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Expression of a fungal phytase gene in Nicotiana tabacum improves phosphorus nutrition of plants grown in amended soils

Expression of a fungal phytase gene in Nicotiana tabacum improves phosphorus nutrition of plants... <h1>Introduction</h1> Phosphorus (P) is an essential plant nutrient which limits agricultural production on a global scale. Inorganic P fertilizers are a scarce resource in much of the world and, where they are applied to promote crop yields, the majority of the added P is transformed into inorganic and organic forms which are of limited availability to plants ( Sanyal and De Datta, 1991 ). Only 10%−20% of fertilizer P is available for utilization by crops in the first year after application ( Holford, 1997 ). For this reason, it is paramount that P use efficiency by agricultural plants is improved. More efficient use of fertilizer P is also required to avoid adverse environmental effects as a result of P contamination of the wider environment ( Tunney et al ., 1997 ). Moreover, at current rates of consumption, known reserves of high-grade rock phosphate are predicted to be exhausted before the end of this century ( Runge-Metzger, 1995 ). Plants have developed a range of mechanisms to enhance their ability to acquire P from soil ( Marschner, 1995 ; Vance et al ., 2003 ). The most important are root morphological characteristics ( Lynch, 1995 ; Gahoonia and Nielsen, 1997 ; Ge et al ., 2000 ). However, mycorrhizal symbioses ( Smith and Read, 1997 ) and the production of root exudates, such as organic anions ( Ryan et al ., 2001 ) and phosphatase enzymes, may also enhance P acquisition ( Tadano et al ., 1993 ; Li et al ., 1997 ; George et al ., 2002 ). For P to be available to plants, it needs to be present as orthophosphate in soil solution. Therefore, the exudation of phosphatases which catalyse the hydrolysis of organic P, is a potentially important way for plants to enhance P availability, particularly as a large proportion of soil P (up to 80%) occurs in organic forms ( Richardson et al ., 2004 ). Phytases, enzymes that hydrolyse derivatives of inositol penta- and hexa kis -phosphates (phytate), are of particular interest because phytate constitutes up to 50% of the total organic P in soil ( Anderson, 1980 ; Turner et al ., 2002 ). Paradoxically, many plants have limited capacity to obtain P directly from phytate when grown under controlled conditions ( Hayes et al ., 2000b ; Richardson et al ., 2001b ). This is because the availability of phytate in soil is low (due to sorption and precipitation reactions; Anderson and Malcolm, 1974 ; Shang et al ., 1992 ; Celi et al ., 2001 ) and because the capacity of plants to exude phytase to the rhizosphere ( Hayes et al ., 1999 ; Richardson et al ., 2000 ) is limited. In low P-sorbing growth media (agar or sand), the ability of plants to use P from phytate is improved when they are inoculated with soil microorganisms which possess phytase activity, or when purified phytase is added to the medium ( Findenegg and Nelemans, 1993 ; Hayes et al ., 2000b ; Richardson et al ., 2001b ; Idriss et al ., 2002 ). Transgenic Arabidopsis thaliana , subterranean clover ( Trifolium subterraneum L.) and potato ( Solanum tuberosum L.), that express a phytase gene ( phyA ) from Aspergillus niger , release extracellular phytase and are able to utilize P supplied as phytate in agar ( Richardson et al ., 2001a ; Mudge et al ., 2003 ; Zimmermann et al ., 2003 ; George et al ., 2004 ). Improved growth and P nutrition of plants that express phytase have not been demonstrated for soil-grown plants. In this paper, the growth and P nutrition of transgenic lines of tobacco ( Nicotiana tabacum L.) expressing phyA were assessed in two P-deficient Australian soils which potentially contained endogenous phytate ( Williams and Anderson, 1968 ; Hayes et al ., 2000a ). Although growth and P accumulation by transgenic tobacco were not increased in unamended soils, P nutrition was improved in soils which had been amended with various combinations of phytate, phosphate and lime. <h1>Results</h1> <h2>Expression of ex::phyA in N. tabacum and utilization of P from phytate</h2> Tobacco plants were transformed with a constitutively expressed phyA gene from A. niger containing a 5′ signal peptide ( ex ) from carrot extensin for extracellular secretion of phytase ( Figure 1A ). Three of 12 independently transformed lines generated were tested, and the expression of ex::phyA in shoots was verified using Northern blot analysis. phyA mRNA was present in the shoot tissue of all transgenic lines ( ex::phyA-1 to ex::phyA-3 ) and absent in plants transformed with the vector alone (vector control) and in wild-type plants ( Figure 1B ). The amount of mRNA in shoots varied ( P < 