ROUGH SHEATH2: A Myb Protein That Represses knox Homeobox Genes in Maize Lateral Organ Primordia

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Science  02 Apr 1999:
Vol. 284, Issue 5411, pp. 151-153
DOI: 10.1126/science.284.5411.151


The regulation of members of the knotted1-like homeobox (knox) gene family is required for the normal initiation and development of lateral organs. The maize rough sheath2(rs2) gene, which encodes a Myb-domain protein, is expressed in lateral organ primordia and their initials. Mutations in thers2 gene permit ectopic expression of knox genes in leaf and floral primordia, causing a variety of developmental defects. Ectopic KNOX protein accumulation in rs2 mutants occurs in a subset of the normal rs2-expressing cells. This variegated accumulation of KNOX proteins in rs2 mutants suggests that rs2 represses knox expression through epigenetic means.

Regulation of knoxgene expression determines the emergence of lateral organs from shoot meristems. In maize, the KNOTTED-1 (KN1) homeodomain protein accumulates in cells of the shoot apex and maintains the meristematic properties of the cells (1). Recruitment of leaf founder cells (and the down-regulation of kn1) begins at a single site on the flank of the shoot apex and continues laterally around the circumference of the apex (1, 2). Otherknox family members, including rough sheath1(rs1), are also implicated in leaf initiation and patterning (3–5).

The knox gene products are absent in normal leaf and floral primordia (1, 3, 6). Ectopic knoxexpression during organogenesis interferes with organ determination and cell differentiation along the adaxial/abaxial and proximodistal axes (5, 7, 8). In maize, dominant mutations in knoxgenes cause the distal displacement of sheath, auricle, and ligule tissues (5). In dicot species, overexpression ofknox genes results in the development of filamentous and lobed leaves and in the formation of ectopic meristems (8).

In maize, recessive mutations that affect knox gene repression have also been identified. The narrow sheath andleafbladeless1 mutations affect kn1down-regulation at leaf initiation, resulting in deletion of the leaf margins and development of radially symmetrical, abaxialized leaves, respectively (9). Mutations in rs2 result in proximodistal patterning defects that are due to rs1 andkn1 expression in leaf primordia (10). Recessive mutations in the PHANTASTICA (PHAN) gene ofAntirrhinum, which encodes a Myb-domain protein, exhibit phenotypes that resemble the phenotypes of these maize mutants (6, 11). Therefore, we investigated the phanhomolog from maize (12).

The maize phan homolog encodes a 370–amino acid protein with a 106–amino acid NH2-terminal Myb domain consisting of two Myb-like repeats (Fig. 1A). The Myb domain and COOH-terminus share a high degree of amino acid identity with PHAN proteins fromAntirrhinum and Arabidopsis. The DNA recognition helices of PHAN share little homology with the other large class of plant Myb proteins (13), suggesting that the PHAN proteins regulate a different class of target genes. However, the PHAN proteins do not contain motifs that suggest a direct transcriptional function. A single intron in the 5′ untranslated region (UTR), ∼50 nucleotides upstream of the translation initiation codon, indicates a structural relation between the Antirrhinum and maize phangenes (Fig. 1B) (12).

Figure 1

rs2 is the maize homolog of theAntirrhinum PHAN gene. (A) Amino acid sequence comparison of RS2 and the PHAN proteins from Antirrhinum andArabidopsis (GenBank accession numbers AJ005586 andAC004684, respectively). Dots indicate gaps in the alignment. Residues that are highlighted in black show identity, and conserved residues are highlighted in gray. (B) Schematic diagram of the rs2 locus. White boxes, untranslated exons; gray box, translated region; dark gray box, the Myb domain. Triangles mark the positions of the Mu1.4 and MuDR transposable elements in the rs2-mum1 and rs2-mum2 alleles, respectively. rs2-R corresponds to a deletion of the Myb domain and the 5′ region.

The maize phan homolog was mapped to a region on chromosome arm 1S that contains a potential knox gene regulator, the rs2 gene (14). A comparison of the structure of the phan locus in wild type and in three mutant alleles of rs2 confirmed thatrs2 is the maize homolog of phan (Fig. 1B) (15). The rs2 mutations cause leaf and floral phenotypic alterations analogous to the phenotypes induced by mutations that alter the regulation of knox genes during lateral organ initiation or development (5, 10).

We compared the pattern of KNOX protein expression in wild-type andrs2 mutant apices by immunolocalization with an antibody specific to KNOX proteins, including KN1 and RS1 (10,16). KNOX proteins accumulated in the shoot apex and stem of wild-type plants but were absent at leaf initiation sites on the apex and in leaf primordia (1) (Fig. 2, A and C). In rs2mutants, KNOX proteins accumulated normally in the meristem and stem, but they also accumulated at the base of leaf primordia and near major lateral veins in the leaf (Fig. 2, B and D). The ectopic accumulation of KNOX protein in patches with sharp lateral boundaries suggests that the leaves were clonal mosaics of knox+ andknox– sectors (Fig. 2B). Sectors expressing KNOX proteins varied among leaves and did not correlate with normal developmental domains. The down-regulation of knoxexpression at the initial site of founder cell recruitment near the center of the new leaf occurred normally in rs2 mutants (Fig. 2D), although the number of founder cells that were recruited laterally was variably reduced.

Figure 2

rs2 is required to repressknox gene expression in lateral organs. Immunolocalization of KNOX protein in (A and C) wild-type and (B and D) rs2-R shoot apices. The transverse (A and B) and longitudinal (C and D) sections are shown. Solid arrows denote some sectors that ectopically accumulate KNOX proteins. Open arrows mark the site on the flank of the shoot apex at which founder cell recruitment has initiated and KN1 protein is no longer present. Scale bar, 100 μm.

