Planta

, Volume 222, Issue 1, pp 27–36

Transgenic expression of a novel M. truncatula phytase gene results in improved acquisition of organic phosphorus by Arabidopsis

Authors

  • Kai Xiao
    • Forage Improvement Division, The Samuel Roberts Noble Foundation
    • College of Agronomy, Hebei Agricultural University
  • Maria J. Harrison
    • Boyce Thompson Institute for Plant Research
    • Forage Improvement Division, The Samuel Roberts Noble Foundation
Original Article

DOI: 10.1007/s00425-005-1511-y

Cite this article as:
Xiao, K., Harrison, M.J. & Wang, Z. Planta (2005) 222: 27. doi:10.1007/s00425-005-1511-y

Abstract

A full-length cDNA encoding an extracellular form of phytase was isolated from the model legume Medicago truncatula. The phytase cDNA (MtPHY1) has an open reading frame of 1,632 bp predicted to encode 543 amino acids including an N-terminal signal peptide of 27 amino acids. The MtPHY1 gene is 5,151 bp in length, containing 7 exons and 6 introns. MtPHY1 transcripts were detected in leaves and roots and levels elevated in roots during growth in low phosphate conditions. Transgenic Arabidopsis lines expressing MtPHY1 under the control of the root-specific MtPT1 promoter or the constitutive CaMV35S promoter were created. Phytase activities in root apoplast of the transgenic Arabidopsis were 12.3- to 16.2-fold higher than those of the control plants. The expressed phytase was secreted into the rhizosphere as demonstrated by enzyme activity staining and HPLC analysis of phytate degradation by root exudates. Transgenic expression of the MtPHY1 led to significant improvement in organic phosphorus utilization and plant growth. When phytate was supplied as the sole source of phosphorus, dry weight of the transgenic Arabidopsis lines were 3.1- to 4.0-fold higher than the control plants and total phosphorus contents were 4.1- to 5.5-fold higher than the control. Transgenic expression of phytase genes of plant origin has great potential for improving plant phosphorus acquisition and for phytoremediation.

Keywords

ArabidopsisMedicago truncatulaPhosphorus acquisitionPhytase genePhytateTransgenic plant

Abbreviations

EST

Expression sequence tag

GFP

Green fluorescent protein

GMO

Genetically modified organism

GUS

Glucuronidase

MtPHY1

Medicago truncatula phytase 1

MtPT1

Medicago truncatula phosphate transporter 1

ORF

Open reading frame

P

Phosphorus

PAP

Purple acid phosphatase

Pi

Inorganic phosphate

PPT

Phosphinothricin

RACE

Rapid amplification of cDNA ends

Introduction

Phosphorus (P) is an essential but often limiting nutrient for plant growth and development. Large amounts of P fertilizers are applied to cropland each year, but only 10% to 20% of the fertilizer P applied in soil is readily utilized by plants (Holford 1997). The major portion of the fertilizer phosphorus that is applied to soil rapidly becomes immobilized into inorganic and organic fractions, which are largely unavailable to plant roots (Richardson et al. 2001; Vance et al. 2003). Thus the large input of P fertilizer is not only expensive but also potentially polluting and nonsustainable (Abelson 1999; Miller et al. 2001).

The P cycle can be characterized as the flow of P among plants, animals, microorganisms and solid phases of the soil (Iyamuremye and Dick 1996). A significant proportion of the soil P is in organic forms, either as specific organic P compounds or as organic compounds to which inorganic P is linked (Larsen 1967; Bieleski 1973). Organic P generally makes up 20% to 80% of the total P in the surface layer of the soil, which, after mineralization, can contribute considerably to the P nutrition of plants (Dalal 1977; Iyamuremye and Dick 1996). The predominant form of organic P is phytate (inositol hexa- and penta-phosphates), which constitutes up to 60% of soil organic P and is poorly utilized by plants (Iyamuremye and Dick 1996; Mudge et al. 2003).

Phytate can be hydrolyzed to inorganic phosphate (Pi) and myo-inositol through the action of phytase enzymes (Mudge et al. 2003). In the study of phytases, much attention has been paid to the use of phytases as an animal feed additive, because phytate in plant seeds is largely indigestible by monogastric animals (reviewed by Wodzinski and Ullah 1996; Brinch-Pedersen et al. 2002; Vohra and Satyanarayana 2003). Phytases have been commercially produced based on the filamentous fungus Aspergillus niger (Brinch-Pedersen et al. 2002). By comparison, phytases in plant roots have received much less attention; the potential for producing phytase in plant roots for improved P uptake has only been recognized in recent years. Application of a fungal phytase to sterile cultures of subterranean clover (Trifolium subterraneum) enabled the seedlings to use phytate as the only source of P (Hayes et al. 2000). Ectopic expression of a fungal phytase gene (Richardson et al. 2001; Mudge et al. 2003) or a synthetic phytase gene (Zimmermann et al. 2003) resulted in increased P acquisition and biomass production in transgenic plants.

