Molecular Analysis of Heavy Metal Uptake and Accumulation Using a Metal

Hyperaccumulating Plant Species

Leon V. KochianU.S. Plant, Soil and Nutrition Laboratory

USDA-ARSCornell University

Thlaspi CaerulescensA Zn/Cd Hyperaccumulator

This species in the Brassicaceae has been the focus of botanicalresearch for over a century because of its ability to colonize

metalliferous soils.

Growth Medium [Zn] of Medium Shoot [Zn] Phytotoxicity(ppm) (ppm) Symptoms

Soil 1800 52000 Severe

Soil 178 18000 None

Hydroponics 206 26000 None

Hydroponics 6.5 6200 None

T. caerulescens is a small, slow growing plant – Thus it’s utility is as an interesting model system for understanding how plants accumulate and tolerate high levels of metals

SUMMARYPhysiology of Zinc Hyperaccumulation

A number of Zn transport sites are altered in T. caerulescens

• Stimulated Zn2+ influx across the root-cell PM

• Reduced Zn sequestration in the root-cell vacuole

• Stimulated Zn transport into the xylem (?)

• Stimulated Zn2+ influx into leaf mesophyll cells

Plant Zn2+ Transport Gene was Cloned by Complementation

wt zhy3

pZNT1

High Zn Low Zn

T. caerulescenscDNA library

zhy3, yeast zinc transporter

gene mutant

Screen on low zinc medium

transforminto

ZNT1 containingcolony

ZNT1 is a Member of the ZIP Family of Micronutrient Transporters

M - - - - - - - - - - - - - - - - A S S P T K I L C D A G E S D L C R D D A A A F L L K F V A I A S1M - - - - - - - - - - - - - - - - A S S T T K I L C D A G E S D L C R D D S A A F L L K F V A I A S1M K T I F L V L I F V S F A I S P A T S T A P E E C G S E S A N P C V N K A K A L P L K V I A I F V1

I L L A G A A G V A I P L I G K N R R F L Q T E G N L F V A A K A F A A G V I L A T G F V H M L A G35I L L A G A A G V A I P L I G R N R R F L Q T E G N L F V A A K A F A A G V I L A T G F V H M L A G35I L I A S M I G V G A P L F S R N V S F L Q P D G N I F T I I K C F A S G I I L G T G F M H V L P D51

G T E A L T N P C L P D Y P W S K F P F P G F F A M V A A L I T L L V D F M G T Q Y Y E S K Q Q R N85G T E A L S N P C L P D F P W S K F P F P G F F A M V A A L A T L L V D F M G T Q Y Y E R K Q E R N85S F E M L S S I C L E E N P W H K F P F S G F L A M L S G L I T L A I D S M A T S L Y T S K N A - -101

E V A G G G E A A V V E E T S S V L P V V V E R G N D S K A F G E E D G G G M H I V G I R A H A A H135Q A A T - - E A A A G S E E I A V V P V V G E R V T D N K V F G E E D G G G I H I V G I R A H A A H135- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V G I M P H G - -149

H N H S H S N A H G T F D G H A H G Q S H G H V H V H G S H D V E N G A - - R H V V V S Q I L E L G185H R H S H S N S H G T C D G H A H G H S H G H M H - - G N S D V E N G A - - R H V V V S Q I L E L G183- - - - - - - - - - - - H G H G H G P A N D V T L P I K E D D S S N A Q L L R Y R V I A M V L E L G156

I V S H S I I I G L S L G V S Q S P C T I R P L I A A L S F H Q F F E G F A L G G C I S Q A Q F K N233I V S H S I I I G L S L G V S Q S P C T I R P L I A A L S F H Q F F E G F A L G G C I S Q A Q F R N229I I V H S V V I G L S L G A T S D T C T I K G L I A A L C F H Q M F E G M G L G G C I L Q A E Y T N194

