Co-expression network analysis for identification of key long non-coding RNA and mRNA modules associated with alkaloid biosynthesis in Catharanthus roseus

Aram, Fazaneh; Marashi, Seyed Hassan; Tahmasebi, Ahmad; Seifi, Alireza; Shahin Kaleybar, Behzad

doi:10.22058/jpmb.2023.2008918.1282

Co-expression network analysis for identification of key long non-coding RNA and mRNA modules associated with alkaloid biosynthesis in Catharanthus roseus

Document Type : Original research paper

Authors

Fazaneh Aram ¹

Seyed Hassan Marashi ²

Ahmad Tahmasebi ¹

Alireza Seifi ²

Behzad Shahin Kaleybar ³

¹ Institute of Biotechnology, Shiraz University, Shiraz, Iran.

² Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran.

³ Department of Plant Breeding and Biotechnology, SANRU, Sari, Iran.

10.22058/jpmb.2023.2008918.1282

Abstract

Catharanthus roseus, produces a diverse array of specialized metabolites known as monoterpene indole alkaloids (MIAs) through an extensive and intricately branched metabolic pathway. It is imperative to unravel the intricate regulatory networks and relationships between the genes involved in the production of these metabolites. Long non-coding RNAs (lncRNAs) are emerging as significant regulatory factors in various biological processes. In this study, 4303 out of 86726 transcripts were identified as potential lncRNAs in C. roseus. Subsequently, we identified coding genes that exhibited a high correlation with CrlncRNA, designating them as potential target genes for collectively modulating the MIA pathway using Weighted Gene Co-Expression Network Analysis (WGCNA), leading to the identification of crucial gene clusters associated with the biosynthesis of MIAs. Based on the findings, three modules (dark turquoise, magenta, and orange) and hub genes were pinpointed as being linked to MIAs. Additionally, the most prominent known coding genes were 10-hydroxygeraniol oxidoreductase, GATA-like transcription factor (GATA1), 7-deoxyloganetic acid UDP-glucosyltransferase (7DLGT), desacetoxyvindoline 4-hydroxylase (DH4), MYC2, and MPK6. The unknown target genes were related to stress response and the intricate process of hormone transduction. ORCA, MYC2, and GATA1 are crucial in regulating the MIA pathway, likely requiring cooperation with CrlncRNAs.

Keywords

lncRNAs

specialized metabolites

transcription factors (TFs)

medicinal plants

gene modules

Ai, G., Li, T., Zhu, H., Dong, X., Fu, X., Xia, C., Pan, W., Jing, M., Shen, D., and Xia, A. (2022). BPL3 binds the long non-coding RNA nalncFL7 to suppress FORKED-LIKE7 and modulate HAI1-mediated MPK3/6 dephosphorylation in plant immunity. Plant Cell 35(1): 598-616. doi: https://doi.org/10.1093/plcell/koac311.

Ariel, F., Lucero, L., Christ, A., Mammarella, M.F., Jegu, T., Veluchamy, A., Mariappan, K., Latrasse, D., Blein, T., Liu, C., Benhamed, M., and Crespi, M. (2020). R-Loop mediated trans action of the APOLO long noncoding RNA. Mol Cell 77(5): 1055-1065 e1054. doi: 10.1016/j.molcel.2019.12.015.

Borah, P., Das, A., Milner, M.J., Ali, A., Bentley, A.R., and Pandey, R. (2018). Long non-coding RNAs as endogenous target mimics and exploration of their role in low nutrient stress tolerance in plants. Genes 9(9): 459.

Bouvier, F., Rahier, A., and Camara, B. (2005). Biogenesis, molecular regulation and function of plant isoprenoids. Prog Lipid Res 44(6): 357-429. doi: 10.1016/j.plipres.2005.09.003.

Brown, S., Clastre, M., Courdavault, V., and O'Connor, S.E. (2015). De novo production of the plant-derived alkaloid strictosidine in yeast. Proc Natl Acad Sci USA 112(11): 3205-3210. doi: 10.1073/pnas.1423555112.

Campalans, A., Kondorosi, A., and Crespi, M. (2004). Enod40, a short open reading frame-containing mRNA, induces cytoplasmic localization of a nuclear RNA binding protein in Medicago truncatula. Plant Cell 16(4): 1047-1059. doi: 10.1105/tpc.019406.

