Improved Method for HPLC Analysis of Polyamines, Agmatine and Aromatic Monoamines in Plant Tissue

Slocum, Robert D.; Flores, Hector E.; Galston, Arthur W.; Weinstein, Leonard H.

doi:10.1104/pp.89.2.512

Cited by 106 publications

(60 citation statements)

References 22 publications

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“…We have developed the first method to quantify pavettamine, the toxic agent causing gousiekte, Unprotonated amines (NH 2 ) react with carbonic or sulfonic acid chlorides at alkaline pH to form amide derivatives (Slocum et al, 1989). These derivatized polyamines have good retention on reverse phase columns.…”

Section: A New Methods To Analyze Pavettamine and Other Polyaminesmentioning

confidence: 99%

Production of toxic pavettamine and pavettamine conjugates in the gousiekte-causing Fadogia homblei plant and its relation to the bacterial endosymbiont

Elst¹,

Wyk²,

Schultz³

et al. 2013

Phytochemistry

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Plant poisoning of livestock is responsible for considerable economic losses in southern Africa.Six plant species of the Rubiaceae family are known to cause gousiekte, a cardiac syndrome of ruminants induced by ingestion of the toxic compound pavettamine. Progress in understanding the aetiology of this disease is largely hampered by the variable toxicity of the plants and the -2 -absence of a quantification method for pavettamine. The pavettamine concentration in leaf samples of Fadogia homblei, a known gousiekte causing plant, was analyzed by massspectrometry. In the most apical leaf pair, the highest concentration of pavettamine was detected. Distal leaves contained progressively less pavettamine. Besides a significant amount of free pavettamine, most pavettamine was found to occur in a conjugated form. To which molecules the pavettamine is conjugated remains unknown as is the function of conjugated pavettamine in the development of gousiekte. All know gousiekte-causing plants contain symbiotic bacteria in their leaves; it was hypothesized that these bacteria might be involved in the production of pavettamine. However, analysis of in vitro cultures of the Fadogia homblei endosymbiont revealed no production of pavettamine. Pavettamine is therefore not produced by the bacteria alone. It is either the product of the interaction with the plant or solely produced by the host.

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Section: A New Methods To Analyze Pavettamine and Other Polyaminesmentioning

confidence: 99%

Production of toxic pavettamine and pavettamine conjugates in the gousiekte-causing Fadogia homblei plant and its relation to the bacterial endosymbiont

Elst¹,

Wyk²,

Schultz³

et al. 2013

Phytochemistry

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“…Then the supernatants from the two centrifugations were combined. For determination of free and conjugated PA levels, 250 ll of the supernatant was hydrolyzed with 12 mol dm -3 HCl and heated at 110°C for 18 h. After acidic hydrolysis the supernatant was evaporated and the residue was resuspended in 200 ll 10 % PCA and used for benzoylation reaction according to method of Slocum et al (1989) and Cvikrová et al (1999) with 1,7-diaminoheptane (Sigma Algrich, USA) as an internal standard. Polyamine standards (Put, Spd and Spm; Sigma, USA) were prepared similarly to plant samples.…”

Section: Soluble Carbohydrate Measurementsmentioning

confidence: 99%

Relationships between polyamines, ethylene, osmoprotectants and antioxidant enzymes activities in wheat seedlings after short-term PEG- and NaCl-induced stresses

et al. 2012

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Contents of ethylene, osmoprotectants, levels and forms of polyamines (PAs) and activities of antioxidant enzymes in the leaves and roots were investigated for five wheat cultivar seedlings (differing in drought tolerance) exposed to osmotic stress (-1.5 MPa). Stress was induced by 2-day-long treatment of plants with polyethylene glycol 6000 (PEG) or NaCl added to hydroponic cultures. Nawra, Parabola and Manu cv. (drought tolerant) showed a marked increase in osmoprotectors (proline and soluble carbohydrates, mainly glucose, saccharose and maltose), free PAs (putrescine Put, spermidine Spd and spermine Spm) and Spd-conjugated levels, in both leaves and roots, after PEGtreatments. Radunia and Raweta (drought sensitive) exhibited smaller changes in the content of these substances. The analysis of enzymes involved in proline metabolism revealed the glutamate as a precursor of proline synthesis in PEGinduced stress conditions. The increase in the activity of antioxidative enzymes, especially catalase and peroxidases, was characteristic for tolerant wheat plants, but for sensitive ones, a decrease in superoxide dismutase and an increase in mainly glutathione reductase activities were observed. After NaCl-treatment smaller changes of all biochemical parameters were registered in comparison with PEG-induced stress. Exceptions were the higher values of ethylene content and a significant increase in saccharose, raffinose and maltose levels (only in stress sensitive plants). The proline synthesis pathway was stimulated from both glutamate and ornithine precursors. These results suggest that the accumulation of inorganic ions in NaCl-stressed plants may be involved in protective mechanisms as an additional osmoregultor. Thus, a weaker stressogenic effect as determined as water deficit by leaf relative water content and relative dry weight increase rate and differences in metabolite synthesis in comparison with PEG stress was observed. Proline seems to be the most important osmo-protector in osmotic stress initiated by both PEG and NaCl. The synthesis of sugars and PAs may be stimulated in a stronger stress conditions (PEG).

