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The auxin–PLETHORA 5 module regulates wood fibre development in Populus tomentosa

Nature Plants volume 11, pages 580–594 (2025)Cite this article

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Abstract

Auxin, as a vital phytohormone, is enriched in the vascular cambium, playing a crucial role in regulating wood formation in trees. While auxin’s influence on cambial stem cells is well established, the molecular mechanisms underlying the auxin-directed development of cambial derivatives, such as wood fibres, remain elusive in forest trees. Here we identified a transcription factor, AINTEGUMENTA-like 5 (AIL5)/PLETHORA 5 (PLT5) from Populus tomentosa, that is specifically activated by auxin signalling within the vascular cambium. PLT5 regulated both cell expansion and cell wall thickening in wood fibres. Genetic analysis demonstrated that PLT5 is essential for mediating the action of auxin signalling on wood fibre development. Remarkably, PLT5 specifically inhibits the onset of fibre cell wall thickening by directly repressing SECONDARY WALL-ASSOCIATED NAC DOMAIN 1 (SND1) genes. Our findings reveal a sophisticated auxin–PLT5 signalling pathway that finely tunes the development of wood fibres by controlling cell wall thickening.

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Fig. 1: Auxin signalling regulates wood fibre development in P. tomentosa.
Fig. 2: PLT5s are the vascular cambium-specific auxin-responsive genes.
Fig. 3: PLT5s regulate wood fibre development.
Fig. 4: PLT5 proteins mediate the regulation of vascular cambium-localized auxin signalling on wood fibre development.
Fig. 5: The auxin–PLT5 signalling cascade coordinates cell wall thickening of developing fibres.
Fig. 6: PLT5 directly represses SND1 expression.
Fig. 7: SND1 acts downstream of PLT5 during wood fibre development.

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Data availability

The data supporting the findings of this study are available within the Article and its Supplementary Information. The GenBank accession numbers of the P. tomentosa genes are as follows: PLT5a (OR250663), PLT5b (OR250664), IAA9 (MH345700), ARF5.1 (MH352401), SND1-A1 (OR514110) and UBQ (PQ155116). The AspWood datasets are available in the supplementary data of ref. 39 (https://academic.oup.com/plcell/article/29/7/1585/6099151). Source data are provided with this paper.

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Acknowledgements

We thank V. L. Chiang and W. Li (Northeast Forestry University, China), H. Lu (Beijing Forestry University, China), and Y. Zhang (Southwest University, China) for helpful comments. This work was supported by grants from the National Key Research and Development Program (2021YFD2200204 to K.L.), the National Natural Science Foundation of China (32271906 to C.X. and 32201579 to X.F.) and the Opening Project of State Key Laboratory of Tree Genetics and Breeding (K2022102 to C.X.).

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Author notes

  1. These authors contributed equally: Shuai Liu, Xiaokang Fu.

Authors and Affiliations

  1. Chongqing Key Laboratory of Tree Germplasm Innovation and Utilization, Integrative Science Center of Germplasm Creation, School of Life Sciences, Southwest University, Chongqing, China

    Shuai Liu, Xiaokang Fu, Yue Wang, Xuelian Du, Lianjia Luo, Dong Chen, Chunzhao Liu, Jian Hu, Keming Luo & Changzheng Xu

  2. Key Laboratory of Eco-environments of Three Gorges Reservoir Region, Ministry of Education, School of Life Sciences, Southwest University, Chongqing, China

    Xiaokang Fu, Keming Luo & Changzheng Xu

  3. Center for Computational Biology, Beijing Forestry University, Beijing, China

    Changjian Fa

  4. Beijing Institute of Mathematical Sciences and Applications, Beijing, China

    Rongling Wu

  5. Yau Mathematical Sciences Center, Tsinghua University, Beijing, China

    Rongling Wu

  6. National Key Laboratory of Plant Molecular Genetics and CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China

    Laigeng Li

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Contributions

C.X. and K.L. conceived the research and designed the experiments. S.L., X.F., Y.W., X.D., L. Luo, D.C., C.L. and J.H. performed the experiments. C.X., S.L. and X.F. analysed the data. C.F. and R.W. conducted the modelling and contributed to the statistical analysis. C.X. and K.L. prepared the manuscript. L. Li contributed to the revision of the manuscript and provided valuable comments.

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Correspondence to Keming Luo or Changzheng Xu.

