Functional analysis of HMA genes in tobacco

Dr. Françoise GOSTI

Pr. Pierre BERTHOMIEU

Dr. Victor HERMAND

Dr. François DORLHAC de BORNE

Dr. Emilie JULIO

Association for Researchon Nicotianeae

& production of low-cadmium tobacco lines

Cadmium is a toxic pollutant

Its concentration is raising in soils

Dguimi et al, 2009

1-2 µg Cd in every cigarette

Tobacco plants accumulate comparatively high contents of Cd in the leaves

Jarüp et al, 1998

Smokers have 4-5 times higher Cd concentration in blood than non smokers

It is desirable to produce low-cadmium tobacco varieties

Kidney

HormoneBone

Cadmium

Introduction

Cd content(µmol / g-1 DW) 50

40

30

20

10

0

60

root shoot

Lung

Soil

Cd, Fe, Zn, Mn

?Vacuole

Cd-PC CdFeMn

Cd, ZnMn, Ca

HMA3CAX2,4

ABC1-2NRAMP

3-4-6

Cytosol

Cd, Ca, Zn? Cd, Zn

IRT1

HMA2,4

Epidermis Cortex Endodermis Pericycle Xylem

Mesophylle

Casparian strip

What are the transporters responsible for Cd accumulation ?

Wong et al, 2009

Cd content in the shoot

*

*

In A. thaliana, HMA2 & HMA4 are critical for cadmium accumulation in the shoot

(nmol Cd.g-1 DW)

20

15

10

5

0

25

WT hma2- hma4- hma2-

hma4-

Aims of the work

To identify, clone and characterise tobacco orthologues to AtHMA2 et AtHMA4

To develop tobacco lines accumulating low levels of Cd in leaves

Two tobacco orthologues to AtHMA2 & AtHMA4

NtHMAα

NtHMAβ from ATG to STOP: 16.5 kb

95% a.a. similarity

NtHMA 709 aaNtHMA 673 aa

length of cytosolic C-term tail:

AtHMA4 478 aa

AtHMA4

vs. 7.5 kb>65% <45%

AtHMA2 267 aa

Phylogenetic relationships within the Zn-Cd P1B-ATPases family

Zorrig et al. 2011 CR Biol 334:863

transcriptaccumulation

Expression of NtHMAα & NtHMAβ

a

b

c c

0.5

0.4

0.2

0.1

0

0.6

rootsshoots

0.3

relative to actin

NtHMA

NtHMA

300 µm

300 µm

5 mm

relative geneexpression

Making of mutant and silenced NtHMA lines

WT

amiRNA9 line

amiRNA constructs

screening of EMS mutagenised linesusing a tilling approach inside

outside

NtHMA

NtHMA

for NtHMA

for NtHMA

E327KP294SW265*G515RR529*

HMAHMAHMAHMA

-silenced lines

1µM Cd in the medium

NthmaβNthmaα

b

a

b b b bCd contentin shoots

Cd accumulation is reduced in NtHMA mutant & silenced plants

ppm

WT

-silenced lines

b

c

b

a

control

Cd contentin shoots

ppm

– 50-60 %

– 80 %

a a a a

Zn but not Fe accumulation is also impairedin NtHMA-silenced plants

b

aa

a

Zn contentin shoots

ppm

Fe contentin shoots

ppm

-silenced lines

control

control

WT amiRNA9 line

+100µM CdCl2

Silencing NtHMAα & NtHMAβ increases cadmium tolerance

Is 'protecting the shoot against Cd accumulation' the key factor ?

Hussain et al, 2004

N. tabacumhmaα hmaβ

(W265* E529*)

A. thalianahma2 hma4

Nthmaα Nthmaβ double mutants are dwarfand unable to complete their life cycle

Inactivation of NtHMAs leads to low fertility

WT

1cm

WT

amiRNA9line

amiRNA9line

which is due to defective pollen

Seed production

aa a a

a a

b

amiRNA9line

WT NtHMAor NtHMA

silenced lines

Viability ofthe pollen

% Germination

48.5%

1.4%

WT

amiRNA9line

Pollen of silenced plants is viable but it cannot germinate

80

60

20

0

100

amiRNA9line

WT

40Alexandertest

(%)

Zn concentrationin the germination medium

Bringing Zn improves pollen germination

Germinationof amiRNA9

pollen%

20

15

5

0

25

10

0 1.5 15 150 1.5 15 150

mMM

Mn Fe

Zn

*

*

Metalcontent

WT

amiRNA9line

WT

amiRNA9line

Analysis of Mn, Fe & Zn in seeds

Conclusions

Two orthologues to AtHMA4 were identified in tobacco

Inactivating these genes induces a 50 to 80% reduction in Cd accumulation in the shoot

Hermand et al. (2012) International patent WO/2012/041913

NtHMA & NtHMA appear to be functionally redundant, at least as far as Cd accumulation is considered

Silencing NtHMA & NtHMA also induces Zn deficiency, particularly in the pollen, which prevents its germination

Inactivating only one of the NtHMA genes is compatible withsufficient Zn nutrition and with a 50% reduction in shoot Cd content

Reducing the shoot Cd content to lower level may require to targetother genes, or to improve of the Zn/Cd selectivity of the NtHMA genes

they have a particularly long C-terminal cytosolic tail

RemerciementsAcknowledgments

Dr. Françoise GOSTI

Pr. Pierre BERTHOMIEU

Dr. Victor HERMAND

Dr. François DORLHAC de BORNE

Dr. Emilie JULIO

Dr. Hélène BERGES

Association for Researchon Nicotianeae

Dr. Felipe RICACHENEVSKY

Dr. Mary-Lou GUERINOT

N. TabacumA. thaliana A. halleri

Répartition du cadmium chez différentes espèces Expression3/3

5µM de Cd0,5µM de Cd 100µM de Cd

µmol

de

cadm

ium

par

g d

e m

atièr

e sè

che

Dguimi et al, 2009 Adapté de Wong et al, 2009 Adapté de Hanikenne et al, 2008