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Uncovering the transcriptional regulatory network involved in boosting wheat regeneration and transformation

Nature Plants volume 9, pages 908–925 (2023)Cite this article

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An Author Correction to this article was published on 05 January 2024

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Abstract

Genetic transformation is important for gene functional study and crop improvement. However, it is less effective in wheat. Here we employed a multi-omic analysis strategy to uncover the transcriptional regulatory network (TRN) responsible for wheat regeneration. RNA-seq, ATAC-seq and CUT&Tag techniques were utilized to profile the transcriptional and chromatin dynamics during early regeneration from the scutellum of immature embryos in the wheat variety Fielder. Our results demonstrate that the sequential expression of genes mediating cell fate transition during regeneration is induced by auxin, in coordination with changes in chromatin accessibility, H3K27me3 and H3K4me3 status. The built-up TRN driving wheat regeneration was found to be dominated by 446 key transcription factors (TFs). Further comparisons between wheat and Arabidopsis revealed distinct patterns of DNA binding with one finger (DOF) TFs in the two species. Experimental validations highlighted TaDOF5.6 (TraesCS6A02G274000) and TaDOF3.4 (TraesCS2B02G592600) as potential enhancers of transformation efficiency in different wheat varieties.

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Fig. 1: A transcriptional and epigenomic atlas of wheat regeneration.
Fig. 2: Chromatin dynamics coordinate transcription profile during wheat regeneration.
Fig. 3: Auxin signalling promotes wheat regeneration.
Fig. 4: TRN governing wheat regeneration.
Fig. 5: Comparative analysis between wheat and Arabidopsis.
Fig. 6: DOFs promote wheat regeneration.
Fig. 7: Working model for epigenetic-associated transcriptional regulation of wheat regeneration.

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Data availability

The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive80 of the National Genomics Data Center81, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA008502 and CRA010204) and are publicly accessible at https://ngdc.cncb.ac.cn/gsa. Quantitative results of RNA-seq, ATAC-seq and CUT&Tag data have been uploaded to the Figshare database (https://doi.org/10.6084/m9.figshare.21378990).

Code availability

Code used for all processing and analysis is available in GitHub (https://github.com/xmliu01/Uncovering-the-TRN-involved-in-boosting-wheat-regeneration-and-transformation).

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Acknowledgements

This research was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA24010204 to J.X.), the National Natural Sciences Foundation of China (31730008 to X.S.Z.), the National Key Research and Development Program of China (2021YFD1201500 to J.X.) and the Major Basic Research Program of Shandong Natural Science Foundation (ZR2021ZD31 to X.S.Z.).

Author information

Author notes

  1. These authors contributed equally: Xuemei Liu, Xiao Min Bie, Xuelei Lin.

Authors and Affiliations

  1. Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    Xuemei Liu, Xuelei Lin, Hongzhe Wang, Xiaoyu Zhang, Yiman Yang & Jun Xiao

  2. National Key Laboratory of Wheat Improvement, College of Life Sciences, Shandong Agricultural University, Tai’an, China

    Xiao Min Bie, Menglu Li, Chunyan Zhang & Xian Sheng Zhang

  3. University of Chinese Academy of Sciences, Beijing, China

    Xiaoyu Zhang & Jun Xiao

  4. Nanjing Agricultural University, Nanjing, China

    Yiman Yang

  5. CAS-JIC Centre of Excellence for Plant and Microbial Science (CEPAMS), Institute of Genetics and Developmental Biology, CAS, Beijing, China

    Jun Xiao

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Contributions

J.X., X.S.Z. and X. Liu designed and supervised the research and wrote the manuscript. X.M.B. did the sample collection and in situ hybridization; M.L. performed plasmid construction and RT–qPCR. X.M.B. and C.Z. conducted wheat transformation; X. Lin and X.M.B. performed CUT&Tag, ATAC-seq and RNA-seq experiments; H.W. and X.Z. conducted the reporter assay; X. Liu performed data analysis. X. Liu, X.M.B., Y.Y. and J.X. prepared all the figures. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Xian Sheng Zhang or Jun Xiao.

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Extended data

Extended Data Fig. 1 Overview of epigenome data of wheat regeneration.

a, Principal component analysis of H3K27me3, H3K4me3 and H3K27ac. b, Pearson correlation analysis of all epigenomic data. PCC: Pearson correlation coefficient. c, Epigenetic profiles on gene sets with different expression levels (data from DAI 6 stage); No, Low, Middle and High represent the level of gene expression; TSS: transcription start site, TES: transcription end site. d, Number and proportion of differentially marked peaks by H3K27me3, H3K27ac and H3K4me3 among different induction stages.

Extended Data Fig. 2 Chromatin dynamic during wheat regeneration.

a, Transcription and epigenetic modification tracks for TaLEC2_3D, TaWOX11_2A TaE2Fa_6A, TaBBM_3A, TaLBD17_4B and TaWOX5_3D. Gene expression data shown as mean values +/− s.d. of 3 replicates. b, Upset plot shows DEGs regulated by chromatin accessibility, H3K27me3 and H3K4me3 in C3 and C4. c, Heatmap shows transcriptional, chromatin accessibility, H3K27me3 and H3K4me3 dynamic of 1,116 DEGs. ## represent DEGs that is simultaneously regulated by chromatin accessibility, H3K27me3 and H3K4me3. d, Heatmap shows transcriptional, chromatin accessibility, and H3K4me3 dynamic of 4,254 DEGs. ### represent DEGs that is regulated by both chromatin accessibility and H3K4me3. e, GO enrichment analysis of 4,254 DEGs. FE: fold enrichment.

