Comparison of reversed-phase high-pressure liquid chromatography with sephadex LH-20 for cytokinin analysis of tomato root pressure exudate

A sensitive, specific enzyme immunoassay (EIA) for trans-zeatin riboside (ZR) and trans-zeatin (Z) in the 03 to 30 picomole range has been described. The reliability of the method for measuring ZR + Z in partially purified extracts of Zea mays L. tissues was verified by highperformance liquid chromatography. EIA measurements showed that there was a concentration gradient of ZR + Z along the length of the Zea stem. The topmost internodes, internodes 7 and 8 counting from the coleoptilar node, had the highest concentration (=130 picomoles per gram fresh weight). Moving basipetally, the concentration dropped =10-fold to a minimum at internode 4, and then increased slightly in internodes 2 and 3. There were also gradients within each internode. The five lowest internodes contained the highest concentrations toward their apical end, the region which included the node; this asymmetry was less pronounced near the top of the plant.The cytokinins Z,' DHZ, and the corresponding ribosides and glucosides have been found in immature maize kernels (7,9,15). Although there is evidence that the most abundant of these cytokinins, Z and ZR, are produced in the root tip (4, 14), little is known about the distribution of cytokinins in maize tissues at different stages of development. In this report, we describe a sensitive, specific EIA for ZR + Z and use the assay to measure cytokinin concentrations along the stem of individual maize plants.The principle underlying EIAs of the type we have developed is reviewed by Schuurs and Van Weemen (12). In brief, the method depends upon binding specific, anti-ZR antibody to a plastic surface and allowing the antibody to react sequentially with an unknown amount of ZR in a plant extract, and then with ZR coupled to alkaline phosphatase. Because the free and coupled ZR compete for the same sites, the amount of enzyme activity bound to the plastic surface is inversely related to the amount of ZR in the sample.We will show that EIA provides a reliable assay for ZR + Z and that there is a striking concentration gradient of these cytokinins along the length of the stem and within individual internodes of the maize plant.'Abbreviations: Z, trans-zeatin; DHZ, DL-dihydrozeatin; ZR, transzeatin riboside; EIA, enzyme immunoassay; PBS, phosphate-buffered saline; IgG, immunoglobulin G; CZ, cis-zeatin; CZR, cis-zeatin riboside; KIN, kinetin; IPA, isopentenyladenosine; IP, isopentenyladenine; RIA, radioimmunoassay.MATERIALS AND METHODS Chemicals. Trans-zeatin riboside, DL-dihydrozeatin and ciszeatin riboside were purchased from Calbiochem-Behring; kinetin, trans-zeatin, isopentenyladenine, isopentenyladenosine, N6-benzylaminopurine, adenine, adenosine, guanosine, BSA ("essentially globulin free" grade), and p-nitrophenylphosphate ("Sigma 104" grade) were purchased from Sigma; alkaline phosphatase from calf intestine (Grade I, sp. activity >2,500 U mg-') was purchased from Boehringer; Freund's complete-and incomplete-adjuvant were purchased from Difco; protein A-sepharose CL-4B was purchased from Ph...

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confidence: 99%

Concentration Gradients of trans-Zeatin Riboside and trans-Zeatin in the Maize Stem

Hansen¹,

Wenzler²,

Meins³

1984

“…Traditional methods of column chromatography and TLC are timeconsuming and greatly limit the number of extracts which can be processed in any given period. In recent years, the speed and efficiency of HPLC in the purification of plant extracts and its utility in the study of endogenous plant growth substances have begun to be recognized by plant physiologists (2,3,12,15). The two methods (8,14) which have been published so far on HPLC of GAs appear to be of little practical use for the routine purification of crude plant extracts (see under "Discussion").…”

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confidence: 99%

Fractionation of Gibberellins in Plant Extracts by Reverse Phase High Performance Liquid Chromatography

