PE (Prickly Eggplant) encoding a cytokinin-activating enzyme responsible for the formation of prickles in eggplant

Abstract

Eggplant is one of the most important vegetables worldwide, with some varieties displaying prickles. These prickles, present on the leaves, stems, and fruit calyxes, posing challenges during cultivation, harvesting, and transportation, making them an undesirable agronomic trait. However, the genetic mechanisms underlying prickle morphogenesis in eggplant remain poorly understood, impeding genetic improvements. In this study, genetic analyses revealed that prickle morphogenesis is governed by a single dominant nuclear gene, termed PE (Prickly Eggplant). Subsequent bulk segregant RNA-sequencing (BSR-seq) and linkage analysis preliminarily mapped PE to chromosome 6. This locus was then fine mapped to a 9233 bp interval in a segregating population of 1109 plants, harboring only one candidate gene, SmLOG1, which encodes a LONELY GUY (LOG)-family cytokinin biosynthetic enzyme. Expression analyses via transcriptome and qRT-PCR demonstrate that SmLOG1 is predominantly expressed in immature prickles. CRISPR-Cas9 knockout experiments targeting SmLOG1 in prickly parental line ‘PI 381159’ abolished prickles across all tissues, confirming its critical role in prickle morphogenesis. Sequence analysis of SmLOG1 pinpointed variations solely within the non-coding region. We developed a cleaved amplified polymorphic sequences (CAPS) marker from a distinct SNP located at −735-bp within the SmLOG1 promoter, finding significant association with prickle variation in 190 eggplant germplasms. These findings enhance our understanding of the molecular mechanisms governing prickle development in eggplant and facilitate the use of marker-assisted selection (MAS) for breeding prickleless cultivars.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Nos 32102378 and 32101732), the Young Elite Scientist Sponsorship Program by Jiangsu Association for Science and Technology (No. JSTJ-2022-051), the Natural Science Foundation of Xuzhou City (No. KC23053), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). We thank Prof. Hanhui Kuang, Prof. Xin Wang, and Dr. Qun Hu for their critical review and editing of this article.

Author contributions

L.Z. and W.Q.H. conceived and designed the research. L.Z., W.Q.H., R.Z.Z., P.Y., L.Q.Z., W.W.Z., H.Q.F., and R.Y.M. performed the research and analysed the data. L.Z. and W.Q.H. wrote the manuscript. All authors read and approved the manuscript.

Data availability

All relevant data can be found within the article and its supporting materials.

Conflict of interest statement

The authors declare no competing interests.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Yang X, Cheng Y-F, Deng C. et al. Comparative transcriptome analysis of eggplant (Solanum melongena L.) and Turkey berry (Solanum torvum Sw.): phylogenomics and disease resistance analysis. BMC Genomics. 2014; 15:412

[2] Page A, Gibson J, Meyer RS. et al. Eggplant domestication: pervasive gene flow, feralisation and transcriptomic divergence. Mol Biol Evol. 2019; 36:1359-72

[3] Miyatake K, Saito T, Nunome T. et al. Fine mapping of a major locus representing the lack of prickles in eggplant revealed the availability of a 0.5-kb insertion/deletion for marker-assisted selection. Breed Sci. 2020; 70:438-48

[4] Coverdale TC. Defence emergence during early ontogeny reveals important differences between spines, thorns and prickles. Ann Bot. 2019;124:iii-v

[5] Pei H, Wu Y, Wu W. et al. A review of the types, functions and regulatory mechanisms of plant spines. Plant Sci. 2024; 341:112010

[6] Simpson MG. 9 - Plant Morphology. In: Simpson MG (ed.), Plant Systematics. 2nd edn. San Diego: Academic Press, 2010, 451-513

[7] Wang X, Shen C, Meng P. et al. Analysis and review of trichomes in plants. BMC Plant Biol. 2021; 21:70

[8] Ma ZY, Wen J, Ickert-Bond SM. et al. Morphology, structure, and ontogeny of trichomes of the grape genus (Vitis, Vitaceae). Front Plant Sci. 2016; 7:704

[9] Swarnkar MK, Kumar P, Dogra V. et al. Prickle morphogenesis in rose is coupled with secondary metabolite accumulation and governed by canonical MBW transcriptional complex. Plant Direct. 2021; 5:e00325

[10] Khadgi A, Weber CA. Morphological characterization of prickled and prickle-free Rubus using scanning electron microscopy. HortScience. 2020; 55:676-83

