A reassessment of substrate specificity and activation of phytochelatin synthases from model plants by physiologically relevant metals

Abstract

Phytochelatin synthases (PCS) catalyze phytochelatin (PC) synthesis from glutathione (GSH) in the presence of certain metals. The resulting PC-metal complexes are transported into the vacuole, avoiding toxic effects on metabolism. Legumes have the unique capacity to partially or completely replace GSH by homoglutathione (hGSH) and PCs by homophytochelatins (hPCs). However, the synthesis of hPCs has received little attention. A search for PCS genes in the model legume Lotus (Lotus japonicus) resulted in the isolation of a cDNA clone encoding a protein (LjPCS1) highly homologous to a previously reported homophytochelatin synthase (hPCS) of Glycine max (GmhPCS1). Recombinant LjPCS1 and Arabidopsis (Arabidopsis thaliana) PCS1 (AtPCS1) were affinity purified and their polyhistidine-tags removed. AtPCS1 catalyzed hPC synthesis from hGSH alone at even higher rates than did LjPCS1, indicating that GmhPCS1 is not a genuine hPCS and that a low ratio of hPC to PC synthesis is an inherent feature of PCS1 enzymes. For both enzymes, hGSH is a good acceptor, but a poor donor, of gamma-glutamylcysteine units. Purified AtPCS1 and LjPCS1 were activated (in decreasing order) by Cd2+, Zn2+, Cu2+, and Fe3+, but not by Co2+ or Ni2+, in the presence of 5 mm GSH and 50 microm metal ions. Activation of both enzymes by Fe3+ was proven by the complete inhibition of PC synthesis by the iron-specific chelator desferrioxamine. Plants of Arabidopsis and Lotus accumulated (h)PCs only in response to a large excess of Cu2+ and Zn2+, but to a much lower extent than did with Cd2+, indicating that (h)PC synthesis does not significantly contribute in vivo to copper, zinc, and iron detoxification.

Figure 1.

Phylogenetic analysis of PCS proteins from cyanobacteria, nematodes, fungi, and plants. The unrooted tree was constructed using the neighbor-joining method (ClustalW) with 1,000 bootstrap replicates. Branch lengths are proportional to genetic distance, which is indicated by a bar (0.1 substitutions per site). GenBank accession numbers are as follows (in parentheses): Asat (Allium sativum, AAO13809), Atha1 (AtPCS1, AAD41794), Atha2 (AtPCS2, AAK94671), Ayok (Athyrium yokoscense, BAB64932), Bjun (Brassica juncea, CAC37692), Cdac (Cynodon dactylon, AAO13810), Cele (Caenorhabditis elegans, NP496475), Gmax (GmhPCS1, AAL78384), Ljap1 (LjPCS1, AY633847), Ntab (Nicotiana tabacum, AAO74500), Nostoc (Nostoc, NP485018), Osat (Oryza sativa, AAO13349), Pmar (Prochlorococcus marinus, NP894844), Taes (Triticum aestivum, AAD50592), Tjap (Thlaspi japonicum, BAB93119), Tlat (Typha latifolia, AAG22095), Spom (S. pombe, CAA92263), and Stub (Solanum tuberosum, CAD68109).

Figure 2.

Purification of heterologously expressed PCS enzymes from model plants by immobilized metal affinity chromatography. A, SDS-PAGE analysis of fractions obtained during purification of AtPCS1 and LjPCS1. Gels were stained conventionally with Coomassie Brilliant Blue R-250. Lanes: 1, desalted crude extract of recombinant AtPCS1; 2, proteins eluted with 50 mm imidazole; 3, AtPCS1 eluted with 250 mm imidazole; 4, AtPCS1 preparation used to obtain 5, prior to thrombin digestion; 5, AtPCS1 after tag removal with thrombin; 6, desalted crude extract of recombinant LjPCS1; 7, LjPCS1 eluted with 250 mm imidazole; and 8, LjPCS1 after tag removal with thrombin. Lanes were loaded with 20 μg of protein. B, Immunoblot analysis of fractions shown in Figure 2A, using anti-polyHis monoclonal antibody and an alkaline phosphatase-based detection system (Sigma). Lanes were loaded with 2 μg of protein.

