The floury2 (fl2) mutation is characterized by a soft, starchy kernel that is highly susceptible to mechanical damage and pathogen infestation. The mutation is expressed semi-dominantly and is associated with elevated levels of BiP, an ER resident chaperone protein. Zein accumulation is generally reduced and the protein body development is severely affected. The mutation maps to 4S within a cluster of alpha-zein genes. We have cloned an alpha-zein gene that is tightly linked to the fl2 locus and encodes a polypeptide that accumulates as a precursor. Failure to process the protein is due to a defect in the signal peptide that targets the molecule to the ER. Specifically, there is an alanine to valine substitution at the C-terminus, or -1 position, of the signal peptide. The product of this gene bears homology to the 22-kDa alpha-zeins, but has an apparent molecular weight of 24-kDa on SDS-polyacrylamide gels. In an analysis of a backcross population the 24-kDa alpha-zein was detected in 64 fl2/+/+ kernels but was absent in 64 +/+/+ kernels. The gene was cloned as a segment of a 7.8 kb SstI RFLP identified by bulked segregant analysis of an F2 population using a 22-kDa alpha-zein gene probe. Linkage of this RFLP to the fl2 locus was assessed by Southern analysis, showing that the band was absent in 79 normal progeny of an F2 population. The deduced amino acid sequence of the gene contained in the RFLP matches perfectly with the first 45 amino acids of the N-terminus of the 24-kDa alpha-zein protein. Isolation and N-terminal sequencing of the protein were done by Dr. Jeff Gillikin in Dr. Becky Boston's lab at North Carolina State University, Raleigh, North Carolina.

Failure to cleave the signal peptide from the 24-kDa alpha-zein would be expected to result in the attachment of the protein to the lumenal surface of the ER membrane, and subsequent disruption of protein translocation. Such a perturbation may explain the elevation in BiP accumulation and the abnormal protein body development. The fact that there has been only one fl2 allele identified, and that it is expressed semi-dominantly, suggests that it is a unique, gain-of-function mutation. This concept is consistent with the defect in the signal peptide of the 24-kDa alpha-zein protein arising from a unique point mutation. Epistasis of the zein regulatory gene O2 to fl2 can be explained by the presence in the promoter region of the 24-kDa alpha-zein gene of the 5'-TCCACGTAGA-3' motif common to genes regulated by the O2 transcriptional activator. Taken together, these data provide compelling evidence that the 24-kDa alpha-zein gene is responsible for the fl2 phenotype. 

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