Figure 1.
SL structures present in rice root exudates or tested in a seed germination or hyphal branching bioassay.
(1) (−)−orobanchol; (2) ent-2'-epi-5-deoxystrigol; (3) orobanchyl acetate and (4) proposed structure of 7-oxoorobanchyl acetate [23]; (5) proposed structure of methoxy-5-deoxystrigol isomers (R1 = OMe; R2 = H) or the methyl ether of orobanchol (R1 = H; R2 = OMe); (6) (+)-ent-2'-epi-orobanchol; (7) sorgomol; (8a–d) stereoisomers of strigol (R1 = CH3; R2 = OH); 5-deoxystrigol (R1 = CH3; R2 = H), stereochemical configurations 8a and 8c are natural (strigol-type and orobanchol-type, respectively) while configurations 8b and 8d are not naturally occurring; (9a–d) stereoisomers of SL analogue GR24.
Figure 2.
Activity profiles of rice root exudates.
Germination of S. hermonthica (one biological replicate; the other two are shown in ) obtained with crude exudates and exudate fractions from rice plants (A) treated with full nutrition (black bars); phosphate starvation (grey bars) and phosphate starvation plus 0.01 µM fluridone (white bars). Water and SL analogue GR24 (0.005, 0.05 and 0.5 µM) were used as controls. The error bars represent the standard error of 3 technical replicates. Significance levels between treatments as determined using a X2 test are indicated: */+ = P<0.05; **/++ = P<0.01; ***/+++ = P<0.001; n.s. = P>0.05; * = control vs. phosphate starvation treatment; + = phosphate starvation vs. phosphate starvation plus fluridone treatment. When germination values are close to zero the statistical test cannot be performed, which is indicated with “−”. AM fungal hyphal branching induced by crude exudates (B) and exudate fractions (C) of rice treated with full nutrition (black bars) and phosphate starvation (white bars) in germinating Gi. rosea spores. The assay was performed with pooled samples of three biological replicates. GR24 (0.005, 0.05 and 0.5 µM) and 10% acetonitrile in water were used as controls. The bars represent the mean of the total number of new branches, the error bar the standard error of the mean (n = 20). Significance values comparing means between control treatments and phosphate starvation treatment are indicated above the bars. (* = P<0.05, ** = P<0.01, *** = P<0.001).
Figure 3.
SL analysis of rice root exudates.
Root exudates from rice plants grown under phosphate starvation were analyzed with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using multiple reaction monitoring (MRM). Chromatograms of (A) transitions 347.2 > 233 and (B) 347.2>96.8 for orobanchol; (C) transitions 331.2> 234 and (D) 331.2>96.8 for 2'-epi-5-deoxystrigol; (E) transitions 361.2>247 and (F) 361.2>96.8 for three putative methoxy-5-deoxystrigol isomers; (G) the total ion count (TIC) showing of all measured transitions and where orobanchol (8.05 min), ent-2'-epi-5-deoxystrigol (12.51 min) and the three putative methoxy-5-deoxystrigol isomers (9.87; 10.33; 10.86 min) are visible.
Figure 4.
Abundance of (−)−orobanchol, ent-2'-epi-5-deoxystrigol and putative SL-like compounds in phosphate starvation and fluridone treatments.
Peak areas obtained with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis using multiple reaction monitoring (MRM) of root exudates of rice. (A) (−)−orobanchol (MRM transition 347.2 >96.8; black bars) and ent-2'-epi-5-deoxystrigol (MRM transition 331.2>234; hatched bars); (B–D) three putative SL-like compounds measured in crude exudates with the retention times: rt = 9.87 (B); rt = 10.3 and (C) rt = 10.9 (D) and the MRM transitions 361>96.8 (black bars) and 361>247 (white bars). All measurements taken from crude exudates of plants grown in different treatments: phosphate starvation (−P); phosphate starvation combined with fluridone (−P+F) and control treatment with full nutrient supply (C). The error bars represent the standard error of 3 biological replicates. Significance values are indicated with * (for ent-2'-epi-orobanchol and for 361>96.8 transition) and + (for 2'-epi-5-deoxystrigol and for 331.2>234 transition) and compare phosphate starvation (-P) treatment vs. phosphate starvation with fluridone (-P+F) and -P vs. full nutrition (C) (*/+ = P<0.05, **/++ = P<0.01, ***/+++ = P<0.001).
Figure 5.
Characterization of rice mutant d10 exudate.
Germination assay with S. hermonthica seeds on exudate fractions of full nutrition (black bars) and phosphate starvation (white bars) treated plants (A). Water and GR24 (0.33, 3.3 and 33 µM) were used as controls. Peak areas of ent-2'-epi-5-deoxystrigol, (−)−orobanchol and putative methoxy-5-deoxystrigol isomers 2 and 3 (B) and putative methoxy-5-deoxystrigol isomer 1 (C) obtained with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis using multiple reaction monitoring (MRM) of root exudates of d10-2 (black bars) and WT (white bars) under phosphate starvation treatment.
Table 1.
Striga hermonthica germination and Gigaspora margarita hyphal branching in the presence of SL standards.
Figure 6.
MS/MS spectra of putative SL-like compounds.
The spectra were measured at the retention time of each isomer: 9.87 min (A), 10.35 min (B) and 10.95 min (C).