University of Missouri
--Allen D. Wright
The accumulation of a UV-light stimulated blue fluorescent compound has been observed in the seedlings of orange pericarp, a mutation caused by the duplicate recessive genes orp1 and orp2. This compound was identified as a glycoside of anthranilic acid (Wright and Neuffer, J. Hered. in press). For this reason, the mutant seedlings might have been expected to have a fragrance similar to that reported for Bf/Bf (Coe, MNL 59:39-40). Instead of the characteristic pleasing odor of anthranilic acid, however, seedlings of the duplicate homozygote had a less pleasing odor which was identified as that of indole. The odor can not be detected in plants that are homozygous recessive for only one of the orp factors. Anthranilate synthetase appears to be the control point for tryptophan biosynthesis in higher plants (e.g. see Singh and Widholm, Physiol Plant 32:240, 1974). The accumulation of anthranilate might therefore be related to the inability of this mutant to synthesize tryptophan from indole.
Indole was extracted from the above-ground portion of 12 day old greenhouse-grown seedlings by partitioning a methanol extract (2ml) with chloroform (2ml) and water (1ml). An aliquot of the chloroform layer was reacted with Ehrlich's reagent (p-dimethylamino benzaldehyde in ethanolic HCl) for the colorimetric determination of indole (Yanofsky, Meth. Enzymol. 2:233, 1955), correcting for background absorbance using a sample prepared in a similar manner without p-dimethylamino benzaldehyde. The mutant leaf tissue had an indole concentration (mean of six reps ± SE) of 935±330µg/gfw while that of the normal sibs was 67±12µg/gfw. The material for the above study was obtained from a self pollination which segregated 3:1, indicating homozygosity for one of the factors and heterozygosity for the other. It is conceivable that a lack of all recessive factors could further lower the concentration of indole. At present the lack of near-isogenic stocks makes quantitative assessment of dosage effects difficult.
An interesting feature of this mutant is the orange pigmentation associated with the duplicate homozygous recessive kernels of a segregating ear. Because the pericarp is maternal tissue, a segregation for pericarp color could only be expected to result from direct interaction with the underlying filial tissue. Indole evidently plays a direct role in the formation of this color. Non-mutant kernels or isolated pericarps will turn orange in a few days if covered with an aqueous solution of indole. This reaction is non-enzymatic, as similar results were produced using heat-treated pericarps. Oxygen appears to be required, as the orange coloration develops first in pericarps nearest to the surface of the indole solution of non-shaken vessels. At least one of the orange compounds produced in this manner has properties in TLC (with three solvent systems) and UV visible spectra properties identical to an orange compound isolated from mutant pericarps. This fortunate circumstance has enabled the production and isolation by preparative TLC of a quantity of the compound sufficient for identification (in progress).
The substance which reacts with indole to produce the orange compound is not unique to the pericarp. The orange compound has also been isolated from the scutella of mutant immature embryos, which are normal in appearance but turn orange (presumably due to indole accumulation plus exposure to air) during callus culture attempts.
Culture attempts on tryptophan-free media have failed. A slow-growing
callus has been successfully initiated from the mutant on tryptophan-supplemented
media, which should provide useful material for further biochemical assessment.
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