Transcriptional modules and hormonal metabolic pathways reveal the critical role of TgHB12-like in the regulation of flower opening and petal senescence in Tulipa gesneriana

Abstract

Tulips ( Tulipa gesneriana) are one of the most widely cultivated bulbous plants with substantial ornamental value. However, the lack of well-documented reference genomes has limited the research progress and molecular breeding of tulips. In the present study, a full-length transcriptome of a commercial tulip cultivar was obtained using single-molecule long-read sequencing (PacBio Iso-Seq). In total, 244,357 full-length transcripts were identified, which had an average length of 2,044 bp and an N50 value of 3,861; 67,350 of these were annotated to databases. An inaugural integrated analysis of the transcriptome and phytohormone profiles during flower opening and petal senescence was performed using Illumina RNA-seq, coupled with Mfuzz (an R pakage, http://mfuzz.sysbiolab.eu) and weighted gene coexpression network analysis (WGCNA). A total of 16 gene coexpression and six transcription factor (TF) modules were constructed. Additionally, 26 hormone analogs were comprehensively profiled. Finally, a prominently novel gene, Tulipa gesneriana Homeobox12-like ( TgHB12-like), which encodes an homeodomain–leucine zipper (HD-zip) TF, was identified as a pivotal regulator of petal senescence. Overall, this work facilitates the identification of hormones and TFs in plants related to flower opening and senescence in tulips. It also provides an important and valuable genetic basis for further research in them.

Keywords

Tulipa gesneriana / Full-length transcriptome / Flower opening and senescence / Transcription factor / Phytohormone profiles / TgHB12-like

