Genetic dissection of the Arabidopsis thaliana ionome

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1 Genetic dissection of the Arabidopsis thaliana ionome Genome Ionome Landscape distribution David E Salt Purdue University, USA

2 What is the Ionome Environment Transcriptome Proteome Ionome The elemental composition of an organism, tissue or cell Metabolome Genome Salt et al Ann Rev Plant Biol 2008

3 High-throughput ionomic analysis of Arabidopsis thaliana Organismal networks Organismal Network Genome Ionome

4 e-laboratory controlling workflow and data acquisition Baxter et al., Plant Physiol 2007

5 e-laboratory Sample analysis and data upload Baxter et al., Plant Physiol 2007

6 e-laboratory Data visualization and download Baxter et al., Plant Physiol 2007

7 Community ionomics HUB to share data 2,746 unique visitors

8 Identification of ionomic mutants Fast neutron population (6000 M2) Lahner et al., Nat Biotech 2003 EMS Population Low Fe (2000 M2) EMS Population Low P (2000 M2)

microarraybased BSA and deletion-mapping to At2g28670 Enhanced

9 Mapping of low shoot Ca ionomic mutants K = 47% Ca = -46% Fe = -39% Ni = -38% 145:01 K Ca Fe Ni Mutation mapped by DNA microarraybased BSA and deletion-mapping to At2g28670 Enhanced Suberin1 (ESB1 ) Baxter et al., PLoS Genetics 2009 Lahner et al., Nat Biotech 2003

10 ESB1 acts in the root to establish the ionomic phenotype of esb1 Grafting Grafted at 5-days PCA of ionomic phenotype Principal Com mponent Col-0 sg Col-0/esb1-2 esb1-2 sg esb1-2/col-0 Analyzed at 5 -weeks Principal Component 1 Baxter et al., PLoS Genetics (2009)

ESB1 expressed in the endodermis and esb1 has elevated aliphatic suberin Total suberin (µg mg -1 dw) 60 50 40 30 20 10 0 * * Total lignin (µg mg -1 dw) 250 200 150

Expression Database. www.arexdb.org Baxter et al.

11 ESB1 expressed in the endodermis and esb1 has elevated aliphatic suberin Total suberin (µg mg -1 dw) * * Total lignin (µg mg -1 dw) Total aliphatic suberin double in esb1 but total lignin not changed ω-hydroxyacids ESB1 endodermal expression Gene expression: AREX: The Arabidopsis Gene Expression Database. Baxter et al., PLoS Genetics (2009) suberin monomers (µg mg -1 ) acids C20 C22 C24 alcohols C18 C20 C22 C16 C18 C18:1 C20 C22 C24 α,ω-diacids C16 C18 C18:1 C20 C22 Ferulic acid All aliphatic suberin components doubled in esb1 Suberin analysis performed by Rochus Benni Franke, University of Bonn

12 esb1 also shows reduced transpiration and increased resistance to wilting Increased resistance to wilting in esb1 Col-0 watered Col-0 8d drought Tra anspiration rate (mmol H 2 O m -2 s -1 ) Lights on Time (min) Reduced transpiration rates in esb1 dir10-1 8d drought dir10-2 8d drought Stomatal pore width (µm) * * Reduced stomatal aperture in esb1 Baxter et al., PLoS Genetics (2009)

Stomatal closure and reduced water loss epidermis cortex endodermis Ca

13 Root suberin forms an extracellular barrier that affect water relations and mineral nutrition Reduced Ca translocation and water deficit signal Stomatal closure and reduced water loss epidermis cortex endodermis Ca 2+ H 2 O suberin suberin ROOT LEAF Baxter et al., PLoS Genetics (2009)

14 Networks connecting genes, the ionome and the landscape Genome Cellular and Organismal Network Ionome Environmental Network Landscape distribution

15 Natural variation in ionome of A. thaliana Shoot ionome in 84 accession compared to Col % difference from Col-0 84 Arabidops sis accessions Li B Na Mg P K Ca Mn Fe Co Ni Cu Zn As Se Mo Cd Ler-0/2 (red lines) cluster together Cvi-0 (blue lines) cluster together Large ionomic variation available for gene and allele discovery

5 4 3 2 1 0 shoot root Col Col Col Ler Ler Col Ler Ler

16 Shoot tissue of Ler-0/2 is low in Mo Ler-0/2 Col-0 and Ler-0 grafts 7 Ler-0 84 % low in Mo Shoot Mo (µg/g dwt) shoot root Col Col Col Ler Ler Col Ler Ler Ler root drives low shoot Mo Baxter et al., PLoS Genetics (2008)

Ler-0 QTL for low shoot Mo Ler-0 x Col-4 RIL population Finer mapping using microarray genotyping Likelihood ratio (LA) probability QTL true/false) ( 250 Chromosome I II III IV V 200 150 100 Shoot 50

