The maize gene liguleless2 encodes a basic leucine zipper protein involved in the establishment of the leaf blade-sheath boundary

Abstract

The blade and sheath of a maize leaf are separated by a linear epidermal fringe, the ligule, and two wedge-like structures, the auricles. In plants homozygous for the null mutation, liguleless2-reference (lg2-R), the ligule and auricles are often absent or positioned incorrectly and the blade-sheath boundary is diffuse. This phenotype is in contrast to that of liguleless1-reference (lgl-R) mutant plants, which have a more defined boundary even in the absence of the ligule and auricles. Additionally, mosaic analysis indicates the lg2-R phenotype is cell-nonautonomous and the lg1-R phenotype is cell-autonomous. Using scanning electron microscopy we show that lg2-R mutant plants are affected before the first visible sign of ligule and auricle formation. We have cloned the Lg2+ gene through a Mutator-8 transposon insertion allele, and verified it with five independently derived alleles. The comparison of genomic DNA and cDNA sequences reveals an open reading frame encoding a protein of 531 amino acids with partial homology to a subclass of plant basic leucine zipper (bZIP) transcription factors. Although a large body of molecular and biochemical characterization exists on this subclass of bZIP proteins, our work represents the first report of a mutant phenotype within this group. A specific reverse transcriptase (RT)-PCR assay shows LG2 mRNA expression in meristem/developing ligule regions. RT-PCR also shows that LG2 mRNA accumulation precedes that of LG1 mRNA. The mutant phenotype and expression analysis of lg2 suggest an early role in initiating an exact blade-sheath boundary within the young leaf primordia.

Figure 1

Wild-type ligule and auricle development. (A) Leaf 7, plastochron 7: Anticlinal cell divisions giving rise to the ligule and auricle have not been initiated. In the top third of the image, the outer cell files begin to curve toward the margin. The ligule will form below this region. The primordium base can be seen in the lower right-hand corner. (B) Leaf 6, plastochron 8: The preligule band (anticlinal divisions) is formed across the leaf primordium. (C) Leaf 5, plastochron 10: Periclinal divisions are well advanced, causing the ligule ridge to protrude from the adaxial leaf surface. Above the ligule ridge, notice the undulating surface reflecting the underlying venation. The arrow under the word margin indicates the direction of the leaf margin.

Figure 2

lg2-R mutant leaf primordium ligule and auricle development. (A) Leaf 5, plastochron 9: The preligule band (anticlinal divisions) is incompletely formed across the primordium width. (B) The transition area boxed in A is displayed at higher magnification. (C) Leaf 4, plastochron 10: The ligule ridge is not developed equally across the width of the leaf primordium. A small displacement is shown in brackets. Notice the undulating surface reflecting the underlying venation in the blade above the ligule (cf. Fig. 1C) and how this pattern continues above the displacement. The arrow under the word margin indicates the direction of the leaf margin.

Figure 3

lg2-R mutant plant mature leaves. (A) Leaf 1, plastochron 13: A small ligule/auricle vestige is found at the leaf margin, as indicated by the arrow. Where this structure is missing the transition from blade to sheath is diffuse; long, rectangular sheath cells gradually yield to the short, square blade cells. (B) Leaf 4, plastochron 10: Ligule and auricle are evident at the leaf margin. These mature structures are most developed at the margin and become decreasingly less developed toward the midrib. A region of cells is displaced along a lateral vein, as shown in brackets. The arrow under the word margin indicates the direction of the leaf margin.

Figure 4

(A) Five putative Mu transposon-induced liguleless2 alleles differ from their progenitor alleles. (a–d) Restricted DNA (a and c, BamHI; b, XhoI; d, EcoRI) from putative Mu-tagged plants and their respective wild-type siblings hybridized to the lg2.2757.EB probe. (e) EcoRI-restricted DNA from a lg2-902 homozygous plant and plants homozygous for its progenitor alleles hybridized to the lg2.2757.EB probe. The arrows point to the tagged fragments. (B) Multiple independently derived alleles map to the same genomic locus. The positions of lesions are shown on a restriction map of the progenitor genomic locus. Known insertion positions are indicated by triangles and the lg2-2757 Mu8 insertion is shaded. Restriction mapped insertions and/or rearrangements are indicated by a triangle beneath a line spanning the possible region. The minimum extent of the deletion allele (lg2-219) is shown by a shaded line. (C) Cloned fragments from lg2-2757 and B73. The Mu8 position within the lg2-2757 clone is indicated by a triangle (D) DNA fragments used as radiolabeled probes.

Figure 5

The nucleotide and predicted amino acid sequence of LG2. Numbers indicate amino acid residues. The putative TATA and CAAT boxes are underscored. The basic region (224–248) is indicated in bold and the three leucines within the leucine zipper (248, 255, and 262) are in bold italics. (▪) The stop codon; (▾) intron positions. Intron sizes are given as number of base pairs. The first and last nucleotides of the sequenced cDNA are in boldface type. Polyadenylation sites are indicated by asterisks.

Figure 6

(A) The LG2 bZIP domain is compared with closely related plant proteins (Katagiri et al. 1989; Tabata et al. 1991; Ehrlich et al. 1992; Foley et al. 1993; Feltkamp et al. 1994; Xiang et al. 1995). (B) The LG2 bZIP domain is compared with other bZIP-containing plant proteins (Hartings et al. 1989; Schmidt et al. 1990; Schindler et al. 1992a). (C) The LG2 bZIP domain is compared with two mammalian bZIP proteins (Hoeffler et al. 1988; Bohman et al. 1987). Identical amino acids are indicated by dashes, and the leucine residues within the leucine zipper by asterisks and boldface type.

Figure 7

RT–PCR analysis of LG2 mRNA and LG1 mRNA abundance in developing ligule regions of wild-type and mutant plants. The plant genotypes are indicated to the left. The lg2-219 and lg1-R alleles are molecular deletions. A and B show PCR fragments of LG2 RT–PCR and LG1 RT–PCR, respectively. The numbers 1, 2, and 3 refer to the RNA samples. (1) P6-8 marginal region; (2) P6-8 midrib region; (3) meristem and P1-5; (P) Plastochron.