Ectopic expression of the maize homeobox gene liguleless3 alters cell fates in the leaf

Abstract

The semidominant mutation Liguleless3-O (Lg3-O) causes a blade-to-sheath transformation at the midrib region of the maize (Zea mays L.) leaf. We isolated a full-length lg3 cDNA containing a knotted1-like family homeobox. Six Lg3-O partial revertant alleles caused by insertion of a Mutator (Mu) transposon and two deletion derivatives were isolated and used to verify that our knotted1-like cDNA corresponds to the LG3 message. In wild-type plants the LG3 mRNA is expressed in apical regions but is not expressed in leaves. In mutant plants harboring any of three dominant lg3 alleles (Lg3-O, -Mlg, and -347), LG3 mRNA is expressed in leaf sheath tissue, indicating that the Lg3 phenotype is due to ectopic expression of the gene. The Lg3-O revertant alleles represent two classes of Lg3 phenotypes that correlate well with the level of ectopic Lg3 expression. High levels of ectopic LG3 mRNA expression results in a severe Lg3 phenotype, whereas weak ectopic Lg3 expression results in a mild Lg3 phenotype. We propose that ectopic Lg3 expression early in leaf development causes the blade-to-sheath transformation, but the level of expression determines the extent of the transformation.

Figure 1

Wild-type and Lg3 leaves. A, Wild-type maize leaf. B, Lg3-O maize leaf. a, Auricle; b, blade; l, ligule; m, midrib; s, sheath.

Figure 3

Nucleotide and predicted amino acid sequence of Lg3. The numbers indicate amino acid residues. The single underline in the 5′ untranslated region indicates a deleted portion of the gene (between nucleotides 90 and 140) for the Lg3-Or81 allele. The single underline with the asterisk indicates a deleted portion of the gene (between nucleotides 96 and 145) for the Lg3-Or422 allele. The single underline with two asterisks indicates a deleted portion of the gene (between nucleotides 80 and 147) for the Lg3-Or211 allele. The double underline in the 5′ untranslated region indicates a short open reading frame. The small inverted triangles indicate the positions of the introns. The large inverted triangles indicate the positions of the Mu transposons for each allele. The outlined region shows the homeodomain. The arrows are the nucleotide sequences used for the lg3 RT-PCR; the 5′ arrow is primer Lg3/4 and the 3′ arrow is primer Lg3-3′. The asterisks in the 3′ untranslated region indicate the site of poly(A⁺) addition identified in the cDNAs. The accession number is AF100455.

Figure 2

DNA gel-blot analysis of two families segregating for the Lg3-O mutation in a B73 inbred line. The genomic DNA samples were digested with BamHI. Lane a, Lg3 plant 1678-7; lanes b (designated by arrows), seven progeny (four Lg3, three wild type) of the cross B73 wild type × plant 1678-7; lane c, plant 1678-8; lanes d (designated by arrows), seven progeny (four Lg3, three wild type) of the cross B73 wild type × plant 1678-8; lane e, B73 inbred tester. The DNA gel blot was probed with the BglII 1.6-kb fragment from the putative lg3 clone. The arrow marks a 10-kb fragment always present in Lg3-O heterozygotes but never present in wild-type siblings. The number scale on the right is in kilobases.

Figure 4

Comparisons of the LG3 amino acid sequence with other KNOX amino acid sequences. A, Comparisons of the predicted maize LG3, RS1, and KN1 and putative rice LG3 amino acid sequences. B, A dendrogram comparison of the plant KNOX-like protein sequences. Entire protein sequences were compared. The maize and rice LG3 proteins (LG3ZM and LG3OS, respectively) form a separate subgroup. The figure was compiled using the software program PileUp (Genetics Computer Group, Madison, WI). Protein sequences in B are from Arabidopsis (KNAT1-5, STM1), Glycine max (SBH1), Hordeum vulgare (HOODED), Lycopersicon esculentum (LET6, TKN1), Oryza sativa (LG3OS, OSH1), Solanum tuberosum (POTH1), and Zea mays (LG3ZM, KN1, RS1).

Figure 5

Lg3 partial revertant alleles. A, Genomic map of the Lg3 gene showing the sites of Mu insertions. B, Lg3-Or81 leaf. C, Lg3-Or671 leaf. D, Lg3-Or331 leaf from a Mu-inactive plant. E, Lg3-Or422 leaf from a Mu-inactive plant. F, Lg3-Or1021 leaf from a Mu-inactive plant. G, Lg3-Or211 leaf from a Mu-inactive plant. The leaves are from plants carrying the alleles in the heterozygous condition. Note the degree of ligule displacement in the alleles. For example, in the Lg3-Or211 allele the ligule is removed and in the Lg3-Or331 allele the ligule is only slightly displaced.

Figure 6

DNA gel blots showing alterations in lg3 genomic sequence associated with a change in Lg3 phenotype. Lanes 1 to 4 were digested with BglII and probed with the entire lg3 cDNA. Lanes 5 to 13 were digested with XbaI and probed with the 5′ untranslated region of the lg3 cDNA. Asterisks indicate polymorphic fragments due to DNA alterations at the lg3 locus due to Mu element insertions into the lg3 gene.

Figure 7

RT-PCR analysis of Lg3 expression in wild-type and mutant tissues. cDNA from different tissues of wild-type and mutant plants was used for amplification of LG3 and ubiquitin sequences. Thirty cycles of PCR were used to amplify the LG3 and ubiquitin sequences. The PCR products were hybridized with the lg3 cDNA and a ubiquitin probe. dap, Days after pollination; Veg., vegetative.

Figure 8

RT-PCR analysis of Lg3 expression in sheath tissue from partially Lg3 revertant alleles. cDNA from sheath tissue from several Lg3 alleles and nonmutant siblings was used for amplification of Lg3 and ubiquitin sequences. Thirty cycles of PCR were used to amplify the Lg3 and ubiquitin sequences. The relative levels of the amplified products were similar at 20 cycles of PCR (data not shown). The PCR products were hybridized with the lg3 cDNA and a ubiquitin probe.

Figure 9

Lg3 leaf phenotypes and ectopic Lg3 expression are represented schematically. The severity connotated for each partially revertant Lg3 allele is shown along with a representation of the amount of ectopic Lg3 expression.