Cis-regulatory effects of carrot miniature inverted-repeat transposable elements on the expression of genes controlled by LHY/RVE transcription factors

Abstract

Transposable elements constitute a large portion of plant genomes and, due to their ability to change their genomic localization, they largely contribute to genome evolution and adaptability. Miniature inverted-repeat transposable elements (MITEs), due to their small size and localization near genes, seem to be a major source of potential functional variability. Effects imposed by MITEs on the expression of associated genes through redistributing cis-regulatory elements have been postulated, but our knowledge in this area still remains limited. We showed that MITEs in the carrot genome are enriched with binding sites for LHY/RVE transcription factors (TFs). Experimental validation using DcLHY-DAP-seq not only confirmed the enrichment of DcLHY binding sites within MITEs but also demonstrated that elements from the DcTourist_15 family likely play a key role in redistributing these TF binding sites. We showed that insertional polymorphisms of DcTourist_15 correspond with changes in the expression of associated genes, both in control conditions and in response to heat stress. In addition to placing individual genes under the control of DcLHY/RVE TFs, DcTourist_15 copies were found in promoters of genes involved in sulfur metabolism and cysteine biosynthesis. The enrichment of rice MITEs in OsLHY binding sites suggests the phenomenon of MITE-driven rewiring of LHY/RVE regulation may be more widespread across the plant kingdom. Carrot MITEs, particularly those from the DcTourist_15 family, drive evolution of the carrot genome, especially in the context of stress responsiveness, as they possibly fine-tune gene expression by redistributing binding sites for TFs from the LHY/RVE family.

Acknowledgements

The research was primarily financed by Polish National Science Center (NCN) project Opus17 no. 2019/33/B/NZ9/00757. The research performed at the University of South Carolina Aiken was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM103499. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Author contributions

A.M.P. and C.N.H designed the research, A.M.P and C.N.H. acquired funding, supervised the work, summarized and interpreted results; A.M.P and K.Z performed analysis and produced the figures; W.W. developed the pipeline for k-mer analysis; E.D., K.K, M.S., Z.B., and Z.M.L. conducted the wet-lab investigation; A.M.P. and D.G. wrote the original draft, A.M.P., D.G. and C.N.H. provided resources, reviewed and edited the manuscript. All authors have read and agreed to the final manuscript.

Data availability

RNA-seq and WGS-seq data have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject PRJNA1234542 (Tables S27 and Table S28).

Conflicts of interest statement

The authors declare that they have no conflict of interests.

Supplementary material

Supplementary material is available at Horticulture Research online.

References

[1] Stelmach K , Macko-Podgórni A , Machaj G , et al. Miniature inverted repeat transposable element insertions provide a source of intron length polymorphism markers in the carrot (Daucus carota L.). Front Plant Sci. 2017; 8: 725

[2] Wei H , Xu H , Su C , et al. Rice CIRCADIAN CLOCK ASSOCIATED 1 transcriptionally regulates ABA signaling to confer multiple abiotic stress tolerance. Plant Physiol. 2022; 190: 1057-73

[3] Akakpo R , Carpentier M , Ie Hsing Y , et al. The impact of transposable elements on the structure, evolution and function of the rice genome. New Phytol. 2020; 226: 44-9

[4] Bennetzen JL , Wang H . The contributions of transposable elements to the structure, function, and evolution of plant genomes. Annu Rev Plant Biol. 2014; 65: 505-30

[5] Buisine N , Quesneville H , Colot V . Improved detection and annotation of transposable elements in sequenced genomes using multiple reference sequence sets. Genomics. 2008; 91: 467-75

[6] Coe K , Bostan H , Rolling W , et al. Population genomics identifies genetic signatures of carrot domestication and improvement and uncovers the origin of high-carotenoid orange carrots. Nat Plants. 2023; 9: 1643-58

[7] International Wheat Genome Sequencing Consortium , Wicker T , Gundlach H , et al. Impact of transposable elements on genome structure and evolution in bread wheat. Genome Biol. 2018; 19: 103

[8] Wicker T , Sabot F , Hua-Van A , et al. A unified classification system for eukaryotic transposable elements. Nat Rev Genet. 2007; 8: 973-82

[9] Hu J , Liu C , Du Z , et al. Transposable elements cause the loss of self-incompatibility in citrus. Plant Biotechnol J. 2024; 22: 1113-31

