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

Abstract

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...

Photoperiodic Control of Gibberellin Metabolism in Spinach

“…2) is known to elute in this particular system (8), the peak was so broad that it appeared likely that several metabolites were present. Therefore, fractions 8 the same elution volume as GA29 (Fig. 3, I), another like that of GA1 ( Fig.…”

“…The plants were maintained under SD conditions in growth chambers as described previously (12) for 6 weeks after sowing. Both SD and LD conditions were identical to those described previously (12 in 25 min and a flow rate of 9.9 ml min-' as described (8,11).…”

“…The plants were maintained under SD conditions in growth chambers as described previously (12) for 6 weeks after sowing. Both SD and LD conditions were identical to those described previously (12 in 25 min and a flow rate of 9.9 ml min-' as described (8,11 Isothermal densed effluent was eluted off the sides of the inner wall of the pipette with acetone into a scintillation vial. The acetone was evaporated under a stream of N2, and the radioactivity was determined as described above.…”

“…The plants were maintained under SD conditions in growth chambers as described previously (12) for 6 weeks after sowing. Both SD and LD conditions were identical to those described previously (12 in 25 min and a flow rate of 9.9 ml min-' as described (8,11).Fractions were collected every min, and the radioactivity present in each fraction was determined by assaying a 1% aliquot by liquid scintillation spectrometry. Counting efficiency was approximately 50%.…”

“…The water-soluble fraction following partitioning of the eluate from charcoal adsorption chromatography was lyophilized, and the chromatographic properties of radioactive products in this fraction were examined by analytical reverse-phase HPLC with a gradient of 10 to 70% methanol in H20 in 30 min (8,11 …”