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

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.Strigolactones (SLs) are allelochemicals, exuded from plant roots, that stimulate seed germination of root parasitic plants, Striga spp., Orobanche spp., and Phelipanche spp. (1). The hyphal branching of the biotrophic arbuscular mycorrhizal (AM) fungi is also induced by SLs in the vicinity of host roots to ensure symbiosis with host plants (2). SLs are not only host recognition signals in the rhizosphere but also play important roles in the SL-producing plants themselves. Since the mid-1990s, the existence of novel hormone-like signals involved in shoot branching inhibition of plants had been proposed following the isolation and analysis of mutants with increased shoot branching, ramosus (rms) of pea (Pisum sativum), decreased apical dominance (dad) of petunia (Petunia hybrida), more axillary growth (max) of Arabidopsis (Arabidopsis thaliana), and dwarf (d) and high tillering dwarf (htd) of rice (Oryza sativa) (3–6). Recently, these mutants have been identified as SL-deficient or -insensitive mutants, providing decisive evidence that SLs function as shoot branch-inhibiting hormones (7, 8). In addition, further characterization of these mutants has shown that SLs affect root growth and development, leaf shape and senescence, internode elongation, secondary growth, and drought and salinity stress responses (9–11).Despite the fact that SLs play important roles in plant growth and development and in the rhizosphere, the biosynthesis pathway of SLs has not fully been elucidated. The natural SLs consist of a tricyclic lactone (ABC ring) connecting to a butenolide group (D ring) via an enol ether bridge. 5-Deoxystrigol (5DS) and ent-2′-epi-5-deoxystrigol [4-deoxyorobanchol (4DO); Fig. 1] are thought to be the precursors of other natural SLs, which have methyl group(s) on the A ring and hydroxyl or acetyloxyl group(s) on the A/B ring (1, 12). Because the mutations in the CCD7 (MAX3/RMS5/HTD1) and CCD8 (MAX4/RMS1/DAD1/D10) genes, both of which encode carotenoid cleavage dioxygenases, result in SL deficiency (7, 8), it has been thought that SLs are synthesized from carotenoids by these enzymes. Recently, it has been demonstrated that the Fe-containing protein D27 catalyzes the isomerization at C-9 of all-trans-β-carotene to produce 9-cis-β-carotene in vitro (13) (Fig. 1). The product 9-cis-β-carotene was a substrate for CCD7 to produce 9-cis-β-apo-10′-carotenal, and this cleavage product was subsequently catalyzed by CCD8 to produce an SL precursor named carlactone (CL) (13) (Fig. 1). More recently, we reported that CL was detected from rice and Arabidopsis, and exogenous CL was converted into SLs in rice, demonstrating that CL is an endogenous precursor for SLs (14). Because CL contains the A and D rings and the enol ether bridge but lacks the B and C rings, additional biosynthetic steps are needed for the conversion of CL to 5DS and 4DO in plants.Open in a separate windowFig. 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.The most probable enzyme catalyzing these reactions is MAX1 (CYP711A1), a cytochrome P450 monooxygenase (5). In reciprocal grafting experiments of Arabidopsis, the hyperbranching phenotype in scions of the max4 (ccd8) mutant was rescued to WT shoot branching patterns when grafted to max1 rootstocks, whereas max4 rootstocks could not restore a WT shoot branching phenotype to max1 scions (5). These results suggested that MAX1 acts on a downstream pathway of CCD8 to produce a mobile signal for shoot branching inhibition. Recently, it was reported that CL could not rescue the max1 phenotype by exogenous application (15), and we found an extreme accumulation of CL in the max1 mutant (14). Hence, CL is the most probable candidate for the substrate of MAX1. In the present study, to elucidate the enzymatic function of MAX1 in SL biosynthesis, we performed in vitro conversion of CL using a recombinant MAX1 protein expressed in yeast microsomes. We then examined if CL is metabolized in a similar manner in vivo by detecting and identifying the CL metabolites in Arabidopsis and rice plants. In addition, to investigate the role of the CL derivatives for shoot branching inhibition, we examined their biological activities and interaction with Arabidopsis thaliana DWARF14 (AtD14), a putative SL receptor.