Isolation and characterization of a new peroxiredoxin from poplar sieve tubes that uses either glutaredoxin or thioredoxin as a proton donor

Abstract

A sequence coding for a peroxiredoxin (Prx) was isolated from a xylem/phloem cDNA library from Populus trichocarpa and subsequently inserted into an expression plasmid yielding the construction pET-Prx. The recombinant protein was produced in Escherichia coli cells and purified to homogeneity with a high yield. The poplar Prx is composed of 162 residues, a property that makes it the shortest plant Prx sequence isolated so far. It was shown that the protein is monomeric and possesses two conserved cysteines (Cys). The Prx degrades hydrogen peroxide and alkyl hydroperoxides in the presence of an exogenous proton donor that can be either thioredoxin or glutaredoxin (Grx). Based on this finding, we propose that the poplar protein represents a new type of Prx that differs from the so-called 2-Cys and 1-Cys Prx, a suggestion supported by the existence of natural fusion sequences constituted of a Prx motif coupled to a Grx motif. The protein was shown to be highly expressed in sieve tubes where thioredoxin h and Grx are also major proteins.

Figure 1

Amino acid sequence alignment of plant Prx. The first three sequences belong to type C Prx, the three following sequences are 2-Cys Prx (type A), and the last three are 1-Cys Prx (type B). This alignment was performed using the Clustal W program, and the accession numbers for these sequences are: Arabidopsis, AAD28242; B. rapa, AAD33602; 2-Cys B. rapa, AF052202; 2-Cys Arabidopsis, BAB08951; 2-Cys Spinacia oleracea, O24364; 1-Cys Hordeum vulgare, CAA65387; 1-Cys Oryza sativa, BAA09947; and 1-Cys Arabidopsis, Y12089. Asterisk, Indicates strict homologies between all the sequences and conserved Cys within a Prx subtype.

Figure 2

Size determination and purity of recombinant poplar Prx. The size marker used is Precision Protein Standards, unstained from Bio-Rad (Hercules, CA). Each lane contains about 3 μg of Prx. Lane 1, SDS and β-mercaptoethanol; lane 2, SDS alone; lane 3, SDS and dithiothreitol (DTT).

Figure 3

Prx-dependent inactivation of DNA cleavage by MCO. Plasmid pLBR19 was added in each reaction mixture after 40 min. Lane 1, pLBR19 alone; lane 2, DTT alone; lane 3, DTT + FeCl₃; lanes 4 through 6, DTT + FeCl₃ + various amounts of Prx (10, 20, and 40 μm); lane 7, DTT + FeCl₃ + bovine serum albumin (BSA; 20 μm); lane 8, ascorbate + FeCl₃. Nicked form (NF) and supercoiled form (SF) of pLBR19 are indicated on the left.

Figure 4

Trx and Prx dependency of H₂O₂ reduction. The concentrations of the other components are as follows: 150 μm NADPH, 1.6 μm NADPH Trx reductase (NTR), and 100 μm H₂O₂. A, Prx concentration is 5 μm; B, Trx concentration is 4 μm.

Figure 5

Grx and Prx dependency of H₂O₂ reduction. The concentrations/amounts of the other components are as follows: 150 μm NADPH, 0.5 units glutathione reductase (GR), 1 mm GSH, and 100 μm H₂O₂. A, Prx concentration is 40 μm; B, Grx concentration is 12 μm. The background activities due to reduced glutathione alone (about 0.1 Δ OD/min) have been substracted.

Figure 6

: Detection of Prx mRNA and polypeptide in planta. L, Leaves, S, stems; R, roots. A, Northern-blot analysis: as a control, 28S rRNA stained by Radiant Red RNA gel stain from Bio-Rad before blotting is shown. B, Western-blot analysis with purified anti-Prx antibody.

Figure 7

Electron micrographs of poplar sieve elements. A, One sieve element (SE) containing sieve element plastids (Pl) and parietal P proteins (PP) shown with companion cell (CC). B, Prx labeling in sieve element plastids.

Figure 8

The two pathways involved in type C Prx catalysis.