Phytases have been identified in roots of plants (Hübel and Beck 1996; Li et al. 1997b; Hayes et al. 1999; Richardson et al. 2000), however, it has been suggested that the activity of these enzymes in roots is inadequate for effective utilization of organic P (Hayes et al. 1999; Richardson et al. 2000; Brinch-Pedersen et al. 2002). The cloning and characterization of plant phytase genes have been reported for maize (Maugenest et al. 1997, 1999) and soybean (Hegeman and Grabau 2001). However, phytases encoded by the soybean or the maize genes do not appear to be secreted or involved in P acquisition of roots from external phytate (Hübel and Beck 1996; Maugenest et al. 1999; Hegeman and Grabau 2001). To date, there have been no reports on improving P uptake by transgenically expressing any phytase genes of plant origin. In this paper, we report the cloning and characterization of a novel phytase gene (MtPHY1) from the model legume Medicago truncatula. Phytase encoded by MtPHY1 is secreted as an extracellular enzyme in roots. Transgenic Arabidopsis thaliana plants expressing MtPHY1 showed significantly increased capacity of P acquisition from phytate, and improved plant growth.

Materials and methods

Cloning of the M truncatula phytase gene, MtPHY1

A tentative consensus (TC91767) with relatively high similarity (66%) to the soybean phytase was identified based on blast search of the M. truncatula gene indices (http://www.tigr.org) using the soybean phytase protein sequence (Hegeman and Grabau 2001). TC91767 is 844 bp and as it is much shorter than the 1,621 bp soybean phytase gene, it likely represents only part of the gene.

To obtain a full-length cDNA sequence, a 5′ part of the gene was amplified using the RACE amplification kit (BD Biosciences, Palo Alto, CA). A fragment of about 1.2 kb was amplified by 5′ RACE with a reverse primer (5′-TCCTCCCGCATTGAAAGAATAAT-3′) specific to an EST clone (NF011E07RT) that represented TC91767. Based on sequence information of the EST clone and the fragment obtained by 5′ RACE, a new pair of primers was designed: forward 5′-AGAAGTTATATGAACCCACTTG-3′ and reverse 5′-AATATAACCAACAGTATACACTG-3′. The full-length cDNA was obtained by RT-PCR using the new primer pair. The sequence of the cDNA, designated MtPHY1, was deposited in Genbank (accession number AY878355). The sequence information of MtPHY1 cDNA was used to blast search genomic sequences of M. truncatula (http://www.genome.ou.edu/medicago.html), provided by the Advanced Center for Genome Technology at the University of Oklahoma (Roe and Kupfer 2004). A genomic clone covering the entire MtPHY1 cDNA was identified. The MtPHY1 cDNA sequence was aligned with the genomic sequence by the DNAstar software and information regarding exons and introns was obtained.

Construction of chimeric transgenes

Two gene constructs, CaMV35S::MtPHY1-GFP and a CaMV35S::GFP control were used to evaluate the subcellular localization of the MtPHY1 protein. The CaMV35S::GFP construct was created by inserting a HindIII-EcoRI fragment from the CaMV35S-sGFP(S65T)-nos plasmid (Chiu et al. 1996) into HindIII-EcoRI digested binary vector pCAMBIA3300. For the construction of phytase-GFP fusion vector (CaMV35S::MtPHY1-GFP), the open reading frame (ORF) of MtPHY1 was PCR amplified using high-fidelity Taq polymerase (Stratagene, LA Jolla, CA). The primers used for the amplification were 5′–T GTCGACAATGGGTTCTGTTTTGG-3′ (forward) and 5′–T CCATGGGACATGTATTATGTGCCT-3′ (reverse), in which a SalI and an NcoI restriction sites (underlined) were introduced at the 5′ and 3′ end, respectively. The PCR amplified product was cloned into TA vector (Promega, Madison, WI), sequenced, double digested by SalI-NcoI, and inserted in front of the GFP of CaMV35S::GFP without frame shift. GFP fluorescence was detected and imaged with the Bio-Rad 1024 ES Confocal Laser Scanning Microscope.

