Progress toward the cloning of genes responsible for carotenoid biosynthesis

The leaves of all green plants contain carotenoid pigments, one function of which is to protect chlorophyll pigments from photooxidation. Yellow-seeded corn also contains carotenoid pigments in the endosperm. The carotenoid pigments in the green plant parts and endosperm are thought to be synthesized by a similar or identical biochemical pathway that is controlled by the same genes. The genetics of many mutants involved in carotenoid biosynthesis in maize has been analyzed, including y1, vp5, vp9, ps, w3, vp2, al, lw1, lw2, lw3, lw4, y9, cl, and y10. Information has been obtained about the relationship of several of these genes to proposed steps in the biosynthetic pathway of carotenoids. Kernels homozygous for these mutant alleles are pale yellow or white, and are deficient in beta-carotene. When these kernels are germinated, they give rise to pale green or albino seedlings, also deficient in beta-carotene (a notable exception is the y1 locus, discussed below). Some of these mutants accumulate carotenoid precursors in the leaves of mutant plants (e.g. w3, phytotene and phytofluene; vp5, phytoene, phytofluene and zeta-carotene; ps, phytoene, phytofluene and lycopene). Accumulation of precursors has also been demonstrated in endosperms of some of these mutants. We are attempting to clone several of the genes responsible for carotenoid biosynthesis in corn.

The gene product of the Y1 locus is thought to be a regulatory element in the carotenoid biosynthetic pathway in maize. We have analyzed 28 families segregating for plants heterozygous for different putative Mu-induced y1 mutations (Y1/y1 -Mum). DNA of 40 such heterozygous plants selected from these families was isolated. The approximate number of Mu1 -homologous elements in each plant was established by Southern blot analyses. The plants with low Mu copy numbers (6 to 12) were outcrossed to a white endosperm stock homozygous for the standard y1 allele, producing seeds of two types: yellow (Y1/y1), and white (y1 /y1 -Mum). DNA was isolated from plants grown from each type of seed, digested with restriction endonucleases that cleave outside the Mu1 element, electrophoresed, Southern blotted and hybridized with Mu1 DNA. The EcoRl hybridization profile of the outcross progeny of one heterozygous plant, 2045-10, contained a 5.9 Kbp band that appeared in all plants derived from white seeds, but was absent in all plants derived from yellow seeds. It is expected that the y1 gene, carrying a Mu element insertion, would segregate exclusively with mutant white-seeded individuals. DNA from a single white-seeded progeny of plant number 2045-10 was digested with EcoRI, and the DNA fragments between 5.5 and 6.0 Kbp in length were isolated. This fraction of DNA was shown to contain a 5.9 Kbp Mu1 -hybridizing fragment. No other Mu1 -homologous DNA fragments were present. The size-fractionated DNA was ligated into EcoRI-digested lambda-gt10, packaged, and transfected into E. coli. A single Mu1-hybridizing plaque was isolated. This clone was purified and shown to contain a Mu element equivalent in size to Mu1 as well as another sequence(s) repetitive in both the clone and the maize genome. To demonstrate that the cloned DNA is homologous to the 5.9 Kbp EcoRI fragment shown to segregate with the white endosperm phenotype, a nonrepetitive sequence from the cloned DNA will be used as a hybridization probe of the EcoRI Southern segregation analysis. To identify a nonrepetitive sequence, the DNA is being subcloned and restriction mapped.

In addition to our efforts to clone the Y1 gene, we have also transposon tagged other genes involved in the carotenoid biosynthetic pathway. Putative Mu-induced mutants allelic to y9, w3, vp5, vp9 and ps have been isolated. Plants that are recessive for any one of these loci have known patterns of accumulation of carotenoid precursors. We have not yet begun a molecular analysis of these mutants. We also have numerous additional Mu-induced white-kernel/albino-seedling mutants that have not yet been tested for allelism.

Some white endosperm inbred lines (e.g., A188) are homozygous for a dominant white endosperm gene as well as for y1. Mutator stocks homozygous for a dominant white endosperm gene (nonallelic to y1) have been produced. These stocks will be used next summer for isolating a Mu-induced mutant at this locus. We are also in the process of testing our dominant white stocks for allelism with the dominant white cap allele (Wc).

Brent Buckner and Donald S. Robertson

Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of the authors.

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