20 



KNOWLEDGE. 



January, 1913. 



in the coloured parent race. This conclusion is confirmed by 

 the genetical behaviour of the whites of the F- 2 generation ; 

 such extracted whites breed true to flower-character, and give 

 rise to white-flowered offspring only. White-flowered races 

 which behave in this way are termed recessive whites. In the 

 second case, where the Fi generation consists of white-flowered 

 offspring, the Fa generation, from selfed or intercrossed Fi 

 plants, consists of three white to one coloured. The coloured 

 offspring breeds true ; of the three whites, one breeds true to 

 whiteness and the other two give rise, like the white Fi genera- 

 tion, to three white : one coloured. White races which thus 

 impose their whiteness on the offspring of their union with a 

 coloured race are known as dominant whites. Mendelians 

 account for the genetical behaviour of dominant whites by 

 assuming that they carry the character for colour, and also a 

 character for colour-inhibition. This hypothesis, though novel 

 to Biology, is amply justified by genetical results, and it pro- 

 pounds a series of questions to the physiologist and biochemist. 



Until recently, knowledge of the processes of pigmentation 

 has advanced along two main and independent lines: — (1) that 

 followed by students of genetics, which has led to a wealth of 

 exact knowledge concerning the factors and characters which 

 determine coloration ; (2) that pursued by biochemists, which 

 has resulted in a great increase of our understanding of the 

 biochemistry of pigmentation. The first to combine the 

 genetical with the biochemical method was Miss Wheldale, to 

 whom we owe a good working theory of the nature of the 

 processes involved in pigment-formation. 



Palladin has shown that respiration consists of a sequence 

 of enzyme-like actions, the later of which result in oxidation 

 and are ascribed to the enzymes (ferments) called oxydases ; 

 that chromogens play a part in the oxidations set up by 

 oxydases, and that these colourless chromogens may undergo 

 either alternate oxidation and reduction and so take a contin- 

 uous part in oxydase action, or undergo permanent oxidation 

 and so constitute the pigments of the plant. Chodat and Bach 

 have suggested that oxydases are of dual nature, the complete 

 oxydase consisting of two parts — a peroxydase and an organic 

 peroxide. An oxydase reacts with oxidisable reagents, such 

 as guiacum, to produce a characteristically coloured product ; 

 hence these reagents may be termed oxydase-reagents. 

 Peroxydases react with oxydase reagents only if there be 

 added, as a substitute for the organic peroxide of the complete 

 oxydase, a source of active oxygen in the form of hydrogen 

 peroxide. Both oxydases and peroxydases occur in the cells 

 of plants, and may be identified in extracts therefrom. 

 Gortner's work on the pigments of insects confirms the view 

 that pigments are the product of the action of oxydase on 

 chromogens ; he has shown that the black or brown melanin 

 of the integuments of insects is produced by the action of an 

 oxydase called tyrosinase upon some such product of protein- 

 hydrolysis as ty rosin. 



Miss Wheldale's theory is that the anthocyan pigments of 

 plants are the outcome of a series of chemical changes of the 

 following order : — Glucosides on being hydrolysed by the 

 ferment emulsin yield chromogens which, acted upon by oxy- 

 dases, give rise to anthocyan pigments. The difficulty in the 

 way of further advance lay in the unsatisfactory nature of the 

 methods for identifying oxydases derived from plant tissues. 

 When Professor Keeble and Dr. E. F. Armstrong began their 

 work on this subject they found, after trials of various known 

 reagents, that a naphthol and benzidine are each suited 

 admirably for the purpose of locating oxydases, and by means 

 of these reagents they have been able to map out the distri- 

 bution of oxydase and peroxydase in the flowers and other 

 parts of various plants. Their results confirm Miss Wheldale's 

 hypothesis of the mode of formation of anthocyan pigments ; 

 but this confirmation was made possible only by reason of 

 the fact that they worked with races of plants bred on 

 Mendelian lines and therefore of known genetic constitutions. 

