The AaBBX21–AaHY5 module mediates light-regulated artemisinin biosynthesis in Artemisia annua L.

Abstract

The sesquiterpene lactone artemisinin is an important anti-malarial component produced by the glandular secretory trichomes of sweet wormwood (Artemisia annua L.). Light was previously shown to promote artemisinin production, but the underlying regulatory mechanism remains elusive. In this study, we demonstrate that ELONGATED HYPOCOTYL 5 (HY5), a central transcription factor in the light signaling pathway, cannot promote artemisinin biosynthesis on its own, as the binding of AaHY5 to the promoters of artemisinin biosynthetic genes failed to activate their transcription. Transcriptome analysis and yeast two-hybrid screening revealed the B-box transcription factor AaBBX21 as a potential interactor with AaHY5. AaBBX21 showed a trichome-specific expression pattern. Additionally, the AaBBX21–AaHY5 complex cooperatively activated transcription from the promoters of the downstream genes AaGSW1,  AaMYB108, and AaORA, encoding positive regulators of artemisinin biosynthesis. Moreover, AaHY5 and AaBBX21 physically interacted with the A. annua E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1). In the dark, AaCOP1 decreased the accumulation of AaHY5 and AaBBX21 and repressed the activation of genes downstream of the AaHY5–AaBBX21 complex, explaining the enhanced production of artemisinin upon light exposure. Our study provides insights into the central regulatory mechanism by which light governs terpenoid biosynthesis in the plant kingdom.

Keywords

AaBBX21 / AaHY5 / AaCOP1 / Artemisia annua L. / artemisinin biosynthesis / light

References

[1] An, J.P.,Wang, X.F.,Zhang, X.W.,Bi, S.Q.,You, C.X., and Hao, Y.J. (2019). MdBBX22 regulates UV-B-induced anthocyanin biosynthesis through regulating the function of MdHY5 and is targeted by MdBT2 for 26S proteasome-mediated degradation. Plant Biotechnol. J. 17:2231–2233.

[2] Ang, L.H.,Chattopadhyay, S.,Wei, N.,Oyama, T.,Okada, K.,Batschauer, A., and Deng, X.W. (1998). Molecular interaction between COP1 and HY5 defines a regulatory switch for light control of Arabidopsis development. Mol. Cell 1:213–222.

[3] Bai, S.,Tao, R.,Tang, Y.,Yin, L.,Ma, Y.,Ni, J.,Yan, X.,Yang, Q.,Wu, Z.,Zeng, Y., et al. (2019a). BBX16, a B-box protein, positively regulates light-induced anthocyanin accumulation by activating MYB10 in red pear. Plant Biotechnol. J. 17:1985–1997.

[4] Bai, S.,Tao, R.,Yin, L.,Ni, J.,Yang, Q.,Yan, X.,Yang, F.,Guo, X.,Li, H., and Teng, Y. (2019b). Two B-box proteins, PpBBX18 and PpBBX21, antagonistically regulate anthocyanin biosynthesis via competitive association with Pyrus pyrifolia ELONGATED HYPOCOTYL 5 in the peel of pear fruit. Plant J. 100:1208–1223.

[5] Bouwmeester, H.J.,Wallaart, T.E.,Janssen, M.H.,van Loo, B.,Jansen, B.J.,Posthumus, M.A.,Schmidt, C.O.,De Kraker, J.W.,König, W.A., and Franssen, M.C. (1999). Amorpha-4, 11-diene synthase catalyses the first probable step in artemisinin biosynthesis. Phytochemistry 52:843–854.

[6] Brown, G.D., and Sy, L.K. (2004). In vivo transformations of dihydroartemisinic acid in Artemisia annua plants. Tetrahedron 60:1139–1159.

[7] Brown, G.D., and Sy, L.K. (2007). In vivo transformations of artemisinic acid in Artemisia annua plants. Tetrahedron 63:9548–9566.

[8] Bursch, K.,Toledo-Ortiz, G.,Pireyre, M.,Lohr, M.,Braatz, C., and Johansson, H. (2020). Identification of BBX proteins as rate-limiting cofactors of HY5. Nat. Plants 6:921–928.

[9] Cashmore, A.R. (2003). Cryptochromes: Enabling plants and animals to determine circadian time. Cell 114:537–543.

[10] Chattopadhyay, S.,Ang, L.H.,Puente, P.,Deng, X.W., and Wei, N. (1998). Arabidopsis bZIP protein HY5 directly interacts with light-responsive promoters in mediating light control of gene expression. Plant Cell 10:673–683.

