A point mutation at the Miniature1 seed locus reduces levels of the encoded protein, but not its mRNA, in maize

We report here on the molecular nature of an EMS-induced mutant, mnl-89, a leaky semidominant allele of the Miniaturel (Mnl) seed locus that encodes a seed-specific cell wall invertase, INCW2. The mnl-89 locus specifies norma l levels of the Incw2 transcript but extremely low levels (about 6% of norm al) of the protein and enzyme activity are expressed. Sequence analysis of Incw2 clones derived from the parental Mnl and the mutant genotypes shows a C to T transition in the mnl-89 allele, leading to a single amino acid alt eration (proline to leucine) near the C-terminus of the mutant INCW2 protei n. Although this change is not in the catalytic domain, putative N-glycosyl ation sites, or the beta-fructosidase motif, it does lie in a motif that is well conserved among all plant invertases and related fructosyltransferase s. On the basis of these genetic in planta data, we believe we have identif ied a proline residue in a hitherto unknown GPFG motif as critical for the stability of such proteins. The single base change (C to T) also leads to t he elimination of a BglI restriction site in the mutant allele. Indeed, Bgl I restriction digests of genomic DNAs from mnl-89 and Mnl genotypes show on e and two fragments, respectively. Sequence analysis of RT-PCR-derived endo sperm Incw clones from mnl-1 (the reference allele) seeds predict five amin o acid substitutions relative to Mnl. Whether or not these sequences are en coded by the mnl-1 locus or another non-allelic Incw gene in the maize geno me remains to be elucidated.