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Comprehensive transcriptome analysis provides insights into metabolic and gene regulatory networks in trichomes of Nicotiana tabacum

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Comprehensive transcriptome analysis suggested that the primary metabolism is modulated to augment the supply of substrates towards secondary metabolism operating in the glandular trichomes of Nicotiana tabacum. The comparative gene expression and co-expression network analysis revealed that certain members of transcription factor genes belonging to the MYB, HD-ZIP, ERF, TCP, SRS, WRKY and DOF families may be involved in the regulation of metabolism and/other aspects in the glandular trichomes of N. tabacum

Abstract

The glandular trichomes of Nicotiana tabacum are highly productive in terms of secondary metabolites and therefore have been projected to be used as a prognostic platform for metabolic engineering of valuable natural products. For obvious reasons, detailed studies pertaining to the metabolic and gene regulatory networks operating in the glandular trichomes of N. tabacum are of pivotal significance to be undertaken. We have carried out next-generation sequencing of glandular trichomes of N. tabcaum and investigated differential gene expression among different tissues, including trichome-free leaves. We identified a total of 37,269 and 37,371 genes, expressing in trichome free leaf and glandular trichomes, respectively, at a cutoff of FPKM ≥ 1. The analysis revealed that different pathways involved with the primary metabolism are modulated in glandular trichomes of N. tabacum, providing a plausible explanation for the enhanced biosynthesis of secondary metabolism in the glandular trichomes. Further, comparative gene expression analysis revealed several genes, which display preferential expression in the glandular trichomes and thereby seem to be potential candidate genes for future studies in connection to the discovery of novel trichome specific promoters. The present study also led to the comprehensive identification of 1750 transcription factor genes expressing at a cutoff of FPKM ≥ 1 in the glandular trichomes of N. tabacum. The clustering and co-expression analysis suggested that transcription factor genes belonging to HD-ZIP, ERF, WRKY, MYB, TCP, SRS and DOF families may be the major players in the regulation of gene expression in the glandular trichomes of N. tabacum. To the best of our knowledge, the present work is the first effort towards detailed identification of genes, especially regulatory genes expressing in the glandular trichomes of N. tabacum. The data resource and the empirical findings from present work in all probability must, therefore, provide a reference and background context for future work aiming at deciphering molecular mechanism of regulation of secondary metabolism and gene expression in the glandular trichomes of N. tabacum.

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References

  • Aharoni A, Dixit S, Jetter R, Thoenes E, van Arkel G, Pereira A (2004) The SHINE clade of AP2 domain transcription factors activates wax biosynthesis, alters cuticle properties, and confers drought tolerance when overexpressed in Arabidopsis. Plant Cell 16:2463–2480

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Amme S, Rutten T, Melzer M, Sonsmann G, Vissers JP, Schlesier B, Mock HP (2005) A proteome approach defines protective functions of tobacco leaf trichomes. Proteomics 5:2508–2518

    Article  CAS  PubMed  Google Scholar 

  • Balcke GU, Bennewitz S, Bergau N, Athmer B, Henning A, Majovsky P, Jimenez-Gomez JM, Hoehenwarter W, Tissier A (2017) Multi-omics of tomato glandular trichomes reveals distinct features of central carbonmetabolism supporting high productivity of specialized metabolites. Plant Cell 29:960–983

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chang WC, Song H, Liu HW, Liu P (2013) Current development in isoprenoid precursor biosynthesis and regulation. Curr Opin Chem Biol 17:571–579

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Choi YE, Lim S, Kim HJ, Han JY, Lee MH, Yang Y, Kim JA, Kim YS (2012) Tobacco NtLTP1, a glandular-specific lipid transfer protein, is required for lipid secretion from glandular trichomes. Plant J 70:480–491

    Article  CAS  PubMed  Google Scholar 

  • Crouzet J, Roland J, Peeters E, Trombik T, Ducos E, Nader J, Boutry M (2013) NtPDR1, a plasma membrane ABC transporter from Nicotiana tabacum, is involved in diterpene transport. Plant Mol Biol 82:181–192