Jones

Metzger²,

Zeevaart³

1980

In studies on endogenous plant gibbereliins (GAs), reverse phase (Bondapak C18) high performance liquid chromatography (HPLC) has proved to be a useful method for the fractionation of plant extracts. The behavior of 18 authentic GAs in such a chromatographic system is described. The main factors determining chromatographic behavior are the degree and the position of hydroxylation of the GA. Generally, dihydroxylated GAs elute before monohydroxylated GAs, whereas 13-hydroxylated GAs elute before 3-hydroxylated GAs. The number of carboxyl groups and the degree of saturation of the A-ring have little effect. For 20-carbon GAs, the oxidation state at C-20 is only relevant insofar as GAs having a methyl group at this position elute later than those with other groups (lactone, aldehyde, or carboxyl).As an illustration of the use of reverse phase HPLC, the endogenous GAs of immature seeds of Pharbitis nil L., strain "Violet," were reinvestigated. The presence of gibberellins A3, A6, A17, A20, and A2, was confirmed by gas-liquid chromatography-mass spectrometry. In addition, two other GAs, A1, and A4, were also identified in extracts of this material.The levels of endogenous GAs3 in vegetative tissues are generally very low (1-10 ,ug/kg fresh weight). Consequently, plant extracts must be extensively purified before reliable analysis by bioassay or physicochemical means can be undertaken. Traditional methods of column chromatography and TLC are timeconsuming and greatly limit the number of extracts which can be processed in any given period. In recent years, the speed and efficiency of HPLC in the purification of plant extracts and its utility in the study of endogenous plant growth substances have begun to be recognized by plant physiologists (2, 3, 12, 15). The two methods (8, 14) which have been published so far on HPLC of GAs appear to be of little practical use for the routine purification of crude plant extracts (see under "Discussion"). We report here on the use of a reverse phase HPLC system for this purpose, and describe the behavior of 18 authentic GAs in this system. To illustrate the effectiveness of this chromatographic system, we have reexamined the endogenous GAs of immature seeds of Pharbitis nil. Bioassays. Gibberellins A1, A3, A4, A5, A7, As, and A20 in the eluted fractions were determined using a modified lettuce hypocotyl assay (6), in which the beakers used to collect fractions from the HPLC were also used to grow the lettuce seedlings. Gibberellins A12, A18, A23, A36, and A37 were assayed with the d-5 corn assay (18).GLC. The positions of gibberellins A8, A13, A14, A17, A25, and A29 in the HPLC eluate were determined by GLC. Fractions from the HPLC were methylated with ethereal diazomethane and chromatographed on a glass column 183 x 0.3 cm i.d., packed with 4% SE-33 as described previously (19). Column temperature was 220 C (240 C for Me-GA8) and detection was by flame ionization.Seeds of P. niL Seeds were harvested from plants of P. nil L., strain "Violet," 20-22 days after anthesis, froz...

“…To determine whether ABA was entrapped in the poorly dissolved components preventing complete methylation of all of the ABA, methanol was added initially to dissolve FIG. 3. Absorbance (254 nm) of standard ABA (500 ng) g±Bondapak-NH2 (B) analytical HPLC columns.…”

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confidence: 99%

Rapid Separation and Quantification of Abscisic Acid from Plant Tissues Using High Performance Liquid Chromatography

Ciha

Brenner²,

Brun³

1977

(v/v) methanol (20 ml methanol/ g fresh weight of tissue) at 4 C, transferred to a 250-ml Erlenmeyer flask, and shaken for 24 hr at 4 C. The homogenate was centrifuged at 1,545g for 10 min and the supernatant filtered through methanol-washed Miracloth (Chicopee Mills, Inc.), and taken to dryness in vacuo at 35 C. The sample was then frozen and stored at -20 C. PARTITION COEFFICIENTS FOR ABAThe determination of ABA from plant tissues has been timeconsuming in the past. It usually consists of a preparative and an analytical procedure. The preparative procedure normally involves the partitioning of a plant extract against a number of organic solvents (6, 11) and is followed by either TLC and/or paper chromatography (1,10,13, 20). The analytical procedure has been achieved either by bioassay (12,17,18) or by GLC (4,8,15 Although several organic solvents have been used for partitioning ABA, to our knowledge the literature does not contain a direct comparison of the effectiveness of such solvents. To achieve such a comparison, the partition coefficients were determined for the partitioning of ABA between water at four pH values and seven organic solvent systems commonly used for this purpose.ABA standards (1 ,tg/ml) in charcoal-filtered, deionized, distilled H20 were adjusted to either pH 2.5, 5, 7, or 9 with 0.1 N HCI or 0.1 N KOH. Ten ml of the ABA standards were placed in 60-ml culture tubes and partitioned once against an equal volume of glass-distilled organic solvent. The organic fractions were removed and dried in vacuo at 40 C. The ABA in the control (no partitioning) was determined by taking to dryness a 10-ml ABA standard for each pH treatment. All samples were reconstituted in 1 ml of 5% (v/v) methanol in 0.2 N acetic acid. A 200-,lI aliquot was injected into a 316 stainless steel column (30 cm x 4 mm i.d.) packed with ,uBondapak C18 (Waters Associates). The sample was eluted with 0.2 N acetic acid in 45% (v/v) methanol at a flow rate of 4.5 ml/min, and a pressure of 262 to 290 bars. The ABA was detected with a fixed wavelength UV detector at 254 nm. The instrumentation used in this HPLC procedure is further described below.The ABA eluted with a very narrow peak width which permitted quantification based on peak height.Partition coefficients were determined as: