Transcriptional analysis of flower and thorn development in Citrus

Abstract

Although both thorns and flowers originate from determinate shoot meristems, they develop into morphologically distinct structures with different biological functions. However, the molecular mechanisms underlying their early developmental divergence remain largely unknown. In this study, we performed comparative transcriptomic analysis of young thorns, young flowers, and adjacent stems from citrange trees to investigate the regulatory programs associated with these two determinate shoot types. We identified 480 flower-preferential and 400 thorn-preferential genes. Flower-preferential genes include canonical MADS-box genes involved in flower development (e.g., APETALA3 (CsAP3), PISTILLATA (CsPI), AGAMOUS (CsAG), and SEPALLATA3 (CsSEP3)), the floral meristem identity gene LEAFY (CsLFY), and known regulators of stamen (e.g., DYSFUNCTIONAL TAPETUM 1 (CsDYT1) and ABORTED MICROSPORES (CsAMS)) and fruit development (e.g., CRABS CLAW (CsCRC)). Thorn-preferential genes include the thorn identity genes TI1, TI2, and TI3, along with the thorn tip sclerification regulatory factor SHORT AND SOFT THORN 1 (SST1) and its downstream targets (e.g., NAC secondary wall thickening promoting factor 1 (CsNST1) and secondary wall-associated NAC domain protein 1 (CsSND1)). Surprisingly, the A-class flower development genes CsAP1 and CsAP2.1 were preferentially expressed in young thorns, and several MADS-box genes, including SHORT VEGETATIVE PHASE (SVP) homologs and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1)-like genes, also exhibited thorn-preferential expression. Functional analysis using CRISPR-Cas9-mediated knockout demonstrated that loss of CsAP1 resulted in significantly shortened thorns, indicating a role for this floral identity gene in thorn growth. Collectively, our findings provide new insights into the developmental programs of thorns and flowers and offer candidate genes for targeted modification of these shoot types in Citrus.

Keywords

Citrus / Thorn / Flower / Determinate growth / MADS-box

References

[1] Appleton AD, Kramer EM. Diversifying floral organ identity. Curr Opin Plant Biol, 2024, 80. 102550

[2] Azeez A, Miskolczi P, Tylewicz S, Bhalerao RP. A tree ortholog of APETALA1 mediates photoperiodic control of seasonal growth. Curr Biol, 2014, 24: 717-724.

[3] Bowman JL, Moyroud E. Reflections on the ABC model of flower development. Plant Cell, 2024, 36: 1334-1357.

[4] Bradley D, Ratcliffe O, Vincent C, Carpenter R, Coen E. Inflorescence commitment and architecture in Arabidopsis. Science, 1997, 275: 80-83.

[5] Carles CC, Ha CM, Jun JH, Fiume E, Fletcher JC. Analyzing shoot apical meristem development. Methods Mol Biol, 2010, 655: 105-129.

[6] Chen S. Ultrafast one-pass FASTQ data preprocessing, quality control, and deduplication using fastp. iMeta, 2023, 2. e107

[7] Chen C, Wu Y, Li J, Wang X, Zeng Z, Xu J, et al. TBtools-II: a "one for all, all for one" bioinformatics platform for biological big-data mining. Mol Plant, 2023, 16: 1733-1742.

[8] Gu Z. Complex Heatmap Visualization Imeta, 2022, 1. e43

[9] Hu T, Li X, Du L, Manuela D, Xu M. LEAFY and APETALA1 down-regulate ZINC FINGER PROTEIN 1 and 8 to release their repression on class B and C floral homeotic genes. Proc Natl Acad Sci U S A, 2023, 120. e2221181120

[10] Huang X, Effgen S, Meyer RC, Theres K, Koornneef M. Epistatic natural allelic variation reveals a function of AGAMOUS-LIKE6 in axillary bud formation in Arabidopsis. Plant Cell, 2012, 24: 2364-2379.

[11] Huang T, Hodgens C, Prakash S, Marconi M, Wabnik K, Sozzani R, et al. A negative feedback loop between TERMINAL FLOWER1 and LEAFY protects inflorescence indeterminacy. Science, 2026, 391. eadv5429

[12] Irish VF. The flowering of Arabidopsis flower development. Plant J, 2010, 61: 1014-1028.

[13] Jin R, Klasfeld S, Zhu Y, Fernandez Garcia M, Xiao J, Han SK, et al. LEAFY is a pioneer transcription factor and licenses cell reprogramming to floral fate. Nat Commun, 2021, 12: 626.

[14] Khojayori FN, Ponraj U, Buch K, Zhao Y, Herrera-Ubaldo H, Glover BJ. Evolution and development of complex floral displays. Development, 2024, 151. dev203027

[15] Kim D, Paggi JM, Park C, Bennett C, Salzberg SL. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol, 2019, 37: 907-915.

[16] Lai X, Blanc-Mathieu R, GrandVuillemin L, Huang Y, Stigliani A, Lucas J, et al. The LEAFY floral regulator displays pioneer transcription factor properties. Mol Plant, 2021, 14: 829-837.

[17] Lin C-YI, Irish VF. Meristem fate: to terminate, or not?. Front Plant Sci, 2026, 16. 1739587

[18] Liu C, Zhou J, Bracha-Drori K, Yalovsky S, Ito T, Yu H. Specification of Arabidopsis floral meristem identity by repression of flowering time genes. Development, 2007, 134: 1901-1910.

