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Updated: May 24, 2026

The Use of Induced Somatic Sector Analysis (ISSA) for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development
Published on: October 5, 2016
Conserved 4-coumarate 3-hydroxylase/ascorbate peroxidase bifunctionality coordinates lignin deposition and plant
Weiwei Zhu1, Harmeet Singh-Bakala1, Bo Liu1
1Division of Plant Science and Technology, College of Agriculture Food and Natural Resources and Interdisciplinary Plant Group, University of Missouri, Columbia, MO 65211, United States.
Abstract:
Cytosolic ascorbate peroxidases (APXs) have been proposed to have bifunctional 4-coumarate 3-hydroxylase (C3H) activity, linking redox regulation to lignin biosynthesis in plants. Although this dual role has been shown in vitro, in vivo validation remains limited. Here, we used CRISPR/Cas9 gene editing to knock out cytosolic C3H/APX genes in Brachypodium distachyon and poplar (Populus tremula × Populus alba). In Brachypodium, BdC3H/APX1 catalyzed the ascorbate-dependent hydroxylation of 4-coumarate to caffeate in vitro. Loss of BdC3H/APX1 function led to reduced lignin content, altered monomer composition, elevated H2O2 levels, and impaired growth, while double monoallelic knockouts of BdC3H/APX1 combined with a biallelic BdC3H/APX2 mutation (Bdc3h/apx1&2) exhibited severe developmental defects. Exogenous caffeate and ferulate rescued the growth and lignin phenotype of the Bdc3h/apx1 knockout mutants, whereas catalase reduced H2O2 without restoring plant growth. Similarly, CRISPR/Cas9-mediated PtC3H/APX1 knockout in poplar resulted in stunted growth and altered lignin composition, while the double Ptc3h/apx1&2 mutants were unable to regenerate from tissue culture. These results provide in vivo evidence of C3H/APX bifunctionality, suggesting that perturbed lignin biosynthesis is the primary cause of the growth defects typically observed in C3H/APX-deficient plants.
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