K S A I I M A C F F A L T A P I G I G I G T A V A S S F N S H S P G A L V T E G I L D S L S A G I L283K S A T I M A C F F A L T T P L G I G I G T A V A S S F N S H S P G A L V T E G I L D S L S A G I L279M K K F V M A F F F A V T T P F G I A L G I A L S T V Y Q D N S P K A L I T V G L L N A C S A G L L244

T Y M A L V D L I A A D F L S K R M S C N V R L Q V V S Y V M L F L G A G L M S A L A I W A . 333V Y M A L V D L I A A D F L S K R M S C N L R L Q V V S Y V M L F L G A G L M S A L A I W A . 329I Y M A L V D L L A A E F M G P K L Q G S I K M Q F K C L I A A L L G C G G M S I I A K W A . 294

Decoration 'Decoration #2': Shade (with solid black) residues that match the Consensus exactly.�

ZNT1AtZIP4AtIRT1

Strategies for Characterizing Members of Ion Transporter Gene Families

Tissue and cell specific localization of gene expression (Northerns, in situ mRNA localization, promoter-reporter gene constructs)Membrane localization of transport protein (via YFP-protein fusions)

Investigate transport properties using heterologous systems (yeast, oocytes, mammalian cell lines) and via knockout mutants (currently developing stable transformation system)

Full Length88ZIP5ZNT5

Full Length88IRT3ZNT4

Missing 200 bp at 5’ end

88ZIP3ZNT3

Missing 28 bp at 5’ end

88ZIP2ZNT2

Full Length88ZIP4ZNT1

Length of Thlaspi clone

% IdentityArabidopsishomolog

Thlaspi caerulescens

ZIP Transporters in Thlaspi caerulescens

We are currently screening for ZIP1, 7 and 9 homologs in Thlaspi

A Number of Other Micronutrient Transporter Gene Families Have Recently

Been Identified in Plants

• Nramp family of broad specificity micronutrient transporters

• CAX family of vacuolar membrane divalent cation/H+ transporters

• P-type heavy metal ATPases

• ZAT family of tonoplast and PM micronutrient/heavy metal transporters (members of the CDF superfamily)

• Subset of ABC transporter super family

Characterization of ZNT1

Is It a “Super” Transporter?

Zn in

flux

(pm

oles

min

-1 1

0-6 c

ells

)

0

2

4

6 pZNT1pFL61saturable ZNT1 kinetics

Cd

Zn or Cd concentration in solution (µM)0 20 40 60 80

Cd

influ

x(p

mol

es m

in-1

10-6

cel

ls)

0

3

6

9

ZNT1-Mediated Zn and Cd Influx in Yeast

Mediation of Zn uptake by RIT1 and ZNT1

[Zn], µM0 20 40 60 80

Zn

influ

x, p

mol

es/1

06ce

lls/m

in

0

1

2

3

4

5

ZNT1-dependent uptakeRIT1-dependent uptake

RIT1 4.0 4.0ZNT1 7.5 2.2

KM VMAX

ZNT1

ZNT1-arvense

Tc Ta

+ - + - + - + -Shoot Root

Zn:

Northern Analysis of ZNT1 Expression

Shoot Root

Membrane Localization of ZNT1 Using ZNT1-GFP Construct

35S GFP pAVA120

c35S GFP pZNT1-GFPZNT1

Transient transformation by particle bombardment into onion, Thlaspi and Arabidopsis and via PEG transformation of protoplasts

ZNT1-GFP Expression in Onion Cells

pZNT1-GFPpAVA120

REGULATION OF HEAVY METAL GENE EXPRESSION: DOES HYPEREXPRESSION

DRIVE HYPERACCUMULATION?

ZNT1

ZNT1-arvense

Tc Ta

+ - + - + - + -Shoot Root

Zn:

Northern Analysis of ZNT1 Expression

Shoot Root

Increasing Plant Zn Status Decreases ZNT1 Expression

0 0 1 1 10 50 µM

A C A C A C

ZNT1 caer. probe

ZNT1 arvense probe

[Zn] in growth solution

Km

Vmax

2 4 2 6 2 5

80 240 40 270 40 80

Altered Regulation of ZIP Transportersin T. caerulescens

• In “normal” plants there is a low level of ZIP expressionthat is upregulated by Zn deficiency.

