The characterization of novel mycorrhiza-specific phosphate transporters from Lycopersicon esculentum and Solanum tuberosum uncovers functional redundancy in symbiotic phosphate transport in solanaceous species.

Solanaceous species are among the >200 000 plant species worldwide forming a mycorrhiza, that is, a root living in symbiosis with soil-borne arbuscular-mycorrhizal (AM) fungi. An important parameter of this symbiosis, which is vital for ecosystem productivity, agriculture, and horticulture, is the transfer of phosphate (Pi) from the AM fungus to the plant, facilitated by plasma membrane-spanning Pi transporter proteins. The first mycorrhiza-specific plant Pi transporter to be identified, was StPT3 from potato [Nature414 (2004) 462]. Here, we describe novel Pi transporters from the solanaceous species tomato, LePT4, and its orthologue StPT4 from potato, both being members of the Pht1 family of plant Pi transporters. Phylogenetic tree analysis demonstrates clustering of both LePT4 and StPT4 with the mycorrhiza-specific Pi transporter from Medicago truncatula [Plant Cell, 14 (2002) 2413] and rice [Proc. Natl Acad. Sci. USA, 99 (2002) 13324], respectively, but not with StPT3, indicating that two non-orthologous mycorrhiza-responsive genes encoding Pi transporters are co-expressed in the Solanaceae. The cloned promoter regions from both genes, LePT4 and StPT4, exhibit a high degree of sequence identity and were shown to direct expression exclusively in colonized cells when fused to the GUS reporter gene, in accordance with the abundance of LePT4 and StPT4 transcripts in mycorrhized roots. Furthermore, extensive sequencing of StPT4-like clones and subsequent expression analysis in potato and tomato revealed the presence of a close paralogue of StPT4 and LePT4, named StPT5 and LePT5, respectively, representing a third Pi transport system in solanaceous species which is upregulated upon AM fungal colonization of roots. Knock out of LePT4 in the tomato cv. MicroTom indicated considerable redundancy between LePT4 and other Pi transporters in tomato.

[1]  M. J. Harrison,et al.  Phosphate transport in Arabidopsis: Pht1;1 and Pht1;4 play a major role in phosphate acquisition from both low- and high-phosphate environments. , 2004, The Plant journal : for cell and molecular biology.

[2]  P. Hansen,et al.  Phosphate pool dynamics in the arbuscular mycorrhizal fungus Glomus intraradices studied by in vivo31 P NMR spectroscopy. , 2004, The New phytologist.

[3]  I. Jakobsen,et al.  Functional diversity in arbuscular mycorrhizal (AM) symbioses: the contribution of the mycorrhizal P uptake pathway is not correlated with mycorrhizal responses in growth or total P uptake , 2004 .

[4]  P. Zimmermann,et al.  The Expression of an Extensin-Like Protein Correlates with Cellular Tip Growth in Tomato1 , 2002, Plant Physiology.

[5]  James B. Hicks,et al.  A plant DNA minipreparation: Version II , 1983, Plant Molecular Biology Reporter.

[6]  P. Martínez,et al.  Physiological Regulation of the Derepressible Phosphate Transporter in Saccharomyces cerevisiae , 1998, Journal of bacteriology.

[7]  C. Rausch,et al.  Molecular mechanisms of phosphate transport in plants , 2002, Planta.

[8]  M. Bucher,et al.  Symbiotic phosphate transport in arbuscular mycorrhizas. , 2005, Trends in plant science.

[9]  Matthew Hurles,et al.  Gene Duplication: The Genomic Trade in Spare Parts , 2004, PLoS biology.

[10]  M. J. Harrison,et al.  A Chloroplast Phosphate Transporter, PHT2;1, Influences Allocation of Phosphate within the Plant and Phosphate-Starvation Responses Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.002220. , 2002, The Plant Cell Online.

[11]  I. Jakobsen,et al.  The relative contribution of hyphae and roots to phosphorus uptake by arbuscular mycorrhizal plants, measured by dual labelling with 32P and 33P , 1993 .

[12]  L. Willmitzer,et al.  Two cDNAs from potato are able to complement a phosphate uptake-deficient yeast mutant: identification of phosphate transporters from higher plants. , 1997, The Plant cell.

[13]  M. J. Harrison,et al.  The arbuscular mycorrhizal symbiosis : an underground association , 1997 .

[14]  N. Amrhein,et al.  Evolutionary conservation of a phosphate transporter in the arbuscular mycorrhizal symbiosis. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[15]  F. W. Smith,et al.  Characterization of two phosphate transporters from barley; evidence for diverse function and kinetic properties among members of the Pht1 family , 2003, Plant Molecular Biology.