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“…Benzoylated putrescine, benzoylated spermidine, and benzoylated spermine were separated by the method of Slocum and co-workers (22). A ternary gradient HPLC system was used with a V4 variable wavelength detector each dish, and at 48 h postinfection, the rickettsiae were isolated by N2 cavitation and Renografin.…”

Section: Methodsmentioning

confidence: 99%

Acquisition of polyamines by the obligate intracytoplasmic bacterium Rickettsia prowazekii

Speed

Winkler

1990

J Bacteriol

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Both the polyamine content and the route of acquisition of polyamines by Rickettsia prowazekii, an obligate intracellular parasitic bacterium, were determined. The rickettsiae grew normally in an ornithine decarboxylase mutant of the Chinese hamster ovary (C55.7) cell line whether or not putrescine, which this host cell required in order to grow, was present. The rickettsiae contained approximately 6 mM putrescine, 5 mM spermidine, and 3 mM spermine when cultured in the presence or absence of putrescine. Neither the transport of putrescine and spermidine by the rickettsiae nor a measurable rickettsial ornithine decarboxylase activity could be demonstrated. However, we demonstrated the de novo synthesis of polyamines from arginine by the rickettsiae. Arginine decarboxylase activity (29 pmol of 14Co2 released per h per 108 rickettsiae) was measured in the rickettsiae growing within their host cell. A markedly lower level of this enzymatic activity was observed in cell extracts of R. prowazekii and could be completely inhibited with 1 mM difluoromethylarginine, an irreversible inhibitor of the enzyme. R. prowazekii failed to grow in C55.7 cells that had been cultured in the presence of 1 mM difluoromethylarginine. After rickettsiae were grown in C55.7 cells in the presence of labeled arginine, the specific activities of arginine in the host cell cytoplasm and polyamines in the rickettsiae were measured; these measurements indicated that 100% of the total polyamine content of R. prowazekii was derived from arginine.Rickettsia prowazekii, the etiological agent of epidemic typhus, is an obligate intracytoplasmic bacterium. R. prowazekii grows free in the cytoplasm of its eucaryotic host cell unbounded by any internal membranes of host cell origin. If the rickettsiae have evolved appropriate transport systems, then they would be able to scavenge from this rich external milieu many of the end products and intermediate metabolites needed for growth rather than expending energy synthesizing these metabolites via complex pathways. For example, the enzymes of the glycolytic and hexose monophosphate pathways are absent in typhus group rickettsiae (9) and the essential metabolites generated by these pathways must be obtained from the cytoplasm of the host cell. Rickettsiae can also employ a dual strategy for the acquisition of metabolites: they can both transport and synthesize the same compound. This can be seen most clearly in the acquisition of ATP. The enzymes of the tricarboxylic acid cycle (9), oxidative phosphorylation (7, 12), and an ATPase (37) are present in the rickettsiae to synthesize ATP. However, the rickettsiae also have a transport system to obtain ATP from the cytoplasm of the host cell (32).Virtually all cells contain significant amounts of some or all of the polyamines putrescine, spermidine, and spermine. Proposed functions for these highly basic amines include, but are not limited to, the stabilization of nucleic acids and the stimulation of protein biosynthesis in both eucaryotic and procaryotic organis...

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Improved Method for HPLC Analysis of Polyamines, Agmatine and Aromatic Monoamines in Plant Tissue

Cited by 106 publications

References 22 publications

Production of toxic pavettamine and pavettamine conjugates in the gousiekte-causing Fadogia homblei plant and its relation to the bacterial endosymbiont

Production of toxic pavettamine and pavettamine conjugates in the gousiekte-causing Fadogia homblei plant and its relation to the bacterial endosymbiont

Relationships between polyamines, ethylene, osmoprotectants and antioxidant enzymes activities in wheat seedlings after short-term PEG- and NaCl-induced stresses

Acquisition of polyamines by the obligate intracytoplasmic bacterium Rickettsia prowazekii

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