Strigolactone biosynthesis in rice can occur via a 9‐cis‐3‐OH‐10′‐apo‐β‐carotenal intermediate

Strigolactones (SLs) play a crucial role in regulating plant architecture and mediating rhizosphere interactions. They are synthesized from all‐trans‐β‐carotene converted into the intermediate carlactone (CL) via the intermediate 9‐cis‐β‐apo‐10′‐carotenal. Recent studies indicate that plants can also synthesize 3‐OH‐CL from all‐trans‐β‐zeaxanthin via the intermediate 9‐cis‐3‐OH‐β‐apo‐10′‐carotenal. However, the question of whether plants can form bioactive SLs from 9‐cis‐3‐OH‐β‐apo‐10′‐carotenal remains elusive. In this study, we supplied the 13C‐labeled 9‐cis‐3‐OH‐β‐apo‐10′‐carotenal to rice seedlings and monitored the synthesis of SLs using liquid chromatography‐mass spectrometry (LC–MS) and Striga bioassay. We further validated the biological activity of 9‐cis‐3‐OH‐β‐apo‐10′‐carotenal‐derived SLs using the ccd7/d17 SL‐deficient mutant, which demonstrated increased Striga seed‐germinating activity and partial rescue of tiller numbers and plant height. Our results establish 9‐cis‐3‐OH‐β‐apo‐10′‐carotenal as a significant SL biosynthetic intermediate with implications for understanding plant hormonal functions and potential applications in agriculture.

Strigolactones (SLs) play a crucial role in regulating plant architecture and mediating rhizosphere interactions.They are synthesized from all-trans-bcarotene converted into the intermediate carlactone (CL) via the intermediate 9-cis-b-apo-10 0 -carotenal.Recent studies indicate that plants can also synthesize 3-OH-CL from all-trans-b-zeaxanthin via the intermediate 9-cis-3-OH-bapo-10 0 -carotenal.However, the question of whether plants can form bioactive SLs from 9-cis-3-OH-b-apo-10 0 -carotenal remains elusive.In this study, we supplied the 13 C-labeled 9-cis-3-OH-b-apo-10 0 -carotenal to rice seedlings and monitored the synthesis of SLs using liquid chromatography-mass spectrometry (LC-MS) and Striga bioassay.We further validated the biological activity of 9-cis-3-OH-b-apo-10 0 -carotenal-derived SLs using the ccd7/d17 SL-deficient mutant, which demonstrated increased Striga seed-germinating activity and partial rescue of tiller numbers and plant height.Our results establish 9-cis-3-OH-b-apo-10 0 -carotenal as a significant SL biosynthetic intermediate with implications for understanding plant hormonal functions and potential applications in agriculture.
To address this knowledge gap, we fed [11-13 C]9-cis-3-OH-b-apo-10 0 -carotenal to hydroponically grown rice seedlings of WT and the SL-free d17 mutant, which lacks CCD7 activity and cannot synthesize 9-cis-3-OH-b-apo-10 0 -carotenal.We analyzed root exudates of fed plants using LC-MS and evaluated their activity to induce seed germination in Striga hermonthica.Moreover, we evaluated the activity of 9-cis-3-OH-b-apo-10 0 -carotenal as a precursor of SL(s) that can regulate plant architecture.

Plant material and growth conditions
Oryza sativa Nipponbare d17 [25] and WT rice plants were grown under controlled conditions (a 12 h photoperiod, 200-lmol photons m À2 Ás À1 and day/night temperature of 27/25 °C).All rice seeds were first surface-sterilized in a 50% sodium hypochlorite solution with 0.01% Tween-20 for 15 min, then rinsed with sterile water, before being germinated in the dark overnight.The pregerminated seeds were placed on Petri dishes containing half-strength liquid Murashige and Skoog (MS) medium and incubated in a growth chamber for 7 days.Thereafter, the seedlings were transferred into 50-mL black falcon tubes filled with half-strength modified Hoagland nutrient solution with an adjusted pH of 5.8.The nutrient solution consisted of 5. 6  [11-13 C]-9-cis-3-OH-b-apo-10 0 -carotenal feeding experiments One-week-old rice seedlings were transferred into 50-mL falcon tubes (one seedling per tube) containing low-Pi of half-strength modified Hoagland nutrient solution in the growth cabinet for 2 weeks.On the day of collection, seedlings were transferred to new 50-mL falcon tubes with low-Pi half-strength modified Hoagland nutrient solution with or without compound.After 24-h incubation, root exudates were collected from each tube for LC-MS/MS analysis and Striga bioassays.Synthetic [11-13 C]-9-cis-3-OH-b-apo-10 0carotenal was purchased (customized synthesis) from Buchem B.V. (Apeldoorn, The Netherlands).

