3-Hydroxycarlactone, a Novel Product of the Strigolactone Biosynthesis Core Pathway

Abstract

Strigolactones (SLs) are a class of carotenoid-derived plant hormones that regulate shoot branching among other developmental processes (Gomez-Roldan et al., 2008Gomez-Roldan V. Fermas S. Brewer P.B. Puech-Pages V. Dun E.A. Pillot J.-P. Letisse F. Matusova R. Danoun S. Portais J.-C. et al.Strigolactone inhibition of shoot branching.Nature. 2008; 455: 189-194Crossref PubMed Scopus (1508) Google Scholar, Umehara et al., 2008Umehara M. Hanada A. Yoshida S. Akiyama K. Arite T. Takeda-Kamiya N. Magome H. Kamiya Y. Shirasu K. Yoneyama K. et al.Inhibition of shoot branching by new terpenoid plant hormones.Nature. 2008; 455: 195-200Crossref PubMed Scopus (1393) Google Scholar, Al-Babili and Bouwmeester, 2015Al-Babili S. Bouwmeester H.J. Strigolactones, a novel carotenoid-derived plant hormone.Annu. Rev. Plant Biol. 2015; 66: 161-186Crossref PubMed Scopus (464) Google Scholar). In addition, SLs are released by roots as a chemical signal attracting symbiotic arbuscular mycorrhizal fungi. However, this signal is also perceived by seeds of root parasitic weeds, announcing the presence of a host and triggering germination (Al-Babili and Bouwmeester, 2015Al-Babili S. Bouwmeester H.J. Strigolactones, a novel carotenoid-derived plant hormone.Annu. Rev. Plant Biol. 2015; 66: 161-186Crossref PubMed Scopus (464) Google Scholar). Natural SLs consist of a butenolide ring (D ring) connected by an enol ether bridge to a tricyclic lactone (ABC rings) in canonical SLs, such as strigol, and to a variable, second moiety in non-canonical SLs, such as methyl carlactonoate. There are around 25 known natural SLs (Xie, 2016Xie X. Structural diversity of strigolactones and their distribution in the plant kingdom.J. Pestic. Sci. 2016; 41: 175-180Crossref Scopus (47) Google Scholar) that differ in the stereochemistry of the B/C junction and in modifications of the ABC-ring of canonical SLs, and in the structure of the second moiety of the non-canonical ones (Al-Babili and Bouwmeester, 2015Al-Babili S. Bouwmeester H.J. Strigolactones, a novel carotenoid-derived plant hormone.Annu. Rev. Plant Biol. 2015; 66: 161-186Crossref PubMed Scopus (464) Google Scholar). Carlactone (CL), the central intermediate in SL biosynthesis, is a C19 molecule derived from all-trans-β-carotene by the sequential action of three enzymes through a stereospecific pathway. The isomerase DWARF27 (D27) converts all-trans-β-carotene into 9-cis-β-carotene, which is cleaved by the stereospecific carotenoid cleavage dioxygenase 7 (CCD7) into 9-cis-β-apo-10′-carotenal and β-ionone. The next enzyme, CCD8, catalyzes a combination of reactions that yield CL and x-OH-(4-CH3)heptanal (Alder et al., 2012Alder A. Jamil M. Marzorati M. Bruno M. Vermathen M. Bigler P. Ghisla S. Bouwmeester H. Beyer P. Al-Babili S. The path from β-carotene to carlactone, a strigolactone-like plant hormone.Science. 2012; 335: 1348-1351Crossref PubMed Scopus (587) Google Scholar, Bruno et al., 2017Bruno M. Vermathen M. Alder A. Wüst F. Schaub P. van der Steen R. Beyer P. Ghisla S. Al-Babili S. Insights into the formation of carlactone from in-depth analysis of the CCD8-catalyzed reactions.FEBS Lett. 2017; 591: 792-800Crossref PubMed Scopus (34) Google Scholar). Cytochrome P450 enzymes (CYP711; MAX1 in Arabidopsis) convert CL into non-canonical; e.g., carlactonoic acid in Arabidopsis, and canonical SLs, such as 4-deoxyorobanchol in rice (Jia et al., 2018Jia K.P. Baz L. Al-Babili S. From carotenoids to strigolactones.J. Exp. Bot. 2018; 69: 2189-2204Crossref PubMed Scopus (111) Google Scholar). 