Discovery of strigol synthase from cotton (Gossypium hirsutum): The enzyme behind the first identified germination stimulant for Striga

Abstract

Societal Impact Statement

Parasitic witchweeds (Striga species) pose a serious threat to food security in Africa, attacking cereal grains and legumes. Chemicals released from the host roots that initiate the life cycle of Striga are known as germination stimulants, predominantly strigolactones (SLs). Strigol, the first identified SL, was isolated from the root exudates of cotton (Gossypium hirsutum), a false host of Striga, over 50 years ago. The identification of strigol synthase in cotton establishes the complete biosynthesis pathway of this emblematic SL. This discovery has the potential to advance our understanding of SL‐mediated rhizosphere interactions and enhance cotton's effectiveness as a trap crop.

INTRODUCTION

Strigol, a plant specialized metabolite, was first discovered as a germination stimulant for the root parasitic weed Striga lutea, from root exudates of the cotton plants, Gossypium hirsutum (Cook et al., 1966) (Figure 1a). Cotton can induce germination of Striga seeds but not function as a host. This false host is employed as a trap crop to diminish the seed bank of Striga. The relative structure of strigol was determined using spectroscopic and X-ray crystallographic data (Cook et al., 1972). A racemic mixture of strigol (a mixture of two enantiomers, strigol and ent-strigol) was synthesized and optically resolved, enabling the unequivocal establishment of the absolute structure of natural strigol (Brooks et al., 1985). Following the discovery of strigol, structural analogs with the germination-stimulating activity for Striga were identified in the root exudates of host plants. Butler (1995) coined the term strigolactone (SL) to refer to these strigol analogs. SLs are now recognized as exhibiting diverse functions, including rhizosphere signaling that induces hyphal branching in arbuscular mycorrhizal fungi (Akiyama et al., 2005) and endogenous plant hormones that inhibit shoot branching (Gomez-Roldan et al., 2008; Umehara et al., 2008). The potential of SLs to mitigate the adverse effects caused by root parasitic weeds on plant production prompted the development of synthetic strigol analogs, including GR compounds (Johnson et al., 1981). It is noteworthy that most of these structural developments had been accomplished soon after the identification of strigol. These analogs have had a tremendous impact on SL research, not only for the management of root parasitic weeds, but also for the study of plant–microbe communication in the rhizosphere and the control of plant architecture.

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Over 30 distinct naturally occurring SLs have been identified in a variety of plants. Their common biosynthetic precursor carlactone (CL) is synthesized from β-carotene through the core SL biosynthesis pathway involving sequential reactions of DWARF27 (D27), CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7), and CCD8 (Alder et al., 2012). The biosynthesis of SLs downstream of CL has become increasingly clear. In Arabidopsis, cytochrome P450 (CYP) AtCYP711A1, encoded by MORE AXILLARY GROWTH 1 (MAX1), catalyzes the conversion of CL to carlactonoic acid (CLA) (Abe et al., 2014; Seto et al., 2014). This enzymatic conversion was found to be a conserved function of CYP711A/MAX1 homologs in various plants (Yoneyama et al., 2018). In rice (Oryza sativa), two MAX1 homologs convert CL to 4-deoxyorobanchol (4DO) via CLA and 4DO to orobanchol (Yoneyama et al., 2018; Zhang et al., 2014). The cotton plants, G. hirsutum and G. arboreum, produce 5-deoxystrigol (5DS) (Iseki et al., 2018), a stereoisomer of 4DO. In G. arboreum, the conversion of CLA to 5DS is catalyzed by another CYP family enzyme, GaCYP722C (Wakabayashi et al., 2020). The conversion to 5DS is also catalyzed by the CYP722C subfamily in other 5DS-producing dicot plants (Wu et al., 2021). SLs sharing the same planar carbon skeleton as 4DO and 5DS have a characteristic tricyclic lactone (ABC-ring) that connects to a butenolide D-ring through an enol ether bridge. These are collectively known as canonical SLs. Both strigol and sorgomol are hydroxylated compounds of 5DS, and contain a hydroxy group at C-5 and C-9, respectively (Figure 1a). In sorghum (Sorghum bicolor), SbCYP728B35 catalyzes the introduction of a hydroxy group at C-9 of 5DS to form sorgomol (Wakabayashi et al., 2021); however, the complete biosynthesis pathway for strigol, a prototypical canonical SL, in G. hirsutum remains elusive, despite more than half a century since its discovery.

