Combination of 3D chromatin architecture and omics analysis provides insight into anthocyanin regulation in Actinidia arguta

Abstract

Actinidia arguta has become popular with consumers recently because of its edible and colorful fruit skin. The 3D spatial organization of its genome plays a key role in the formation of various biological traits. However, the function of 3D genome reorganization during fruit skin color formation is poorly understood in A. arguta. In this study we constructed the 3D genome of the red-skinned A. arguta cultivar ‘Zhonghongbei’ (ZHB) and the green-skinned cultivar ‘Zhonglvbei’ (ZLB), and performed chromatin structure comparisons between them at compartment, topologically associating domain (TAD), and loop levels. Global compartment comparisons at whole 3D genome level between red-skinned and green-skinned A. arguta showed that A-B compartment transition specifically occurred in chromosome 7 and chromosome 16, based on which all genes within 3 Mb upstream and downstream of A-B compartment transition were retrieved to construct a four-way Venn diagram, which showed that AaCBP60B-like, encoding calmodulin-binding protein 60 B-like, is the key candidate gene negatively correlating with fruit color. Exogenous calcium chloride treatments enhancing AaCBP60B-like expression to repress anthocyanin biosynthesis proved a negative role of AaCBP60B-like in anthocyanin biosynthesis. Overexpression and virus-induced gene silencing assays of AaCBP60B-like revealed the inhibition of anthocyanin biosynthesis derived from differential expression of AaCBP60B-like resulting from a 346-bp InDel variation located at the AaCBP60B-like promoter resulting in activity differences in red-and green-skinned A. arguta. ATAC-seq results proved that the 346-bp InDel variation affects 3D genome organization. Our study provides the first 3D chromosome organization in red-and green-skinned A. arguta, based on which a candidate gene, AaCBP60B-like, involved in anthocyanin regulation is identified.

Acknowledgments

This work was supported by grants from the Agricultural Science and Technology Innovation Program (CAAS-ASTIP-ZFRI-03-202502), National Key Research and Development Program of China (2022YFD1200503), National Natural Science Foundation of China (32202435), China Agriculture Research System of MOF and MARA (CARS-26), National Key Research and Development Program of China (2022YFD1600700), Major Science and Technology Projects of Henan Province (221100110400), Technical System of Bulk Fruit Industry in Henan Province (HARS-22-09-S), Agricultural Science and Technology Innovation Program, and Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2023-ZFRI-03).

Data availability

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

Conflict of interest

The authors declare no competing interests.

Supplementary data

Supplementary data are available at Horticulture Research online.

References

[1] Li Y, Fang J, Qi X. et al. A key structural gene, AaLDOX, is involved in anthocyanin biosynthesis in all red-fleshed kiwifruit (Actini-dia arguta) based on transcriptome analysis. Gene. 2018; 648: 31-41

[2] Wang WQ, Moss SMA, Zeng L. et al. The red flesh of kiwifruit is differentially controlled by specific activation-repression sys-tems. New Phytol. 2022; 235:630-45

[3] LiY, CuiW, QiX. et al. MicroRNA858 negatively regulates anthocyanin biosynthesis by repressing AaMYBC1 expression in kiwifruit (Actinidia arguta). Plant Sci. 2020; 296:110476

[4] Peng Y, Thrimawithana AH, Cooney JM. et al. The proanthocyanin-related transcription factors MYBC1 and WRKY44 regulate branch points in the kiwifruit anthocyanin pathway. Sci Rep. 2020; 10:14161

[5] Liu Y, Lv G, Yang Y. et al. Interaction of AcMADS68 with tran-scription factors regulates anthocyanin biosynthesis in red-fleshed kiwifruit. Hortic Res. 2022;10:uhac252

[6] Li W, Ding Z, Ruan M. et al. Kiwifruit R2R3-MYB transcription factors and contribution of the novel AcMYB75 to red kiwifruit anthocyanin biosynthesis. Sci Rep. 2017; 7:16861

[7] Liu Y, Zhou B, Qi Y. et al. Expression differences of pigment struc-tural genes and transcription factors explain flesh coloration in three contrasting kiwifruit cultivars. Front Plant Sci. 2017; 8:1507