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Extended data

Extended Data Fig. 1 Phenotypes of secondary xylem, DF cell layers and wood fibres in WOX4apro:IAA9m transgenics.

a, Quantification of secondary xylem cell layer number at the 7th, 8th and 9th internodes of 3-month-old WT and WOX4apro:IAA9m transgenics (#2 and #3). b, c, Observation (b) and quantification (c) of DF phenotypes on the cross-sections of the 8th internodes of 3-month-old WT and WOX4apro:IAA9m transgenics (#2 and #3). d, percentage of DF cell layer number compared to the total number of secondary xylem cell layers. e, Cellular morphology of fibres isolated from the 10th internodes of 3-month-old WT and WOX4apro:IAA9m transgenics (#2 and #3). In b and e, cross-sections (b) and fibre cells (e) were stained with toluidine blue. In b, the DSX domains are indicated in yellow. In a and c, cell layer measurements for secondary xylem (a) and DF (c) were obtained from 10 radial cell files in cross-sections of the 7th, 8th and 9th internodes. In d, the percentage was calculated from the data of total secondary xylem cell layer number (a) and DF cell layer number (c). Each biological replicate included 5 cross-sections, with 6 biological replicates (independent plants) analyzed for WT and each transgenic line. The data for all 30 cross-sections across 6 replicates are shown in boxes and whisker plots, with boxes indicating the median and 5th to 95th percentile, and whiskers representing the data range excluding outliers. One-way ANOVA analysis followed by Dunnett test for pairwise comparison was performed to evaluate significant differences between WT and transgenic lines. The average values of 5 cross-sections from a biological replicate were used for statistical analyses (n = 6). **, P < 0.01; *, P < 0.05; n.s., not significant. VC: vascular cambium; Ph: phloem; SXy: secondary xylem. Scale bars: 30 μm (b) and 100 μm (e).

Source data

Extended Data Fig. 2 Identification of PLT5a and PLT5b genes from the transcriptome datasets of IAA9m-overexpressing P. tomentosa lines.

a, Network diagram of the genes that are repressed by IAA9m overexpression and specifically expressed in vascular cambium. Transcriptome data of 35Spro:IAA9m are sourced from our previous study35. The data of genes specifically expressed in vascular cambium are from AspWood database39. The network is constructed with Gephi software. Ca, cambium; Down, down-regulated genes by IAA9m overexpression. The numbers in the diagram represent the number of genes in different clusters. b, Enrichment of GO terms of the genes that are down-regulated by IAA9m overexpression and specifically expressed in vascular cambium. c, Venn diagram of the transcription factors (TFs) that are down-regulated by IAA9m overexpression and specifically expressed in vascular cambium versus those identified as vascular cambium-specific (VCS) TFs38. d, The families of 15 identified vascular cambium specific TFs that are responsive to auxin. The number of identified members from each family is indicated. e, The phylogenetic analysis of PLT proteins from Arabidopsis and poplar. The PLT5 homologs are indicated in red. In (b), the Chi-square test of independence is employed to evaluate the significance of difference between the expected frequencies of genes annotated with GO terms and their observed frequencies.

Extended Data Fig. 3 ARF5 directly targets PLT5a to activate its expression.

a, Distribution of predicted auxin response factor (ARF) TF-binding sites within the promoter of PLT5a and ChIP-qPCR analyses. b, The binding capacity of ARF5.1 protein to the PLT5a promoter fragment (PLT5apro-III) examined by EMSA assays. c, Luciferase-based effector-reporter assays of ARF5.1-activated PLT5a expression in transiently transfected tobacco leaf cells. d, Quantification of luciferase activities. In c and d, PLT5apro-Im and PLT5apro-IIIm indicate the promoter regions of PLT5a with impaired binding sites of ARF5.1 proteins generated by site-directed mutagenesis. In a and d, data are shown as mean ± S.D.; dots represent the values from independent biological replicates (n = 3). Student’s t-test (two-tailed) was performed to evaluate significant differences. *, P < 0.05; **, P < 0.01; n.s., not significant. In b, the experiments were repeated independently three times, with similar results.

