ZmHox: a novel class of maize homeobox genes.

Clones of two highly related genes, ZmHox2a and ZmHox2b (Zea mays homeobox), were isolated from maize embryo cDNA libraries by screening with the ZmHox1a homeobox sequence. The genes map to chromosomes 3 and 8, respectively, and encode mRNA transcripts of 6kb. The encoded proteins, ZmHox2a and b, share 84% sequence identity and exhibit a modular structure with several novel plant-specific protein domains. Interestingly, each ZmHox2a, gene product contains two complete homeodomains which, for Zmhox2a, were both shown to be functional DNA-binding motifs in vitro. Not only probes encoding the homeobox but also DNA fragments corresponding to other ZmHox2 domains hybridize to multiple bands in genomic Southern blots, indicating that related protein domains may be conserved in other maize genes. The ZmHox2a/b genes, therefore, are members of a novel and large class of maize genes, some of which can be expected to encode new transcription factors.

Transposon tagging of the maize Glossy2 locus with the transposable element En/Spm.

The Glossy2 (Gl2) locus of maize is required for the formation of the epicuticular wax layer of young plants. gl2 mutant seedlings can be visually identified because of their glossy leaf surface which is different from the dull surface of wild-type seedlings. The Gl2 locus was isolated by transposon tagging. Seven unstable mutations, gl2-m2 to gl2-m8, were induced in a parental strain carrying an active transposable Activator (Ac) element in the unstable wx-m7 allele. Genetic tests on the gl2-m2 allele indicated that it was not caused by the Ac element but by the insertion of the transposable element Enhancer/Suppressor-Mutator (En/Spm). A Sa/l restriction fragment segregating with the mutant phenotype was identified, by Southern analysis, using sequences from the En/Spm element as a probe. Part of the fragment was cloned and was shown to carry part of the unstable gl2-m2 allele. These gl2 sequences were used to identify a genomic fragment carrying the wild-type allele and to isolate its corresponding cDNA sequence. The predicted Glossy2 protein consists of 426 amino acids. No similar amino acid sequence was found in protein data banks and the biochemical function of the Gl2 gene product is still unknown. The wild-type Gl2 transcript is found predominantly in juvenile leaves. The transcript level in the leaves of seedlings homozygous for a stable recessive gl2-ref allele is hardly detectable.

The combination of a novel stimulatory element in the first exon of the maize Shrunken-1 gene with the following intron 1 enhances reporter gene expression up to 1000-fold.

Both exon 1 and intron 1 of the maize Shrunken-1 (Sh1) gene individually stimulate expression of reporter genes in transient gene expression experiments if present within the transcription unit. The Sh1 exon 1 mediates a 10-fold increase in activity when inserted at the 5' end of the bacterial chloramphenicol transacetylase (CAT) marker gene in both monocot and dicot protoplasts. The Sh1 intron 1 enhances chimeric gene expression in rice and maize protoplasts approximately 100-fold but inhibits CAT expression in tobacco protoplasts. In combination, the stimulatory effects of Sh1 exon 1 and intron 1 are multiplicative in monocot protoplasts resulting in a final enhancement of up to 1000-fold compared to the unmodified CAT or luciferase marker genes.