For transgenic expression of MtPHY1, chimeric genes were constructed under the control of the constitutive CaMV35S promoter and the root-specific MtPT1 promoter, respectively. For the construction of MtPHY1 under the control of CaMV35S promoter (CaMV35S::MtPHY1), the ORF of MtPHY1 were PCR amplified using primers 5′–T CCATGGGTTCTGTTTTGGTGCAT-3′ (forward) and 5′- A GGTAACCTGAAATGTCAGGGATGA-3′ (reverse). Restriction sites NcoI and BstEII were introduced in the amplified fragment. The fragment was cloned into TA vector, sequenced, double digested by NcoI-BstEII and then introduced into NcoI-BstEII digested binary vector pCAMBIA3301. The root-specific promoter, MtPT1, was isolated from the phosphate transporter 1 gene. For the construction of MtPHY1 under the control of the MtPT1 promoter (MtPT1::MtPHY1), the promoter fragment was PCR amplified from M. truncatula genomic DNA with primers 5′-T GGATCCATGCATGGGCTGGAGTT-3′ (forward) and 5′-T CCATGGCTGAATTTGTTACCTAGT-3′ (reverse). Restriction sites BamHI and NcoI were introduced in the amplified promoter fragment. The amplified MtPT1 promoter fragment was cloned into a TA vector, double digested by BamHI-NcoI and then inserted into BamHI-NcoI digested CaMV35S::MtPHY1 to replace the CaMV35S promoter.

Genetic transformation of Arabidopsis

DNA of the newly constructed binary vectors was transferred into the Agrobacterium tumefaciens strain C58 by the freeze-thaw method (Chen et al. 1994). Transgenic Arabidopsis (ecotype Columbia) plants were produced following the floral dip method (Clough and Bent 1998). Single-copy trangenic plants were identified by Southern hybridization analysis. T3 homozygous lines were obtained after selfing and phosphinothricin (PPT) selection. An empty vector transgenic line, which showed no difference to wild-type plants, was used as control.

Growth conditions

Medicago truncatula (ecotype A17) was grown under conditions as described by Liu et al. (1998). Briefly, the seeds were treated with concentrated H2SO4 for 10 min, rinsed three times in sterile water and germinated in pots filled with sterilized fine sand. Seedlings were fertilized with half-strength Hoagland’s solution containing either 10 μM or 2 mM KH2PO4 three times a week. After three weeks, the roots, leaves and stems were harvested, frozen in liquid nitrogen and stored at −80°C for RNA isolation.

Seeds of transgenic and control Arabidopsis lines were surface sterilized for 7 min in 50% bleach containing 0.02% Triton X-100 with gentle agitation. After rinsing three times in sterile water, the seeds were spread evenly onto normal MS agar medium (Murashige and Skoog 1962) in petri dishes and germinated for eight days. The seedlings were then carefully transferred onto a modified MS medium in which Pi was replaced by phytate (250 μM myo-inositol hexaphosphoric acid dodecasosium salt from rice, Sigma, St. Louis, MO). Phytate (InsP6) from rice was used as organic P throughout the experiments. The seedlings and plants were grown at 24°C under fluorescent light (240 μE  m−2 s−1) at a photoperiod of 16 h in a growth room. Roots of the 22-day-old (8 days on normal MS medium, two weeks on phytate containing MS medium) transgenic Arabidopsis lines were collected and used for RNA isolation and APase activity analysis. P concentration, fresh weight and dry weight were measured from 15-, 30- and 45-day-old plants.

Southern and northern blot hybridization analyses

Twenty μg of M. truncatula genomic DNA was digested with restriction enzymes BamHI, EcoRI, NcoI and SalI and separated through a 0.8% agarose gel. DNA gel blotting was carried out following standard protocols (Sambrook et al. 1989). To avoid cross hybridization of MtPHY1 with other similar sequences, the 3′ untranslated region (UTR) was [32 P] dCTP labeled and used as the probe. Southern hybridizations were performed following the QuikHyb Hybridization protocols (Stratagene, La Jolla, CA).

