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The Korean Society of Phycology (한국조류학회(藻類))
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A zeaxanthin epoxidase homolog from the intertidal red alga Pyropia haitanensis: evolutionary divergence and stress-responsive expression

  • Zhujun Zhu (Collaborative Innovation Center for Zhejiang Marine High-efficiency and Healthy Aquaculture, Ningbo University) ;
  • Tingting Niu (Collaborative Innovation Center for Zhejiang Marine High-efficiency and Healthy Aquaculture, Ningbo University) ;
  • Yanrong Li (Collaborative Innovation Center for Zhejiang Marine High-efficiency and Healthy Aquaculture, Ningbo University) ;
  • Juanjuan Chen (Collaborative Innovation Center for Zhejiang Marine High-efficiency and Healthy Aquaculture, Ningbo University) ;
  • Xiaojun Yan (College of Marine Science and Technology, Zhejiang Ocean University) ;
  • Haimin Chen (Collaborative Innovation Center for Zhejiang Marine High-efficiency and Healthy Aquaculture, Ningbo University)
  • Received : 2025.07.18
  • Accepted : 2026.05.17
  • Published : 2026.06.19
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Abstract

Zeaxanthin epoxidase (ZEP) plays a central role in carotenoid metabolism, but its evolution and function in red algae remain unclear. Here, we comprehensively identified ZEP homologs across red algal lineages through phylogenetic reconstruction, conserved motif analysis, structural comparison, and branch-specific selection pressure analyses to uncover adaptive evolutionary patterns. The ZEP homolog from Pyropia haitanensis (PhZEP) was selected for functional characterization, including promoter cis-element analysis, heterologous expression, in vitro enzymatic assays, and expression profiling under high light and desiccation-rehydration stress. Thirteen ZEP homologs were identified and clustered into three clades, indicating early divergence and a cyanobacterial origin. Lineage-specific structural divergence and purifying selection with relaxed constraints in Bangiophyceae were observed. Recombinant PhZEP specifically converted zeaxanthin to antheraxanthin, but not further to violaxanthin. PhZEP expression was moderately induced by high light and strongly upregulated during desiccation, peaking at 9.52-fold at 4.8% relative water content. These results demonstrate that PhZEP exhibits specific zeaxanthin epoxidase activity and may be associated with abiotic stress responses, providing insights into carotenoid pathway evolution and photoprotection strategies in red algae.

Keywords

Acknowledgement

This work was supported by the Zhejiang Provincial Natural Science Foundation of China (LQ23C190003), the National Natural Science Foundation of China (32503155, 32373099), the Major Scientific and Technological Project of Zhejiang Province (2021C02069-9), the Major Scientific and Technological Project of Ningbo (2021Z004, 2021Z103), the Ningbo Natural Science Foundation (2024J175, 2024J463), and the China Agriculture Research System.

