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Background <p>During drought stress, one of the physiological challenges plants face is the embolism and collapse of xylem vessels. Lignin biosynthetic enzymes form complexes during lignification to facilitate metabolic channeling. Although cinnamyl alcohol dehydrogenase (CAD)-mediated lignin biosynthesis is essential for reinforcing cell wall mechanical strength and maintaining water transport capacity under stress, the molecular mechanisms by which <i>CAD</i> genes confer drought tolerance in the valuable timber species <i>Phoebe zhennan</i> remain elusive.</p> Results <p>In this study, we identified six <i>PzCAD</i> genes within the <i>P. zhennan</i> genome, characterized by an uneven chromosomal distribution and a localized tandem duplication event on chromosome 2. Phylogenetic and structural analyses revealed that these members are distributed across three distinct evolutionary clades and exhibit significant exon–intron divergence, while their promoters are universally enriched in stress- and hormone-responsive elements. Protein interaction assays, including yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), and luciferase complementation imaging (LCI), demonstrated that <i>PzCAD2-1</i> and <i>PzCAD2-2</i> assemble into stable homodimers. Structural and transcriptional analyses showed that while virus-induced gene silencing (VIGS) of Clade I (<i>PzCAD1</i>) and Clade II (<i>PzCAD2</i>) members failed to alter bulk lignin accumulation, the Clade IV orthologs (<i>PzCAD4-1</i>, <i>PzCAD4-2</i>, <i>PzCAD4-3</i>) were profoundly upregulated under PEG-simulated drought. Comprehensive <i>in planta</i> functional validations in transgenic hairy root systems revealed that overexpressing the <i>PzCAD4</i> clade markedly enhanced drought tolerance by augmenting lignin deposition, elevating antioxidant enzyme activities, accumulating osmoprotectants, and attenuating reactive oxygen species (ROS) bursts. Additionally, the overexpression lines synergistically accumulated defense-associated phytohormones (salicylic acid and jasmonic acid) alongside moderately elevated H₂O₂, which more likely functioned as a signaling molecule under drought stress. In stark contrast, CRISPR/Cas9-mediated knockouts of individual <i>PzCAD4</i> genes severely repressed lignin biosynthesis, exacerbating cellular oxidative damage and precipitating pronounced drought responses.</p> Conclusions <p>We conclude that <i>PzCAD4</i> is a key positive regulator of lignin biosynthesis under drought stress that improves PEG-induced drought tolerance mainly by reinforcing secondary cell walls, accompanied by coordinated changes in antioxidant metabolism and defense phytohormone accumulation. These findings comprehensively elucidate the functional landscape of the <i>P. zhennan CAD</i> gene family, providing theoretical foundations and genetic targets for the molecular breeding of drought-resilient timbers.</p>

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Genome-wide analysis of Phoebe zhennan CADs identifies PzCAD4 as a key contributor to drought response via lignin biosynthesis

  • 期刊论文

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  • 发表日期：

    2026年7月22日

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  • Beibei Chen,
  • Lingfan Yu,
  • Zhenqi Liao,
  • Yingxuan Luo,
  • Yunjie Gu,
  • Lihua Wang,
  • Yuan Li,
  • Min Ye,
  • Zhongbiao Ding,
  • Weiyi Liu,
  • Shengjia Huang

摘要

Background

During drought stress, one of the physiological challenges plants face is the embolism and collapse of xylem vessels. Lignin biosynthetic enzymes form complexes during lignification to facilitate metabolic channeling. Although cinnamyl alcohol dehydrogenase (CAD)-mediated lignin biosynthesis is essential for reinforcing cell wall mechanical strength and maintaining water transport capacity under stress, the molecular mechanisms by which CAD genes confer drought tolerance in the valuable timber species Phoebe zhennan remain elusive.

Results

In this study, we identified six PzCAD genes within the P. zhennan genome, characterized by an uneven chromosomal distribution and a localized tandem duplication event on chromosome 2. Phylogenetic and structural analyses revealed that these members are distributed across three distinct evolutionary clades and exhibit significant exon–intron divergence, while their promoters are universally enriched in stress- and hormone-responsive elements. Protein interaction assays, including yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), and luciferase complementation imaging (LCI), demonstrated that PzCAD2-1 and PzCAD2-2 assemble into stable homodimers. Structural and transcriptional analyses showed that while virus-induced gene silencing (VIGS) of Clade I (PzCAD1) and Clade II (PzCAD2) members failed to alter bulk lignin accumulation, the Clade IV orthologs (PzCAD4-1, PzCAD4-2, PzCAD4-3) were profoundly upregulated under PEG-simulated drought. Comprehensive in planta functional validations in transgenic hairy root systems revealed that overexpressing the PzCAD4 clade markedly enhanced drought tolerance by augmenting lignin deposition, elevating antioxidant enzyme activities, accumulating osmoprotectants, and attenuating reactive oxygen species (ROS) bursts. Additionally, the overexpression lines synergistically accumulated defense-associated phytohormones (salicylic acid and jasmonic acid) alongside moderately elevated H₂O₂, which more likely functioned as a signaling molecule under drought stress. In stark contrast, CRISPR/Cas9-mediated knockouts of individual PzCAD4 genes severely repressed lignin biosynthesis, exacerbating cellular oxidative damage and precipitating pronounced drought responses.

Conclusions

We conclude that PzCAD4 is a key positive regulator of lignin biosynthesis under drought stress that improves PEG-induced drought tolerance mainly by reinforcing secondary cell walls, accompanied by coordinated changes in antioxidant metabolism and defense phytohormone accumulation. These findings comprehensively elucidate the functional landscape of the P. zhennan CAD gene family, providing theoretical foundations and genetic targets for the molecular breeding of drought-resilient timbers.