Total RNA was isolated using TRI reagent (Molecular Research Center, Inc., Cincinnati, OH) and RNA gel blotting was carried out according to standard protocols (Sambrook et al. 1989). For analyzing transcript levels in different organs of M. truncatula, the 3′ UTR of MtPHY1 was used as the probe. For analyzing expression levels of transgenes in Arabidopsis root, the coding sequence of MtPHY1 was used as the probe. Northern hybridizations were performed using [32 P] dCTP labeled probes following the QuikHyb Hybridization protocols (Stratagene, La Jolla, CA).

Assay of phytase activity in root and root apoplast

Measurement of phytase activity of T3 transgenic Arabidopsis lines essentially followed the procedure described by Richardson et al. (2001). Samples of whole root tissues (four replicates, 30 plants per replicate) were ground in a mortar and pestle with three volumes (v/w) of MES/Ca buffer (15 mM MES buffer with 0.5 mM CaCl2, pH 5.5) containing 1 mM EDTA. Extracts were then centrifuged for 10 min at 12,000g; 250 μl of the crude extract was added to a total volume of 500 μl MES/Ca buffer containing 2 mM phytate. The reaction was incubated at 27°C for 60 min and was terminated by the addition of an equal volume of 10% TCA. Phytase activity was calculated from the release of Pi over the incubation period by spectrophotometry at 882 nm using the molybdate-blue procedure (Murphy and Riley 1962). The protein concentrations were determined using Bio-Rad Dc protein assay reagent (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin as standard. Enzyme activity was calculated as mU per mg protein, where 1 unit (U) releases 1 μmol Pi min−1 under the assay conditions described (Richardson et al. 2001).

For the measurement of phytase activity in root apoplast, 5 μl of apoplast sap obtained from roots by the centrifugation method (Yu et al. 1999) was added to a total volume of 100 μl of MES/Ca buffer containing 2 mM phytate. The reaction conditions and the calculations of the enzyme activity were the same as the procedure used for whole root extract as described above.

Histochemical analysis of recombinant phytase activity

Root staining for phosphomonoesterase activity was done by adding staining solution to petri dishes with transgenic plants. The staining solution consisted of 50 mM tri-sodium-citrate (TSC) buffer (pH 5.5), 37.5 mM α-naphthyl phosphate and 2.7 mM Fast Red TR (Zimmermann et al. 2003). The roots were stained for 2 h at room temperature and then photographed.

HPLC analysis of the hydrolysis of phytate by root exudates

Seeds of the transgenic lines as well as the empty vector control line were sown on normal MS agar medium. Fifteen-day-old seedlings were carefully transferred to 50 ml plastic tubes containing 20 ml liquid modified MS medium in which Pi was replaced by phytate. The tubes were fixed in a rack and the seedlings were grown for seven more days in a shaker at 40 rpm. The roots were then harvested, washed with deionized water and incubated in 50 ml of 5 mM maleate buffer, pH 5.5, containing 2 mM CaCl2, 0.01% protease inhibitor cocktail (Sigma, St. Louis, MO) and 2 mM InsP6 (Sigma, St. Louis, MO). One milliliter was sampled at time points 0, 12 and 24 h, and the enzyme was inactivated by the addition of 0.5 ml 15% TCA. Samples were analyzed by HPLC. Myo-inositol 1,3,4,5,6-pentakisphosphate, myo-inositol 1,3,4,5-tetrakisphosphate, myo-1,4,5-triphosphate, myo-inositol 4,5-biphosphate and myo-inositol-4-monophosphate (all from Sigma, St. Louis, MO) were used as standard for InsP5, InsP4, InsP3, InsP2 and InsP1, respectively. The sum of InsP2, InsP1 and Ins was calculated as the difference between the total initial InsP6 and the sum of measured values for the other InsP forms (Zimmermann et al. 2003).

Assay of inorganic phosphate and total phosphorus

For the assay of inorganic phosphate, plant samples were ground to fine powder in liquid nitrogen and suspended in 1% glacial acetic acid. After incubation at 42°C for 30 min, the samples were centrifuged, and the supernatant was assayed at OD820 as described by Ames (1966).

For the assay of total phosphorus, samples were collected in glass tubes containing 0.03 ml Mg(NO3)2 solution. The samples were dried and ashed by shaking over strong flames. After adding HCl and assay mix, total phosphate was determined by colorimetric assay at OD820 (Ames 1966).