Source data

Extended Data Fig. 4 Phenotypes of DF cell layers and wood fibres caused by plt5a plt5b double mutations and WOX4apro:PLT5a transgene.

a, Anatomical sections of the 7th, 8th and 9th internodes of 3-month-old WT, plt5a plt5b mutants (#4 and #5) and WOX4apro:PLT5a transgenics (#1 and #2). b, Quantification of DF cell layer number at the 7th, 8th and 9th internodes of WT, mutant and transgenic lines corresponding to (a). c, Cellular morphology of fibres isolated from the 10th internodes of 3-month-old WT, plt5a plt5b mutants (#4 and #5) and WOX4apro:PLT5a transgenics (#1 and #2). d and e, The length (d) and width (e) of fibres isolated from the 10th internodes of WT, mutant and transgenic lines corresponding to (c). In a and c, cross-sections (a) and fibre cells (c) were stained with toluidine blue. In a, DSX domains are indicated in yellow. In b, average values for DF cell layers were determined from 10 radial cell files from cross-sections of the 7th, 8th and 9th internodes, with 5 cross-sections per biological replicate. Date from 6 biological replicates (independent plants) were analyzed, encompassing 30 cross-sections in total. Boxes and whisker plots represent these values. In d and e, 25 fibre cells isolated were quantified for each biological replicate. Six biological replicates (independent plants). The values from all 150 fibre cells from 6 biological replicates were included in box and whisker plots. In b, d and e, boxes show the median and 5th to 95th percentile, and whiskers represent the value range excluding outliers. One-way ANOVA analysis followed by Dunnett test for pairwise comparison was performed to evaluate significant differences between WT and mutant or transgenic lines. The average values of 5 cross-sections (b) or 25 fibre cells (d, e) from a biological replicate were used for statistical analyses (n = 6). **, P < 0.01. DF, developing fibre; SXy, secondary xylem; VC, vascular cambium. Scale bars, 30 μm (a) and 100 μm (c).

Source data

Extended Data Fig. 5 Cell wall thickness of developing fibres affected by PLT5.

a, SEM analyses of DF cell layers at the DSX domain of the 8th internodes from 3-month-old WT, plt5a plt5b mutants (#4 and #5) and WOX4apro:PLT5a transgenics (#1 and #2). b, SEM analyses of developing fibre cells at the DSX domain of the 8th internodes from 3-month-old WT and PLT5apro:PLT5a-YFP (#1) and PLT5apro:PLT5a-YFP×3 (#1) transgenics. c, Quantification of cell wall thickness of the 1st to 5th DF cell layers at the DSX domain of WT and PLT5apro:PLT5a-YFP×3 (#1) transgenics. d, SEM analyses of DF cell layers at the DSX domain of the 8th internodes from 3-month-old WT and WOX4apro:IAA9m (#3), PLT5apro:PLT5a-YFP > > WOX4apro:IAA9m (#1) and PLT5apro:PLT5a-YFP×3 > >WOX4apro:IAA9m (#2) transgenics. In c, cell wall thickness between neighboring DF cell layers was measured in 10 radial cell files per biological replicate. Data from 6 biological replicates (independent plants) for WT and transgenic lines were analyzed, including 60 radial cell files in total. Boxes represent the median and 5th to 95th percentile, while whisker plots indicate the range excluding outliers. One-way ANOVA followed by Dunnett’s test for pairwise comparison was used to assess differences between WT and transgenic lines. Average values from 10 radial cell files per replicate were used for statistical analysis (n = 6). n.s., not significant. The numbers in red (a, b and d) indicate the developing fibre cell layers. CW, cell wall. Scale bars: 15 μm (a), (b) and (d).

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Extended Data Fig. 6 Morphology and length distribution of fibre cells in different developmental classes for plt5a plt5b and WOX4apro:IAA9m transgenics.

a and b, cellular morphology (a) and length quantification (b) of fibre cells isolated from the 10th internodes of 3-month-old WT, plt5a plt5b mutants and WOX4apro:IAA9m transgenics classified into four categories. In a, fibres were stained with toluidine blue. In b, the length of isolated fibres was measured using ImageJ software. A total of 72 fibre cells from 3 biological replicates (independent plants) were measured. Data are shown as mean ± S.D. (n = 3). Scale bars, 100 μm (a).

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Extended Data Fig. 7 The fitness of growth equation to the cell wall thickness that changes with increasing fibre cell layers.

a and b, The fitness of growth equation to cell wall thickness that changes with increasing fibre cell layers in the 8th internodes of plt5a plt5b (#4) mutants and WOX4apro:PLT5a (#1) transgenics (a) and WOX4apro:IAA9m (#3) and PLT5apro:PLT5a-YFP >>WOX4apro:IAA9m (#1) transgenics (b). Dots denote observations of cell wall thickness at different layers. The timing of inflection point tI is indicated in each case.