References

[1]	Altschul SF, Madden TL, Sch?ffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped blast and PSI-BLAST: a new generation of protein database search programs. Nucl Acids Res. 1997;25:3389–402. https://doi.org/10.1093/nar/25.17.3389.
[2]	Anders S, Huber W. Differential expression analysis for sequence count data. Genome Biol. 2010;11:R106. https://doi.org/10.1186/gb-2010-11-10-r106.
[3]	Arrom L, Munné-Bosch S. Hormonal changes during flower development in floral tissues of Lilium. Planta. 2012;236:343–54. https://doi.org/10.1007/s00425-012-1615-0.
[4]	Azad AK, Ishikawa T, Sawa Y, Shibata H. Intracellular energy depletion triggers programmed cell death during petal senescence in tulip. J Exp Bot. 2008;59:2085–95. https://doi.org/10.1093/jxb/ern066.
[5]	Baharudin NF, Osman NI. Plant development, stress responses, and secondary metabolism under ethylene regulation. Plant Stress. 2023;7:100146. https://doi.org/10.1016/j.stress.2023.100146.
[6]	Bu D, Luo H, Huo P, Wang Z, Zhang S, He Z, et al. KOBAS-i: intelligent prioritization and exploratory visualization of biological functions for gene enrichment analysis. Nucl Acids Res. 2021;49:W317–25. https://doi.org/10.1093/nar/gkab447.
[7]	Chang X, Donnelly L, Sun D, Rao J, Reid MS, Jiang CZ. A petunia homeodomain-leucine zipper protein, PhHD-Zip, plays an important role in flower senescence. PLoS One. 2014;9:e88320. https://doi.org/10.1371/journal.pone.0088320.
[8]	Chen WH, Li PF, Chen MK, Lee YI, Yang CH. Forever young flower negatively regulates ethylene response DNA-binding factors by activating an ethylene-responsive factor to control Arabidopsis floral organ senescence and abscission. Plant Physiol. 2015;168:1666–83. https://doi.org/10.1104/pp.15.00433.
[9]	Cubría-Radío M, Arrom L, Puig S, Munné-Bosch S. Hormonal sensitivity decreases during the progression of flower senescence in Lilium longiflorum. J Plant Growth Regul. 2017;36:402–12. https://doi.org/10.1007/s00344-016-9648-4.
[10]	Gao Y, Xi F, Zhang H, Liu X, Wang H, Zhao L, et al. Single-molecule real-time (SMRT) isoform sequencing (Iso-Seq) in plants: the status of the bioinformatics tools to unravel the transcriptome complexity. Curr Bioinform. 2019;14:566–73. https://doi.org/10.2174/1574893614666190204151746.
[11]	Grosskinsky DK, Syaifullah SJ, Roitsch T. Integration of multi-omics techniques and physiological phenotyping within a holistic phenomics approach to study senescence in model and crop plants. J Exp Bot. 2018;69:825–44. https://doi.org/10.1093/jxb/erx333.
[12]	Hackl T, Hedrich R, Schultz J, F?rster F. Proovread: large-scale high-accuracy PacBio correction through iterative short read consensus. Bioinformatics. 2014;30:3004–11. https://doi.org/10.1093/bioinformatics/btu392.
[13]	Hunter DA, Ferrante A, Vernieri P, Reid MS. Role of abscisic acid in perianth senescence of daffodil (Narcissus pseudonarcissus ‘Dutch Master’). Physiol Plant. 2004;121:313–21. https://doi.org/10.1111/j.0031-9317.2004.0311.x.
[14]	Hur YS, Um JH, Kim S, Kim K, Park HJ, Lim JS, et al. Arabidopsis thaliana homeobox 12 (ATHB12), a homeodomain-leucine zipper protein, regulates leaf growth by promoting cell expansion and endoreduplication. New Phytol. 2015;205:316–28. https://doi.org/10.1111/nph.12998.
[15]	Jin J, Tian F, Yang DC, Meng YQ, Kong L, Luo J, et al. PlantTFDB 4.0: toward a central hub for transcription factors and regulatory interactions in plants. Nucl Acids Res. 2017;45:D1040-5. https://doi.org/10.1093/nar/gkw982.
[16]	Kanehisa M, Sato Y, Kawashima M, Furumichi M, Tanabe M. KEGG as a reference resource for gene and protein annotation. Nucl Acids Res. 2016;44:D457–62. https://doi.org/10.1093/nar/gkv1070.
[17]	Kumar L, Futschik ME. Mfuzz: a software package for soft clustering of microarray data. Bioinformation. 2007;2:5–7. https://doi.org/10.6026/97320630002005.
[18]	Kumar M, Singh VP, Arora A, Singh N. The role of abscisic acid (ABA) in ethylene insensitive Gladiolus (Gladiolus grandiflora Hort.) flower senescence. Acta Physiol Plant. 2014;36:151–9. https://doi.org/10.1007/s11738-013-1395-6.
