Diversity and Distribution of Plant Metallothioneins

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 (25M 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 & salt stress 11

Elsholtzia haichowensis Y Y Strong in roots & weaker in leaves Up by Cu, heat & H2O2 12 Festuca rubra Y Y Up by Cu, Cd 13 Glycine max Y Y Low Strong in roots & weak in leaves Down by Cd exposure (short-term, then 14 up at 40hrs 1 Gossypium hirsutum Y Strongly at root tip 15 Hordeum vulgare Y Up in Fe-deficient conditions 16 Mimulus guttatus Y Low Not by Cu 17 Nelumbo nucifera Y Y Low in Strong in roots, mature stage of developing Up in embryonic axes with salinity stress; 18 stems seeds & early germination; absent in seeds Up by H2O2, methyl viologen, Cu; Down by exposed to heat stress Zn; Not by Cd & Pb Oryza sativa (1b) Y Y Present in seed development; young & Down by Pb; Up by cold & salinity 19 mature leaves; late pannicle development stresses; Not by drought stress Oryza sativa (1c) Y Down by Pb, cold & salinity stress; Not by drought stress Oryza sativa (1d) Y Down by Pb, salt & drought stress; Up after M. grisea infection Oryza sativa (1e) Y Y Late stage embryo development Down by Pb, cold, drought and salinity stresses Oryza sativa(1f) Y Down by Pb, NaCl, drought Oryza sativa (1b) Y Y Y Flower Weak in leaves & shoots Slightly up by H. seropedicae infection; Up 20, 21 by Zn, PEG Oryza sativa (1a) Y Y Present in seed development & young Down by Pb; Up by cold stress; Not by 19 leaves NaCl, drought Pisum sativum Y Low - Modified MRE 22 Populus alba Y Up by Zn 23

Prosopis julifora Y UpbyCu,ABA,H2O2; Slightly up by Zn, Cd CuRE, ERE, STRE, 24 No MRE, ABRE, OSE Saccharum spp. Y Y Up by Cd 25 Electronic Supplementary Material (ESI) for Metallomics This journal is © The Royal Society of Chemistry 2013 Solanum nigra Y Low Strong in shoots & weaker in roots Down by Ni -roots; Not induced by Ni - 26 shoots Triticum aestivum Y Y Up by wounding, Cu, Ga, In; Slightly up by 27 Al, Fe, Cd, Zn, La; Up by Ca deficiency; Not by P or Mg deficiency Trifolium repens Node - 28 Vicia faba Y Y Stem Young leaves Not by Cu, Zn, Fe, Cd 29 Zea mays Y Low - Up by wounding, glucose starvation, salt No MRE 30,31 & heavy metal stress † ABA – abscisic acid; PEG – Polyethyene glycol; ET – ethephon; MeJa – methyl jasomonate; SA – salicylic acid; PQ - paraquat ‡ ABRE = abscisic acid responsive elements; ARE = antioxidant responsive element; CuRE = copper-respopnsive element; ERE = ethylene responsive element; MeJaRE = methyl jasmonate responsive element; MRE = metal responsive element; ORE = organ-specific responsive element; RSE = root-specific element; SARE = salicylic acid response element; STRE = stress responsive element; Electronic Supplementary Material (ESI) for Metallomics This journal is © The Royal Society of Chemistry 2013

Table S2. Expression profiles of Type 2 pMTs. Spatial and temporal expression of is provided, as well as the experimentally observed up- or down- regulation of pMT transcripts as a result of exposure to exogenous compunds. 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‡ Abutilon theophrastii Y Up by C. coccodes infection 77 Actinidia deliciosa (Kiwi) Low Low Y Low Early fruit development 78 Allium sativum Y Up by Cd 32 Arabidopsis thaliana(2a/b) Y Y Y Flowers; Developing embryo 2a: Up by Cu; 2b: constitutive 1, 3, 4 Stem