[10] Lu C , Chen J , Zhang Y , et al. Miniature inverted-repeat transposable elements (MITEs) have been accumulated through amplification bursts and play important roles in gene expression and species diversity in Oryza sativa. Mol Biol Evol. 2012; 29: 1005-17

[11] Xu L , Yuan K , Yuan M , et al. Regulation of rice tillering by RNA-directed DNA methylation at miniature inverted-repeat transposable elements. Mol Plant. 2020; 13: 851-63

[12] Arce AL , Mencia R , Cambiagno DA , et al. Polymorphic inverted repeats near coding genes impact chromatin topology and phenotypic traits in Arabidopsis thaliana. Cell Rep. 2023; 42: 112029

[13] Song Y , Bu C , Chen P , et al. Miniature inverted repeat transposable elements cis-regulate circular RNA expression and promote ethylene biosynthesis, reducing heat tolerance in Populus tomentosa . Gibbs D (ed.). J Exp Bot. 2021; 72: 1978-94

[14] Oki N , Yano K , Okumoto Y , et al. A genome-wide view of miniature inverted-repeat transposable elements (MITEs) in rice, Oryza sativa ssp. japonica. Genes Genet Syst. 2008; 83: 321-9

[15] Shen J , Liu J , Xie K , et al. Translational repression by a miniature inverted-repeat transposable element in the 3′ untranslated region. Nat Commun. 2017; 8: 14651

[16] Niu XM , Xu YC , Li ZW , et al. Transposable elements drive rapid phenotypic variation in Capsella rubella . Proc Natl Acad Sci. 2019; 116: 6908-13

[17] Macko-Podgórni A , Machaj G , Grzebelus D . A global landscape of miniature inverted-repeat transposable elements in the carrot genome. Genes. 2021; 12: 859

[18] Hénaff E , Vives C , Desvoyes B , et al. Extensive amplification of the E2F transcription factor binding sites by transposons during evolution of Brassica species . Plant J. 2014; 77: 852-62

[19] Morata J , Marín F , Payet J , et al. Plant lineage-specific amplification of transcription factor binding motifs by miniature inverted-repeat transposable elements (MITEs). Genome Biol Evol. 2018; 10: 1210-20

[20] Roquis D , Robertson M , Yu L , et al. Genomic impact of stress-induced transposable element mobility in Arabidopsis. Nucleic Acids Res. 2021; 49: 10431-47

[21] Zhang Y , Li Z , Liu J , et al. Transposable elements orchestrate subgenome-convergent and -divergent transcription in common wheat. Nat Commun. 2022; 13: 6940

[22] Maistriaux LC , Laurent MJ , Jeanguenin L , et al. Genetic variability of aquaporin expression in maize: from eQTLs to a MITE insertion regulating PIP2;5 expression . Plant Physiol. 2024; 196: 368-84

[23] Kamioka M , Takao S , Suzuki T , et al. Direct repression of evening genes by CIRCADIAN CLOCK-ASSOCIATED1 in the Arabidopsis circadian clock. Plant Cell. 2016; 28: 696-711

[24] Hsu PY , Devisetty UK , Harmer SL . Accurate timekeeping is controlled by a cycling activator in Arabidopsis. eLife. 2013; 2: 473

[25] Li W , Tian Y , Li J , et al. A competition-attenuation mechanism modulates thermoresponsive growth at warm temperatures in plants. New Phytol. 2022; 237: 177-91

[26] Du H , Wang YB , Xie Y , et al. Genome-wide identification and evolutionary and expression analyses of MYB-related genes in land plants. DNA Res. 2013; 20: 437-48

[27] Bian Y , Song Z , Liu C , et al. The BBX7/8-CCA1/LHY transcription factor cascade promotes shade avoidance by activating PIF4. New Phytol. 2025; 245: 637-52

[28] de Leone MJ , Yanovsky MJ . The circadian clock and thermal regulation in plants: novel insights into the role of positive circadian clock regulators in temperature responses. J Exp Bot. 2024; 75: 2809-18

[29] Hughes CL , Harmer SL . Myb-like transcription factors have epistatic effects on circadian clock function but additive effects on plant growth. Plant Direct. 2023; 7: e533

