An histidine covalent receptor and butenolide complex mediates strigolactone perception

Abstract

Strigolactone plant hormones control plant architecture and are key players in both symbiotic and parasitic interactions. They contain an ABC tricyclic lactone connected to a butenolide group, the D ring. The DWARF14 (D14) strigolactone receptor belongs to the superfamily of α/β-hydrolases, and is known to hydrolyze the bond between the ABC lactone and the D ring. Here we characterized the binding and catalytic functions of RAMOSUS3 (RMS3), the pea (Pisum sativum) ortholog of rice (Oryza sativa) D14 strigolactone receptor. Using new profluorescent probes with strigolactone-like bioactivity, we found that RMS3 acts as a single-turnover enzyme that explains its apparent low enzymatic rate. We demonstrated the formation of a covalent RMS3-D-ring complex, essential for bioactivity, in which the D ring was attached to histidine 247 of the catalytic triad. These results reveal an undescribed mechanism of plant hormone reception in which the receptor performs an irreversible enzymatic reaction to generate its own ligand.

Figure 1. RMS3 can interact with and hydrolyze GR24 enantiomers and is stabilized by these compounds

(a) Chemical structures of one natural strigolactone, (±)-solanacol, and GR24 stereoisomers. (b) Melting temperature curves of RMS3 and mutant proteins in the presence of different concentrations of (±)-GR24, as assessed by differential scanning fluorimetry (DSF). Each line represents the average protein melt curve for three technical replicates and the experiment was carried out twice. (c) Titration of RMS3 interaction with (±)-GR24, monitored by intrinsic fluorescence at 340 nm. Changes in fluorescence were used to calculate the dissociation coefficient (K_d) of (±)-GR24 with RMS3. Each data point is the mean of two technical replicates. (d-f) Elution profile of the enzymatic assay with buffer, RMS3 or RMS3^S96A and GR24 analogs. UPLC-DAD (200–400 nm) analysis showing the formation of ABC and an unknown derivative (MW 270) (confirmed by mass spectrometry analyses) from (d) (±)-GR24, (e) (±)-2’-epi-GR24, and (f) (+)-GR24 and (−)-GR24. AU, absorbance unit. UPLC-DAD, ultra-performance liquid chromatography method with diode array detection. The chromatograms show representative results observed in two independent experiments with two technical replicates.

Figure 2. Characterization of the profluorescent probe (±)-GC242

(a) Chemical structures and principle of the profluorescent probe. (b) Length of the axillary bud for rms1-10 and rms3-5 pea plants, 8 days following direct application of (±)-GC242. Data are means ± SE (≥ 20 plants) (c) Axillary bud length for rms1-10 pea plants, 8 days following direct application of (±)-GR24, (+)-GC242, (−)-GC242. Data are means ± SE (≥ 18 plants). Asterisks indicate significant differences from control values (***p < 0.001, ** p < 0.01, Kruskal-Wallis rank sum test). (d) Number of axillary shoots after hydroponic treatment of max4-1 Arabidopsis plants with (±)-GR24, (±)-GC242 (1 μM). Data are means ± SE (≥ 19 plants). (***p < 0.001, Kruskal-Wallis rank sum test). All experiments (b,c,d) were repeated twice. (e) Arabidopsis hypocotyl length in response to (±)-GR24 or (±)-GC242 of Col-0 (WT), Atd14-1, htl-3, Atd14-1 htl-3, and max2-1 Arabidopsis mutants. Data are means ± SE (≥ 10 plants). Asterisks indicate significant difference from corresponding acetone treatment (CTL) (***p < 0.001, ** p < 0.01, * p < 0.05, Student’s t test). The experiments were repeated twice. (f) Melting temperature curves for RMS3 and mutant proteins at varying concentrations of (±)-GC242, as assessed by DSF. Each line represents the average protein melt curve for three replicate samples run in parallel. (g) Titration of RMS3 interaction with (±)-GC242 monitored by fluorescence. Each data point is the mean ± SD of three technical replicates and three or four independent experiments, which gave similar results.