• In T. caerulescens, ZNT1 expression is very high underZn deficient and sufficient conditions.

• Only when T. caerulescens is grown on very high levels ofZn is ZNT1 expression reduced.

• Regulation of Zn transporter expression is altered in T. caerulescens, contributing to hyperaccumulation.

Zn Homeostasis in Yeast

ZREZrt1Zrt2etc

+Zn+Zn

-Zn

-Zn

Zrt1Zrt2

Zn

[ Zn ]Zrt1/ Zrt2 degradation

+ Zn

Zrt3

Zrc1Cot1

/

VACUOLE

NUCLEUS

Low Zn

High Zn

From MacDiarmid et al (2000) EMBO 19:2845

Zap1Zap1

Zap1

MetalloproteinsOther Organelles

Zrt3Zrt2

Zrt1

αZip13

Zip2Zip9Irt3

Znt1Zip4

β

Zip6

Zip1Zip14

Zip3γ

Zip5

Znt5

Znt4

δ

Zip7

Zip10Zip12Rit1LeIrt1

LeIrt2

Zip8

Irt1

Irt2

ZIP FamilyPhylogenetic Tree δ

γ

β

α

5’-ATG(A/T)CGA(A/C)A(C/T)-3’

ZIP promoters contain ppZRE(s)

α

β

γ

δ

ZIP4*, ZIP9, IRT3

ZIP1, ZIP3*, ZIP5

IRT1, IRT2, ZIP12

ZIP2, ZIP13

ZIP6, ZIP14

ZIP7, ZIP8

ZIPsubfamily

One or moreppZREs

NoppZREs

Zrt3Zrt2

Zrt1

αZip13

Zip2Zip9Irt3

Znt1Zip4

β

Zip6

Zip1Zip14

Zip3γ

Zip5

Znt5

Znt4

δ

Zip7

Zip10Zip12Rit1LeIrt1

LeIrt2

Zip8

Irt1

Irt2

ZIP familyPhylogenetic

Tree

δ

γ

β

α

degenerate ppZRE

palindromic ppZRE

Is Expression of Other Metal Transport Genes Altered in T. caerulescens?

Fourteen ZIP and three IRT homologues have been identified in ArabidopsisWe have cloned 5 ZNT homologs in Thlaspi with more on the way

What are their roles in hyperaccumulation and micronutrient homeostasis?

Other micronutrient/heavy metal transporter families:CAX family of tonoplast divalent cation/H+ antiporters

ZAT family of tonoplast and PM micronutrient/heavy metal transporters (members of the CDF superfamily)

Nramp family of broad specificity micronutrient/heavy metal transporters

Do any of these play a role in hyperaccumulation or tolerance?

