38 SUBCELLULAR PARTICLES 



embryo and root tissues were employed. Thus, phenol added to peroxidase-HsOa 

 yields orange-brown, water-soluble products which finally become black, highly 

 insoluble substances; phenol supplied to slices of embryonic bean hypocotyl to- 

 gether with peroxide yields instead yellow, pink, and finally gray-brown products 

 progressing from the soluble to the highly insoluble condition. The product of 

 /7-toluidine peroxidation is deep red-orange when formed with the isolated en- 

 zyme, but yellow when formed in the slices themselves. Similar results were 

 obtained with a number of peroxidase substrates, including eugenol, i -hydroxy-, 

 2-methoxy-, 4-allyl-benzene, whose in vitro and in situ products differed prin- 

 cipally in the far lower solubility of the latter in ethanol and chloroform. 

 Eugenol and related hydroxyphenylpropane derivatives have long been re- 

 garded as similar to the fundamental building stones of lignin (4), and indeed, 

 have been isolated from native lignins by reductive degradation procedures. With 

 knowledge of the generally accepted relationship between eugenol and lignin, 

 application of the standard phloroglucinol-HCl test for the latter followed. This 

 test, which yields a red to magenta color with lignins, was positive for the 

 ethanol- and chloroform-insoluble peroxidation products of eugenol. 



Biochemical Characteristics of the Lignifying System. Examination of a 

 number of plant parts and species showed that the capacity for conversion 

 of eugenol to lignin was not limited, but internodes of the aquatic angiosperm, 

 Elodea, normally unlignified (through loss of lignifying ability, presumably), ex- 

 hibited an exceptionally high rate of lignin deposition. In the study of lignin 

 deposition in Elodea tissue, the Klason technique for isolation of the product 

 was employed; the procedure involved depends upon the insolubility of lignins 

 in strong ( 70-75 ^r) sulfuric acid and hot dilute mineral acids, both of which 

 are applied in the removal of cellulose and other wall and protoplast com- 

 ponents (24). 



Lignin is formed rapidly in moderately acid media, especially in the region of 

 pH 4.5. At pH 3 or below, and above pH 8.5, no lignin is produced (fig. i; 

 ref. 28). In the alkaline range, the autoxidation of eugenol competes with the 

 tissue lignifying system. 



Qualitatively, detectable amounts of lignin were formed from io-6o°C, al- 

 though in greatest yield in the region of 20-35 °C. 



No lignin is formed in the absence of peroxide, and in general the peroxide 

 requirement exceeds the eugenol supply. Using young pea root tips, another 

 active tissue in lignin formation, it was found that the yield of lignin increased 

 linearly with increasing peroxide until attainment of a ratio of about 2 moles 

 of peroxide per mole of precursor. Measurement of eugenol consumption con- 

 currently with peroxide disappearance yielded molar ratios of somewhat less 

 than 2:1. A possible objection to these approximations, that catalatic peroxide 

 decomposition may increase the calculated mole ratio, can be countered with the 



