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Biomedical subjects

Jorge M Vivanco

Publications and source records attributed to Jorge M Vivanco.

At least 37 records · Page 2Linked to original sources
PubMed2004

Proton-transfer-reaction mass spectrometry as a new tool for real time analysis of root-secreted volatile organic compounds in Arabidopsis.

Plant roots release about 5% to 20% of all photosynthetically-fixed carbon, and as a result create a carbon-rich environment for numerous rhizosphere organisms, including plant pathogens and symbiotic microbes. Although some characterization of root exudates has been achieved, especially of secondary metabolites and proteins, much less is known about volatile organic compounds (VOCs) released by roots. In this communication, we describe a novel approach to exploring these rhizosphere VOCs and their induction by biotic stresses. The VOC formation of Arabidopsis roots was analyzed using proton-transfer-reaction mass spectrometry (PTR-MS), a new technology that allows rapid and real time analysis of most biogenic VOCs without preconcentration or chromatography. Our studies revealed that the major VOCs released and identified by both PTR-MS and gas chromatography-mass spectrometry were either simple metabolites, ethanol, acetaldehyde, acetic acid, ethyl acetate, 2-butanone, 2,3,-butanedione, and acetone, or the monoterpene, 1,8-cineole. Some VOCs were found to be produced constitutively regardless of the treatment; other VOCs were induced specifically as a result of different compatible and noncompatible interactions between microbes and insects and Arabidopsis roots. Compatible interactions of Pseudomonas syringae DC3000 and Diuraphis noxia with Arabidopsis roots resulted in the rapid release of 1,8-cineole, a monoterpene that has not been previously reported in Arabidopsis. Mechanical injuries to Arabidopsis roots did not produce 1,8-cineole nor any C6 wound-VOCs; compatible interactions between Arabidopsis roots and Diuraphis noxia did not produce any wound compounds. This suggests that Arabidopsis roots respond to wounding differently from above-ground plant organs. Trials with incompatible interactions did not reveal a set of compounds that was significantly different compared to the noninfected roots. The PTR-MS method may open the way for functional root VOC analysis that will complement genomic investigations in Arabidopsis.

Animals↗
PubMed2003

Pseudomonas aeruginosa-plant root interactions. Pathogenicity, biofilm formation, and root exudation.

Pseudomonas aeruginosa is an opportunistic human pathogen capable of forming a biofilm under physiological conditions that contributes to its persistence despite long-term treatment with antibiotics. Here, we report that pathogenic P. aeruginosa strains PAO1 and PA14 are capable of infecting the roots of Arabidopsis and sweet basil (Ocimum basilicum), in vitro and in the soil, and are capable of causing plant mortality 7 d postinoculation. Before plant mortality, PAO1 and PA14 colonize the roots of Arabidopsis and sweet basil and form a biofilm as observed by scanning electron microscopy, phase contrast microscopy, and confocal scanning laser microscopy. Upon P. aeruginosa infection, sweet basil roots secrete rosmarinic acid (RA), a multifunctional caffeic acid ester that exhibits in vitro antibacterial activity against planktonic cells of both P. aeruginosa strains with a minimum inhibitory concentration of 3 microg mL(-1). However, in our studies RA did not attain minimum inhibitory concentration levels in sweet basil's root exudates before P. aeruginosa formed a biofilm that resisted the microbicidal effects of RA and ultimately caused plant mortality. We further demonstrated that P. aeruginosa biofilms were resistant to RA treatment under in vivo and in vitro conditions. In contrast, induction of RA secretion by sweet basil roots and exogenous supplementation of Arabidopsis root exudates with RA before infection conferred resistance to P. aeruginosa. Under the latter conditions, confocal scanning laser microscopy revealed large clusters of dead P. aeruginosa on the root surface of Arabidopsis and sweet basil, and biofilm formation was not observed. Studies with quorum-sensing mutants PAO210 (DeltarhlI), PAO214 (DeltalasI), and PAO216 (DeltalasI DeltarhlI) demonstrated that all of the strains were pathogenic to Arabidopsis, which does not naturally secrete RA as a root exudate. However, PAO214 was the only pathogenic strain toward sweet basil, and PAO214 biofilm appeared comparable with biofilms formed by wild-type strains of P. aeruginosa. Our results collectively suggest that upon root colonization, P. aeruginosa forms a biofilm that confers resistance against root-secreted antibiotics.