4-Deoxyorobanchol is the parent molecule of orobanchol-like canonical SLs and is converted in rice by a MAX1 homolog, the orobanchol synthase, into orobanchol (Zhang et al., 2014Zhang Y. van Dijk A.D. Scaffidi A. Flematti G.R. Hofmann M. Charnikhova T. Verstappen F. Hepworth J. van der Krol S. Leyser O. et al.Rice cytochrome P450 MAX1 homologs catalyze distinct steps in strigolactone biosynthesis.Nat. Chem. Biol. 2014; 10: 1028-1033Crossref PubMed Scopus (237) Google Scholar). Normal shoot branching in Arabidopsis requires SL(s) arising by hydroxylation of methyl carlactonoate, which is catalyzed by Lateral Branching Oxidoreductase (reviewed in Jia et al., 2018Jia K.P. Baz L. Al-Babili S. From carotenoids to strigolactones.J. Exp. Bot. 2018; 69: 2189-2204Crossref PubMed Scopus (111) Google Scholar). It is supposed that 5-deoxystrigol, the parent molecule of strigol-like canonical SLs, which shows an opposite stereo-configuration at the B/C junction, is also formed from CL (Jia et al., 2018Jia K.P. Baz L. Al-Babili S. From carotenoids to strigolactones.J. Exp. Bot. 2018; 69: 2189-2204Crossref PubMed Scopus (111) Google Scholar). CCD7 enzymes show strict stereo- and wide substrate-specificity in vitro by cleaving hydroxylated 9-cis-carotenoids, such as 9-cis-zeaxanthin and -lutein, leading to 9-cis-3-OH-β-apo-10′-carotenal and 9-cis-3-OH-α-apo-10′-carotenal, respectively (Bruno et al., 2014Bruno M. Hofmann M. Vermathen M. Alder A. Beyer P. Al-Babili S. On the substrate- and stereospecificity of the plant carotenoid cleavage dioxygenase 7.FEBS Lett. 2014; 588: 1802-1807Crossref PubMed Scopus (47) Google Scholar). This raised the question of whether CCD8 enzymes can convert other apocarotenals produced by CCD7 into CL-like compounds that may be precursors of as yet unidentified hydroxylated SLs or SLs with ε-ionone rings (Jia et al., 2018Jia K.P. Baz L. Al-Babili S. From carotenoids to strigolactones.J. Exp. Bot. 2018; 69: 2189-2204Crossref PubMed Scopus (111) Google Scholar). To answer this question, we pursued an in vitro approach by incubating crude lysates of Escherichia coli cells expressing Arabidopsis, rice, or pea thioredoxin-CCD8 fusion protein with the CCD7 products 9-cis-3-OH-α-, 9-cis-α-, or 9-cis-3-OH-β-apo-10′-carotenal, followed by high-performance liquid chromatography (HPLC) analysis. This test showed that CCD8 enzymes do not convert 9-cis-α- or 9-cis-3-OH-α-apo-10′-carotenal (Supplemental Figure 1). However, all three enzymes formed a novel product from 9-cis-3-OH-β-apo-10′-carotenal (Figure 1A), which was tentatively identified as 3-OH-carlactone (3-H-CL), based on elution pattern, UV–visible spectrum, and accurate mass (liquid chromatography–tandem mass spectrometry [LC–MS/MS] analysis; Supplemental Figure 2). To verify the nature of the CCD8 product by nuclear magnetic resonance (NMR), we purified around 1 mg from in vitro assays and also synthesized 3-H-CL to be used as a reference (Supplemental Figure 11). Both compounds were then subjected to one-dimensional 1H and two-dimensional (2D) homo- and heteronuclear NMR measurements. The 1H and 13C chemical shift values measured for isolated and synthesized 3-H-CL are summarized in Supplemental Table 1. In addition, the values obtained from chemical shift prediction and experimental values reported for CL (Alder et al., 2012Alder A. Jamil M. Marzorati M. Bruno M. Vermathen M. Bigler P. Ghisla S. Bouwmeester H. Beyer P. Al-Babili S. The path from β-carotene to carlactone, a strigolactone-like plant hormone.Science. 2012; 335: 1348-1351Crossref PubMed Scopus (587) Google Scholar) are given in Supplemental Table 1. A good agreement between the isolated and synthesized 3-H-CL was found. The experimental chemical shift values matched well with the predicted ones. Compared with CL, 3-H-CL gave rise to a typical multiplet at 3.951 ppm, which was assigned to the 3-CH group of 3-H-CL carrying the hydroxy group. This assignment was confirmed by the 1H-1H correlated spectroscopy (COSY) spectrum of the isolated 3-H-CL displayed in Figure 1B. The COSY spectrum shows homonuclear coupling of the 3-CH proton to the neighboring 4-CH and 2-CH protons of the β-ionone ring (Figure 1B, right). Likewise, COSY cross peaks between 7-CH and 8-CH protons could be observed (Figure 1B, left), proving all expected homonuclear couplings in accordance with the structure of 3-H-CL. The 2D 1H-13C heteronuclear single quantum coherence experiment (Supplemental Figure 7) was used to differentiate and assign the different carbon types (-CH3, -CH, -CH2) and to support the proton assignment. The 2D 1H-13C-HMBC experiment, in which long-range couplings