150 PHOTOCONTROL OF GROWTH 



a cork borer or section cutter and floated on a GA solution. Generally 

 four discs per leaflet could be obtained, one disc being placed in each 

 of four petri dishes containing a different GA solution. In general, 

 application of GA to the entire plant produced the greater effects on 

 inhibitor level, though similar results were obtained with aU techniques. 

 Sample data are presented in Fig. 4. The results appear to permit the 

 conclusion that GA somehow raises the inhibitor level, perhaps by 

 itself being converted to inhibitor. 



CHEMICAL NATURE OF THE INHIBITOR 



Although we cannot yet present detailed information concerning 

 the chemi'cal nature of the inhibitor, considerable progress has been 

 made in the preparation and fractionation of active extracts. This 

 work has used as its starting point the leaflets of 14-day-old Alaska pea 

 seedlings grown under continuous light, at a temperature of 17°C. 

 Such leaflets, as mentioned previously, are relatively rich sources of 

 the inhibitor. 



The leaves are added to boiling water (1 g/10 ml), boiled vigor- 

 ously for 20 min, and the supernatant is then decanted and cooled. 

 The inhibitor activity is extremely stable, and such crude extracts have 

 been stored in the frozen state for several months. For assay purposes, 

 the inhibitor is added to a reaction mixture including inhibitor-free 

 enzyme prepared from the third internodes of 7-day-old etiolated pea 

 epicotyls, fortified with optimal concentrations (ca. 10~^M) MnCl- 

 and 2,4-dichlorophenol. lAA at 2 X 10"^ is added at zero time, and 

 aliquots removed for colorimetric assay at 15-min intervals. 



The first step in the purification involves adsorption onto Darco-G- 

 60 charcoal previously acid-washed and heated to 500 °C. Smafl 

 amounts of the active charcoal are added stepwise to the inhibitor solu- 

 tion until assay of the supernatant revealed essentially complete ad- 

 sorption of the inhibitor. The charcoal can then be washed with 30% 

 ethanol and water without loss of inhibitor, and the inhibitor finally 

 eluted with a mixture of 50% ethanol-O.OlM NH3. The elution solvent 

 is removed in vacuo, and the residue is taken up in water. Such a 

 purified aqueous solution can be satisfactorily stored in the frozen 



