADD.html.
[Show abstract][Hide abstract] ABSTRACT: The medlar fruit (Mespilus germanica L) has been gaining commercial importance in recent years, attracting research on its chemical composition. In this study, we have investigated how the degree of ripeness affects the concentrations and proportions of phenolic acids. The DPPH scavenging activity and the total phenolic content (TPC) were determined. Five stages of fruit maturity were studied and eight phenolic acids (protocatechuic, 4-hydroxybenzoic, syringic, 3-hydroxybenzoic, caffeic, salicylic, 4-coumaric and sinapic) were determined by HPLC-MS. The concentrations of phenolic acids mostly decreased as the fruit ripening progressed, except for insoluble ester-bound phenolics, which increased at the early stages of maturity and decreased only during the ripe to over-ripe stage of maturity. The DPPH scavenging activity also decreased during fruit maturation, suggesting a decrease of natural antioxidants in fruit. A strong correlation between TPC and antioxidant capacity measured by the DPPH method was found. The data presented demonstrates the influence of ripening on the phenolic acid content and antioxidant properties of medlar fruit. (C) 2010 Elsevier Ltd. All rights reserved.