Arachis hypogaea Y Y Stems Strong - mature leaves; weak - young Up by Cu, Cd, ABA, H2O2, heat; Not by PEG 33, 34 leaves; Strong in young roots; absent - or Zn; Up by salt stress – roots; Up by C. mature roots personatum infection Avicennia germinans Y Not by Cu; Up by Cd 35 Avicennia marina Y Y Stem Up by Zn, Cu, Pb 36, 37 Azolla filiculoides Y Up by Cd, Zn, Ni, Cu Brassica campestris (rapa) Y 38

Brassica rapa Seedlings Down by ABA, methyl viologen, H2O2; Up ERE, ARE, LTR, 6 by ET, NaCl; Not by Cu, PEG; Slightly up defence & STRE by Fe, Zn, Mn 2 Brassica campestris Low Y Stem (l) Strong in leaves Up by Cd - roots & leaves; Not by Cu 5 Brassica juncea Seedling Down by Cu 39 Brassica napa X Y Senescing leaves 40 Brassica oleracea Florets 41 Brugueria gymnorrhiza Y Up by Zn, Cu, Pb, Cd 42, 43 Cicer microphyllum Y Y Y Weak in young leaves, but strong in roots Up by cold, ABA, PEG, Zn 44 Citrullus lanatus Y Up by drought 45 Citrus unshiu Y Y Y Y Flower Senescing leaves; early ripening Not by Zn, Cu 46 Coffea arabica Y 47 Eichhornia crassipes Y 48 Fragaria ananassa Y Fruit ripening 49 Fagus sylvatica Y 50 Glycine max Low Y Low Strong in leaves; Weaker in roots Down by Cu, Up by Cd, but not in roots 51, 14 Helianthus tuberosus Node Down by Cu 52

Hevea brasiliensis Low Y Bark; Latex Strong - young leaves & latex; Weak - roots Up by ethephon & H2O2 – latecifers 53 & bark Hordeum vulgare X Y Up in drought stress 54 Ipomoea batatas Y Y Stem Senescing leaves Up by ethylene 55 Kandelia candel Low Y Stem Strong - young leaves; Weak - roots & stems Up by Zn, Cu, Cd, Pb - leaves 56 Lycopersicon esculentum Y Y Y Y Flowers Up by Zn & Fe deficiency MRE 57, 58 (1)(4) Lycopersicon esculentum (2) Y Down by Cu Lycopersicon esculentum (3) Y Low Flower Down by diamide Lycopersicon esculentum Y Y Up by B 59 Malus domestica Y Y Peel Senescing leaves Up in cooling, ripening fruit 60 Musa acuminata Y Early ripening (2a); Late ripening (2b) Up by ET (2a); Up by Zn, Cd, Cu (2b) 61 Electronic Supplementary Material (ESI) for Metallomics This journal is © The Royal Society of Chemistry 2013 Nicotiana glutinosa Y Up by pathogen, wounding 62

Nelumbo nucifera YYY Stems (l) Strong - developing seed; Up in early Up by NaCl, H2O2, methyl viologen – 18 Flowers (l) imbibition of germinating seeds; Weak in embryonic axes; Up by Cu, Pb; Not by Cd; other tissues Slightly down by Zn Oryza sativa (MT2c) Y Y Y Node Young leaves; Up by metal ions – stems; Down by metal 63, 19 Present in seed development; mature ions – roots; Not by Pb, drought; Down by leaves not young leaves; mid & late stage cold, NaCl pannicle development Oryza sativa (MT2a) Y Y Present in seed development; young & Up by Pb - roots; Up by drought, NaCl - 19 mature leaves; mid & late stage panicle seedlings; Not by cold stress; Up by M. development grisea infection Oryza sativa (MT2b) Y Y Present in seed development; young & Down by Pb; Up by drought, NaCl - mature leaves; mid & late stage panicle seedlings; Not by cold stress; Up by M. development grisea infection Low X Y Weak in young root, mature stem, ovary & Down by cold - young roots & shoots; Up ABRE, GARE, 64, 65 stigma; Strong in panicle & germinating by Fe, Zn, IAA; down by Cu, 6-BA, KT, LTRE, HSE, W- embryos NaCl, ZT - roots; Up with Mn, Cu but box, GT-1 motif, downby 6-BA & KT, Fe, GA - shoots I-box, DRE, PRE1, HMRE Persea americana Y Up with Phytophthora infection 66 Populus alba Y Low Up by Zn 23 Populus euphratica (2a) Y Up by drought stress 95 Populus euphratica (2b) Low Y Young leaves Up by drought stress Posidonia oceanica (2b) Y Y Y Young leaves 67 Posidonia oceanica (2f) Low Y Y Flowers