between protons and carbons (2,3JCH) is detected, served to assign the signals of quaternary carbons and to independently prove the assignments of the proton-bearing carbons. Taken together, the NMR analysis demonstrates that the product formed by OsCCD8 from 9-cis-3-OH-β-apo-10′-carotenal is 3-H-CL. To confirm that 3-H-CL can be formed in planta, we transiently expressed the cDNAs of OsD27, OsCCD7, and OsCCD8 in Nicotiana benthamiana leaves and analyzed the leaf extracts using LC-MS/MS. As shown in Figure 1C, we detected two signals that correspond to hydroxylated CL, including one that co-eluted with 3-H-CL (13.57 min) produced in vitro. In addition, the comparison of the accurate mass and MS/MS patterns of 3-H-CL from plant leaves and from in vitro reaction confirmed the identification of 3-H-CL in N. benthamiana. We assume that the second peak is an isomer of 3-H-CL. Similar results were obtained upon expressing the corresponding cDNAs from Arabidopsis in N. benthamiana leaves (Supplemental Figure 12). As expected, we also detected CL that was present in higher amounts than 3-H-CL (Figure 1D), which is consistent with the in vitro observation that OsCCD8 converts the CL precursor 9-cis-β-apo-10′-carotenal at a much higher rate than 9-cis-3-OH-β-apo-10′-carotenal (data not shown). The formation of 3-H-CL in N. benthamiana leaves required the co-expression of the three cDNAs involved in CL formation; i.e. OsD27, OsCCD7, and OsCCD8. Expression of single cDNA or combinations of two cDNAs did not lead to detectable amounts of 3-H-CL (Supplemental Figure 13). These results suggest that 3-H-CL is formed in planta by the same enzymes mediating CL synthesis. It is likely that 9-cis-3-OH-β-apo-10′-carotenal is produced from 9-cis-zeaxanthin that can arise directly from 9-cis-β-carotene by hydroxylation of the β-ionone rings (Figure 1E). To assess the role of β-carotene hydroxylases (BCHs), which are required for zeaxanthin formation, in determining the amount of formed 3-H-CL, we co-expressed a cDNA coding for one of the two rice BCHs, OsBCH100 (NCBI: XM_015758320) and OsBCH500 (NCBI: XM_015773287.1), with the three cDNAs required for CL synthesis. However, we did not observe any significant change in the zeaxanthin/β-carotene ratio in infiltrated leaves (Supplemental Figure 14) or in the amounts of 3-H-CL (Figure 1D). Next, we checked the presence of 3-H-CL in rice by analyzing roots of the SL perception mutant d14, which is known to accumulate the SL intermediate CL (Seto et al., 2014Seto Y. Sado A. Asami K. Hanada A. Umehara M. Akiyama K. Yamaguchi S. Carlactone is an endogenous biosynthetic precursor for strigolactones.Proc. Natl. Acad. Sci. USA. 2014; 111: 1640-1645Crossref PubMed Scopus (208) Google Scholar). Results obtained (Supplemental Figure 15) demonstrate that 3-H-CL is an endogenous rice compound that likely acts as an intermediate of SL biosynthesis. To test whether 3-H-CL can act as a parasitic seed germination stimulant, we applied different concentrations of the in vitro produced 3-H-CL on Striga seeds and determined the germination rates, in comparison with the common SL analog GR24. Although at rates lower than those of GR24, 3-H-CL showed considerable germination activity inducing, at 1 μM concentration, the germination of 22% of Striga seeds, which is around 50% of the activity observed with GR24 at the same concentration (Figure 1F). This result suggests that 3-H-CL or 3-H-CL derivative(s) have SL activity in stimulating root parasitic seed germination. We also tested the hormone activity of 3-H-CL by determining its effect on tiller growth of the rice SL-deficient mutant d10 (ccd8). After 2 weeks of treatment at a concentration of 3.5 μM, in vitro-produced 3-H-CL inhibited the outgrowth of the second tiller (Figure 1H), restoring the phenotype of Shiokari wild-type (Figure 1G). This 3-H-CL effect was comparable with that of GR24 at a concentration of 2.5 μM. In summary, our study identifies 3-H-CL as a metabolite produced by the SL core pathway, which is likely the starting point of a new branch in SL biosynthesis and the precursor of as-yet unidentified SLs.