SummaryHigher plants contain plant-specific peroxidases (class III peroxidase; Prxs) that exist as large multigene families. Reverse genetic studies to characterize the function of each Prx have revealed that Prxs are involved in lignification, cell elongation, stress defense and seed germination. However, the underlying mechanisms associated with plant phenotypes following genetic engineering of Prx genes are not fully understood. This is because Prxs can function as catalytic enzymes that oxidize phenolic compounds while consuming hydrogen peroxide and/or as generators of reactive oxygen species. Moreover, biochemical efforts to characterize Prxs responsible for lignin polymerization have revealed specialized activities of Prxs. In conclusion, not only spatiotemporal regulation of gene expression and protein distribution, but also differentiated oxidation properties of each Prx define the function of this class of peroxidases.

Plant class III heme peroxidases catalyze lignin polymerization. Previous reports have shown that at least three Arabidopsis thaliana peroxidases, AtPrx2, AtPrx25 and AtPrx71, are involved in stem lignification using T-DNA insertion mutants, atprx2, atprx25, and atprx71. Here, we generated three double mutants, atprx2/atprx25, atprx2/atprx71, and atprx25/atprx71, and investigated the impact of the simultaneous deficiency of these peroxidases on lignins and plant growth. Stem tissue analysis using the acetyl bromide method and derivatization followed by reductive cleavage revealed improved lignin characteristics, such as lowered lignin content and increased arylglycerol-b-aryl (b-O-4) linkage type, especially b-O-4 linked syringyl units, in lignin, supporting the roles of these genes in lignin polymerization. In addition, none of the double mutants ○exhibited severe growth defects, such as shorter plant stature, dwarfing, or sterility, and their stems had improved cell wall degradability. This study will contribute to progress in lignin bioengineering to improve lignocellulosic biomass.Keywords: Arabidopsis; knockout mutant; lignin biosynthesis; plant peroxidase Citation: Shigeto J, Itoh Y, Hirao S, Ohira K, Fujita K, Tsutsumi Y (2015) Simultaneously disrupting AtPrx2, AtPrx25 and AtPrx71 alters lignin content and structure in Arabidopsis stem.

Permeability is a crucial trait that affects seed longevity and is regulated by different polymers including proanthocyanidins, suberin, cutin and lignin located in the seed coat. By testing mutants in suberin transport and biosynthesis, we demonstrate the importance of this biopolymer to cope with seed deterioration. Transcriptomic analysis of cog1‐2D, a gain‐of‐function mutant with increased seed longevity, revealed the upregulation of several peroxidase genes. Reverse genetics analysing seed longevity uncovered redundancy within the seed coat peroxidase gene family; however, after controlled deterioration treatment, seeds from the prx2 prx25 double and prx2 prx25 prx71 triple mutant plants presented lower germination than wild‐type plants. Transmission electron microscopy analysis of the seed coat of these mutants showed a thinner palisade layer, but no changes were observed in proanthocyanidin accumulation or in the cuticle layer. Spectrophotometric quantification of acetyl bromide‐soluble lignin components indicated changes in the amount of total polyphenolics derived from suberin and/or lignin in the mutant seeds. Finally, the increased seed coat permeability to tetrazolium salts observed in the prx2 prx25 and prx2 prx25 prx71 mutant lines suggested that the lower permeability of the seed coats caused by altered polyphenolics is likely to be the main reason explaining their reduced seed longevity.

The final step of lignin biosynthesis, which is catalyzed by a plant peroxidase, is the oxidative coupling of the monolignols to growing lignin polymers. Cationic cell-wall-bound peroxidase (CWPO-C) from poplar callus is a unique enzyme that has oxidative activity for both monolignols and synthetic lignin polymers. This study shows that putative CWPO-C homologues in Arabidopsis , AtPrx2, AtPrx25, and AtPrx71, are involved in lignin biosynthesis. Analysis of stem tissue using the acetyl bromide method and derivatization followed by the reductive cleavage method revealed a significant decrease in the total lignin content of ATPRX2 and ATPRX25 deficient mutants and altered lignin structures in ATPRX2, ATPRX25, and ATPRX71 deficient mutants. Among Arabidopsis peroxidases, AtPrx2 and AtPrx25 conserve a tyrosine residue on the protein surface, and this tyrosine may act as a substrate oxidation site as in the case of CWPO-C. AtPrx71 has the highest amino acid identity with CWPO-C. The results suggest a role for CWPO-C and CWPO-C-like peroxidases in the lignification of vascular plant cell walls.

Protein tyrosine nitration is a selective process, as revealed in studies of animals. However, evidence for selective protein nitration in plants is scarce. In this study, Arabidopsis plants were exposed to air with or without nitrogen dioxide at 40 ppm for 8 h in light. Proteins extracted from whole leaves or isolated chloroplasts were subjected to 2D PAGE followed by SYPRO Ruby staining and immunoblotting using an anti-3-nitrotyrosine antibody. We determined the relative intensity of a spot on an immunoblot (designated RISI), and relative intensity of the corresponding spot on SYPRO Ruby gel (designated RISS). Proteins that exhibited a high RISI value and/or a high RISI/RISS ratio were considered selectively nitrated. In whole leaf proteins from exposed plants, all immunopositive spots were identified as PsbO1, PsbO2 or PsbP1 by PMF. Thus, nitration was exclusive to PsbO and PsbP, extrinsic proteins of photosystem II (PSII). Their RISI/RISS ratio was ≤1.5. Non-exposed plants showed very faint nitration. In purified chloroplast proteins, PsbO and PsbP accounted for >80% of the total RISI values, while four non-PSII proteins, including peroxiredoxin II E, exhibited high RISI/RISS ratios (2.5∼6.6). Tyr(9) of PsbO1 was identified as a nitration site. Thus, nitration is selective for two PSII and four non-PSII proteins in Arabidopsis.