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Title: Attempting to monitor the incorporation of deuterium into indole-3-acetic acid and tryptophan in Zea mays grown on deuterium oxide labeled water

Abstract

We are attempting to determine when seedlings of Zea mays sweet corn, var. Silver Queen begin de novo biosynthesis of tryptophan and indole-3-acetic acid (IAA). We wish to use the general precursor, deuterium labeled water, to minimize assumptions as to the biosynthetic route. Protium in positions 2, 4, 5, 6 7 of the indole ring are non-exchangeable. IAA and tryptophan synthesized via the shikimic acid pathway would contain deuterium in one or more of these positions . The protium on the indene nitrogen, the carboxyl, the amino group, or the protium alpha to the carboxyl exchange readily and so are removed prior to analysis by base catalyzed exchange. The IAA, or trypotophan, is then purified by DEAE, Dowex 50, and two HPLC steps. IAA is methylated with diazomethane and analyzed by GC/MS. Trytophan is acetylated with triethylamine-acetic anhydride and then methylated with diazomethane and analyzed by GC/MS. Results of these studies on plants grown for varying lengths of time and under various light and nutrient conditions will be reported.

Authors:
;  [1]
  1. (Michigan State Univ., East Lansing (USA))
Publication Date:
OSTI Identifier:
6610935
Alternate Identifier(s):
OSTI ID: 6610935
Resource Type:
Journal Article
Resource Relation:
Journal Name: Plant Physiology, Supplement; (USA); Journal Volume: 89:4
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; AUXINS; BIOSYNTHESIS; TRYPTOPHAN; BIOLOGICAL PATHWAYS; DEUTERIUM; FRACTIONATION; GAS CHROMATOGRAPHY; HEAVY WATER; LIQUID COLUMN CHROMATOGRAPHY; MAIZE; MASS SPECTROSCOPY; TRACER TECHNIQUES; AMINO ACIDS; AROMATICS; AZAARENES; AZOLES; CARBOXYLIC ACIDS; CEREALS; CHROMATOGRAPHY; GRASS; HETEROCYCLIC ACIDS; HETEROCYCLIC COMPOUNDS; HYDROGEN COMPOUNDS; HYDROGEN ISOTOPES; INDOLES; ISOTOPE APPLICATIONS; ISOTOPES; LIGHT NUCLEI; LILIOPSIDA; MAGNOLIOPHYTA; NUCLEI; ODD-ODD NUCLEI; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; OXYGEN COMPOUNDS; PLANT GROWTH REGULATORS; PLANTS; PYRROLES; SEPARATION PROCESSES; SPECTROSCOPY; STABLE ISOTOPES; SYNTHESIS; WATER 550201* -- Biochemistry-- Tracer Techniques

