Effect of Mosaic Knockout of Phytoene Desaturase Gene NtPDS on Biosynthesis of Carotenoids in Nicotiana tabacum L.

A. V. Nezhdanova; Нежданова А. В.; M. A. Slugina; Слугина М. А.; A. V. Kulakova; Кулакова А. В.; A. M. Kamionskaya; Камионская А. М.; E. Z. Kochieva; Кочиева Е. З.; A. V Shchennikova; Щенникова А. В.

doi:10.31857/S001533032360033X

Effect of Mosaic Knockout of Phytoene Desaturase Gene NtPDS on Biosynthesis of Carotenoids in Nicotiana tabacum L.

Authors: Nezhdanova A.V.¹, Slugina M.A.¹, Kulakova A.V.¹, Kamionskaya A.M.¹, Kochieva E.Z.¹, Shchennikova A.V¹
Affiliations:
1. Institute of Bioengineering, Federal Research Center of Biotechnology, Russian Academy of Sciences
Issue: Vol 70, No 6 (2023)
Pages: 601-611
Section: ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
URL: https://journals.rcsi.science/0015-3303/article/view/232770
DOI: https://doi.org/10.31857/S001533032360033X
EDN: https://elibrary.ru/CXCKHG
ID: 232770

Cite item

Full Text

Open Access
Restricted Access

Access granted
Restricted Access

Subscription Access

Abstract
About the authors
References
Supplementary files
Statistics

Abstract

Carotenoids are essential participants in photosynthesis and photo protection as well as growth, development, and stress response in plants. Phytoene desaturase (PDS; EC 1.3.5.5) is an enzyme that catalyzes the first stage of desaturation of 15-cis-phytoene (a precursor to all carotenoids). In this work, we examined for the first time the effect of PDS knockout in the genome of Nicotiana tabacum L. using the CRISPR-Cas9 system on activity of downstream genes involved in biosynthesis of carotenoids. Nine transgenic lines of tobacco were obtained with a mosaic editing of gene NtPDS; three versions of indels (350^351→ins^g#, 350^351→ins^t#, and t351→del(1nt)#) in the exon II resulting in the synthesis of a shortened non-functional version of the protein. The lines were characterized by spotted, green-white pigmentation of the leaves, altered flowering time and morphology as well as a reduced content of carotenoids and chlorophylls in the leaf tissue. A rise in the level of transcripts of phytoene synthase gene NtPSY2 was shown in late-flowering lines as compared to control material. Edited line L29 with the latest flowering showed a considerable increase in the level of transcripts of downstream structural genes of carotenogenesis. A reduction in the carotenoid content in the leaves with mosaic editing was accompanied by a decrease in the level of expression of MADS-box gene NtSEP1, the product of which presumably participates in regulation of transcription of the carotenoid biosynthesis genes. The obtained results may be used for further studies of regulation of biosynthesis of carotenoids and apocarotenoids in Solanaceae.

Keywords

Nicotiana tabacum, CRISPR-Cas9, biosynthesis of carotenoids, phytoene desaturase, gene expression