Colinas, M., Pollier, J., Vaneechoutte, D., Malat, D., Schweizer, F., De Clercq, R., Guedes, J., Martínez-Cortés, T., Molina Hidalgo, F., and Sottomayor, M. (2020). A modular system regulates specialized metabolite pathway branch choice in the medicinal plant Catharanthus roseus. BioRxiv. doi: https://doi.org/10.1101/2020.05.04.075671.

Csorba, T., Questa, J.I., Sun, Q., and Dean, C. (2014). Antisense COOLAIR mediates the coordinated switching of chromatin states at FLC during vernalization. Proc Natl Acad Sci 111(45): 16160-16165.

Cui, J., Jiang, N., Meng, J., Yang, G., Liu, W., Zhou, X., Ma, N., Hou, X., and Luan, Y. (2019). LncRNA33732‐respiratory burst oxidase module associated with WRKY1 in tomato‐Phytophthora infestans interactions. Plant J 97(5): 933-946.

De Luca, V., Salim, V., Thamm, A., Masada, S.A., and Yu, F. (2014). Making iridoids/secoiridoids and monoterpenoid indole alkaloids: progress on pathway elucidation. Curr Opin Plant Biol 19: 35-42. doi: 10.1016/j.pbi.2014.03.006.

Fedak, H., Palusinska, M., Krzyczmonik, K., Brzezniak, L., Yatusevich, R., Pietras, Z., Kaczanowski, S., and Swiezewski, S. (2016). Control of seed dormancy in Arabidopsis by a cis-acting noncoding antisense transcript. Proc Natl Acad Sci U S A 113(48): E7846-E7855. doi: 10.1073/pnas.1608827113.

Fox, E.R., and Unguru, Y. (2020). Oncology drug shortages in the USA—business as usual. Nat Rev Clin Oncol 17(3): 128-129.

Franco-Zorrilla, J.M., Valli, A., Todesco, M., Mateos, I., Puga, M.I., Rubio-Somoza, I., Leyva, A., Weigel, D., Garcia, J.A., and Paz-Ares, J. (2007). Target mimicry provides a new mechanism for regulation of microRNA activity. Nat Genet 39(8): 1033-1037. doi: 10.1038/ng2079.

Gershenzon, J., and Dudareva, N. (2007). The function of terpene natural products in the natural world. Nat Chem Biol 3(7): 408-414. doi: 10.1038/nchembio.2007.5.

Gongora-Castillo, E., Childs, K.L., Fedewa, G., Hamilton, J.P., Liscombe, D.K., Magallanes-Lundback, M., Mandadi, K.K., Nims, E., Runguphan, W., Vaillancourt, B., Varbanova-Herde, M., Dellapenna, D., McKnight, T.D., O'Connor, S., and Buell, C.R. (2012). Development of transcriptomic resources for interrogating the biosynthesis of monoterpene indole alkaloids in medicinal plant species. PLoS One 7(12): e52506. doi: 10.1371/journal.pone.0052506.

Heo, J.B., and Sung, S. (2011). Vernalization-mediated epigenetic silencing by a long intronic noncoding RNA. Science 331(6013): 76-79. doi: 10.1126/science.1197349.

Hisanaga, T., Okahashi, K., Yamaoka, S., Kajiwara, T., Nishihama, R., Shimamura, M., Yamato, K.T., Bowman, J.L., Kohchi, T., and Nakajima, K. (2019). A cis-acting bidirectional transcription switch controls sexual dimorphism in the liverwort. EMBO J 38(6): e100240. doi: 10.15252/embj.2018100240.

Kellner, F., Kim, J., Clavijo, B.J., Hamilton, J.P., Childs, K.L., Vaillancourt, B., Cepela, J., Habermann, M., Steuernagel, B., Clissold, L., McLay, K., Buell, C.R., and O'Connor, S.E. (2015). Genome-guided investigation of plant natural product biosynthesis. Plant J 82(4): 680-692. doi: 10.1111/tpj.12827.

