Electronic Supplementary Material (ESI) for Metallomics This journal is © The Royal Society of Chemistry 2013 Diversity and Distribution of Plant Metallothioneins: A review of structure, properties and functions Oksana I. Leszczyszyn, Hasan T. Imam and Claudia A. Blindauer* Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK Supplementary Information Materials and Methods for Section 5.4. Figure S1. Distribution of plant metallothionein and metallothionein-like nucleotide sequences within the families of land plant phyla. Figure S2. Mass spectral data for metal-free A. thaliana MT4a and MT4b. Table S1. Expression profiles of Type 1 pMTs. Table S2. Expression profiles of Type 2 pMTs. Table S3. Expression profiles of Type 3 pMTs. Table S4. Expression profiles of Type 4 pMTs. Table S5. Spatial expression profiles of EST transcripts of Type 4 pMTs. Electronic Supplementary Material (ESI) for Metallomics This journal is © The Royal Society of Chemistry 2013 S1. Materials and methods S1.1 Recombinant protein expression and purification Expression plasmids for MT4a and MT4b were provided by Professor Peter Goldsbrough (Purdue University, USA). Both genes were cloned into the pET-28a vector using NcoI and BamHI restriction sites, without any affinity tags, and kanamycin was used as selective antibiotic. MT4a and MT4b were overexpressed in E. coli (Rosetta 2(DE3) pLysS) cells (Novagen). Transformation was performed according to the protocol provided by the manufacturer. Expression cultures, typically 0.4-0.6 L, were induced by IPTG (isopropylthiogalactoside; 0.5 mM) at an OD600 of ca. 0.7-0.75. At induction, 0.5 mM ZnSO4 was also added. Protein expression was carried out at 37°C, and cells were harvested after 5 hours of induction by centrifugation at 3000×g. The cell pellet was stored at -20°C. Sonication buffer (3ml/gram per cell weight, 50 mM Tris-Cl, 0.1 M KCl, 3 mM dithiothreitol (DTT), 1 mM ZnSO4, pH 8.5) was added to the thawed cell pellet and cells were ruptured by sonication. Streptomycin solution (10%, 0.375ml/gcw) was added to the translucent suspension before centrifugation at 30000×g at 4°C for 50 minutes. The supernatant was collected and subjected to chemical precipitation. In brief, 0.5 volumes of an ice cold ethanol:chloroform (100:8) mixture (1 volume equivalent to total supernatant volume) was added drop-wise to the supernatant with constant stirring in an ice bath, and the precipitated proteins were collected by centrifugation at 5000×g for 10 minutes at 4°C and stored at -20°C. Another 3.5 volumes of the ethanol:chloroform (100:8) mixture were added to the supernatant and the protein pellet was collected by centrifugation at 5000×g for 10 minutes at 4°C and stored at -20°C. The precipitate from the 3.5 vol fraction was re-dissolved in ammonium bicarbonate buffer (20 mM, pH 7.45) and filtered (0.2μm-0.4μm, Minisart®). Purification was achieved by FPLC (Pharmacia Äkta Purifier) using a size exclusion column (HiLoad 16/60 Superdex 75, Amersham Biosciences) and eluting isocratically in ammonium bicarbonate buffer (20 mM, pH 7.45). The elution of protein fractions was monitored at 220 nm and 280 nm. Selected fractions containing the protein were concentrated using Amicon Ultra-4 (3000 MWCO) filter devices. Protein concentration was determined by ICP-OES or by Ellman’s Test, and ESI-MS spectra were recorded on 25 M samples. Electronic Supplementary Material (ESI) for Metallomics This journal is © The Royal Society of Chemistry 2013 S1.2 Preparation of Cd loaded MT4a and MT4b Zn6MT4a (2.0 mL, 34 μM, 20 mM ammonium bicarbonate) was incubated with dithiothreitol (250 μL, 100 mM) in ice for 1h. Hydrochloric acid (250 μL, 1 M) was added to the sample to lower the pH to ca. 1. The sample was then loaded under N2 onto a PD-10 column (Sephadex G-25; GR Healthcare) pre-equilibrated with 0.01 M HCl. Elution of the apo-protein was achieved by addition of HCl (3.5 mL, 0.01 M) under N2. To the collected apo-protein, CdCl2 (8 molar equivalents) was added and the pH of the samples was raised to 8.5 by addition of NH3 solution (1 M). The volume of the protein samples was reduced to 2.5 ml by ultrafiltration and then buffer exchanged (10 mM ammonium bicarbonate) using a gel filtration column (GE Healthcare PD-10). The mass spectrum of the sample (25 M) was recorded. Reconstitution of MT4b with Cd(II) was achieved following the same procedure. S1.3 Electrospray Ionisation Mass Spectrometry Mass spectra were recorded on a Bruker Daltonics MicroTOF spectrometer with electrospray ionisation source at a source temperature of 468 K. The samples (25M in 10 mM NH4HCO3, pH 7.0-7.5, 10% MeOH for metallated proteins), were directly infused into the spectrometer by a syringe pump with a flow rate of 240 μL/hr. Data were