In the present study, we selected candidate strigol synthase genes in a transcriptome analysis of G. hirsutum roots by identifying upregulated genes under conditions that promote SL production. Screening of candidate genes via heterologous expression in the Nicotiana benthamiana transient expression system revealed GhCYP71AH as a promising candidate. Enzyme assays were performed using purified recombinant GhCYP71AH expressed in Escherichia coli to determine its substrate specificity and stereoselective hydroxylation of 5DS. These results confirm the function of GhCYP71AH as a strigol synthase (Figure 1b).

EXPLORATION OF CANDIDATE CYPs FOR STRIGOL SYNTHASE

In a previous study, we demonstrated that the respective administration of CL, CLA, and 5DS in hydroponic solutions of G. hirsutum resulted in the formation of strigol (Iseki et al., 2018). The conversion of the immediate precursor 5DS to strigol was inhibited by the CYP inhibitor uniconazole-P in a dose-dependent manner (Ueno et al., 2018), suggesting that CYP catalyzes the introduction of a hydroxy group at C-5 of 5DS. We also demonstrated that under phosphate-deficient conditions, the expression of SL biosynthesis genes was upregulated in G. arboreum, another Gossypium sp., resulting in increased SL production (Iseki et al., 2018; Wakabayashi et al., 2020). In the present study, we confirmed high strigol exudation from the roots of G. hirsutum grown hydroponically under phosphate-deficient conditions (Figure S1), which is consistent with our previous report (see Method S1 for the detailed methods employed in this study) (Iseki et al., 2018). We hypothesized that the expression of the biosynthesis gene responsible for the conversion of 5DS to strigol is also induced by phosphate deficiency in G. hirsutum. To identify candidate CYP genes involved in the synthesis of strigol, we performed a comparative transcriptome analysis by RNA-seq using the above-mentioned G. hirsutum roots grown under phosphate-rich and phosphate-deficient conditions (Figure S1). Eleven transcripts, annotated as encoding CYP, were differentially up-regulated under phosphate-deficient conditions (Table S1). These transcripts include GhCYP722C (Gh_A12G067300.1), an ortholog of GaCYP722C in G. arboreum which converts CLA to 5DS (Wakabayashi et al., 2020). As described below, we confirmed the same function of GhCYP722C as GaCYP722C. Accordingly, 10 transcripts, excluding GhCYP722C, were selected as candidates for strigol synthase.

EVALUATION OF THE CONVERSION ACTIVITY OF 5DS TO STRIGOL BY CANDIDATE CYPs IN N. benthamiana

Reconstruction of the SL biosynthesis pathway in N. benthamiana through the transient co-expression of relevant genes represents a robust approach to determine the function of additional unknown biosynthesis genes (Wang et al., 2022). Prior to pathway reconstruction, we confirmed the function of GhMAX1 (Gh_D05G044100) (Tian et al., 2022), a homolog of MAX1, using an in vitro enzyme assay as previously reported (Wakabayashi et al., 2020). Recombinant GhMAX1, which was expressed in a baculovirus-insect cell expression system, catalyzed the conversion of CL to CLA (Figure S2), thus verifying its conserved function (Yoneyama et al., 2018). In the reconstruction of the 5DS biosynthesis pathway in N. benthamiana, we co-expressed the Arabidopsis genes AtD27, AtCCD7, and AtCCD8, which are necessary for CL biosynthesis, along with the G. hirsutum genes, GhMAX1 and above-mentioned GhCYP722C. LC–MS/MS analysis of leaf extracts co-expressing these genes revealed 5DS production, which confirmed the successful reconstruction of the 5DS biosynthesis pathway (Figures 1c and S3). The generation of 5DS by GhCYP722C provides further evidence of the function of CYP722C in 5DS-producing dicot plants.

