2015 Volume 40 Issue 4 Pages 214-216
Strigolactones (SLs) mainly produced in roots move upward to shoots and inhibit axillary bud outgrowth. However, SLs were not detected by LC-MS/MS in xylem saps collected from rice, tomato, cucumber, tobacco, sorghum, and Arabidopsis. d1-Orobanchol and d6-4-deoxyorobanchol that were fed to roots of rice plants were detected in shoots harvested 20 hr after treatment, although not in the xylem sap. These results imply that both endogenous and exogenous SLs are transported from roots to shoots, although not through the xylem.
Strigolactones (SLs) are carotenoid-derived plant secondary metabolites.1–3) SLs released from plant roots into the rhizosphere promote symbiotic interactions with arbuscular mycorrhizal fungi4) and root nodule bacteria5,6); they also trigger parasitic interactions with the root parasitic weeds witchweeds (Striga spp.) and broomrapes (Orobanche and Phelipanche spp.).2,3) In plants, SLs as a novel class of plant hormones regulate plant growth and development through crosstalk with other hormones.2,3,7,8) It is generally accepted that SLs are mainly produced in the roots, move upward to shoots, and inhibit axillary bud outgrowth.1,2,9) In fact, root-applied SLs restore the shoot phenotype in SL-deficient mutants of Arabidopsis, rice, etc.8) Since orobanchol was detected in xylem saps from Arabidopsis and tomatoes, it has been proposed that SLs are transported through the xylem.10) However, there have been no other reports supporting xylem transport of SLs.
In the present study, we first examined whether xylem saps collected from several plant species contain SLs. Then, we monitored the transport of root-applied SLs at two different time-points, 2 and 20 hr after treatment.
Xylem saps (approximate total volumes) collected from rice (1 L), tomato (13 L), cucumber (20 L), tobacco (7 L), sorghum (1 L), and Arabidopsis (2.5 mL) were immediately extracted with ethyl acetate. After evaporation of the solvent, the extracts were analyzed by LC-MS/MS. No signals attributable to known SLs and their isomers were detected in these xylem sap extracts from rice (Fig. 1A–I), tomato (Supplemental Fig. S1), cucumber (Supplemental Fig. S2), tobacco (Supplemental Fig. S3), sorghum (Supplemental Fig. S4) and Arabidopsis plants (Supplemental Fig. S5). Furthermore, carlactone11) and its oxidized metabolites including carlactonoic acid and methyl carlactonoate,12) were below the detection limit (Fig. 1J–L, Supplemental Figs. S1–S5). In these LC-MS/MS analyses with a linear ion-trap system, the detection limit for a typical SL, orobanchol, is well below 10 fg. Therefore, it is likely that SLs are not transported through the xylem, at least not in these plant species.
The absence of SLs in xylem saps suggests that directed cell-to-cell transport of SLs from roots to shoots occurs and is mediated by an active transport system. To confirm this, 6′[2H]orobanchol and 3a,4,4,5,5,6′[2H]6-4-deoxyorobanchol were applied to the roots of rice plants, and shoots were cut at 2 cm above the shoot/root junction 2 and 20 hr after treatment. Harvested shoots were cut into small pieces and extracted with ethyl acetate. d1-Orobanchol and d6-4-deoxyorobanchol were used, as they are major SLs produced by rice plants. Although d1-orobanchol and d6-4-deoxyorobanchol were not detected in shoots harvested 2 hr after treatment (Fig. 1M, N), clear peaks for these deuterated SLs were observed for the sample harvested 20 hr after treatment (Fig. 1O, P), indicating that these SLs move from the roots to shoots rather slowly. Under the experimental conditions, levels of endogenous SLs, including orobanchol in the shoot tissues, were below the detection limit. In general, the SL content in shoots is less than 1/1000 that of roots13) and is higher in the basal parts of shoots. In a separate experiment, xylem saps collected 2 hr and 20 hr after treatment for a period of 2 hr were analyzed by LC-MS/MS; however, neither the d1(6)-SL nor known SLs were detected (data not shown). These results clearly demonstrate that both exogenous and endogenous SLs are transported from the roots to shoots not through the xylem but probably through hypodermis passage cells, as in petunias, where polar and asymmetric localizations of an ABC transporter, Petunia axillaris PLEITROPIC DRUG RESISTANCE 1 (PaPDR1), have been shown to mediate directional (shootward) SL transport as well as localized exudation into the rhizosphere.14,15) However, our results do not exclude the possibility that unknown SL-related compounds that move through the xylem are involved in the root-to-shoot SL signaling pathway. Transport of root-applied deuterium-labeled SLs in various plant species is being examined to clarify structural specificities and stereospecificities of this process in plants.
