Confirming stereochemical structures of strigolactones produced by rice and tobacco

Abstract

Major strigolactones (SLs) produced by rice (Oryza sativa L. cv. Nipponbare) and tobacco (Nicotiana tabacum L. cv. Michinoku No. 1) were purified and their stereochemical structures were determined by comparing with optically pure synthetic standards for their NMR and CD data and retention times and mass fragmentations in ESI-LC/MS and GC-MS. SLs purified from root exudates of rice plants were orobanchol, orobanchyl acetate, and ent-2'-epi-5-deoxystrigol. In addition to these SLs, 7-oxoorobanchyl acetate and the putative three methoxy-5-deoxystrigol isomers were detected by LC-MS/MS. The production of 7-oxoorobanchyl acetate seemed to occur in the early growth stage, as it was detected only in the root exudates collected during the first week of incubation. The root exudates of tobacco contained at least 11 SLs, including solanacol, solanacyl acetate, orobanchol, ent-2'-epi-orobanchol, orobanchyl acetate, ent-2'-epi-orobanchyl acetate, 5-deoxystrigol, ent-2'-epi-5-deoxystrigol, and three isomers of putative didehydro-orobanchol whose structures remain to be clarified. Furthermore, two sorgolactone isomers but not sorgolactone were detected as minor SLs by LC-MS/MS analysis. It is intriguing to note that rice plants produced only orobanchol-type SLs, derived from ent-2'-epi-5-deoxystrigol, but both orobanchol-type and strigol-type SLs, derived from 5-deoxystrigol were detected in tobacco plants.

Figure 1.

Structures of Natural Strigolactones.

Figure 2.

Characterization of Strigolactones Exuded by Rice Plants.(A) Five-channel multiple reaction monitoring (MRM) chromatogram of rice root exudate, where the transitions of m/z 425 > 268, 411 > 254, 383 > 286, 369 > 272, and 353 > 256 were monitored for 7-oxoorobanchyl acetate, orobanchyl acetate, methoxy-5-deoxystrigol, orobanchol, and 5-deoxystrigol and their isomers, respectively.(B) Distribution of germination stimulation activity on Orobanche minor after reversed-phase high-performance liquid chromatography (RP–HPLC) separation of the root exudate. Data are means ± SE (n = 3).

Figure 3.

Stereoisomers of 5-Deoxystrigol and Orobanchol.

Figure 4.

Chiral–LC–MS/MS Analyses of Orobanchol and 5-Deoxystrigol Isomers Isolated from Rice Root Exudate.(A) Synthetic standards of orobanchol (Rt: 8.59min), ent-orobanchol (Rt: 9.41min), 2’-epi-orobanchol (Rt: 8.39min), and ent-2’-epi-orobanchol (Rt: 9.74min).(B, D) Rice root exudate.(C) Synthetic standards of 5-deoxystrigol (Rt: 11.02min), ent-5-deoxystrigol (Rt: 9.70min), 2’-epi-5-deoxystrigol (Rt: 9.38min), and ent-2’-epi-5-deoxystrigol (Rt: 10.00min).

Figure 5.

Characterization of Strigolactones Exuded by Tobacco Plants.(A) Five-channel multiple reaction monitoring (MRM) chromatogram of tobacco root exudate, where the transitions of m/z 411 > 254, 407 > 250, 369 > 272, 365 > 268, and 353 > 256 were monitored for orobanchyl acetate, solanacyl acetate, orobanchol, solanacol, and 5-deoxystrigol and their isomers, respectively.(B) Distribution of germination stimulation activity on Orobanche minor after reversed-phase high-performance liquid chromatography (RP–HPLC) separation of the root exudate. Data are means ± SE (n = 3).

Figure 6.

Chiral–LC–MS/MS Analyses of Orobanchol and 5-Deoxystrigol Isomers Isolated from Tobacco Root Exudate.(A) Combined MRM chromatogram monitoring orobanchol and its isomers. Orobanchol (Rt: 8.58min) and ent-2’-epi-orobanchol (Rt: 9.74min) were separately analyzed by chiral–LC–MS/MS and chromatograms were combined.(B) MRM chromatogram monitoring 5-deoxystrigol isomers. The peaks correspond to 5-deoxystrigol (Rt: 11.02min) and ent-2’-epi-5-deoxystrigol (Rt: 10.00min). Retention times of these stereoisomers are shown in Figure 4.