ZAT1tc

MT2a

µM Zn

AtNramp2A C A C A C

ZNT4µM Zn0 1 10/50

Root

A C A C A C

Znt2

Znt3

Znt5

0 1 10/50

Root

Heavy Metal-Related Gene Expression in Roots of T. caerulescens vs. T.arvense

Zn Homeostasis in Yeast

ZREZrt1Zrt2etc

+Zn+Zn

-Zn

-Zn

Zrt1Zrt2

Zn

[ Zn ]Zrt1/ Zrt2 degradation

+ Zn

Zrt3

Zrc1Cot1

/

VACUOLE

NUCLEUS

Low Zn

High Zn

From MacDiarmid et al (2000) EMBO 19:2845

Zap1Zap1

Zap1

MetalloproteinsOther Organelles

Zhy6 Wt

pE2F1 pE2F2

High Zinc Low Zinc

E2F1 and E2F2 Confer Growth on Low Zinc for the ZAP Deletion Mutant, ZHY6

Nuclear Localization Signal

DNA binding domain

Leucine Zipper

Marked Box

Rb binding domain

Significant Sequence Homology among Plant E2F-like proteins

Wt Zhy6 E2F1 E2F2 Wt Zhy6 E2F1 E2F2 Wt E2F1 E2F2

E2F Induced Zrt1 Expression in Yeast

High Zinc Sufficient Zinc Low Zinc

0 1 10/50

Root Shoot

Ta Tc Ta Tc Ta Tc Ta Tc Ta Tc Ta Tc

0 1 10/50 µM Zn

E2F1

E2F2

E2F1/E2F2 are members of a family of transcription factors involved in regulation of the cell cycle via a complex involvingcyclins and CDKs. How might such a protein be involved in regulation of plant mineral nutrition?

PHO Regulon in Yeast:Molecular Regulation of P Nutrition/Acquisition

Ogawa, DeRisi and Brown. 2000. Molecular Biology of the Cell 11:4309.

PHO Regulon in Yeast:Pho regulatory system showing the 5 main regulator proteins (Pho 2, 4, 80, 81, and 85)

Ogawa, DeRisi and Brown. 2000. Molecular Biology of the Cell 11:4309.

Elucidating mechanisms of Zn-dependent gene regulation in Thlaspi caerulescens

Promoter Analysis

• We have cloned Thlaspi ZNT1 promoter via genome walking and Arabidopsis ZIP4 promoter via PCR(ZNT1 promoter contains the ppZRE found in ZIP4)

• Expression analysis using promoter-reporter gene constructs and protoplast transient expression assay –reciprocal assays

• Promoter deletion analysis to identify functional ZREs

AACTCCCCATCTTA-CAAAGTT-ACCGTCCTTTTAGCTTAAGCTGCCTACTTCTCATCCTT-TTTCAGCTTAAGCTACTCC---------------TAATC--ATCCTT--TTAAACCTA 1 pAtZp4promoterGACTGAAGATGGCAGCAATGTGGAGAGGGAAGAGAGGAAGAGCTTTCTGGAAATATTAATTATGTTGGTTGGGAATATACCAAGGGAGAGGTGAAACAATCCAATCCTTAACCCAAGCTA 1 pZnprom_typeI_F

CGGCTT--TAAGTTTTTTTTTAACTC---ATATAATCTTCTGCAGTAGACTTGACTTAATCGGATTTTCTGTTTCATGAACTTGTTGGTAGTGTG-GAACAAATGGGAAAATGAATATTT 99 pAtZp4promoterCCTCCTAATAATCCTTTTTTTAGCTTCCGGTTTAGTGTGTTGAAGTTGTTAAACTCCTTTTATAACCGTTGTTATGTAATCGTTTTTGTATTTCATGAACAAATGGATGAA-GAATATTT 121 pZnprom_typeI_F

T---TGGAACAAATTGATTTTCTGTTTCATAT-TAAGTTAAATCATTCTGTTTCCACTGAAATAAATTGTTTTCCAAAAATCACTCCGTTTATTATGTCTTTGTTTTTAAGAAATAAAAG 213 pAtZp4promoterTAGTTGGAACCA-TCATTACAATATTTTACTTGTCAACTAAAA-ATGTAAATGCAATTAGCACTCTCCATCTACAATCTGTCAACCT-TGTGCAATCAATAAAAATGTAGATAATAATAC 240 pZnprom_typeI_F

TGAGAAAACAGAATAACGCGAAAATGTCGACATATTTGGCTAAGTATAGACAAGATTGGGAAGCTCTGTTTAGTTAT--GCGT---CAGTC--TCTCATCAGTGTTCAACTGCCACGGAG 329 pAtZp4promoterTAGCATCTTTTAACTTTAATATTATTTTCCAAATTTTGTGTGGAAAAAAATGAAAAGAGAGAATAACGTGAAAATGTCGACATAATTAGCCAAATATACAAGTGTACAAGTGCCACGGAG 357 pZnprom_typeI_F