    Article  CAS  PubMed  Google Scholar 

  • Cui H, Zhang ST, Yang HJ, Ji H, Wang XJ (2011) Gene expression profile analysis of tobacco leaf trichomes. BMC Plant Biol 11:76

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29:15–21

    Article  CAS  PubMed  Google Scholar 

  • Ebersbach P, Stehle F, Kayser O, Freier E (2018) Chemical fingerprinting of single glandular trichomes of Cannabis sativa by coherent anti-stokes Raman scattering (CARS) microscopy. BMC Plant Biol 18:275

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Edwards KD, Fernandez-Pozo N, Drake-Stowe K, Humphry M, Evans AD, Bombarely A, Allen F, Hurst R, White B, Kernodle SP, Bromley JR, Sanchez-Tamburrino JP, Lewis RS, Mueller LA (2017) A reference genome for Nicotiana tabacum enables map-based cloning of homeologous loci implicated in nitrogen utilization efficiency. BMC Genomics 18(1):448

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ennajdaoui H, Vachon G, Giacalone C, Besse I, Sallaud C, Herzog M, Tissier A (2010) Trichome specific expression of the tobacco (Nicotiana sylvestris) cembratrien-ol synthase genesis controlled by both activating and repressing cis-regions. Plant Mol Biol 73:673–685

    Article  CAS  PubMed  Google Scholar 

  • Fatland BL, Ke J, Anderson MD, Mentzen WI, Cui LW, Allred CC, Johnston JL, Nikolau BJ, Wurtele ES (2002) Molecular characterization of a heteromeric ATP-citrate lyase that generates cytosolic acetyl-coenzyme A in Arabidopsis. Plant Physiol 130:740–756

    Article  PubMed  PubMed Central  Google Scholar 

  • Fatland BL, Nikolau BJ, Wurtele ES (2005) Reverse genetic characterization of cytosolic acetyl-CoA generation by ATP-citrate lyase in Arabidopsis. Plant Cell 17:182–203

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Glas JJ, Schimmel BC, Alba JM, Escobar-Bravo R, Schuurink RC, Kant MR (2012) Plant glandular trichomes as targets for breeding or engineering of resistance to herbivores. Int J Mol Sci 13:17077–17103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Harada E, Kim JA, Meyer AJ, Hell R, Clemens S, Choi YE (2010) Expression profiling of tobacco leaf trichomes identifies genes for biotic and abiotic stresses. Plant Cell Physiol 51:1627–1637

    Article  CAS  PubMed  Google Scholar 

  • Huchelmann A, Boutry M, Charles H (2017) Plant glandular trichomes: natural cell factories of high biotechnological interest. Plant Physiol 75:6–22

    Article  CAS  Google Scholar 

  • Jin J, Panicker D, Wang Q, Kim MJ, Liu J, Yin JL, Wong L, Jang IC, Chua NH, Sarojam R (2014) Next generation sequencing unravels the biosynthetic ability of spearmint (Mentha spicata) peltate glandular trichomes through comparative transcriptomics. BMC Plant Biol 14:292

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Johnson SR, Lange I, Srividya N, Lange BM (2017) Bioenergetics of monoterpenoid essential oil biosynthesis in nonphotosynthetic glandular trichomes. Plant Physiol 175:681–695

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Julien P, Brawand D, Soumillon M, Necsulea A, Liechti A, Schütz F et al (2012) Mechanisms and evolutionary patterns of mammalian and avian dosage compensation. PLoS Biol 10:e1001328. https://doi.org/10.1371/journal.pbio.100132

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ke J, Behal RH, Back SL, Nikolau BJ, Wurtele ES, Oliver DJ (2000) The role of pyruvate dehydrogenase and acetyl-coenzyme A synthetase in fatty acid synthesis in developing Arabidopsis seeds. Plant Physiol 123:497–508

