Metabolism and mode of action 



(c) The cells may acquire the capacity for lignin synthesis, probably by 



virtue of the induced formation of a peroxidase involved in the 



conversion of hydroxyphenylpropane-type precursors into lignin. 



I propose during the remainder of my paper to discuss in some detail each 



of the above-mentioned consequences of lAA-administration to plant cells, 



THE EXPERIMENTAL COUPLING OF lAA TO PROTEIN 



Several years ago, Dr. S. M. Siegel and I were examining the proteins of pea 

 root apices which had been exposed for several hours to extremely high 

 (10-3 M) concentrations of lAA (Siegel and Galston, 1953). We noticed 

 that the trichloroacetic-acid (TCA) precipitates of the brei of roots previously 

 exposed to lAA had a faint but distinctly pink colour when compared with 

 that of roots incubated in buffer alone as a control. After demonstrating to 

 ourselves that TCA applied to lAA on filter paper gave a pink colour, we 

 naturally suspected that the colour of the protein might be a consequence 

 of lAA bound to it. This conclusion was borne out by the application of the 

 Salkowski reagent (Tang and Bonner, 1947) which gives a characteristic 

 pink colour with lAA and some related compounds. This reagent turned the 

 protein a bright pink to red. Unlike free lAA, which turns pink gradually 

 and then fades when treated with Salkowski reagent, the lAA-protein 

 became coloured quickly and retained the same colour intensity for at least 

 several days under refrigeration. This fact permitted the quantitative 

 estimation of the lAA on the protein. 



The major conclusions which Dr. Siegel and I derived from our study 

 (Siegel and Galston, 1953) of this lAA protein formed in pea root apices are: 



(a) lAA placed in the medium may be detected in the tissues in the free 

 form within 2-3 minutes. Significant quantities of protein-bound lAA may 

 be noted within 5-10 minutes, the amount of the material increasing 

 rapidly for 30 minutes and less rapidly for several hours after that {Figure 1). 



{b) The higher the concentration of lAA supplied, the greater is the 

 amount of lAA bound to protein. Under the most favourable conditions, 

 however, only 10 per cent as much lAA is coupled to protein as is destroyed 

 by the oxidase. Assuming a molecular weight for the protein of 100,000, 

 then it appears that of the order of 0-1 to 1-0 mole of lAA is bound per mole 

 of protein. 



[c) The lAA-protein appears to be localized in the 'non-particulate' 

 phase of the cytoplasm. 



{d) The binding of I AA to protein occurs only under conditions of active 

 aerobic respiration linked to the synthesis of energy-rich compounds such 

 as ATP. It is prevented by oxygen deprivation, or by the inhibitors cyanide, 

 azide, iodacetate, and 2 :4-dinitrophenol {Figure 2). 



{e) Prior treatment of the roots with some other auxin, such as 2:4- 

 dichlorophenoxyacetic acid or a-naphthalene-acetic acid, interferes with lAA 

 binding to the protein. The greater the concentration of analogue, the less 

 lAA is bound {Figure 3). This implies some general affinity of the protein for 

 auxin-type molecules. 



(/) In vitro, lAA may be coupled to the proteins in an acetone-powder of 

 pea roots with the aid of ATP. Coenzyme A, which we expected might aid 



220 



