Purification and characterization of peroxidases correlated with lignification in poplar xylem

Abstract

Lignin is an integral cell wall component of all vascular plants. Peroxidases are widely believed to catalyze the last enzymatic step in the biosynthesis of lignin, the dehydrogenation of the p-coumaryl alcohols. As the first stage in identifying lignin-specific peroxidase isoenzymes, the classical anionic peroxidases found in the xylem of poplar (Populus trichocarpa Trichobel) were purified and characterized. Five different poplar xylem peroxidases (PXP 1, PXP 2, PXP 3-4, PXP 5, and PXP 6) were isolated. All five peroxidases were strongly glycosylated (3.6% to 4.9% N-glucosamine), with apparent molecular masses between 46 and 54 kD and pI values between pH 3.1 and 3.8. Two of the five isolated peroxidases (PXP 3-4 and PXP 5) could oxidize the lignin monomer analog syringaldazine, an activity previously correlated with lignification in poplar. Because these isoenzymes were specifically or preferentially expressed in xylem, PXP 3-4 and PXP 5 are suggested to be involved in lignin polymerization.

Figure 1

Anionic peroxidase activities from poplar (P. trichocarpa Trichobel). Extracts from root bark (1), stem bark (2), leaf (3), root xylem (4), and stem xylem (5) were analyzed on a native gel. Peroxidases were visualized with DAB and H₂O₂ as described in Methods. The letters a through f were chosen arbitrarily to represent the xylem gel activities. SM, Slow migrating; FM, fast migrating. The protein migration was from − to +.

Figure 2

Mono-Q separation of the anionic peroxidase isoenzymes. The elution of the Mono-Q column was recorded at 404 nm (lower chromatogram, lower scale to the left) and at 280 nm (upper chromatogram, scale to the right). The upper line represents the NaCl concentration in the elution gradient (upper scale to the left). The horizontal scale represents the elution volume. The eluting peaks are designated PXP 1, PXP 2, PXP 3, PXP 4, PXP 5, and PXP 6.

Figure 3

Native gel analysis of the separated isoenzymes. The isoenzymes were separated in a native gel and visualized with DAB and H₂O₂ as in Figure 1. The numbers above the lanes correspond to the isoenzyme names given in Figure 2. The letters a through e represent the xylem gel activities, as described in the legend to Figure 1. The protein migration was from − to +.

Figure 4

Analysis of the purified isoenzymes on a denaturing gel. SDS-PAGE (10%) analysis of the purified isoenzymes (50 pmol), stained with Coomassie blue. Apparent molecular mass markers (M) are given with mass indications in kD. The numbers above the lanes correspond to the isoenzyme names given in Figure 2.

Figure 5

Analysis of the substrate specificity of the peroxidase isoenzymes. The rate of oxidation for the three substrates ABTS, DAB, and SYR was measured for the six purified isoenzymes as described in Methods. The activity of the most active isoenzyme for each substrate was set to 100. The measurements were repeated 10 times. The means ± sd values are indicated. Enzyme activity measurements were performed using 10 pmol of peroxidase isoenzyme in a 200-μL reaction volume. Gray bars, DAB; black bars, ABTS; white bars, SYR.

Figure 6

Analysis of the peroxidases by trypsin digestion. Comparison of the reversed-phase HPLC separation chromatograms of the trypsin-generated peptides from the four most abundant peroxidases. mAU, Milli-absorbance unit.

Figure 7

Amino acid sequences of the trypsin- and cyanogen bromide-generated peptides. Underlined sequences are putative glycosylation sites (NXT/S or 0XT/S, where X represents any amino acid except P). If N was linked to a glycan, it was not detected during sequencing (indicated by 0). Sequences placed at the end (double underlined) showed no homology to other peptide sequences obtained.

Figure 8

Correlation of peroxidase isoenzymes, gel activities, and SYR oxidation. The top panel represents a stem xylem lane from Figure 1. The bottom panel represents the 404-nm chromatogram from Figure 2. The dashed lines indicate the relation between gel activities and the Mono-Q-separated peroxidase isoenzymes. PXP 1 to PXP 6 represent the gene products determined from the molecular characterization. The SYR-oxidizing isoenzymes are indicated.