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kroumova AB, Zaitlin D, Wagner GJ (2016) Natural variability in acyl moieties of sugar esters produced by certain tobacco and other Solanaceae species. Phytochemistry 130:218–227

    Article  CAS  PubMed  Google Scholar 

  • Kruger NJ, von Schaewen A (2003) The oxidative pentose phosphate pathway: structure and organisation. Curr Opin Plant Biol 6:236–246

    Article  CAS  PubMed  Google Scholar 

  • Kryuchkova-Mostacci N, Robinson-Rechavi M (2016) A benchmark of gene expression tissue-specificity metrics. Brief Bioinform 8:205–214

    Google Scholar 

  • Kundan M, Gani U, Nautiyal AK, Misra P (2019) Molecular biology of glandular trichomes and their functions in environmental stresses. In: Singh S, Upadhyay S, Pandey A, Kumar S (eds) Molecular approaches in plant biology and environmental challenges, 1st edn. Springer, Singapore, pp 365–393

    Chapter  Google Scholar 

  • Laterre R, Pottier M, Remacle C, Boutry M (2017) Photosynthetic Trichomes Contain a Specific Rubisco with a Modified pH-Dependent Activity. Plant Physiol 173:2110–2120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lee SB, Kim HU, Suh MC (2016) MYB94 and MYB96 Additively Activate Cuticular Wax Biosynthesis in Arabidopsis. Plant Cell Physiol 57:2300–2311

    Article  CAS  PubMed  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)). Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Lohse M, Nagel A, Herter T, May P, Schroda M, Zrenner R, Tohge T, Fernie AR, Stitt M, Usadel B (2014) Mercator: a fast and simple web server for genome scale functional annotation of plant sequence data. Plant Cell Environ 37:1250–1258

    Article  CAS  PubMed  Google Scholar 

  • Maeda H, Dudareva N (2012) The shikimate pathway and aromatic amino Acid biosynthesis in plants. Annu Rev Plant Biol 63:73–105

    Article  CAS  PubMed  Google Scholar 

  • Misra P, Pandey A, Tewari M, Chandrashekhar K, Siddhu OP, Asif MH, Chakrabarty D, Singh PK, Nath P, Trivedi PK, Tuli R (2010) Modulation of transcriptome and metabolome by AtMYB12 transcription factor leads to insect tolerance. Plant Physiol 152:2258–2268

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nazarov PV, Muller A, Khutko V (2010) Co-expression analysis of large microarray data sets using CoExpress software tool. In: Conference: WCSB

  • Pandey A, Misra P, Bhambhani S, Bhatia C, Trivedi PK (2014) Expression of Arabidopsis MYB transcription factor, AtMYB111, in tobacco requires light to modulate flavonol content. Sci Rep 4:5018

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rather GA, Sharma A, Pandith SA, Kaul V, Nandi U, Misra P, Lattoo SK (2018) De novo transcriptome analyses reveals putative pathway genes involved in biosynthesis and regulation of camptothecin in Nothapodytes nimmoniana (Graham) Mabb. Plant Mol Biol 96:197–215

    Article  CAS  PubMed  Google Scholar 

  • Sallaud C, Giacalone C, Topfer R, Goepfert S, Bakaher N, Rosti S, Tissier A (2012) Characterization of two genes for the biosynthesis of the labdane diterpene Z-abienol in tobacco (Nicotiana tabacum) glandular trichomes. Plant J. 72:1–17

    Article  CAS  PubMed  Google Scholar 

  • Sallets A, Beyaert M, Boutry M, Champagne A (2014) Comparative proteomics of short and tall glandular trichomes of Nicotiana tabacum reveals differential metabolic activities. J Proteome Res 13:3386–3396

    Article  CAS  PubMed  Google Scholar 

  • Schubert M, Lindgreen S, Orlando L (2016) AdapterRemoval v2: rapid adapter trimming, identification, and read merging. BMC Res Notes 9:88

