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Biochemical genetics of the anthocyanins of barley (Hordeum vulgare L.) Mullick, Dharam Bir

Abstract

Patterns of flavonoid and melanic pigmentation were studied by visual and qualitative chemical means in 20 varieties of cultivated barley. Two new techniques, one manual and one chemical, were developed which made possible the study of the pigments in the separate tissues of the caryopsis. The observation that anthocyanins are localized in the spermoderm and not in pericarp is an example of the usefulness of the techniques. Environmental modifications of pigmentation patterns in varieties and in individual plants are large and biochemical and genetical analyses of patterns are thereby greatly complicated. For example, pelargonidin derivatives were regularly produced in field grown plants but not in greenhouse grown plants. Genes which control pigment production and disappearance during development add to the complexity of pigment patterns. Colors which are visually very similar may be biochemically dissimilar. Leucoanthocyanins occur in aleurone and endosperm but do not occur at any stage in pericarp, spermoderm, or other maternal tissues. In aleurone, which is alkaline, anthocyanins occur as anhydro bases. In aleurone, also, and in certain other tissues, such as the hood veins of the black variety Gatami, anthocyanins may occur as pseudo bases. In young aleurone tissue, anthocyanins are largely free and minor amounts are tissue bound; later in development most are tissue bound. At any stage of development anthocyanins in aleurone are difficult to extract because the nucellar epidermis, alone or with the aleurone envelope, is highly impermeable to most solvents. Biochemical differentiation of anthocyanins, using accepted techniques of extraction and processing, and using paper chromatography, was undertaken for varieties, lines and tissues. Support and extension of earlier genetical study is given. Biochemical phenotypes could be differentiated from basal leaf sheath extracts when visual phenotypes could not be. In varieties such as Black Hulless, anthocyanins from aleurone were largely delphinidin and petunidin derivatives while those from spermoderm were largely derivatives of cyanidin and peonidin. Anthocyanin patterns from different maternal tissues of a single plant were similar but some seasonal variations were recorded. Anthocyanin patterns in varieties, with very different breeding such as Gopal and Black Hulless, even when grown under a variety of environments, were similar and pointed to parallel genetical control of color. Three new anthocyanins with novel characteristics were found in young, but not in mature, caryopses; an outstanding property was their rapid movement on chromatopaper when BAW was the solvent. Whether or not their frequent disappearance was due to inherent instability or was a normal feature of development was uncertain. In some varieties and tissues at least it was established that cyanidin derivatives form first and pelargonidin and/or peonidin derivatives form later. Qualitative associations in development of polyphenols, anthocyanins and melanins were noted. In future, studies of anthocyanins in development would be greatly aided if the experimental barley was grown in constant environments. Detailed characterization of the anthocyanins of barley was made difficult by the complexities of the color patterns and by their instability when commonly accepted processing procedures were used. Most of the anthocyanins of barley were complex and split readily into simpler components. Ninety-three isolates were obtained from basal leaf sheaths and caryopses; some isolates split into as many as 5 components while others did not split. The anthocyanidins of 63 anthocyanin isolates, run against known anthocyanidins, were identified by Rf values obtained in 7 solvents and by spectra; 39 isolates were hydrolyzed for sugar analyses and 25 were hydrolyzed partially for structural elucidation. Many anthocyanins were crystallized and many were rather fully characterized. Many of the anthocyanins of barley are new and had not been reported for other species. New techniques were devised to achieve anthocyanin stability and to enable work with micro amounts of tissue from single hybrid plants. It was shown that evaporation to dryness of extracts in 1% methanolic HC1, a common procedure, is a very serious, but not the single, cause of anthocyanin degradation; certain anthocyanins show a degradative spectral peak ca. 360 mu attributed provisionally to that of chalcones and most show splittings. Clamping and sewing of chromatobands directly to new paper avoided elution and flash evaporation in methanolic HC1. Modification of existing spectral technique also permitted the examination of even very weak anthocyanin bands on paper. Employment of very weak (0.03%) methanolic HCl in procedures was useful. Although the problems of anthocyanin lability were reduced by modifying older procedures and devising new techniques, they were not eliminated. Evidence was obtained finally which showed that anthocyanins are best extracted in neutral methanol as pseudo bases rather than, as is customary, in acidified methanol, as flavylium salts. Not only were techniques devised for anthocyanin stability, they were also devised for handling micro extracts from single plants. Time and materials were conserved in techniques for hydrolysis, concentration, purification and characterization on paper. Complementary equipment for banding and hydrolysis on paper was developed. New solvents for anthocyanidin chromatography greatly aided characterization. Hydrolyses of eluates both from anthocyanins and blank chromatopaper yielded glucose, galactose, xylose and arabinose; a method to remove paper-derived sugar artefacts from chromatographically purified anthocyanins is presented. Additionally, it was found that glycosidic hydrolysis of anthocyanins occurs even on chromatopaper and that the so-called irreversibly adsorbed anthocyanins of many investigators are anthocyanidins. Moreover 'glycosidic' hydrolysis of certain barley anthocyanins, even in crystalline state, to simpler anthocyanins, is the basis of anthocyanin splitting and explains the appearance of the complex chromatoband patterns. Other investigators have also found in other species complex patterns but have assumed them to be in vivo patterns. If biochemical responses to gene action and biogenesis of anthocyanins are to be studied precisely in barley, procedures must avoid artefact production during extraction and processing and yield anthocyanins in their in vivo state.

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