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First published online May 28, 2004
doi: 10.1242/10.1242/dev.01164

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The maize duplicate genes narrow sheath1 and narrow sheath2 encode a conserved homeobox gene function in a lateral domain of shoot apical meristems

Judith Nardmann^1,*, Jiabing Ji^2,*, Wolfgang Werr^1, and Michael J. Scanlon^2,

¹ Institut für Entwicklungsbiologie, Universität zu Köln, Gyrhofstr 17, D-50923 Köln, Germany
² Department of Plant Biology, University of Georgia, Athens, GA 30602, USA

View larger version (55K): [in a new window]   Fig. 1. The narrow sheath mutant phenotype is a deletion of a lateral compartment that includes the leaf margins. (A) Model depicting the recruitment of maize founder cells in two distinct compartments, corresponding to the central domain (green) and the ns lateral domain (yellow). (B) Cartoon of a transverse section through the maize founder cells. The model predicts that NS functions to recruit the lateral founder-cell domains from two distinct foci (red arrows), one corresponding to each side of the leaf. Cartoons model the ns mutant (D) and the non-mutant leaf primordium (C). Non-mutant leaves comprise at least two mediolateral compartments, the central domain (green) and the ns lateral domain (yellow). The ns mutant leaf exhibits a deletion of the lateral domain, which includes the margins of the leaf blade, the leaf sheath and internode. Note that the central compartment includes the midrib and the leaf tip, domains that are intact in ns mutant leaves. The ns mutant phenotype is a duplicate factor trait, dependent upon mutations in both ns1 and ns2. (E) Mature maize leaves from plants homozygous for non-mutant alleles of Ns1 and Ns2. (F) ns mutant leaves homozygous for mutations in both ns1 and ns2. Non-mutant leaves from plants of the complimentary genotypes Ns1, ns2 (G), and ns1, Ns2 (H). SAM, shoot apical meristem.

View larger version (55K): [in a new window]   Fig. 2. (A) NS1 and NS2 protein sequence compared with PRS of Arabidopsis and the closest rice relative. Note the very high similarity of the homeodomain (underlined) and the high sequence conservation between the maize and rice proteins at the C terminus. (B) Phylogeny of NS/PRS proteins. The NS proteins are more similar to PRS than they are to WUSCHEL, or to other WUSCHEL-like proteins from Arabidopsis thaliana. The unrooted tree was generated using the neighbor-joining method from a CLUSTALW alignment of the homeodomain. Bootstrap values were calculated from 1000 replicates. The maximal parsimony method yielded a phylogenetic tree with identical topology (bootstrap values calculated from 1000 replicates are shown in parentheses). AtPRS (Arabidopsis thaliana, BAB79446), AtWUS (Arabidopsis thaliana, CAA09986), AtHD (Arabidopsis thaliana, NP_188428), OsHD1 (Oryza sativa, CL042143.26.34) and OsHD2 (Oryza sativa, BAA90492).

View larger version (53K): [in a new window]   Fig. 3. Analyses of ns alleles. (A) A hybridization probe (probe 1; Materials and methods) derived from a PRS-homologous, maize genomic clone identifies two distinct DraI restriction fragments linked to the ns mutant phenotype in F2 (ns-RxB73) segregating progeny. Note that no internal DraI restriction sites are present within the sequence of probe 1 obtained from either ns mutant or B73 individuals. (B) Composite gene map of the ns duplicate genes, each of which comprises two exons and a single intron. Exons are boxed, the positions of the homeodomain (HD), the glutamine-rich region (Q), the histidine-rich region (H) and the PLK domain are indicated. Solid lines indicate introns and untranslated regions. The position of the extra G in ns2-R and of the CACTA element insertion in ns1-R are indicated above the drawing. The dashed line below the drawing indicates regions of the ns2 locus that are deleted in the ns2-Mu1 allele. (C) Newly identified ns1-Mu* alleles are deletions. Active mutator lines Ns1/Ns1; ns2-R/ns2-R (ns1-Mu6/7 Mu parent) were pollinated with ns1-R/ns1-R; ns2-R/ns2-R (ns-R parent) pollen and analyzed for phenotypic progeny in the M1 generation. Southern analysis of ns mutant progeny (shown are ns1-Mu1 or ns1-Mu3) showed lack of the ns1 wild-type (6 kb) band but presence of the ns1-R (4.5 kb) fragment when hybridized to probe 2 (Materials and methods), whereas wild-type siblings (ns1-Mu1/3 sibs) always contained both bands (6 kb + 4.5 kb). Note that probe 2 hybridizes more weakly to ns2 because it includes 5' UTR sequence specific to the ns1 locus. (D) Plants homozygous for the ns2-*Mu1 mutant allele exhibit no hybridizing restriction fragment corresponding to the ns2 locus, whereas non-mutant siblings exhibit a 3.5 kb band linked to ns2. (E) NS protein does not accumulate in young ears obtained from ns1-R, ns2-R mutant plants. Polyclonal antibodies raised against an oligopeptide that is conserved in the predicted NS1 and NS2 proteins recognize a protein (arrow) in homozygous non-mutant ears, as well as in non-mutant ears from plants that contain a single non-mutant allele of Ns1 but are homozygous for the ns2-R mutant allele. This 29 kDa band is absent in ears from ns mutant plants, and corresponds to the predicted molecular weight of the NS proteins. Note that the NS polyclonal antibody is not specific for NS proteins.

