Transcriptional regulatory framework for vascular cambium development in Arabidopsis roots

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Abstract

Vascular cambium, a lateral plant meristem, is a central producer of woody biomass. Although a few transcription factors have been shown to regulate cambial activity1, the phenotypes of the corresponding loss-of-function mutants are relatively modest, highlighting our limited understanding of the underlying transcriptional regulation. Here, we use cambium cell-specific transcript profiling followed by a combination of transcription factor network and genetic analyses to identify 62 new transcription factor genotypes displaying an array of cambial phenotypes. This approach culminated in virtual loss of cambial activity when both WUSCHEL-RELATED HOMEOBOX 4 (WOX4) and KNOTTED-like from Arabidopsis thaliana 1 (KNAT1; also known as BREVIPEDICELLUS) were mutated, thereby unlocking the genetic redundancy in the regulation of cambium development. We also identified transcription factors with dual functions in cambial cell proliferation and xylem differentiation, including WOX4, SHORT VEGETATIVE PHASE (SVP) and PETAL LOSS (PTL). Using the transcription factor network information, we combined overexpression of the cambial activator WOX4 and removal of the putative inhibitor PTL to engineer Arabidopsis for enhanced radial growth. This line also showed ectopic cambial activity, thus further highlighting the central roles of WOX4 and PTL in cambium development.

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Fig. 1: Identification and validation of cambium TFs during radial growth in Arabidopsis roots.
Fig. 2: A transcriptional network based on inducible overexpression analysis largely predicts the severity of cambial loss-of-function mutants.
Fig. 3: Among the TFs studied, WOX4 and KNAT1 emerged as major regulators of radial growth.
Fig. 4: Activation of positive transcriptional regulators and removal of negative regulator synergistically stimulate cambial activity.

Data availability

Gene accession numbers are as follows: ARR15, AT1G74890; CYCD3;1, AT4G34160; ANT, AT4G37750; WOX14, AT1G20700; KNAT1 (also known as BP), AT4G08150; PTL, AT5G03680; SVP, AT2G22540; AGL24, AT4G24540; LBD4, AT1G31320; SCL7, AT3G50650; AGL14, AT4G11880; ATHB53, AT5G66700; ATHB5, AT5G65310; WRI3, AT1G16060; AHL11, AT3G61310; MYB47, AT1G18710; MYB95, AT1G74430; MYB87, AT4G37780; STY2, AT4G36260; RAS1, AT1G09950; AtERF019, AT1G22810; AtERF021, AT1G71450; AtERF029, AT4G25490; AtERF032, AT1G63030; AtERF043, AT4G32800; AtERF071, AT2G47520; AtERF072, AT3G16770; WOX4, AT1G46480; WOX13, AT4G35550; STM, AT1G62360; KNAT2, AT1G70510; KNAT6, AT1G23380; BOP1, AT3G57130; BOP2, AT2G41370; EDA31, AT3G10000; LBD3 (also known as ASL9), AT1G16530; LBD1, AT1G07900; SCL4, AT5G66770; SCL3, AT1G50420; SCL28, AT1G63100; ANAC015 (also known as BRN1), AT1G33280; ANAC070 (also known as BRN2), AT4G10350; SMB, AT1G79580; ANAC042 (also known as JUNGBRUNNEN 1), AT2G43000; ANAC042-like, AT3G12910; MYB3R4, AT5G11510; MYB3R1, AT4G32730; PXY (also known as TDR), AT5G61480; ERECTA/ER, AT2G26330. Raw data of Affymetrix ATH1 GeneChip (accession no. GSE125244) and RNA-seq data can be found in NCBI (accession no. PRJNA523600). Source data related to LithoGraphX analysis (Fig. 3c,d; Supplementary Fig. 11) and ANOVA analysis (Fig. 4d; Supplementary Figs. 8e and 11–13) can be found in Supplementary Datasets. The data that support the findings of this study are available from the corresponding authors upon request. Mutants generated in this study will be deposited to NASC.

Code availability

The code used for network construction and LithoGraphX analysis can be accessed at Github (https://github.com/Zhangcambium2019/Zhang2019). R-codes for boxplot, half-violin plot, median calculation, average calculation and P value calculation can be accessed at Github.