Anti-Bacterial Agents↗
PubMed2003

Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production.

Relatively little is known about the exact mechanisms used by Bacillus subtilis in its behavior as a biocontrol agent on plants. Here, we report the development of a sensitive plant infection model demonstrating that the bacterial pathogen Pseudomonas syringae pv tomato DC3000 is capable of infecting Arabidopsis roots both in vitro and in soil. Using this infection model, we demonstrated the biocontrol ability of a wild-type B. subtilis strain 6051 against P. syringae. Arabidopsis root surfaces treated with B. subtilis were analyzed with confocal scanning laser microscopy to reveal a three-dimensional B. subtilis biofilm. It is known that formation of biofilms by B. subtilis is a complex process that includes secretion of surfactin, a lipopeptide antimicrobial agent. To determine the role of surfactin in biocontrol by B. subtilis, we tested a mutant strain, M1, with a deletion in a surfactin synthase gene and, thus, deficient in surfactin production. B. subtilis M1 was ineffective as a biocontrol agent against P. syringae infectivity in Arabidopsis and also failed to form robust biofilms on either roots or inert surfaces. The antibacterial activity of surfactin against P. syringae was determined in both broth and agar cultures and also by live-dead staining methods. Although the minimum inhibitory concentrations determined were relatively high (25 microg mL(-1)), the levels of the lipopeptide in roots colonized by B. subtilis are likely to be sufficient to kill P. syringae. Our results collectively indicate that upon root colonization, B. subtilis 6051 forms a stable, extensive biofilm and secretes surfactin, which act together to protect plants against attack by pathogenic bacteria.

Agar↗
PubMed2003

Isolation and characterization of an RIP (ribosome-inactivating protein)-like protein from tobacco with dual enzymatic activity.

Ribosome-inactivating proteins (RIPs) are N-glycosidases that remove a specific adenine from the sarcin/ricin loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, a protein termed tobacco RIP (TRIP) was isolated from tobacco (Nicotiana tabacum) leaves and purified using ion exchange and gel filtration chromatography in combination with yeast ribosome depurination assays. TRIP has a molecular mass of 26 kD as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed strong N-glycosidase activity as manifested by the depurination of yeast rRNA. Purified TRIP showed immunoreactivity with antibodies of RIPs from Mirabilis expansa. TRIP released fewer amounts of adenine residues from ribosomal (Artemia sp. and rat ribosomes) and non-ribosomal substrates (herring sperm DNA, rRNA, and tRNA) compared with other RIPs. TRIP inhibited translation in wheat (Triticum aestivum) germ more efficiently than in rabbit reticulocytes, showing an IC50 at 30 ng in the former system. Antimicrobial assays using highly purified TRIP (50 microg mL(-1)) conducted against various fungi and bacterial pathogens showed the strongest inhibitory activity against Trichoderma reesei and Pseudomonas solancearum. A 15-amino acid internal polypeptide sequence of TRIP was identical with the internal sequences of the iron-superoxide dismutase (Fe-SOD) from wild tobacco (Nicotiana plumbaginifolia), Arabidopsis, and potato (Solanum tuberosum). Purified TRIP showed SOD activity, and Escherichia coli Fe-SOD was observed to have RIP activity too. Thus, TRIP may be considered a dual activity enzyme showing RIP-like activity and Fe-SOD characteristics.

Adenine↗
PubMed2003

Allelopathy and exotic plant invasion: from molecules and genes to species interactions.

Here we present evidence that Centaurea maculosa (spotted knapweed), an invasive species in the western United States, displaces native plant species by exuding the phytotoxin (-)-catechin from its roots. Our results show inhibition of native species' growth and germination in field soils at natural concentrations of (-)-catechin. In susceptible species such as Arabidopsis thaliana, the allelochemical triggers a wave of reactive oxygen species (ROS) initiated at the root meristem, which leads to a Ca2+ signaling cascade triggering genome-wide changes in gene expression and, ultimately, death of the root system. Our results support a "novel weapons hypothesis" for invasive success.

Arabidopsis↗
PubMed2003

Molecular and biochemical characterization of an enzyme responsible for the formation of hypericin in St. John's wort (Hypericum perforatum L.).