References

  1. Baroli, I., Do, A. D., Yamane, T. & Niyogi, K. K. 2003. Zeaxan‑thin accumulation in the absence of a functional xantho‑phyll cycle protects Chlamydomonas reinhardtii from photo‑oxidative stress. Plant Cell 15:992–1008. doi.org/10.1105/tpc.010405
  2. Büch, K., Stransky, H. & Hager, A. 1995. FAD is a further essential cofactor of the NAD(P)H and O2-depen‑dent zeaxanthin-epoxidase. FEBS Lett. 376:45–48. doi.org/10.1016/0014-5793(95)01243-9
  3. Bugos, R. C., Hieber, A. D. & Yamamoto, H. Y. 1998. Xantho‑phyll cycle enzymes are members of the lipocalin family, the first identified from plants. J. Biol. Chem. 273:15321–15324. doi.org/10.1074/jbc.273.25.15321
  4. Cao, X., Kong, F., Sun, B., et al. 2022. A toolbox for constructing a stable genetic transformation platform allowing foreign fragment integration in the genome of Neopyropia yezoensis. Front. Mar. Sci. 9:856790. doi.org/10.3389/fmars.2022.856790
  5. Chen, H., Chu, J. S.-C., Chen, J., et al. 2022. Insights into the ancient adaptation to intertidal environments by red algae based on a genomic and multiomics investigation of Neoporphyra haitanensis. Mol. Biol. Evol. 39:msab315. doi.org/10.1093/molbev/msab315
  6. Coesel, S., Oborník, M., Varela, J., Falciatore, A. & Bowler, C. 2008. Evolutionary origins and functions of the carotenoid biosynthetic pathway in marine diatoms. PLoS ONE 3:e2896. doi.org/10.1371/journal.pone.0002896
  7. Contreras-Porcia, L., Thomas, D., Flores, V. & Correa, J. A. 2011. Tolerance to oxidative stress induced by desiccation in Porphyra columbina (Bangiales, Rhodophyta). J. Exp. Bot. 62:1815–1829. doi.org/10.1093/jxb/erq364
  8. Dautermann, O. & Lohr, M. 2017. A functional zeaxanthin epoxidase from red algae shedding light on the evolution of light-harvesting carotenoids and the xanthophyll cycle in photosynthetic eukaryotes. Plant J. 92:879–891. doi.org/10.1111/tpj.13725
  9. Denoeud, F., Godfroy, O., Cruaud, C., et al. 2024. Evolutionary genomics of the emergence of brown algae as key com‑ponents of coastal ecosystems. Cell 187:6943–6965. doi.org/10.1016/j.cell.2024.10.049
  10. Eilers, U., Dietzel, L., Breitenbach, J., Büchel, C. & Sandmann, G. 2016. Identification of genes coding for functional zeaxanthin epoxidases in the diatom Phaeodactylum tricornutum. J. Plant Physiol. 192:64–70. doi.org/10.1016/j.jplph.2016.01.006
  11. Esteban, R., Martınez, B., Fernandez-Marin, B., Becerril, J. M. & García-Plazaola, J. I. 2009. Carotenoid composi‑tion in Rhodophyta: insights into xanthophyll regula‑tion in Corallina elongata. Eur. J. Phycol. 44:221–230. doi.org/10.1080/09670260802439109
  12. Fernández-Marín, B., Míguez, F., Becerril, J. M. & García-Pla‑zaola, J. I. 2011. Activation of violaxanthin cycle in dark‑ness is a common response to different abiotic stresses: a case study in Pelvetia canaliculata. BMC Plant Biol. 11:181. doi.org/10.1186/1471-2229-11-181
  13. Giossi, C. E., Wünsch, M. A., Dautermann, O., et al. 2025. Both major xanthophyll cycles present in nature promote non‑photochemical quenching in a model diatom. Plant Physi‑ol. 199:kiaf371. doi.org/10.1093/plphys/kiaf371
