Genetic and molecular analysis of the gl3-m3 allele of maize
 --Donini, G; Maddaloni, M; Salamini, F; Motto, M

Epicuticular waxes from the outermost layer of aerial plant organs are thought to confer resistance to insect herbivore, fungal pathogens and drought (Martin and Juniper, The cuticle of plants, Edward Arnold, Ltd., 1970). The production of plant epicuticular wax is a biologically complex process involving a host of synthetic and transport mechanisms (von Wettstein-Knowles;, In RJ Hamilton, ed, Waxes: Chemistry, Molecular Biology and Functions, The Oily Press, Dundee, UK, pp 91-129, 1995). In maize the biosynthesis and deposition of epicuticular waxes in the first five or six leaves of young plants is affected by at least 20 genetic loci (Coe, MNL 67:133-166, 1993). In this plant cuticular waxes are a complex mixture of very long chain fatty acids and their derivatives (Bianchi et al., Maydica 30:179-198, 1985). From genetic and biochemical analyses of maize plants carrying different mutations affecting wax synthesis and depositions, it was hypothesised that two pathways can give rise to different kind of leaf waxes (Bianchi et al., Maydica 30:179-198, 1985). One pathway is responsible for wax synthesis in the first five or six juvenile leaves, whereas the second pathway produces waxes during the whole life cycle of the maize plant. The availability of mutants affecting cuticular wax accumulation and the isolation of the corresponding genes will assist in the elucidation of cuticular wax accumulation and the molecular mechanism by which its production is regulated. Therefore, a molecular genetic approach, based on transposable elements, is being used to isolate, clone and characterise the Glossy3 (Gl3) gene.

The Gl3 locus is required for the biosynthesis of the epicuticular wax layer of the first five or six leaves of young plants. From genetic analyses and biochemical examination of the epicuticular wax composition of plants carrying a recessive mutation at the Gl3 gene, it was concluded that the Gl3 gene product may be involved in the elongation step from C28-C30 (Bianchi et al., Maydica 22:9-17, 1977). Mutated gl3 plants produce approximately four times less wax than wild-type Gl3 plants in the first five to six leaves and numerous variations in the distribution chain length within each class of wax compounds were found (Bianchi et al., Maydica 22:9-17, 1977).

A maize strain carrying the Ac transposable element inserted into the Waxy locus (wx-m7) was crossed as a male to the inbred line WF9 carrying the double mutation gl3-gl8. Out of 55,000 F1 seedlings scored, we have identified three unstable alleles at the Gl3 locus; these alleles were named gl3-m1,-m2 and-m3. Despite the use of wx-m7 strain, we were not able to find Ac activity associated with the mutability at the Gl3 locus. This finding was not surprising because, by using the wx-m7 strains, we generated several En/Spm unstable alleles at the Opaque-2 locus (Michel et al., Mol. Gen. Genet. 248:287-292, 1995) and at least one unstable allele at the Gl2 locus (Tacke et al., The Plant J. 8:907-917, 1995). Therefore, a detailed molecular and genetic analysis was undertaken to characterise the transposable element inserted in each gl3 mutable allele. The gl3-mutable alleles were backcrossed twice to the WF9 gl3 strain and seeds of the second backcross generation were germinated; at the seedlings stage variegated and glossy stable shoots were bulked separately. The DNA extracted from each bulk was restricted with methylation-sensitive restriction enzymes and probed with an En/Spm specific probe. Results from Southern blot analyses were particularly encouraging with respect to the gl3-m3 allele, as fragments between 8 to 9 kb cosegregating with the mutable phenotype, were detected with each of the enzymes SalI, PstI, and PvuI. In addition gl3-m3/gl3 plants were crossed to the c2-m1 En/Spm tester strain to establish an association between the mutability at the gl3-m3 locus and the appearance of coloured spots in the aleurone. The recorded data are in favour of the presence of an En/Spm element in the gl3-m3 allele.

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