[19]	Langfelder P, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics. 2008;9:559. https://doi.org/10.1186/1471-2105-9-559.
[20]	Langmead B, Salzberg SL. Fast gapped-read alignment with bowtie 2. Nat Methods. 2012;9:357–9. https://doi.org/10.1038/NMETH.1923.
[21]	Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-seq data with or without a reference genome. BMC Bioinformatics. 2011;12:323. https://doi.org/10.1186/1471-2105-12-323.
[22]	Liu X, Mei W, Soltis PS, Soltis DE, Barbazuk WB. Detecting alternatively spliced transcript isoforms from single-molecule long-read sequences without a reference genome. Mol Ecol Resources. 2017;17:1243–56. https://doi.org/10.1111/1755-0998.12670.
[23]	Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods. 2001;25:402–8. https://doi.org/10.1006/meth.2001.1262.
[24]	Lombardi L, Arrom L, Mariotti L, Battelli R, Picciarelli P, Kille P, et al. Auxin involvement in tepal senescence and abscission in Lilium: a tale of two lilies. J Exp Bot. 2015;66:945–56. https://doi.org/10.1093/jxb/eru451.
[25]	Lü P, Zhang C, Liu J, Liu X, Jiang G, Jiang X, et al. RhHB1 mediates the antagonism of gibberellins to ABA and ethylene during rose (Rosa hybrida) petal senescence. Plant J. 2014;78:578–90. https://doi.org/10.1111/tpj.12494.
[26]	Ma N, Ma C, Liu Y, Shahid MO, Wang C, Gao J. Petal senescence: a hormone view. J Exp Bot. 2018;69:719–32. https://doi.org/10.1093/jxb/ery009.
[27]	Marasek-Ciolakowska A, Ramanna MS, Arens P, Van Tuyl JM. Breeding and cytogenetics in the genus tulipa. Floric Ornam Biotechnol. 2012;6:90–7.
[28]	Meng L, Yang H, Yang J, Wang Y, Ye T, Xiang L, Chan Z, Wang Y. Tulip transcription factor TgWRKY75 activates salicylic acid and abscisic acid biosynthesis to synergistically promote petal senescece. J Exp Bot. 2024;75:2435–50. https://doi.org/10.1093/jxb/erae021.
[29]	Meng L, Yang H, Xiang L, Wang Y, Chan Z. NAC transcriptionfactor TgNAP promotes tulip petal senescence. Plant Physiol. 2022;190:1960–77. https://doi.org/10.1093/plphys/kiac351.
[30]	Müller GL, Drincovich MF, Andreo CS, Lara MV. Role of photosynthesis and analysis of key enzymes involved in primary metabolism throughout the lifespan of the tobacco flower. J Exp Bot. 2010;61:3675–88. https://doi.org/10.1093/jxb/erq187.
[31]	Panavas T, Walker EL, Rubinstein B. Possible involvement of abscisic acid in senescence of daylily petals. J Exp Bot. 1998;49:1987–97. https://doi.org/10.1093/jxb/49.329.1987.
[32]	Pourkhaloee A, Khosh-Khui M, Arens P, Salehi H, Razi H, Niazi A, et al. Molecular analysis of genetic diversity, population structure, and phylogeny of wild and cultivated tulips (Tulipa L.) by genic microsatellites. Horticul Environ Biotech. 2018;59:875–88. https://doi.org/10.1007/s13580-018-0055-6.
[33]	Prinsi B, Negri AS, Quattrocchio FM, Koes RE, Espen L. Proteomics of red and white corolla limbs in petunia reveals a novel function of the anthocyanin regulator anthocyanin1 in determining flower longevity. J Proteomics. 2016;131:38–47. https://doi.org/10.1016/j.jprot.2015.10.008.
[34]	Rogers HJ. Programmed cell death in floral organs: how and why do flowers die? Annals Bot. 2006;97:309–15. https://doi.org/10.1093/aob/mcj051.
[35]	Rogers HJ. From models to ornamentals: how is flower senescence regulated? Plant Mol Biol. 2013;82:563–74. https://doi.org/10.1007/s11103-012-9968-0.
[36]	Sexton R, Laird G, van Doorn WG. Lack of ethylene involvement in tulip tepal abscission. Physiol Plantarum. 2000;108:321–9. https://doi.org/10.1034/j.1399-3054.2000.108003321.x.
[37]	Shahin A, van Kaauwen M, Esselink D, Visser RGF, van Tuyl JM, Arens P, et al. Generation and analysis of expressed sequence tags in the extreme large genomes Lilium and Tulipa. BMC Genomics. 2012;13:640. https://doi.org/10.1186/1471-2164-13-640.
[38]	Sharon D, Tilgner H, Grubert F, Snyder M. A single-molecule long-read survey of the human transcriptome. Nat Biotechnol. 2013;31:1009–14. https://doi.org/10.1038/nbt.2705.
