Brassinosteroid biosynthesis and signalling in Petunia hybrida

Abstract

Brassinosteroids (BRs) are steroidal plant hormones that play an important role in the growth and development of plants. The biosynthesis of sterols and BRs as well as the signalling cascade they induce in plants have been elucidated largely through metabolic studies and the analysis of mutants in Arabidopsis and rice. Only fragmentary details about BR signalling in other plant species are known. Here a forward genetics strategy was used in Petunia hybrida, by which 19 families with phenotypic alterations typical for BR deficiency mutants were identified. In all mutants, the endogenous BR levels were severely reduced. In seven families, the tagged genes were revealed as the petunia BR biosynthesis genes CYP90A1 and CYP85A1 and the BR receptor gene BRI1. In addition, several homologues of key regulators of the BR signalling pathway were cloned from petunia based on homology with their Arabidopsis counterparts, including the BRI1 receptor, a member of the BES1/BZR1 transcription factor family (PhBEH2), and two GSK3-like kinases (PSK8 and PSK9). PhBEH2 was shown to interact with PSK8 and 14-3-3 proteins in yeast, revealing similar interactions to those during BR signalling in Arabidopsis. Interestingly, PhBEH2 also interacted with proteins implicated in other signalling pathways. This suggests that PhBEH2 might function as an important hub in the cross-talk between diverse signalling pathways.

Keywords: BES1; BIN2; BRI1; BZR1; Petunia hybrida; SEC.; brassinosteroids; compact disc; dwarf.

Fig. 1.

Biosynthesis of steroids and BRs, and the Arabidopsis enzymes involved. The metabolic products are shown in boxes and the enzymes involved in the pathway are depicted in red. The predominant pathway to synthesize BL runs from the campestanol (CN)-independent pathway and late C-6 oxodation pathway and is indicated by the red arrows. The CN-dependent pathway is represented by the green arrows. Compounds measured in this study are indicated in a green box.

Fig. 2.

Petunia BR biosynthesis and signalling mutants. (A) Phenotype of 4-week-old Petunia hybrida wild-type (left) and cd1 ^W503, cd2 ^P2015, cd3 ^D2213, and cd9 ^P2023 plants. (B) Top view of a cd1 ^W503 mutant. (C) Complementation of the cd2 ^P2013 mutant but not of the cd10 ^P2020 mutant by spraying with 1 μM 24-epibrassinolide (+BL). Mock indicates plantlets treated with a solution without 24-epibrassinolide.

Fig. 3.

Molecular analysis of two BR biosynthesis mutant families. (A) Mutant phenotype of a cd2 ^W2254 plant. (B) A cd2 ^W2254 plant that develops a revertant shoot (cd2 ^M2194-4). (C) PCR with CYP90A1-specific primers on genomic DNA extracted from plants carrying various CYP90A1 alleles. (D) Schematic drawing of the petunia CYP90A1 gene and mutant alleles. (E) Schematic drawing of the petunia CYP85A1 gene and mutant alleles. In D and E, boxes represent exons and the thin lines represent introns. The triangles and line illustrate the dTph1 transposon insertions and footprint in the indicated alleles. (F) Sequence analysis of the dTph1 insertions in various CYP90A1 and CYP85A1 alleles. The red sequence indicates the target site duplication.

Fig. 4.

Characterization of the BRI1 receptor from petunia. (A) Phylogenetic tree constructed using derived amino acid sequences of the BRI1 homologue isolated from petunia and various other species [At, Arabiopsis thaliana; Hv, Hordeum vulgare; Os, Oryzia sativa; Le, Lycoperscicon esculentum (Solanum lycopersicum); Nb, Nicotiana benthamiana; Nt, Nicotiana tabacum; Ps, Pisum sativum; and Ph, Petunia hybrida]. The petunia BRI1 protein (PhBRI1) is underlined. CLAVATA1 from Arabidopsis was used as an outgroup. GenBank accession numbers are provided in Supplementary Table S2 at JXB online. (B) Schematic drawing of PhBRI1 with its predicted functional domains. The triangle indicates the dTph1 transposon insertion in cd10 ^P2020. (C) PCR with BRI1-specific primers on genomic DNA extracted from cd10 ^P2020 and wild-type plants. (D) Sequence analysis of the dTph1 insertion in cd10. The underlined sequence indicates the target site duplication.

Fig. 5.

Characterization of PhBEH2 and the identification of binding partners by a yeast two-hybrid screen. (A) Phylogenetic tree constructed using derived amino acid sequences of PhBEH2 and the BES1/BZR1 family from Arabidopsis. GenBank accession numbers are provided in Supplementary Table S2 at JXB online. (B) The full-length sequence of PhBEH2 was fused to the yeast GAL4-binding domain and screened against a cDNA library made from young petunia inflorescences. Interactions were measured by growth on medium lacking histidine (–LTH) or histidine and adenine (–LTHA), and by blue colouring using 5-bromo-4-chloro-3-indolyl-β-d-galactosidase (–LT X-gal). (C) Classification of the positive clones from B. As indicated, for some proteins, a homologue with known function is found in Arabidopsis. (D–I) Verification of interactions in petunia protoplasts by BiFC (Hu et al., 2002). (D) Free YFP; (E) PhBEH2–GFP; (F) PhBEH2–YFPN and PSK8–YFPC; (G) PhBEH2–YFPN and 14-3-3κ–YFPC; (H) PSK8–YFPN and PhBEH2–YFPC; (I) PhSEC–YFPN and PhBEH2–YFPC. All constructs are driven by the 35S promoter. All panels show a transformed as well as an untransformed protoplast.

Fig. 6.

Phylogenetic analysis of PSK8/9 and the identification of PSK8 binding partners by a yeast two-hybrid screen. (A) Phylogenetic tree constructed of derived amino acid sequences of PSK8/9 and GSK3-like kinases from Arabidopsis and petunia. MPK1 from Arabidopsis was used as an outgroup to construct the tree, but is not shown in this tree. GenBank accession numbers are shown in Supplementary Table S2 at JXB online. (B) Interaction of PSK8 with eight different clones in a yeast two-hybrid assay. Interactions were measured by growth on medium lacking histidine (–LTH) or histidine and adenine (–LTHA), and by blue colouring using 5-bromo-4-chloro-3-indolyl-β-d-galactosidase (–LT X-gal). (C) Classification of the positive clones from B. When a clear homologue in Arabidopsis is found, their AGI identifier is listed.

Fig. 7.

The BES1/BZR1 family and group II GSK3/SHAGGY-like kinases from petunia and Arabidopsis are functionally homologous. Full-length sequences of AtBIN2, PSK8, and PSK9 were fused to the GAL4-binding domain, and AtBZR1, AtBES1, and PhBEH2 to the GAL4 activation domain, and tested for interaction. Interactions were measured by growth on medium lacking histidine (–LTH) or histidine and adenine (–LTHA).