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First published online 16 February 2005
doi: 10.1242/dev.01671

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thick tassel dwarf1 encodes a putative maize ortholog of the Arabidopsis CLAVATA1 leucine-rich repeat receptor-like kinase

Peter Bommert^1,*, China Lunde², Judith Nardmann¹, Erik Vollbrecht^2,, Mark Running^2,, David Jackson³, Sarah Hake² and Wolfgang Werr^1,

¹ Institut für Entwicklungsbiologie, Universität zu Köln, Gyrhofstrasse 17, D-50923 Köln, Germany
² Plant Gene Expression Center, Agricultural Research Service - USDA, 800 Buchanan St, Albany, CA 94710, USA
³ Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA

View larger version (78K): [in a new window]   Fig. 1. Tassel development in td1 mutants. (A) Tassel rachis of normal sibling with some spikelets removed at the base. (B) Tassel rachis of td1-Ref mutant with spikelets removed. (C) A single spikelet of td1-Ref with three glumes (numbered). (D) td1-Ref spikelet with two florets, one of which contains four stamens (arrowed). (E) Paired spikelets from a normal tassel. (F) Normal spikelet opened to reveal two florets, each with three stamens. Images are from td1-Ref introgressed into W22 and the normal siblings in an F2 family.

View larger version (77K): [in a new window]   Fig. 2. Ear development in td1 mutants. (A) Comparison of normal (left) and td1-Ref ear (right) at 20 days after pollination. (B) Scanning electron micrograph (SEM) of normal ear. (C-F) SEMs of td1 mutant ears. The regular arrangement of the SPMs is disturbed, especially at the tip of the ear. Occasionally, SPMs give rise to three SMs (E, numbered asterisks). SPMs are frequently larger than normal (F; arrows). (G) In-situ hybridization with kn1 on normal ear or (H) td1-Ref ear. Scale bars: 500 µm in B,C,E; 100 µm in D,F.

View larger version (116K): [in a new window]   Fig. 7. Ear morphology in td1/fea2 double mutants compared with single mutant and normal siblings. (A) SEM of a td1/fea2 double mutant ear compared with td1- (B) and fea2- (C) single mutant ears. (D) td1/fea2 double mutant ears exhibit an additive and synergistic phenotype as they produce more kernel rows in a highly irregular pattern (see also Table 3). In addition ears were shorter than their single mutant and normal siblings. Double heterozygote ears show no defects compared with normal siblings. Scale bars: 1 mm.

View larger version (44K): [in a new window]   Fig. 3. The CLAVATA1 protein and phylogeny. (A) Schematic of predicted domains in the CLV1 protein. The 21 LRR-motifs are indicated in gray, the kinase domain is depicted in orange; SP: signal peptide; TM: transmembrane domain. Black arrows indicate the position of the degenerate primers. (B) Phylogenetic analysis with LRR-RLK sequences obtained from NCBI or DDBJ databases. The kinase domain sequences were aligned using the CLUSTALW program (http://www.ebi.ac.uk/clustalw/-index.html). The phylogenetic reconstruction was performed with the Minimum Evolution algorithm provided by the MEGA2.1 software (Kumar et al., 2001). The tree indicates the existence of two closely related CLV1-like subfamilies, including the newly isolated maize sequences ZmKin1, 2, 3 and 5. Arabidopsis thaliana: CLV1 gi:15222877, RLK1 gi:30698151, RLK2 gi:15229189, RLK3 gi:15235366, RLK4 gi:15225805, RLK5 gi:15220056; Oryza sativa: OsLRK1 gi:8132685, FON1 AB182388, RLK2 gi:34915202 Glycine max: GmRLK1 gi:9651941, GmRLK2 gi:9651943, GmRLK3 gi:9651945, GmClv1a gi:11260266, GmNARK gi:25732530; Pisum sativum: RLK gi:24940244; Lotus japonicus: RLK gi:24940156; Streptocarpus duneii: RLK AY061836.

View larger version (37K): [in a new window]   Fig. 5. td1 transcripts are decreased in a td1-Mu allele. (A) RNA gel blot of 2 µg polyA(+) RNA from immature ears of td1-glf and normal siblings was hybridized to a td1 specific probe and revealed an abnormal sized transcript (asterisk). The same membrane was stripped and re-probed with the kn1 cDNA as a loading control. (B) Real-time RT-PCR also showed that the level of td1 RNA was reduced in td1-glf (20%) relative to wild type, whereas the level of the kn1 meristem marker was slightly increased.

View larger version (86K): [in a new window]   Fig. 4. Molecular characterization of td1. (A) CLUSTALW alignment of TD1 (from B73 inbred line; top line) and CLV1 (below, GenBank Accession No.: 15222877). Identical residues are outlined in black, similar in gray; dashes represent gaps introduced to optimize the alignment. The black arrow indicates the position of the single intron. Predictions of transmembrane and signal sequences are by SMART (http://smart.embl-heidelberg.de/) for TD1 and from Clark et al. (Clark et al., 1997) for CLV1. Domains are labeled below the respective sequences; yellow, signal peptide; gray, extracellular LRR domain, numbering of single LRR motifs; blue, transmembrane domain; red, kinase domain (the 12 subdomains are labeled SDI-SDXII). Cysteine pairs are marked by asterisks above the respective sequences. The defect in the polypeptide encoded by td1-Ref is shown above TD1. (B) Schematic representation of the Mu element insertion sites within the different td1 alleles.

View larger version (77K): [in a new window]   Fig. 6. Organ- and tissue-specific expression analysis of td1. Organ-specific analysis of td1 transcription was carried out by quantitative real-time RT-PCR using endogenous ubiquitin transcript levels for normalization. Highest td1 transcript level is detected in the vegetative apex arbitrarily set to 100%. The RNA level is lower in the female inflorescence (58%). Low td1 transcript levels are detected in the embryo (11%) or the root (11%). A significant td1 transcript level is also detected in young leaves (63%). (B-F) In-situ expression pattern of td1 in shoot meristems at different stages of development. (B) Late vegetative shoot apex, showing td1 expression in developing leaves but also absence of detectable td1 expression in the SAM. (C) Tip of an ear primordium, showing weak td1 expression in the three outermost layers of the IM and td1 expression within SMs. (D) Tip of an ear primordium showing td1 expression in adaxial parts of the emerging SPMs. (E) Developing SM (stage F), showing expression within the SM and also in developing inner and outer glume and outer lemma primordia. (F) Upper and lower floral meristem (stage H): td1 is expressed strongly within the lower FM, but also in the developing ovule, silk and stamen primordia of the upper FM. Gi, inner glume; Go, outer glume; L, leaf; Lo, outer lemma; St, stamen primordium; S, silk primordium; O, ovule primordium; Pi, inner palea; LFM, lower floral meristem.