Prosopis julifora Y Up by Zn, ABA & H2O2; Down by Cu, Cd No MRE, ABRE, 24 OSE Prunus armeniaca Y 68 Prunus persica Y 69

Quercus suber Y Y Y Stem Up by H2O2, paraquat 70 Ricinus communis Cotyledon 71 Saccharum spp. YY 25 Salicornia brachiata Seedlings Up by Zn, Cu, Cd, NaCl, heat, cold & 72 drought stresses Sambucus nigra L. Y Leaflets Present in abscission zone; absent in non- Up by ET - abscission zone; possibly 73 abscission zone responds to wounding Silene nicaeensis Y Y Stem Strong in young leaves; present in mature 67 leaves & stem dividing cells Silene vulgaris Y - Not by metals; constitutive 74 Solanum nigra Seedlings 26 Solanum nigra Y Up by Ni –shoots Solanum nigra Y Y Up by Ni Thlaspi caerulescens (2a) Y Y Up by Zn 75 Trifolium repens Node 28 Typha latifolia Y Y Down by Cu; Up by Cd 76 Vitis vinifera var. Shiraz Y Early ripening 105 † ABA – abscisic acid; ET – ethephon; GA – giberellic acid; IAA – indole-3-acetic acid (auxin); KT – kinetin (a cytokinin); MeJa – methyl jasomonate; PEG – polyethyene glycol; ZT - zeatin; 6-BA – 6-benzylaminopurine ‡ ABRE = abscisic acid responsive elements; ARE = antioxidant responsive element; ERE = ethylene responsive element;MRE = metal responsive element; ORE = organ-specific responsive element; RSE = root-specific element; STRE = stress responsive element; LTE = light responsive element; GARE = gibrellic acid responsive element; LTRE = low temperature responsive element; DRE = drought responsive responsive element; PRE1 = pollen responsiv element; HMRE = high metal responsive element Electronic Supplementary Material (ESI) for Metallomics This journal is © The Royal Society of Chemistry 2013 Table S3. Expression profiles of Type 3 pMTs. Spatial and temporal expression of is provided, as well as the experimentally observed up- or down- regulation of pMT transcripts as a result of exposure to exogenous compunds. 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 Abutilon theophrastii Y Up by C. Coccodes infection 77 Actinidia deliciosa (Kiwi) X X Y Low in Late stage fruit development & ripening; 78 flowers Weaker in ripe fruit Alstroemeria var. Rebecca Petal Up in flower senescence 79 Arabidopsis thaliana Y Y Y Y Flower; Senescing leaves; developing embryo Up by Cu 3 Stem; Buds Brassica rapa Seedling Down by ABA, methyl viologen; Up by Mn, 6 ET, MeJA, H2O2, NaCl, cold; Not by Cu, PEG; Slightly Up by Fe, Zn Carica papaya Y 80

Citrus unshiu Y Y Not flower; Not by metal ions 81, 46, Seedling 82 Elaeis guineensis (3a) X Low Y X Y Senescing leaves; early ripening 83 Elaeis guineensis (3b) X Y X X Y Late ripening RSE, ERE, MRE Fagopyrum esculentum Y Y Young & senescing leaves Up by Cu, Zn – leaves; Up by Zn – seeds 84 Fagopyrum esculentum Y Y Y Early to mid maturation stages of 85 developing seed; Weak - roots. Strongly – leaves