[16]  M. Sudol,et al.  The WW domain of Yes-associated protein binds a proline-rich ligand that differs from the consensus established for Src homology 3-binding modules. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[17]  Sarah Melamed,et al.  A new model system for tomato genetics , 1997 .

[18]  Amos Bairoch,et al.  ScanProsite: a reference implementation of a PROSITE scanning tool. , 2002, Applied bioinformatics.

[19]  M. J. Harrison,et al.  A phosphate transporter from the mycorrhizal fungus Glomus versiforme , 1995, Nature.

[20]  T. Chiou,et al.  The spatial expression patterns of a phosphate transporter (MtPT1) from Medicago truncatula indicate a role in phosphate transport at the root/soil interface. , 2001, The Plant journal : for cell and molecular biology.

[21]  G. Borst-Pauwels Ion transport in yeast. , 1981, Biochimica et biophysica acta.

[22]  U. Paszkowski,et al.  Rice phosphate transporters include an evolutionarily divergent gene specifically activated in arbuscular mycorrhizal symbiosis , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[23]  B. Dell,et al.  Nutrient uptake in mycorrhizal symbiosis , 1994, Plant and Soil.

[24]  Thierry Vermat,et al.  Integral membrane proteins of the chloroplast envelope: Identification and subcellular localization of new transporters , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[25]  F. Skoog,et al.  A revised medium for rapid growth and bio assays with tobacco tissue cultures , 1962 .

[26]  B. Persson,et al.  Functional analysis and cell-specific expression of a phosphate transporter from tomato , 1998, Planta.

[27]  K. Raghothama,et al.  Tomato phosphate transporter genes are differentially regulated in plant tissues by phosphorus. , 1998, Plant physiology.

[28]  N. Claassen,et al.  Availability in Soil and Acquisition by Plants as the Basis for Phosphorus and Potassium supply to Plants , 1989 .

[29]  F. Gaymard,et al.  Tissue-specific expression of Arabidopsis AKT1 gene is consistent with a role in K+ nutrition. , 1996, The Plant journal : for cell and molecular biology.

[30]  Mark C. Brundrett,et al.  Coevolution of roots and mycorrhizas of land plants. , 2002, The New phytologist.

[31]  K. Raghothama,et al.  Transcriptional regulation of plant phosphate transporters. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[32]  M. Bevan,et al.  GUS fusions: beta‐glucuronidase as a sensitive and versatile gene fusion marker in higher plants. , 1987, The EMBO journal.

[33]  D. Shibata,et al.  Overexpression of an Arabidopsis thaliana high-affinity phosphate transporter gene in tobacco cultured cells enhances cell growth under phosphate-limited conditions. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[34]  N. Amrhein,et al.  Pht2;1 Encodes a Low-Affinity Phosphate Transporter from Arabidopsis , 1999, Plant Cell.

[35]  I. Maldonado-Mendoza,et al.  A phosphate transporter gene from the extra-radical mycelium of an arbuscular mycorrhizal fungus Glomus intraradices is regulated in response to phosphate in the environment. , 2001, Molecular plant-microbe interactions : MPMI.

[36]  I. Jakobsen,et al.  Mycorrhizal Fungi Can Dominate Phosphate Supply to Plants Irrespective of Growth Responses1 , 2003, Plant Physiology.

[37]  F. W. Smith,et al.  Expression analysis suggests novel roles for members of the Pht1 family of phosphate transporters in Arabidopsis. , 2002, The Plant journal : for cell and molecular biology.

[38]  A. Sandelin,et al.  Identification of conserved regulatory elements by comparative genome analysis , 2003, Journal of biology.

[39]  M. J. Harrison,et al.  Cloning and characterization of two phosphate transporters from Medicago truncatula roots: regulation in response to phosphate and to colonization by arbuscular mycorrhizal (AM) fungi. , 1998, Molecular plant-microbe interactions : MPMI.

[40]  J. Jansa,et al.  Long-distance transport of P and Zn through the hyphae of an arbuscular mycorrhizal fungus in symbiosis with maize , 2003 .

[41]  P. Zimmermann,et al.  Engineering the root-soil interface via targeted expression of a synthetic phytase gene in trichoblasts. , 2003, Plant biotechnology journal.

[42]  D. Schachtman,et al.  Phosphorus Uptake by Plants: From Soil to Cell , 1998, Plant physiology.

[43]  M. Bevan,et al.  Binary Agrobacterium vectors for plant transformation. , 1984, Nucleic acids research.

