Quantitative extraction of pericarp pigments
--O. Prem Das, Margaret Morales and Joachim Messing
Several genes, including P, R and A1, control pigmentation in kernel pericarp, and for P, alleles that vary over a wide range in phenotype are known. For example, mutable alleles of the P-vv type can give rise to different degrees of variegation and background pigmentation: stable alleles can also condition different degrees and patterns of pigmentation. In addition, we (MNL 67; Das and Messing, Genetics, in press) have described an allele (termed P-pr), originating from epimutation of P-rr, that can generate a range of variegated pigmentation on its own, as well as in heterozygotes with P-rr (see note below). A quantitative measure of pigmentation would be useful in the characterization of these alleles and their interactions, and therefore, we have developed the following simple method for pigment extraction from pericarp.
Kernels (5 per ear) are soaked in water for 2 hrs or more, and pericarps are manually peeled and placed in a tared Eppendorf tube with a pierced cap. Tissue is dried by overnight lyophilization, and the tube is weighed to determine dry tissue weight (~50mg). Then 0.2 ml conc. HCl is added to this tube, followed by 0.8 ml dimethyl sulfoxide (DMSO). These reagents should be added sequentially with vigorous vortex mixing after each addition. The tube is centrifuged briefly to clarify the suspension, and 0.05-0.2 ml, depending on the intensity of color, is diluted with 1 ml methanol for absorbance readings (-max = 510 nm for P pigments, 530 nm for r-ch pigments).
The extracted pigment is stable; there is only a slight increase (<10%) in the absorbance and little change in the absorption spectrum of the extract upon overnight extraction compared to a 0.5 hr treatment, and a similar increase in the absorbance of the methanol-diluted sample. More than 90% of the pigment is recovered in a one-hour extraction. Grinding of the tissue slightly increases extraction efficiency, but is unnecessary for routine comparative analysis. Pigments that depend on the P gene can be recovered after workup in acetone for further analysis, which we have not pursued. It is likely that the strong acid treatment affects the chemical structure of these pigments. However, treatment with milder acid does not give quantitative extraction of P pigments, and results in two absorption maxima at 510 and 570 nm. Longer treatment or stronger acid conditions result in a progressive loss of the 570 nm peak, and a concomitant increase in OD at 510 nm, indicating a precursor-product relationship between the species that give rise to these peaks. Under the conditions given above, the 570 nm peak is present, if at all, as a slight shoulder. The r-ch pigment gives a simple, narrow absorption spectrum in the visible range, suggesting that one or a closely related set of chromophores are present. The absorption spectrum of the P pigment is more complex, with strong absorbance into the near UV. For both, absorbance accurately reflects pigmentation, as shown by testing mixtures of colorless and colored pericarps in known ratios. Furthermore, extraction is nearly quantitative, since the residue after one or two extractions is almost colorless. It is possible that this simple method may apply to the quantitation of pigmentation conditioned by other genes, and in other tissues.
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