We successfully cloned six of the 10 CYP candidates (Gh_D07G061000.1, GhCYP71AH; Gh_D04G121500.1, GhCYP76A; Gh_D12G259300.1, GhCYP78A; Gh_A01G219900.1, GhCYP712A; Gh_D01G076100.1, GhCYP728B; Gh_A02G027800.1, GhCYP749A) (see Table S2 for primer sequences used for cloning). Sequencing results showed that the translated amino acid sequences of cloned GhCYP78A and GhCYP728B matched with those recorded in the database, but the others were found to be, in part, different from the database sequences (Figure S4). We transiently co-expressed each of them together with the 5DS biosynthesis pathway. Of these six CYPs, the co-expression of only GhCYP71AH resulted in a significant reduction in 5DS. The generation of a product with a retention time of 11.67 min was observed, which was consistent with that of authentic rac-strigol (Figure 1d,e). These results suggest that GhCYP71AH is a potential candidate for strigol synthase in G. hirsutum. Sorgomol was not detected in the leaf extracts of N. benthamiana co-expressing GhCYP728B (Figure S5), which belongs to the same subfamily as SbCYP728B35 catalyzing the conversion of 5DS to sorgomol in sorghum (Wakabayashi et al., 2021).

IDENTIFICATION OF GhCYP71AH AS STRIGOL SYNTHASE

To confirm the function of GhCYP71AH as strigol synthase, we performed an in vitro enzyme assay using purified recombinant GhCYP71AH. Recombinant GhCYP71AH was expressed in E. coli as a truncated form of the N-terminal transmembrane domain and purified (Figure S6). For the enzyme assay, we focused on the substrate specificity and stereoselectivity of the C-5 hydroxylation of 5DS. The 5DS stereoisomers, 5DS, ent-5DS, 2′-epi-5DS, and ent-2′-epi-5DS (4DO), were used as substrates. Eight stereoisomers of strigol were considered as enzyme reaction products, depending on the configuration of the introduced hydroxy group at C-5 of each 5DS stereoisomer (Figure 2a). When 5DS was used as a substrate, a peak with a retention time consistent with that of authentic rac-strigol (mixture of strigol and ent-strigol) was observed. In contrast, no products consistent with the corresponding strigol stereoisomer standards were detected with the other substrates. (Figure 2b). Because 5DS contains a C-2′R configuration in the D-ring, the enzyme reaction product consistent with rac-strigol using 5DS as a substrate must be identical to strigol (Figure 2). The results indicate that GhCYP71AH catalyzes the stereoselective introduction of a hydroxy group at C-5 of 5DS to form strigol.

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In the present study, the identification of strigol synthase signifies a noteworthy advancement in understanding the complete biosynthesis pathway of strigol, a prototypical and emblematic SL, at the molecular and biochemical levels. Enhancing the strigol biosynthesis pathway in cotton may confer useful traits as a trap crop, producing high levels of SL resulting in more Striga suicidal germination. On the other hand, it should be noted that the possibility that four uncloned candidates, whose inability to be cloned remains obscure, encode strigol synthase cannot be ruled out. These findings will further advance the use of strigol in SL research and increase our understanding of the rhizosphere interaction between plants and neighboring organisms.

AUTHOR CONTRIBUTIONS

Takatoshi Wakabayashi, Masaharu Mizutani, and Yukihiro Sugimoto planned and designed research. Takatoshi Wakabayashi, Megumi Nakayama, Yurie Kitano, and Masato Homma conducted experiments. Kenji Miura helped to construct the plasmids for the transient expression experiment. Hirosato Takikawa performed the synthesis of SLs. Takatoshi Wakabayashi and Yukihiro Sugimoto wrote the manuscript. All authors have read and approved the manuscript.