    Article  PubMed  PubMed Central  Google Scholar 

  • Sierro N, Battey JN, Ouadi S, Bakaher N, Bovet L, Willig A, Goepfert S, Peitsch MC, Ivanov NV (2014) The tobacco genome sequence and its comparison with those of tomato and potato. Nat Commun 5:3833

    Article  CAS  PubMed  Google Scholar 

  • Soetaert SS, Van Neste CM, Vandewoestyne ML, Head SR, Goossens A, Van Nieuwerburgh FC, Deforce DL (2013) Differential transcriptome analysis of glandular and filamentous trichomes in Artemisia annua. BMC Plant Biol 13:220

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Spyropoulou EA, Haring MA, Schuurink RC (2014) Expression of Terpenoids 1, a glandular trichome-specific transcription factor from tomato that activates the terpene synthase 5 promoter. Plant Mol Biol 84:345–357

    Article  CAS  PubMed  Google Scholar 

  • Stracke R, Ishihara H, Huep G, Barsch A, Mehrtens F, Niehaus K, Weisshaar B (2007) Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling. Plant J 50:660–677

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sturn A, Quackenbush J, Trajanoski Z (2002) Genesis: cluster analysis of microarray data. Bioinformatics 18:207–208

    Article  CAS  PubMed  Google Scholar 

  • Taneja M, Upadhyay SK (2018) Molecular characterization and differential expression suggested diverse functions of P-type IICa2 + ATPases in Triticum aestivum L. BMC Genomics 19:389

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Tissier A (2012) Glandular trichomes: what comes after expressed sequence tags? Plant J 70:51–68

    Article  CAS  PubMed  Google Scholar 

  • Tissier A (2018) Plant secretory structures: more than just reaction bags. Curr Opin Biotechnol 49:73–79

    Article  CAS  PubMed  Google Scholar 

  • To A, Joubes J, Barthole G, Lecureuil A, Scagnelli A, Jasinski S, Lepiniec L, Baud S (2012) WRINKLED transcription factors orchestrate tissue-specific regulation of fatty acid biosynthesis in Arabidopsis. Plant Cell 24:5007–5023

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Usadel B, Nagel A, Steinhauser D, Gibon Y, Blasing OE, Redestig H, Sreenivasulu N, Krall L, Hannah MA, Poree F, Fernie AR, Stitt M (2006) PageMan: an interactive ontology tool to generate, display, and annotate overview graphs for profiling experiments. BMC Bioinform 7:535

    Article  CAS  Google Scholar 

  • Van Cutsem E, Simonart G, Degand H, Faber AM, Morsomme P, Boutry M (2011) Gel-based and gel-free proteomic analysis of Nicotiana tabacum trichomes identifies proteins involved in secondary metabolism and in the (a) biotic stress response. Proteomics 11:440–454

    Article  PubMed  CAS  Google Scholar 

  • Wagner GJ (1991) Secreting glandular trichomes: more than just hairs. Plant Physiol 96:675–679

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang E, Wang R, DeParasis J, Loughrin JH, Gan S, Wagner GJ (2001) Suppression of a P450 hydroxylase gene in plant trichome glands enhances natural-product-based aphid resistance. Nat Biotechnol 19:371–374

    Article  CAS  PubMed  Google Scholar 

  • Wang E, Gan S, Wagner GJ (2002) Isolation and characterization of the CYP71D16 trichome-specific promoter from Nicotiana tabacum L. J Exp Bot 53:1891–1897

    Article  CAS  PubMed  Google Scholar 

  • Wang Z, Guhling O, Yao R, Li F, Yeats TH, Rose JK, Jetter R (2011) Two oxidosqualene cyclases responsible for biosynthesis of tomato fruit cuticular triterpenoids. Plant Physiol 155:540–552

    Article  CAS  PubMed  Google Scholar 

  • Zhang Y, Butelli E, Alseekh S, Tohge T, Rallapalli G, Luo J, Kawar PG, Hill L, Santino A, Fernie AR, Martin C (2015) Multi-level engineering facilitates the production of phenylpropanoid compounds in tomato. Nat Commun 6:8635