References

Maoka T. Carotenoids as natural functional pigments // J. Nat. Med. 2020. V. 74. P. 1. https://doi.org/10.1007/s11418-019-01364-x
Brausemann A., Gemmecker S., Koschmieder J., Ghisla S., Beyer P., Einsle O. Structure of phytoene desaturase provides insights into herbicide binding and reaction mechanisms involved in carotene desaturation. Structure // 2017. V. 25. P. 1222. https://doi.org/10.1016/j.str.2017.06.002
Demmig-Adams B., Adams W.W. Antioxidants in photosynthesis and human nutrition // Science. 2002. V. 298. P. 2149. https://doi.org/10.1126/science.1078002
Chen K., Li G.J., Bressan R.A., Song C.P., Zhu J.K., Zhao Y. Abscisic acid dynamics, signaling, and functions in plants // J. Integr. Plant Biol. 2020. V. 62. P. 25. https://doi.org/10.1111/jipb.12899
Mashiguchi K., Seto Y., Yamaguchi S. Strigolactone biosynthesis, transport and perception // Plant J. 2021. V. 105. P. 335. https://doi.org/10.1111/tpj.15059
Babu R., Rojas N.P., Gao S., Yan J., Pixley K. Validation of the effects of molecular marker polymorphisms in LcyE and CrtRB1 on provitamin A concentrations for 26 tropical maize populations // Theor. Appl. Genet. 2013. V. 126. P. 389. https://doi.org/10.1007/s00122-012-1987-3
Wagner T., Windhövel U., Römer S. Bansformation of tobacco with a mutated cyanobacterial phytoene desaturase gene confers resistance to bleaching herbicides // Z. Naturforsch. C. J. Biosci. 2002. V. 57. P. 671. https://doi.org/10.1515/znc-2002-7-821
Naing A.H., Kyu S.Y., Pe P.P.W., Park K.I., Lee J.M., Lim K.B., Kim C.K. Silencing of the phytoene desaturase (PDS) gene affects the expression of fruit-ripening genes in tomatoes // Plant Methods. 2019. V. 15: 110. https://doi.org/10.1186/s13007-019-0491-z
Dong T., Chen G., Tian S., Xie Q., Yin W., Zhang Y., Hu Z. A non-climacteric fruit gene CaMADS-RIN regulates fruit ripening and ethylene biosynthesis in climacteric fruit // PLoS One. 2014. V. 9: e95559. https://doi.org/10.1371/journal.pone.0095559
Fujisawa M., Nakano T., Ito Y. Identification of potential target genes for the tomato fruit-ripening regulator RIN by chromatin immunoprecipitation // BMC Plant Biol. 2011. V. 11: 26. https://doi.org/10.1186/1471-2229-11-26
Li S., Xu H., Ju Z., Cao D., Zhu H., Fu D., Grierson D., Qin G., Luo Y., Zhu B. The RIN-MC fusion of MADS-box transcription factors has transcriptional activity and modulates expression of many ripening genes // Plant Physiol. 2018. V. 176. P. 891. https://doi.org/10.1104/pp.17.01449
Chen L., Li W., Katin-Grazzini L., Ding J., Gu X., Li Y., Gu T., Wang R., Lin X., Deng Z., McAvoy R.J., Gmitter F.G., Deng Z., Zhao Y., Li Y. A method for the production and expedient screening of CRISPR/Cas9-mediated non-transgenic mutant plants // Hortic. Res. 2018. V. 5: 13. https://doi.org/10.1038/s41438-018-0023-4
Bánfalvi Z., Csákvári E., Villányi V., Kondrák M. Generation of transgene-free PDS mutants in potato by Agrobacterium-mediated transformation // BMC Biotechnol. 2020. V. 20: 25. https://doi.org/10.1186/s12896-020-00621-2
Xu H., Xu L., Yang P., Cao Y., Tang Y., He G., Yuan S., Ming J. Tobacco rattle virus-induced PHYTOENE DESATURASE (PDS) and Mg-chelatase H subunit (ChlH) gene silencing in Solanum pseudocapsicum L. // PeerJ. 2018. V. 6: e4424. https://doi.org/10.7717/peerj.4424
Zhang L., Gase K., Baldwin I., Gális I. Enhanced fluorescence imaging in chlorophyll-suppressed tobacco tissues using virus-induced gene silencing of the phytoene desaturase gene // Biotechniques. 2010. V. 48. P. 125. https://doi.org/10.2144/000113345
DeBlasio S.L., Rebelo A.R., Parks K., Gray S.M., Heck M.C. Disruption of chloroplast function through downregulation of phytoene desaturase enhances the systemic accumulation of an aphid-borne, phloem-restricted virus // Mol. Plant Microbe Interact. 2018. V. 31. P. 1095. https://doi.org/10.1094/MPMI-03-18-0057-R
Jacobs T.B., LaFayette P.R., Schmitz R.J., Parrott W.A. Targeted genome modifications in soybean with CRISPR/Cas9 // BMC Biotechnology. 2015. V. 15: 16. https://doi.org/10.1186/s12896-015-0131-2
