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First published online 23 February 2005
doi: 10.1242/dev.01708

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Functional diversification of MYB23 and GL1 genes in trichome morphogenesis and initiation

Victor Kirik¹, Myeong Min Lee^2,*, Katja Wester¹, Ullrich Herrmann¹, Zhengui Zheng³, David Oppenheimer³, John Schiefelbein² and Martin Hulskamp^1,

¹ Botanical Institute, University of Cologne, Gyrhofstrasse 15, 50931 Cologne, Germany
² Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA
³ Department of Botany, UF Genetics Institute, University of Florida, 220 Bartram Hall, PO Box 118526, Gainesville, FL 32611-8526, USA

View larger version (44K): [in a new window]   Fig. 1. Molecular analysis and trichome phenotype of myb23 alleles. (A) Schematic illustration of the T-DNA insertions positions in the myb23-1 and myb23-2 alleles. (B) RT-PCR was conducted using MYB23 gene-specific primers for 30, 35 and 40 cycles. After 40 cycles, a weak band is visible in the myb23-2 mutant. Control RT-PCR was carried out with primers specific for the translation elongation factor EF1A4. (C) Three- and four-branched trichomes on wild-type (Col) leaf. (D) Two- and three-branched trichomes on the myb23-1 leaf.

View larger version (77K): [in a new window]   Fig. 2. MYB23 and GL1 proteins are functionally equivalent during trichome initiation. (A) Redundant function of MYB23 and GL1 during trichome initiation. Trichomes at the leaf edges and petioles present in gl1 are absent in the gl1 myb23-2 double mutant. (B) Cis-regulatory sequences, but not protein-coding regions, specify the functions of the MYB23 and GL1 genes in trichome initiation. Schematic presentations of the constructs used for rescue experiment are depicted on the left. Nucleotide numbers indicate the length of the used 5' and 3' flanking sequences before the start and after the stop codon respectively. Phenotypes of the gl1 mutant and gl1 myb23-2 double mutant transformed with the corresponding construct are shown on the right.

View larger version (80K): [in a new window]   Fig. 3. Comparison of the MYB23 and GL1 expression during leaf development. (A) RT-PCR using MYB23 gene-specific primers (35 cycles, top row) or using GL1 gene-specific primers (35 cycles, middle row). RT-PCR using primers for translation elongation factor EF1A4 (20 cycles) were used as a cDNA control. The gl1-1 allele has a deletion of the entire protein-coding region, resulting in the absence of GL1 mRNA (Oppenheimer et al., 1991). (B) Comparison of MYB23::GUS and GL1::GUS expression (blue) in leaves of wild type (WT), ttg1 and gl1 mutants.

View larger version (137K): [in a new window]   Fig. 4. Distribution of the MYB23 mRNA in the shoot. (A,B) Longitudinal section through a vegetative apex hybridized with a MYB23-specific antisense probe. (C) Control hybridization with the labelled MYB23 sense strand probe.

View larger version (144K): [in a new window]   Fig. 5. Genetic analysis of the MYB23 gene functions during trichome differentiation. (A-G) DAPI-stained trichomes on the leaves of wild-type (Wassilewskija) (A), myb23-2 (B), gl3 (C), myb23-2 gl3 (D), try (E), try myb23-1 (F) and GL2::GL3 (G) plants. (H) Trichomes on the gl2 mutant leaf surface. (I) Trichomes on the gl2 myb23-2 leaf. There is no trichome outgrowth at the leaf edge. (J) Trichomes at the leaf edges of the gl2. (K) Trichome at the leaf edges of GL2::MYB23 gl2 plants. (L) gl2 stem trichomes. (M) Stem trichomes in GL2::MYB23 gl2 plants. Scale bars: 100 µm in A-G.

View larger version (17K): [in a new window]   Fig. 6. Relative DNA content of DAPI stained trichomes. Fluorescence intensity was normalized to the mean fluorescence of wild-type trichomes (see Material and methods). RFU, relative fluorescence units; percent, indicates the fraction of the nuclei measured for each line.

View larger version (16K): [in a new window]   Fig. 7. Protein interactions between MYB23, GL1, TTG1, TRY and GL3 in yeast two-hybrid assays. lacZ reporter activity in yeast cells for the pair-wise combination of plasmids is shown. The activity of the negative control (yeast containing plasmids pACT and pAS) represents the background level in yeast cells. Interaction between GL3, GL3-96 and TTG1 demonstrates that the 96 amino acids N-terminal truncated version of GL3 is capable of interaction with TTG1 and is consistent with previous data (Payne et al., 2000). Error bars show standard deviation. AD, GAL4 transcriptional activation domain; BD, GAL4 DNA binding domain.