Abstract
Phosphorus (P) is an essential macronutrient for crop development and production. Phosphate starvation response 1 (PHR1) acts as the central regulator for Pi-signaling and Pi-homeostasis in plants by binding to the cis-element PHR1 binding sequence (P1BS; GNATATNC). However, how phosphate starvation-induced gene expression is regulated remains obscure. In this work, we investigated the DNA binding affinity of the PHR1 ortholog OsPHR2 to its downstream target genes in Oryza sativa (rice). We confirmed that a combination of P1BS and P1BS-like motifs are essential for stable binding by OsPHR2. Furthermore, we report that variations in P1BS motif bases affected the binding affinity of OsPHR2 and that the highest affinity motif was GaATATtC (designated the A–T-type P1BS). We also found that a combination of two A–T-type P1BS elements in tandem, namely HA-P1BS, was very efficient for binding of OsPHR2. Using the cis-regulator HA-P1BS, we modified the promoters of Transporter Traffic Facilitator 1 (PHF1), a key factor controlling endoplasmic reticulum-exit of phosphate transporters to the plasma membrane, for efficient uptake of phosphorous in an energetically neutral way. Transgenic plants with the modified promoters showed significantly enhanced tolerance to low phosphate stress in both solution and soil conditions, which provides a new strategy for crop improvement to enhance tolerance of nutrient deficiency.
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References
Aung K, Lin SI, Wu CC, Huang YT, Cl Su, Chiou TJ (2006) pho2, a phosphate overaccumulator, is caused by a nonsense mutation in a microRNA399 target gene. Plant Physiol 141:1000–1011
Bari R, Datt Pant B, Stitt M, Scheible WR (2006) PHO2, microRNA399, and PHR1 define a phosphate-signaling pathway in plants. Plant Physiol 141:988–999
Bayle V, Arrighi JF, Creff A, Nespoulous C, Vialaret J, Rossignol M, Gonzalez E, Paz-Ares J, Nussaume L (2011) Arabidopsis thaliana high-affinity phosphate transporters exhibit multiple levels of posttranslational regulation. Plant Cell 23:1523–1535
Bustos R, Castrillo G, Linhares F, Puga MI, Rubio V, Perez-Perez J, Solano R, Leyva A, Paz-Ares J (2010) A central regulatory system largely controls transcriptional activation and repression responses to phosphate starvation in Arabidopsis. PLoS Genet 6:e1001102
Calvenzani V, Testoni B, Gusmaroli G, Lorenzo M, Gnesutta N et al (2012) Interactions and CCAAT-binding of Arabidopsis thaliana NF-Y subunits. PLoS One 7(8):e42902. doi:10.1371/journal.pone.0042902
Chen JY, Liu Y, Ni J, Wang YF, Bai YH, Shi J, Gan J, Wu ZC, Wu P (2011) OsPHF1 regulates the plasma membrane localization of low- and high-affinity inorganic phosphate transporters and determines inorganic phosphate uptake and translocation in rice. Plant Physiol 157:269–278
Chiou TJ, Lin SI (2011) Signaling network in sensing phosphate availability in plants. Annu Rev Plant Biol 62:185–206
Duan K, Yi KK, Dang L, Huang HT, Wu W, Wu P (2008) Characterization of a sub-family of Arabidopsis genes with the SPX domain reveals their diverse functions in plant tolerance to phosphorus starvation. Plant J 54:965–975
Franco-Zorrilla JM, González E, Bustos R, Linhares F, Leyva A, Paz-Ares J (2004) The transcriptional control of plant responses to phosphate limitation. J Exp Bot 55:285–293
Franco-Zorrilla JM, Valli A, Todesco M, Mateos I, Puga MI, Rubio-Somoza I, Leyva A, Weigel D, García JA, Paz-Ares J (2007) Target mimicry provides a new mechanism for reg-ulation of microRNA activity. Nat Genet 39:1033–1037
González E, Solano R, Rubio V, Leyva A, Paz-Ares J (2005) Phosphate transporter traffic facilitator1 is a plant-specific SEC12-related protein that enables the endoplasmic reticulum exit of a high-affinity phosphate transporter in Arabidopsis. Plant Cell 17:3500–3512