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Extended Data Fig. 8 Expression levels of SND1 genes in IAA9m- and PLT5-associated transgenic lines assayed by qRT-PCR.

a and b, Expression levels of SND1-A1, SND1-A2, SND1-B1 and SND1-B2 in stems of WOX4pro:IAA9m transgenics (a), plt5a plt5b mutants, PLT5apro:PLT5a-YFP and PLT5apro:PLT5a-YFP×3 transgenics (b). The 8th internodes of 3-month-old WT and transgenic lines were collected for RNA extraction followed by qRT-PCR assays. Data are shown as mean ± S.D. from 3 biological replicates (independent plants); n = 3. One-way ANOVA analysis followed by Dunnett test for pairwise comparison was performed to test significant differences among WT and mutant or transgenic lines. **, P < 0.01; *, P < 0.05; n.s., not significant.

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Extended Data Fig. 9 ChIP-qPCR and effector-reporter assays of PLT5-bindiing capacity to SND1-A2/B1/B2 promoters.

a, b and c, Distribution of predicted PLT5a-binding sites within the promoter of SND1-A2 (a), SND1-B1 (b) and SND1-B1 (c) and ChIP-qPCR analysis for the promoter of SND1-A2 (a), SND1-B1 (b) and SND1-B1 (c). d, f and h, Luciferase-based effector-reporter assays of PLT5a-suppressed SND1-A2 (d), SND1-B1 (f) and SND1-B2 (h) expression in transiently transfected tobacco leaf cells. e, g and i, Quantification of luciferase activity of PLT5a-suppressed SND1-A2 (e), SND1-B1 (g) and SND1-B2 (i) expression in transiently transfected tobacco leaf cells. In d to i, SND1-A2pro-Im, SND1-B1pro-Im and SND1-B1pro-IIm indicate the promoter regions of SND1-A2, SND1-B1 and SND1-B2 with impaired binding sites of PLT5a proteins generated by site-directed mutagenesis. Data are shown as mean ± S.D.; dots represent the values from independent biological replicates (n = 3). Student’s t-test (two-tailed) was performed to evaluate significant differences. **, P < 0.01; *, P < 0.05; n.s., not significant.

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Extended Data Fig. 10 Phenotypes of DSX and wood fibres caused by SND1-A1 and PLT5a-associated transgenes.

a, Anatomical sections of the 8th internodes of 3-month-old WT and 35Spro:SND1-A1 (#2), WOX4apro:PLT5a (#1) and 35Spro:SND1-A1 > > WOX4apro:PLT5a (#2) transgenics. b, Cellular morphology of fibres isolated from the 10th internodes of 3-month-old WT and 35Spro:SND1-A1 (#2), WOX4apro:PLT5a (#1) and 35Spro:SND1-A1 > > WOX4apro:PLT5a (#2) transgenics. c, d, The length (c) and width (d) of fibres isolated from the 10th internodes of WT and transgenic lines corresponding to (b). e, The percentages of four classes of fibres cells isolated from the 10th internodes of WT and transgenic lines corresponding to (b). f, SEM analyses of DF cell layers at the DSX domain of the 8th internodes from 3-month-old WT and 35Spro:SND1-A1 (#2), WOX4apro:PLT5a (#1) and 35Spro:SND1-A1 > > WOX4apro:PLT5a (#2) transgenics. In a and b, cross-sections (a) and isolated fibre cells (b) were stained with toluidine blue. In a, the DSX domains are indicated in yellow. In c and d, 25 fibre cells isolated from the 10th internodes were quantified for each biological replicate. Six biological replicates (independent plants) were analyzed for WT and transgenic lines. The box and whisker plots represent values from 150 fibre cells across 6 biological replicates, with boxes showing the median and 5th to 95th percentile, and whiskers indicating the range excluding outliers. One-way ANOVA followed by Tukey test was used to assess significant differences between WT and transgenic lines. Statistical analyses were based on the average values of 25 fibre cells per biological replicate (n = 6). **, P < 0.01; *, P < 0.05; n.s., not significant. The numbers in red (f) indicate DF cell layers at DSX domain. DSX, developing secondary xylem; Ph, phloem; SXy, secondary xylem; VC, vascular cambium. Scale bars, 30 μm (a), 100 μm (b) and 15 μm (f).

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Liu, S., Fu, X., Wang, Y. et al. The auxin–PLETHORA 5 module regulates wood fibre development in Populus tomentosa. Nat. Plants 11, 580–594 (2025). https://doi.org/10.1038/s41477-025-01931-z

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