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Acknowledgements

We thank O. Smetana for providing the root cross-section sketches for making vector images in Fig. 1a; S. Miyashima for providing the pBI-nlsYFP-GUS vector for the cloning of pLBD4::nYFP-GUS; H. Fukuda, J. Murray, D. Smyth, U. Fischer, H. Yu, T. Mizuno, J. Lim, B. Scheres, M. Ito, S. Hepworth, V. Pautot, M. Kater and S. Turner for providing published seeds (Supplementary Table 4a,b); K. Kainulainen and M. Herpola for technical assistance; O. Smetana and L.-L. Ye for providing help on finalizing the figures and S. El-Showk for language correction. This work was supported by the Finnish Centre of Excellence in Molecular Biology of Primary Producers (Academy of Finland CoE programme 2014–2019, decision no. 271832), the Gatsby Foundation (no. GAT3395/PR3), the University of Helsinki (award no. 799992091) and the European Research Council Advanced Investigator Grant SYMDEV (no. 323052 to Y.H.); Academy of Finland (grants nos. 132376, 266431 and 271832), University of Helsinki HiLIFE fellowship to A.P.M. and National Research Foundation of Korea (nos. 2018R1A5A1023599 and 2016R1A2B2015258 to J.-Y.L.).

Author information

J.Z., J.-Y.L., A.P.M. and Y.H. designed the experiments. J.Z. carried out most of the experiments with help from other co-authors. K.N. constructed the pARR15::GFP line for FACS. K.N., A.E. and J.-Y.L. performed FACS and microarray hybridization. J.-Y.L. and J.-G.J. analysed the microarray data from the FACS. J.Z. generated and analysed all transgenic lines with input from A.E. on GUS reporter lines. W.Y. and J.Z. carried out the RNA in situ analysis. M.K. conducted the qRT–PCR and analysed the data for network construction. S.E.A. constructed the network and carried out perturbation analysis. J.Z. and G.E. generated combinatorial mutants. J.-Y.Y. and J.-H.L. produced the asl9 CRISPR mutant. J.Z., G.E., J.X. and T.D. performed mutant genotyping. J.Z., with assistance from J.X., G.E. and J.A.-S., characterized the mutant and overexpression line phenotypes. J.Z., G.E. and J.A.-S. conducted quantification and statistical analyses for phenotypic examination of mutants and overexpression lines. J.A.-S. ran the LithoGraphX analysis with input from G.E., L.R. and P.B.R. J.Z. performed the RNA-seq experiment and analysed the RNA-seq data with S.E.A. G.E. drew all box plot figures. J.Z., J.-Y.L., A.P.M. and Y.H. wrote the manuscript with help from the co-authors. All authors read and commented on the manuscript.

Correspondence to Ji-Young Lee or Ari Pekka Mähönen or Ykä Helariutta.

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Competing interests

University of Helsinki, University of Cambridge, Natural Resources Institute Finland (Luke) and Seoul National University have filed one patent application (application no. FI0195659) on the use of constructs and methods that are described here to increase radial growth and biomass in plants, in which J.Z., G.E., J. A.-S., M.K., K.N., J.-Y.L., A.P.M. and Y.H. are listed as inventors. The remaining authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–13, Supplementary Tables 1–8, Supplementary Datasets 1–4, Supplementary References and Supplementary Note.

Reporting Summary

Supplementary Table 1

Cambium enriched gene and gene module identification in Arabidopsis roots.

Supplementary Table 2

Identification of cambium transcription factors.

Supplementary Table 3

Transcript profiling for network and the mutant phenotype prediction.

Supplementary Table 4

Plant materials, cloning vectors and primers used in this study.

Supplementary Table 5

Vascular phenotype characterization in overexpression lines.

Supplementary Table 6

Vascular phenotype characterization of mutants.

Supplementary Table 7

Differentially expressed genes in mutants.

Supplementary Table 8

Cambium genes are perturbed in mutants.

Supplementary Dataset 1

Source data for correlation analyses.

Supplementary Dataset 2

ANOVA output data.

Supplementary Dataset 3

LithoGraphX Cell classifier raw data.

Supplementary Dataset 4

RNA-seq read counts of wild-type (Col) and mutants.

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Zhang, J., Eswaran, G., Alonso-Serra, J. et al. Transcriptional regulatory framework for vascular cambium development in Arabidopsis roots. Nat. Plants 5, 1033–1042 (2019) doi:10.1038/s41477-019-0522-9

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