HOMBRES DESPIERTOS

Another problem during extraction and purification of IAA is spontaneous oxidation of IPyA to IAA, which can

introduce error into determination of IAA in plant tissues. Conversion was shown to occur during paper chromatography

(Bentley e_t aT 1956) and IAA extraction procedure (Atsumi ej: al 1976).

Polyamide TLC was highly effective in separating IAA from IPyA. Two y g IPyA was developed using polyamide TLC with solvent system I (Table 1), the IAA zone was eluted, and the IAA in it was determined by the fluorescence method

[BHT was added to the IPyA sample (8 m g ), to the developing solvent (100 mg/1), and to the eluting solvent (1.5/ml)]. The IAA amounts determined were 4 to 6 ng, which represents only 0.4% of the IPyA amount on a molar basis. Separation of IAA and IPyA on polyamide TLC is also adequate: the Rf values of IAA and IPyA are 0.33 and 0.12, respectively. It can be concluded that there is no IPyA in the IAA fraction

after the TLC step.

Analyses of the operations commonly involved in IAA extraction procedure showed that (a) the conversion is greater in aqueous solution that in organic solvents, (b)

IPyA is more stable in phosphate buffer than in bicarbonate buffer, (c) pH of the buffer solution (pH 2.5-8.5) has no

significant effect on the conversion. In these experiments,

IAA was determined by the fluorescence method following

polyamide TLC. The conversion of IPyA to IAA in phosphate

buffer could be reduced further by lowering the temperature

of the solution and adding BHT (Table 7-B, C ) . The

conversion was at its lowest when IPyA was extracted with minimal delay from the phosphate buffer by ether (Table 7- D), indicating that acidification of the aqueous solution and evaporation of the ether solution to dryness (the ether was water-washed, BHT-supplemented, and passed through

cotton) caused essentially no conversion. These results

show that the conversion of IPyA to IAA during the

extraction procedure can be drastically reduced by lowering the temperature, adding BHT, and shortening the duration of extraction.

5. Proposed Scheme for IAA Estimation in Plant Tissues and

Application to Maize Seedling Shoots.

Based on the findings described above, a scheme for the estimation of IAA in plant tissues is proposed (Fig. 7). Essential features of the scheme include: (a) minimizing the period of extraction of ground tissues with aqueous acetone - this period must be optimized for each plant material

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(b) application of the aqueous extract directly to a PVP column without prior concentration - further elution is carried out with 0.1 M K2HPO4 at pH 8.0 (practically all of a sample of [14(3] IAA was recovered in a 10 to 65 ml

fraction); (c) partitioning the acidified aqueous solution three times against a 1/3 volume of ether (recovery of

[14c]IAA more than 99.5%) - partitioning with petroleum spirit prior to this partitioning (Kamisaka and Larsen 1977, Knegt and Bruinsma 1973) is not included; (d) addition of BHT and pretreatments of solvents (Figure 7); (e) carrying out all manipulations under subdued light.

IAA in etiolated shoots of maize was estimated using

the proposed scheme. As shown in Table 8, 18 ng/g fresh

weight of IAA was obtained. Recovery of radioactivity from

[14c]IAA added at the beginning of the extraction was over 90%.

Although radioactive IAA is the internal standard most commonly used to correct for losses (Kengt and Bruinsma 1973, Little e_t al 1978 , Sweetser and Swarzfager 1978), it should be recognized that the radioactivity recovered may not totally represent the authentic compound originally

added (see McDougall and Hillman 1978). Reliability of the

proposed scheme in correcting losses of IAA has been

assessed by the following experiment. About 20 ng

[14c]IAA was processed through the entire procedure in a sham extraction, and the recovery was determined by both radioactivity and fluorimetric assays; the recovery

determined based on either parameter was exactly the same. One possibility which remains is that, in the presence of

actual plant extracts, radioactive derivatives of labelled IAA which are co-purified with IAA may be produced.

To achieve a more realistic appraisal of the losses and estimation of IAA, the following suggestions are made: (a) as an internal standard, [1^C] IAA seems to be the most suitable radioactive form because evolved upon

decarboxylation, one of the possible degradative processes, will not remain in the residues; (b) assays of IAA should be made as soon as possible following the extraction - when storage of samples is inevitable, it is advisable to store them as assay aliquots with added authentic carrier IAA

because nonlinearity of the calibration line provides a good indication of breakdown of IAA during the storage period; (c) all aliquots used for the fluorimetric assay and

scintillation counting must be taken simultaneously from the sample, freshly extracted and dissolved only once in

methanol, because each further evaporation to dryness and dissolution may cause decomposition of IAA.

It has been suggested by Mousdale et a_l ( 1978) that the background values of fluorescence should be subtracted from that of the reacting solution after complete destruction of indolo-a-pyrone by light. With the extracts here, there was no significant difference between background values obtained by adding 90% acetic acid before the addition of the mixture of acetic anhydrous and TFAA or after photo-decomposition of

indolo-a-pyrone (Table 9 ). Nor were there significant increases in background level in extracts (in Table 9,

compare Blanks I and II). Although etiolated maize shoot is said to be one of the most difficult materials to work with

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owing to reaction-inhibiting impurities in the extracts (Mousdale et _al 1978), the inhibition of fluorescence was less than 20%. Thus, IAA samples obtained by the proposed scheme are effectively free of contaminants affecting

fluorimetric assay.

The steps in the extraction procedure (Fig. 7) are

designed to obtain high recovery of IAA but also to minimize the conversion of IPyA to IAA. However, 8.2 + 0.2% (mean of three replicates with SE) of IPyA was still converted to IAA when 2 yg IPyA was processed through the entire procedure. This degree of conversion seems unavoidable. Atsumi ejt jal

(1976) have reported a new liquid partitioning system for IAA (water-Cl2CH2 system), which theoretically can

remove 82% of IPyA, and recover 91% of IAA. Using this system, IPyA not already converted to IAA before and during the partitioning will be effectively removed.

The aim here was to establish a reliable method of IAA extraction and determination which can be easily utilized for physiological experiments. As it stands, the improved indolo-a-pyrone fluorescence method coupled with the

improved extraction method is particularly suitable for such experiments. Minute amounts of IAA in plant extracts (as little as 1 ng) can be determined, and the extraction procedure can be scaled according to the amount of plant material, which can be as little as 0.1 g fresh weight in the case of maize shoots; the entire procedure from

homogenization to the determination of IAA can be completed within 10 h, and several samples can be processed