Fagopyrum esculentum Y Y Young & mature leaves; Developing embryo Up by drought, H2O2 86 3 Fragaria vesca Low Low Y Low in Present during late stage fruit development Up by wounding 87 flowers; & max during fruit ripening Stem Glycine max Y Y Y Strong in leaves. Weak in roots. Not induced by Cd – roots 14 Gossypium hirsutum cotyledon 88

Gossypium hirsutum cotyledon Up by Cu, Zn, ABA, PEG, ET, H2O2, PQ, 89 NaCl, cold Ipomoea batatas Y Y Stem Not by developmental stages 55 Hordeum vulgare Y 90 Malus domestica Y Y Y Not flower; Senescing leaves; Down in ripening fruit Up in cooling 60 Peel Musa acuminata Y Late ripening Up by Zn, Cd, Cu 61

Nelumbo nucifera Y Y Stems; Low Absent in developing seed, but present after Up by NaCl, H2O2, Zn, Cu, Cd, Pb - 18 in flowers germination embryonic axes Oryza sativa Y Y Present in seed development; young & Up by Pb - roots; Down by cold, NaCl, 19, 91 mature leaves drought; Up after M. grisea infection Oryza sativa X Y Not in Mature & senescing leaves Up by Cu, PEG, cold, NaCl - leaves; Down 92 buds; Stem by Pb (l) Picea glauca Y Not by ABA 93 Picea abies Needles Up by pollution damage 94 Populus alba Y Fruit ripening 23 Populus euphratica Y Up by drought stress 95 Electronic Supplementary Material (ESI) for Metallomics This journal is © The Royal Society of Chemistry 2013

Populus deltoids Stem Down by drought stress 96 Populus deltoids Stem Up by drought stress Populus x canadensis Stem Down by drought stress Populus x canadensis Stem Not by drought stress Populus tremula Y Weak - young leaves; Strong - senscing 97 leaves

Prosopis julifora Y Up by ABA, Cd, Zn, Cu, H2O2 No MRE; 24 ABRE;OSE Porteresia coarctata Y Y Abundant in roots & leaves Up by Zn, Cd, Cu – leaves RSE, ABRE, STRE 98 (Oryza coarctata) Prunus armeniaca Y Up in response to phytoplasma infection 99 Prunus avium Y 100 Ribes nigrum L. Y Y Y Stem Strong in fruit ripening; Plants producing 101 fruit – roots & stem (weak); Plants not producing fruit - leaves only Rubus idaeus X Low Not stem Strongly expressed in late devlopment and 102 in ripening fuit; Weaker in ripe fruit Saccharum ssp. Y 25 Solanum nigra Y Not constitutively expressed - roots and Up in shoots with Ni exposure 26 shoots Solanum nigra Not constitutively expressed - roots and shoots Tamarix hispida Y Y Weak in roots & leaves Up by Cd, Cu, Zn, NaCl - leaves; Up by Cd, 103 NaCl – roots Thlaspi caerulescens Y Low Up by Cu; Not by Cd 104 Vitis vinifera var. Shiraz Y Late ripening 105 † ABA – abscisic acid; MeJa – methyl jasmonate; PEG – Polyethyene glycol; ET – ethephon ‡ ABRE = abscisic acid responsive elements; ARE = antioxidant responsive element; ERE = ethylene responsive element;MRE = metal responsive element; ORE = organ-specific responsive element; RSE = root-specific element; STRE = stress responsive element Electronic Supplementary Material (ESI) for Metallomics This journal is © The Royal Society of Chemistry 2013