CGAACCGATTCCTAATTGCAACGTCCCGAGTCCATAGAATGTCGACACTCTTTCACTCTTTCTCCAAGTTGCCTCCTTTGAGTCCTTTCTCATATTTTATAGACTCACTTTCTGTTTCTT 442 pAtZp4promoterCGAGCGAGCGGTT--CCGCAAC-TCCAACTTGCTGAGAATGTCGACACTCTTTCGCTCGTC-----GATT-CCTCCTTT---TTTCTAATC-TGCTTT-TCGGTTAGCTTTCTGCTCCTT 477 pZnprom_typeI_F

GATCCCGAGGAAGAAGAAGAATAAACTCTT-GTTCCCATGATCTTCGTCGATGTTAGT---------ACCTTGAGATTCGATT--CGTCTCTCCGATTCTTTATAATCG-GAATTTATTG 562 pAtZp4promoterGATCCCGAGAAA-AAGAAGAAGACACTCTTTGTTCCCATGATCATCGCCGATGTTAGTCATATATCCACCTTGGGATTCGTTTTCCGTCTCTCCACTTTTCTATATTCTTGAATTTACTG 583 pZnprom_typeI_F

GTTT--------GTGGAATCTGATTTT-GGGTTCTCTTCTATTGCTCTTATAGGTTCTTTGGAAATTGTTTCCTCTATACTCGTTTGGATCAGGAAGAGACTCTCTCTCAGGTATTTCAT 669 pAtZp4promoterTTTTCTCTTTTTGTGGAATCTGATTTTCGAATTCTCTTCTCTTGGTCTTA--GCTTCTTTGGAAATCGTTTCCTCTATACTTGTTCAGATCAGGAAGAGATTCTCTCTCAGGTATTTCGT 702 pZnprom_typeI_F

CGAACACTTACCCTACAATTTAGTGAAATTTCTGTGTGATGTAGAATTCTCTAGATTCTTCTAATCGAGTTTTAGCTGAGAAAATTAGTGAAATGAAATGAGATTTTCCATTGATTGCT- 780 pAtZp4promoterCCAACACTTGTTTCAT--TTCAACC---TCACAATTCAGTG-AGA---CTCTG--TTTCACCGCTCAA---TTATCTGTCTTCTTCCATCT--TCCAATCGAATTTCCCCTTGATTGCTC 820 pZnprom_typeI_F

-AGATTCCAAAACTTTAGCTTGTTGTTCTGAATTGCACTTATTTGTTGTGTTGTATGGACCATGGAAACATCCAAAAGAGTCCATTTTGCAGA--------TCATTCCC---GA------ 899 pAtZp4promoterTAGATTCCGAAACTGTAA---ATTGTTCTGAATTGCCCTC-TCTGTTGC--TCTATGGACCATGAAAGC-CGAAAAAGAGTCCATTTTGCAGACAATGGCTTCATCTCCCACGAAAATCC 924 pZnprom_typeI_F

-----GACA 1001 pAtZp4promoterTCTGTGATG 1037 pZnprom_typeI_F

ZNT1 promoter ZRE

ZIP4 promoter ZRE

34% identity between ZIP4 and ZNT1 promoters

Elucidating mechanisms of Zn-dependent gene regulation in Thlaspi caerulescensTranscription factor analysis – E2F1/2:

•Test E2F1/2 DNA binding to ZNT1 promoter viaDNAse I footprinting and electromobility shiftassays

•Characterize T-DNA knock-out line for AtE2F2

•Search for other trans-factors using yeast 1-hybrid analysis

A Novel Quantitative in situ mRNAHybridization Technique for Rapidly

Determining Cell Specific Heavy Metal Transporter Gene Expression

Overview of in situ mRNA hybridization method

cut out

max 2x2 mm

vacuum infiltrate with alkaline fixation solution

dehydrate

extract hydrophobic compounds

digest proteins

(denature)

hybridize with fluorescently labeled oligonucleotides

record images in CLSMquantify

rehydrate

postfixate

Compartmentation of metals in leaves of Thlaspi and AlyssumZn/Cd/Ni accumulation in epidermal vacuoles