Results

Cloning, structure and expression analysis of a phytase gene, MtPHY1, from M truncatula

A full-length cDNA coding for phytase was isolated from M. truncatula by 5′ RACE and RT-PCR. The cloned cDNA is 2,067 bp in length, containing an open reading frame (ORF) of 1,632 bp capable of encoding 543 amino acids. A signal peptide of 27 amino acids at the N’ end was predicted by SignalP (Nielsen et al. 1997; Bendtsen et al. 2004). Analysis of the predicted MtPHY1 protein sequence by TargetP (Emanuelsson et al. 2000) revealed a secretion pathway score of 0.967, indicating that the protein is likely to be secreted to the outside of the cell.

The predicted MtPHY1 protein shares 71.9% and 71.6% identities with the soybean phytase (AAK49438.1) and an Arabidopsis putative purple acid phosphatase (AF448726), respectively. Genomic sequence analysis indicates that the MtPHY1 gene is 5,151 bp long and includes 7 exons interrupted by 6 introns (Fig. 1a). Southern hybridization analysis indicated that there are 1–2 copies of this gene in M. truncatula genome (data not shown). The expression pattern of the MtPHY1 gene was analyzed by northern hybridization with RNA isolated from different tissues of M. truncatula. Under high-Pi (2 mM) growth conditions, higher level of MtPHY1 transcripts accumulated in the leaf than in the root (Fig. 1b). However, under low-Pi (10 μM) conditions, the transcript level was increased in the root, with the strongest hybridization signal detected in the root (Fig. 1c).
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Fig. 1

a Diagram of the structure of the MtPHY1 gene based on analysis of the genomic DNA sequences. Exons a, b, c, d, e, f and g are at positions 1–556, 557–751, 752–1084, 1085–1300, 1301–1491, 1492–1777 and 1778–1893 of the cDNA, respectively. Introns 1, 2, 3, 4, 5 and 6 are 1,447 bp, 1,054 bp, 140 bp, 81 bp, 80 bp and 365 bp in length, respectively. b Northern hybridization analysis of different organs of M. truncatula under high (2 mM) Pi conditions. c Northern hybridization analysis of different organs of M. truncatula under low (10 μM) Pi conditions. The 3′ UTR of MtPHY1 cDNA was used as probe

Subcellular localization of the phytase-GFP fusion protein

To localize the translated product of the cloned phytase gene, a binary vector (CaMV35S::MtPHY1-GFP) containing an in-frame fusion of MtPHY1 ORF and GFP under the control of CaMV35S promoter was constructed. Transgenic Arabidopsis plants were generated with CaMV35S::MtPHY1-GFP and CaMV35S::GFP and were grown in MS agar medium with phytate as the sole source of P. In lines carrying CaMV35S::MtPHY1-GFP, green fluorescence was mainly detected in the root apoplast (Fig. 2a), whereas green fluorescence was freely distributed in cells of the transgenic lines carrying CaMV35S::GFP (Fig. 2b). The results indicated that the phytase-GFP fusion protein was transported across the cell membrane and accumulated in the apoplast. Thus the phytase encoded by MtPHY1 is an extracellular protein.
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Fig. 2

a Subcellular localization of MtPHY1-GFP fusion protein in roots of transgenic Arabidopsis carrying the gene construct CaMV35S::MtPHY1-GFP. Arrow shows the detection of green fluorescence in the apoplast. b transgenic root carrying the gene construct CaMV35S::GFP as control. Green fluorescence is evident in the cytoplasm and nucleus

Transgenic expression of chimeric MtPHY1 gene constructs in Arabidopsis

Two chimeric transgenes (MtPT1::MtPHY1, CaMV35S::MtPHY1) were constructed by placing MtPHY1 ORF under the control of the root-specific MtPT1 promoter and the constitutive CaMV35S promoter, respectively (Figs. 3a, c). Ten T3 homozygous lines carrying MtPT1::MtPHY1 and eight T3 homozygous lines carrying CaMV35S::MtPHY1 were used for further analyses. Northern blot hybridization analysis revealed large differences in transcript levels between the independent transgenic lines (Figs. 3b, d). High levels of transgene expression were observed in independent transgenic lines 2, 8 and 11 (Figs. 3b, 3d), and these were chosen for further detailed analysis. Lines 2 and 8 carried MtPHY1 under the control of the MtPT1 promoter, line 11 carried MtPHY1 under the control of CaMV35S promoter.
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Fig. 3

a Schematic illustration of the MtPT1::MtPHY1 gene construct used for generating transgenic Arabidopsis plants. b Northern hybridization analysis of transgenic Arabidopsis carrying the gene construct MtPT1::MtPHY1. c Schematic illustration of the CaMV35S::MtPHY1 gene construct used for generating transgenic Arabidopsis plants.d Northern hybridization analysis of transgenic Arabidopsis carrying the gene construct CaMV35S::MtPHY1