[11] Kellogg AA, Branaman TJ, Jones NM. et al. Morphological studies of developing Rubus prickles suggest that they are modified glandular trichomes. Botany. 2011; 89:217-26

[12] Zhou N, Simonneau F, Thouroude T. et al. Morphological studies of rose prickles provide new insights. Hort Res. 2021; 8:221

[13] Zhou NN, Tang KX, Jeauffre J. et al. Genetic determinism of prickles in rose. TAG Theoretical Applied Genet. 2020; 133:3017-35

[14] Zhang Y, Zuo M, Li R. et al. Morphology, structure and development of glandular prickles in the genus Rosa. Sci Hortic. 2024; 326:112763

[15] Zhang L, Sun H, Xu T. et al. Comparative transcriptome analysis reveals key genes and pathways involved in prickle development in eggplant. Genes. 2021; 12:341

[16] Huang X, Yi P, Liu Y. et al. RrTTG1 promotes fruit prickle development through an MBW complex in Rosa roxburghii. Front Plant Sci. 2022; 13:939270

[17] Pattanaik S, Patra B, Singh SK. et al. An overview of the gene regulatory network controlling trichome development in the model plant, Arabidopsis. Front Plant Sci. 2014; 5:00259

[18] Li S, He Y, Li D. et al. Fine mapping an AUXIN RESPONSE FACTOR, SmARF18, as a candidate gene of the PRICKLE LOCUS that controls prickle absence/presence on various organs in eggplant (Solanum melongena L.). Sci Hortic. 2024; 327:112874

[19] Doganlar S, Frary A, Daunay MC. et al. Conservation of gene function in the solanaceae as revealed by comparative mapping of domestication traits in eggplant. Genetics. 2002; 161:1713-26

[20] Frary A, Frary A, Daunay M-C. et al. QTL hotspots in eggplant (Solanum melongena) detected with a high resolution map and CIM analysis. Euphytica. 2014; 197:211-28

[21] Portis E, Barchi L, Toppino L. et al. QTL mapping in eggplant reveals clusters of yield-related loci and orthology with the tomato genome. PLoS One. 2014; 9:e89499

[22] Portis E, Cericola F, Barchi L. et al. Association mapping for fruit, plant and leaf morphology traits in eggplant. PLoS One. 2015; 10:e0135200

[23] Mangino G, Plazas M, Vilanova S. et al. Performance of a set of eggplant (Solanum melongena) lines with introgressions from its wild relative S. incanum under open field and Screenhouse conditions and detection of QTLs. Agronomy. 2020; 10:10040467

[24] Wei Q, Wang W, Hu T. et al. Construction of a SNP-based genetic map using SLAF-Seq and QTL analysis of morphological traits in eggplant. Front Genet. 2020; 11:178

[25] Qian Z, Zhang B, Chen H. et al. Identification of quantitative trait loci controlling the development of prickles in eggplant by genome re-sequencing analysis. Front Plant Sci. 2021; 12:731079

[26] Chen L, Jameson GB, Guo Y. et al. The LONELY GUY gene family: from mosses to wheat, the key to the formation of active cytokinins in plants. Plant Biotechnol J. 2022; 20:625-45

[27] Kuroha T, Tokunaga H, Kojima M. et al. Functional analyses of LONELY GUY cytokinin-activating enzymes reveal the importance of the direct activation pathway in Arabidopsis. Plant Cell. 2009; 21:3152-69

[28] Tokunaga H, Kojima M, Kuroha T. et al. Arabidopsis lonely guy (LOG) multiple mutants reveal a central role of the LOG-dependent pathway in cytokinin activation. Plant J. 2012; 69:355-65

[29] Hua L, Wang DR, Tan L. et al. LABA1, a domestication gene associated with long, barbed awns in wild rice. Plant Cell. 2015; 27:1875-88

[30] Gu B, Zhou T, Luo J. et al. An-2 encodes a cytokinin synthesis enzyme that regulates awn length and grain production in rice. Mol Plant. 2015; 8:1635-50

[31] Kurakawa T, Ueda N, Maekawa M. et al. Direct control of shoot meristem activity by a cytokinin-activating enzyme. Nature. 2007; 445:652-5

[32] Kieber JJ, Schaller GE. Cytokinins. Arabidopsis Book. 2014; 12:e0168

[33] Pils B, Heyl A. Unraveling the evolution of cytokinin signaling. Plant Physiol. 2009; 151:782-91

