5. Experiments on Gene Action in Anthocyanin Synthesis. In those genotypes which normally produce anthocyanin in the root, excised roots cultured on media containing glucose and mineral nutrients produce anthocyanin abundantly. Anthocyanin therefore may be synthesized by the cell from externally supplied glucose, without the intercession of other substances derived from the overground parts of the plant. The genes essential for root color in the dark are A (or Ab), A2, Pl, and a suitable R allele (Rch, rch, and some but not all Rr's and rr's). B is not essential and does not replace Rr.

It may be possible to learn something of the course of synthesis of anthocyanin, and of the role of various genes affecting it, by physiological experiments with excised tissues, testing the effects of postulated intermediates between glucose and anthocyanin, of specific enzyme inhibitors, of diffusible substances extracted from plants of contrasting genotype, etc.

Experiments with intermediates supplied in place of glucose cannot well be made with excised root-tip cultures, because the addition of some glucose or fructose is necessary to keep the roots growing. An intermediate would have to replace glucose in general metabolism as well as in anthocyanin synthesis to give positive results. Minimal quantities of sugar will maintain slow growth with little or no anthocyanin production, and experiments may be made with intermediates added to increase the anthocyanin yield.

A more satisfactory technique is to use sections of mesocotyl or leaf blade from young seedlings, since cell division is not a factor and since differentiated cells capable of anthocyanin production are present from the start. These sections remain alive for several days in buffer solutions, dilute, salt solutions, or pure water. In suitable genotypes, they fail to produce anthocyanin unless sugar is added, while with added glucose or fructose they produce anthocyanin abundantly. Although these sections may contain reserve carbohydrate which may be used in the synthesis of anthocyanin, they cannot complete the synthesis without something which they obtain from added glucose.

Leaf blades from mature plants also serve very well in rch stocks (with A b Pl), and quite well in Rch. Anthocyanin is produced poorly in mature leaf tissues with the best of the Rr and rr alelles tested, and not at all with some. Mature leaves are convenient material, especially for producing the quantities of pigment required for chemical analysis.

Several preliminary experiments of this type were performed this winter, and some of the results are summarized below.

Galactose, which does not support the growth of excised root tips, may be substituted for glucose in the production of anthocyanin in leaf or mesocotyl tissue. On the contrary, mannose, 1-sorbose, and 1-rhamnose give no anthocyanin.

The pentoses, xylose and lyxose, give a good yield of anthocyanin, while arabinose (both d- and l- forms) and ribose fail.

Some modifications of the C1 and C6 groups in the glucose molecule may be made without preventing the production of anthocyanin. Sorbitol and glueuronic acid yield anthocyanin; -methyl-glucoside and gluconic acid do not.

The trioses, glyceraldehyde and dihydroxyacetone, in phosphorylated form, are produced from glucose in the normal course of respiration. Either glyceraldehyde or dihydroxyacetone (unphosphorylated), supplied in place of glucose, will permit the production of some anthocyanin, more in the case of glyceraldehyde than of dihydroxyacetone.

Various specific enzyme inhibitors or poisons have been supplied over a range of concentration extending to the toxic limit, without producing a distinct reduction in the yield of anthocyanin from glucose. These include cyanide, azide, iodoacetate, fluoride, malonic acid, urethane and maleic acid. Certain other inhibitors show possible effects which are still under study. The only substance which in catalytic concentrations shows inhibition of the production of anthocyanin from glucose, in the trials made so far, is 2-4-dinitrophenol. This is a well-known stimulant of respiration and glycolysis, and may reduce anthocyanin synthesis competitively by diverting glucose to other channels. At concentrations of the order of 10-5 molar it inhibits anthocyanin production, and at lower concentrations it reduces materially the quantity of anthocyanin produced.

A possible hypothesis is that anthocyanin is produced by condensation of two phenol derivatives, related to phloroglucinol and catechol, with a 3C unit derived from glyceraldehyde. The effect of A would be a reduction in the 3C unit, which might occur either before or after the condensation. If the reduced 3C substance in A stocks were glyceraldehyde itself, it might be possible to produce anthocyanin in tissue lacking the A gene by supplying this substance. This was tried, unsuccessfully, with a, ap, Alt, and a2. Similar trials with dihydroxyacetone, glycerol, and hydroxypyruvic aldehyde (all of which produce some anthocyanin in A tissue) also failed. Experiments in this direction with various 3C substances are being continued, together with analogous experiments with catechol derivatives and 6C-3C compounds in relation to the Pr effect.

The experiments mentioned are of course merely exploratory trials, made chiefly to test the feasibility of the general approach and to determine which aspects, if any, have sufficient promise to justify more intensive study. Obviously, neither the positive nor the negative effects of specific substances upon anthocyanin production may be interpreted in terms of the place of these substances in biosynthesis, without careful study of their other physiological effects.

L. J. Stadler