1. 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 Research on Nicotianeae & production of low-cadmium tobacco lines

2. 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 rootshoot Lung

3. Soil Cd, Fe, Zn, Mn ? Vacuole Cd-PC Cd Fe Mn Cd, Zn Mn, Ca HMA3 CAX2,4 ABC1-2 NRAMP 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 ?

4. 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 -

5. 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

6. Two tobacco orthologues to AtHMA2 & AtHMA4 NtHMAα NtHMAβ from ATG to STOP: 16.5 kb 95% a.a. similarity NtHMA  709 aa NtHMA  673 aa length of cytosolic C-term tail: AtHMA4478 aa AtHMA4 vs. 7.5 kb >65%<45% AtHMA2267 aa

7. Phylogenetic relationships within the Zn-Cd P 1B -ATPases family Zorrig et al. 2011 CR Biol 334:863

8. transcript accumulation 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 5 mm

9. relative gene expression Making of mutant and silenced NtHMA lines WT amiRNA9 line amiRNA constructs screening of EMS mutagenised lines using a tilling approach inside outside NtHMA  NtHMA  for NtHMA  for NtHMA  E327K P294S W265* G515R R529* HMA  HMA  HMA  HMA  -silenced lines

10. 1µM Cd in the medium NthmaβNthmaα b a b b b b Cd content in shoots Cd accumulation is reduced in NtHMA mutant & silenced plants ppm WT -silenced lines b c b a control Cd content in shoots ppm – 50-60 % – 80 %

11. a a a a Zn but not Fe accumulation is also impaired in NtHMA-silenced plants b a a a Zn content in shoots ppm Fe content in shoots ppm -silenced lines control

12. WTamiRNA9 line +100µM CdCl 2 Silencing NtHMAα & NtHMAβ increases cadmium tolerance Is 'protecting the shoot against Cd accumulation' the key factor ?

13. Hussain et al, 2004 N. tabacum hmaα hmaβ (W265* E529*) A. thaliana hma2 hma4 Nthmaα Nthmaβ double mutants are dwarf and unable to complete their life cycle

14. Inactivation of NtHMAs leads to low fertility WT 1cm WT amiRNA9 line amiRNA9 line which is due to defective pollen Seed production a a a a a a b amiRNA9 line WT NtHMA  or NtHMA  silenced lines

15. Viability of the pollen % Germination 48.5% 1.4% WT amiRNA9 line Pollen of silenced plants is viable but it cannot germinate 80 60 20 0 100 amiRNA9 line WT 40 Alexander test (%)

16. Zn concentration in the germination medium Bringing Zn improves pollen germination Germination of amiRNA9 pollen % 20 15 5 0 25 10 01.5151501.515150 mM MM

17. MnFe Zn * * Metal content WT amiRNA9 line WT amiRNA9 line Analysis of Mn, Fe & Zn in seeds

18. 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 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 they have a particularly long C-terminal cytosolic tail

19. Remerciements Acknowledgments Dr. Françoise GOSTI Pr. Pierre BERTHOMIEU Dr. Victor HERMAND Dr. François DORLHAC de BORNE Dr. Emilie JULIO Dr. Hélène BERGES Association for Research on Nicotianeae Dr. Felipe RICACHENEVSKY Dr. Mary-Lou GUERINOT

21. N. TabacumA. thalianaA. halleri Répartition du cadmium chez différentes espèces Expression 3/3 5µM de Cd 0,5µM de Cd 100µM de Cd µmol de cadmium par g de matière sèche Dguimi et al, 2009Adapté de Wong et al, 2009 Adapté de Hanikenne et al, 2008