Functional analysis of HMA genes in tobacco
& production of low-cadmium tobacco lines
Association for Research
on Nicotianeae
Dr. Victor HERMAND
Dr. Françoise GOSTI
Pr. Pierre BERTHOMIEU
Dr. Emilie JULIO
Dr. François DORLHAC de BORNE
Introduction
Kidney
Lung
Cadmium
Cadmium is a toxic pollutant
Bone
Hormone
Its concentration is raising in soils
Cd content
Tobacco plants accumulate comparatively
high contents of Cd in the leaves
(µmol / g-1 DW)
60
50
40
30
20
1-2 µg Cd in every cigarette
10
0
Smokers have 4-5 times higher Cd concentration
in blood than non smokers
Jarüp et al, 1998
It is desirable to produce low-cadmium tobacco varieties
root shoot
Dguimi et al, 2009
What are the transporters responsible for Cd accumulation ?
ABC1-2
Vacuole
Cd
Fe
Mn
Cd-PC
Cd, Zn
Mn, Ca
HMA3
CAX2,4
Soil
NRAMP
3-4-6
?
Mesophylle
Cytosol
IRT1
Cd, Fe,
Zn, Mn
HMA2,4
Cd, Zn
?
Cd, Ca, Zn
Epidermis
Cortex
Endodermis Pericycle
Casparian strip
Xylem
In A. thaliana, HMA2 & HMA4 are critical
for cadmium accumulation in the shoot
25
Cd content
in the shoot
20
15
(nmol Cd.g-1 DW)
*
10
5
*
0
WT
hma2-
hma4-
hma2hma4Wong et al, 2009
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
95% a.a. similarity
NtHMAα
NtHMAβ
from ATG to STOP: 16.5 kb
>65%
<45%
vs. 7.5 kb
AtHMA4
length of cytosolic C-term tail:
NtHMAa
NtHMAb
AtHMA2
AtHMA4
709 aa
673 aa
267 aa
478 aa
Phylogenetic relationships within the Zn-Cd P1B-ATPases family
Zorrig et al. 2011 CR Biol 334:863
Expression of NtHMAα & NtHMAβ
transcript
accumulation
relative to actin
0.6
a
0.5
b
0.4
300 µm
0.3
NtHMAa
NtHMAb
0.2
0.1
c
c
0
shoots
roots
300 µm
5 mm
Making of mutant and silenced NtHMA lines
outside
screening of EMS mutagenised lines
using a tilling approach
for NtHMAa
for NtHMAb
inside
E327K
P294S
W265*
G515R
R529*
amiRNA constructs
relative gene
expression
NtHMAa
NtHMAb
HMAa HMAb HMAa WT
HMAb
-silenced lines
amiRNA9 line
Cd accumulation is reduced in NtHMA mutant & silenced plants
a
150
Cd content
in shoots
ppm
– 50-60 %
100
b
b
b
b
P294S
W265*
G515R
R529*
b
50
0
WT
Sauvage
E387K
a
Nthmaα
Nthmaβ
200
Cd content
in shoots
150
ppm
50
b
100
b
– 80 %
c
0
control
Vecteur
vide
1µM Cd in the medium
NtHMAα
NtHMAβ
-silenced lines
NtHMAαβ
Zn but not Fe accumulation is also impaired
in NtHMA-silenced plants
Zn content
in shoots
4000
ppm
2000
a
3000
a
a
b
1000
0
3000
Fe content
in shoots
2000
ppm
1000
0
control
Vecteur
vide
a
control
Vecteur
vide
NtHMAα
NtHMAβ
NtHMAαβ
a
a
a
NtHMAα
NtHMAβ
NtHMAαβ
-silenced lines
Silencing NtHMAα & NtHMAβ increases cadmium tolerance
+100µM CdCl2
WT
amiRNA9 line
Is 'protecting the shoot against Cd accumulation' the key factor ?
Nthmaα Nthmaβ double mutants are dwarf
and unable to complete their life cycle
N. tabacum
hmaα hmaβ
(W265* E529*)
A. thaliana
hma2 hma4
Hussain et al, 2004
Inactivation of NtHMAs leads to low fertility
Seed production
3000
2500
WT
a
a
a
a
a
amiRNA9
line
a
WT
2000
1500
1000
500
b
amiRNA9
line
0
WT
NtHMAa
or NtHMAb
silenced lines
amiRNA9
line
which is due to defective pollen
1cm
Pollen of silenced plants is viable but it cannot germinate
% Germination
100
Viability of
the pollen
80
(%)
60
Alexander
test
WT
48.5%
40
20
0
WT
amiRNA9
line
amiRNA9
line
1.4%
Bringing Zn improves pollen germination
25
Germination
of amiRNA9
pollen
%
20
15
10
5
0
0 1.5 15 150 1.5 15 150
mM
mM
Zn concentration
in the germination medium
Analysis of Mn, Fe & Zn in seeds
WT
Mn
Fe
amiRNA9
line
WT
200
Metal
content 150
Zn
*
100
amiRNA9
line
*
50
0
Mn
Fe
Zn
Conclusions
Two orthologues to AtHMA4 were identified in tobacco
they have a particularly long C-terminal cytosolic tail
Inactivating these genes induces a 50 to 80% reduction in Cd
accumulation in the shoot
Hermand et al. (2012) International patent WO/2012/041913
NtHMAa & NtHMAb appear to be functionally redundant,
at least as far as Cd accumulation is considered
Silencing NtHMAa & NtHMAb also induces Zn deficiency,
particularly in the pollen, which prevents its germination
Inactivating only one of the NtHMA genes is compatible with
sufficient Zn nutrition and with a 50% reduction in shoot Cd content
Reducing the shoot Cd content to lower level may require to target
other genes, or to improve of the Zn/Cd selectivity of the NtHMA genes
Acknowledgments
Remerciements
Association for Research
on Nicotianeae
Dr. Victor HERMAND
Dr. Françoise GOSTI
Pr. Pierre BERTHOMIEU
Dr. Hélène BERGES
Dr. Emilie JULIO
Dr. François DORLHAC de BORNE
Dr. Felipe RICACHENEVSKY
Dr. Mary-Lou GUERINOT
Expression
3/3
Répartition du cadmium chez différentes espèces
A. thaliana
µmol de cadmium par g de matière sèche
45
A. halleri
N. Tabacum
3
60
2.5
50
2
40
1.5
30
1
20
0.5
10
40
35
30
25
20
15
10
5
0
0
Racines Feuilles
0,5µM de Cd
Adapté de Wong et al, 2009
Racines
Feuilles
5µM de Cd
Adapté de Hanikenne et al, 2008
0
Racines Feuilles
100µM de Cd
Dguimi et al, 2009