  14. Goss, R. & Latowski, D. 2020. Lipid dependence of xantho‑phyll cycling in higher plants and algae. Front. Plant Sci. 11:455. doi.org/10.3389/fpls.2020.00455
  15. Goss, R. & Lepetit, B. 2015. Biodiversity of NPQ. J. Plant Physi‑ol. 172:13–32. doi.org/10.1016/j.jplph.2014.03.004
  16. He, H., Yang, X., Zeb, A., et al. 2024. Cloning and functional analysis of a zeaxanthin epoxidase gene in Ulva prolifera. Biology 13:695. doi.org/10.3390/biology13090695
  17. Hieber, A. D., Bugos, R. C. & Yamamoto, H. Y. 2000. Plant li‑pocalins: violaxanthin de-epoxidase and zeaxanthin epoxi‑dase. Biochim. Biophys. Acta 1482:84–91. doi.org/10.1016/s0167-4838(00)00141-2
  18. Holt, N. E., Fleming, G. R. & Niyogi, K. K. 2004. Toward an understanding of the mechanism of nonphotochemical quenching in green plants. Biochemistry 43:8281–8289. doi.org/10.1021/bi0494020
  19. Huang, Y., Weng, Z., Li, S., et al. 2024. The photosynthetic per‑formance and photoprotective role of carotenoids response to light stress in intertidal red algae Neoporphyra haitanensis. J. Phycol. 60:942–955. doi.org/10.1111/jpy.13480
  20. Jahns, P. & Holzwarth, A. R. 2012. The role of the xanthophyll cycle and of lutein in photoprotection of photosystem II. Biochim. Biophys. Acta Bioenerg. 1817:182–193. doi.org/10.1016/j.bbabio.2011.04.012
  21. Kim, M., Cazzaniga, S., Jang, J., et al. 2024. Photoautotrophic cultivation of a Chlamydomonas reinhardtii mutant with zeaxanthin as the sole xanthophyll. Biotechnol. Biofuels Bioprod. 17:41. doi.org/10.1186/s13068-024-02483-8
  22. Kobayashi, Y., Ando, H., Hanaoka, M. & Tanaka, K. 2016. Ab‑scisic acid participates in the control of cell cycle initiation through heme homeostasis in the unicellular red alga Cyanidioschyzon merolae. Plant Cell Physiol. 57:953–960. doi.org/10.1093/pcp/pcw054
  23. Koizumi, J., Takatani, N., Kobayashi, N., et al. 2018. Carot‑enoid profiling of a red seaweed Pyropia yezoensis: insights into biosynthetic pathways in the order Bangiales. Mar. Drugs 16:426. doi.org/10.3390/md16110426
  24. Kuczyńska, P., Latowski, D., Niczyporuk, S., et al. 2012. Zea‑xanthin epoxidation: an in vitro approach. Acta Biochim. Pol. 59:105–107. doi.org/10.18388/abp.2012_2182
  25. Kumar, M., Gupta, V., Trivedi, N., et al. 2011. Desiccation induced oxidative stress and its biochemical responses in intertidal red alga Gracilaria corticata (Gracilariales, Rhodo‑phyta). Environ. Exp. Bot. 72:194–201. doi.org/10.1016/j.envexpbot.2011.03.007
  26. Kumar, S., Stecher, G. & Tamura, K. 2016. MEGA7: molecu‑lar evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33:1870–1874. doi.org/10.1093/molbev/msw054
  27. Küster, L., Lücke, R., Brabender, C., Bethmann, S. & Jahns, P. 2023. The amount of zeaxanthin epoxidase but not the amount of violaxanthin de-epoxidase is a critical determinant of zeaxanthin accumulation in Arabidopsis thaliana and Nicotiana tabacum. Plant Cell Physiol. 64:1220–1230. doi.org/10.1093/pcp/pcad091
  28. Li, Z., Peers, G., Dent, R. M., et al. 2016. Evolution of an atypi‑cal de-epoxidase for photoprotection in the green lineage. Nat. Plants 2:16140. doi.org/10.1038/nplants.2016.140