Kim, D.H., and Sung, S. (2017). Vernalization-triggered intragenic chromatin loop formation by long noncoding RNAs. Dev Cell 40(3): 302-312 e304. doi: 10.1016/j.devcel.2016.12.021.

Kindgren, P., Ard, R., Ivanov, M., and Marquardt, S. (2018). Transcriptional read-through of the long non-coding RNA SVALKA governs plant cold acclimation. Nat Commun 9(1): 4561. doi: 10.1038/s41467-018-07010-6.

Langfelder, P., and Horvath, S. (2008). WGCNA: an R package for weighted correlation network analysis. BMC Bioinform 9(1): 559. doi: 10.1186/1471-2105-9-559.

Li, C., Wood, J.C., Vu, A.H., Hamilton, J.P., Rodriguez Lopez, C.E., Payne, R.M., Serna Guerrero, D.A., Yamamoto, K., Vaillancourt, B., and Caputi, L. (2022). Single-cell multi-omics enabled discovery of alkaloid biosynthetic pathway genes in the medical plant Catharanthus roseus. BioRxiv: 2022.2007. 2004.498697.

Liu, Y., Patra, B., Pattanaik, S., Wang, Y., and Yuan, L. (2019). GATA and phytochrome interacting factor transcription factors regulate light-induced vindoline biosynthesis in Catharanthus roseus. Plant Physiol 180(3): 1336-1350. doi: 10.1104/pp.19.00489.

Martino, E., Casamassima, G., Castiglione, S., Cellupica, E., Pantalone, S., Papagni, F., Rui, M., Siciliano, A.M., and Collina, S. (2018). Vinca alkaloids and analogues as anti-cancer agents: Looking back, peering ahead. Bioorganic Med Chem Lett 28(17): 2816-2826.

Menke, F.L., Champion, A., Kijne, J.W., and Memelink, J. (1999). A novel jasmonate- and elicitor-responsive element in the periwinkle secondary metabolite biosynthetic gene Str interacts with a jasmonate- and elicitor-inducible AP2-domain transcription factor, ORCA2. EMBO J 18(16): 4455-4463. doi: 10.1093/emboj/18.16.4455.

Miettinen, K., Dong, L., Navrot, N., Schneider, T., Burlat, V., Pollier, J., Woittiez, L., van der Krol, S., Lugan, R., Ilc, T., Verpoorte, R., Oksman-Caldentey, K.M., Martinoia, E., Bouwmeester, H., Goossens, A., Memelink, J., and Werck-Reichhart, D. (2014). The seco-iridoid pathway from Catharanthus roseus. Nat Commun 5(1): 3606. doi: 10.1038/ncomms4606.

Ngo, Q.A., Roussi, F., Cormier, A., Thoret, S., Knossow, M., Guenard, D., and Gueritte, F. (2009). Synthesis and biological evaluation of vinca alkaloids and phomopsin hybrids. J Med Chem 52(1): 134-142. doi: 10.1021/jm801064y.

O'Connor, S.E., and Maresh, J.J. (2006). Chemistry and biology of monoterpene indole alkaloid biosynthesis. Nat Prod Rep 23(4): 532-547. doi: 10.1039/b512615k.

Pan, Q., Wang, Q., Yuan, F., Xing, S., Zhao, J., Choi, Y.H., Verpoorte, R., Tian, Y., Wang, G., and Tang, K. (2012). Overexpression of ORCA3 and G10H in Catharanthus roseus plants regulated alkaloid biosynthesis and metabolism revealed by NMR-metabolomics. PLoS One 7(8): e43038. doi: 10.1371/journal.pone.0043038.

Patra, B., Pattanaik, S., Schluttenhofer, C., and Yuan, L. (2018). A network of jasmonate-responsive bHLH factors modulate monoterpenoid indole alkaloid biosynthesis in Catharanthus roseus. New Phytol 217(4): 1566-1581. doi: 10.1111/nph.14910.

Paul, P., Singh, S.K., Patra, B., Sui, X., Pattanaik, S., and Yuan, L. (2017). A differentially regulated AP 2/ERF transcription factor gene cluster acts downstream of a MAP kinase cascade to modulate terpenoid indole alkaloid biosynthesis in Catharanthus roseus. New Phytol 213(3): 1107-1123.

