Strigolactone perception and deactivation by a hydrolase receptor DWARF14

Abstract

The perception mechanism for the strigolactone (SL) class of plant hormones has been a subject of debate because their receptor, DWARF14 (D14), is an α/β-hydrolase that can cleave SLs. Here we show via time-course analyses of SL binding and hydrolysis by Arabidopsis thaliana D14, that the level of uncleaved SL strongly correlates with the induction of the active signaling state. In addition, we show that an AtD14^D218A catalytic mutant that lacks enzymatic activity is still able to complement the atd14 mutant phenotype in an SL-dependent manner. We conclude that the intact SL molecules trigger the D14 active signaling state, and we also describe that D14 deactivates bioactive SLs by the hydrolytic degradation after signal transmission. Together, these results reveal that D14 is a dual-functional receptor, responsible for both the perception and deactivation of bioactive SLs.

Fig. 1

Chemical structures of SL-related compounds and a scheme for their biosynthesis and signaling pathways. a Structures of SL-related compounds. b The scheme for the SL biosynthesis and signaling pathways. Red, blue, orange, and green characters indicate genes of Arabidopsis, rice, petunia, and pea, respectively. Black arrows indicate the biosynthetic steps, and a white arrow indicates the signaling step. (CCD; carotenoid cleavage dioxygenase)

Fig. 2

Evaluation of the AtD14-SL interaction using hydrolysis and DSF assays. a Melting temperature curves of AtD14 in the presence of various SLs and analogs. The names in red and blue denote biologically active and inactive (or weakly active) compounds, respectively. b and d, Monitoring by LC-MS/MS of the AtD14 hydrolysis reaction of GR24 (b) and CN-PMF (d). HBN; Hydroxybenzenenitrile. Data are the means ± SD (n = 3). c and e Melting temperature curves of AtD14 pre-incubated with GR24 (c) and CN-PMF (e) for indicated time period. Source data are provided as a Source Data file

Fig. 3

In vitro and in vivo functional analysis of catalytic triad mutants of AtD14. a Hydrolysis activities of catalytic triad mutants of AtD14 using 1 μM of 5DS as a substrate. Data are the means ± SD (n = 3). The control reaction (Cont.) is for MBP protein only. b No. of axillary shoots (over 5 mm) of Arabidopsis transgenic plants expressing each catalytic triad mutant of AtD14 in the atd14-2 mutant background. Data are the means ± SD (n = 5–10, Different letters indicate significant differences at P < 0.05 with Tukey-kramer multiple comparison test.). c Phenotypes of Arabidopsis transgenic plants expressing each catalytic triad mutant of AtD14. Mature 50 days old plants phenotypes (upper panel) and leaf morphology phenotypes of 25 days old plants (lower panel) are shown. Scale bars = 5 cm (upper panel), 1 cm (lower panel). d Y2H analysis of the interaction between SMXL7 and each catalytic triad mutant of AtD14. Yeast transformants were spotted onto the control medium (SD−Leu/−Trp (−TL)) and selective medium (SD−Leu/−Trp/−His (−TLH)) in the absence or presence of SLs (10 μM rac-MeCLA or 10 μM 5DS). Control (Cont.) is acetone only. e Shoot branching inhibition assays of the Arabidopsis transgenic lines expressing AtD14^WT and AtD14^D218A, respectively, in the atd14 max4 double mutants background. The bars indicate the No. of axillary shoots (over 5 mm) in the presence (+) or absence (−) of GR24 at 5 μM (left panel) and 0.5 μM (right panel), respectively. Data are the means ± SD (n = 3–13, Different letters indicate significant differences at P < 0.05 with Tukey-kramer multiple comparison test). Source data are provided as a Source Data file

Fig. 4

In vitro and in vivo functional analysis of OsD14^R233H/AtD14^R133H. a Phenotypes of 2 weeks old seedlings (left) and mature plants (right) of the rice d14-2 mutant. The white arrow in the left picture indicates the outgrowing tiller. Scale bars = 5 cm (left panel), 20 cm (right panel). b Hydrolase activities of OsD14^R233H and AtD14^R133H mutants using 1 μM 5DS as a substrate. Data are the means ± SD (n = 3–4). c No. of tillers of rice transgenic plants overexpressing OsD14^R233H in the WT (Nipponbare) background. Empty vector expressing plants are indicated as EV. Data are the means ± SD (n = 3–5). d Phenotypes of 42 days old transgenic plants overexpressing OsD14^R233H (OsD14^R233HOE). Scale bars = 10 cm. e Quantitative analysis of 4DO in the root exudates (left panel) and extracts (right panel) of OsD14^R233H overexpressing (OsD14^R233HOE) plants. Data are the means ± SD (n = 3–4). Different letters in c and e indicate significant differences at P < 0.05 with Tukey-kramer multiple comparison test. Source data are provided as a Source Data file

Fig. 5

A proposed working model of D14 in the SL signaling pathway. A bioactive SL molecule induces the protein conformational changes of D14, which triggers complex formation with the signaling partners. After the degradation of D53/SMXLs and transmission of the SL signal, D14 reconstructs the catalytic triad to hydrolytically decompose the bioactive SL