recorded for 2 min in positive mode over a mass range of 500-5000 m/z. The raw data were smoothed and deconvoluted onto a true mass scale using the CompassTM Data Analysis program (v. 4.0, Bruker Daltonics, Bremen). If necessary, protein solutions were concentrated using Amicon® Ultra-4 filters (3000 MWCO) prior to the addition of 10% methanol. The apo forms of the proteins were generated by addition of formic acid (2% v/v) to the same mixture, and a second mass spectrum was acquired. S1.4 UV absorption spectroscopy pH titrations of Zn(II) and Cd(II) loaded proteins were carried out using a Cary 50 Bio UV- Visible spectrophotometer (Varian) in the range of 200-300 nm at room temperature. Respective protein samples (10 μM, in 1 mM Tris buffer, pH 7.92) were placed into a quartz Electronic Supplementary Material (ESI) for Metallomics This journal is © The Royal Society of Chemistry 2013 cuvette and titrated with dilute HCl (0.01-0.5 M, ~1-1.5 µL). Sample dilution due to addition of HCl was negligible. The pH of the samples was measured before and after the UV-spectrum was recorded. The pH(1/2) values were estimated by recording maximum and minimum absorbance, and reading the respective pH value relating to (Amax-Amin)/2. Although the curves shown in Fig. 13 are not fitted lines, there are a sufficient number of points in the relevant regions to permit this procedure for obtaining an estimate. Electronic Supplementary Material (ESI) for Metallomics This journal is © The Royal Society of Chemistry 2013 Gymnosperms Pterophytes Coniferophyta Ginkgophyta Cycadophyta Gnetoophyta Lycophyta Pterophyta Salviniaceae (1) Pinaceae (73) Gnetaceae Sellaginellaceae (6) Cycadaceae Dennstadtiaceae Cupressaceae (1) Ginkgoaceae Ephedraceae Lycopodiaceae Zamiaceae Adiantaceae Taxodiaceae Welwitschaceae Isoetaceae Osmundaceae Angiosperms Bryophytes Eudicots Monocots Magnoliidae Bryophyta Hepatophyta Anthocerophyta Brassicales (112) Poales (348) Laurales (2) Funariaceae Fabales (80) Alismatakes (42) Piperales Pottiaceae Jungermanniaceae Solanales (53) Zingiberales (11) Magnoliales Grimmiaceae Malpighiales (52) Asparagales (8) Rosales (32) Commelinales (8) Caryophyllales (27) Arecales (6) Lamiales (26) Lilales (4) Vitales (25) Pandanales (2) Cucurbitales (23) Sapindales (11) Ericales (11) Gentianales (10) Asterales (8) Fagales (7) Myrtales (6) Malvales (5) Apiales (5) Proteales (3) Dipsacaes (1) Aquifoliales (1) Salifrigales (1) Ranunculales (1) Broaginaceae (1) Figure S1. Distribution of plant metallothionein and metallothionein-like nucleotide sequences within the families of land plant phyla. Those in grey and dashed lines refer to taxa in which pMT sequences are available in the expressed sequence tag (EST) database at NCBI. Electronic Supplementary Material (ESI) for Metallomics This journal is © The Royal Society of Chemistry 2013 1360.7 8158.2 100 A Apo MT4a C pH 2.37 6+ Apo 80 60 1632.6 5+ 40 M1 20 0 8306.3 80 B 1385.2 Apo MT4b D pH 2.76 6+ Apo 60 40 1662.0 5+ 20 M1 0 1300 1400 1500 1600 1700 7900 8100 8300 8500 8700 Mass (Da) m/z Figure S2. ESI-MS spectra of Arabidopsis thaliana MT4a and MT4b at low pH, giving the apo forms of the proteins (25 M protein, 10 mM NH4HCO3, 10% MeOH, 2% formic acid). Charge state spectra are shown in (A) MT4a and (B) MT4b, and deconvoluted spectra giving the neutral species are shown in (C) for MT4a and (D) for MT4b.Theoretical masses are 8159.1 for MT4a and 8306.1 for MT4b. In both cases, the N- terminal methionine is absent. Electronic Supplementary Material (ESI) for Metallomics This journal is © The Royal Society of Chemistry 2013 Table S1. Expression profiles of Type 1 pMTs. Spatial and temporal expression information is provided, as well as the experimentally observed up- or down- regulation of pMT transcripts as a result of exposure to exogenous compounds. Potential regulatory elements are provided if available. Organ of expression Regulatory Plant Species Developmental expression Up or Down Regulation† Ref. Shoot Root Leaf Embryo Fruit Other element‡ Arabidopsis thaliana Y Y Y Y Flowers; Senescing leaves; Developing embryo Up by Cu No MRE 1, 2, 3, (1a & c) Buds; Stem 4 Brassica campestris Y Low Low in Strong in roots Up by Cd - roots & leaves; up by Cu -roots 5 stems Brassica rapa Seedlings Up by ABA, ET, Mn, SA, MeJa, H2O2, cold & ABRE, ARE, 6 methyl viologen; Strongly up by PEG, MeJaRE, SARE, NaCl; Not by Fe; Slightly up by Zn, Cu ERE, JARE, defence, stress, wounding Cajanus cajan Seedlings Up by Cu, Cd, Zn 7 Catharanthus roseus Y Not by S. citri infection 8 Casuarina glauca Low Y Y Nitrogen Young leaves Not by Cu, Zn, Cd; No MRE; ARE 9, 10 fixing Promoter: up by X. campestris present; G- & H- nodules box; Nodule- specific element Chloris virgata swartz Y Up by Cu, Zn, Co, paraquat &