Citation Formats

Jensen, P.J., and Bandurski, R.S. Attempting to monitor the incorporation of deuterium into indole-3-acetic acid and tryptophan in Zea mays grown on deuterium oxide labeled water. United States: N. p., 1989. Web.
Jensen, P.J., & Bandurski, R.S. Attempting to monitor the incorporation of deuterium into indole-3-acetic acid and tryptophan in Zea mays grown on deuterium oxide labeled water. United States.
Jensen, P.J., and Bandurski, R.S. Sat . "Attempting to monitor the incorporation of deuterium into indole-3-acetic acid and tryptophan in Zea mays grown on deuterium oxide labeled water". United States. doi:.
@article{osti_6610935,
title = {Attempting to monitor the incorporation of deuterium into indole-3-acetic acid and tryptophan in Zea mays grown on deuterium oxide labeled water},
author = {Jensen, P.J. and Bandurski, R.S.},
abstractNote = {We are attempting to determine when seedlings of Zea mays sweet corn, var. Silver Queen begin de novo biosynthesis of tryptophan and indole-3-acetic acid (IAA). We wish to use the general precursor, deuterium labeled water, to minimize assumptions as to the biosynthetic route. Protium in positions 2, 4, 5, 6 7 of the indole ring are non-exchangeable. IAA and tryptophan synthesized via the shikimic acid pathway would contain deuterium in one or more of these positions . The protium on the indene nitrogen, the carboxyl, the amino group, or the protium alpha to the carboxyl exchange readily and so are removed prior to analysis by base catalyzed exchange. The IAA, or trypotophan, is then purified by DEAE, Dowex 50, and two HPLC steps. IAA is methylated with diazomethane and analyzed by GC/MS. Trytophan is acetylated with triethylamine-acetic anhydride and then methylated with diazomethane and analyzed by GC/MS. Results of these studies on plants grown for varying lengths of time and under various light and nutrient conditions will be reported.},
doi = {},
journal = {Plant Physiology, Supplement; (USA)},
number = ,
volume = 89:4,
place = {United States},
year = {Sat Apr 01 00:00:00 EST 1989},
month = {Sat Apr 01 00:00:00 EST 1989}
}
  • Either 5-(/sup 3/H)indole-3-acetic acid (IAA) or 5-(/sup 3/H)indole-3-acetyl-myoinositol was applied to the endosperm of kernels of dark-grown Zea mays seedlings. The distribution of total radioactivity, radiolabeled indole-3-acetic acid, and radiolabeled ester conjugated indole-3-acetic acid, in the shoots was then determined. Differences were found in the distribution and chemical form of the radiolabeled indole-3-acetic acid in the shoot depending upon whether 5-(/sup 3/H)indole-3-acetic acid or 5-(/sup 3/H)indole-3-acetyl-myo-inositol was applied to the endosperm. We demonstrated that indole-3-acetyle-myo-inositol applied to the endosperm provides both free and ester conjugated indole-3-acetic acid to the mesocotyl and coleoptile. Free indole-3-acetic acid applied to the endosperm suppliesmore » some of the indole-3-acetic acid in the mesocotyl but essentially no indole-3-acetic acid to the coleoptile or primary leaves. It is concluded that free IAA from the endosperm is not a source of IAA for the coleoptile. Neither radioactive indole-3-acetyl-myo-inositol nor IAA accumulates in the tip of the coleoptile or the mesocotyl node and thus these studies do not explain how the coleoptile tip controls the amount of IAA in the shoot.« less
  • The authors present evidence that the role of tryptophan and other potential intermediates in the pathways that could lead to indole derivatives needs to be reexamined. Two lines of Lemna gibba were tested for uptake of ({sup 15}N-indole)-labeled tryptophan isomers and incorporation of that label into free indole-3-acetic acid (IAA). Both lines required levels of L-({sup 15}N)tryptophan 2 to 3 orders of magnitude over endogenous levels in order to obtain measurable incorporation of label into IAA. Labeled L-tryptophan was extractable from plant tissue after feeding and showed no measurable isomerization into D-tryptophan. D-({sup 15}N)trytophan supplied to Lemna at rates ofmore » approximately 400 times excess of endogenous D-tryptophan levels (to yield an isotopic enrichment equal to that which allowed detection of the incorporation of L-tryptophan into IAA), did not result in measurable incorporation of label into free IAA. These results demonstrate that L-tryptophan is a more direct precursor to IAA than the D isomer and suggest (a) that the availability of tryptophan in vivo is not a limiting factor in the biosynthesis of IAA, thus implying that other regulatory mechanisms are in operation and (b) that L-tryptophan also may not be a primary precursor to IAA in plants.« less
  • Syntheses are described for tetra and pentadeutero indole-3-acetic acid (IAA) labeled in positions 4,5,6,7 or 2,4,5,6,7 of the indole moiety. Polydeuterated IAA is proposed as an internal standard for gas chromatographic-mass spectrometric analysis of IAA by selected ion monitoring. Nanogram amounts of IAA may be assayed by monitoring the base peak of IAA at m/z = 130 (134 for d/sub 4/-IAA) and the molecular ion of the methyl ester of IAA at 189 (193 for d/sub 4/-IAA). Deuterium in positions 4,5,6, and 7 and, to only a slightly lesser extent, that in position 2 of IAA is retained during alkalimore » treatment, thus permitting use of these compounds as internal standards for assay of IAA released by alkaline hydrolysis of ester and amide conjugates. The use of polydeutero internal standards separates the standards from the isotope cluster caused by the normal abundance of heavy isotopes and also permits use of reduced mass resolution, thus leading to a 10-fold increase in sensitivity. Tetradeutero IAA was used as an internal standard for determining free plus ester IAA in alkaline hydrolysates of Zea mays, and showed exact agreement between estimates based on the molecular ion of the methyl ester and those based upon base peak. Application of the method to measuring free IAA in the upper and lower halves of geotropically stimulated Zea shoots showed 61 +- 4% of the free IAA to be on the lower side.« less
  • Previous studies from this laboratory have demonstrated an increase within 3 min in both free and ester indole-3-acetic acid (IAA) on the lower side of the mesocotyl cortex of a gravity stimulated Zea mays seedling. Since both free and ester IAA are being transported from endosperm to shoot through the stele these results suggest that the gravity stimulus affects movement of IAA and/or its esters from stele to cortex. To test this postulate they injected 5-(/sup 3/H)-IAA into the endosperm and, after a 30 min period with the plants held vertically, severed the kernel from the shoot and placed themore » plants in a horizontal position. After 60 min the distribution of radioactivity in the mesocotyl cortex was 55 + 3% in the lower half and 45 + 3% in the upper half. These results support the working theory that a target for the gravity stimulus is the gating mechanism for the movement of hormone from stele to cortex.« less
  • A study was undertaken using the photoaffinity labeling agent, tritiated 5-azidoindole-3-acetic acid (({sup 3}H),5-N{sub 3}IAA), to identify cells in the etiolated maize (Zea mays L.) shoot which transport auxin. Transport of ({sup 3}H),5-N{sub 3}IAA was shown to be polar, inhibited by 2,3,5-triiodobenzoic acid (TIBA) and essentially freely mobile. There was no detectable radiodecomposition of ({sup 3}H),5-N{sub 3}IAA within tissue kept in darkness for 4 hours. Shoot tissue which had taken up ({sup 3}H),5-N{sub 3}IAA was irradiated with ultraviolet light to covalently fix the photoaffinity labeling agent within cells that contained it at the time of photolysis. Subsequent microautoradiography showed thatmore » all cells contained radioactivity; however, the amount of radioactivity varied among different cell types. Epidermal cells contained the most radioactivity per area, approximately twofold more than other cells. Parenchyma cells in the mature stelar region contained the next largest amount and cortical cells, sieve tube cells, tracheary cells, and all cells in the leaf base contained the least amount of the radioactive label. Two observations suggest that the auxin within the epidermal cells is transported in a polar manner: (a) the amount of auxin in the epidermal cells is greatly reduced in the presence of TIBA, and (b) auxin accumulates on the apical side of a wound in the epidermis and is absent on the basal side. While these results indicate that auxin in the epidermis is polarly transported, this tissue cannot be the only pathway since the epidermis is only a small fraction of the shoot volume.« less