Genome-wide expression atlas of tomato flower buds revealed the SllncERF162-SlERF162 module associated with basal thermotolerance

Abstract

High temperatures impair pollen viability and reduce fruit set, ultimately affecting the yield of crops. Understanding the genetic components involved in the heat stress (HS) response is essential for developing climate-resilient crop varieties. However, the regulatory mechanisms governing HS responses during pollen development in tomato (Solanum lycopersicum) remain unexplored. In this study, we identified the microspore mother cell stage as the most heat-sensitive phase in tomato pollen development. Furthermore, we generated a comprehensive RNA expression profile of tomato flower buds under HS, encompassing 8051 mRNAs, 5738 lncRNAs, 62 circRNAs, and 24 miRNAs. Comparative analysis of these RNAs revealed three distinct response phases, early, late, and dual, and enabled the identification of coexpression modules comprising both coding and noncoding transcripts. Among these, SlERF162 was identified as a key regulatory gene that promotes pollen thermotolerance. We further identified the lncRNA TCONS_00023929 (designated SllncERF162) as a positive regulator of SlERF162 expression. Both SlERF162 and SllncERF162 contributed to maintaining pollen viability under HS. Additional experiments demonstrated that the SllncERF162-SlERF162 regulatory module enhances basal thermotolerance by directly targeting and activating the heat-responsive genes SlHsfB1 and SlsHSP. Overall, this study provides a high-resolution expression atlas of RNAs under HS and uncovers a novel noncoding RNA-mediated regulatory network that promotes thermotolerance during tomato pollen development.

Acknowledgements

We thank members of Tao Lab for stimulating discussion on this project. We thank Dr Yanglin Ding (China Agricultural University, China) for excellent advice. This work was supported by grants from the National Natural Science Foundation of China (32072571), the 111 Project (B17043), and the Construction of Beijing Science and Technology Innovation and Service Capacity in Top Subjects (CEFF-PXM2019_014207_000032).

Author contributions

T.L., Y.F.Z and N.Z. conceived the research and designed the experiments. Q.Q.Y. and X.L.G. analyzed the data and performed the experiments. H.W.L., Y.Q.C., Y.E.C., Y.Y. and M.Z. performed the part of the experiments. Q.Q.Y. wrote the manuscript. T.L., N.Z., and Z.Y.Z. revised the manuscript. All authors of this paper read and approved the final manuscript.

Data availability

All data supporting this study are included in the manuscript and Supplementary Materials. Raw sequencing data have been deposited in the Genome Sequence Archive (GSA) of the Beijing Institute of Genomics (BIG) Data Center (https://bigd.big.ac.cn/) under accession number CRA018919.

Conflict of interest statement

The authors declare no competing interests.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Khan AH, Min L, Ma Y. et al. High-temperature stress in crops: male sterility, yield loss and potential remedy approaches. Plant Biotechnol J. 2023; 21:680-97

[2] Chaturvedi P, Wiese AJ, Ghatak A. et al. Heat stress response mechanisms in pollen development. New Phytol. 2021; 231: 571-85

[3] Ayenan MAT, Danquah A, Hanson P. et al. Accelerating breeding for heat tolerance in tomato (Solanum lycopersicum L.): an inte-grated approach. Agronomy. 2019; 9:720

[4] Ding Y, Yang S. Surviving and thriving: how plants perceive and respond to temperature stress. Dev Cell. 2022; 57:947-58

[5] Ohama N, Sato H, Shinozaki K. et al. Transcriptional regulatory network of plant heat stress response. Trends Plant Sci. 2017; 22: 53-65

[6] Hahn A, Bublak D, Schleiff E. et al. Crosstalk between Hsp90 and Hsp70 chaperones and heat stress transcription factors in tomato. Plant Cell. 2011; 23:741-55

[7] Fragkostefanakis S, Simm S, El-Shershaby A. et al. The repres-sor and co-activator HsfB1 regulates the major heat stress transcription factors in tomato. Plant Cell Environ. 2019; 42: 874-90

[8] Liu HC, Liao HT, Charng YY. The role of class A1 heat shock factors (HSFA1s) in response to heat and other stresses in Ara-bidopsis. Plant Cell Environ. 2011; 34:738-51

[9] Yoshida T, Ohama N, Nakajima J. et al. Arabidopsis HsfA1 tran-scription factors function as the main positive regulators in heat shock-responsive gene expression. Mol Gen Genomics. 2011; 286: 321-32

[10] Fragkostefanakis S, Mesihovic A, Simm S. et al. HsfA2 controls the activity of developmentally and stress-regulated heat stress protection mechanisms in tomato male reproductive tissues. Plant Physiol. 2016; 170:2461-77

[11] Kan Y, Mu XR, Gao J. et al. The molecular basis of heat stress responses in plants. Mol Plant. 2023; 16:1612-34

[12] Licausi F, Ohme-Takagi M, Perata P. APETALA2/ethylene respon-sive factor (AP2/ERF) transcription factors: mediators of stress responses and developmental programs. New Phytol. 2013; 199: 639-49