ACKNOWLEDGMENTS

This work was supported, in part, by Science and Technology Research Partnership for Sustainable Development (SATREPS), Japan Science and Technology Agency (JST)/Japan International Cooperation Agency (JICA) (No. JPMJSA1607 to YS), by ACT-X, JST (No. JPMJAX20BM to TW), by PRESTO, JST (No. JPMJPR22DA to TW), and by Japan Society for the Promotion of Science (JSPS) KAKENHI (Nos. 25292065 to YS and 20K15459 to TW).

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no competing interest.

DATA AVAILABILITY STATEMENT

The methods applied in this study are described in Supporting Information. Primary RNA-seq data were deposited to the DDBJ (DNA Data Bank of Japan) Sequence Read Archive (accession number DRA016503). The accession numbers for the sequences of the GhMAX1, GhCYP722C, and GhCYP71AH are LC770317, LC770318, and LC770319, respectively. The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

Abe, S., Sado, A., Tanaka, K., Kisugi, T., Asami, K., Ota, S., Kim, H. I., Yoneyama, K., Xie, X., Ohnishi, T., Seto, Y., Yamaguchi, S., Akiyama, K., Yoneyama, K., & Nomura, T. (2014). Carlactone is converted to carlactonoic acid by MAX1 in Arabidopsis and its methyl ester can directly interact with AtD14 in vitro. Proceedings of the National Academy of Sciences, 111, 18084–18089. https://doi.org/10.1073/pnas.1410801111

Akiyama, K., Matsuzaki, K., & Hayashi, H. (2005). Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi. Nature, 435, 824–827. https://doi.org/10.1038/nature03608

Alder, A., Jamil, M., Marzorati, M., Bruno, M., Vermathen, M., Bigler, P., Ghisla, S., Bouwmeester, H., Beyer, P., & al‐Babili, S. (2012). The path from β‐carotene to carlactone, a strigolactone‐like plant hormone. Science, 335, 1348–1351. https://doi.org/10.1126/science.1218094

Brooks, D. W., Bevinakatti, H. S., & Powell, D. R. (1985). The absolute structure of (+)‐strigol. The Journal of Organic Chemistry, 50(20), 3779–3781. https://doi.org/10.1021/jo00220a020

Butler, L. G. (1995). Chemical communication between the parasitic weed Striga and its crop host. ACS Symposium Series, 582, 158–168. https://doi.org/10.1021/bk-1995-0582.ch012

Cook, C. E., Whichard, L. P., Turner, B., Wall, M. E., & Egley, G. H. (1966). Germination of witchweed (Striga lutea Lour.): Isolation and properties of a potent stimulant. Science, 154(3753), 1189–1190. https://doi.org/10.1126/science.154.3753.1189

Cook, C. E., Whichard, L. P., Wall, M., Egley, G. H., Coggon, P., Luhan, P. A., & McPhail, A. T. (1972). Germination stimulants. II. Structure of strigol, a potent seed germination stimulant for witchweed (Striga lutea Lour). Journal of the American Chemical Society, 94(17), 6198–6199. https://doi.org/10.1021/ja00772a048

Gomez‐Roldan, V., Fermas, S., Brewer, P. B., Puech‐Pagès, V., Dun, E. A., Pillot, J.‐P., Letisse, F., Matusova, R., Danoun, S., Portais, J. C., Bouwmeester, H., Bécard, G., Beveridge, C. A., Rameau, C., & Rochange, S. F. (2008). Strigolactone inhibition of shoot branching. Nature, 455, 189–194. https://doi.org/10.1038/nature07271

Iseki, M., Shida, K., Kuwabara, K., Wakabayashi, T., Mizutani, M., Takikawa, H., & Sugimoto, Y. (2018). Evidence for species‐dependent biosynthetic pathways for converting carlactone to strigolactones in plants. Journal of Experimental Botany, 69(9), 2305–2318. https://doi.org/10.1093/jxb/erx428

Johnson, A. W., Gowada, G., Hassanali, A., Knox, J., Monaco, S., Razavi, Z., & Rosebery, G. (1981). The preparation of synthetic analogues of strigol. Journal of the Chemical Society, Perkin Transactions, 1, 1734–1743. https://doi.org/10.1039/P19810001734