These patterns of KNOX protein accumulation were compared to the distribution of rs2 and kn1 transcripts (17). In wild-type apices, rs2transcripts accumulated throughout the P1 leaf primordium, but in later stages of leaf development (P2 through P5), rs2expression became more restricted to the major vascular bundles and leaf margins (Fig. 3A). rs2was not expressed in the meristem, but expression was observed late during founder cell recruitment, at the transition from the P0 to the P1 stage. In contrast, kn1 was expressed in meristematic cells of the shoot apex but was absent in early leaf founder cells (early P0 stage) (Fig. 3B). No rs2 transcripts were detected in the reference allele of rs2 (rs2-R) mutant shoot apices (Fig. 3C), but the kn1 expression pattern in the meristem was unaltered. As in vegetative apices, kn1expression was limited to meristematic cells in flowers and was down-regulated in floral organ primordia and their initials (Fig. 3, E and H). rs2 transcripts accumulated relatively early in founder cells of floral organ primordia (Fig. 3, D and G), andrs2 expression persisted in young floral organ primordia, including both pistil and stamen (Fig. 3G). No rs2expression was observed in silk (styles), but rs2transcripts were detected during the later stages of stamen development in the tassel. The rs2 expression pattern in normal leaf and floral organ primordia is consistent with the rs2 mutant phenotype and with the pattern of ectopic KNOX protein accumulation inrs2 mutant apices. The differentiation of auricle and sheathlike tissue occurs in sectors along the major lateral veins in the blade region of rs2 mutant leaves. In addition,rs2 mutations frequently affect founder cell recruitment and development of the leaf margins (10).

Figure 3

rs2 and kn1 are expressed in mutually exclusive domains in vegetative and floral apices. (A and B) In situ hybridization of wild-type shoot apices with probes for rs2 (A) or kn1 (B). (C) rs2-R shoot apex hybridized with ars2-specific probe. (D through F) Spikelets with young glume primordia probed with rs2 (D),kn1 (E), or a sense probe derived from rs2 (F). Arrow in (D) marks the accumulation of rs2 transcripts in the P0 initials of the inner and outer lemmas. Arrow in (E) marks the absence of kn1 transcripts in the corresponding positions. (G and H) Comparable sections through a spikelet that has initiated the lower floret, showing the accumulation ofrs2 (G) but not of kn1 (H) in the pistil. The arrows in (G) and (H) mark the adaxial cells on the lower floret that no longer express kn1 but that do express rs2. P, pistil primordium; F2, secondary or lower floret. Scale bar, 50 μm.

The amino acid sequence conservation between RS2 and PHAN and the resemblance of the phan mutant phenotype to that of maize mutants defective in knox gene regulators (6,9–11) suggest that RS2 and PHAN have analogous functions. Consistent with this, expression of the Antirrhinum homeobox gene SHOOTMERISTEMLESS (AmSTM) (6) is restricted to meristematic tissues in wild-type Antirrhinum but was observed in corollas and young leaves of the phan mutant (Fig. 4) (18). However, unlikers2, phan mutants exhibit adaxial/abaxial polarity defects (11). This suggests that knoxexpression in leaves has different effects on leaf development in maize and Antirrhinum. KNOX proteins delay the transition from cell proliferation to differentiation (5, 8). The spatial pattern of this transition in leaves differs among plant species, resulting in different leaf morphologies. In maize, this pattern is basipetal, such that ectopic knox expression results in a distal displacement of features (5). The development of abaxial features on the adaxial leaf surface of phan mutant leaves suggests that this transition in Antirrhinum is deferred along both the proximodistal and adaxial/abaxial axes. Alternatively, PHAN may affect the regulation of additional target genes, or aspects of the rs2 gene function may be masked by the action of other maize genes with partially redundant functions.

Figure 4

Ectopic AmSTM expression in lateral organs of the Antirrhinum phan mutant. Reverse transcriptase–PCR analysis of the expression levels of theknox gene, AmSTM, and the Antirrhinum UBIQUITIN gene in vegetative shoot apices and lateral organs (lanes 2 through 4 and 6 through 8) of either wild-type (lanes 1 through 4) or phan-mutant (lanes 5 through 8) plants. lvs, leaves.

Our observations suggest that rs2 and PHAN act directly or indirectly to maintain knox genes in an “off” state, preventing their expression in leaf and floral primordia and their founder cells. Thus, the rs2- andPHAN-encoded Myb-domain proteins have a function that is analogous to the CURLY LEAF gene of Arabidopsis, which encodes a Polycomb-like factor that suppresses the expression of floral homeotic genes in vegetative parts of the plant (19). In organisms such as Drosophila and yeast, key homeobox genes are spatially regulated by chromatin remodeling factors that confer “cellular memory” (20). The patchiness of phenotypic effects in rs2 mutant leaves, together with the apparent clonal sectors of KNOX protein accumulation, is suggestive of an imperfect silencing of knox gene activity that is clonally propagated during primordium development. Although the rs2 alleles that we analyzed are null alleles,rs2 mutant leaves appear as mosaics of normal and abnormal cell differentiation, and KNOX proteins accumulate only in a variable subset of the normal rs2-expressing cells. In addition, thers2 and phan mutants exhibit phenotypes that are dependent on temperature and on genetic background (6, 10,11). These observations suggest that rs2 acts onknox genes as an epigenetic regulator (21).

  • * To whom correspondence should be addressed. E-mail: timothy.nelson{at}


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