[40] Isolation of cytokinins by immunoaffinity chromatography and analysis by high-performance liquid chromatography radioimmunoassay

Macdonald, Elizabeth; Morris, R. O.

doi:10.1016/s0076-6879(85)10093-5

Cited by 82 publications

(55 citation statements)

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“…Eluates were dried in vacuo, resuspended in 400 l of dimethyl sulfoxide, and diluted with 40 mM ammonium acetate (pH 7.0) to obtain 40-ml preparations. Samples were passed through DEAE-cellulose (DE-52; bed volume, 10 ml; Whatman) connected in tandem to immunoaffinity columns, each containing 0.5 to 1.0 ml of microcrystalline cellulose (Whatman) to which purified clone 16 (anti-trans-zeatin and anti-trans-ribosylzeatin) monoclonal antibody (44) had been conjugated (29). The columns were each equilibrated with 120 ml of immunoaffinity buffer and, after sample application, were washed with an additional 20 ml of this buffer passed through both columns and 10 ml passed through only the immunoaffinity column.…”

Section: Methodsmentioning

confidence: 99%

tRNA Is the Source of Low-Level trans- Zeatin Production in Methylobacterium spp

2002

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Pink-pigmented facultatively methylotrophic bacteria (PPFMs), classified as Methylobacterium spp., are persistent colonizers of plant leaf surfaces. Reports of PPFM-plant dialogue led us to examine cytokinin production by PPFMs. Using immunoaffinity and high-performance liquid chromatography (HPLC) purification, we obtained 22 to 111 ng of trans-zeatin per liter from culture filtrates of four PPFM leaf isolates (from Arabidopsis, barley, maize, and soybean) and of a Methylobacterium extorquens type culture originally recovered as a soil isolate. We identified the zeatin isolated as the trans isomer by HPLC and by a radioimmunoassay in which monoclonal antibodies specific for trans-hydroxylated cytokinins were used. Smaller and variable amounts of trans-zeatin riboside were also recovered. trans-Zeatin was recovered from tRNA hydrolysates in addition to the culture filtrates, suggesting that secreted trans-zeatin resulted from tRNA turnover rather than from de novo synthesis. The product of the miaA gene is responsible for isopentenylation of a specific adenine in some tRNAs. To confirm that the secreted zeatin originated from tRNA, we mutated the miaA gene of M. extorquens by single exchange of an internal miaA fragment into the chromosomal gene. Mutant exconjugants, confirmed by PCR, did not contain zeatin in their tRNAs and did not secrete zeatin into the medium, findings which are consistent with the hypothesis that all zeatin is tRNA derived rather than synthesized de novo. In germination studies performed with heat-treated soybean seeds, cytokinin-null (miaA) mutants stimulated germination as well as wild-type bacteria. While cytokinin production may play a role in the plant-PPFM interaction, it is not responsible for stimulation of germination by PPFMs.Although the best-studied plant-bacterium relationships involve pathogenesis or symbiosis, plants interact constantly with phylloplane bacteria and with the bacteria in the soil around their roots (rhizosphere). While it is not widely believed that epi-and endophytic bacteria have overt effects on plants, there is evidence (13) that plants interact with pink-pigmented facultatively methylotrophic bacteria (PPFMs), which are inhabitants of the phylloplane and rhizosphere. PPFMs are gram-negative members of the alpha subclass of the class Proteobacteria that belong to the genus Methylobacterium and are known to inhabit the leaf surfaces of a wide variety of plant species (11). One intriguing aspect of the plant-PPFM relationship is the possibility that PPFMs may provide cytokinins to the plant host or have cytokinin-like effects. Corpe and Basile (12) demonstrated that callus from Streptocarpus prolixus (gracilis) (Cape primrose) regenerated plantlets within 15 days when it was cultured cobiotically with PPFMs. PPFM-free controls exhibited no plant development after 30 days. However, Corpe and Basile did not indicate whether specific phytohormone regimens could mimic the PPFM effects or whether PPFMs produce phytohormones. We demonstrated previously that heat ...

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Section: Methodsmentioning

confidence: 99%

tRNA Is the Source of Low-Level trans- Zeatin Production in Methylobacterium spp

2002

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“…If immunoaffinity prepurification were not used, it was expected that other labeled materials would obscure the radioactive cytokinin peaks. However, because of the specificity of the immunoaffinity procedure, which is capable of purifying cytokinins to analytical homogeneity in a single step (13), it was anticipated that meaningful data could be obtained.…”

Section: Resultsmentioning

confidence: 99%

“…The problem can be alleviated by combining immunoaffinity chromatography, to purify the cytokinins from nonspecific interfering substances (13), with HPLC to separate the individual components. RIA or ELISA can then be used to detect each cytokinin in the appropriate HPLC fraction (7,12 …”

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confidence: 99%

Rapid Identification of Cytokinins by an Immunological Method

Morris

Jameson²,

Laloue³

et al. 1991

Plant Physiol.

