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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
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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
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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 ?
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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-
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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
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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
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Phylogenetic relationships within the Zn-Cd P1B-ATPases family
Zorrig et al. 2011 CR Biol 334:863
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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
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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
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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 %
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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
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WT amiRNA9 line
+100µM CdCl2
Silencing NtHMAα & NtHMAβ increases cadmium tolerance
Is 'protecting the shoot against Cd accumulation' the key factor ?
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Hussain et al, 2004
N. tabacumhmaα hmaβ
(W265* E529*)
A. thalianahma2 hma4
Nthmaα Nthmaβ double mutants are dwarfand unable to complete their life cycle
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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
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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
(%)
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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
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Mn Fe
Zn
*
*
Metalcontent
WT
amiRNA9line
WT
amiRNA9line
Analysis of Mn, Fe & Zn in seeds
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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
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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
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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