Variation in Molybdenum Content Across Broadly Distributed Populations of Arabidopsis thaliana Is Controlled by a Mitochondrial Molybdenum Transporter (MOT1) Ivan Baxter 1 , Balasubramaniam Muthukumar 2 , Hyeong Cheol Park 2 , Peter Buchner 3 , Brett Lahner 2 , John Danku 2 , Keyan Zhao 4 , Joohyun Lee 5 , Malcolm J. Hawkesford 3 , Mary Lou Guerinot 5 , David E. Salt 1,2 * 1 Bindley Bioscience Center, Purdue University, West Lafayette, Indiana, United States of America, 2 Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana, United States of America, 3 Rothamsted Research, Harpenden, Hertfordshire, United Kingdom, 4 Molecular and Computational Biology, University of Southern California, Los Angeles, California, United States of America, 5 Biological Sciences, Dartmouth College, Hanover, New Hampshire, United States of America Abstract Molybdenum (Mo) is an essential micronutrient for plants, serving as a cofactor for enzymes involved in nitrate assimilation, sulfite detoxification, abscisic acid biosynthesis, and purine degradation. Here we show that natural variation in shoot Mo content across 92 Arabidopsis thaliana accessions is controlled by variation in a mitochondrially localized transporter ( Molybdenum Transporter 1 - MOT1) that belongs to the sulfate transporter superfamily. A deletion in the MOT1 promoter is strongly associated with low shoot Mo, occurring in seven of the accessions with the lowest shoot content of Mo. Consistent with the low Mo phenotype, MOT1 expression in low Mo accessions is reduced. Reciprocal grafting experiments demonstrate that the roots of Ler-0 are responsible for the low Mo accumulation in shoot, and GUS localization demonstrates that MOT1 is expressed strongly in the roots. MOT1 contains an N-terminal mitochondrial targeting sequence and expression of MOT1 tagged with GFP in protoplasts and transgenic plants, establishing the mitochondrial localization of this protein. Furthermore, expression of MOT1 specifically enhances Mo accumulation in yeast by 5-fold, consistent with MOT1 functioning as a molybdate transporter. This work provides the first molecular insight into the processes that regulate Mo accumulation in plants and shows that novel loci can be detected by association mapping. Citation: Baxter I, Muthukumar B, Park HC, Buchner P, Lahner B, et al. (2008) Variation in Molybdenum Content Across Broadly Distributed Populations of Arabidopsis thaliana Is Controlled by a Mitochondrial Molybdenum Transporter (MOT1). PLoS Genet 4(2): e1000004. doi:10.1371/journal.pgen.1000004 Editor: Joy Bergelson, University of Chicago, United States of America Received October 31, 2007; Accepted January 17, 2008; Published February 29, 2008 Copyright: ß 2008 Baxter et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by the National Institutes of Health, National Institute of General Medicine (R01 GM078536-01A1), the National Science Foundation Arabidopsis 2010 program (IOB 0419695), and the Indiana 21st Century Research and Technology Fund (912010479). KZ is supported by National Institutes of Health Center of Excellence in Genomic Science grant P50 HG002790. Rothamsted Research receives grant-aided support from the Biotechnology and Biological Science Research Council (BBSRC) of the UK. The funding agencies had no role in: the design and conduct of the study, the collection, analysis, and interpretation of the data, nor in the preparation, review, or approval of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: dsalt@purdue.edu Introduction Plants have developed complex biochemical and regulatory pathways to acquire mineral nutrients from the soil environment and distribute them to appropriate tissues. Natural populations of Arabidopsis thaliana (Arabidopsis) provide an excellent system to study how plants have adapted their mineral nutrient and trace element uptake pathways to thrive under different environmental conditions. Molybdenum (Mo) is an important micronutrient for plants, being incorporated into molybdopterin, an essential cofactor for enzymes involved in nitrate assimilation, sulfite detoxification, abscisic acid biosynthesis and purine degradation [1]. Molybdenum in either deficiency or excess has been demonstrated to inhibit plant growth and agricultural productivity [2]. The genes comprising the biochemical pathway that synthesizes the molybdopterin cofactor have been identified in plants, animals and microbes, but to date, a Mo transporter in plants has not been found [2]. The first committed step in molybdopterin biosynthesis occurs in the mitochondria [3], confirming the predicted sub-cellular localization of the enzymes [1]. The remaining 3 steps are thought to occur in the cytoplasm [3]. While a substantial amount is known about the biochemistry, enzymology and underlying genetics of molybdopterin biosynthe- sis, very little is known about the mechanisms for Mo uptake, distribution and accumulation in plants. In this study, natural variation in whole plant Mo accumulation has been coupled with genomics techniques and genetics to identify a mitochondrial Mo transporter (MOT1) that regulates whole plant Mo content in Arabidopsis. Alleles of this gene are demonstrated to be responsible for low Mo accumulation across a diversity collection of 92 Arabidopsis accessions. All soil grown plant ionomic data from this study is freely available at www.purdue.edu/dp/ ionomics [4]. Results Shoot tissue from a geographically diverse panel of 98 Arabidopsis accessions (93 from [5] plus Bu-15, Col-4, Kas-1, PLoS Genetics | www.plosgenetics.org 1 2008 | Volume 4 | Issue 2 | e1000004