[11] Chen, C.,Chen, H.,Zhang, Y.,Thomas, H.R.,Frank, M.H.,He, Y., and Xia, R. (2020). TBtools: An integrative toolkit developed for interactive analyses of big biological data. Mol. Plant 13:1194–1202.

[12] Chen, G.,He, W.,Guo, X., and Pan, J. (2021a). Genome-wide identification, classification and expression analysis of the MYB transcription factor family in Petunia. Int. J. Mol. Sci. 22:4838.

[13] Chen, H.,Huang, X.,Gusmaroli, G.,Terzaghi, W.,Lau, O.S.,Yanagawa, Y.,Zhang, Y.,Li, J.,Lee, J.H.,Zhu, D., et al. (2010). Arabidopsis CULLIN4-damaged DNA binding protein 1 interacts with CONSTITUTIVELY PHOTOMORPHOGENIC1-SUPPRESSOR OF PHYA complexes to regulate photomorphogenesis and flowering time. Plant Cell 22:108–123.

[14] Chen, M.,Yan, T.,Shen, Q.,Lu, X.,Pan, Q.,Huang, Y.,Tang, Y.,Fu, X.,Liu, M.,Jiang, W., et al. (2017). GLANDULAR TRICHOME-SPECIFIC WRKY 1 promotes artemisinin biosynthesis in Artemisia annua. New Phytol. 214:304–316.

[15] Chen, T.,Li, Y.,Xie, L.,Hao, X.,Liu, H.,Qin, W.,Wang, C.,Yan, X.,Wu-Zhang, K.,Yao, X., et al. (2021b). AaWRKY17, a positive regulator of artemisinin biosynthesis, is involved in resistance to Pseudomonas syringae in Artemisia annua. Hort. Res 8:217.

[16] Chen, T.T.,Liu, H.,Li, Y.P.,Yao, X.H.,Qin, W.,Yan, X.,Wang, X.Y.,Peng, B.W.,Zhang, Y.J.,Shao, J., et al. (2023). AaSEPALLATA1 integrates jasmonate and light-regulated glandular secretory trichome initiation in Artemisia annua. Plant Physiol. 192:1483–1497.

[17] Datta, S.,Johansson, H.,Hettiarachchi, C.,Irigoyen, M.L.,Desai, M.,Rubio, V., and Holm, M. (2008). LZF1/SALT TOLERANCE HOMOLOG3, an Arabidopsis B-box protein involved in light-dependent development and gene expression, undergoes COP1-mediated ubiquitination. Plant Cell 20:2324–2338.

[18] Delabays, N.,Simonnet, X., and Gaudin, M. (2001). The genetics of artemisinin content in Artemisia annua L. and the breeding of high yielding cultivars. Curr. Med. Chem. 8:1795–1801.

[19] Dondorp, A.M.,Nosten, F.,Yi, P.,Das, D.,Phyo, A.P.,Tarning, J.,Lwin, K.M.,Ariey, F.,Hanpithakpong, W.,Lee, S.J., et al. (2009). Artemisinin resistance in Plasmodium falciparum malaria. N. Engl. J. Med. 361:455–467.

[20] Fu, X.,Peng, B.,Hassani, D.,Xie, L.,Liu, H.,Li, Y.,Chen, T.,Liu, P.,Tang, Y.,Li, L., et al. (2021). AaWRKY9 contributes to light-and jasmonate-mediated to regulate the biosynthesis of artemisinin in Artemisia annua. New Phytol. 231:1858–1874.

[21] Greenwood, B., and Mutabingwa, T. (2002). Malaria in 2002. Nature 415:670–672.

[22] Hao, X.,Zhong, Y.,Fu, X.,Lv, Z.,Shen, Q.,Yan, T.,Shi, P.,Ma, Y.,Chen, M.,Lv, X., et al. (2017). Transcriptome analysis of genes associated with the artemisinin biosynthesis by jasmonic acid treatment under the light in Artemisia annua. Front. Plant Sci. 8:971.

[23] Hao, X.,Zhong, Y.,Nï Tzmann, H.W.,Fu, X.,Yan, T.,Shen, Q.,Chen, M.,Ma, Y.,Zhao, J.,Osbourn, A., et al. (2019). Light-Induced artemisinin biosynthesis is regulated by the bZIP transcription factor AaHY5 in Artemisia annua. Plant Cell Physiol. 60:1747–1760.