Phytase activity and extracellular secretion of the enzyme in transgenic plants

The three transgenic lines (2, 8, 11) were grown in agar medium containing phytate as the sole source of P. Phytase activities in whole root extracts of the transgenic lines were only 22% to 36% higher than that of the control plants (Fig. 4a). In contrast, phytase activities in apoplast of roots were 12.3- to 16.2-fold of that in the empty vector control plants (Fig. 4b). The results further confirmed that much of the phytase produced was secreted into apoplast. Phytase activity of transgenic line 8 (MtPT1::MtPHY1) was significantly higher than that of transgenic line 11, which carried MtPHY1 under the control of CaMV35S promoter. Phytase activity of another MtPT1 promoter driven MtPHY1 line, line 2, was similar to the CaMV35S driven MtPHY1 line.
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Fig. 4

a Phytase activities in whole root extracts of transgenic Arabidopsis lines growing in agar medium with phytate as the sole source of P. b Phytase activities in root apoplast of transgenic Arabidopsis lines growing in agar medium with phytate as the sole source of P. CTRL: empty vector control, lines 2 and 8 carried the transgene MtPT1::MtPHY1, line 11 carried the transgene CaMV35S::MtPHY1. Data are presented as the mean ± SE of three individual assays

Transgenic as well as empty vector control plants were also grown in liquid MS medium with phytate as the sole P source, and the intermediates of phytate (InsP6) degradation were analyzed by HPLC. The exudates from control roots degraded InsP6 at a low level (Fig. 5a), whereas root exudates from the transgenic lines degraded InsP6 rapidly with a concomitant accumulation of InsP5, InsP4, InsP3, InsP2, InsP1 and Ins (Fig. 5b). Most of the InsP6 was degraded after incubating transgenics for 24 h in liquid medium (Fig. 5b). Thus, the phytase secreted from the transgenic roots was able to degrade phytate in the liquid medium.
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Fig. 5

a, b Intermediates of phytate (InsP6) degradation by root exudates of empty vector control (a) and transgenic line 8 (b) in liquid culture medium with phytate as the sole source of P. Data are presented as the mean ± SE of three individual assays.

Active phytase protein was visualized by staining for phosphomonoesterase activity. When plants were grown in agar with phytate as the sole source of P, roots of transgenic plants were stained darker than those of control plants (Figs. 6a–c), indicating that the recombinant phytase was secreted into the rhizosphere directed by the native signal peptide.
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Fig. 6

a–c Staining of phosphomonoesterase activity in roots of Arabidopsis growing in agar medium with phytate as the sole source of P. a Empty vector control. b Transgenic line 8 carrying the gene construct MtPT1::MtPHY1. c Transgenic line 11 carrying the gene construct CaMV35S::MtPHY1. The dark, purple color indicates enzyme activity in roots and root exudates. d Phenotype of transgenic Arabidopsis plants growing in MS agar medium with phytate as the sole source of P. CTRL: empty vector control, lines 2 and 8 carried the transgene MtPT1::MtPHY1, line 11 carried the transgene CaMV35S::MtPHY1. The plants from different lines were firstly germinated and grown on normal MS agar medium for 8 days and then transferred to modified MS medium in which Pi was replaced by phytate and grown for two more weeks.

Plant growth, biomass production and P uptake of transgenic Arabidopsis

Ectopic expression of MtPHY1 in Arabidopsis did not result in phenotypic difference when plants were grown in agar medium with sufficient Pi (2 mM). Dry matter weight, P concentration and total P content in the transgenics were similar to the control plants under Pi sufficient conditions (data not shown). However, large differences in plant growth, biomass production and P uptake were evident when the plants were supplied with phytate as the sole source of P (Fig. 6d).