[30] Macko-Podgórni A , Stelmach K , Kwolek K , et al. Stowaway miniature inverted repeat transposable elements are important agents driving recent genomic diversity in wild and cultivated carrot. Mob DNA. 2019; 10: 47

[31] Mizoguchi T , Wheatley K , Hanzawa Y , et al. LHY and CCA1 are partially redundant genes required to maintain circadian rhythms in Arabidopsis. Dev Cell. 2002; 2: 629-41

[32] Kyung J , Jeon M , Jeong GM , et al. The two clock proteins CCA1 and LHY activate VIN3 transcription during vernalization through the vernalization-responsive cis-element . Plant Cell. 2021; 34: 1020-37

[33] Jiang Z , Xu G , Jing Y , et al. Phytochrome B and REVEILLE1/2-mediated signalling controls seed dormancy and germination in Arabidopsis. Nat Commun. 2016; 7: 7

[34] Rawat R , Schwartz J , Jones MA , et al. REVEILLE1, a Myb-like transcription factor, integrates the circadian clock and auxin pathways. Proc Natl Acad Sci. 2009; 106: 16883-8

[35] Xu G , Guo H , Zhang D , et al. REVEILLE1 promotes NADPH:protochlorophyllide oxidoreductase A expression and seedling greening in Arabidopsis. Photosynth Res. 2015; 126: 331-40

[36] Tian Y , Li W , Wang M , et al. REVEILLE 7 inhibits the expression of the circadian clock gene EARLY FLOWERING 4 to fine-tune hypocotyl growth in response to warm temperatures . J Integr Plant Biol. 2022; 64: 1310-24

[37] Gilman IS , Moreno-Villena JJ , Lewis ZR , et al. Gene co-expression reveals the modularity and integration of C4 and CAM in Portulaca . Plant Physiol. 2022; 189: 735-53

[38] Turcotte K , Srinivasan S , Bureau T . Survey of transposable elements from rice genomic sequences. Plant J. 2001; 25: 169-79

[39] Rolling WR , Ellison S , Coe K , et al. Combining genome-wide association and genomic prediction to unravel the genetic architecture of carotenoid accumulation in carrot. Plant Genome. 2025; 18: e20560

[40] Stelmach K , Macko-Podgórni A , Allender CJ , et al. Genetic diversity structure of western-type carrots. BMC Plant Biol. 2021; 21: 200

[41] Baduel P , Leduque B , Ignace A , et al. Genetic and environmental modulation of transposition shapes the evolutionary potential of Arabidopsis thaliana. Genome Biol. 2021; 22: 138

[42] Kribelbauer JF , Rastogi C , Bussemaker HJ , et al. Low-affinity binding sites and the transcription factor specificity paradox in eukaryotes. Annu Rev Cell Dev Biol. 2019; 35: 357-79

[43] Monroe JG , Srikant T , Carbonell-Bejerano P , et al. Mutation bias reflects natural selection in Arabidopsis thaliana. Nature. 2022; 602: 101-5

[44] Lu Z , Cui J , Wang L , et al. Genome-wide DNA mutations in Arabidopsis plants after multigenerational exposure to high temperatures. Genome Biol. 2021; 22: 160

[45] Borredá C , Leduque B , Colot V , et al. Transposable element products, functions, and regulatory networks in Arabidopsis. 2024; BioRxiv: 2024.04.02.587720

[46] Kidokoro S , Hayashi K , Haraguchi H , et al. Posttranslational regulation of multiple clock-related transcription factors triggers cold-inducible gene expression in Arabidopsis . Proc Natl Acad Sci. 2021; 118: e2021048118

[47] Castanera R , Morales-Díaz N , Gupta S , et al. Transposons are important contributors to gene expression variability under selection in rice populations. eLife. 2023; 12: RP86324

[48] Castanera R , Vendrell-Mir P , Bardil A , et al. Amplification dynamics of miniature inverted-repeat transposable elements and their impact on rice trait variability. Plant J. 2021; 107: 118-35

[49] McKenna A , Hanna M , Banks E , et al. The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010; 20: 1297-303

[50] Biscarini F , Cozzi P , Gaspa G , et al. Detect runs of homozygosity and runs of heterozygosity in diploid genomes [R package detectRUNS version 0.9.6]. R-Proj. 2019

[51] Danecek P , Auton A , Abecasis G , et al. The variant call format and VCFtools. Bioinformatics. 2011; 27: 2156-8