Pauw, B., Hilliou, F.A., Martin, V.S., Chatel, G., de Wolf, C.J., Champion, A., Pré, M., van Duijn, B., Kijne, J.W., and van der Fits, L. (2004). Zinc finger proteins act as transcriptional repressors of alkaloid biosynthesis genes in Catharanthus roseus. J Biol Chem 279(51): 52940-52948.

Peebles, C.A., Sander, G.W., Hughes, E.H., Peacock, R., Shanks, J.V., and San, K.Y. (2011). The expression of 1-deoxy-D-xylulose synthase and geraniol-10-hydroxylase or anthranilate synthase increases terpenoid indole alkaloid accumulation in Catharanthus roseus hairy roots. Metab Eng 13(2): 234-240. doi: 10.1016/j.ymben.2010.11.005.

Prakash, P., Ghosliya, D., and Gupta, V. (2015). Identification of conserved and novel microRNAs in Catharanthus roseus by deep sequencing and computational prediction of their potential targets. Gene 554(2): 181-195.

Rigo, R., Bazin, J., Romero‐Barrios, N., Moison, M., Lucero, L., Christ, A., Benhamed, M., Blein, T., Huguet, S., and Charon, C. (2020). The Arabidopsis lnc RNA ASCO modulates the transcriptome through interaction with splicing factors. EMBO Rep 21(5): e48977.

Rizvi, N.F., Weaver, J.D., Cram, E.J., and Lee-Parsons, C.W. (2016). Silencing the transcriptional repressor, ZCT1, illustrates the tight regulation of terpenoid indole alkaloid biosynthesis in Catharanthus roseus hairy roots. PLoS One 11(7): e0159712. doi: 10.1371/journal.pone.0159712.

Rohrig, H., John, M., and Schmidt, J. (2004). Modification of soybean sucrose synthase by S-thiolation with ENOD40 peptide A. Biochem Biophys Res Commun 325(3): 864-870. doi: 10.1016/j.bbrc.2004.10.100.

Seo, J.S., Diloknawarit, P., Park, B.S., and Chua, N.H. (2019). ELF18-Induced long noncoding RNA 1 evicts fibrillarin from mediator subunit to enhance pathogenesis-releated gene 1 (PR1) expression. New Phytol 221(4): 2067-2079. doi: 10.1111/nph.15530.

Shen, E.M., Singh, S.K., Ghosh, J.S., Patra, B., Paul, P., Yuan, L., and Pattanaik, S. (2017). The miRNAome of Catharanthus roseus: identification, expression analysis, and potential roles of microRNAs in regulation of terpenoid indole alkaloid biosynthesis. Sci Rep 7: 43027. doi: 10.1038/srep43027.

Sibéril, Y., Benhamron, S., Memelink, J., Giglioli-Guivarc'h, N., Thiersault, M., Boisson, B., Doireau, P., and Gantet, P. (2001). Catharanthus roseus G-box binding factors 1 and 2 act as repressors of strictosidine synthase gene expression in cell cultures. Plant Mol Biol 45: 477-488.

Singh, S.K., Patra, B., Paul, P., Liu, Y., Pattanaik, S., and Yuan, L. (2020). Revisiting the ORCA gene cluster that regulates terpenoid indole alkaloid biosynthesis in Catharanthus roseus. Plant Sci 293: 110408.

Sousa, C., Johansson, C., Charon, C., Manyani, H., Sautter, C., Kondorosi, A., and Crespi, M. (2001). Translational and structural requirements of the early nodulin gene enod40, a short-open reading frame-containing RNA, for elicitation of a cell-specific growth response in the alfalfa root cortex. Mol Cell Biol 21(1): 354-366. doi: 10.1128/MCB.21.1.354-366.2001.

Sui, X., Singh, S.K., Patra, B., Schluttenhofer, C., Guo, W., Pattanaik, S., and Yuan, L. (2018). Cross-family transcription factor interaction between MYC2 and GBFs modulates terpenoid indole alkaloid biosynthesis. J Exp Bot 69(18): 4267-4281.

