functional analysis of hma genes in tobacco dr. françoise gosti pr. pierre berthomieu dr. victor...
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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
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