A major gene termed Hyp-1 encoding for hypericin (HyH) biosynthesis was cloned and characterized from Hypericum perforatum (St. John's wort) cell cultures. H. perforatum leaves are widely used as an herbal remedy in the treatment of mild to moderate depression. Hypericin, a photosensitive and red-colored naphthodianthrone, has been reported as the bioactive compound responsible for reversing the depression symptoms. In this study a novel red-color-based colony screening method for examining a cDNA library (lambda-TriplEX2) derived from H. perforatum cell cultures revealed the gene responsible for hypericin biosynthesis after the administration of emodin, a precursor of hypericin. The selected clones were expressed in Escherichia coli (BM 25.8 line) and were further screened for biosynthesis of emodin to hypericin, which resulted in an 84.6% conversion. The full-length cDNA sequence of Hyp-1 is 782 nucleotides in length with an open reading frame of 477 nucleotides coding for a protein of 159 amino acids, with a 45.1% homology to Bet.v.1 class allergens. Reverse transcriptase-PCR analysis showed high levels of Hyp-1 transcripts in dark-grown cell cultures compared with the levels in light-grown cell cultures and leaves. Southern blot analysis showed the presence of a single Hyp-1 gene in H. perforatum. Furthermore, Hyp-1 was expressed with a His6 affinity tag linked to its N terminal region using the expression vector pET-28a, and the recombinant Hyp-1 protein was able to convert HyH from emodin under in vitro conditions. HyH product inhibition was observed with emodin analogues, rhein, rhein methyl ester, and DNA3-55-1. Our results demonstrate a direct and complex conversion of emodin to HyH that is solely catalyzed by Hyp-1, a Bet.v.1 class allergen from H. perforatum.

Amino Acid Sequence↗
PubMed2003

Molecular characterization and post-transcriptional regulation of ME1, a type-I ribosome-inactivating protein from Mirabilis expansa.

Ribosome-inactivating proteins (RIPs) are N-glycosidases that remove a specific adenine from the sarcin/ricin (S/R) loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, ME1, a type-1 RIP, was cloned and sequenced from storage roots of Mirabilis expansa (Ruiz & Pavon). The full-length cDNA sequence of ME1 has 1,129 nucleotides with an open reading frame of 951 nucleotides representing 317 amino acids. Nucleotide analysis revealed that the N-terminal region of ME1 was cleaved, and the mature protein started at amino acid 34. ME1 showed very close similarities to MAP and MAP-4 from Mirabilis jalapa. Southern blot analysis revealed the presence of two homologous genes for ME1 cDNA in M. expansa. Northern blot analysis showed high levels of ME1 transcripts in primary and storage roots. Interestingly, jasmonic acid induced ME1 transcript expression in cell suspension cultures of M. expansa; however, the production of ME1 protein was not enhanced as observed by Western blot analysis. Our data suggest that ME1 has the ability to depurinate its own mRNA, thus inhibiting its translation. These observations suggest a possible mechanism by which ME1 protein levels are post-transcriptionally regulated.

Amino Acid Sequence↗
PubMed2003

Metabolic profiling of root exudates of Arabidopsis thaliana.

In addition to accumulating biologically active chemicals, plant roots continuously produce and secrete compounds into their immediate rhizosphere. However, the mechanisms that drive and regulate root secretion of secondary metabolites are not fully understood. To enlighten two neglected areas of root biology, root secretion and secondary metabolism, an in vitro system implementing root-specific elicitation over a 48-day time course was developed. After roots of Arabidopsis thaliana had been elicited with salicylic acid, jasmonic acid, chitosan, and two fungal cell wall elicitors, the secondary metabolites subsequently secreted were profiled. High-performance liquid chromatography was used to metabolically profile compounds in the root exudates, and 289 possible secondary metabolites were quantified. The chemical structures of 10 compounds were further characterized by (1)H and (13)C NMR: butanoic acid, trans-cinnamic acid, o-coumaric acid, p-coumaric acid, ferulic acid, p-hydroxybenzamide, methyl p-hydroxybenzoate, 3-indolepropanoic acid, syringic acid, and vanillic acid. Several of these compounds exhibited a wide range of antimicrobial activity against both soil-borne bacteria and fungi at the concentration detected in the root exudates.

Anti-Bacterial Agents↗
PubMed2003

Structure-dependent phytotoxicity of catechins and other flavonoids: flavonoid conversions by cell-free protein extracts of Centaurea maculosa (spotted knapweed) roots.

