Carlactone is converted to carlactonoic acid by MAX1 inArabidopsisand 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.strigolactone | biosynthesis | cytochrome P450 | Arabidopsis | rice S trigolactones (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 SLproducing 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 branchinhibiting 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).Despit...

Nitro-Phenlactone, a Carlactone Analog with Pleiotropic Strigolactone Activities

“…SLs are synthesized from carotenoids via carlactone, which lacks the B/C-ring (Alder et al, 2012). In Arabidopsis, carlactone ( Figure 1A) is converted by MAX1, a member of CYP711 family, into carlactonoic acid that can, after methylation, bind to SL receptor (Abe et al, 2014). In rice, a MAX1-homolog, the carlactone oxidase, introduces B/C-rings into carlactone, yielding 4-deoxyorobanchol (Supplemental Figure 1) (Zhang et al, 2014), the precursor of canonical orobanchol-like SLs.…”

“…In Arabidopsis, carlactone is converted by the biosynthetic enzyme MAX1 before exerting SL activities (Abe et al, 2014). To test the effect of rac-N-PL on shoot branching and to determine whether the activity is MAX1-dependent, we treated the biosynthetic mutants max1 and max4, and the SL perception mutant d14 with this compound in hydroponic assays, using rac-GR24 as a positive control.…”

LATERAL BRANCHING OXIDOREDUCTASEacts in the final stages of strigolactone biosynthesis inArabidopsis

“…A second MAX1 enzyme oxidizes 4DO to make orobanchol (4). In Arabidopsis, the situation is less clear, because it has only a single MAX1 gene, and the encoded protein oxidizes C19 of CL to produce carlactonoic acid (CLA), but it does not apparently catalyze formation of B and C rings (3,4,6) (Fig. 1).…”

“…1). Based on interaction with receptor D14, it was proposed that MeCLA is bioactive, whereas CL and CLA are not bioactive (6). Grafting experiments in Arabidopsis suggest that both the upstream and downstream products of MAX1 are graft-transmissible: that is, the MAX1 substrate and products can move from the rootstock to the shoot (5).…”

From little things big things grow: karrikins and new directions in plant development

“…Mutation of the catalytic triad residues renders D14 nonfunctional as an enzyme, and also as a transducer of strigolactone signalling in planta (Hamiaux et al 2012). A critical observation is that binding of strigolactone to D14 induces a marked change in protein melting temperature, suggesting that the ligand-protein interaction imparts a change in D14 conformation (Hamiaux et al 2012;Zhao et al 2013;Abe et al 2014;Waters et al 2015b). Consistent with this interpretation, crystallography demonstrated the ligand-dependent interaction of D14 and D3/MAX2, together with the Arabidopsis SCF.…”