PagKNAT5a promotes plant growth by enhancing xylem cell elongation and secondary wall formation in poplar

Abstract

Investigating the regulatory mechanisms that govern plant growth is crucial for developing high-yield wood varieties. In this context, the KNOX gene family has been identified as a significant regulator of plant growth. Our study focuses on PagKNAT5a, a class II member of the KNOX gene family, which has been found to promote the growth of poplar. Transgenic plants overexpressing PagKNAT5a exhibited significant increases in both plant height and stem diameter compared to wild-type controls. Histochemical analyses revealed that these overexpression lines had elongated xylem vessels and fiber cells, which correlated with elevated auxin levels. Additionally, we observed thickened secondary cell walls (SCWs) and increased lignin content in the fiber cells of these transgenic lines. Further protein interaction assays indicated that PagKNAT5a physically interacts with MYB46, a crucial regulator of SCW biosynthesis. This interaction activates downstream secondary wall MYB-responsive elements (SMREs), leading to the upregulation of lignin biosynthesis genes driven by these cis-acting elements. Moreover, the increased photosynthetic rate observed in the overexpression lines is likely to significantly support overall plant development. Our findings suggest that PagKNAT5a facilitates the longitudinal elongation of vascular cells by modulating auxin levels while simultaneously promoting the radial growth of xylem tissue through the activation of the MYB46-mediated lignin biosynthesis pathway. The functional analysis of PagKNAT5a highlights its potential for improving wood yield in forestry applications.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (32201582), the Zhejiang Provincial Natural Science Foundation of China (LQ22C160008), the National Key Research and Development Program of China (2021YFD2200205), the Key Scientific and Technological Grant of Zhejiang for Breeding New Agricultural Varieties (2021C02070-1), and the Research Foundation of Zhejiang A&F University (2018FR013).

Author contributions

J.Z., M.L., and L.H. conceived this study. L.H., J.L., X.T., Y.L., Y.C., Y.A., C.J., and N.C. performed the experiments. L.H., M.L., and J.Z. wrote the manuscript.

Data availability

The RNA-Seq data has been uploaded to National Genomics Data Center (https://bigd.big.ac.cn/bioproject) with the BioProject accession number: CRA020529.

Conflict of interest statement

The authors declare no conflicts of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Hay A, Tsiantis M. KNOX genes: versatile regulators of plant development and diversity. Development. 2010; 137:3153-65

[2] Nookaraju A, Pandey SK, Ahlawat YK. et al. Understanding the modus operandi of class II KNOX transcription factors in sec-ondary cell wall biosynthesis. Plants. 2022; 11:493

[3] Hamant O, Pautot V. Plant development: a TALE story. CRBiol. 2010; 333:371-81

[4] Hake S, Smith HM, Holtan H. et al. The role of knox genes in plant development. Annu Rev Cell Dev Biol. 2004; 20:125-51

[5] Liebsch D, Sunaryo W, Holmlund M. et al. Class I KNOX transcrip-tion factors promote differentiation of cambial derivatives into xylem fibers in the Arabidopsis hypocotyl. Development. 2014; 141: 4311-9

[6] Woerlen N, Allam G, Popescu A. et al. Repression of BLADE-ON-PETIOLE genes by KNOX homeodomain protein BREVIPEDI-CELLUS is essential for differentiation of secondary xylem in Arabidopsis root. Planta. 2017; 245:1079-90

[7] Du J, Mansfield SD, Groover AT. The Populus homeobox gene ARBORKNOX2 regulates cell differentiation during secondary growth. Plant J. 2009; 60:1000-14

[8] Zhao K, Zhang X, Cheng Z. et al. Comprehensive analysis of the three-amino-acid-loop-extension gene family and its tissue-differential expression in response to salt stress in poplar. Plant Physiol Biochem. 2019; 136:1-12

[9] Li E, Bhargava A, Qiang W. et al. The class II KNOX gene KNAT7 negatively regulates secondary wall formation in Arabidopsis and is functionally conserved in Populus. New Phytol. 2012; 194: 102-15

[10] Wang S, Yamaguchi M, Grienenberger E. et al. The class II KNOX genes KNAT3 and KNAT7 work cooperatively to influence depo-sition of secondary cell walls that provide mechanical support to Arabidopsis stems. Plant J. 2020; 101:293-309