Invasive plants are believed to succeed in part by secretion of allelochemicals, thus displacing competing plant species. Centaurea maculosa (spotted knapweed) provides a classic example of this process. We have previously reported that spotted knapweed roots secrete (+/-)-catechin and that (-)-catechin, but not (+)-catechin, is phytotoxic and hence may be a major contributor to C. maculosa's invasive behavior in the rhizosphere. In this communication, we explore both structure/activity relationships for flavonoid phytotoxicity and possible biosynthetic pathways for root production of (+/-)-catechin. Kaempferol and dihydroquercetin were shown to be phytotoxic, while quercetin was not. Kaempferol was converted to dihydroquercetin and (+/-)-catechin when treated with total root protein extracts from C. maculosa, but quercetin was not. This finding suggests an alteration in the standard flavonoid biosynthetic pathway in C. maculosa roots, whereby kaempferol is not a dead-end product but serves as a precursor to dihydroquercetin, which in turn leads to (+/-)-catechin production.

Catechin↗
PubMed2003

7,8-Benzoflavone: a phytotoxin from root exudates of invasive Russian knapweed.

Root exudates from Acroptilon repens (Russian knapweed) were found to be phytotoxic and the phytotoxin in the exudate was identified as 7,8-benzoflavone (alpha-naphthoflavone), (1), not previously known as a natural product. In tests on growing seedlings both 1 and its isomer 5,6-benzoflavone (2) were phytotoxic. Flavone, a structural analog of 1 and a known granular leaf and stem exudate of other plant species, was also phytotoxic and more potent than 1 or 2.

Arabidopsis↗
PubMed2003

In vitro activities of Maesa lanceolata extracts against fungal plant pathogens.

In vitro tests were carried out using extracts of Maesa lanceolata var. goulungensis weir against a broad range of fungal plant pathogens such as Phytophthora cryptogea, Trichoderma virens, Aspergillus niger, Phoma sp., Fusarium oxysporium, Pythium ultimum, Cochliobolus heterostrophus, Rhizoctonia solani, Sclerotium rolfsii and Pyrenophora teres. M. lanceolata extracts were very active against all the pathogens tested except P. ultimum and R. solani.

Antifungal Agents↗
PubMed2003

Intraspecific and interspecific interactions mediated by a phytotoxin, (-)-catechin, secreted by the roots of Centaurea maculosa (spotted knapweed).

Centarea maculosa Lam. (spotted knapweed) is one of the most destructive invasive weeds in the western United States, particularly in pastures and rangelands. One of the components that may contribute to its invasiveness is the naturally produced, root-secreted allelochemical (-)-catechin. This compound has been shown to have broad-spectrum phytotoxic activity, possibly assisting C. maculosa in displacing native plant communities. As a recently characterized phytochemical, little is known about the specific effect of (-)-catechin on either C. maculosa or other plant species. We have found that, in vitro, C. maculosa begins to secrete phytotoxic levels of (-)-catechin within 2-3 weeks of seedling emergence. Furthermore, (-)-catechin concentrations consistent with those naturally secreted by C. maculosa were sufficient to inhibit germination in all species tested, including C. maculosa. These concentrations were also often either phytotoxic or growth inhibitory to seedlings in a range of plant species, while having no negative effects on the growth of C. maculosa seedlings. However, our results also indicate that different levels of resistance and susceptibility to (-)-catechin exist in plant populations, suggesting that the capability of C. maculosa to invade an area through allelochemistry may be dependent on the age and species composition of plants in that area.

Catechin↗
PubMed2002

Enzymatic specificity of three ribosome-inactivating proteins against fungal ribosomes, and correlation with antifungal activity.