 On treating coloured flowers of Primula sinensis with 

 each of the two reagents, it is found that the actions of 

 a naphthol and benzidine are in considerable measure supple- 

 mentary one of the other. Thus, the lilac-blue a naphthol 

 reaction is confined to the veins of the corolla ; the brown 



benzidine reaction is shown by the superficial (epidermal) cells 

 and also by the veins. The peroxydases are therefore termed 

 epidermal peroxydase and bundle oxydase, the former occur- 

 ring in the epidermis and hairs, the latter in the bundle-sheath 

 which accompanies the veins. Similarly, the stem of P. sinensis 

 contains a superficial peroxydase and a deep-seated peroxy- 

 dase. The distribution of peroxydase coincides broadly with 

 the distribution of pigment ; that is, the peroxydase framework 

 for pigmentation occurs throughout the species, and the 

 building of the several colour varieties is determined by the 

 activity of the factor for chromogen production, and if we 

 conceive of this factor as administered in a series of doses 

 we have a picture of the mode of evolution of the series of 

 varieties characterised by increasing or decreasing amount of 

 pigmentation of their parts. 



The application of these reagents to recessive white races 

 shows that these white-flowered races, though lacking the 

 factor for colour, contain in the flower both epidermal and 

 bundle peroxydase, as might be expected from analogy with 

 the peroxydases of the stem. 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, and 

 this conclusion conforms with that arrived at previously by 

 Mendelian methods, which show that anthocyan pigmentation 

 of the flower of P. sinensis depends on the presence of one 

 factor only, and that the absence of pigmentation characteris- 

 tic of recessive whites is due to the absence of that single 

 colour-factor. 



The investigation of the peroxydases of dominant white 

 flowers gives a very different result ; for these show no sign of 

 peroxydase either in epidermis or in bundles. Hence such 

 flowers either lack peroxydase or else they contain a substance 

 which inhibits peroxydase from exercising its oxidising action 

 on the oxydase reagents. It is known that the addition of 

 certain phenolic compounds (orcin, resorcin, and so on) prevents 

 tyrosinase from exercising its characteristic action upon 

 tyrosin. Assuming that an inhibitor of peroxydase exists in 

 dominant white flowers, it may act either by destroying the 

 peroxydase or by setting up conditions under which the 

 activity of peroxydase is arrested ; and if the latter is the 

 mode of action, it follows that if by some means the inhibitor 

 can be removed, the peroxydase will be free to effect the 

 oxidation of the reagents used. This train of reasoning led 

 Keeble and Armstrong to the discovery that hydrogen cyanide 

 forms a means of removing peroxydase inhibition ; if dominant 

 white flowers are immersed in a dilute solution of hydrogen 

 cyanide and then treated with either of the two oxydase 

 reagents together with hydrogen peroxide, pronounced peroxy- 

 dase reactions are obtained both in the epidermal and bundle 

 tissues of the corolla. 



To test this hypothesis further, a race of Primulas was 

 used in which the flowers are blue with white patches on each 

 petal, the known ancestry of this race indicating the proba- 

 bility that the white patches are produced by a localised 

 inhibitor. On treating corollas of these flowers with the two 

 oxydase reagents and then with hydrogen peroxide, the parts 

 originally blue are stained lilac-blue or brown according to 

 the reagent used, and the inhibitory patches stand out as in 

 the intact flower as white areas on the coloured ground. If, 

 however, these parti-coloured flowers are treated first with 

 hydrogen cyanide, then with the reagent and subsequently 

 with hydrogen peroxide, the peroxydase reaction is produced 

 over blue and white areas alike — the inhibition located in the 

 white areas has been removed. Hence the Mendelian 

 hypothesis of the inhibitory nature of dominant whites is 

 confirmed by biochemical methods ; these methods, moreover, 

 prove that the inhibitor acts not by destroying but by 

 preventing the action of oxydase upon the chromogen. 



This fruitful line of investigation has given various other 

 results and has raised many questions for further research. 

 For instance, the close proximity in the flower of the 

 superficial and deep-seated oxydases suggests that the latter 

 may cooperate with the former in producing flower-pigments. 

 This possibility entails the hypothesis of a translocation of 

 oxydase from the region in which it is secreted to that in 



