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On the substrate specificity of the rice strigolactone biosynthesis enzyme DWARF27

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Abstract

Main conclusion

The β-carotene isomerase OsDWARF27 is stereo- and double bond-specific. It converts bicyclic carotenoids with at least one unsubstituted β-ionone ring. OsDWARF27 may contribute to the formation of α-carotene-based strigolactone-like compounds.

Strigolactones (SLs) are synthesized from all-trans-β-carotene via a pathway involving the β-carotene isomerase DWARF27, the carotenoid cleavage dioxygenases 7 and 8 (CCD7, CCD8), and cytochrome P450 enzymes from the 711 clade (MAX1 in Arabidopsis). The rice enzyme DWARF27 was shown to catalyze the reversible isomerization of all-trans- into 9-cis-β-carotene in vitro. β-carotene occurs in different cis-isomeric forms, and plants accumulate other carotenoids, which may be substrates of DWARF27. Here, we investigated the stereo and substrate specificity of the rice enzyme DWARF27 in carotenoid-accumulating E. coli strains and in in vitro assays performed with heterologously expressed and purified enzyme. Our results suggest that OsDWARF27 is strictly double bond-specific, solely targeting the C9–C10 double bond. OsDWARF27 did not introduce a 9-cis-double bond in 13-cis- or 15-cis-β-carotene. Substrates isomerized by OsDWARF27 are bicyclic carotenoids, including β-, α-carotene and β,β-cryptoxanthin, that contain at least one unsubstituted β-ionone ring. Accordingly, OsDWARF27 did not produce the abscisic acid precursors 9-cis-violaxanthin or -neoxanthin from the corresponding all-trans-isomers, excluding a direct role in the formation of this carotenoid derived hormone. The conversion of all-trans-α-carotene yielded two different isomers, including 9′-cis-α-carotene that might be the precursor of strigolactones with an ε-ionone ring, such as the recently identified heliolactone.

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Abbreviations

CCD:

Carotenoid cleavage dioxygenase

MBP:

Maltose binding protein

SL:

Strigolactone

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Acknowledgments

We thank Dr. Peter Beyer, University of Freiburg, Germany, for valuable discussions and Dr. Hansgeorg Ernst, BASF, Germany, for providing the synthetic apocarotenoid substrates. The research reported in this publication was supported by funding from King Abdullah University of Science and Technology (KAUST) and the EU (METAPRO; FP7 KBBE-2009-3-1-01).

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Authors and Affiliations

  1. BESE Division, King Abdullah University of Science and Technology (KAUST), 4700, 23955-6900, Thuwal, Kingdom of Saudi Arabia

    Salim Al-Babili

  2. Faculty of Biology, Albert-Ludwigs University of Freiburg, Schaenzlestr. 1, 79104, Freiburg, Germany

    Mark Bruno & Salim Al-Babili

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  1. Mark Bruno
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Correspondence to Salim Al-Babili.

Electronic supplementary material

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425_2016_2487_MOESM1_ESM.pptx

Suppl. Fig. S1 SDS PAGE of maltose binding protein (MBP) purified D27-fusion protein. Lanes represent: M, molecular marker (size in kDa on the left). a Solubilized total lysate of control cells expressing MBP after French press. b Total lysate of MBP-OsD27 producing cells. c Supernatant from (b) after centrifugation. d Flow through obtained after (C) binding to amylose resin. e Eluate of control, (2) indicating the expressed MBP. f Eluate from amylose resin of OsD27-fusion protein; (1) pMAL-OsD27 fusion protein (PPTX 99 kb)

425_2016_2487_MOESM2_ESM.pptx

Suppl. Fig. S2 HPLC analysis of in vitro assays with apocarotenoids: We did not observe any isomerization of all-trans β-apo-8′-carotenal (a) or for all-trans β-apo-10′-carotenal (b). UV/Vis spectra of substrates are summarized in Suppl. Fig. S5, for chemical structures see Fig. 1. HPLC system 1 was employed for separation (PPTX 67 kb)

425_2016_2487_MOESM3_ESM.pptx

Suppl. Fig. S3 In vitro activity of MBP-OsD27 with linear and monocyclic carotenes: No conversion was observed when purified MBP-fusion protein was incubated with the linear all-trans-lycopene (a) or the monocyclic γ-carotene (b). For separation, we used systems 2 (a) and 1 (b). UV/Vis spectra of substrates are summarized in Suppl. Fig. S5, for chemical structures see Fig. 1 (PPTX 68 kb)

425_2016_2487_MOESM4_ESM.pptx

Suppl. Fig. S4 In vitro activity of MBP-OsD27 with xanthophylls: The MBP-OsD27 did not convert the dihydroxylated all-trans zeaxanthin (a) and all-trans-lutein (b), nor the epoxydized all-trans-viola- (c) and neoxanthin (d). UV/Vis spectra of substrates are summarized in Suppl. Fig. S5, for chemical structures see Fig. 1. We used HPLC system 1 for analysis (PPTX 106 kb)

425_2016_2487_MOESM5_ESM.pptx

Suppl. Fig. S5 UV/Vis spectra of substrates and products: (I) all-trans-β-carotene, (II) 9-cis-β-carotene, (III) 13-cis-β-carotene, (IV) 15-cis-β-carotene, (V) all-trans-α-carotene, (VI) 9-cis-α-carotene, (VII) 9′-cis-α-carotene, (VIII) all-trans-ε,ε-carotene, (IX) all-trans-β,β-cryptoxanthin, (X) 9-cis-β,β-cryptoxanthin, (XI) all-trans-zeaxanthin, (XII) all-trans-lutein, (XIII) all-trans-violaxanthin, (XIV) all-trans-neoxanthin, (XV) all-trans-lycopene, (XVI) all-trans-γ-carotene, (XVII) all-trans-β-apo-8′-carotenal, (XVIII) β-apo-10′-carotenal (PPTX 92 kb)

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Bruno, M., Al-Babili, S. On the substrate specificity of the rice strigolactone biosynthesis enzyme DWARF27. Planta 243, 1429–1440 (2016). https://doi.org/10.1007/s00425-016-2487-5

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