0.05) between the transgenic lines. A line with transgene insertions at multiple loci ( ex::phyA-2 ) had 2.9-fold greater relative expression of phyA than line ex::phyA-1 , and 6.3-fold greater relative expression of phyA than line ex::phyA-3 ( Figure 1C ). Shoot phytase activity was increased by 2.0–4.8-fold ( P < 0.001) in transgenic plants that expressed the chimeric ex::phyA gene. Most importantly, all ex::phyA lines showed substantially greater extracellular phytase activities than controls ( P < 0.001), which had no detectable extracellular activity. The relative level of gene expression of the transgenic lines was apparent in the extracellular phytase activity, in that the line ( ex::phyA-3 ) which showed the lowest expression of phyA mRNA also had 2.8-fold lower extracellular phytase activity ( P < 0.05) ( Figure 1 ). The effectiveness of the expression of ex::phyA by plants was demonstrated by comparing the shoot biomass and P nutrition of the lowest expressing transgenic line ( ex::phyA-3 ) with vector control plants when grown in sterile agar supplied with P solely as phytate ( Figure 2 ). Control treatments included plants grown without P and those supplied with P as orthophosphate. Line ex::phyA -3 showed similar shoot biomass to the vector controls in all P treatments ( Figure 2A ). However, when P was provided as phytate, plants that expressed ex::phyA-3 showed a 1.8-fold greater shoot biomass than when no P was added ( P < 0.05), but no difference was observed for the vector control. Despite this, P nutrition was improved by expression of the phytase gene ( Figure 2B ). When plants were supplied with P as phytate, the ex::phyA-3 line accumulated fourfold more P than vector control plants ( P < 0.05) and 29-fold more P than plants grown in the absence of phosphorus ( P < 0.05). However, ex::phyA plants supplied with phytate did not accumulate as much P as equivalent plants supplied with the same amount of P as orthophosphate ( P < 0.05). <h2>Growth and P nutrition in soil of plants that release extracellular phytase</h2> Transgenic line ex::phyA-3 was also grown in an Alfisol soil which had been collected from plots (both unamended and fertilized) in a field fertilizer trial (Hall soil, Table 1 ). The shoot biomass and P nutrition of ex::phyA-3 plants were compared with those of the vector control after 45 days of growth. In soil taken from an unfertilized plot, the growth of both plant lines was restricted and no differences in biomass or P nutrition were evident between lines ( Table 2 ). The growth and P nutrition of all plants were greatly enhanced, by between 10- and 19-fold, when plants were grown in soil that had received P fertilizer in the field. Significantly, the shoot biomass in the phyA -expressing line ( ex::phyA-3 ) was increased by 38% when compared with the control plants grown in the soil collected from fertilized plots ( P < 0.05) ( Table 2 ; Figure 3 ). P accumulation by transgenic plants expressing ex::phyA was also significantly improved by 34% compared with control plants in this soil ( P < 0.05). Further experiments were undertaken to validate the growth and P nutrition response observed for the ex::phyA-3 line of transgenic tobacco in field-fertilized Hall soil. Three independently transformed lines ( ex::phyA-1 to ex::phyA-3 ) were grown in two soils, the Hall soil (an Alfisol) and Tilba soil (a Spodosol), with moderate and low P sorption characteristics, respectively. Soils were either left unamended or supplemented with P as either calcium phytate (Ca phytate) or orthophosphate, with or without the addition of lime to increase the soil pH. P accumulation in shoots of the transgenic lines was compared with that in vector control and wild-type plants. Liming resulted in a pH increase in both soils ( P < 0.05) ( Table 1 ), and generally reduced the growth and P nutrition of all plants (Figure 4). With the addition of both phosphate and phytate, P availability increased ( P < 0.05), as measured by resin extraction, whilst NaOH-extractable inorganic and organic P pools were also greater. P availability was increased to a greater extent ( P < 0.05) with the addition of orthophosphate than with the addition of the same amount of P as Ca phytate ( Table 1 ). When grown in unamended soils, plant growth was severely restricted and all plants accumulated only small amounts of P. This was coincident with relatively low concentrations of resin-extractable P ( Table 1 ). In both the Tilba and Hall soils, no significant differences in shoot P accumulation were evident for the ex::phyA plant lines compared with the vector control or wild-type plants ( Figure 4A ). This was despite the presence of a pool of soil P that was amenable to hydrolysis by a crude phytase preparation (NaOH-Pphos; Table 1 ). Liming the