SL identification in root exudates
Collection, extraction, and analysis of SLs in rice root exudates were followed the published protocol [26].Briefly, root exudates were extracted with a C 18 -Fast Reversed-SPE column (500 mg/3 mL), preconditioned with 3 mL of methanol and followed with 3 mL of water.After washing with 3 mL of water, SLs were eluted with 5 mL of acetone.Thereafter, SL-containing fraction was concentrated to SL aqueous solution ( $ 500 lL), followed by 1 mL of ethyl acetate extraction.750 lL of SL enriched fraction was dried under vacuum.The final extract was redissolved in 100 lL of acetonitrile : water (25 : 75, v : v) and filtered through a 0.22-lm filter for LC-MS/MS analysis.
The identification of SLs was performed using UHPLC-Orbitrap ID-X Tribrid Mass Spectrometer (Thermo Scientific TM , Germering, Germany) with a heated-electrospray ionization source.Chromatographic separation was achieved on the Hypersil GOLD C18 Selectivity HPLC Columns (150 9 4.6 mm; 3 lm; Thermo Scientific TM ) with mobile phases consisting of water (A) and acetonitrile (B), both containing 0.1% formic acid, and the following linear gradient (flow rate, 0.5 mLÁmin À1 ): 0-15 min, 25-100% B, followed by washing with 100% B and equilibration with 25% B for 3 min.The injection volume was 10 lL, and the column temperature was maintained at 30 °C for each run.The MS conditions were as follows: positive mode, ion source of H-ESI, spray voltage of 3500 V, sheath gas flow rate of 60 arbitrary units, auxiliary gas flow rate of 15 arbitrary units, sweep gas flow rate of 2 arbitrary units, ion transfer tube temperature of 350 °C, vaporizer temperature of 400 °C, Slens RF level of 60, resolution of 120 000 for MS; stepped HCD collision energies of 10-50% and resolution of 30 000 for MS/MS.The mass accuracy of the identified compounds (accurate mass AE 5 ppm mass tolerance) was acquired using the XCALIBUR software version 4.1 (Germering, Germany).
Phenotyping of exogenous applications of 9-cis-3-OH-10 0 -apo-b-carotenal For investigating the effect of 9-cis-3-OH-b-apo-10 0carotenal on rice seedlings, 1-week-old seedlings were grown hydroponically in half-strength Hoagland nutrient solution containing 0.4 mM K 2 HPO 4 Á2H 2 O (+Pi), 5 lM 9-cis-3-OH-b-apo-10 0 -carotenal, or the corresponding volume of the solvent (mock; acetone) for 2 weeks.The solution was changed three times per week, adding the chemical at each renewal.Root length, shoot length, and tiller numbers were recorded at the end of the experiment.