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A method for rapid identification of bacterial cytokinins has been developed in which cultures are fed [3H]adenine, the cytokinins (including 3H-labeled cytokinins) are isolated by immunoaffinity chromatography, and analyzed by HPLC with on-line scintillation counting. Analysis of Agrobacterium tumefaciens strains showed that some produced primarily trans-zeatin, whereas others produced primarily trans-zeatin riboside. Pseudomonas syringae pv savastanoi produced mixtures of transzeatin, dihydrozeatin, 1"-methyl-trans-zeatin riboside, and other unknown cytokinin-like substances. Corynebacterium fascians, produced cis-zeatin, isopentenyladenine and isopentenyladenosine. The technique is designed for qualitative rather than quantitative studies and allows ready identification of bacterial cyto- 'Abbreviations: RIA, radioimmunoassay; "MeZR, 1 "-methyl trans-zeatin riboside; cZ, cis-zeatin; cZR, cis-zeatin riboside; DZ, dihydrozeatin; DZR, dihydrozeatin riboside; iP, isopentenyladenine; iPA, isopentenyladenosine; Z, trans-zeatin; ZR, trans-zeatin riboside.Unfortunately, direct analysis of phytohormones in unfractionated plant extracts by RIA or ELISA can lead to erroneous results. In some cases, crude extracts may contain compounds which cause nonspecific interference; their presence leads to generation of false positives. For example, Cohen et al. (6) showed that ELISA ofIAA in even semipurified plant samples led to high estimates of the IAA content. As successively more pure samples were analyzed, the apparent IAA values approached those determined by rigorous mass spectrometric analysis. Nonspecific interference is, however, not the only problem. When several cross-reacting phytohormone species are present, a composite result will be obtained. A case in point is the analysis of bacterially produced cytokinins. The primary cytokinin produced by Agrobacterium tumefaciens strain C58 has been shown to be zeatin (9). Small amounts of iP are also present, but not in sufficient quantity to interfere with zeatin-specific RIA of supernatants (20). Zeatin determination by ELISA or RIA is therefore likely to give reasonably accurate results. The same is not true for Pseudomonas syringae pv savastanoi strains. Some strains secrete Z, ZR, and 1 "MeZR and, late in stationary phase, these are present in the medium in approximately equal quantities (14). Since the relative cross reactivities of Z, ZR and 1 "MeZR with anti-ZR antibody are approximately 10:50:1 (unpublished data), assays ofunfractionated supernatants will not give meaningful data. For most plant samples, where several cytokinins are usually present, the same considerations apply.The problem can be alleviated by combining immunoaffinity chromatography, to purify the cytokinins from nonspecific interfering substances (13), with HPLC to separate the individual components. RIA or ELISA can then be used to detect each cytokinin in the appropriate HPLC fraction (7,12

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“…The phytohormones can be recovered from the immunosorbent in sufficiently pure form for direct analysis by high resolution techniques such as HPLC, GLC, or GC-MS. Immunoaffinity methodologies have been described for cytokinins (7,8,14,16), abscisic acid (7), 3-indole acetic acid (17), and for GA3 (12).…”

mentioning

confidence: 99%

Immunoaffinity Techniques Applied to the Purification of Gibberellins from Plant Extracts

Durley¹,

Sharp²,

Maki³

et al. 1989

Plant Physiol.

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The use of immunoaffinity columns containing anti-gibberellin (GA) antibodies for the selective purification of GAs in plant extracts is described. GA1, GA3, GA4, GAs, GA7, and GA, conjugates to bovine serum albumin were synthesized and used to elicit anti-GA polyclonal antibodies (Abs) in rabbits. Protein A purified rabbit serum, containing a mixture of anti-GA Abs, was immobilized on matrices of Affi-gel 10 or Fast-Flow Sepharose 4B. Columns of these immunosorbents retained a wide range of C-19 GA methyl esters, but no C-20 GA methyl esters. Quantitative recovery of C-19 GA methyl esters was achieved from the columns, which, after reequilibration in buffer, could be reused up to 500 times. The immunosorbents were tested by examination of extracts from immature soybean and pea seeds. GAs were initially purified by passing the extracts through DEAE-cellulose and concentrating them on octadecylsilica. The extracts were methylated and further purified on the mixed anti-GA immunoaffinity columns. GAs were detected and quantified as methyl esters or methyl ester trimethylsilyl ethers by gas chromatography-mass spectrometry-selected ion monitoring. GA7 was found in soybean seeds, 17 days after anthesis, at low levels (8.8 nanograms per gram fresh weight). C-19 GAs were examined in cotyledons, embryonic axes, and testae of G2 pea seeds harvested 20 days after anthesis. High levels of GA2o and GA29 were found in cotyledons (3580 and 310 nanograms per gram fresh weight, respectively) and embryonic axes (5375 and 1430 nanograms per gram) fresh weight, respectively). Lower levels of GA9 were found in cotyledons and embryonic axes (147 and 161 nanograms per gram fresh weight, respectively). GA, was the major GA of testae at levels of 195 nanograms per gram fresh weight. Trace quantities of GA2, and GA,1 were also observed in testae.

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[40] Isolation of cytokinins by immunoaffinity chromatography and analysis by high-performance liquid chromatography radioimmunoassay

Cited by 82 publications

References 5 publications

tRNA Is the Source of Low-Level trans- Zeatin Production in Methylobacterium spp

tRNA Is the Source of Low-Level trans- Zeatin Production in Methylobacterium spp

Rapid Identification of Cytokinins by an Immunological Method

Immunoaffinity Techniques Applied to the Purification of Gibberellins from Plant Extracts

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