Figure 3. Enzymatic kinetics reveal that the serine and histidine of the catalytic triad are essential for RMS3 and AtD14 function

(a,d-e) Comparison of enzymatic kinetics when RMS3, AtD14, AtHTL, and different RMS3 mutant proteins were incubated with (±)-GC242. (a) Progress curves during (±)-GC242 hydrolysis. RMS3, RMS3^S96A, RMS3^S96C and RMS3^H247A catalyzed hydrolysis with 400 nM of protein (and 1700 nM for RMS3^S96C) and 500 nM of (±)-GC242. (b-c) Complementation of the Atd14-1 mutant by different D14 mutant proteins. The Atd14-1 mutant was transformed with a chimeric construct consisting of the native D14 promoter fused to either the WT D14 coding sequence and a 6xHA tag (pD14::D14-6xHA), or an otherwise identical coding sequence in which Ser97 was mutated to Ala (pD14::D14^S97A-6xHA) or to Cys (pD14::D14^S97C-6xHA), and H247 was mutated to Ala (pD14::D14^H247A-6xHA). T2 segregating seeds were grown for 40 days. Data are means ± SE of at least 10 plants. Asterisks indicate significant difference from WT Col 0 (***p < 0.001, Student’s t test). Further methodological details are provided in Methods. Scale bar = 4 cm. The experiments were repeated twice. (d) Progress curves during (±)-GC242 hydrolysis. RMS3, AtD14, and AtHTL catalyzed hydrolysis with 400 nM of protein and 500 nM of (±)-GC242. (e) RMS3 and AtD14 pre-steady-state kinetics reaction velocity with (±)-GC242. (a,d) The progress curves show representative results observed in three independent experiments with three technical replicates. (e) Each data point is the mean ± SE of three technical replicates.

Figure 4. RMS3 acts as single turnover enzyme

(a-d) DiFMU concentration progress curves during (±)-GC242 hydrolysis. The release of DiFMU was monitored (λ_em 460 nm) at 25 °C. The progress curves show representative results observed in two independent experiments with two technical replicates. (a) RMS3-catalyzed hydrolysis of (±)-GC242 with different substrate concentrations in the presence of 400 nM protein. (b) RMS3-catalyzed hydrolysis with different protein concentrations and (±)-GC242 (500 nM). (c) (±)-GC242 (500 nM) hydrolysis with 2 successive additions of RMS3 protein (400 nM). Black arrows indicate protein additions. (d) (±)-GC486 (1 μM) hydrolysis with RMS3 protein (330 nM) versus (±)-GC242 and (±)-GC240 (1 μM) hydrolysis with RMS3 protein. (e) Effect of (±)-ABC tricycle on the enzyme kinetics of RMS3 shown as a Lineweaver–Burk plot. (f) Effect of (±)-GR24 on the enzyme kinetics of RMS3 shown as a Lineweaver–Burk plot. These plots were used to determine the K_i value. (e-f) Each data point is the mean ± SE of three technical replicates.

Figure 5. Formation of a stable RMS3-D-ring complex

(a) Mass spectra of RMS3 and RMS3-D-ring complex. Deconvoluted electrospray mass spectra of RMS3 before and after addition of (±)-GR24 (500 μM final concentration) in denaturant and native conditions. Peaks with an asterisk correspond to RMS3 covalently bound to D-ring (RMS3-D). The mass increment of 98 ± 2 Da in denaturant condition and 96 ± 2 Da in native condition measured for RMS3-GR24. Bold circle indicates RMS3 modified with a-N-Gluconoylation. Different unannotated peaks correspond to Na⁺ adducts. (b) Mass spectra of the peptides (GHLPHLSAPSYLAHQLE (266-281) (246-262 in RMS3)) obtained after digestion of RMS3-HIS, RMS3-HIS + (±)-GR24, RMS3-HIS + (±)-GC242 by endoproteinase gluC. [a.u.], arbitrary unit. (c) Proposed kinetic mechanism for SL perception. [ ] = complex, black dot (?) symbolizes a covalent interaction and white dot (○) a non–covalent interaction. For RMS3_x, subscript indicates the amino acid involved in the covalent interaction, k_y = association constant and k_-y = dissociation constant. Double arrows indicate equilibrium and single arrows an irreversible reaction. Dotted arrows indicate hypothetical step. The grey arrow indicates the hypothetical product inhibition. The release of the ABC tricycle has been arbitrarily indicated during the first step of the hydrolysis.