Gutjahr C, Banba M, Croset V, An K, Miyao A, An G, Hirochika H, Imaizumi-Anraku H, Paszkowski U (2008) Arbuscular mycorrhiza-specific signaling in rice transcends the common symbiosis signaling pathway. Plant Cell 20:2989–3005
Higuchi R, Krummel B, Saiki RK (1988) A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucl Acids Res 16:7351–7367
Holford ICR (1997) Soil phosphorus: its measurement, and its uptake by plants. Aust J Soil Res 35:227–239
Huang TK, Han CL, Lin SI, Chen YJ, Tsai YC, Chen YR, Chen JW, Lin WY, Chen PM, Liu TY, Chen YS, Sun CM, Chiou TJ (2013) Identification of downstream components of ubiquitin-conjugating enzyme PHOSPHATE2 by quantitative membrane proteomics in Arabidopsis roots. Plant Cell 25:4044–4060
Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907
Kant S, Peng M, Rothstein SJ (2011) Genetic regulation by NLA and microRNA827 for maintaining nitrate-dependent phosphate homeostasis in Arabidopsis. PLoS Genet 7:e1002021
Kobae Y, Hata S (2010) Dynamics of periarbuscular membranes visualized with a fluorescent phosphate transporter in arbuscular mycorrhizal roots of rice. Plant Cell Physiol 51:341–353
Lin WY, Huang TK, Chiou TJ (2013) Nitrogen limitation adaptation, a target of microRNA827, mediates degradation of plasma membrane-localized phosphate transporters to maintain phosphate homeostasis in Arabidopsis. Plant Cell 25:4061–4074
Liu F, Wang ZY, Ren HY, Shen CJ, Li Y, Ling HQ, Wu CY, Lian XM, Wu P (2010) OsSPX1 suppresses the function of OsPHR2 in the regulation of expression of OsPT2 and phosphate homeostasis in shoots of rice. Plant J 62:508–517
Lv QD, Zhong YJ, Wang YG, Wang ZY, Zhang L, Shi J, Wu ZC, Liu Y, Mao CZ, Yi KK, Wu P (2014) SPX4 negatively regulates phosphate signaling and homeostasis through its interaction with PHR2 in rice. Plant Cell 26:1586–1597
Marschner H (1995) Mineral nutrition of higher plants. Academic Press, London
Miura K, Rus A, Sharkhuu A, Yokoi S, Karthikeyan AS, Raghothama KG, Baek D, Koo YD, Jin JB, Bressan RA, Yun DJ, Hasegawa PM (2005) The Arabidopsis SUMO E3 ligase SIZ1 controls phosphate deficiency responses. Proc Natl Acad Sci 102:7760–7765
Oropeza-Aburto A, Cruz-Ramírez A, Acevedo-Hernández GJ, Pérez-Torres CA, Caballero-Pérez J, Herrera-Estrella L (2012) Functional analysis of the Arabidopsis PLDZ2 promoter reveals an evolutionarily conserved low-Pi-responsive transcriptional enhancer element. J Exp Bot 63:2189–2202
Pao SS, Paulsen IT, Saier MH Jr (1998) Major facilitator superfamily. Microbiol Mol Biol Rev 62:1–34
Park BS, Seo JS, Chua NH (2014) Nitrogen limitation adaptation recruits phosphate2 to target the phosphate transporter PT2 for degradation during the regulation of Arabidopsis phosphate homeostasis. Plant Cell 26:454–464
Paszkowski U, Kroken S, Roux C, Briggs SP (2002) Rice phosphate transporters include an evolutionarily divergent gene specifically activated in arbuscular mycorrhizal symbiosis. Proc Natl Acad Sci 99:13324–13329
Poirier Y, Bucher M (2002) Phosphate transport and homeostasis in Arabidopsis. The Arabidopsis Book 1:e0024. doi:10.1199/tab.0024
Raghothama KG (1999) Phosphate acquisition. Annu Rev Plant Physiol 50:665–693
Rajkumar AS, Dénervaud N, Maerkl SJ (2013) Mapping the fine structure of a eukaryotic promoter input–output function. Nat Genet 45:1207–1215
Rouached H, Stefanovic A, Secco D, Bulak Arpat A, Gout E, Bligny R, Poirier Y (2011) Uncoupling phosphate deficiency from its major effects on growth and transcriptome via PHO1 expression in Arabidopsis. Plant J 65:557–670
Rubio V, Linhares F, Solano R, Martin AC, Iglesias J, Leyva A, Paz-Ares J (2001) A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plants and in unicellular algae. Genes Dev 15:2122–2133