[52] Purcell S , Neale B , Todd-Brown K , et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet. 2007; 81: 559-75

[53] Wickham H . Ggplot2: Elegant Graphics for Data Analysis . New York: Springer; 2009

[54] Love MI , Huber W , Anders S . Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15: 550

[55] Wickham H . ggplot2: Elegant Graphics for Data Analysis . Springer-Verlag, New York, 2016: 11-31

[56] Tang Y , Horikoshi M , Li W . Ggfortify: unified Interface to visualize statistical results of popular R packages. R J. 2016; 8: 474

[57] R Core Team . R: A Language and Environment for Statistical Computing. 2024

[58] Machaj G , Bostan H , Macko-Podgórni A , et al. Comparative transcriptomics of root development in wild and cultivated carrots. Genes. 2018; 9: 431-1

[59] Bryant DM , Johnson K , DiTommaso T , et al. A tissue-mapped axolotl de novo transcriptome enables identification of limb regeneration factors. Cell Rep. 2017; 18: 762-76

[60] Chen J , Wrightsman TR , Wessler SR , et al. RelocaTE2: a high resolution transposable element insertion site mapping tool for population resequencing. PeerJ. 2017; 5: e2942-2

[61] Marçais G , Kingsford C . A fast, lock-free approach for efficient parallel counting of occurrences of k-mers. Bioinformatics. 2011; 27: 764-70

[62] Mathelier A , Fornes O , Arenillas DJ , et al. JASPAR 2016: a major expansion and update of the open-access database of transcription factor binding profiles. Nucleic Acids Res. 2015; 44: D110-5

[63] Gupta S , Stamatoyannopoulos JA , Bailey TL , et al. Quantifying similarity between motifs. Genome Biol. 2007; 8: R24

[64] Consortium TU . UniProt: the universal protein knowledgebase. Nucleic Acids Res. 2018; 46: 2699-9

[65] Bartlett A , O’Malley RC , Huang SC , et al. Mapping genome-wide transcription-factor binding sites using DAP-seq. Nat Protoc. 2017; 12: 1659-72

[66] Grabherr MG , Haas BJ , Yassour M , et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011; 29: 644-52

[67] Langmead B , Salzberg SL . Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012; 9: 357- 9

[68] Zhang Y , Liu T , Meyer CA , et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 2008; 9: R137

[69] Bailey TL , Johnson J , Grant CE , et al. The MEME suite. Nucleic Acids Res. 2015; 43: W39-49

[70] Quinlan AR , Hall IM . BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010; 26: 841-2

[71] Bu D , Luo H , Huo P , et al. KOBAS-i: intelligent prioritization and exploratory visualization of biological functions for gene enrichment analysis. Nucleic Acids Res. 2021; 49: W317-25

[72] Kanehisa M , Goto S . KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 2000; 28: 27-30

[73] Ashburner M , Ball CA , Blake JA , et al. Gene ontology: tool for the unification of biology. Nat Genet. 2000; 25: 25-9

[74] Pertea G , Pertea M . GFF utilities: GffRead and GffCompare. F1000Res. 2020; 9: 304

[75] Rubin DB . Randomization analysis of experimental data: the Fisher randomization test comment. J Am Stat Assoc. 1980; 75: 591

[76] Harkness WL . Properties of the extended hypergeometric distribution. Ann Math Stat. 1965; 36: 938-45

[77] Benjamini Y , Hochberg Y . On the adaptive control of the false discovery rate in multiple testing with independent statistics. J Educ Behav Stat. 2000; 25: 60-83

[78] Krzywinski M , Schein J , Birol I , et al. Circos: an information aesthetic for comparative genomics. Genome Res. 2009; 19: 1639-45

[79] Deplancke B , Mukhopadhyay A , Ao W , et al. A gene-centered C. elegans protein-DNA interaction network. Cell. 2006; 125: 1193-205

[80] Katoh K , Standley DM . MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013; 30: 772-80

[81] Kimura M . A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol. 1980; 16: 111-20

[82] Kumar S , Stecher G , Li M , et al. MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Battistuzzi FU (ed.). Mol Biol Evol. 2018; 35: 1547-9

[83] Saitou N , Nei M . The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987; 4: 406-25

[84] Hillis DM , Bull JJ . An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Syst Biol. 1993; 42: 182