Compartmentation in leaves of Thlaspi caerulescens

Zn Mg P S Cl K0

2

4

6

8mature leaves

mM

/ mM

Ca

Zn Mg P S Cl K0

2

4

6

8 young leaves

mM

/ mM

Ca

upper epidermis upper mesophylllower mesophyll lower epidermis

Concentrations of elementsin leaf tissues

Zn K α line scan and dot map of a leaf

15 20 25 30 35 40 45 500

2

4

6

8

Linear Regression: Y = A + B * XParameter Value Error-----------------------------------------------A -0.057 1.027B 0.153 0.034-----------------------------------------------R SD N P-----------------------------------------------0.816 1.079 12 0.00118-----------------------------------------------

mM

Zn /

mM

Ca

cell width

regression lineconfidence limits

data points

Correlation between cell size and zinc concentration

Küpper H, Zhao F, McGrath SP (1999) Plant Physiology 119, 305-11

ZNT1 mRNA localization in young leaves of Thlaspi caerulescens:Mesophyll, antisense probe

Overlay of green autofluorescence and red oligonucleotide fluorescence

Red oligonucleotide fluorescence

ZNT1 mRNA localization in young leaves of Thlaspi caerulescens:Mesophyll, control with sense strand probe

Overlay of green autofluorescence and red oligonucleotide fluorescence

Red oligonucleotide fluorescence

ZNT1 in young leaves of Thlaspi caerulescens Prayon:mesophyll, antisense probe

overlay of green autofluorescence and red oligonucleotide fluorescence

red oligonucleotide fluorescence

ZNT1 mRNA localization in young leaves of Thlaspi caerulescens:Epidermis, antisense probe

Overlay of green autofluorescence and red oligonucleotide fluorescence

Red oligonucleotide fluorescence

ZNT1 mRNA localization in young leaves of Thlaspi caerulescens:Stomata, antisense probe

Overlay of green autofluorescence and red oligonucleotide fluorescence

Red oligonucleotide fluorescence

Summary of ZNT1 Leaf Localization Studies

1) ZNT1 expressed primarily in photosynthetic cells - guard cells in epidermis and mesophyll cells in leaf interior (high stringency & high affinity conditions)

2) ZNT1 appears to be involved in “normal” leaf Zn nutrition and not hyperaccumulation

3) Related members of Thlaspi ZIP family expressed in specialized large epidermal cells (metal accumulating cells?) and around veins (transfer from xylem to leaf?)

ZNT1 mRNA localization in roots of Thlaspi caerulescens:Starting at tip of mature root, antisense probe

Series of optical cross sections moving back from root tip -overlays of green autofluorescence and red oligonucleotide fluorescence

Susceptible and Tolerant yeast growing on metal enriched SD plates.

Searching for Thlaspi Cadmium Tolerance Genes

• Yeast transformed with T. caerulescens cDNA library are grown on high Zn or Cd plates.

• Tolerance phenotype is confirmed by replating; insert is isolated and identified.