[34] Castro-Mondragon JA, Riudavets-Puig R, Rauluseviciute I. et al. JASPAR 2022: the 9th release of the open-access database of transcription factor binding profiles. Nucleic Acids Res. 2022;50:D165-73

[35] Yang Z, Gu S, Wang X. et al. Molecular evolution of the CPP-like gene family in plants: insights from comparative genomics of Arabidopsis and Rice. J Mol Evol. 2008; 67:266-77

[36] Liu Z, Running MP, Meyerowitz EM. TSO 1 functions in cell division during Arabidopsis flower development. Development. 1997; 124:665-72

[37] Hauser BA, He JQ, Park SO. et al. TSO1 is a novel protein that modulates cytokinesis and cell expansion in Arabidopsis. Development. 2000; 127:2219-26

[38] Wang W, Sijacic P, Xu P. et al. Arabidopsis TSO1 and MYB3R1 form a regulatory module to coordinate cell proliferation with differentiation in shoot and root. Proc Natl Acad Sci USA. 2018;115:E3045-54

[39] Hibrand Saint-Oyant L, Ruttink T, Hamama L. et al. A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits. Nature Plants. 2018; 4:473-84

[40] Pandey S, Goel R, Bhardwaj A. et al. Transcriptome analysis provides insight into prickle development and its link to defense and secondary metabolism in Solanum viarum Dunal. Sci Rep. 2018; 8:17092

[41] Osugi A, Sakakibara H. Q&A: how do plants respond to cytokinins and what is their importance? BMC Biol. 2015; 13:102

[42] Hnatuszko-Konka K, Gerszberg A, Weremczuk-Jeżyna I. et al. Cytokinin signaling and de novo shoot organogenesis. Genes. 2021; 12:265

[43] Hai NN, Chuong NN, Tu NHC. et al. Role and regulation of cytokinins in plant response to drought stress. Plan Theory. 2020; 9:422

[44] Mortier V, Wasson A, Jaworek P. et al. Role of LONELY GUY genes in indeterminate nodulation on Medicago truncatula. New Phytol. 2014; 202:582-93

[45] Villao-Uzho L, Chávez-Navarrete T, Pacheco-Coello R. et al. Plant promoters: their identification, characterization, and role in gene regulation. Genes. 2023; 14:1226

[46] Ruan B, Shang L, Zhang B. et al. Natural variation in the promoter of TGW2 determines grain width and weight in rice. New Phytol. 2020; 227:629-40

[47] Jia D, Wu P, Shen F. et al. Genetic variation in the promoter of an R2R3-MYB transcription factor determines fruit malate content in apple (Malus domestica Borkh.). Plant Physiol. 2021; 186:549-68

[48] Zhang L, Qian J, Han Y. et al. Alternative splicing triggered by the insertion of a CACTA transposon attenuates LsGLK and leads to the development of pale-green leaves in lettuce. Plant J. 2022; 109:182-95

[49] Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30:2114-20

[50] Wei Q, Wang J, Wang W. et al. A high-quality chromosome-level genome assembly reveals genetics for important traits in eggplant. Hortic Res. 2020; 7:153

[51] Dobin A, Davis CA, Schlesinger F. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013; 29:15-21

[52] Li H, Handsaker B, Wysoker A. et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009; 25:2078-9

[53] Untergasser A, Nijveen H, Rao X. et al. Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res. 2007;35:W71-4

[54] Thompson JD, Higgns DG, Gibson Toby J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994; 22:4673-80

[55] Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016; 33:1870-4

[56] Gantasala NP, Papolu PK, Thakur PK. et al. Selection and validation of reference genes for quantitative gene expression studies by real-time PCR in eggplant (Solanum melongena L). BMC Res Notes. 2013; 6:312

[57] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001; 25:402-8

[58] Deng L, Wang H, Sun C. et al. Efficient generation of pink-fruited tomatoes using CRISPR/Cas9 system. J Genet Genomics. 2018; 45:51-4

[59] Liu H, Ding Y, Zhou Y. et al. CRISPR-P 2.0: an improved CRISPR-Cas9 tool for genome editing in plants. Mol Plant. 2017; 10:530-2

[60] Naito Y, Hino K, Bono H. et al. CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics. 2015; 31:1120-3

[61] Khatun M, Borphukan B, Alam I. et al. An improved Agrobacterium mediated transformation and regeneration protocol for successful genetic engineering and genome editing in eggplant. Sci Hortic. 2021; 293:110716