Although tryptophan is generally believed to be the endogenous precursor of IAA in higher plants (see e.g. Schneider and Wightman 1978), it has been regarded by some as not the precursor in etiolated coleoptiles (Winter 1966, Thimann and Grochowska 1968, Bandurski 1980). Nevertheless, incorporation of radioactivity into IAA from isotopically
/ V
Black and Hamilton 1971, Erdmann and Schiewer 1971, and Heerkloss and Libbert 1976). Muir and Chang (1974) were able to increase the yield of diffusible IAA from the
coleoptile tip by applying tryptophan. Tryptophan is likely to be at least one of the IAA precursors in the coleoptile.
Incorporation of tritium from [^h]tryptophan into IAA was first investigated. Excised coleoptile tips were
incubated for a few hours in the medium containing
[^H]tryptophan, and an ether extract was obtained from the incubation medium (for details, see legend to Fig. 6). The extract was developed on polyamide TLC, and radioactivity on the plate was scanned. As shown in Fig 6-A, only one peak was found, its Rf value corresponding to that of the
authentic IAA marker. No such peak was found in the ether extract obtained from the medium alone, minus incubating tissue (Fig. 6-B). Radioactivity in the blanks measured by scintillation counting (see Materials and Method) was not detected by the scanner. The ether extract, methylated by diazomethane, was developed on TLC. A peak was again found at the position of the authentic Me-IAA marker (Fig. 7). The results support the view that the radioactive peak on TLC is due to [^H]IAA, produced from [^H]tryptophan.
At the position of tryptophan on the TLC plate (the origin), there was no apparent peak of radioactivity
(Fig. 6). Thus the extraction procedure employed is very effective in removing most of the [^h]tryptophan present
in the incubation medium, and after the ether extraction, arrests its auto-decomposition to produce [^H]IAA or other radioactive compounds which might interfere with measurement
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(see Materials and Methods).
The ether extract, developed on TLC, contains acidic and neutral compounds, but the only radioactive peak found was at the position of IAA. This probably means that, of the many metabolites produced from tryptophan, including IAA intermediates, IAA is selectively exported from the tissue.
The rates of [^HjlAA production during a few hours incubation were examined. There was some incorporation of tritium into IAA in the first 30 min, and the rate increased with time (Fig. 8). This result is taken as evidence that the [^H]IAA produced was not due to contaminating
bacteria. If it were, the rate of [^H]IAA production
would show no such increase in the course of the renewal of the medium, which contained streptomycin. The most probable explanation for the increase in the rate of production is the tissue content of [^h]tryptophan, which is expected to increase by continuous uptake.
The time course of red light effects on the formation of [^H]IAA from [^H]tryptophan was then examined.
Intact seedlings were irradiated with red light, in the usual manner, for 10 min. After scheduled dark intervals, coleoptile tips were excised and incubated with
[^H]tryptophan. After 30 min the medium was replaced with a fresh aliquot, and the tips were incubated for a further 1 h. [^H]IAA present in the second incubation medium was determined. Effects due to excision of the coleoptile tip on the IAA production are considered to be minimal within this incubation period, since the initial rate of IAA
excision (lino and Carr 1982, Chapter 4; see also the dark
control in Fig. 5). As shown in Fig. 9-A, red light reduced
the yield of [^h]IAA, with a time course very similar to
that obtained for diffusible IAA (Fig. 2 -A).
In the present studies, the [^HjlAA was recovered
from the incubation medium, not from the tissue. From the
determination of diffusible and free IAA (Figs. 2-A and 3-B), it can be concluded that, following exposure to red light, the level of diffusible IAA parallels that of free
IAA. Moreover, the content of free IAA in the coleoptile
tip is low (0.2 ng per/3 mm-long unirradiated tip) compared to the yield of diffusible IAA (1.0 ng/tip/h) (lino and Carr 1982, Chapter 4; see also Fig. 2-A and Fig. 5 for diffusible
IAA). The rate of [^H]IAA diffusion probably reflects
very closely the rate of [3h]IAA production in the tissue.
It should also be noted that the radioactivity of [^H]IAA diffusing out of an unirradiated coleoptile tip is about 200 dpm, corresponding to only 5.6 pg of [^H]IAA (calculated
from the specific activity of the [^H]tryptophan used). The assumption is made that the exogenously supplied
[^h]tryptophan does not, e.g. by increasing the pool size
of tryptophan, alter the status quo of IAA production. The possibility that the decrease in [^H]IAA
formation might be caused by inhibition of [^h]tryptophan
uptake by the tissue is negated by the fact that
radioactivity in the coleoptile tips, collected at the end of the incubation period was considerably increased by red
light (Fig. 9-B). Thus red light can affect uptake of
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These results confirm that red light acts to reduce the yield of diffusible IAA, or the level of free IAA, by changing the metabolism of IAA. Although stimulation or induction of IAA decomposition cannot be totally excluded from the possibilities, the most likely metabolic process on which red light acts is the synthesis of IAA, as already discussed (see Red light and endogenous IA A ) . It should also be noted that the results obtained here support the view that tryptophan is a physiologically important
precursor of IAA in the maize coleoptile tip.