Seto, Y., Sado, A., Asami, K., Hanada, A., Umehara, M., Akiyama, K., & Yamaguchi, S. (2014). Carlactone is an endogenous biosynthetic precursor for strigolactones. Proceedings of the National Academy of Sciences, 111, 1640–1645. https://doi.org/10.1073/pnas.1314805111

Tian, Z., Zhang, Y., Zhu, L., Jiang, B., Wang, H., Gao, R., Friml, J., & Xiao, G. (2022). Strigolactones act downstream of gibberellins to regulate fiber cell elongation and cell wall thickness in cotton (Gossypium hirsutum). The Plant Cell, 34(12), 4816–4839. https://doi.org/10.1093/plcell/koac270

Ueno, K., Nakashima, H., Mizutani, M., Takikawa, H., & Sugimoto, Y. (2018). Bioconversion of 5‐deoxystrigol stereoisomers to monohydroxylated strigolactones by plants. Journal of Pesticide Science, 43(3), 198–206. https://doi.org/10.1584/jpestics.D18-021

Umehara, M., Hanada, A., Yoshida, S., Akiyama, K., Arite, T., Takeda‐Kamiya, N., Magome, H., Kamiya, Y., Shirasu, K., Yoneyama, K., Kyozuka, J., & Yamaguchi, S. (2008). Inhibition of shoot branching by new terpenoid plant hormones. Nature, 455, 195–200. https://doi.org/10.1038/nature07272

Wakabayashi, T., Ishiwa, S., Shida, K., Motonami, N., Suzuki, H., Takikawa, H., Mizutani, M., & Sugimoto, Y. (2021). Identification and characterization of sorgomol synthase in sorghum strigolactone biosynthesis. Plant Physiology, 185(3), 902–913. https://doi.org/10.1093/plphys/kiaa113

Wakabayashi, T., Shida, K., Kitano, Y., Takikawa, H., Mizutani, M., & Sugimoto, Y. (2020). CYP722C from Gossypium arboreum catalyzes the conversion of carlactonoic acid to 5‐deoxystrigol. Planta, 251(5), 97. https://doi.org/10.1007/s00425-020-03390-6

Wang, Y., Durairaj, J., Suárez Duran, H. G., van Velzen, R., Flokova, K., Liao, C.‐Y., Chojnacka, A., MacFarlane, S., Schranz, M. E., Medema, M. H., van Dijk, A. D. J., Dong, L., & Bouwmeester, H. J. (2022). The tomato cytochrome P450 CYP712G1 catalyses the double oxidation of orobanchol en route to the rhizosphere signalling strigolactone, solanacol. New Phytologist, 235(5), 1884–1899. https://doi.org/10.1111/nph.18272

Wu, S., Ma, X., Zhou, A., Valenzuela, A., Zhou, K., & Li, Y. (2021). Establishment of strigolactone‐producing bacterium‐yeast consortium. Science Advances, 7, eabh4048. https://doi.org/10.1126/sciadv.abh4048

Yoneyama, K., Mori, N., Sato, T., Yoda, A., Xie, X., Okamoto, M., Iwanaga, M., Ohnishi, T., Nishiwaki, H., Asami, T., Yokota, T., Akiyama, K., Yoneyama, K., & Nomura, T. (2018). Conversion of carlactone to carlactonoic acid is a conserved function of MAX1 homologs in strigolactone biosynthesis. New Phytologist, 218(4), 1522–1533. https://doi.org/10.1111/nph.15055

Zhang, Y., van Dijk, A. D. J., Scaffidi, A., Flematti, G. R., Hofmann, M., Charnikhova, T., Verstappen, F., Hepworth, J., van der Krol, S., Leyser, O., Smith, S. M., Zwanenburg, B., al‐Babili, S., Ruyter‐Spira, C., & Bouwmeester, H. J. (2014). Rice cytochrome P450 MAX1 homologs catalyze distinct steps in strigolactone biosynthesis. Nature Chemical Biology, 10, 1028–1033. https://doi.org/10.1038/nchembio.1660