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

(Molecular Plant 11(10):1312-1314; October 2018; https://doi.org/10.1016/j.molp.2018.06.008) The amounts of CL and 3-H-CL shown in the original Figure 1D and Supplementary Figure 13 are inaccurate, due to an erroneous preparation and mixing up of samples. In addition, there is an error in the designation of the spectra of two peaks in the original Figure 1A, in which the spectrum designated as I should belong to peak II, and vice versa. We have repeated the related experiments and quantification, and the corrected Figure 1 and Supplementary Figure 13 are shown below. Based on new results, the original text related to Figure 1D “However, we did not observe any significant change in the zeaxanthin/β-carotene ratio in infiltrated leaves (Supplementary Figure 14) or in the amounts of 3-H-CL (Figure 1D)” is corrected as “However, we did not observe any significant change in the zeaxanthin/β-carotene ratio in infiltrated leaves (Supplementary Figure 14), but a clear decrease in the amounts of CL and 3-H-CL (Figure 1D)”. The responsible authors apologize for not detecting these errors prior to publication and for any inconvenience that may have caused.Supplemental Figure 13. 3-H-CL and CL quantification in tobacco (N. benthamiana) leaves infiltrated with different combinations of cDNAs of the rice SL biosynthesis enzymes OsD27, OsCCD7 and OsCCD8. Empty vector (EV) was taken as negative control. ND, not detectable. Data are means ± SE (n = 4-6).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Moreover, we provide the detailed methods used to detect CL and 3-H-CL as follows. Detection of CL and 3-H-CL in Nicotiana benthamiana For CL and 3-H-CL analysis, around 50 mg of lyophilized plant material was grinded. The samples were extracted with ethyl acetate containing D3-3-OH-β-apo-13-carotenone (2 ng) and D3-β-apo13-carotenone (2 ng) (Buchem, the Netherlands) as an internal standards in an amber glass vial. The samples were vortexed and sonicated for 15 min in a Branson 5510 ultrasonic bath (Branson Ultrasonics). Samples were centrifuged, and the solvent was transferred to a clean glass vial. The pellets were re-extracted with ethyl acetate. The combined ethyl acetate extracts were dried using Labconco RapidVap. To purify the sample, crude extracts were dissolved in 2 mL of n-hexane and loaded into HyperSep silica 500 mg/3 mL SPE column preconditioned with ethyl acetate followed by n-hexane. Subsequently, columns were washed with n-hexane, and CL and 3-H-CL were eluted with ethyl acetate. Samples were evaporated to dryness, and the residues were dissolved in (acetonitrile: water, 50:50, v/v). The samples were filtered through 0.22 μm PVDF filters before LC-MS/MS analysis. As a standard, we used 13C-3-H-CL produced in vitro from the labeled substrate 13C-3-OH-9-cis-β-apo-10’-carotenal purchased from BUCHEM (the Netherlands). Analysis of CL and 3-H-CL in plant material was performed on a Dionex Ultimate 3000 UHPLC system coupled with a Q-Exactive plus MS (Orbitrap detector, Thermo Scientific) with an electrospray source. UHPLC method I: chromatographic separation was carried out on a Phenomenex Gemini C18 (150 × 2.0 mm, 5 μm) column, at 35°C. The mobile phases A and B was (0.1% formic acid- 95% acetonitrile - 5% water) and (0.1% formic acid- 95% water- 5% acetonitrile), respectively. The gradient used was 20 min, 25%-100% A; 20-25 min, 100% A; 25-26 min, 100%-25% A; 26-36 min, 25% A. UHPLC method II: Chromatographic separation was carried out on an ACQUITY UPLC BEH C18 column (100 × 2.1 mm, 1.7 μm) with an UPLC BEH C18 guard column (5 × 2.1 mm, 1.7 μm). The mobile phases A (Water containing 0.1% formic acid) and B (Acetonitrile containing 0.1% formic acid) were employed for the chromatographic separation of targeted compounds with the gradient program: 0-5 min, 25%-55% B; 5-16 min, 55%-100% B; followed by washing with 100% B and equilibration with 25% B. The flow rate was 0.2 mL/min. The MS was operated in positive ionization mode. The conditions were as follows: spray voltage 4.0 kV, capillary temperature 320°C, auxiliary gas heater temperature 310°C, sheath gas flow rate 30 arbitrary units, auxiliary gas flow rate 10 arbitrary units, and PRM (HCE 15 eV). UHPLC method I was employed for the qualitative data acquisition of 3-H-CL (Figure 1C and Supplemental Figures 2, 12 and 15). UHPLC method II was used for the quantification of CL and 3-H-CL shown in Figure 1D and Supplemental Figure 13. 3-Hydroxycarlactone, a Novel Product of the Strigolactone Biosynthesis Core PathwayBaz et al.Molecular PlantJuly 2, 2018In BriefStrigolactones (SLs) are a class of carotenoid-derived plant hormones that regulate shoot branching among other developmental processes (Gomez-Roldan et al., 2008; Umehara et al., 2008; Al-Babili and Bouwmeester, 2015). 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, 2015). 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. Full-Text PDF Open Archive