OSTERHOUT: THE NUCLEUS AS A CENTER OF OXIDATION 345 



The darkening which occurs is due in part to free oxygen left in the 

 leaf and in part to oxygen in compounds from which it can be spHt 

 off for the oxidation of the chromogen (analogous to anaerobic 

 respiration). 



2. If leaves are torn in two or crushed at once, dropped into boiling 

 water, o.i M HCl, o.i M NaOH, o.i M KCN, or 3 percent H2O2 the 

 darkening does not occur. These agents are inhibitors of oxidation in 

 living tissues. Hydrogen peroxide may inhibit at high concentration, 

 but accelerate at low concentrations. In NaOH and KCN the leaf 

 becomes pale yellow: this seems to be due to the action of hydroxyl 

 ions. 



3. The chromogen may be extracted by placing stems in 0.1 M 

 NaOH in a bottle completely filled (so as to exclude air) and tightly 

 stoppered (with a glass stopper coated with vaseline). The solution 

 becomes pale yellow (or slightly reddish) and may be kept in this 

 condition for months. On opening the bottle and pouring out the 

 solution into a shallow dish it at once becomes red as the result of 

 oxidation. The behavior seems to be analogous to that of pyrogallol, 

 which is easily oxidized by the air in alkaline solution, but not in 

 neutral solution except under the influence of oxidases (from plants or 

 animals) or other catalyzers. 



That the darkening of the nucleus is due to oxidation taking place 

 in the nucleus itself and not to the taking up by the nucleus of a stain 

 produced in the cytoplasm or vacuoles is shown by the following 

 experiment. Plants were ground in a mortar and allowed to stand 

 until they became black. The juice was squeezed out and centrifuged, 

 giving an inky fluid. In this were placed pieces of leaves which had 

 been treated with 0.1 KCN and afterward with water. The solution 

 was allowed to stand until it became concentrated by evaporation : 

 it then appeared black. It was found that where the nuclei had been 

 squeezed out of the cut cells by the knife they had taken up some stain 

 but not more than the cytoplasm. In cells which were merely cut 

 open there was little or no staining of the nucleus. 



We must therefore conclude that oxidation occurs more rapidly in 

 the nucleus than elsewhere in the cell. The only way to escape this 

 conclusion would be by assuming that at the moment of injur}' there 

 is a sudden migration into the nucleus of some or all of the substances 

 necessary for the oxidation. This is not only very improbable from a 

 theoretical standpoint, but observation shows that it can not be the 

 case, for in this migration the substances would mingle and produce 

 the pigment cither outside the nucleus or at its surface before any 

 pigment appeared in the interior of the nucleus. Observation of the 

 nucleus shows that the pigment appears as soon in the interior of the 

 nucleus as at its surface. 



