Regulation and a conserved intron sequence of liguleless3/4 knox class-I homeobox genes in grasses

The nine class-I maize (Zea mays L.) knox genes are putative transcription factors normally expressed in shoot apices, but not in leaves. knotted1 (kn1) seems to function in shoot apical meristem maintenance, and rough sheath1 (rs1)-like genes may act in internode elongation. The function of liguleless3 (lg3)-type genes is still unknown. Here, we characterized lg3 as well as the two most closely related genes liguleless4a (lg4a, formerly knox11) and liguleless4b (lg4b, formerly knox5). We termed this subclass of knox genes lg3/4 genes. We studied the expression patterns of lg3/4 genes and compared their sequences. We obtained knockout mutants of lg3 by finding Mu transposon insertions into exons. Our results show that lg3 was not essential for plant development, and that lg4a and lg4b were likely to encode the redundant function. In addition, lg4a but not lg4b was ectopically expressed in the Lg4-O mutant, suggesting that this mutant was affected at the lg4a locus. We found that the lg3 gene was unique among knox genes as it was co-induced in the leaves of leaf mutants that ectopically expressed knox genes in the leaves. The leaf phenotype expressed in the dominant Rs1-O mutant was not altered when lg3 function was removed using the knockout. Genomic sequence comparisons of lg3, lg4a and lg4b from maize and the two homologous genes, osh6 and osh71, from rice revealed a 14-bp phylogenetic footprint in intron II. This sequence was conserved in nucleotide composition, position and polarity in the lg3/4 genes of divergent grasses representing six Gramineae subfamilies. In an independent experiment, this same conserved sequence was found in a yeast reverse one-hybrid screen for putative binding sites of the LG3 homeodomain protein. Distribution of this 14-bp sequence was examined within the public rice database. The possible function of this sequence in regulation of lg3/4 genes is discussed.