Carlactone is converted to carlactonoic acid by MAX1 in Arabidopsis and its methyl ester can directly interact with AtD14 in vitro

Abstract

Strigolactones (SLs) stimulate seed germination of root parasitic plants and induce hyphal branching of arbuscular mycorrhizal fungi in the rhizosphere. In addition, they have been classified as a new group of plant hormones essential for shoot branching inhibition. It has been demonstrated thus far that SLs are derived from carotenoid via a biosynthetic precursor carlactone (CL), which is produced by sequential reactions of DWARF27 (D27) enzyme and two carotenoid cleavage dioxygenases CCD7 and CCD8. We previously found an extreme accumulation of CL in the more axillary growth1 (max1) mutant of Arabidopsis, which exhibits increased lateral inflorescences due to SL deficiency, indicating that CL is a probable substrate for MAX1 (CYP711A1), a cytochrome P450 monooxygenase. To elucidate the enzymatic function of MAX1 in SL biosynthesis, we incubated CL with a recombinant MAX1 protein expressed in yeast microsomes. MAX1 catalyzed consecutive oxidations at C-19 of CL to convert the C-19 methyl group into carboxylic acid, 9-desmethyl-9-carboxy-CL [designated as carlactonoic acid (CLA)]. We also identified endogenous CLA and its methyl ester [methyl carlactonoate (MeCLA)] in Arabidopsis plants using LC-MS/MS. Although an exogenous application of either CLA or MeCLA suppressed the growth of lateral inflorescences of the max1 mutant, MeCLA, but not CLA, interacted with Arabidopsis thaliana DWARF14 (AtD14) protein, a putative SL receptor, as shown by differential scanning fluorimetry and hydrolysis activity tests. These results indicate that not only known SLs but also MeCLA are biologically active in inhibiting shoot branching in Arabidopsis.

Keywords: Arabidopsis; biosynthesis; cytochrome P450; rice; strigolactone.

Fig. 1.

Proposed biosynthesis pathway for SL from carotenoid. The conversion from β-carotene to CL by D27, CCD7, and CCD8 enzymes has been confirmed previously by in vitro assay (13). The conversion from CL to CLA by MAX1 and the existence of CLA and MeCLA in Arabidopsis were shown in this study.

Fig. 2.

Identification of 19-hydroxy-CL and CLA produced from CL by recombinant MAX1. rac-CL was incubated with MAX1 microsomes. The extracts of the microsomes and authentic standards were analyzed by LC-MS/MS [a triple quadrupole/linear ion trap instrument (QTRAP)]. SRM chromatograms (Left) and product ion spectra (Right) derived from the precursor ion [M+H–H₂O]⁺ (m/z 301) of (A) 19-hydroxy-CL and [M–H]^– (m/z 331) of (B) CLA are shown.

Fig. 3.

Conversions of stereoisomers 11R-CL and 11S-CL by recombinant MAX1. 11R-CL and 11S-CL were incubated with MAX1 microsomes and the extracts were analyzed using LC-MS/MS (QTRAP). SRM chromatograms of products, 19-hydroxy-CL (Left) and CLA (Right), in MAX1 microsomes and authentic standards are shown.

Fig. 4.

Endogenous analysis of CLA in Arabidopsis. Endogenous CLA was detected in WT, the max2 and atd14 mutants by LC-MS/MS [a quadrupole/time-of-flight instrument (QTOF)]. (A) Ion traces from the LC-MS/MS analysis (Left) and product ion spectra (Right) derived from respective precursor ions of [1-¹³CH₃]-labeled (internal standard) and endogenous CLA extracted from max2 roots as a representative data and those of unlabeled authentic standard are shown. (B) Endogenous CLA was quantified in root extracts of WT, max1, max4, atd14, and max2 using [1-¹³CH₃]CLA as an internal standard by LC-MS/MS (QTOF). Data are the means ± SD (n = 3). n.d., not detectable.

Fig. 5.

Identification of endogenous MeCLA in Arabidopsis. Endogenous SL-LIKE1 was identified as MeCLA using LC-MS/MS (QTOF). Ion traces from the LC-MS/MS analysis (Left) and product ion spectra (Right) derived from respective precursor ions of [10-²H₁]MeCLA (internal standard) and endogenous SL-LIKE1 extracted from roots of Arabidopsis atd14 mutant and those of unlabeled authentic MeCLA are shown.

Fig. 6.

Inhibitory effects of CL derivatives on increased lateral inflorescences of Arabidopsis max mutants. Each 10 μM solution of CL, 19-hydroxy-CL, CLA, and MeCLA (all racemic mixtures) was applied onto the basal region of the primary inflorescences of max1, max4, and max2 every second day for 2 wk, and then the number of lateral inflorescences from rosette leaves was counted. The 0.5% (vol/vol) acetonitrile solution without authentic sample was used as a mock treatment. Values are mean ± SEM (n = 15). The number of lateral inflorescence was 1.4 ± 0.3 in WT treated with the mock solution. Different letters indicate significant differences tested by the Holm-Sidak method under ANOVA (P < 0.05).

Fig. 7.

Biochemical analysis of the interaction between AtD14 and CL derivatives. DSF assays of AtD14 were carried out in the presence of CL, CLA, MeCLA, and GR24 (all racemic mixtures). The melting temperature curves of AtD14 are shown. atd14:S97A, the mutant protein having a substitution change in the active site.