Because of the P reserves, dry weight of 15-day-old (eight days on Pi sufficient agar medium, then seven days on phytate only medium) transgenic plants were only 14–20% higher than that of the control plants (Fig. 7a). The effects of transgene expression on the utilization of organic P became more evident following longer periods of growth on the phytate only medium. For 30-day-old plants, dry weight of the transgenics was 3.1- to 3.6-fold higher than the control plants (Fig. 7a). For 45-day-old plants, dry weight of the transgenics was 3.1- to 4.0-fold higher than the control plants (Fig. 7a). P concentrations of 30-day and 45-day plants increased 38.5–46.0% and 25.7 –47.3%, respectively (Fig. 7b). Because of the drastic increase in total dry matter, total P contents in 30-day-old transgenics increased 4.1- to 4.9-fold, and total P contents in 45-day-old transgenics increased 4.1- to 5.5-fold (Fig. 7c).
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Fig. 7

a Dry weight of the shoots of the transgenic Arabidopsis lines growing in agar medium with phytate as the sole source of P.b Pi concentration of transgenic Arabidopsis lines growing in agar medium with phytate as the sole source of P. c Total P content of transgenic Arabidopsis lines growing in agar medium with phytate as the sole source of P. CTRL empty vector control, lines 2 and 8 carried the transgene MtPT1::MtPHY1, line 11 carried the transgene CaMV35S::MtPHY1. The plants from different lines were first germinated and grown on normal MS agar medium for 8 days and then transferred to a modified MS medium in which Pi was replaced by phytate. Measurements were recorded when the plants were 15-, 30- and 45-days old. Data are presented as the mean ± SE of six replicates per line

Discussion

The predicted MtPHY1 protein shares relatively high identity with soybean phytase (Hegeman and Grabau 2001). Both the soybean phytase and MtPHY1 lack the active site motif, RHGXRXP, previously reported for fungal and maize phytases (Ullah and Dischinger 1993; Maugenest et al. 1999; Hegeman and Grabau 2001). While soybean phytase contains all the five conserved blocks of metal-ligating residues characteristic of purple acid phosphatases (PAPs), the MtPHY1 does not contain a complete set of these blocks. The introduction of the MtPHY1 transgene into Arabidopsis confirmed that the enzyme can effectively catalyze phytate degradation.

To be able to hydrolyze P from organic P substrates, phytase must be extracellularly secreted from the root into the rhizosphere. The phytase gene cloned from M. truncatula, MtPHY1, was predicted to contain a signal sequence for secretion. Analyses of the localization of the MtPHY1-GFP fusion protein and the activity of phytase enzyme in transgenic Arabidopsis showed the accumulation of recombinant phytase in the apoplast. HPLC analysis of root exudates, staining for active phytase enzyme and growth analysis of the transgenics revealed the secretion of active phytase from the root into agar medium. Although the cloning and characterization of plant phytase genes has been reported in soybean and maize, these genes do not seem to be secreted or involved in P uptake from organic P (Hübel and Beck 1996; Maugenest et al. 1999; Hegeman and Grabau 2001; Richardson et al. 2001). Thus, MtPHY1 is a novel gene that encodes an extracellular form of phytase that could effectively hydrolyze phytate and release Pi for plant nutrition.

Transgenic strategies to improve P acquisition by the overexpression of phytases must consider the final location of the phytase enzyme. The expression of Bacillus subtilis phytase gene in the cytoplasm of tobacco cells resulted in phenotypic changes, such as increased flower numbers and reduced seed size (Yip et al. 2003). To avoid altering phosphate metabolism inside plant cells, researchers wishing to express fungal or bacterial phytase genes in plants, had to add a plant signal peptide to direct the transgene product to apoplastic locations (Brinch-Pedersen et al. 2002). The addition of potato patatin signal sequence or carrot extension signal sequence allowed the production of active Aspergillus phytase in transgenic soybean cell suspension cultures (Li et al. 1997a) or Arabidopsis plants (Richardson et al. 2001). In the case of MtPHY1, no extra signal is required for the production and secretion of active phytase in transgenic plants.

Constitutive expression of certain genes may result in metabolic disorders or undesirable side effects (Kasuga et al. 1999). a root-specific or trichoblast-specific promoter is expected to be more desirable for transgenic expression of phytase genes. In the present study, P acquisition was improved in the MtPHY1 transgenic plants driven either by the root-specific promoter or by the CaMV35S promoter. No obvious phenotypic change was observed when the MtPHY1 gene was driven by the CaMV35S promoter. This is probably because the phytase produced was mainly accumulated in apoplast.