[19] Liu C, Xi W, Shen L, Tan C, Yu H. Regulation of floral patterning by flowering time genes. Dev Cell, 2009, 16: 711-722.

[20] Liu X, Huang J, Parameswaran S, Ito T, Seubert B, Auer M, et al. The SPOROCYTELESS/NOZZLE gene is involved in controlling stamen identity in Arabidopsis. Plant Physiol, 2009, 151: 1401-1411.

[21] Liu X, Kim YJ, Müller R, Yumul RE, Liu C, Pan Y, et al. AGAMOUS terminates floral stem cell maintenance in Arabidopsis by directly repressing WUSCHEL through recruitment of Polycomb Group proteins. Plant Cell, 2011, 23: 3654-3670.

[22] Liu Q, Wang C, Jiao X, Zhang H, Song L, Li Y, et al. Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR/Cas systems. Sci China Life Sci, 2019, 62: 1-7.

[23] Liu H, Wang X, Liu S, Huang Y, Guo YX, Xie WZ, et al. Citrus Pan-Genome to Breeding Database (CPBD): A comprehensive genome database for citrus breeding. Mol Plant, 2022, 15: 1503-1505.

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

[25] Machida C, Nakagawa A, Kojima S, Takahashi H, Machida Y. The complex of ASYMMETRIC LEAVES (AS) proteins plays a central role in antagonistic interactions of genes for leaf polarity specification in Arabidopsis. Wires Dev Biol, 2015, 4: 655-671.

[26] Mateos JL, Madrigal P, Tsuda K, Rawat V, Richter R, Romera-Branchat M, et al. Combinatorial activities of SHORT VEGETATIVE PHASE and FLOWERING LOCUS C define distinct modes of flowering regulation in Arabidopsis. Genome Biol, 2015, 16. 31

[27] Maurya JP, Miskolczi PC, Mishra S, Singh RK, Bhalerao RP. A genetic framework for regulation and seasonal adaptation of shoot architecture in hybrid aspen. Proc Natl Acad Sci U S A, 2020, 117: 11523-11530.

[28] Orbović V, Ravanfar SA, Acanda Y, Narvaez J, Merritt BA, Levy A, et al. Stress-inducible Arabidopsis thaliana RD29A promoter constitutively drives Citrus sinensis APETALA1 and LEAFY expression and precocious flowering in transgenic Citrus spp. Transgenic Res, 2021, 30: 687-699.

[29] Peña L, Martín-Trillo M, Juárez J, Pina JA, Navarro L, Martínez-Zapater JM. Constitutive expression of Arabidopsis LEAFY or APETALA1 genes in citrus reduces their generation time. Nat Biotechnol, 2001, 19: 263-267.

[30] Pertea M, Kim D, Pertea GM, Leek JT, Salzberg SL. Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. Nat Protoc, 2016, 11: 1650-1667.

[31] Ren J, Duan Y, Li R, Zhang X, Shi Y, Zhou S, et al. Transcriptional regulation of thorn tip sclerification in plants. Proc Natl Acad Sci U S A, 2025, 122. e2510775122

[32] Siegfried KR, Eshed Y, Baum SF, Otsuga D, Drews GN, Bowman JL. Members of the YABBY gene family specify abaxial cell fate in Arabidopsis. Development, 1999, 126: 4117-4128.

[33] Sun B, Xu Y, Ng KH, Ito T. A timing mechanism for stem cell maintenance and differentiation in the Arabidopsis floral meristem. Genes Dev, 2009, 23: 1791-1804.

[34] Sun B, Looi LS, Guo S, He Z, Gan ES, Huang J, et al. Timing mechanism dependent on cell division is invoked by Polycomb eviction in plant stem cells. Science, 2014, 343. 1248559

[35] Wang J, Liao X, Wu Z, Sane S, Han S, Chen Q, et al. Genetic control of seasonal meristem arrest in trees. Proc Natl Acad Sci U S A, 2025, 122. e2505641122

[36] Yu YD, Duan YY, Zhao RY, Zhang F. A single TCP transcription factor modulates leaf size in Citrus. Hortic Plant J, 2026.

[37] Zhang F, LeBlanc C, Irish VF, Jacob Y. Rapid and efficient CRISPR/Cas9 gene editing in Citrus using the YAO promoter. Plant Cell Rep, 2017, 36: 1883-1887.

[38] Zhang F, Rossignol P, Huang T, Wang Y, May A, Dupont C, et al. Reprogramming of stem cell activity to convert thorns into branches. Curr Biol, 2020, 30: 2951-2961.

[39] Zhang F, Wang Y, Irish VF. Centroradialis maintains shoot meristem indeterminacy by antagonizing thorn identity1 in Citrus. Curr Biol, 2021, 31: 2237-2242.

[40] Zheng Y, Zhang K, Guo L, Liu X, Zhang Z. Auxin response factor3 plays distinct role during early flower development. Plant Signal Behav, 2018, 13. e1467690

[41] Zheng Q, Zhou S, Irish VF, Zhang F. Thorn specification in Citrus plants by an SHI/STY family transcription factor. Curr Biol, 2026, 36: 888-901.e4.

Funding

National Key Research and Development Program of China(2022YFF1003100)

the National Natural Science Foundation of China(32172541)

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