Physiologia Plantarum 143: 344–354. 2011 Copyright © Physiologia Plantarum 2011, ISSN 0031-9317 Characterization of upregulated genes associated with high phosphorus accumulation in cucumber Priya Padmanabhan a,† , Perumal Venkatachalam b,‡ and Shivendra V Sahi a,∗ a Department of Biology, Western Kentucky University, Bowling Green, KY 42101, USA b Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA Correspondence *Corresponding author, e-mail: shiv.sahi@wku.edu Received 24 February 2011; revised 22 June 2011 doi:10.1111/j.1399-3054.2011.01512.x Excessive application of phosphorus (P)-rich manures to agricultural lands often results in P-accumulation in soils leading to water pollution through runoffs and leaching. Use of suitable plant species that can extract and sequester excess P from soil into their biomass is an effective method of remediation of P-contaminated soils. Knowledge on the molecular responses of plants to high P-accumulation and tolerance is lacking. Therefore, a sup- pression subtractive hybridization (SSH) strategy was employed to identify and elucidate the pattern of gene expression related to P-tolerance and accu- mulation in cucumber (Cucumis sativus L.), a P-accumulator plant. RNA isolated from cucumber grown in high P was used for ‘tester’ cDNA synthesis and SSH library preparation. A total of 63 cDNAs were identified as showing upregulated expression in this plant in response to high P. No putative func- tion could be assigned to 7 (11%) of the 63 upregulated high P-modulated genes and 11 expressed sequence tags (ESTs) (17%) did not match database entries. The remaining 45 ESTs were grouped into five functional classes. The majority of these ESTs belonged to three groups: ‘metabolism’, ‘protein synthesis/degradation and signaling’ and ‘cell structure/cell wall’. Only six ‘stress/defense’-related ESTs were identified from this library. The results of reverse northern blot analysis was further confirmed and validated through semi-quantitative RT-PCR carried out with representative ESTs identified in this study. The research reported here may contribute to a preliminary under- standing of the high P-related gene expression in this P-accumulating plant. Introduction Phosphorus (P) is an important nutrient limiting plant growth and development. So, P-fertilizers and animal manures are applied to soil to enhance P availability. Inorganic P-sources are often added to animal feeds to Abbreviations – C4H, cinnamate 4-hydroxylase; CAF1, CCR4 associated factor 1-related protein; CS, cysteine synthase; ESTs, expressed sequence tags; GRP, glycine-rich protein; GTPBP, small GTP-binding protein; MBF1, transcriptional co-activator multiprotein bridging factor; MJ, methyl jasmonate; MT, metallothionein; P, phosphorus; PCR, polymerase chain reaction; PME, pectin methylesterase; PP2, Phloem protein 2; PRP, proline-rich protein; RT-PCR, reverse transcriptase polymerase chain reaction; SAT, serine acetyltransferase; SSH, suppression subtractive hybridization. † Present address: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA ‡ Present address: Department of Biotechnology, Periyar University, Salem, Tamil Nadu 636011, India ensure adequate nutrition to the livestock, but most of this dietary P passes through livestock and is excreted in animal manures. Utilization of animal manure as a fertilizer on crops and agricultural lands is a well- known practice to recycle nutrients (Khaleel et al. 1981). Thus, in areas with intensive livestock farming, the soil 344 Physiol. Plant. 143, 2011