[11] Zhong R, Lee C, Zhou J. et al. A battery of transcription factors involved in the regulation of secondary cell wall biosynthesis in Arabidopsis. Plant Cell. 2008; 20:2763-82

[12] Gong SY, Huang GQ, Sun X. et al. Cotton KNL1, encoding a class II KNOX transcription factor, is involved in regulation of fibre development. JExp Bot. 2014; 65:4133-47

[13] Wang S, Yang H, Mei J. et al. Rice homeobox protein KNAT7 inte-grates the pathways regulating cell expansion and wall stiffness. Plant Physiol. 2019b; 181:669-82

[14] Wang L, Lu W, Ran L. et al. R2R3-MYB transcription factor MYB6 promotes anthocyanin and proanthocyanidin biosynthesis but inhibits secondary cell wall formation in Populus tomentosa. Plant J. 2019a; 99:733-51

[15] Pandey SK, Nookaraju A, Fujino T. et al. Virus-induced gene silencing (VIGS)-mediated functional characterization of two genes involved in lignocellulosic secondary cell wall formation. Plant Cell Rep. 2016; 35:2353-67

[16] Ahlawat YK, Nookaraju A, Harman-Ware AE. et al. Genetic mod-ification of KNAT7 transcription factor expression enhances saccharification and reduces recalcitrance of woody biomass in poplars. Front Plant Sci. 2021; 12:762067

[17] Li E, Wang S, Liu Y. et al. OVATE FAMILY PROTEIN4 (OFP4) interaction with KNAT7 regulates secondary cell wall formation in Arabidopsis thaliana. Plant J. 2011; 67:328-41

[18] Liu Y, You S, Taylor-Teeples M. et al. BEL1-LIKE HOMEODOMAIN6 and KNOTTED ARABIDOPSIS THALIANA7 interact and regulate secondary cell wall formation via repression of REVOLUTA. Plant Cell. 2014; 26:4843-61

[19] Qin W, Yin Q, Chen J. et al. The class II KNOX transcription factors KNAT3 and KNAT7 synergistically regulate monolignol biosynthesis in Arabidopsis. JExp Bot. 2020; 71:5469-83

[20] Robischon M, Du J, Miura E. et al. The Populus class III HD ZIP, popREVOLUTA, influences cambium initiation and patterning of woody stems. Plant Physiol. 2011; 155:1214-25

[21] Bhargava A, Ahad A, Wang S. et al. The interacting MYB75 and KNAT7 transcription factors modulate secondary cell wall deposition both in stems and seed coat in Arabidopsis. Planta. 2013; 237:1199-211

[22] Bhargava A, Mansfield SD, Hall HC. et al. MYB75 func-tions in regulation of secondary cell wall formation in the Arabidopsis inflorescence stem. Plant Physiol. 2010; 154: 1428-38

[23] Sundell D, Street NR, Kumar M. et al. AspWood: high-spatial-resolution transcriptome profiles reveal uncharacterized mod-ularity of wood formation in Populus tremula. Plant Cell. 2017; 29: 1585-604

[24] Shi R, Wang JP, Lin Y-C. et al. Tissue and cell-type co-expression networks of transcription factors and wood component genes in Populus trichocarpa. Planta. 2017; 245:927-38

[25] Yamaji N, Huang CF, Nagao S. et al. A zinc finger transcription factor ART1 regulates multiple genes implicated in aluminum tolerance in rice. Plant Cell. 2009; 21:3339-49

[26] Chen B, Sun Y, Tian Z. et al. GhGASA10-1 promotes the cell elongation in fiber development through the phytohormones IAA-induced. BMC Plant Biol. 2021; 21:448

[27] Claisse G, Charrier B, Kreis M. The Arabidopsis thaliana GSK3/shaggy like kinase AtSK3-2 modulates floral cell expan-sion. Plant Mol Biol. 2007; 64:113-24

[28] Goda H, Sawa S, Asami T. et al. Comprehensive comparison of auxin-regulated and brassinosteroid-regulated genes in Ara-bidopsis. Plant Physiol. 2004; 134:1555-73

[29] Han S, Jiao Z, Niu M-X. et al. Genome-wide comprehensive analy-sis of the GASA gene family in Populus. Int J Mol Sci. 2021; 22:12336

[30] Zhang J, Xie M, Tuskan GA. et al. Recent advances in the tran-scriptional regulation of secondary cell wall biosynthesis in the woody plants. Front Plant Sci. 2018; 9:1535