  29. Liang, C., Zhao, F., Wei, W., Wen, Z. & Qin, S. 2006. Carot‑enoid biosynthesis in cyanobacteria: structural and evo‑lutionary scenarios based on comparative genomics. Int. J Biol. Sci. 2:197–207. doi.org/10.7150/ijbs.2.197
  30. Liu, M., Ding, W., Pan, Y., Hu, H. & Liu, J. 2023. Zeaxanthin epoxidase is involved in the carotenoid biosynthesis and light-dependent growth of the marine alga Nannochloropsis oceanica. Biotechnol. Biofuels Bioprod. 16:74. doi.org/10.1186/s13068-023-02326-y
  31. Marin, E., Nussaume, L., Quesada, A., et al. 1996. Molecular identification of zeaxanthin epoxidase of Nicotiana plumbaginifolia, a gene involved in abscisic acid biosynthesis and corresponding to the ABA locus of Arabidopsis thaliana. EMBO J. 15:2331–2342. doi.org/10.1002/j.1460-2075.1996.tb00589.x
  32. Müller, P., Li, X.-P. & Niyogi, K. K. 2001. Non-photochemical quenching: a response to excess light energy. Plant Physi‑ol. 125:1558–1566. doi.org/10.1104/pp.125.4.1558
  33. Nambara, E. & Marion-Poll, A. 2005. Abscisic acid biosynthe‑sis and catabolism. Annu. Rev. Plant Biol. 56:165–185. doi.org/10.1146/annurev.arplant.56.032604.144046
  34. Nègre, D., Aite, M., Belcour, A., et al. 2019. Genome-scale met‑abolic networks shed light on the carotenoid biosynthesis pathway in the brown algae Saccharina japonica and Cladosiphon okamuranus. Antioxidants 8:564. doi.org/10.3390/antiox8110564
  35. Nisar, N., Li, L., Lu, S., Khin, N. C. & Pogson, B. J. 2015. Ca‑rotenoid metabolism in plants. Mol. Plant 8:68–82. doi.org/10.1016/j.molp.2014.12.007
  36. Oritani, T. & Kiyota, H. 2003. Biosynthesis and metabolism of abscisic acid and related compounds. Nat. Prod. Rep. 20:414–425. doi.org/10.1039/b109859b
  37. Ruban, A. V., Berera, R., Ilioaia, C., et al. 2007. Identification of a mechanism of photoprotective energy dissipation in higher plants. Nature 450:575–578. doi.org/10.1038/na‑ture06262
  38. Schaller, S., Wilhelm, C., Strzałka, K. & Goss, R. 2012. Inves‑tigating the interaction between the violaxanthin cycle enzyme zeaxanthin epoxidase and the thylakoid mem‑brane. J. Photochem. Photobiol. B Biol. 114:119–125. doi.org/10.1016/j.jphotobiol.2012.05.019
  39. Schubert, N. & García-Mendoza, E. 2008. Photoinhibition in red algal species with different carotenoid profiles. J. Phycol. 44:1437–1446. doi.org/10.1111/j.1529-8817.2008.00590.x
  40. Schubert, N., García-Mendoza, E. & Pacheco-Ruiz, I. 2006. Carotenoid composition of marine red algae. J. Phycol. 42:1208–1216. doi.org/10.1111/j.1529-8817.2006.00274.x
  41. Shin, Y. J., Lim, J.-M., Park, J. H., et al. 2016. Characteriza‑tion of PyGUS gene silencing in the red macroalga, Pyropia yezoensis. Plant Biotechnol. Rep. 10:359–367. doi.org/10.1007/s11816-016-0408-5
  42. Sun, Y., Pri-Tal, O., Michaeli, D. & Mosquna, A. 2020. Evolu‑tion of abscisic acid signaling module and its perception. Front. Plant Sci. 11:934. doi.org/10.3389/fpls.2020.00934
  43. Suyama, M., Torrents, D. & Bork, P. 2006. PAL2NAL: robust conversion of protein sequence alignments into the corre‑sponding codon alignments. Nucleic Acids Res. 34:W609–W612. doi.org/10.1093/nar/gkl315
  44. Takaichi, S. 2011. Carotenoids in algae: distributions, bio‑syntheses and functions. Mar. Drugs 9:1101–1118. doi.org/10.3390/md9061101
