Rough sheath2 encodes a MYB-domain protein that negatively regulates meristem-specific KNOTTED1-like homeobox proteins in the leaf
--Timmermans, M, Nelson, T

Characterization of the leafbladeless1 (lbl1) mutant phenotype suggested that Lbl1 is required to establish adaxial cell identity in leaves and leaf-like lateral organs (Timmermans et al., MNL 71:66, 1997; Timmermans et al., manuscript in preparation). In the absence of LBL1 activity, cells obtain an abaxial identity which results in the formation of radially symmetric, abaxialized leaves. Less severe leaf phenotypes include the formation of ectopic laminar outgrowths at the boundaries of abaxialized sectors on the adaxial leaf surface, and the bifurcation of leaves. Immunolocalization using a KNOTTED1 (KN1) specific antibody on transverse sections of wild type and lbl1 mutant apices at the level of the incipient leaf showed that the region in which cells do not accumulate KN1 protein is far smaller in lbl1 mutant apices. Therefore, Lbl1 or the establishment of the adaxial/abaxial axis is required for the downregulation of KN1 in the meristem, i.e. for the propagation of founder cell recruitment. Whether lbl1 affects kn1 expression directly or indirectly is not known.

The Phantastica (phan) mutant in Antirrhinum exhibits similar defects in adaxial/abaxial patterning and results in the loss of adaxial cell identity in leaves, sepals and petals (Waites and Hudson, Development 121:2143, 1995). The most extreme manifestation is the development of radially symmetric, abaxialized leaves, whereas less severely affected phan leaves frequently develop ectopic laminar outgrowths on the adaxial leaf surface surrounding patches of abaxial epidermis. In addition, the phan mutation results in the misregulation of kn1-like homeobox genes in Antirrhinum (A. Hudson, personal communication). The Phan gene has recently been cloned and was shown to encode a MYB-domain protein (A. Hudson, personal communication). We cloned the Phan homolog from maize, because the similarities between the lbl1 and phan mutant phenotypes suggested that the phan homolog may either be lbl1 or a gene functioning in a similar developmental pathway.

We screened a cDNA library derived from vegetative apices and young leaf primordia using the region of the Phan cDNA encoding the MYB-domain as a probe. Two positive cDNAs were isolated from 1-2x106 plaques screened. One cDNA clone is 1.4 kb in length and potentially encodes a full length protein. The 107 aa MYB-domain is highly conserved between PHAN in Antirrhinum and maize, 93% aa identity. The non-MYB region of the protein is less conserved, but contains several conserved motifs in the N-terminal portion of this domain and the C-terminal 120 aa have 68% identity between maize and Antirrhinum. Probes derived from the region encoding the MYB-domain hybridize predominantly to a single locus in the maize genome and less strongly to a second locus. Interestingly, the fragment of the maize Phan cDNA clone encoding the non-MYB region is highly repetitive within the maize genome.

The Phan homologs were mapped using the BNL recombinant inbred populations. Neither homolog mapped near the lbl1 locus on chromosome 6S. The major band mapped on chromosome arm 1S in close proximity to the RFLP marker npi598, whereas the minor band mapped to chromosome 8L. Comparison of the RI-line-based map with the genetic map, placed the phan locus in close proximity to the recessive leaf mutant rough sheath2 (rs2). Southern analysis using DNA from plants homozygous for the rs2-R allele showed that these plants carried a deletion in the phan locus. Similar analysis of plants homozygous for the rs2*-90 allele, which was isolated in a directed tagging experiment with Mutator (R. Schneeberger and M. Freeling, unpublished results), showed the presence of a MuDR insertion near the C-terminus of the MYB-domain. Additional alleles are currently being analyzed, but these observations indicate that Rs2 is the maize homolog of the Antirrhinum Phan gene.

Recessive mutations at rs2 lead to a roughening of the auricle and to expression of sheath-like characters along lateral veins in the blade. These phenotypes strongly resemble the phenotypes induced by dominant mutations in the homeobox gene Rough sheath1. Additional phenotypes observed in rs2 mutant plants resemble the morphological aberrations caused by dominant mutations in other homeobox genes (e.g. Hairy sheath frayed1 and Kn1). The most severe manifestation of the rs2 mutation is the development of extremely narrow leaves, which appear nearly radially symmetric and express mostly midrib-like characters. Whether these narrow leaves result from a defect in founder cell recruitment or from a failure to establish leaf identity throughout the incipient primordium remains to be determined. However, rs2 causes cells within the leaf to maintain a less determined fate.

In situ hybridization on wild type shoot apices using the MYB-domain encoding fragment of the Rs2 cDNA as a probe showed the accumulation of Rs2 transcripts throughout the P1 primordium and along the major vascular bundles in P2 to P4 primordia. Rs2 transcripts could not be detected in the apex and in leaf founder cells. Absence of transcripts in the P0 incipient leaf is unexpected because leaf initiation is affected in severe rs2 mutant plants. The accumulation of Rs2 transcripts in the vasculature is consistent with the rs2 mutant phenotype, in which only cells overlying the lateral vascular bundles adopt less determined fates. Taken together, these observations suggest a role for the MYB-domain protein encoded by Rs2 in the negative regulation of one or more homeobox proteins in the leaf. We are currently testing this hypothesis by analyzing the expression patterns of kn1-like homeobox genes in shoot apices of wild type and rs2 mutant seedlings.

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