17 Ler-0 QTL for low shoot Mo Ler-0 x Col-4 RIL population Finer mapping using microarray genotyping Likelihood ratio (LA) probability QTL true/false) ( 250 Chromosome I II III IV V Shoot 50 Seed cm Low Mo RILs 81 gene interval Candidate screening QTL for low Mo in both shoot and seed on chromosome II MOT1 is annotated as a sulphate Transporter. No functional data mot1-1 is 95% Low in Mo Baxter et al., PLoS Genetics (2008)

Cloning of Ler low Mo QTL Genetic complementation 52 bp deletion in promoter in Ler Ler MOT1 Col-0 MOT1 TAATTTTGATAAGTTTAAGGCAAATTTTTTCTAACACACAATAAAGAAT C

-----------------------------------TCGATACAAACCACA GATACGATATAAAGAGATACGCTTATTGCTCTGTTTCGATACAAACCACA ColxLer Colxmot1-1 Ler MOT1 Col-0 MOT1 AAACAGAAACAATGGAGTCTCAGTCTCAGAGAGGTCAACACGAAACCCCG

18 Cloning of Ler low Mo QTL Genetic complementation 52 bp deletion in promoter in Ler Ler MOT1 Col-0 MOT1 TAATTTTGATAAGTTTAAGGCAAATTTTTTCTAACACACAATAAAGAAT C TAATTTTGATAAGTTTAAGGCAAATTTTTTCTAACACACAATAAAGAATC Ler MOT1 Col-0 MOT1 AGATACTGTCGCCATCAAGGTTTTGCTTTATT AGATACTGTCGCCATCAAGGTTTTGCTTTATTGTCAACGCTTTGGTTTTT Ler MOT1 Col-0 MOT TCGATACAAACCACA GATACGATATAAAGAGATACGCTTATTGCTCTGTTTCGATACAAACCACA ColxLer Colxmot1-1 Ler MOT1 Col-0 MOT1 AAACAGAAACAATGGAGTCTCAGTCTCAGAGAGGTCAACACGAAACCCCG AAACAGAAACAATGGAGTCTCAGTCTCAGAGAGGTCAACACGAAACCCCG Ler-2 Col-0 mot1-1 Lerxmot1-1 mot1-1 cannot complement low Mo in Ler Low expression of MOT1-1 in roots drives low shoot Mo in Ler Relative Expression (2 - CT ) shoot root mot1-1 Ler-2 Col-0 Baxter et al., PLoS Genetics (2008)

19 MOT1 promoter deletion is associated with reduced Mo across 92 diverse Arabidopsis accessions All accession with lowest Mo have loss-offunction MOT1 alleles including Sha and Kly-2 10 Frequency Shoot Mo (ppm) Baxter et al., PLoS Genetics (2008)

20 What is the function of MOT1? yeast MOT1 625 µm 300 µm MitoTracker vector MOT1 Increases Mo accumulation when expressed in yeast 300 µm Expressed in protodermis, epidermis, cortex and vascular 1.25 mm Merged Mitochondrial localization Baxter et al., PLoS Genetics (2008)

21 What is the function of MOT1? PM First committed step in molybdopterin biosynthesis is in the mitochondria Mo Mo MOT1 Mo Perhaps mitochondria is the site for sensing Mo levels.

22 Association of MOT1 allelic variation with soil and geographic location Does MOT1 determine the landscape distribution of Arabidopsis?

Acknowledgments Salt Laboratory Dr Daiyin Chao Dr Ivan Baxter Dr Ana Rus Dr Muthukumar Dr

Dr John Danku (yeast) Analytical Chemists Dr Mourad Ouzzani (Purdue University Discovery Park

Chicago) Dr Magnus Nordborg (University Southern California) Dr Keyan Zhao Dr Olivier Loudet

23 Acknowledgments Salt Laboratory Dr Daiyin Chao Dr Ivan Baxter Dr Ana Rus Dr Muthukumar Dr Hyeong Cheol Park Prashant Hosmani Elena Yakubov Marina Tikhonova Brett Lahner (Arabidopsis) Dr John Danku (yeast) Analytical Chemists Dr Mourad Ouzzani (Purdue University Discovery Park Cyber Center) Brad Kennedy, Gemez Marshall, Maged Zereba Dr Justin Borevitz (University of Chicago) Dr Magnus Nordborg (University Southern California) Dr Keyan Zhao Dr Olivier Loudet (INRA, France) Dr Edgar Cahoon (University of Nebraska-Lincoln) Dr Mary Lou Guerinot (Dartmouth College)

Mapping connections between the genome, ionome and the physical landscape. Photo by Bruce Bohm. David E Salt Purdue University, USA

Mapping connections between the genome, ionome and the physical landscape Photo by Bruce Bohm David E Salt Purdue University, USA What is the Ionome Environment Transcriptome Proteome Ionome The elemental