[31] Zhong R, Ye ZH. MYB46 and MYB83 bind to the SMRE sites and directly activate a suite of transcription factors and secondary wall biosynthetic genes. Plant Cell Physiol. 2012; 53:368-80

[32] Ko JH, Jeon HW, Kim WC. et al. The MYB46/MYB83-mediated transcriptional regulatory programme is a gatekeeper of sec-ondary wall biosynthesis. Ann Bot. 2014; 114:1099-107

[33] Kerstetter R, Vollbrecht E, Lowe B. et al. Sequence analysis and expression patterns divide the maize knotted1-like homeobox genes into two classes. Plant Cell. 1994; 6:1877-87

[34] Meisel L, Lam E. The conserved ELK-homeodomain of KNOTTED-1 contains two regions that signal nuclear localization. Plant Mol Biol. 1996; 30:1-14

[35] Nagasaki H, Sakamoto T, Sato Y. et al. Functional analysis of the conserved domains of a rice KNOX homeodomain protein, OSH15. Plant Cell. 2001; 13:2085-98

[36] Vollbrecht E, Veit B, Sinha N. et al. The developmental gene Knotted-1 is a member of a maize homeobox gene family. Nature. 1991; 350:241-3

[37] Zhong R, Richardson EA, Ye ZH. The MYB46 transcription factor is a direct target of SND1 and regulates secondary wall biosyn-thesis in Arabidopsis. Plant Cell. 2007; 19:2776-92

[38] Hazen S, Zhong R, McCarthy R. et al. The poplar MYB master switches bind to the SMRE site and activate the secondary wall biosynthetic program during wood formation. PLoS One. 2013; 8:e69219

[39] Han X, Zhao Y, Chen Y. et al. Lignin biosynthesis and accumu-lation in response to abiotic stresses in woody plants. For Res. 2022; 2:0

[40] Ou R, Xie Y, Wolcott MP. et al. Morphology, mechanical prop-erties, and dimensional stability of wood particle/high density polyethylene composites: effect of removal of wood cell wall composition. Mater Des. 2014; 58:339-45

[41] Kim WC, Ko JH, Han KH. Identification of a cis-acting regulatory motif recognized by MYB46, a master transcriptional regulator of secondary wall biosynthesis. Plant Mol Biol. 2012; 78:489-501

[42] Fukuda H. Xylogenesis: initiation, progression, and cell death. Annu Rev Plant Physiol Plant Mol Biol. 1996; 47:299-325

[43] Meents MJ, Watanabe Y, Samuels AL. The cell biology of sec-ondary cell wall biosynthesis. Ann Bot. 2018; 121:1107-25

[44] Tsuda K, Hake S. Diverse functions of KNOX transcription fac-tors in the diploid body plan of plants. Curr Opin Plant Biol. 2015; 27:91-6

[45] Scanlon MJ, Henderson DC, Bernstein B. SEMAPHORE 1 functions during the regulation of ancestrally duplicated knoxgenes and polar auxin transport in maize. Development. 2002; 129:2663-73

[46] Bolduc N, Yilmaz A, Mejia-Guerra MK. et al. Unraveling the KNOTTED1 regulatory network in maize meristems. Genes Dev. 2012; 26:1685-90

[47] Yan F, Deng W, Pang X. et al. Overexpression of the KNOX gene Tkn4 affects pollen development and confers sensitivity to gibberellin and auxin in tomato. Plant Sci. 2019; 281:61-71

[48] Yang Q, Cong T, Yao Y. et al. KNOX genes were involved in regu-lating axillary bud formation of chrysanthemum × morifolium. Int J Mol Sci. 2023; 24:7081

[49] Zhao YQ, Song XQ, Guo W. et al. Promotes shoot branching by attenuating auxin-strigolactone signalling in poplar. Plant Cell Environ. 2024; 47:3920-35

[50] Nakazawa M, Yabe N, Ichikawa T. et al. DFL1, an auxin-responsive GH3 gene homologue, negatively regulates shoot cell elongation and lateral root formation, and positively regulates the light response of hypocotyl length. Plant J. 2008; 25:213-21

[51] Staswick PE, Serban B, Rowe M. et al. Characterization of an Ara-bidopsis enzyme family that conjugates amino acids to indole-3-acetic acid. Plant Cell. 2005; 17:616-27