Improving phosphorus acquisition of white clover (Trifolium repens L.) by transgenic expression of plant-derived phytase and acid phosphatase genes Xue-Feng Ma 1 , Elane Wright 2 , Yaxin Ge, Jeremey Bell, Yajun Xi 3 , Joseph H. Bouton, Zeng-Yu Wang * Forage Improvement Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, USA 1. Introduction Phosphorus (P) is not only a vital component of a number of macromolecules such as nucleic acids, phospholipids and sugar phosphates in plants, but also an integral part of energy metabolism and major biological processes including photosynth- esis, respiration and membrane transportation [1]. Plants obtain P nutrition through root uptake from soil. Although multiple forms of P exist in soil, only the inorganic form (Pi) in soil solution is readily available for plant root uptake. However, the natural concentration of orthophosphate in soil solution is often lower than the level required for normal plant growth. Furthermore, the slow rate of Pi diffusion in soil also limits the availability of Pi in the rhizosphere [2]. In agriculture, this is accommodated by costly applications of P fertilizer. However, the efficiency of plants acquiring P fertilizer is low because up to 80% of the applied P fertilizer could be immobilized rapidly into forms other than orthophosphate [3]. Soil accumulates a large amount of organic phosphate (Po), which accounts for 50–80% of the total P in soil [4]. The most dominant species of Po in soil is inositol hexakisphosphates, also known as phytate [5]. Phytate is synthesized in terrestrial ecosystems by plants and highly accumulated in soil, but poorly utilized by plants [6,7]. Hydrolytic enzymes that catalyze the stepwise phosphate splitting of phytate (or phytic acid) are collectively known as phytases [8]. Based on amino acid sequence similarities and catalytic mechanisms, at least four phytase families have been classified, including histidine acid phosphatase (HAP, EC 3.1.3.8), purple acid phosphatase (PAP, EC 3.1.3.2), b-propeller phytase (BPP, EC 3.1.3.8), and cysteine phosphatases [9,10]. There has been increasing interest in overexpressing phytase genes in plant roots to promote P uptake from soil. Most of the work has focused on genes cloned from microorganisms, such as the HAP phytase gene (phyA) from Aspergillus niger and the BPP phytase gene from Bacillus subtilis (168phyA). Transgenic expres- sion of phyA in Arabidopsis, tobacco (Nicotiana tabacum L.) and subterranean clover (Trifolium subterraneum L.) resulted in increased biomass and total P accumulation when Po was supplied Plant Science 176 (2009) 479–488 ARTICLE INFO Article history: Received 18 November 2008 Received in revised form 30 December 2008 Accepted 2 January 2009 Available online 20 January 2009 Keywords: White clover Phytase Acid phosphatase Organic phosphorus Phosphate acquisition Transgenic plant ABSTRACT Phosphate is one of the least available macronutrients restricting crop production in many ecosystems. A phytase gene (MtPHY1) and a purple acid phosphatase gene (MtPAP1), both isolated from the model legume Medicago truncatula, were introduced into white clover (Trifolium repens L.) by Agrobacterium- mediated transformation. The transgenes were driven by the constitutive CaMV35S promoter or the root-specific MtPT1 promoter. Transcripts were detected in roots of the transgenic plants. Phytase or acid phosphatase (APase) activities in root apoplasts of the transgenic plants were increased up to three- fold compared to the wild type control. After the plants were grown 80 days in sand pots supplied with organic phosphorus (Po) as the sole P source, dry weights of shoot tissues of the best performing transgenic plants almost doubled that of the control and were comparable to the counterparts supplied with inorganic phosphorus (Pi). Relative biomass production of the transgenics under Po treatment was over 90% and 80% of that from the Pi treatment when the plants were grown in hydroponics (40 days) and sand pots (80 days), respectively. In contrast, biomass of the wild type controls under Po treatment was only about 50% of the Pi treatment in either hydroponic cultures or sand pots. In addition, shoot P concentrations of the transgenic plants were significantly increased compared to the control. Transgenic plants accumulated much higher amounts of total P (up to 2.6-fold after 80 days of growth) than the control in Po supplied sand pots. The results showed that transgenic expression of MtPHY1 or MtPAP1 in white clover plants increased their abilities of utilizing organic phosphorus in response to P deficiency. ß 2009 Elsevier Ireland Ltd. All rights reserved. * Corresponding author. Tel.: +1 580 224 6830; fax: +1 580 224 6802. E-mail address: zywang@noble.org (Z.-Y. Wang). 1 Current address: Ceres, Inc., Thousand Oaks, CA 91320, USA. 2 Current address: Oklahoma State University, Stillwater, OK 74078, USA. 3 Current address: College of Agriculture, Northwest A and F University, Yangling, Shanxi 712100, China. Contents lists available at ScienceDirect Plant Science journal homepage: www.elsevier.com/locate/plantsci 0168-9452/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2009.01.001