Striga hermonthica seed germination bioassays
Striga seed germination bioassay was carried out based on the protocol [26].Briefly, 10-day-old preconditioning Striga seeds were supplied with 50 lL of extracted root exudates of different rice genotypes.After application, Striga seeds were incubated at 30 °C in the dark for 24 h.Germinated (seeds with radicle) and nongerminated seeds were counted under a binocular microscope to calculate germination rate (%) by using the SEEDQUANT software [27].
In case of conversion, this precursor should lead to SLs labeled in the D-ring [17,24].After 24 h, we collected and analyzed the root exudates using LC-MS (accurate masses AE5 ppm) and compared them with endogenous Oro.As shown in (Fig. 2A), a putative [2 0 -13 C]6-OH-4DO was detected; however, neither [2 0 -13 C]6-OH-Oro nor [2 0 -13 C]3-OH methyl 4-oxocarlactononate (4-oxo-MeCLA) were identified (data not shown) [21,29].This result suggests that 6-OH-4DO might be the final product of 3-OH-CL conversion in the canonical SL pathway.Rice does not form a keto-group, when hydroxylated at the C3 position of the A-ring, that is, in 3-OH-CL for the production of 6-oxo-4DO (Fig. 1).However, we cannot exclude the possibility that this and other reactions occurred with low efficiency, preventing the detection of products in the canonical SL biosynthesis.Moreover, 18-OHcarlactononic acid (CLA) is an intermediate product during canonical SL formation [30,31], suggesting that the 3,18-diOH-CLA might be the intermediate of putative 6-OH-4DO formation in rice; however, this reaction must be validated in future studies.
To further confirm the structure of [2 0 -13 C]6-OH-4DO, we performed tandem MS analysis and successfully identified the 13 C-labeled D-ring (98.031AE 5 ppm) fragment and the nonlabeled D-ring (97.028AE 5 ppm) in fed and control plants, respectively (Fig. 2A).Although, we demonstrated the conversion of 3-OH-9-cis-b-apo-10 0 -carotenal into a downstream canonical SL, the conversion level of [2 0 -13 C]6-OH-4DO was low compared with the endogenous Oro signal, suggesting that 9-cis-3-OH-b-apo-10 0 -carotenal might not be the preferred substrate in the canonical SL biosynthesis.Nevertheless, our result demonstrates that 9-cis-3-OH-b-apo-10 0 -carotenal is an endogenous substrate in SL biosynthesis and confirms via metabolism tracking the ability of plant roots to take up long-chain apocarotenoids [17,24].
Subsequently, we supplied the exudates collected from d17 fed with labeled 9-cis-3-OH-b-apo-10 0 -carotenal and from the corresponding control to Striga seeds, to determine the biological activity of the produced SL(s) in inducing Striga seed germination.Notably, the Striga seed germination rate was approximately 20% after the application of the exudates of 9-cis-fed d17 seedlings, but was approximately 0% with the mock exudates (Fig. 4A), confirming the presence of bioactive SLs in the root exudates of fed d17.
Finally, to gain insights into the biological function of SLs derived from 9-cis-3-OH-b-apo-10 0 -carotenal in plants, we applied this apocarotenoid at a 5 lM concentration to WT and d17 rice seedlings.We observed that the tiller numbers and plant height of d17 were partially rescued by 9-cis-3-OH-b-apo-10 0 -carotenal (Fig. 4B), indicating that the SL(s) originating from this apocarotenoid exerts SL hormonal functions [3].

Conclusion
Taken together, our findings unequivocally establish 9-cis-3-OH-b-apo-10 0 -carotenal as an additional SL biosynthetic intermediate and a precursor of rice SLs.This observed activity may explain why CCD7 produces this apocarotenoid when exposed to the corresponding precursors, 9-cis-zeaxanthin or 9 0 -cis-lutein, in vitro [16].A thorough assessment of the biological significance of this metabolic pathway requires the generation of a plant mutant with a CCD7 lacking the capability to cleave hydroxylated carotenoids or a CCD8 mutation that does not produce 3-OH-CL.

572FEBS
Letters 598 (2024) 571-578 ª 2024 The Authors.FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Fig. 2 .
Fig. 2. LC-MS identification of SLs in root exudates.(A) Identification of endogenous Oro (EIC: 347.1620;Retention time: 11.38) in the mock condition and [2 0 -13 C]6-OH-4DO (EIC: 348.1490;Retention time: 11.29) after 24 h feeding with 13 C-9-cis-3-OH-b-apo-10 0 -carotenal in WT.MS/MS fragmentations of the precursor ion (m/z 347.1620 [M + H] + in positive mode) and endogenous Oro was characterized by Dring at m/z 347.1620 > 97.02834.[2 0 -13 C]6-OH-4DO were identified using ions pairs at m/z 348.1490 > 98.0316 ( 13 C-D-ring).Red color represents the ion transition of the endogenous Oro in the mock group, whereas the 13 C-3-OH-4DO identified in the fed sample is denoted in blue.The orange flag indicates the 13 C-D-ring ion.(B) EIC chromatograms of SL identification in d17 root exudates (Upper panel) fed with labeled compound.Identification of [2 0 -13 C]6-OH-4DO (Retention time: 11.31) using accurate mass at m/z 348.2169 ([M + H] + in positive mode; red color) in the root exudates of d17 (Upper panel) and WT(Middle panel) fed with labeled substrate.Red arrows indicate the peak of [2 0 -13 C]6-OH-4DO identified in the chromatograms.The orange flag in the lower panel indicates the MS/MS fragmentations of the 13 C-Dring presented in the WT fed with the labeled substrate.