Suttipanta, N., Pattanaik, S., Kulshrestha, M., Patra, B., Singh, S.K., and Yuan, L. (2011). The transcription factor CrWRKY1 positively regulates the terpenoid indole alkaloid biosynthesis in Catharanthus roseus. Plant Physiol 157(4): 2081-2093. doi: 10.1104/pp.111.181834.

Swiezewski, S., Liu, F., Magusin, A., and Dean, C. (2009). Cold-induced silencing by long antisense transcripts of an Arabidopsis Polycomb target. Nature 462(7274): 799-802. doi: 10.1038/nature08618.

Van der Fits, L., and Memelink, J. (2000). ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism. Science 289(5477): 295-297. doi: 10.1126/science.289.5477.295.

Van Moerkercke, A., Steensma, P., Schweizer, F., Pollier, J., Gariboldi, I., Payne, R., Vanden Bossche, R., Miettinen, K., Espoz, J., Purnama, P.C., Kellner, F., Seppanen-Laakso, T., O'Connor, S.E., Rischer, H., Memelink, J., and Goossens, A. (2015). The bHLH transcription factor BIS1 controls the iridoid branch of the monoterpenoid indole alkaloid pathway in Catharanthus roseus. Proc Natl Acad Sci U S A 112(26): 8130-8135. doi: 10.1073/pnas.1504951112.

Wang, J., Yu, W., Yang, Y., Li, X., Chen, T., Liu, T., Ma, N., Yang, X., Liu, R., and Zhang, B. (2015). Genome-wide analysis of tomato long non-coding RNAs and identification as endogenous target mimic for microRNA in response to TYLCV infection. Sci Rep 5(1): 1-16.

Wang, P., Dai, L., Ai, J., Wang, Y., and Ren, F. (2019). Identification and functional prediction of cold-related long non-coding RNA (lncRNA) in grapevine. Sci Rep 9(1): 6638. doi: 10.1038/s41598-019-43269-5.

Wang, Q., Yuan, F., Pan, Q., Li, M., Wang, G., Zhao, J., and Tang, K. (2010). Isolation and functional analysis of the Catharanthus roseus deacetylvindoline-4-O-acetyltransferase gene promoter. Plant Cell Rep 29(2): 185-192. doi: 10.1007/s00299-009-0811-2.

Wierzbicki, A.T., Blevins, T., and Swiezewski, S. (2021). Long noncoding RNAs in plants. Annu Rev Plant Biol 72: 245-271. doi: 10.1146/annurev-arplant-093020-035446.

Yamamoto, K., Takahashi, K., Caputi, L., Mizuno, H., Rodriguez-Lopez, C.E., Iwasaki, T., Ishizaki, K., Fukaki, H., Ohnishi, M., Yamazaki, M., Masujima, T., O'Connor, S.E., and Mimura, T. (2019). The complexity of intercellular localisation of alkaloids revealed by single-cell metabolomics. New Phytol 224(2): 848-859. doi: 10.1111/nph.16138.

Yang, Y., Ding, L., Zhou, Y., Guo, Z., Yu, R., and Zhu, J. (2023). Establishment of recombinant Catharanthus roseus stem cells stably overexpressing ORCA4 for terpenoid indole alkaloids biosynthesis. Plant Physiol Biochem 196: 783-792. doi: 10.1016/j.plaphy.2023.02.039.

Yu, Y., Zhang, Y., Chen, X., and Chen, Y. (2019). Plant noncoding RNAs: Hidden players in development and stress responses. Annu Rev Cell Dev Biol 35: 407-431. doi: 10.1146/annurev-cellbio-100818-125218.

Zhang, H., Hedhili, S., Montiel, G., Zhang, Y., Chatel, G., Pré, M., Gantet, P., and Memelink, J. (2011). The basic helix‐loop‐helix transcription factor CrMYC2 controls the jasmonate‐responsive expression of the ORCA genes that regulate alkaloid biosynthesis in Catharanthus roseus. Plant J 67(1): 61-71.

Zhang, X., Dong, J., Deng, F., Wang, W., Cheng, Y., Song, L., Hu, M., Shen, J., Xu, Q., and Shen, F. (2019). The long non-coding RNA lncRNA973 is involved in cotton response to salt stress. BMC Plant Biol 19: 1-16.