Ribosome-inactivating proteins (RIPs) are enzymes that cleave a specific adenine base from the highly conserved sarcin/ricin (S/R) loop of the large ribosomal RNA, thus arresting protein synthesis at the translocation step. In the present study, we employed three RIPs to dissect the antifungal activity of RIPs as plant defense proteins. We measured the catalytic activity of RAT (the catalytic A-chain of ricin from Ricinus communis L.), saporin-S6 (from Saponaria officinalis L.), and ME (RIP from Mirabilis expansa R&P) against intact ribosomal substrates isolated from various pathogenic fungi. We further determined the enzymatic specificity of these three RIPs against fungal ribosomes, from Rhizoctonia solani Kuhn, Alternaria solani Sorauer, Trichoderma reesei Simmons and Candida albicans Berkhout, and correlated the data with antifungal activity. RAT showed the strongest toxicity against all tested fungal ribosomes, except for the ribosomes isolated from C. albicans, which were most susceptible to saporin. RAT and saporin showed higher enzymatic activity than ME against ribosomes from all of the fungal species assayed, but did not show detectable antifungal activity. In contrast, ME showed substantial inhibitory activity against fungal growth. Using N-hydroxysuccinimide-fluorescein labeling of RIPs and fluorescence microscopy, we determined that ME was targeted to the surface of fungal cells and transferred into the cells. Thus, ME caused ribosome depurination and subsequent fungal mortality. In contrast, saporin did not interact with fungal cells, correlating with its lack of antifungal activity.

Catalysis↗
PubMed2002

Jasmonic acid-induced hypericin production in cell suspension cultures of Hypericum perforatum L. (St. John's wort).

Hypericum perforatum L. (St. John's wort) is an herbal remedy widely used in the treatment of mild to moderate depression. Hypericin, a photosensitive napthodianthrone, is believed to be the compound responsible for reversing the depression symptoms. In this study, novel in vitro cell culture systems of H. perforatum were used to monitor the effect of elicitation on cell growth and production of hypericin. A dramatic increase in cell growth and hypericin production was observed after exposure to jasmonic acid (JA). However, other elicitors such as salicylic acid (SA) and fungal cell wall elicitors failed to show any stimulatory effect on either cell growth or hypericin production. Cell cultures treated with JA and incubated in the dark showed increased growth and hypericin production as compared to the cultures grown under light conditions. Jasmonate induction in dark conditions played an important role in growth and hypericin production in cell suspension cultures, to our knowledge an undocumented observation.

Anthracenes↗
PubMed2002

Exudation of fluorescent beta-carbolines from Oxalis tuberosa L roots.

Root fluorescence is a phenomenon in which roots of seedlings fluoresce when irradiated with ultraviolet (UV) light. Soybean (Glycine max) and rye grass (Elymus glaucus) are the only plant species that have been reported to exhibit this occurrence in germinating seedling roots. The trait has been useful as a marker in genetic, tissue culture and diversity studies, and has facilitated selection of plants for breeding purposes. However, the biological significance of this occurrence in plants and other organisms is unknown. Here we report that the Andean tuber crop species Oxalis tuberosa, known as oca in the highlands of South America, secretes a fluorescent compound as part of its root exudates. The main fluorescent compounds were characterized as harmine (7-methoxy-1-methyl-beta-carboline) and harmaline (3, 4-dihydroharmine). We also detected endogenous root fluorescence in other plant species, including Arabidopsis thaliana and Phytolacca americana, a possible indication that this phenomenon is widespread within the plant kingdom.

Arabidopsis↗
PubMed2002

Isolation and characterization of a novel ribosome-inactivating protein from root cultures of pokeweed and its mechanism of secretion from roots.

Ribosome-inactivating proteins are N-glycosidases that remove a specific adenine from the sarcin/ricin loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, a novel type I ribosome-inactivating protein, termed PAP-H, was purified from Agrobacterium rhizogenes-transformed hairy roots of pokeweed (Phytolacca americana). The protein was purified by anion- and cation-exchange chromatography. PAP-H has a molecular mass of 29.5 kD as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its isoelectric point was determined to be 7.8. Yeast (Saccharomyces cerevisiae) ribosomes incubated with PAP-H released the 360-nucleotide diagnostic fragment from the 26S rRNA upon aniline treatment, an indication of its ribosome-inactivating activity. Using immunofluorescence microscopy, PAP-H was found to be located in the cell walls of hairy roots and root border cells. PAP-H was determined to be constitutively secreted as part of the root exudates, with its secretion enhanced by a mechanism mediated by ethylene induction. Purified PAP-H did not show in vitro antifungal activity against soil-borne fungi. In contrast, root exudates containing PAP-H as well as additional chitinase, beta-1,3-glucanase, and protease activities did inhibit the growth of soil-borne fungi. We found that PAP-H depurinates fungal ribosomes in vitro and in vivo, suggesting an additive mechanism that enables PAP-H to penetrate fungal cells.

Amino Acid Sequence↗