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.

Keywords: BAHD acyltransferase; heterostyly.

Figure 1

Phenotypes of short (S-morph) and long (L-morph) style morphs. (A) Diagram showing reciprocal arrangement of stigma and style (S) and anthers and stamens (A). (B,C) Longitudinal section of a mature flower showing the short morph and long morph of Turnera subulata.

Figure 2

Confirmation of insert in transgenic lines in A. thaliana. Three independent lines of the TsBAHD overexpression construct. Each 35S::TsBAHD line has been confirmed in severe (S), intermediate (I), and weak (W) dwarf phenotypes, represented by the three bands under each 35S::TsBAHD line in corresponding order. Expected fragment size is 683 bp. Each lane contained 5 µL of PCR product and 2 µL of loading dye. LAD was 5 µL of a 1 kb DNA ladder.

Figure 3

Phenotypes and expression of 35S::TsBAHD-25 in A. thaliana normalized to the actin 8 (At1g4920) housekeeping gene. (A) From left to right: six week old plants of wild type (Col-0) compared to three 35S::TsBAHD (line 25) transformants showing weak, intermediate, and most severe dwarf phenotypes. (B) Expression of 35S::TsBAHD lines 25, 54, and 63 in S, I, and W dwarfs compared to the wild type. Wild-type samples were included as a negative control. Errors bars represent standard error.

Figure 4

Relative expression of BR biosynthesis and inactivating genes in 35S::TsBAHD normalized to an actin housekeeping gene. Expressions of DWF4 (A), CPD (B), and BAS1 (C) in six week old 35S::TsBAHD lines 24, 54, and 63. All 35S::TsBAHD transformants showing S, I, and W phenotypes compared to the Col-0 wild type. Each qRT-PCR value is the mean of three replicates. Errors bars represent standard error. p-values comparing 35S::TsBAHD lines to the wild type were calculated using Student’s t-test comparing each line to the wild-type expression * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Hypocotyl response to different concentrations of brassinolide (BL) in light (A,B) and dark (C,D). Seedlings pictured are representatives of wild type and 35S::TsBAHD lines from plates with 0 nM BL. Hypocotyls of 4 day old seedlings were grown on plates at 25 °C in continuous light at an intensity of 80 µE and in complete darkness. Plates contained half strength Linsmaier and Skoog modified basal medium, 0.6% Gellan, and 1.5% sucrose. BL concentrations were 0, 10, and 100 nM. Green dotted lines represent two replicates of three independent lines of 35S::TsBAHD and solid gray line represents the wild type. Errors bars represent standard error. Scale bars = 2 mm.