[8] Wang L, Tang W, Hu Y. et al. A MYB/bHLH complex regulates tissue-specific anthocyanin biosynthesis in the inner pericarp of red-centered kiwifruit Actinidia chinensis cv. Hongyang Plant J. 2019; 99:359-78

[9] Bonev B, Cavalli G. Organization and function of the 3D genome. Nat Rev Genet. 2016; 17:661-78

[10] Davies JO, Oudelaar AM, Higgs DR. et al. How best to identify chromosomal interactions: a comparison of approaches. Nat Methods. 2017; 14:125-34

[11] de Laat W, Duboule D. Topology of mammalian developmental enhancers and their regulatory landscapes. Nature. 2013; 502: 499-506

[12] Dekker J, Mirny L. The 3D genome as moderator of chromosomal communication. Cell. 2016; 164:1110-21

[13] Lieberman-Aiden E, van Berkum NL, Williams L. et al. Com-prehensive mapping of long-range interactions reveals folding principles of the human genome. Science. 2009; 326:289-93

[14] Rowley MJ, Corces VG. Organizational principles of 3D genome architecture. Nat Rev Genet. 2018; 19:789-800

[15] Zhao L, Wang S, Cao Z. et al. Chromatin loops associated with active genes and heterochromatin shape rice genome architec-ture for transcriptional regulation. Nat Commun. 2019; 10:3640

[16] Li E, Liu H, Huang L. et al. Long-range interactions between proximal and distal regulatory regions in maize. Nat Commun. 2019; 10:2633

[17] Ni L, Liu Y, Ma X. et al. Pan-3D genome analysis reveals structural and functional differentiation of soybean genomes. Genome Biol. 2023; 24:12

[18] Kong W, Yu J, Yang J. et al. The high-resolution three-dimensional (3D) chromatin map of the tea plant (Camellia sinensis). Hortic Res. 2023;10:uhad179

[19] Zhang L, Zhao R, Liang J. et al. BL-Hi-C reveals the 3D genome structure of Brassica crops with high sensitivity. Hortic Res. 2024; 11:uhae017

[20] Mascher M, Gundlach H, Himmelbach A. et al. A chromosome conformation capture ordered sequence of the barley genome. Nature. 2017; 544:427-33

[21] Wang M, Wang P, Lin M. et al. Evolutionary dynamics of 3D genome architecture following polyploidization in cotton. Nat Plants. 2018; 4:90-7

[22] Xie T, Zhang FG, Zhang HY. et al. Biased gene retention during diploidization in Brassica linked to three-dimensional genome organization. Nat Plants. 2019; 5:822-32

[23] Liang Z, Zhang Q, Ji C. et al. Reorganization of the 3D chro-matin architecture of rice genomes during heat stress. BMC Biol. 2021; 19:53

[24] Li Y, Song Z, Zhan X. et al. Chromosome-level genome assembly assisting for dissecting mechanism of anthocyanin regulation in kiwifruit (Actinidia arguta). Mol Hortic. 2025; 5:18

[25] Ursu O, Boley N, Taranova M. et al. GenomeDISCO: a concordance score for chromosome conformation capture experiments using random walks on contact map graphs. Bioinformatics. 2018; 34: 2701-7

[26] Liu C, Cheng YJ, Wang JW. et al. Prominent topologically asso-ciated domains differentiate global chromatin packing in rice from Arabidopsis. Nat Plants. 2017; 3:742-8

[27] Zhang F, Wang Y, Lin Y. et al. Haplotype-resolved genome assem-bly provides insights into evolutionary history of the Actinidia arguta tetraploid. Mol Hortic. 2024; 4:4

[28] Zou B, Wan D, Li R. et al. Calmodulin-binding protein CBP60g functions as a negative regulator in Arabidopsis anthocyanin accumulation. PLoS One. 2017; 12:e0173129

[29] Lu XM, Yu XF, Li GQ. et al. Genome assembly of autote-traploid Actinidia arguta highlights adaptive evolution and enables dissection of important economic traits. Plant Commun. 2024; 5:100856