[44]  A. Hoekema,et al.  A small-scale procedure for the rapid isolation of plant RNAs. , 1989, Nucleic acids research.

[45]  Y. Oshima,et al.  Regulation of inorganic phosphate transport systems in Saccharomyces cerevisiae , 1985, Journal of bacteriology.

[46]  D. R. Hoagland,et al.  GENERAL NATURE OF THE PROCESS OF SALT ACCUMULATION BY ROOTS WITH DESCRIPTION OF EXPERIMENTAL METHODS. , 1936, Plant physiology.

[47]  B. Persson,et al.  Identification, cloning and characterization of a derepressible Na+-coupled phosphate transporter in Saccharomyces cerevisiae , 1998, Molecular and General Genetics MGG.

[48]  H. Marschner Mineral Nutrition of Higher Plants , 1988 .

[49]  T. Altmann,et al.  Analysis of phosphate acquisition efficiency in different Arabidopsis accessions. , 2000, Plant physiology.

[50]  S. Harashima,et al.  The PHO84 gene of Saccharomyces cerevisiae encodes an inorganic phosphate transporter , 1991, Molecular and cellular biology.

[51]  J. Lynch,et al.  Regulation of root hair density by phosphorus availability in Arabidopsis thaliana , 2001 .

[52]  S. Dickson,et al.  Transfer of phosphate from fungus to plant in VA mycorrhizas: calculation of the area of symbiotic interface and of fluxes of P from two different fungi to A Allium porrum L. , 1994, The New phytologist.

[53]  T. Davies,et al.  Restricted spatial expression of a high-affinity phosphate transporter in potato roots , 2003, Journal of Cell Science.

[54]  D. Weigel,et al.  Regulatory Elements of the Floral Homeotic Gene AGAMOUS Identified by Phylogenetic Footprinting and Shadowing Online version contains Web-only data. Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.009548. , 2003, The Plant Cell Online.

[55]  J. Cairney,et al.  Efflux of phosphate from the ectomycorrhizal basidiomycete Pisolithus tinctorius: general characteristics and the influence of intracellular phosphorus concentration , 1993 .

[56]  M. Osaki,et al.  Cloning and characterization of four phosphate transporter cDNAs in tobacco , 2002 .

[57]  Kay Hofmann,et al.  Tmbase-A database of membrane spanning protein segments , 1993 .

[58]  E. Ábrahám,et al.  Distribution of 1000 sequenced T-DNA tags in the Arabidopsis genome. , 2002, The Plant journal : for cell and molecular biology.

[59]  G. Cox,et al.  TRANSLOCATION AND TRANSFER OF NUTRIENTS IN VESICULAR-ARBUSCULAR , 1976 .

[60]  P. Tinker,et al.  TRANSLOCATION AND TRANSFER OF NUTRIENTS IN VESICULAR‐ARBUSCULAR MYCORRHIZAS , 1978 .

[61]  Y. Poirier,et al.  Phosphate Transport and Homeostasis in Arabidopsis , 2002, The arabidopsis book.

[62]  Mark W. Chase,et al.  Evolution of the angiosperms: calibrating the family tree , 2001, Proceedings of the Royal Society of London. Series B: Biological Sciences.

[63]  M. J. Harrison,et al.  A Phosphate Transporter from Medicago truncatula Involved in the Acquisition of Phosphate Released by Arbuscular Mycorrhizal Fungi Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.004861. , 2002, The Plant Cell Online.

[64]  J A Raven,et al.  Roots: evolutionary origins and biogeochemical significance. , 2001, Journal of experimental botany.

[65]  E. O’Shea,et al.  Phosphate transport and sensing in Saccharomyces cerevisiae. , 2001, Genetics.

[66]  J. Jansa,et al.  A phosphate transporter expressed in arbuscule-containing cells in potato , 2001, Nature.

[67]  A. Force,et al.  Preservation of duplicate genes by complementary, degenerative mutations. , 1999, Genetics.

[68]  W. Versaw,et al.  A phosphate-repressible, high-affinity phosphate permease is encoded by the pho-5+ gene of Neurospora crassa. , 1995, Gene.

[69]  Y. Elkind,et al.  Technical advance: a high throughput system for transposon tagging and promoter trapping in tomato. , 2000, The Plant journal : for cell and molecular biology.

[70]  P. Zimmermann,et al.  Expression analysis suggests novel roles for the plastidic phosphate transporter Pht2;1 in auto- and heterotrophic tissues in potato and Arabidopsis. , 2004, The Plant journal : for cell and molecular biology.