[24] He, W.,Liu, H.,Li, Y.,Wu, Z.,Xie, Y.,Yan, X.,Wang, X.,Miao, Q.,Chen, T.,Rahman, S., et al. (2023). Genome-wide characterization of B-box gene family in Artemisia annua L. and its potential role in the regulation of artemisinin biosynthesis. Ind. Crops Prod. 199:116736.

[25] Holm, M.,Hardtke, C.S.,Gaudet, R., and Deng, X.W. (2001). Identification of a structural motif that confers specific interaction with the WD40 repeat domain of Arabidopsis COP1. EMBO J. 20:118–127.

[26] Jiao, Y.,Lau, O.S., and Deng, X.W. (2007). Light-regulated transcriptional networks in higher plants. Nat. Rev. Genet. 8:217–230.

[27] Lau, K.,Podolec, R.,Chappuis, R.,Ulm, R., and Hothorn, M. (2019). Plant photoreceptors and their signaling components compete for COP1 binding via VP peptide motifs. EMBO J. 38: e102140.

[28] Li, C.,Pei, J.,Yan, X.,Cui, X.,Tsuruta, M.,Liu, Y., and Lian, C. (2021a). A poplar B-box protein PtrBBX23 modulates the accumulation of anthocyanins and proanthocyanidins in response to high light. Plant Cell Environ. 44:3015–3033.

[29] Li, Y.,Chen, T.,Liu, H.,Qin, W.,Yan, X.,Wu-Zhang, K.,Peng, B.,Zhang, Y.,Yao, X.,Fu, X., et al. (2022). The truncated AaActin1 promoter is a candidate tool for metabolic engineering of artemisinin biosynthesis in Artemisia annua. L. J. Plant Physiol. 274:153712.

[30] Li, Y.,Qin, W.,Fu, X.,Zhang, Y.,Hassani, D.,Kayani, S.I.,Xie, L.,Liu, H.,Chen, T.,Yan, X., et al. (2021b). Transcriptomic analysis reveals the parallel transcriptional regulation of UV-B-induced artemisinin and flavonoid accumulation in Artemisia annua L. Plant Physiol. Biochem. 163:189–200.

[31] Li, Y.,Qin, W.,Liu, H.,Chen, T.,Yan, X.,He, W.,Peng, B.,Shao, J.,Fu, X.,Li, L., et al. (2023). Increased artemisinin production by promoting glandular secretory trichome formation and reconstructing the artemisinin biosynthetic pathway in Artemisia annua. Hort. Res. 10: uhad055.

[32] Liu, H.,He, W.,Yao, X.,Yan, X.,Wang, X.,Peng, B.,Zhang, Y.,Shao, J.,Hu, X.,Miao, Q., et al. (2023b). The light-and jasmonic acid-induced AaMYB108-like positive regulates the initiation of glandular secretory trichome in Artemisia annua L. Int. J. Mol. Sci. 24:12929.

[33] Liu, H.,Li, L.,Fu, X.,Li, Y.,Chen, T.,Qin, W.,Yan, X.,Wu, Z.,Xie, L.,Kayani, S.-l, et al. (2023a). AaMYB108 is the core factor integrating light and jasmonic acid signaling to regulate artemisinin biosynthesis in Artemisia annua. New Phytol. 237:2224–2237.

[34] Lu, X.,Zhang, L.,Zhang, F.,Jiang, W.,Shen, Q.,Zhang, L.,Lv, Z.,Wang, G., and Tang, K. (2013). AaORA, a trichome-specific AP2/ERF transcription factor of Artemisia annua, is a positive regulator in the artemisinin biosynthetic pathway and in disease resistance to Botrytis cinerea. New Phytol. 198:1191–1202.

[35] Lv, Z.,Guo, Z.,Zhang, L.,Zhang, F.,Jiang, W.,Shen, Q.,Fu, X.,Yan, T.,Shi, P.,Hao, X., et al. (2019). Interaction of bZIP transcription factor TGA6 with salicylic acid signaling modulates artemisinin biosynthesis in Artemisia annua. J. Exp. Bot. 70:3969–3979.

[36] Ma, Y.N.,Xu, D.B.,Li, L.,Zhang, F.,Fu, X.Q.,Shen, Q.,Lyu, X.Y.,Wu, Z.K.,Pan, Q.F.,Shi, P., et al. (2018). Jasmonate promotes artemisinin biosynthesis by activating the TCP14-ORA complex in Artemisia annua. Sci. Adv. 4: eaas9357.

