Planteose as a storage carbohydrate required for early stage of germination of Orobanche minor and its metabolism as a possible target for selective control

Abstract

Root parasitic weeds in Orobanchaceae cause serious damage to worldwide agriculture. Germination of the parasites requires host-derived germination stimulants, such as strigolactones, as indicators of host roots within reach of the parasite's radicles. This unique germination process was focused on to identify metabolic pathways required for germination, and to design a selective control strategy. A metabolomic analysis of germinating seeds of clover broomrape, Orobanche minor, was conducted to identify its distinctive metabolites. Consequently, a galactosyl-sucrose trisaccharide, planteose (α-d-galactopyranosyl-(1→6)-β-d-fructofuranosyl-(2→1)-α-d-glucopyranoside), was identified as a metabolite that decreased promptly after reception of the germination stimulant. To investigate the importance of planteose metabolism, the effects of several glycosidase inhibitors were examined, and nojirimycin bisulfite (NJ) was found to alter the sugar metabolism and to selectively inhibit the germination of O. minor. Planteose consumption was similar in NJ-treated seeds and non-treated germinating seeds; however, NJ-treated seeds showed lower consumption of sucrose, a possible intermediate of planteose metabolism, resulting in significantly less glucose and fructose. This inhibitory effect was recovered by adding glucose. These results suggest that planteose is a storage carbohydrate required for early stage of germination of O. minor, and NJ inhibits germination by blocking the supply of essential glucose from planteose and sucrose. Additionally, NJ selectively inhibited radicle elongation of germinated seeds of Orobanchaceae plants (Striga hermonthica and Phtheirospermum japonicum). Thus, NJ will be a promising tool to develop specific herbicides to the parasites, especially broomrapes, and to improve our understanding of the molecular mechanisms of this unique germination.

Keywords: Broomrapes; metabolomics; nojirimycin; planteose; root parasitic weeds; seed germination; selective control; sugar metabolism..

Fig. 1.

Metabolic profiling of germinating seeds of O. minor. (A) Squares (with GR24 treatment) or circles (without GR24 treatment) show time points of sample collection. GR24 was applied on day 8 (arrow). (B) PCA of metabolite profiles of seeds at different stages during conditioning and germination, shown as the combination of the first two PCs (representing 85% of metabolite variance). Each data point is an independent sample. (C) Loading plot showing weight for each data point of the total ion current chromatogram in calculating PC 1.

Fig. 2.

Sugar profiles in O. minor seeds during conditioning and germination. Galactose was not detected in any samples. (A) Total ion current chromatogram of sugars in dry seeds obtained by GC-MS analysis. Fru, fructose; Glc, glucose; Pla, planteose; Suc, sucrose. (B) Changes in the amounts of sugars in seeds without GR24 treatment (mean ± SD, n = 3). Conditioning period was 7 days and distilled water was applied on final day of conditioning, therefore germination was not induced. Asterisks indicate significant differences in each sugar contents between dry seeds and during conditioning seeds (P < 0.05, Tukey-Kramer). (C) Germination rate of O. minor seeds after GR24 treatment. Conditioning period was 7 days; 10 mg·L^-1 (w/v) GR24 was applied on final day of conditioning. Seeds did not germinate without GR24 treatment. Seeds were observed and germinated seeds were counted under a microscope (mean ± SD, n = 3). (D–G) Changes in the amounts of sugars in seeds with GR24 treatment (mean ± SD, n = 3). Conditioning period was 7 days and 10 mg·L^-1 (w/v) GR24 was applied on final day of conditioning. Different letters indicate significant differences in sugar contents during germination (P < 0.05, Tukey-Kramer).

Fig. 3.

Profiles of starch and total fatty acids in germinating O. minor seeds. Amounts of (A) starch and (B) total fatty acids were measured in seeds with (+) or without (-) GR24 treatment (mean ± SD, n = 3). Arrows indicate the day of GR24 treatment.

Fig. 4.

UPLC-ELSD analysis of sugars in seeds of various root parasitic weeds. (A) Authentic standard compounds. (B) O. crenata. (C) P. aegyptiaca. (D) S. hermonthica. Black and grey arrows indicate planteose and sucrose, respectively. DAG and germination rate at that time are shown in upper left of chromatograms. Seeds of each parasitic weed were collected immediately after conditioning and at two time points during germination before reaching maximum germination rate. The dates of collection of seeds were different among species because germination rate of each plant species is different.

Fig. 5.

Effect of NJ on seed germination of parasitic and non-parasitic plants. (A) Effect of NJ at various concentrations on germination rates of O. minor seeds (mean ± SD, n = 3). Germination rate of O. minor seeds decreased in a dose-dependent manner with NJ. (B) Effect of NJ on seed germination rates in other plant species (mean ± SD, n = 3). (C) Morphological changes in radicle elongation of S. hermonthica in the presence of NJ. Staining by crystal violet facilitated visualization of the radicles. Treatments were as follows: (i) 10 mg·L^-1 (w/v) GR24 only; (ii) GR24 + 1 μM NJ; (iii) GR24 + 10 μM NJ; and (iv) GR24 + 100 μM. Bar: 200 μm. (D) Radicle lengths of S. hermonthica treated with NJ at various concentrations. Radicle lengths of 100 germinating seeds were determined using ImageJ software. Radicle elongation was inhibited by NJ in a dose-dependent manner. Different letters indicate significant differences in radicle lengths (P < 0.05, Tukey-Kramer).

Fig. 6.

Changes in sugar contents in O. minor seeds in the presence of NJ. (A) Amounts of sugars in seeds at 3 DAG and at 7 DAG with (+NJ) or without (C, control) NJ treatment (mean ± SD, n = 3). NJ (10 μM) was added along with GR24 after the conditioning period. (B) Magnification of graph showing amounts of sucrose and planteose. Asterisks indicate significant differences in amounts of sugars between control and NJ-treated seeds on the same day (P < 0.05, Student’s t test). Fru, fructose; Glc, glucose; Pla, planteose; Suc, sucrose.

Fig. 7.

Recovery of seed germination rate by simultaneous addition of exogenous sugars and NJ. Seed germination rates were measured at 7 DAG (mean ± SD, n = 3). Asterisks indicate significant differences in germination rates between seeds treated with NJ and those treated with NJ + exogenous sugars (P < 0.05, Student’s t test). Fru, fructose; Gal, galactose; Glc, glucose; Pla, planteose; Suc, sucrose; UDP-Glc, UDP-glucose.

Fig. 8.

Effects of NJ on activities of INVs. (A) Effect of NJ on activities of INVs in vitro. Reaction mixture contained crude enzyme extract from germinating seeds at 5 DAG and NJ at a final concentration of 0.1mM. NJ effect was calculated as ratio of activity with NJ treatment to that without NJ (mean ± SD, n = 3). (B) Effect of NJ on activities of INVs in vivo. Crude enzyme extracts were prepared from germinating and 10 μM NJ-treated seeds of O. minor. Activities of SAI, SNI, and CWI were assayed. Solid and dashed lines show mean values of enzyme activity in non-treated and NJ-treated seeds, respectively (mean ± SD, n = 6 to 8). Enzyme activity is expressed as nmol glucose·min^-1·mg protein^-1. Asterisks indicate significant differences in enzyme activity between non-treated and NJ-treated seeds at same time point (* P < 0.05, ** P < 0.01, Student’s t test).