LC-MS/MS analysis of proton adduct ions was performed with a triple quadrupole/linear ion trap instrument (LIT) (QTRAP5500; AB Sciex) with an electrospray source. The LC-MS/MS analytical conditions were essentially the same as those described previously.12) Details of the LC-MS/MS analytical conditions are described in the Supplementary file.
2. ChemicalsThe optically pure 6′[2H]orobanchol4,16) and 3a,4,4,5,5,6′[2H]6-4-deoxyorobanchol17) were synthesized in accordance with to the reported methods. 7-Hydroxyorobanchyl acetate and 7-oxoorobanchyl acetate were purified from cucumber root exudates.18) Solanacol and solanacyl acetate were generous gifts of Dr. François-Didier Boyer (INRA, France). Orobanchol and orobanchyl acetate were purified from red clover root exudates19) and sorgomol from sorghum root exudates.20) Carlactone and its related compounds were synthesized as reported previously.12,21) The other analytical grade chemicals and HPLC solvents were obtained from Kanto Chemical Co., Ltd. and Wako Pure Chemical Industries, Ltd.
3. Plant materialSeeds of cucumber (Cucumis sativus L. cv. Aonagakei-Jibai), rice (Oryza sativa L. cv. Nipponbare), tomato (Solanum lycopersicum L. cv. Momotaro), and sorghum (Sorghum bicolor (L.) Moench cv. Hybrid) were purchased from a local market. Tobacco seeds (Nicotiana tabacum L. cv. Michinoku No. 1) were a generous gift from Japan Tobacco Inc. Cucumber, tomato, and sorghum were grown in an experimental field of Utsunomiya University. Rice plants were grown in 10-L volume plastic pots placed outdoors. Arabidopsis seedlings were grown in vermiculite. Fertilizers and watering were conducted in accordance with standard growth protocols for each plant species.
4. Collection of xylem sapsXylem saps were collected in accordance with reported protocols.10,22,23) We did not use absorbent cotton balls to collect xylem saps from sorghum and rice; xylem saps were directly pipetted to avoid possible contamination. Xylem saps thus collected were extracted with ethyl acetate as soon as possible because SLs and related compounds in aqueous solution decompose gradually; in addition freezing/thawing of these solutions often accelerates decomposition. The total number of plants used and the volume of xylem saps collected were 200 and 1 L, 43 and 13 L, 120 and 20 L, 350 and 7 L, 70 and 1 L, and 250 and 2.5 mL for rice, tomato, cucumber, tobacco, sorghum, and Arabidopsis, respectively.
5. Feeding experimentsRice seeds (cv. Nipponbare) were surface-sterilized in 70% EtOH for 2 min. After being thoroughly rinsed with sterile Milli-Q water, the seeds were soaked in water at 25°C for 5 days. Fifteen germinated seeds were transferred to a strainer (22×18×7 cm, width×length×height (W×L×H)) lined with a sheet of gauze moistened by placing it in a slightly larger container (22.5×18.5×9 cm, W×L×H) containing 1.5 L of 1/2 Tadano and Tanaka medium24) without phosphate as the culture medium in a growth chamber with a 14/10-hr photoperiod at 120 µmol photons/m2/s at 30/24°C. Rice plants grown hydroponically for 15 days were treated with medium containing d1-orobanchol (1 µM) and d6-4-deoxyorobanchol (1 µM). Shoots were cut at 2 cm above the shoot/root junction to eliminate possible contamination from the media 2 or 20 hr after treatment, and the shoot tissues (ca. 20 g FW) were extracted with acetone at 4°C for 24 hr. After removal of the tissues by filtration, the acetone was evaporated in vacuo. Preparation of the samples for LC-MS/MS analyses was conducted as reported previously.12) The experiment was conducted with three replications.
We thank Dr. François-Didier Boyer (INRA, France) for his generous gifts of solanacol and solanacyl acetate. We also thank Dr. Christopher McErlean (University of Sydney, Australia) for critical reading of the manuscript and fruitful discussions. This work was supported by the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry, by KAKENHI (23880005, 26850069), by a grant from JGC-S Scholarship Foundation to XX, and by the special grant for the UU-COE from Utsunomiya University. Kaori Yoneyama is supported by RPD project (JSPS).