[13] Xie Z, Nolan T, Jiang H. et al. The AP2/ERF transcription fac-tor TINY modulates brassinosteroid-regulated plant growth and drought responses in Arabidopsis. Plant Cell. 2019; 31: 1788-806

[14] Xie Z, Nolan TM, Jiang H. et al. AP2/ERF transcription factor regulatory networks in hormone and abiotic stress responses in Arabidopsis. Front Plant Sci. 2019; 10:228

[15] Schramm F, Larkindale J, Kiehlmann E. et al. A cascade of transcription factor DREB2A and heat stress transcription factor HsfA3 regulates the heat stress response of Arabidopsis. Plant J. 2008; 53:264-74

[16] Yoshida T, Sakuma Y, Todaka D. et al. Functional analysis of an Arabidopsis heat-shock transcription factor HsfA3 in the transcriptional cascade downstream of the DREB2A stress-regulatory system. Biochem Biophys Res Commun. 2008; 368:515-21

[17] Chen H, Hwang JE, Lim CJ. et al. Arabidopsis DREB2C functions as a transcriptional activator of HsfA3 during the heat stress response. Biochem Biophys Res Commun. 2010; 401:238-44

[18] Huang J, Zhao X, Bürger M. et al. Two interacting ethylene response factors regulate heat stress response. Plant Cell. 2021; 33:338-57

[19] Pan C, Ye L, Zheng Y. et al. Identification and expression profiling of microRNAs involved in the stigma exsertion under high-temperature stress in tomato. BMC Genomics. 2017; 18:843

[20] Liu Q, Yang T, Yu T. et al. Integrating small RNA sequencing with QTL mapping for identification of miRNAs and their target genes associated with heat tolerance at the flowering stage in rice. Front Plant Sci. 2017; 8:43

[21] Ding Y, Ma Y, Liu N. et al. microRNAs involved in auxin signalling modulate male sterility under high-temperature stress in cotton (Gossypium hirsutum). Plant J. 2017; 91:977-94

[22] Kumar RR, Pathak H, Sharma SK. et al. Novel and conserved heat-responsive microRNAs in wheat (Triticum aestivum L.). Funct Integr Genomics. 2015; 15:323-48

[23] Stief A, Altmann S, Hoffmann K. et al. Arabidopsis miR156 regulates tolerance to recurring environmental stress through SPL transcription factors. Plant Cell. 2014; 26:1792-807

[24] Wang Y, Sun F, Cao H. et al. TamiR159 directed wheat TaGAMYB cleavage and its involvement in anther development and heat response. PLoS One. 2012; 7:e48445

[25] Zhao J, He Q, Chen G. et al. Regulation of non-coding RNAs in heat stress responses of plants. Front Plant Sci. 2016; 7:1213

[26] Severing E, Faino L, Jamge S. et al. Arabidopsis thaliana ambi-ent temperature responsive lncRNAs. BMC Plant Biol. 2018; 18:145

[27] Zhang P, He R, Yang J. et al. The long non-coding RNA DANA2 pos-itively regulates drought tolerance by recruiting ERF84 to pro-mote JMJ29-mediated histone demethylation. Mol Plant. 2023; 16: 1339-53

[28] Ma H, Yang T, Li Y. et al. The long noncoding RNA MdLNC499 bridges MdWRKY1 and MdERF109 function to regulate early-stage light-induced anthocyanin accumulation in apple fruit. Plant Cell. 2021; 33:3309-30

[29] Yu J, Qiu K, Sun W. et al. A long noncoding RNA functions in high-light-induced anthocyanin accumulation in apple by activating ethylene synthesis. Plant Physiol. 2022; 189:66-83

[30] Gao Z, Li J, Luo M. et al. Characterization and cloning of grape cir-cular RNAs identified the cold resistance-related Vv-circATS1. Plant Physiol. 2019; 180:966-85

[31] Litholdo CG Jr, da Fonseca GC. Circular RNAs and plant stress responses. Adv Exp Med Biol. 2018; 1087:345-53

[32] Franco-Zorrilla JM, Valli A, Todesco M. et al. Target mimicry provides a new mechanism for regulation of microRNA activity. Nat Genet. 2007; 39:1033-7

[33] Su X, Wang B, Geng X. et al. A high-continuity and annotated tomato reference genome. BMC Genomics. 2021; 22:898

[34] Pertea M, Pertea GM, Antonescu CM. et al. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol. 2015; 33:290-5

[35] Li W, Cowley A, Uludag M. et al. The EMBL-EBI bioinformat-ics web and programmatic tools framework. Nucleic Acids Res. 2015;43:W580-4

[36] Memczak S, Jens M, Elefsinioti A. et al. Circular RNAs are a large class of animal RNAs with regulatory potency. Nature. 2013; 495: 333-8

[37] Kalvari I, Nawrocki EP, Ontiveros-Palacios N. et al. Rfam 14: expanded coverage of metagenomic, viral and microRNA fam-ilies. Nucleic Acids Res. 2020;49:D192-200