Plant phytases are generally considered to be 6-phyatses and hydrolyse the phosphate ester bond at the D-4 position of phytic acid, while phytases from microorganisms are often referred to as 3-phytases and hydrolyse the D-3 bond first (Brinch-Pedersen et al. 2002). Despite these differences, the effects of transgenic expression of MtPHY1 and fungal phytase genes share the same trend. When the Aspergillus phytase gene was expressed in Arabidopsis, dry weight of the transgenic plants increased from 23.2% to 65.9% and total P content of the transgenics increased 3- to 4-fold (Richardson et al. 2001; Mudge et al. 2003). Transgenic potato plants carrying a fungal-sequence-based synthetic phytase gene showed about a 30% increase in biomass and a 40% increase in total P content (Zimmermann et al. 2003). Our results showed that dry weight of the transgenic Arabidopsis were 3.1- to 4.0-fold higher than that of the control plants and total P contents in transgenics were 4.1- to 5.5-fold higher than in the control.

Although improvement of phosphate acquisition in crops can be explored by transgenic expression of either the plant phytase gene or the fungal phytase genes, a gene of plant origin has advantages over fungal genes with regard to regulatory and biosafety issues (König 2003; Nielsen 2003). The introduction of genetic material from distantly related organisms is one of the major concerns that causes objections to GMOs (Nielsen 2003). In recent years, public concern about the extent to which transgenic crops differ from their traditionally bred counterparts has resulted in revised molecular strategies and the choice of genes (Rommens et al. 2004). It has been proposed to categorize GMOs into different classes based on genetic distance between the target organism and the source of transgenes (Nielsen 2003). M. truncatula is a forage species that is closely related to certain other legumes (e.g. alfalfa); improvement of P acquisition in legume species is particularly important because they have the capacity of fixing nitrogen. Because of the tremendous cost associated with regulatory requirements in releasing transgenic cultivars, it is imperative to consider carefully the origin of the transgene at the beginning. The phytase gene from M. truncatula, MtPHY1, could be a good candidate for molecular improvement of P acquisition in plants. Data mining based on MtPHY1 sequence could also allow the identification of genes with similar functions from other plant species.

Phosphorus is limiting for crop yield on over 30% of the world’s arable land, and by some estimates, world resources of inexpensive rock phosphate may be depleted by 2050 (Vance et al. 2003). Improvement of plant P uptake capacity will reduce the input of fertilizer and fertilizer’s negative environmental impacts. This approach will thus have significant impacts on sustainable agriculture.

On the other hand, there are areas with too much phosphorus that have caused prominent local problems (Matuszeski 2000; Gaston et al. 2003; Pote et al. 2003). P is a major component of animal manures and is especially significant in poultry litter, which is spread on cropland and pastures in areas with extensive poultry industry (Matuszeski 2000). In recent years, the accumulation of P in soils has received major attention in ecologically sensitive agricultural catchments due to the eutrophication it causes to surrounding water bodies (Pant et al. 2004). Because the transgenic plants expressing MtPHY1 had higher P concentration and drastically increased total P contents, the transgenic approach can also be used in removing excessive organic P from certain land areas.

Phytate is the main storage form of P in many plant seeds that are used as animal feed, but phosphate bound in this form is not available to monogastric animals (Vohra and Satyanarayana 2003; Hong et al. 2004). Thus, besides the potential use for improving P uptake from soil, transgenic expression of phytase in plant seeds offers promising prospects for improving phytate-phosphorus digestibility in monogastric animals. In the past several years, fungal and bacterial phytase genes have been transgenically expressed in seeds of soybean (Denbow et al. 1998), wheat (Brinch-Pedersen et al. 2000, 2003), rice (Lucca et al. 2001a; 2001b; Hong et al. 2004) and canola (Ponstein et al. 2002). The potential usefulness of the MtPHY1 gene for improving P and mineral bioavailability remains to be tested. A study using transgenic wheat materials possessing endogenous 6-phytase and an introduced Aspergillus 3-phytase revealed that the concerted action of endogenous and heterologous phytases are very efficient, resulting in almost complete elimination of InsP6 and lower inositol phosphates (InsP3-InsP5) in wheat flour incubated for less than one hour (Brinch-Pedersen et al. 2003). We speculate that simultaneous expression of MtPHY1 and a fungal phytase gene in transgenic plants may lead to more efficient degradation of phytate, and thus benefit P uptake in roots or bioavailability of P and minerals in seeds.

In summary, consistent and closely related molecular, biochemical and phenotypic data demonstrated for the first time that the transgenic expression of a plant phytase gene resulted in significant improvement in P uptake and plant biomass production when phytate was supplied as the sole source of P. The transgenic approach has great potential for improving plant P acquisition capacity and for phytoremediation.

Acknowledgments

The authors thank Xuefeng Ma for critically reading the manuscript and Caroline Lara for editing it. The work was supported by the Samuel Roberts Noble Foundation.

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