Mutations in genes associated with the pathway of flavonoid biosynthesis can readily be induced in barley and since 1977 more than 700 mutants have been isolated in which the biosynthesis of anthocyanin and/or proanthocyanidin is affected. Diallelic crosses have localized a majority of the mutants to 28 different complementation groups (Ant genes). Mutations in ten of the Ant genes result in a blocked synthesis of proanthocyanidins in the testa layer of the grains, whereas mutations in 18 Ant gene loci only affect anthocyanin synthesis in various organs of the plants. Seven Ant genes have been localized to a chromosome. A number of investigations have been carried out to characterize the ten known Ant genes acting on the biosynthesis of proanthocyanidins. Grains of mutants in different Ant genes and wildtypes were analyzed for contents of flavonoids and other phenolic compounds and the activity levels of several of the enzymes in the flavonoid pathway were studied. The gene locus Ant 18 has been isolated both in the form of cDNA and genomic clones. Ant 17, Ant 18, Ant 19, Ant 22, and Ant 26 are structural genes. Ant 13 is a regulatory gene coding for a transcription factor necessary for transcription of at least three structural genes in the flavonoid pathway. The action of the genes Ant 21, Ant 25, Ant 27, and Ant 28 is not fully understood. They might be regulatory genes.

Mutations in genes associated with the pathway of flavonoid biosynthesis can be easily induced in barley, and more than 600 mutants have been isolated in which the biosynthesis of proanthocyanidins is genetically blocked. Diallelic crosses have localized a majority of the proanthocyanidin-free mutants to nine different ant genes. In order to characterize the ant gene assays for flavanone 3-hydroxylase, flavonoid 3'-hydroxylase and dihydroflavonol reductase activities in protein extracts from developing grains of proanthocyanidin-free mutants in six ant genes and wild-type barley were performed. The presence of chalcone synthase enzyme protein in wild-type barley and the mutant ant 13-152 was investigated by SDS-PAGE, protein blotting, and reaction with an antiserum against parsley chalcone synthase. The results of these experiments combined with earlier studies of flavonoid content in barley grains of different genotypes characterize the functions of six ant genes as follows. The gene ant 13 is a regulatory gene affecting the transcription of structural genes in the flavonoid pathway. The genes ant 17 and ant 22 are probably coding for different components necessary for hydroxylation of flavanones in 3-position. The gene ant 18 is the structural gene coding for dihydroflavonol reductase, and ant 19 is most likely the structural gene coding for leucoanthocyanidin reductase. Ant 21 is probably a regulatory gene affecting the biosynthesis of proanthocyanidins and anthocyanins.

The effects of proanthocyanidins (PA), dehulling, and removal of the pericarp on the rate and extent of barley digestion by ruminal micro‐organisms were studied using a control barley (cv Harrington) and three PA‐free barley lines (Caminant, Ca504316 and Ca802711). Each barley was studied in five preparations: whole grain (W); dehulled kernels (DH); kernels with pericarp removed (DP); dry‐rolled grain (DR), and the pericarp‐testa fraction (PT) produced during preparation of DP. Vanillin‐HCl staining and chemical analysis confirmed that PA were present only in the Harrington barley, and localised in the pericarp‐testa layer. Whole kernels, DH and DP were incubated with diluted ruminal fluid in vitro, and all five preparations were incubated in situ (nylon bag technique). Harrington DR and all four PT fractions were also incubated in vitro in the presence (0.074% w/v) and absence of polyethylene glycol (PEG), which specifically binds PA. The four barleys did not differ in in vitro dry matter disappearance (DMD) or gas production from W, DH or DP preparations, nor in in situ DMD rates from W, DH or DP (P > 0.05). In vitro DMD and gas production among PT fractions from the four barleys were also similar (P > 0.05), as were in vitro DMD from PEG‐present and PEG‐absent DR Harrington. With Harrington PT, in vitro ammonia concentrations were higher (P < 0.05) with PEG than without. For each barley line, in vitro DMD rates were highest (P < 0.01) with DP, followed by DH, and then by W (P < 0.01). In in situ incubations also, DMD rates and effective degradabilities of DR samples exceeded (P < 0.05) those of DH samples. It was concluded that the presence of proanthocyanidins did not affect ruminal digestion of barley grain, and that abrasive milling to breach the hull and pericarp may be a promising method by which to regulate the rate of barley digestion in the rumen. © Minister of Public Works and Government Services Canada 1999

The proanthocyanidin composition of wild-type barley was analyzed by high-performance liquid chromatography. The use of authentic standards allowed tentative identification of procyanidin B-3, prodelphinidin B-3, and catechin as the major constituents. The proanthocyanidin content of different barley varieties (wild types) was found to be qualitatively and quantitatively similar. Mutants belonging to different gene loci and deficient in the biosynthesis of proanthocyanidins and other flavonoids were characterized.

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