--Karen C. Cone, Emily B. Frisch and Thomas E. Phillips

Anthocyanin synthesis in the single-cell aleurone layer of the maize kernel requires a complex interaction between many genes. A number of genetic, biochemical, and molecular studies have established that a1, a2, bz1, bz2, and c2 encode enzymes in the biosynthetic pathway. The c1 and r genes coordinately affect the expression of the biosynthetic genes, and thus are assumed to play regulatory roles. Mutations in other genes, e.g., vp1, dek1, and anl1, result in an anthocyaninless phenotype, but also cause abnormal development of endosperm and/or embryo. The pleiotropic nature of mutations in these three genes suggests that their colorless phenotypes may be secondary to their effects on kernel development.

In at least one case, that of dek1, this idea is substantiated by the morphology of mutant kernels. Kernels homozy-gous for dek1 apparently arrest early in development. Mutant embryos develop only to the proembryo stage, and mutant endosperms are small, colorless, and floury (hence the early designation clf) with no anthocyanin or carotenoids present (Neuffer and Sheridan, Genetics 95:929-944, 1980). In non-mutant kernels, the proembryo stage is normally attained about 8-11 days after pollination (Sheridan and Clark, Trends in Genet. 3:3-6, 1987), and the anthocyanin-producing aleurone layer differentiates from the epidermis of the endosperm when periclinal wall formation ceases approximately 20-22 DAP (Randolph, J. Ag. Research 53:881-916, 1936). Therefore, if dek1 embryo and endosperm arrest simultaneously 8-11 days after pollination, then the anthocyaninless phenotype might be explained simply by the lack of a differentiated aleurone layer.

In fact, Gavazzi and colleagues have reported that dek1 kernels do not have an aleurone (MNL 61:73-74; 62:91-92). To confirm this observation, we examined kernels that were mosaic for dek1. We obtained from G. Neuffer a self-pollinated ear (30 DAP) from a plant with the following markers on chromosome 1S: P-WW dek1/P-VV (Ac) Dek1 Ds-4. Approximately 3/4 of the kernels were mosaic for anthocyanin production; colorless sectors resulted from somatic loss of Dek1, mediated by Ac/Ds-induced chromosome breaks. A number of mosaic kernels were fixed, embedded, and prepared for light microscopy. The accompanying figure shows a cross-section through the pericarp and endosperm of a portion of a mosaic kernel. Purple sectors (Dek1) had normal aleurone cells, but colorless areas (dek1) were devoid of recognizable aleurone cells. A striking feature of the endosperm tissue underlying the aleurone is the lack of the cambium-like organization usually associated with the sub-aleurone tissue in non-mutant kernels. It is possible that this organization was disrupted by lateral expansion of non-mutant endosperm cells to fill "gaps" that resulted from dek1-mediated deficiencies in cell division/expansion. However, without other assayable markers on 1S to distinguish mutant from non-mutant endosperm, it is difficult to say what the origin of the underlying tissue is. The fact that the colorless sectors lack aleurone cells, however, is consistent with earlier observations.

Figure Legend:  Cross-section through pericarp, aleurone, and endosperm of a kernel mosaic for dek1 showing colorless regions missing aleurone cells. P: pericarp. A: aleurone cell. E: endosperm. Kernels were harvested 30 days after pollination and cut into small cubes. The tissue was fixed in 2% glutaraldehyde containing 1 M sucrose, 30 mM HEPES, 70 mM NaCl, 5 mM CaCl2, pH 7.2 and then treated with 1% OsO4 in 30 mM HEPES, 70 mM NaCl, pH 7.2. Samples were dehydrated in ethanol and embedded in Spurr's resin. Semithin (0.5um) sections were stained with 1% toluidine blue in 1 % sodium borate, and photographed with a Zeiss Axiophot microscope. Bar = 100um.
 

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