[39]	Sun L, Luo H, Bu D, Zhao G, Yu K, Zhang C, et al. Utilizing sequence intrinsic composition to classify protein-coding and long non-coding transcripts. Nucl Acids Res. 2013;41:e166. https://doi.org/10.1093/nar/gkt646.
[40]	Sun X, Zhu S, Li N, Cheng Y, Liu T, Qiao X, et al. A chromosome-level genome assembly of garlic (Allium sativum) provides insights into genome evolution and allicin biosynthesis. Mol Plant. 2020;13:1328–39. https://doi.org/10.1016/j.molp.2020.07.019.
[41]	Tang N, Shahin A, Bijman P, Liu J, van Tuyl J, Arens P. Genetic diversity and structure in a collection of tulip cultivars assessed by SNP markers. Sci Hortic. 2013;161:286–92. https://doi.org/10.1016/j.scienta.2013.07.016.
[42]	Tang N, van der Lee T, Shahin A, Holdinga M, Bijman P, Caser M, et al. Genetic mapping of resistance to Fusarium oxysporum f. sp tulipae in tulip. Mol Breed. 2015;35:122. https://doi.org/10.1007/s11032-015-0316-3.
[43]	Trivellini A, Cocetta G, Vernieri P, Mensuali-Sodi A, Ferrante A. Effect of cytokinins on delaying petunia flower senescence: a transcriptome study approach. Plant Mol Biol. 2015;87:169–80. https://doi.org/10.1007/s11103-014-0268-8.
[44]	Valdés AE, Verns E, Johansson H, Rada-Iglesias A, Engstrom P. The homeodomain-leucine zipper (HD-Zip) class I transcription factors ATHB7 and ATHB12 modulate abscisic acid signalling by regulating protein phosphatase 2C and abscisic acid receptor gene activities. Plant Mol Biol. 2012;80:405–18. https://doi.org/10.1007/s11103-012-9956-4.
[45]	van Doorn WG, van Meeteren U. Flower opening and closure: a review. J Exp Bot. 2003;54:1801–12. https://doi.org/10.1093/jxb/erg213.
[46]	Van Scheepen J. Classified list and international register of tulip names. Hillegom: Royal General Bulbgrowers’ Association (KAVB); 1996.
[47]	Van Tuyl JM, Creij MGM. Tulipa gesneriana and T. hybrids. In: Anderson NO, editor. Flower breeding and genetics. Berlin: Springer-Verlag; 2007. p. 623–41. https://doi.org/10.1007/978-1-4020-4428-1.
[48]	Verlinden S. Changes in mineral concentrations in petunia coroll during development and senescence. Hort Sci. 2003;38:71–4. https://doi.org/10.1038/s41438-018-0018-1.
[49]	Wang Q, Cai WJ, Yu L, Ding J, Feng YQ. Comprehensive profiling of phytohormones in honey by sequential liquid–liquid extraction coupled with liquid chromatography–mass spectrometry. J Agri Food Chem. 2017;65:575–85. https://doi.org/10.1021/acs.jafc.6b04234.
[50]	Wang Y, Zhao H, Liu C, Qu L, Bao M, Wang J, et al. Integrating physiological and metabolites analysis to identify ethylene involvement in petal senescence in Tulipa gesneriana. Plant Physiol Biochem. 2020;149:121–31. https://doi.org/10.1016/j.plaphy.2020.02.001.
[51]	Yan S, Li Y, Liu J, Si D, Zhang X. Guideline for extraction, qualitative, quantitative, and stabilityanalysis of anthocyanins. eFood. 2022;4:e59. https://doi.org/10.1002/efd2.59.
[52]	Yin J, Chang X, Kasuga T, Bui M, Reid MS, Jiang CZ. A basic helix-loop-helix transcription factor, PhFBH4, regulates flower senescence by modulating ethylene biosynthesis pathway in petunia. Hortic Res. 2015;2:15059. https://doi.org/10.1038/hortres.2015.59.
[53]	Young MD, Wakefield MJ, Smyth GK, Oshlack A. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol. 2010;11:R14. https://doi.org/10.1186/gb-2010-11-2-r14.
[54]	Yu L, Ding J, Wang YL, Liu P, Feng YQ. Phenylaminomethyl-benzeneboric acid modified tip extraction for determination of brassinosteroids in plant tissues by stable isotope labeling-liquid chromatography mass spectrometry. Anal Chem. 2016;88:1286–93. https://doi.org/10.1021/acs.analchem.5b03720.
[55]	Zhang S, Zhao Q, Zeng D, Xu J, Zhou H, Wang F, et al. RhMYB108, an R2R3-MYB transcription factor, is involved in ethylene- and JA-induced petal senescence in rose plants. Hortic Res. 2019;6:131. https://doi.org/10.1038/s41438-019-0221-8.
[56]	Zonneveld BJ. The systematic value of nuclear genome size for all species of Tulipa L. (Liliaceae). Plant Syst Evol. 2009;281:217–45. https://doi.org/10.1007/s00606-009-0203-7.

Funding

National Natural Science Foundation of China(32170372); Fundamental Research Funds for the Central Universities(2662016QD026)