i8o 



NATURE 



[October io, 19 12 



that chromogens play a part in the oxidations set up 

 bv oxydases, and that these colourless chromogens 

 may undergo either alternate oxidation and reduction 

 and so take a continuous part in oxydase action, or 

 undergo permanent oxidation and so constitute the 

 pigments of the plant. 



("hodat and Bach have given us a serviceable con- 

 ception of the nature of oxydases. According to the 

 C'hodat-Bach hypothesis, oxydases are of dual nature; 

 the- complete oxydase consisting of two parts — a per- 

 oxvdase and an organic peroxide. .\n oxydase reacts 

 with oxidisable reagents, such as guaiacum, to pro- 

 duce a characteristically coloured product. Hence these 

 reagents may be termed oxydase-reagents. Peroxy- 

 dases react with oxydase reagents only if there be 

 added, as a substitute for the organic peroxide of the 

 complete oxydase, a source of active oxj'gen in the 

 form of hydrogen peroxide. Both oxydases and per- 

 oxvdases occur in the cells of plants, and may be 

 identified in extracts therefrom. 



The work of Gortner on the pigments of insects 

 adds confirmation to the view that pigments are the 

 product of the action of oxydase on chromogens. 

 Thus he has shown that the black or brown melanin 

 of the integuments of insects is produced by the action 

 of an oxydase, tyrosinase, on some such product of 

 protein-hydrolysis as tyrosin. 



Miss VVheldale's studies have led her to formulate 

 the hypothesis that the anthocyan pigments of plants 

 are the outcome of a series of chemical changes of the 

 following order : Glucosides hydrolysed by emulsin 

 vield chromogens which, acted on by oxydases, give 

 rise to anthocyan pigments. The difficulty in the way 

 of further advance lay in the unsatisfactory nature of 

 the methods for identifying o.xydases derived from 

 plant tissues. Hence when we turned our attention to 

 this subject Dr. E. F. Armstrong and I made it our 

 first task to search for means whereby we should 

 be able not only to identify, but also to locate, o.xy- 

 dases and peroxydases in plant-tissues. Clarke had 

 tested already numerous oxydase-reagents and found 

 that certain among them are adapted for micro- 

 chemical use. As the result of a considerable number 

 of trials of known reagents we have found that 

 a-naphthol and benzidine are each adapted admirably 

 for the purpose of locating oxydases. By means of 

 these reagents we have been able to map out the 

 distribution of oxydase and peroxydase in the flowers 

 and other parts of various plants, and although the 

 work is laborious and the technique as yet imperfect, 

 the results afford strong confirmation of the current 

 hypothesis of the mode of formation of anthocyan pig- 

 ments. This confirmation, however, was rendered pos- 

 sible only by reason of the fact that we worked with 

 races of plants bred on Mendelian lines, and hence of 

 known genetic constitutions. 



Our method of investigation is briefly as follows. 

 The oxydase-reagent is used in weak alcoholic solu- 

 tion, the part of the plant to be tested is incubated 

 in the solution for a suitable time, and if no oxvdase 

 action takes place — that is, if no characteristic colora- 

 tion of the tissues occur.s — the material is tested for 

 peroxydase bv the addition of hydrogen peroxide. The 

 method may be employed for intact corollas or petals 

 or for sections of plant-tissues. 



It is important to mention that the first result of 

 immersing a sap-pigmented tissue in either reagent is 

 the decolorisation of the tissue. For example, a 

 corolla of a coloured-flowered race of Primula sinensis 

 loses its colour completely after beinsr immersed for 

 .an hour or tw'o in either reagent. The decolorised 

 corolla, which in the case of P. sinensis remains 

 colourless, is treated with hydrogen peroxide, with the 

 result that a well-marked peroxvdase reaction is ob- 



tained. The reaction is confined to the non-chloro- 

 phyllous parts of the corolla, and does not occur, 

 except in the epidermal hairs, in the region of the 

 yellow or green eye, the tissues of which contain 

 chlorophyll. Indeed, there is good reason to believe 

 that chlorophyll inhibits oxydase action. 



By treating similar flowers with each of our two 

 reagents we find that the action of a-naphthol and 

 benzidine are, in a considerable measure, supplemen- 

 tary one of the other. Thus the lilac-blue a-naphthol 

 reaction is confined, or alinost confined, to the veins 

 of the corolla, the brown benzidine reaction is ex- 

 hibited by the superficial (epidermal) cells and also by 

 the veins. In order to emphasise the facts of distri- 

 bution we speak of the peroxydases of P. sinensis 

 as epidermal peroxydase and bundle o-xydase. The 

 former occurs in the epidermis and in the epidermal 

 hairs, the latter in the bundle sheath which accom- 

 panies the veins. 



Similarly, if sections of a stem of P. sinensis be 

 investigated they are found to contain a superficial 

 peroxydase and a deep-seated peroxydase. As the 

 result of investigating the peroxydases, not of any un- 

 known varietv taken at hazard, but of the several 

 varieties characterised by constant differences of depth 

 and extent of pigmentation, we have been able to 

 show that the distribution of peroxydase in any one 

 race coincides broadly with the distribution of pig- 

 ment in the most pigmented races. In other words, 

 in P. sinensis the peroxydase framework for pigment- 

 ation occurs throughout the species, and the building 

 of the several colour varieties is determined by the 

 activity of the factor for chromogen production. If 

 we conceive of this factor as administered in a series 

 of doses we can form a fair picture of the mode of 

 evolution of the series of varieties characterised by 

 increasing or decreasing amount of pigmentation of 

 their vegetative parts. 



On turning to investigate the pero.xydases in white- 

 flowered races of P. sinensis, we shall expect to find 

 from analogy with the peroxydases of the stem that 

 these agents of pigment-formation are not lacking 

 from the corollas of recessive whites. The application 

 of our reagents shows that this expectation is correct, 

 and that those white-flowered races which lack the 

 factor for colour contain epidermal and bundle per- 

 oxydase. Hence we conclude that the absence of 

 colour from recessive white flowers is due, not to the 

 absence of peroxydase, but to absence of chromogen. 

 This conclusion is in conformity with that arrived at 

 previouslv bv Mendelian methods ; for, as we have 

 noted alreadv, these methods demonstrate that antho- 

 cvan pigmentation of the flower of P. sinensis depends 

 on the presence of one factor only, and that the 

 absence of pigmentation which is characteristic of 

 recessive whites is due to the absence of that single 

 colour-factor. 



The result of our investigation of the peroxydases 

 of dominant white flowers is, on the other hand, quite 

 different from that given by recessive whites. When 

 corollas of dominant white races are treated with 

 o-naphthol or benzidine and subsequently with 

 hydrogen peroxide, they show no sign of oeroxydase 

 neither in epidermis nor in bundles. Hence such 

 flowers either lack oeroxydase or else they contain a 

 substance which inhibits peroxvdase from exercising 

 its oxidising action on our oxvdase-indicators. 



That oxvdases mav be inhibited in vitro has been 

 demonstrated alreadv bv Gortner. who has show-n that 

 the addition of certain phenolic compounds — orcin, 

 resorcin, ilc, prevents tyrosinase from exercising its 

 chnraclerislic action upon tyrosin. 



Assuming that an inhibitor of pero.xydase occurs in 

 dominant white flowers, it may be supposed to act 



NO. 



'241, VOL. qo] 