Table S4. Expression profiles of Type 4 pMTs. Spatial and temporal expression of is provided, as well as the experimentally observed up- or down- regulation of pMT transcripts as a result of exposure to exogenous compunds. 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 Mature & dry embryo Up by ABA, MeJa, Man; Down by ZT, GA, 4 cold; Down by IAA - MT4b; Up by NaCl - MT4a Glycine max XXY NotbyCd 14 Hordeum vulgare Y Developing seed 90 Oryza sativa Y ABRE,ERE106 Pseudotsuga menziesii Y Up by metal ions 107 4 Up by osmotic stress Up by ABA Sesamum indicum X X X Y X Developmental 108 Triticum aestivum Y Pollen Strongly in embryonic microspores Up by ABA - seed; ABRE 109, Down by light - pollen; Up by Ca – pollen 110, 111 Xerophyta humilis Y Y Up by drought stress 112 Zea mays X X X Y X 113 † ABA – abscisic acid; GA – giberellic acid; IAA – indole-3-acetic acid (auxin); Man - mannitol; MeJA – methyl jasmonate; ZT - zeatin; ‡ ABRE = abscisic acid responsive elements; ARE = antioxidant responsive element; ERE = ethylene responsive element;MRE = metal responsive element; ORE = organ-specific responsive element; RSE = root-specific element; STRE = stress responsive element Electronic Supplementary Material (ESI) for Metallomics This journal is © The Royal Society of Chemistry 2013 Table S5. Spatial expression profiles of EST transcripts of Type 4 pMTs. Data from different plant phyla were mined from the EST database at NCBI using the Type 4 plant MT from wheat (P30569) as a seed sequence.

Location of expressed EST Plant species and accession number

Seed/ Embryo/ Pod Secale cereale (BE494154); Eleusine coracana (CX264798); Saccharum hybrid cultivar (CA284519); Punica granatum (JZ123610); Theobroma cacao (CU604949); Camellia japonica (JK711196); Beta vulgaris (FG344225); Juglans regia (CB304274); Euonymus alatus (HS256347); Elaeis guineensis (EL695364); Prunus dulcis (BU573735); Phaseolus vulgaris (GW912947); Jatropha curcas (FM893340); Vigna unguiculata (GH620026); Brassica nigra (GT069280); Linum usitatissimum (JG020505); Malus x domestica (CN889956); Panicum virgatum (FE599713); Citrus sinensis (CX049759); Arachis duranensis (GW939883); Citrus trifoliata (EY838538); Lens culinaris (GT620232); Camellia japonica (JK711253); Medicago truncatula (AJ498296); Helianthus annuus (BU030877); Cyamopsis tetragonoloba (EG979287); Limnanthes alba (FD647825); Pinus taeda (DN632024 ); Picea glauca (GE478151); Reproductive organs/ Flowers Secale cereale (BE494154); Cycas rumphii (EX927452); Brassica oleracea (DK510516); Mimulus guttatus (GO976629); Capsicum annuum (GD087665); Petunia x hybrida (AF049937) Fruit Fragaria x ananassa (GT151796); Euphorbia esula (DV114857); Actinidia deliciosa (FG439646); Coffea arabica (GT014567); Coffea racemosa (GT662256); Coffea canephora (EE197409); Vaccinium corymbosum (JK652125); Paullinia cupana (EC764431); Solanum lycopersicum (BP895795); Vegetative tissues Osmunda lancea (FS994081 ); Pinus sylvestris (HE635689); Pteridium aquilinum (GW575141); Adiantum capillus-veneris (DK953870); Marchantia polymorpha (BJ852235); Tropaeolum majus (GH166519); Brassica rapa (FY419340); Tropaeolum majus (GH166519); Gossypium raimondii (CO093479); Aquilegia formosa (JZ010421); Pseudotsuga menziesii (CN637534); Vascular tissues Larix kaempferi (AB251484); Pinus contorta (GT247006); Pinus banksiana (GW738403); Chamaecyparis obtusa (BW987215); Other Cryptomeria japonica (AU066462); Eutrema salsugineum (DN776943); Gossypium arboreum (BF269166); Cicer arietinum (HO063868); Isoetes lacustris (GW574598) Electronic Supplementary Material (ESI) for Metallomics This journal is © The Royal Society of Chemistry 2013 References

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