Zn Tolerance Genes – Melinda KleinCd Tolerance Genes – Ashot Papoyan

Clone Closest Match and Function Accession # GenBank Hit Reference

1 Brassica juncea metallothionein- AY486002 Y10849 Schafer et al. (1998)like protein

2 Arabidopsis thaliana AY486003 L15389 Zhou et al. (1994)metallothionen

3 Arabidopsis thaliana AY486004 U15130 Yeh et al. (1994)metallothionen

4 Arabidopsis thaliana AY486005 NM114760 Town et al.(2003)60S ribosomal protein L13

5 Arabidopsis thaliana putative AY486006 NM_118925 Town et al (2003)protein

6 Sinapis alba subunit of AY486007 Y07498 Wenng et al. ( 1989)of photosystem II

7 Arabidopsis thalina light AY486008 Z12120 Park et al. (1992)regulated kinase

8 Arabidopsis thalina putative AY486009 CAB87795 Rieger et al. (2000) protein

9 Arabidopsis thalina putative AY486001 AF412407 Richaud et al.(2001)heavy metal P-type ATPase

Thlaspi Genes Conferring Cd Tolerance in Yeast

Athma4 ASTSKLNGRKLEGDDDYVVDLEAGLLTKSGNGQCKSSCCGDKKNQENVVMMKPSSKTSSD 776Tchma4 --------KKLEGVDDQGLDLEAGLLSKS---QCNSGCCGDQKSQEKVMLMRPASKTSSD 768 :**** ** :*******:** **:*.****:*.**:*::*:*:****** Athma4 HSHPGCCGDKKEEKVKPLVKDGCCSEKTRKSEGDMVSLSSCKKSSHVKHDLKMKGGSGCC 836 Tchma4 HLHSGCCGEKKQESVK-LVKDSCCGEKSRKPEGDMASLSSCKKS---NNDLKMKGGSSCC 824 * *.****:**:*.** ****.**.**:**.****.******** ::********.** Athma4 ASKNEKGKE-VVAKSCCEKPKQQVESVGDCKSGHCEKKKQAEDIVVPVQIIGHALTHVEI 895 Tchma4 ASKNEKLKEAVVAKSCCED-KEKTEGNVEMQILNLERGSQKK------------------ 865 ****** ** ********. *::.*. : : : *: .* : Athma4 ELQTKETCKTSCCDSKEKVKETGLLLSSENTPYLEKGVLIKDEGNCKSGSENMGTVKQSC 955 Tchma4 ---VGETCKSSCCGDKEKAKETRLLLASEDPSYLEK-------EERQTTEANIVTVKQSC 915 . ****:***..***.*** ***:**:..**** : :: . *: ****** Athma4 HEKGCSDEKQ--TGEITLASE---EETDDQDCSSGCCVN-EGTVKQSFDEKKHSVLVEKE 1009 Tchma4 HEKASLDIETGVTCDLKLVCCGNIEVGEQSDLEKGMKLKGEGQCKSDCCGDEIPLASEED 975 ***.. * : * ::.*.. * ::.* ..* :: ** *.. .: .: *:: Athma4 GLDMETGFC--------------------CDAKLVCCGNTEGEVKEQCR----LEIKKEE 1045 Tchma4 SVDCSSGCCGNKEELTQICHEKTCLDIVSCDSKLVCCGETEVEVREQCDLKKGLQIKNEG 1035 .:* .:* * **:******:** **:*** *:**:* Athma4 HCKSGCCGEEIQTGEITLVSE--EETESTNCSTGCC----------------VDKEEVTQ 1087 Tchma4 QCKSVRCGDEKKTEEITEETDNLKSESGDDCKSPCCGTGLKQEGSSSLVNVVVESGESGS 1095 :*** **:* :* *** :: :. .. :*.: ** *:. * . Athma4 TCHEKPASLVVSG---LEVKKDEHCESSHRAVKVETCCKVKIP--EACASKCRDRAKR-H 1141 Tchma4 SCCSKEGEIVKVSSQSCCASPSDVVLSDLEVKKLEICCKAKKTPEEVRGSKCKETEKRHH 1155 :* .* ..:* . .. .: *. .. *:* ***.* . *. .***:: ** * Athma4 SGKSCCRSYAKELCSHRHHHHHHHHHHHVSA 1172 Tchma4 VGKSCCRSYAKEYCSHRHHHHHHHHHVGAA- 1185 *********** ************* .:

* HMA Domain (GMTCxxC) is missing* Arabidopsis and Thlaspi HMA4s are 71% Identical

Alignment of 3’ ends of Arabidopsis and Thlaspi HMA4’s

0µM Cd

75µM Cd

90µM Cd

OD600

1 0.1 0.01 0.001

ATPase

WT

ATPase

WT

ATPase

WT

Cd Tolerance for WT and TcHMA4 Transformed Yeast

Cd2+ Accumulation

00.0020.0040.0060.0080.01

0.0120.0140.0160.0180.02

WT(5M

in)