[37] Ma, Y.N.,Xu, D.B.,Yan, X.,Wu, Z.K.,Kayani, S.I.,Shen, Q.,Fu, X.Q.,Xie, L.H.,Hao, X.L.,Hassani, D., et al. (2021). Jasmonate-and abscisic acid-activated AaGSW1-AaTCP15/AaORA transcriptional cascade promotes artemisinin biosynthesis in Artemisia annua. Plant Biotechnol. J. 19:1412–1428.

[38] Olsson, M.E.,Olofsson, L.M.,Lindahl, A.L.,Lundgren, A.,Brodelius, M., and Brodelius, P.E. (2009). Localization of enzymes of artemisinin biosynthesis to the apical cells of glandular secretory trichomes of Artemisia annua L. Phytochemistry 70:1123–1128.

[39] Osterlund, M.T.,Hardtke, C.S.,Wei, N., and Deng, X.W. (2000). Targeted destabilization of HY5 during light-regulated development of Arabidopsis. Nature 405:462–466.

[40] Oyama, T.,Shimura, Y., and Okada, K. (1997). The Arabidopsis HY5 gene encodes a bZIP protein that regulates stimulus-induced development of root and hypocotyl. Genes Dev. 11:2983–2995.

[41] Paddon, C.J.,Westfall, P.J.,Pitera, D.J.,Benjamin, K.,Fisher, K.,McPhee, D.,Leavell, M.D.,Tai, A.,Main, A.,Eng, D., et al. (2013). High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 496:528–532.

[42] Podolec, R., and Ulm, R. (2018). Photoreceptor-mediated regulation of the COP1/SPA E3 ubiquitin ligase. Curr. Opin. Plant Biol. 45:18–25.

[43] Ro, D.K.,Paradise, E.M.,Ouellet, M.,Fisher, K.J.,Newman, K.L.,Ndungu, J.M.,Ho, K.A.,Eachus, R.A.,Ham, T.S.,Kirby, J., et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940–943.

[44] Schramek, N.,Wang, H.,Römisch-Margl, W.,Keil, B.,Radykewicz, T.,Winzenhörlein, B.,Beerhues, L.,Bacher, A.,Rohdich, F.,Gershenzon, J., et al. (2010). Artemisinin biosynthesis in growing plants of Artemisia annua. A ¹³CO₂ study. Phytochemistry 71:179–187.

[45] Shen, Q.,Lu, X.,Yan, T.,Fu, X.,Lv, Z.,Zhang, F.,Pan, Q.,Wang, G.,Sun, X., and Tang, K. (2016). The jasmonate-responsive AaMYC2 transcription factor positively regulates artemisinin biosynthesis in Artemisia annua. New Phytol. 210:1269–1281.

[46] Shen, Q.,Zhang, L.,Liao, Z.,Wang, S.,Yan, T.,Shi, P.,Liu, M.,Fu, X.,Pan, Q.,Wang, Y., et al. (2018). The genome of Artemisia annua provides insight into the evolution of Asteraceae family and artemisinin biosynthesis. Mol. Plant 11:776–788.

[47] Tang, Y.,Li, L.,Yan, T.,Fu, X.,Shi, P.,Shen, Q.,Sun, X., and Tang, K. (2018). AaEIN3 mediates the downregulation of artemisinin biosynthesis by ethylene signaling through promoting leaf senescence in Artemisia annua. Front. Plant Sci. 9:413.

[48] Teoh, K.H.,Polichuk, D.R.,Reed, D.W., and Covello, P.S. (2009). Molecular cloning of an aldehyde dehydrogenase implicated in artemisinin biosynthesis in Artemisia annua. Botany 87:635–642.

[49] Teoh, K.H.,Polichuk, D.R.,Reed, D.W.,Nowak, G., and Covello, P.S. (2006). Artemisia annua L. (Asteraceae) trichome-specific cDNAs reveal CYP71AV1, a cytochrome P450 with a key role in the biosynthesis of the antimalarial sesquiterpene lactone artemisinin. FEBS Lett. 580:1411–1416.

[50] Towler, M.J., and Weathers, P.J. (2007). Evidence of artemisinin production from IPP stemming from both the mevalonate and the nonmevalonate pathways. Plant Cell Rep. 26:2129–2136.

[51] World Health Organization. (2023). World malaria report 2023 [WWWdocument]. https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2023

[52] Wu, Z.,Li, L.,Liu, H.,Yan, X.,Ma, Y.,Li, Y.,Chen, T.,Wang, C.,Xie, L.,Hao, X., et al. (2021). AaMYB15, an R2R3-MYB TF in Artemisia annua, acts as a negative regulator of artemisinin biosynthesis. Plant Sci. 308:110920.