Secco D, Wang C, Arpat BA, Wang ZY, Poirier Y, Tyerman SD, Wu P, Shou HX, Whelan J (2012) The emerging importance of the SPX domain-containing proteins in phosphate homeostasis. New Phytol 193:842–851
Segal E, Widom J (2009) From DNA sequence to transcriptional behaviour: a quantitative approach. Nat Rev Genet 10:443–456
Shi J, Hu H, Zhang KM, Zhang W, Yu Y, Wu ZC, Wu P (2014) The paralogous SPX3 and SPX5 genes redundantly modulate Pi homeostasis in rice. J Exp Bot 65:859–870
Shin H, Shin HS, Dewbre GR, Harrison MJ (2004) Phosphate transport in Arabidopsis: Pht1;1 and Pht1;4 play a major role in phosphate acquisition from both low- and high-phosphate environments. Plant J 39:629–642
Vander-Meer JE, Ahituv N (2011) cis-regulatory mutations are a genetic cause of human limb malformations. Dev Dyn 240:920–930
Wang C, Ying S, Huang HJ, Li K, Wu P, Shou HX (2009a) Involvement of OsSPX1 in phosphate homeostasis in rice. Plant J 57:895–904
Wang ZY, Hu H, Huang HJ, Duan K, Wu ZC, Wu P (2009b) Regulation of OsSPX1 and OsSPX3 on expression of OsSPX domain genes and Pi-starvation signaling in rice. J Integr Plant Biol 51:663–674
Wang C, Huang W, Ying YH, Li S, Secco D, Tyerman S, Whelan J, Shou HX (2012) Functional characterization of the rice SPX-MFS family reveals a key role of OsSPX-MFS1 in controlling phosphate homeostasis in leaves. New Phytol 196:139–148
Wang ZY, Ruan WY, Shi J, Zhang L, Xiang D, Yang C, Li CY, Wu ZC, Liu Y, Yu YN, Shou HX, Mo XR, Mao CZ, Wu P (2014) Rice SPX1 and SPX2 inhibit phosphate starvation responses through interacting with PHR2 in a phosphate-dependent manner. Proc Natl Acad Sci 111:14953–14958
Wu ZC, Ren HY, McGrath SP, Wu P, Zhao FJ (2011) Investigating the contribution of the phosphate transport pathway to arsenic accumulation in rice. Plant Physiol 157:498–508
Wu P, Shou HX, Xu GH, Lian XM (2013) Improvement of phosphorus efficiency in rice on the basis of understanding phosphate signaling and homeostasis. Curr Opin Plant Boil 16:1–8
Yang XJ, Finnegan PM (2010) Regulation of phosphate starvation responses in higher plants. Ann Bot (Lond.) 105:513–526
Yang SY, Grønlund M, Jakobsen I, Grotemeyer MS, Rentsch D, Miyao A, Hirochika H, Kumar CS, Sundaresan V, Salamin N, Catausan S, Mattes N, Heuer S, Paszkowski U (2012) Nonredundant regulation of rice arbuscular mycorrhizal symbiosis by two members of the phosphate transporter1 gene family. Plant Cell 24:4236–4251
Zhou X, O’Shea EK (2011) Integrated approaches reveal determinants of genome-wide binding and function of the transcription factor Pho4. Mol Cell 42:826–836
Zhou J, Jiao FC, Wu ZC, Li YY, Wang XM, He XW, Zhong WQ, Wu P (2008) OsPHR2 is involved in phosphate-starvation signaling and excessive phosphate accumulation in shoots of plants. Plant Physiol 146:1673–1686
Acknowledgments
This work was supported by grants from the National Basic Research and Development Program of China (Grant No. 2011CB100303) and Ministry of Science and Technology of China (Grant Nos. 2010DFA31080 and 2012AA10A302).
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Wenyuan Ruan and Meina Guo have contributed equally to this work.
Accession Numbers
Rice Genome Initiative locus identifiers for the genes mentioned in this article are Os07g25710 (OsPHR2) and Os07g09000 (OsPHF1). The GenBank Accession Numbers: AY568759 (OsIPS1), AK240849 (OsIPS2).
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Ruan, W., Guo, M., Cai, L. et al. Genetic manipulation of a high-affinity PHR1 target cis-element to improve phosphorous uptake in Oryza sativa L.. Plant Mol Biol 87, 429–440 (2015). https://doi.org/10.1007/s11103-015-0289-y
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DOI: https://doi.org/10.1007/s11103-015-0289-y