Arabidopsis brassinosteroid-insensitive dwarf12 mutants are semidominant and defective in a glycogen synthase kinase 3beta-like kinase

Abstract

Mutants defective in the biosynthesis or signaling of brassinosteroids (BRs), plant steroid hormones, display dwarfism. Loss-of-function mutants for the gene encoding the plasma membrane-located BR receptor BRI1 are resistant to exogenous application of BRs, and characterization of this protein has contributed significantly to the understanding of BR signaling. We have isolated two new BR-insensitive mutants (dwarf12-1D and dwf12-2D) after screening Arabidopsis ethyl methanesulfonate mutant populations. dwf12 mutants displayed the characteristic morphology of previously reported BR dwarfs including short stature, short round leaves, infertility, and abnormal de-etiolation. In addition, dwf12 mutants exhibited several unique phenotypes, including severe downward curling of the leaves. Genetic analysis indicates that the two mutations are semidominant in that heterozygous plants show a semidwarf phenotype whose height is intermediate between wild-type and homozygous mutant plants. Unlike BR biosynthetic mutants, dwf12 plants were not rescued by high doses of exogenously applied BRs. Like bri1 mutants, dwf12 plants accumulated castasterone and brassinolide, 43- and 15-fold higher, respectively, providing further evidence that DWF12 is a component of the BR signaling pathway that includes BRI1. Map-based cloning of the DWF12 gene revealed that DWF12 belongs to a member of the glycogen synthase kinase 3beta family. Unlike human glycogen synthase kinase 3beta, DWF12 lacks the conserved serine-9 residue in the auto-inhibitory N terminus. In addition, dwf12-1D and dwf12-2D encode changes in consecutive glutamate residues in a highly conserved TREE domain. Together with previous reports that both bin2 and ucu1 mutants contain mutations in this TREE domain, this provides evidence that the TREE domain is of critical importance for proper function of DWF12/BIN2/UCU1 in BR signal transduction pathways.

Figure 1

Phenotypic comparison of the BR biosynthetic mutant dwf4-1, and the insensitive mutants bri1-5, twisted1-60, and dwf12. Plants shown are 25 d old. dwf12 exhibits characteristic BR dwarf phenotypes including short inflorescences, infertility of homozygous mutants, short round leaves, and abnormal de-etiolation. In addition, dwf12 leaves show more severe downward curling than dwf4 and bri1. This phenotype and the mild twisting of inflorescences appear to be weaker version of twd1 (B. Schulz and K. Feldmann, unpublished data). The height of dwf12 heterozygous plants is intermediate between the homozygote and wild type, suggesting that the mutation is semidominant. Unit bar = 2 cm. In the inset, one set each of the adaxial side of a cauline leaf, a rosette leaf, and a silique of Ws-2 wild type, dwf12-1D, dwf12-2D, dwf12-1D/+, and dwf12-2D/+ are shown. Note the extreme downward curling of the leaves. Siliques of the homozygous dwf12 mutants are almost completely sterile and contain few seeds.

Figure 2

A, Dose response of wild type, dwf4-1, bri1-5, and dwf12-1D to epi-BL. Different doses of epi-BL are C, control; −9, 10⁻⁹ m; −8, 10⁻⁸ m; and −7, 10⁻⁷ m. A wild-type seedling at 10⁻⁷ m shows typical BR responses, such as elongated hypocotyls and petioles as well as shortened root length. In addition, the BR biosynthetic mutant dwf4-1 is fully rescued to a wild-type phenotype at a concentration as low as 10⁻⁸ m, showing expanded cotyledons and an elongated hypocotyl. However, dwf12-1D seedlings did not respond to epi-BL regardless of the concentrations, confirming that dwf12-1D is defective in sensing or downstream signaling. Similar insensitivity is shown also in the bri1-5 mutant. Unit bar = 1 cm. B, Quantitative analysis of the hormone dose response tests. Root growth inhibition of wild type is decreased linearly and proportionally to the epi-BL concentration. In contrast, both dwf12-1D and bri1-5 are significantly insensitive to epi-BL at all concentrations tested. The two signaling mutants also show altered responses to abscisic acid (ABA) and auxin in that they are more sensitive to ABA but slightly resistant to auxin. Percent root length represents the ratio of the root length grown on BL-supplemented media over the root length grown on the control media containing the same amount of 95% (v/v) ethyl alcohol used to dilute BL from a 4 mm stock solution.

Figure 3

Endogenous levels of BRs in wild-type (Ws-2), dwf12-1D, and bri1-5 plants. Similar to Arabidopsis bri1 plants, dwf12-1D accumulates significant amounts of BRs, especially typhasterol (TY), castasterone (CS), and BL. BR biosynthetic pathways with major intermediates and enzyme names are shown with their chemical structures. nd, Not detected; na, not analyzed; unit = ng g fresh weight⁻¹.

Figure 4

Map-based cloning of DWF12. A, Chromosome 4 with major genetic markers. In addition to previously available markers, we have developed novel simple sequence length polymorphism (SSLP) markers, including Ga, Na, Da, La, Ma, Ba, Sa, Aa, and Ja (Table III). B, Localization of the mutation between CH42 and AG markers. The number of recombinants found with CH42 and AG and dwf12-1D is two and one of 560 examined chromatids, respectively. C, Geography around the DWF12 gene annotated as F28A21.120 by the Arabidopsis Genome Initiative. D, The schematic of DWF12 with mutations. The gene consists of 10 exons and nine introns. The two dwf12 mutations are in exon 8, changing consecutive Glu residues to Lys. The dwf12-2D mutation abolishes an XhoI site (CTCGAG to CTCGAA). Schematics are drawn to the scales that are depicted on the top right of each diagram.

Figure 5

Multiple sequence alignment of two Arabidopsis GSK3-like protein and GSK3s from human, D. melanogaster, and D. discoideum. DWF12 (GenBank accession no. AY157149) and KGSQ_ARATH (Q96287), possessing the longest amino acid sequence among 10 Arabidopsis GSK3-like proteins, were compared with GSK3β proteins from human (P49841), D. melanogaster (SGG_DROME, P18431), and D. discoideum (KG3H_DICDI, P51136). Alignment was performed using the PileUp program of the Genetics Computer Group software with a gap creation penalty of 3 and a gap extension penalty of 1. The aligned sequences were further annotated using BOXSHADE and Photoshop programs. Annotations are based on structural characterization described by Dajani et al. (2001). Black circles indicate the residues involved in active site formation. White squares identify the residues that are involved in homodimerization. The TREE domain was identified to be a putative Thr phosphorylation site by caseine kinase II, and the two dwf12 mutations were located in this domain. Thick lighter bars delimit an N- or C-terminal variable domain, and a darker bar spans the protein kinase domain. Numbers in parentheses are based on the DWF12 protein sequence. Dashes and dots indicate gaps introduced to maximize alignment. Δ, Truncation of amino acid sequences for better comparison.