[30] Feng S, Cokus SJ, Schubert V. et al. Genome-wide Hi-C analyses in wild-type and mutants reveal high-resolution chromatin inter-actions in Arabidopsis. Mol Cell. 2014; 55:694-707

[31] Ding P, Redkar A. Pathogens suppress host transcription factors for rampant proliferation. Trends Plant Sci. 2018; 23: 950-3

[32] Li LS, Yang YY, Chen YX. et al. CBP60b clade proteins are proto-typical transcription factors mediating immunity. Plant Physiol. 2024; 196:1489-501

[33] Li LS, Ying J, Li E. et al. Arabidopsis CBP60b is a central transcriptional activator of immunity. Plant Physiol. 2021; 186: 1645-59

[34] Zhao L, Liu YD, Chen XM. et al. Visible light induces the PpHYH transcription to promote anthocyanin pigmentation in peach peel. Fruit. Res. 2023; 3:5

[35] Zheng X, Zheng Y. CscoreTool: fast Hi-C compartment analysis at high resolution. Bioinformatics. 2018; 34:1568-70

[36] Crane E, Bian Q, McCord RP. et al. Condensin-driven remodelling of X chromosome topology during dosage compensation. Nature. 2015; 523:240-4

[37] Dixon JR, Selvaraj S, Yue F. et al. Topological domains in mam-malian genomes identified by analysis of chromatin interac-tions. Nature. 2012; 485:376-80

[38] Lupiáñez DG, Kraft K, Heinrich V. et al. Disruptions of topo-logical chromatin domains cause pathogenic rewiring of gene-enhancer interactions. Cell. 2015; 161:1012-25

[39] Flyamer IM, Illingworth RS, Bickmore WA. Coolpup.Py: ver-satile pile-up analysis of Hi-C data. Bioinformatics. 2020; 36: 2980-5

[40] Hsieh TS, Cattoglio C, Slobodyanyuk E. et al. Enhancer-promoter interactions and transcription are largely maintained upon acute loss of CTCF, cohesin, WAPL or YY1. Nat Genet. 2022; 54: 1919-32

[41] Yang H, Luan Y, Liu T. et al. A map of cis-regulatory ele-ments and 3D genome structures in zebrafish. Nature. 2020; 588: 337-43

[42] Emms DM, Kelly S. OrthoFinder: phylogenetic orthology infer-ence for comparative genomics. Genome Biol. 2019; 20:238

[43] Goel M, Sun H, Jiao WB. et al. SyRI: finding genomic rear-rangements and local sequence differences from whole-genome assemblies. Genome Biol. 2019; 20:277

[44] Wang K, Li M, Hakonarson H. ANNOVAR: functional annota-tion of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 2010; 38:e164

[45] 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

[46] Tannenbaum M, Sarusi-Portuguez A, Krispil R. et al. Regula-tory chromatin landscape in Arabidopsis thaliana roots uncov-ered by coupling INTACT and ATAC-seq. Plant Methods. 2018; 14:1-12

[47] Danecek P, Bonfield JK, Liddle J. et al. Twelve years of SAMtools and BCFtools. Gigascience. 2021;10:giab008

[48] Ramírez F, Ryan DP, Grüning B. et al. deepTools2: a next genera-tion web server for deep-sequencing data analysis. Nucleic Acids Res. 2016;44:W160-5

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

[50] Hellens RP, Allan AC, Friel EN. et al. Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods. 2005; 1:1-14

[51] Gao Y, Wei W, Fan Z. et al. Re-evaluation of the nor mutation and the role of the NAC-NOR transcription factor in tomato fruit ripening. JExp Bot. 2020; 71:3560-74

[52] Hu J, Xu Q, Liu C. et al. Downregulated expression of S2-RNase attenuates self-incompatibility in “Guiyou No. 1” pummelo. Hor-tic Res. 2021; 8:199

[53] Bustin SA, Benes V, Garson JA. et al. The MIQE guidelines: mini-mum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009; 55:611-22