Nezhdanova A.V., Slugina M.A., Dyachenko E.A., Kamionskaya A.M., Kochieva E.Z., Shchennikova A.V. Analysis of the structure and function of the tomato Solanum lycopersicum L. MADS-box gene SlMADS5 // Vavilovskii Zhurnal Genet Selektsii. 2021. V. 25. P. 492. https://doi.org/10.18699/VJ21.056
Filyushin M.A., Dzhos E.A., Shchennikova A.V., Kochieva E.Z. Dependence of pepper fruit colour on basic pigments ratio and expression pattern of carotenoid and anthocyanin biosynthesis genes // Russian Journal of Plant Physiology. 2020. V. 67. P. 1054. https://doi.org/10.1134/S1021443720050040
Meugnier E., Rome S., Vidal H. Regulation of gene expression by glucose // Curr. Opin. Clin. Nutr. Metab. Care. 2007. V. 10. P. 518. https://doi.org/10.1097/MCO.0b013e3281298fef
Slugina M.A. Transcription factor RIPENING INHIBITOR and its homologs in regulation of fleshy fruit ripening of various plant species // Russian Journal of Plant Physiology. 2021. V. 68. P. 783. https://doi.org/10.1134/S1021443721050186
Castelán-Muñoz N., Herrera J., Cajero-Sánchez W., Arrizubieta M., Trejo C., García-Ponce B., Sánchez M.P., Álvarez-Buylla E.R., Garay-Arroyo A. MADS-box genes are key components of genetic regulatory networks involved in abiotic stress and plastic developmental responses in plants // Front. Plant Sci. 2019. V. 10: 853. https://doi.org/10.3389/fpls.2019.00853
Zhang Y., Cai W., Wang A., Huang X., Zheng X., Liu Q., Cheng X., Wan M., Lv J., Guan D., Yang S., He S. MADS-box protein AGL8 interacts with chromatin-remodeling component SWC4 to activate thermotolerance and environment-dependent immunity in pepper // J. Exp. Bot. 2023. erad092. https://doi.org/10.1093/jxb/erad092
Cazzonelli C.I., Pogson B.J. Source to sink: regulation of carotenoid biosynthesis in plants // Trends Plant Sci. 2010. V. 15. P. 266. https://doi.org/10.1016/j.tplants.2010.02.003
Quian-Ulloa R., Stange C. Carotenoid Biosynthesis and Plastid Development in Plants: The Role of Light // Int. J. Mol. Sci. 2021. V. 22: 1184. https://doi.org/10.3390/ijms22031184
Yan P., Gao X.Z., Shen W.T., Zhou P. Cloning and expression analysis of phytoene desaturase and ζ-carotene desaturase genes in Carica papaya // Mol. Biol. Rep. 2011. V. 38. P. 785. https://doi.org/10.1007/s11033-010-0167-2
Cui Y., Zhao J., Gao Y., Zhao R., Zhang J., Kong L. Efficient multi-sites genome editing and plant regeneration via somatic embryogenesis in Picea glauca // Front. Plant Sci. 2021. V. 12: 751891. https://doi.org/10.3389/fpls.2021.751891
Shu K., Luo X., Meng Y., Yang W. Toward a molecular understanding of abscisic acid actions in floral transition // Plant Cell Physiol. 2018. V. 59. P. 215. https://doi.org/10.1093/pcp/pcy007
Song Y.H., Ito S., Imaizumi T. Flowering time regulation: photoperiod- and temperature-sensing in leaves // Trends Plant Sci. 2013. V. 18. P. 575. https://doi.org/10.1016/j.tplants.2013.05.003
Yang G., Li W., Fan C., Liu M., Liu J., Liang W., Wang L., Di S., Fang C., Li H., Ding G., Bi Y., Lai Y. Genome-wide association study uncovers major genetic loci associated with flowering time in response to active accumulated temperature in wild soybean population // BMC Genomics. 2022. V. 23: 749. https://doi.org/10.1186/s12864-022-08970-2
Parenicová L., de Folter S., Kieffer M., Horner D.S., Favalli C., Busscher J., Cook H.E., Ingram R.M., Kater M.M., Davies B., Angenent G.C., Colombo L. Molecular and phylogenetic analyses of the complete MADS-box transcription factor family in Arabidopsis: new openings to the MADS world // Plant Cell. 2003. V. 15. P. 1538. https://doi.org/10.1105/tpc.011544
Li X., Yu B., Wu Q., Min Q., Zeng R., Xie Z., Huang J. OsMADS23 phosphorylated by SAPK9 confers drought and salt tolerance by regulating ABA biosynthesis in rice // PLoS Genet. 2021. V. 17: e1009699. https://doi.org/10.1371/journal.pgen.1009699
Zhao P.X., Zhang J., Chen S.Y., Wu J., Xia J.Q., Sun L.Q., Ma S.S., Xiang C.B. Arabidopsis MADS-box factor AGL16 is a negative regulator of plant response to salt stress by downregulating salt-responsive genes // New Phytol. 2021. V. 232. P. 2418. https://doi.org/10.1111/nph.17760