Extended Data Fig. 3 Comprehensive analysis of ARF target genes.

a, Methods to identify ARF and type-B ARR target genes. b, GO enrichment analysis for ARFs target genes. c, Dynamic of chromatin accessibility and histone modification (H3K27me3, H3K27ac, H3K4me3) near AuxRE. The random background consists of randomly selected regions from other TF binding sites.

Extended Data Fig. 4 Construction of TRN of wheat regeneration.

a, Pipeline for TRN construction. b, Location distribution of footprint within the peak of ATAC-seq (data from DAI 6 stage). c, Distribution of the width of TF footprint (data from DAI 6 stage). d, Numbers of footprint at different stages. e, Sequence conservation analysis at footprint sites; The background is randomly selected intergenic regions outside the open chromatin and coding regions; All regions are truncated to 25 bp. f, Protection scores dynamic of differential footprint; Protection score reflects the chromatin accessibility of footprint. g, Overview of the TRN of wheat regeneration. h, Two transcriptional regulatory modes during wheat regeneration. i, A network of functional related TFs in regulatory mode Type I. j, A network of functional related TFs in regulatory mode Type II.

Extended Data Fig. 5 The network of TaBBM and TaWOX5.

a, GO enrichment analysis of TaBBM’s target genes. b, Enriched TF family of TaBBM’s target genes. Front size shows degree of enrichment (–Log10(p.adj)). c, The network of TaBBM. d, GO enrichment analysis of TaWOX5’s target genes. e, Enriched TF family of TaWOX5’s target genes. Front size shows degree of enrichment (–Log10(p.adj)). f, Network of TaWOX5. g, Intersection of TaWOX5 target genes and DEGs caused by TaWOX5 transformation (two sided Fisher’s exact test). NET represents the target genes of TaWOX5 in the TRN. DEG represents the DEGs of TaWOX5 transformation compared GUS transformation at DAI 6 and DAI 9. h, The expression patterns of TaREV and TaSOUL-1 of transformation of GUS and TaWOX5 at DAI 6 and DAI 9 (two sided Student’s t-test). Data shown as mean values +/− s.d. of 3 replicates.

Extended Data Fig. 6 Differences in transcription and chromatin accessibility between Fielder and JM22.

a, Heatmap of 446 TFs in Fielder and JM22. Similar and Distinct refer to the similar or distinct transcription patterns between Fielder and JM22. b, Transcription and chromatin accessibility tracks for TaBBM_3A, TaDOF3.4_2B TaREV_4B and TaSCR_4B. Gene expression data shown as mean values + /- s.d. of 3 replicates. c, Transcription and chromatin accessibility tracks for WOX in Fielder and JM22. Dotted lines indicate significant SNPs associated with callus differentiation rate. Gene expression data shown as mean values +/− s.d. of 3 replicates. d, Callus differentiation rates of two haplotypes divided by three SNPs in Extended Data Fig. 6c (one sided Student’s t-test). Boxplot: median (horizontal line), quartiles (top and bottom boundaries), whiskers (minimum and maximum values excluding outliers), outliers (individual points).

Extended Data Fig. 7 Comparative analysis between wheat and Arabidopsis.

a, Principal component analysis of RNA-seq and ATAC-seq dataset during wheat and Arabidopsis regeneration. b, The expression pattern of AtWOX11, AtWOX12, AtWOX5 and AtWOX7 during Arabidopsis regeneration. Gene expression data shown as mean values +/− s.d. of 3 replicates. c, Motif activity dynamic during regeneration in wheat and Arabidopsis; Motif activity represents the chromatin accessibility at the TF binding sites. d, K-means clustering analysis of DEGs in Arabidopsis. e, TF family enrichment analysis of genes with similar expression pattern in C1. f, The footprint of DOF5.6 in Arabidopsis at different induction stages. g, TF family enrichment analysis of genes in Arabidopsis cluster A3. h, Motif activity dynamic of LBDs in wheat and Arabidopsis; Activity score reflects the chromatin accessibility.

Extended Data Fig. 8 Expression and potential targets of DOFs during regeneration.

a, The expression heatmap of DOF TFs during wheat regeneration. The orthologs in Arabidopsis are shown. b, ATAC-seq footprint of DOF3.4 at different induction stages. c, The functional related target genes of TaDOF5.6 in TRN. d, The functional related target genes of TaDOF3.4 in TRN.

Extended Data Fig. 9 Normal growth of TaDOF5.6 and TaDOF3.4 transgenic wheat plants.

a, The growth status of TaDOF5.6 and TaDOF3.4 transgenic T0 plants with healthy shoots and roots. b, Seeds produced by overexpressing TaDOF5.6 and TaDOF3.4 transgenic T0 plants. c, The TaDOF5.6 and TaDOF3.4 transgenic T1 plants growth normal and fertile. d, Phenotypic statistics of TaDOF5.6 and TaDOF3.4 transgenic plants. Tiller number and flowering time were counted from transgenic T1 plants. Grain length and grain width were measured from transgenic T0 plants (two sided Student’s t-test).

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Liu, X., Bie, X.M., Lin, X. et al. Uncovering the transcriptional regulatory network involved in boosting wheat regeneration and transformation. Nat. Plants 9, 908–925 (2023). https://doi.org/10.1038/s41477-023-01406-z

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