View larger version (92K): [in a new window]   Fig. 4. ns transcripts predominately accumulate in tissues enriched for shoot meristems and young primordia. (A) Schematic drawing of coleoptile development. The coleoptile (col) emerges from the periphery of the SAM and grows to form a coleoptilar ring that eventually encloses the SAM. The frame in the cartoon of the coleoptilar stage embryo on the left indicates the plane of the transverse section depicted to the right. (B) In situ hybridization of transverse sections of developing maize embryos reveal that ns transcripts accumulate in the tips of the emerging coleoptile. (C) Cartoon depicting a median longitudinal section through the shoot apex of a maize seedling 14 days after germination. The midrib regions of leaf primordia (C-H) are designated by plastochron (P) number, such that the incipient primordium (on the SAM flank) is labeled P0, the next oldest leaf is labeled P1, and so on. The margins (r) of the corresponding leaf primordia are found on the flank of the SAM opposing the midrib. Arrows indicate the position of transverse sections shown in D-F; corresponding close-up images are shown in G-I. (D-I) In situ hybridization of serial transverse sections reveal that ns transcripts accumulate in the marginal edges of leaf primordia (r in G-I) and also in two lateral foci in the founder cells of the new leaf primordium (P1 in D and G). (J) Schematic drawing of maize flower development [based on Cheng et al. (Cheng et al., 1983)]. Longitudinal (K,M-P) and transverse (L) sections through female inflorescences or florets at different developmental stages show ns activity in marginal cells of all floral organs: glumes (K,L), lemmas (M-O), paleas (K-M), stamens (N-P) and gynoecium (O,P). Expression of ns in the gynoecium is detected in the gynoecial ridge (O), a small cleft to the ovule primordium is marked by an asterisk. (K) Longitudinal section of a female inflorescence. (L) Transverse section of a spikelet meristem. (M) Longitudinal section of an upper and lower floret meristem. (N) Longitudinal section of a slightly older flower than that shown in M. (O) Longitudinal section of an upper flower. (P) Longitudinal section of a slightly older upper flower than that shown in O. (Q) Schematic drawing of a longitudinal section through a maize flower, with ns1 expression domains depicted in red. IM, inflorescence meristem; SPM, spikelet pair meristem; ig, inner glume; og, outer glume; UFM, upper floret meristem; LFM, lower floret meristem; il, inner lemma; ol, outer lemma; ip, inner palea; op, outer palea; gy, gynoecium; st, stamen. Scale bars: 50 µm.

View larger version (19K): [in a new window]   Fig. 5. Quantitative real-time RT-PCR data of transcript abundance (relative to the level of ns2 transcript in vegetative apices and normalized to ubiquitin transcript levels) in maize tissues employing primers specific for ns1 or ns2 transcripts (see Materials and methods). ns transcripts accumulate in tissues enriched for vegetative shoot meristems and inflorescence meristems (apices, ears and tassels), but are not detected in mature lateral organs (coleoptile, juvenile leaves) or roots.

View larger version (105K): [in a new window]   Fig. 6. Phenotypes of Prs- mutant Arabidopsis leaves and cotyledons. At maturity non-mutant (A) and Prs- mutant (E) rosette leaves have equivalent phenotypes. Whole-mount (B,C,F,G) and scanning electron microscopic (D,H) analyses of non-mutant (B-D) and Prs- mutant (F-H) leaf primordia (L1, L2) reveal that the lateral stipules (S) are deleted from mutant leaves. Note that the size of the emerging trichomes (T) indicates that the lack of stipule development in Prs- mutant primordia is not due to differences in the developmental age of these samples. No obvious Prs- mutant phenotype is noted in comparisons of mature (I,J) or primordial (not shown) non-mutant (I) and Prs- mutant (J) cotyledons. (K-M) Expression of PRS during Arabidopsis embryogenesis. (K) PRS expression is localized to the tips of the prospective cotyledons in the heart stage embryo. In torpedo stage embryos, PRS is expressed at the apices (L) and the lateral margins (M) of the cotyledons (c). PRS-expressing cell layers therefore define a border between adaxial and abaxial side of the cotyledons, similar to the maize coleoptile (Fig. 4B). Scale bars: 50 µm.

View larger version (24K): [in a new window]   Fig. 7. The domain deletions observed in Prs- mutant and ns mutant leaves are consistent with a model describing the differential elaboration of upper versus lower leaf zones during the morphological evolution of monocot and eudicot bifacial leaves. Details are provided in the text; models are adapted from Troll (Troll, 1955), as elaborated by Kaplan (Kaplan, 1973).

View larger version (41K): [in a new window]   Fig. 8. (A) NS activity (red line) in the maize shoot apex and at the margins of emerging leaf primordia (P0-P3) in wild type. (B) Model of competing founder-cell domains in ns mutants.

View larger version (19K): [in a new window]   Fig. 9. Model for a conserved NS/PRS function during the recruitment of a lateral founder-cell domain in vegetative (A,B) and reproductive (C) shoot meristems of maize (A) and Arabidopsis (B,C). NS/PRS function initiates from lateral foci (red arrows) and recruits founder cells in a lateral meristematic domain (yellow). Recruitment of the central domain founder cells (green) occurs prior to recruitment of the lateral domain. In vegetative apices that produce a single leaf per node, recruitment of the central founder-cell domain occurs from a single flank of the apex and proceeds bi-directionally toward the lateral domain foci, whereas in the Arabidopsis floral meristem this recruitment commences simultaneously from both the adaxial and abaxial flanks of the apex. Loss of NS/PRS function causes failure to develop any and all organ domains that are normally derived from the lateral founder-cell domain of the meristem. Further details of this organ domain-blind model of NS/PRS are explained in the text.