    Article  CAS  PubMed  Google Scholar 

  • Zhang H, Zhang S, Yang Y, Jia H, Cui H (2018) Metabolic flux engineering of cembratrien-ol production in both the glandular trichome and leaf mesophyll in Nicotiana tabacum. Plant Cell Physiol 59:566–574

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

PM acknowledges funding support by Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Government of India for the project under “Scheme for Young Scientists” (SB/YS/LS-305/2013). AKN, UG, MK and MF acknowledge CSIR, DST-INSPIRE and UGC, India for Junior and senior research fellowships. The manuscript has institutional publication number CSIR-IIIM/IPR/0099.

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Authors and Affiliations

  1. Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu Tawi, Jammu, 180001, India

    Abhishek Kumar Nautiyal, Umar Gani, Priyanka Sharma, Maridul Kundan, Mohd. Fayaz, Surrinder K. Lattoo & Prashant Misra

  2. Academy of Scientific and Innovative Research (AcSIR), New Delhi, India

    Abhishek Kumar Nautiyal, Umar Gani, Priyanka Sharma, Maridul Kundan, Mohd. Fayaz & Prashant Misra

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  1. Abhishek Kumar Nautiyal
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Contributions

Conceived and designed the experiments: PM; Performed the experiments: AKN, UG, PS, MK, MF; Analyzed the data: PM, AKN. Contributed reagents/materials/analysis tools: SKL, PM; Original draft was prepared by PM. SKL edited the manuscript. PM finalized the manuscript.

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Correspondence to Prashant Misra.

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The raw read data and sample information have been submitted under Bio-project ID PRJNA561900.

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11103_2020_968_MOESM1_ESM.tif

Supplementary file1 (TIF 106 kb) Figure S1: Expression analysis of trichome specific genes using qRT-PCR. The NtUbiquitin gene was used for normalization. The results are the mean of three replicates depicting mean ±SD values. The statistical significance was calculated using t-test. The asterisks *** denotes the significance of the fold change at the p-value less that 0.0001, with respect to the L sample. T, trichome sample, ML, leaf sample with trichomes, L, trichome-free leaf sample

11103_2020_968_MOESM2_ESM.docx

Supplementary file2 (DOCX 14 kb) Supplementary file S1: Tables S1 and S2 detailing the raw read and alignment summary (DOCX 14 kb)

Supplementary file3 (XLSX 243 kb) Supplementary file S2: Top 3000 highly expressing genes in L and T (XLSX 244 kb)

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Supplementary file4 (XLSX 1126 kb) Supplementary file S3: Up and down-regulated genes in T as compared to L (XLSX 1127 kb)

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Supplementary file5 (XLSX 13 kb) Supplementary file S4: Over- and under-representation analysis of differentially expressed genes between T and L samples (XLSX 14 kb)

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Supplementary file6 (XLSX 195 kb) Supplementary file S5: List, expression values, SI values and fold change in expression of the genes belonging to the cluster 19

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Supplementary file7 (XLSX 68 kb) Supplementary file S6: List, expression values, SI values and fold change in expression of the genes belonging to the cluster 6

11103_2020_968_MOESM8_ESM.xlsx

Supplementary file8 (XLSX 151 kb) Supplementary file S7: Co-expression analysis of genes including transcription factor genes

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Supplementary file9 (XLSX 11 kb) Supplementary file S8: Putative orthologs of the selected transcription factors in tomato

Supplementary file10 (XLSX 12 kb) Supplementary file S9: List of the primers used for the quantitative real time PCR

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Nautiyal, A.K., Gani, U., Sharma, P. et al. Comprehensive transcriptome analysis provides insights into metabolic and gene regulatory networks in trichomes of Nicotiana tabacum. Plant Mol Biol 102, 625–644 (2020). https://doi.org/10.1007/s11103-020-00968-2

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