  45. Takaichi, S., Yokoyama, A., Mochimaru, M., Uchida, H. & Murakami, A. 2016. Carotenogenesis diversification in phylogenetic lineages of Rhodophyta. J. Phycol. 52:329–338. doi.org/10.1111/jpy.12411
  46. Wang, H., Xie, X., Gu, W., et al. 2024. Gene editing of eco‑nomic macroalga Neopyropia yezoensis (Rhodophyta) will promote its development into a model species of marine algae. New Phytol. 244:1687–1691. doi.org/10.1111/nph.20123
  47. Wang, Q. 2023. The role of forkhead-associated (FHA)-domain proteins in plant biology. Plant Mol. Biol. 111:455–472. doi.org/10.1007/s11103-023-01338-4
  48. Wang, S., Zhang, L., Chi, S., et al. 2018. Phylogenetic analyses of the genes involved in carotenoid biosynthesis in algae. Acta Oceanol. Sin. 37:89–101. doi.org/10.1007/s13131-0181178-4
  49. Xie, X., Lu, X., Wang, L., He, L. & Wang, G. 2020. High light intensity increases the concentrations of β-carotene and zeaxanthin in marine red macroalgae. Algal Res. 47:101852. doi.org/10.1016/j.algal.2020.101852
  50. Yang, J., Gu, W., Feng, Z., Yu, B., Niu, J. & Wang, G. 2021. Synthesis of abscisic acid in Neopyropia yezoensis and its regulation of antioxidase genes expressions under hyper‑saline stress. Front. Microbiol. 12:775710. doi.org/10.3389/fmicb.2021.775710
  51. Yang, L.-E., Deng, Y.-Y., Xu, G.-P., Russell, S., Lu, Q.-Q. & Bro‑die, J. 2020. Redefining Pyropia (Bangiales, Rhodophyta): four new genera, resurrection of Porphyrella and descrip‑tion of Calidia pseudolobata sp. nov. from China. J. Phycol. 56:862–879. doi.org/10.1111/jpy.12992
  52. Yang, L.-E., Huang, X.-Q., Hang, Y., Deng, Y.-Y., Lu, Q.-Q. & Lu, S. 2014. The P450-type carotene hydroxylase PuCHY1 from Porphyra suggests the evolution of carotenoid me‑tabolism in red algae. J. Integr. Plant Biol. 56:902–915. doi.org/10.1111/jipb.12229
  53. Yang, Z. 2007. PAML 4: phylogenetic analysis by maximum likelihood. Mol. Biol. Evol. 24:1586–1591. doi.org/10.1093/molbev/msm088
  54. Yang, Z., Wong, W. S. W. & Nielsen, R. 2005. Bayes empirical Bayes inference of amino acid sites under positive selec‑tion. Mol. Biol. Evol. 22:1107–1118. doi.org/10.1093/mol‑bev/msi097
  55. Yoon, H. S., Müller, K. M., Sheath, R. G., Ott, F. D. & Bhat‑tacharya, D. 2006. Defining the major lineages of red algae (Rhodophyta). J. Phycol. 42:482–492. doi.org/10.1111/j.1529-8817.2006.00210.x
  56. Zhang, C., Chen, J., Yang, R., et al. 2022. Abscisic acid acti‑vates desiccation tolerance responses in intertidal seaweed Neoporphyra haitanensis. Front. Mar. Sci. 9:1007193. doi.org/10.3389/fmars.2022.1007193
  57. Zhang, J., Wu, Q., Eléouët, M., et al. 2023. CRISPR/LbCa‑s12a-mediated targeted mutation of Gracilariopsis lemaneiformis (Rhodophyta). Plant Biotechnol J. 21:235–237. doi.org/10.1111/pbi.13949
  58. Zhao, J., Xu, P., Chen, J., et al. 2016. Simultaneous analysis of nine phytohormones in Porphyra haitanensis by high performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry. Food Sci. 37:216–222. doi.org/10.7506/spkx1002-6630-201616035
  59. Zheng, Z., He, B., Xie, X. & Wang, G. 2021. Co-suppression in Pyropia yezoensis (Rhodophyta) reveals the role of PyLHCI in light harvesting and generation switch. J. Phycol. 57:160–171. doi.org/10.1111/jpy.13073