The Turnera Style S-Locus Gene TsBAHD Possesses Brassinosteroid-Inactivating Activity When Expressed in Arabidopsis thaliana

Abstract

Heterostyly distinct hermaphroditic floral morphs enforce outbreeding. Morphs differ structurally, promote cross-pollination, and physiologically block self-fertilization. In Turnera the self-incompatibility (S)-locus controlling heterostyly possesses three genes specific to short-styled morph genomes. Only one gene, TsBAHD, is expressed in pistils and this has been hypothesized to possess brassinosteroid (BR)-inactivating activity. We tested this hypothesis using heterologous expression in Arabidopsis thaliana as a bioassay, thereby assessing growth phenotype, and the impacts on the expression of endogenous genes involved in BR homeostasis and seedling photomorphogenesis. Transgenic A. thaliana expressing TsBAHD displayed phenotypes typical of BR-deficient mutants, with phenotype severity dependent on TsBAHD expression level. BAS1, which encodes an enzyme involved in BR inactivation, was downregulated in TsBAHD-expressing lines. CPD and DWF, which encode enzymes involved in BR biosynthesis, were upregulated. Hypocotyl growth of TsBAHD dwarfs responded to application of brassinolide in light and dark in a manner typical of plants over-expressing genes encoding BR-inactivating activity. These results provide empirical support for the hypothesis that TsBAHD possesses BR-inactivating activity. Further this suggests that style length in Turnera is controlled by the same mechanism (BR inactivation) as that reported for Primula, but using a different class of enzyme. This reveals interesting convergent evolution in a biochemical mechanism to regulate floral form in heterostyly.

Pistil Mating Type and Morphology Are Mediated by the Brassinosteroid Inactivating Activity of the S-Locus Gene BAHD in Heterostylous Turnera Species

“…The weak dwarfs (displaying the mildest phenotype) had slightly paler, larger green leaves and longer inflorescences, but were still substantially reduced in size relative to wild-type controls ( Figure 4 A,B). These phenotypes are typical of BR-deficient mutants [ 26 , 27 , 28 , 29 ] and are indistinguishable with those identified for TsBAHD [ 20 ]. Phenotypic variability was also previously observed in transformants of TsBAHD and is likely a result of transgene suppression [ 20 ].…”

“…These phenotypes are typical of BR-deficient mutants [ 26 , 27 , 28 , 29 ] and are indistinguishable with those identified for TsBAHD [ 20 ]. Phenotypic variability was also previously observed in transformants of TsBAHD and is likely a result of transgene suppression [ 20 ]. To confirm that the dwarfism observed in plants transformed with TkBAHD was caused by altered active BR levels, we assessed expression of genes involved in BR homeostasis.…”

“…Ectopic expression in Arabidopsis thaliana has been used a bioassay for brassinosteroid inactivating activity [ 25 ] and we recently used this approach to demonstrate that the acyltransferase encoded by TsBAHD possesses this activity [ 20 ]. We employed this bioassay to investigate whether the mutation in the GN motif in the allele associated with homostyly ( TkBAHD H ) caused a loss brassinosteroid inactivating activity relative to the wild type allele.…”

“…TsYUC6 , which is expressed only in anthers, has been proposed to control pollen incompatibility mating type through auxin synthesis [ 19 ]. The third S -gene, TsBAHD , is the sole S -gene expressed in pistils and has conserved motifs found in BAHD acyltransferases [ 19 , 20 ]. The closest characterized A. thaliana homologs of TsBAHD include BIA1 , a gene reported to acetylate and inactivate BRs [ 21 ].…”

“…When over-expressed in A. thaliana , BIA1 causes a phenotype that mimics mutants defective in BR synthesis, severe dwarfism, shortened stems and petioles, small epinastic, dark green leaves, delayed senescence, and infertility [ 21 ]. We recently took advantage of this phenotype to use A. thaliana as a bioassay to confirm that TsBAHD possesses BR inactivating activity [ 20 ]. As the only S -gene expressed in pistils, BAHD is predicted to control all female characteristics, namely pistil length and female biochemical mating type.…”