Figure 3.

Substrate specificity of PCS enzymes from model plants. A, Desalted crude extracts of AtPCS1 (200 μg of protein) and LjPCS1 (120 μg of protein) were assayed (30 min) with 500 μm Cd²⁺ and the stated concentrations of (h)GSH. B, Purified AtPCS1 (60 μg of protein) and LjPCS1 (60 μg of protein) were assayed (30 min) with 50 μm Cd²⁺ and the stated concentrations of (h)GSH. The polypeptides PC₂ (yellow), PC₃ (orange), PC₄ (red), hPC₂ (light blue), hPC₃ (dark blue), and hPC₄ (green) were quantified by HPLC with postcolumn derivatization. Values are given in nmol of (h)PCs produced (GSH equivalents) per minute per milligram of protein and represent means ± se of four to six enzyme preparations.

Figure 4.

Activation of PCS enzymes from model plants by physiologically relevant metals. A, Desalted crude extracts of AtPCS1 (200 μg of protein) were assayed (30 min) with 500 μm metal ions and 5 mm GSH. Values represent means ± se of three to six enzyme preparations and are expressed in percent relative to the PCS activities with Cd²⁺. The 100% value corresponds to a specific activity of 56 ± 7 nmol of total PCs (GSH equivalents) produced per minute per milligram of protein. B, Purified AtPCS1 (60 μg of protein) and LjPCS1 (60 μg of protein) were assayed (30 min) with 50 μm metal ions and 5 mm GSH. Values represent means ± se of four to eight enzyme preparations and are expressed in percent relative to the PCS activities with Cd²⁺. The 100% values correspond to specific activities of 87 ± 3 (AtPCS1) and 89 ± 14 (LjPCS1) nmol of total PCs (GSH equivalents) produced min⁻¹ mg⁻¹ of protein.

Figure 5.

Activation of PCS enzymes from model plants by Fe. A, Representative HPLC chromatogram showing activation of purified AtPCS1 by 5 mm GSH and either 50 μm Cd²⁺ (solid line) or 50 μm Fe³⁺ (dotted line). The latter reaction is completely inhibited by 500 μm desferrioxamine (dashed line). Note the synthesis of PC_2–4 with Cd²⁺ and of PC_2–3 with Fe³⁺ after the 30-min incubation period. B, Purified AtPCS1 (60 μg of protein) and LjPCS1 (60 μg of protein) were assayed with 50 μm metal ion, 5 mm GSH, and either 0 (white bars) or 500 μm (black bars) of desferrioxamine. AtPCS1 was incubated for 30 min and LjPCS1 for 60 min. All reactions were completely inhibited by 500 μm EDTA. Values are means ± se of two to three enzyme preparations and are expressed in percent relative to the PCS activities with Cd²⁺. The 100% values correspond to specific activities of 91 ± 4 (AtPCS1) and 68 ± 4 (LjPCS1) nmol of total PCs (GSH equivalents) produced per minute per milligram of protein.

Figure 6.

Synthesis of PCs and hPCs in model plants in response to metals. Arabidopsis and Lotus plants were treated for 4 d with 100 μm Cd²⁺, 100 μm Cu²⁺, 100 μm Zn²⁺, or 500 μm Fe³⁺. Figure shows (h)PC contents in whole plants of Arabidopsis and in roots of Lotus. Control (untreated) and Fe³⁺-treated plants had no detectable (h)PCs. Color codes for the (h)PC polypeptides are as in Figure 3. Values are means ± se of three to four plants and are expressed in nanomoles of (h)PCs (GSH equivalents) per gram of fresh weight.