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

[39] Langfelder P, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics. 2008; 9:559

[40] Chen C, Chen H, Zhang Y. et al. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant. 2020; 13:1194-202

[41] Bailey TL, Elkan C. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc Int Conf Intell Syst Mol Biol. 1994; 2:28-36

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

[43] Palos K, Yu L, Railey CE. et al. Linking discoveries, mechanisms, and technologies to develop a clearer perspective on plant long noncoding RNAs. Plant Cell. 2023; 35:1762-86

[44] Li Z, An X, Zhu T. et al. Discovering and constructing ceRNA-miRNA-target gene regulatory networks during anther develop-ment in maize. Int J Mol Sci. 2019; 20:3480

[45] He X, Guo S, Wang Y. et al. Systematic identification and analysis of heat-stress-responsive lncRNAs, circRNAs and miRNAs with associated co-expression and ceRNA networks in cucumber (Cucumis sativus L.). Physiol Plant. 2020; 168:736-54

[46] Guihur A, Rebeaud ME, Goloubinoff P. How do plants feel the heat and survive. Trends Biochem Sci. 2022; 47:824-38

[47] Chen X, Xue H, Zhu L. et al. ERF49 mediates brassinosteroid regulation of heat stress tolerance in Arabidopsis thaliana. BMC Biol. 2022; 20:254

[48] Han J, Xie X, Zhang Y. et al. Evolution of the dehydration-responsive element-binding protein subfamily in green plants. Plant Physiol. 2022; 190:421-40

[49] Li T, Wu Z, Zhang Y. et al. An AP2/ERF member LlERF012 confers thermotolerance via activation of HSF pathway in lily. Plant Cell Environ. 2024; 47:4702-19

[50] Wang Y, Deng XW, Zhu D. From molecular basics to agronomic benefits: insights into noncoding RNA-mediated gene regulation in plants. J Integr Plant Biol. 2022; 64:2290-308

[51] Zhao M, Tian R, Sun X. et al. lncRNA MtCIR2 positively regulates plant-freezing tolerance by modulating CBF/DREB1 gene clus-ters. Plant Cell Environ. 2023; 46:2450-69

[52] Ramírez Gonzales L, Shi L, Bergonzi SB. et al. Potato CYCLING DOF FACTOR 1 and its lncRNA counterpart StFLORE link tuber development and drought response. Plant J. 2021; 105:855-69

[53] Ye X, Wang S, Zhao X. et al. Role of lncRNAs in cis-and trans-regulatory responses to salt in Populus trichocarpa. Plant J. 2022; 110:978-93

[54] JiaY, ZhaoH, NiuY. et al. Long noncoding RNA from Betula platy-phylla, BplncSIR1, confers salt tolerance by regulating BpNAC2 to mediate reactive oxygen species scavenging and stomatal movement. Plant Biotechnol J. 2024; 22:48-65

[55] Cui C, Wan H, Li Z. et al. Long noncoding RNA TRABA suppresses β-glucosidase-encoding BGLU24 to promote salt tolerance in cotton. Plant Physiol. 2023; 194:1120-38

[56] Kalwa M, Hänzelmann S, Otto S. et al. The lncRNA HOTAIR impacts on mesenchymal stem cells via triple helix formation. Nucleic Acids Res. 2016; 44:10631-43

[57] TaoX, LiM, ZhaoT. et al. Neofunctionalization of a polyploidization-activated cotton long intergenic non-coding RNA DAN1 during drought stress regulation. Plant Physiol. 2021; 186:2152-68

[58] Moison M, Pacheco JM, Lucero L. et al. The lncRNA APOLO interacts with the transcription factor WRKY42 to trigger root hair cell expansion in response to cold. Mol Plant. 2021; 14: 937-48

[59] Sun L, Luo H, Bu D. et al. Utilizing sequence intrinsic composi-tion to classify protein-coding and long non-coding transcripts. Nucleic Acids Res. 2013; 41:e166-6

[60] Kalvari I, Nawrocki EP, Argasinska J. et al. Non-coding RNA analysis using the Rfam database. Curr Protoc Bioinformatics. 2018; 62:e51

[61] Langmead B, Trapnell C, Pop M. et al. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009;10:R25

[62] Friedländer MR, Mackowiak SD, Li N. et al. miRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades. Nucleic Acids Res. 2012; 40:37-52

[63] Ma C, Wang X. Application of the Gini correlation coefficient to infer regulatory relationships in transcriptome analysis. Plant Physiol. 2012; 160:192-203

[64] Ma C, Xin M, Feldmann KA. et al. Machine learning-based differ-ential network analysis: a study of stress-responsive transcrip-tomes in Arabidopsis. Plant Cell. 2014; 26:520-37

[65] Mathelier A, Zhao X, Zhang AW. et al. JASPAR 2014:an extensively expanded and updated open-access database of transcription factor binding profiles. Nucleic Acids Res. 2014;42:D142-7