TR(5Min)

WT(30

Min)

TR(30M

in)W

T(70M

in)TR(70

Min)

µMol

Cd/

106 C

ells

Pb2+ Accumulation

00.005

0.010.015

0.020.025

0.030.035

WT(5min)

TR(5min)W

T(30Min)

TR(30Min)

WT(70M

in)TR(70min)

µMol

Pb

/ 10

6 Cel

ls

Cd2+ and Pb2+ Accumulation for WT andTcHMA4 Transformed Yeast

Cd Influx and Efflux

0

5

10

15

20

25

30

35

40

45

Influx Efflux

fmM

olC

d/10

6 cells

/min

Wild TypeATPase

Quantification of Unidirectional 109Cd Influx and Effluxin WT and TcHMA4 Transformed Yeast

Root TcHMA4 Gene Expression in Response to Changesin Plant Zn and Cd Status

0 µM Zn 1 µM Zn 5 µM Zn 100 µM Zn 1 µM Zn 10 µM Cd 100 µM Cd

8.9 273 409 239 273 580 4641

µg Cd / g dwtµg Zn / g dwt

Root

Shoot 29 960 3055 5805 0.2 1228 5680

TcHMA4 gene expression strongly induced both by Zn deficiency and high Cd and Zn

Does TcHMA4 Play a Role in Both Zn Nutrition and Heavy Metal Hyperaccumulation?

•Expressed primarily in the root•Under Zn deficiency does it function to provide n to the shoot?•In high Zn/Cd soils, does it function to translocatemetals to the shoot?•Preliminary results suggest it is expressed near root xylem•Is it an efflux transporter involved in xylem loading and root-to-shoot transport?

384aa ---------------------------------------------MASLSSCKKSNNDLK 15141aa MLMRPASKTSSDHLHSGCCGEKKQESVKLVKDSCCGEKSRKPEGDMASLSSCKKSNNDLK 60 *************** 384aa MKGGSSCCASKNDKLKEVVVAKSCCEDKEKAEGNVEMQILNLEKGSQKKVGETCKSSCCG 75 141aa MKGGSSCCASKNDKLKEVVVAKSCCEDKEKAEGNVEMQILNLEKGSQKKVGETCKSSCCG 120 ************************************************************ 384aa DKEKAKETRLLLASEDPSYLEKEERQTTEANIVTVKQSCHEKASLDIETGVTCDLKLVCC 135 141aa DKEKAKETRLLLASED-------------------------------------------- 141 **************** 384aa GNIEVGEQSDLEKGMKLKGEGQCKSDCCGDEIPLASEEDSVDCSSGCCGNKEELTQICHE 195 141aa ------------------------------------------------------------ 384aa KTCLDIVSCDSKLVCCGETEVEVREQCDLKKGLQIKNEGQCKSVRCGDEKKTEEITEETD 255 384aa NLKSESGDDCKSLCCGTGLKQEGSSSLVNVVVESGESGSSCCSKEGEIVKVSSQSCCASP 315 384aa SDVVLSDLEVKKLEICCKAKKTPEEVRGSKCKETEKRHHVGKSCCRSYAKEYCSHRHHHH 375 384aa HHHHHVGAA 384

Alignment Of Partial Clones of TcHMA4

Cd Tolerance for WT Yeast and Yeast Transformed with Partial TcHMA4 Clones

Cd2+Accumulation for WT Yeast and Yeast ExpressingPartial TcHMA4 Clones

Cd2+ Accumulation

0

0.002

0.004

0.006

0.008

0.01

0.012

0.014

0.016

0.018

1 hour 3hours 5hours

Time

µMol

Cd/M

illio

n Ce

lls

WT141a.a.384a.a.

When expressed in plants, will they confer enhanced Cd toleranceand accumulation for phytoremediation purposes?

Who Actually Did the Work?

Not Shown: Hendrik Kupper and Laura Seib (in situ gene expression techniques), Randy Clark (sorghum Al tolerance genes)