[53] Xiao, Y.,Chu, L.,Zhang, Y.,Bian, Y.,Xiao, J., and Xu, D. (2022). HY5: A pivotal regulator of light-dependent development in higher plants. Front. Plant Sci. 12:800989.

[54] Xu, D. (2020). COP1 and BBXs-HY5-mediated light signal transduction in plants. New Phytol. 228:1748–1753.

[55] Xu, D.,Jiang, Y.,Li, J.,Holm, M., and Deng, X.W. (2018). The B-box domain protein BBX21 promotes photomorphogenesis. Plant Physiol. 176:2365–2375.

[56] Xu, D.,Jiang, Y.,Li, J.,Lin, F.,Holm, M., and Deng, X.W. (2016). BBX21, an Arabidopsis B-box protein, directly activates HY5 and is targeted by COP1 for 26S proteasome-mediated degradation. Proc. Natl. Acad. Sci. U.S.A. 113:7655–7660.

[57] Yan, T.,Chen, M.,Shen, Q.,Li, L.,Fu, X.,Pan, Q.,Tang, Y.,Shi, P.,Lv, Z.,Jiang, W., et al. (2017). HOMEODOMAIN PROTEIN 1 is required for jasmonate-mediated glandular trichome initiation in Artemisia annua. New Phytol. 213:1145–1155.

[58] Yang, G.,Zhang, C.,Dong, H.,Liu, X.,Guo, H.,Tong, B.,Fang, F.,Zhao, Y.,Yu, Y.,Liu, Y., et al. (2022). Activation and negative feedback regulation of SlHY5 transcription by the SlBBX20/21-SlHY5 transcription factor module in UV-B signaling. Plant Cell 34:2038–2055.

[59] Yuan, M.,Shu, G.,Zhou, J.,He, P.,Xiang, L.,Yang, C.,Chen, M.,Liao, Z., and Zhang, F. (2023). AabHLH113 integrates jasmonic acid and abscisic acid signaling to positively regulate artemisinin biosynthesis in Artemisia annua. New Phytol. 237:885–899.

[60] Zhang, D.,Sun, W.,Shi, Y.,Wu, L.,Zhang, T., and Xiang, L. (2018). Red and blue light promote the accumulation of artemisinin in Artemisia annua L. Molecules 23:1329.

[61] Zhang, F.,Fu, X.,Lv, Z.,Lu, X.,Shen, Q.,Zhang, L.,Zhu, M.,Wang, G.,Sun, X.,Liao, Z., et al. (2015). A basic leucine zipper transcription factor, AabZIP1, connects abscisic acid signaling with artemisinin biosynthesis in Artemisia annua. Mol. Plant 8:163–175.

[62] Zhang, H.,He, H.,Wang, X.,Wang, X.,Yang, X.,Li, L., and Deng, X.W. (2011). Genome-wide mapping of the HY5-mediated gene networks in Arabidopsis that involve both transcriptional and post-transcriptional regulation. Plant J. 65:346–358.

[63] Zhang, H.,Zhang, Q.,Zhai, H.,Gao, S.,Yang, L.,Wang, Z.,Xu, Y.,Huo, J.,Ren, Z.,Zhao, N., et al. (2020). IbBBX24 promotes the jasmonic acid pathway and enhances fusarium wilt resistance in sweet potato. Plant Cell 32:1102–1123.

[64] Zhang, Y.,Teoh, K.H.,Reed, D.W.,Maes, L.,Goossens, A.,Olson, D.J.,Ross, A.R., and Covello, P.S. (2008). The molecular cloning of artemisinic aldehyde Delta11(13) reductase and its role in glandular trichome-dependent biosynthesis of artemisinin in Artemisia annua. J. Biol. Chem. 283:21501–21508.

[65] Zheng, H.,Fu, X.,Shao, J.,Tang, Y.,Yu, M.,Li, L.,Huang, L., and Tang, K. (2023). Transcriptional regulatory network of high-value active ingredients in medicinal plants. Trends Plant Sci. 28:429–446.

[66] Zhou, L.,Huang, Y.,Wang, Q., and Guo, D. (2021). AaHY5 ChIP-seq based on transient expression system reveals the role of AaWRKY14 in artemisinin biosynthetic gene regulation. Plant Physiol. Biochem. 168:321–328.

RIGHTS & PERMISSIONS

2024 The Author(s). Journal of Integrative Plant Biology published by John Wiley & Sons Australia, Ltd on behalf of Institute of Botany, Chinese Academy of Sciences.