unfertilized soils had little effect on the availability of soil P pools and did not affect the P nutrition of plant lines in the Hall soil, but caused a significant decrease in P accumulation in plants grown in the Tilba soil. <h2>Growth and P nutrition of plants in soils amended with phytate and lime</h2> When grown in soil supplemented with phytate, transgenic plants that expressed ex::phyA accumulated more P in shoots than did control plants ( Figure 4B ). In Hall soil, the shoot P content of ex::phyA plants was 52% greater than that in control plants and, on average, the plants accumulated 90 µg more P ( Figure 4B ). However, this difference in P content did not result in dry weights that were significantly different between the plant lines (mean shoot dry weight of 85.4 mg/plant across all lines; data not shown). In Tilba soil amended with phytate, only the higher expressing transgenic lines ( ex::phyA-1 and ex::phyA-2 ) accumulated more P (28% or 121 µg P per plant; Figure 4B ) than either of the controls and, in this case, differences in shoot dry weight were significant ( P < 0.05; mean shoot weight of 100.4 mg/plant for the two transgenic lines compared with 76.4 mg/plant for the controls). Soils supplemented with phytate had greater resin P ( P < 0.05) and more organic P extracted by NaOH. Moreover, amendment of both soils with phytate increased, by between 13% and 23%, the amount of NaOH-extractable organic P that was amenable to hydrolysis by a crude phytase preparation ( P < 0.05) ( Table 1 ). In contrast with plant responses in acidic soils, when phytate was added to soils that had been limed, P accumulation by transgenic ex::phyA plant lines was not significantly different from that of the controls, and the P nutrition of all plants was significantly less than in unlimed soils. The addition of phytate to limed soils also increased resin-extractable P, but to a lesser extent than in acidic soils. Unlike under acidic conditions, phytate addition did not increase the NaOH-extractable organic P and did not consistently increase the portion which was amenable to hydrolysis by crude phytase ( Table 1 ). These observations are consistent with the reduced P accumulation by all plants in limed soil after the addition of phytate ( P < 0.001), compared with the unlimed equivalent. <h2>Growth and P nutrition of plants in soils amended with phosphate and lime</h2> Shoot P accumulation was greatest in soils amended with phosphate, with all plants accumulating between three- and 22-fold more P than when the same amount of P was added as Ca phytate ( P < 0.05) ( Figure 4C ). This was coincident with increased ( P < 0.05) resin-extractable P concentrations in both soils. When plants were grown in Hall soil that had received a recent application of phosphate, transgenic ex::phyA -expressing plants accumulated, on average, 228 µg more P in shoots than did the control lines ( P < 0.05). There was no such response in phosphate-fertilized Tilba soil. However, in the presence of lime and phosphate, large increases in P accumulation by plants that expressed phyA were observed relative to the controls in both soils. Under these conditions, ex::phyA plant lines accumulated 597 and 347 µg more P than control plants in Tilba and Hall soils, respectively ( P < 0.001). This was equivalent to a 32% increase in P accumulation in both soils ( Figure 4C ) and, for the Hall soil, resulted in significantly greater shoot dry weights for the transgenic lines that expressed ex::phyA ( P < 0.05) (mean shoot dry weight of 319.3 mg/plant for the ex::phyA transgenic lines compared with 220.9 mg/plant for the controls). Although the addition of phosphate to Hall soil had little effect on the amount of organic P extracted by NaOH, the proportion of this organic P which was hydrolysed by a nonspecific phytase preparation was increased by between 54% and 100%. Comparable increases were not evident in the Tilba soil, although the amount of organic P extracted by NaOH increased, but this was only significant in unlimed soil. When grown in field-fertilized Hall soil, the P nutrition of all plants was improved in relation to equivalent plants grown in unamended soil (cf. Figure 4A and 4D ). This was again coincident with greater resin-extractable P ( P < 0.05). As previously observed ( Table 2 ), the ex::phyA-3 line accumulated significantly more P (35% more) than the vector control plants. However, this transgenic line was not different from the wild-type, and the two other ex::phyA lines showed no improvement in P nutrition compared with either of the controls. The field application of P to Hall soil increased the amount of NaOH-extractable inorganic and organic P ( P < 0.05) and increased the component of organic P that was hydrolysed by the crude phytase preparation, but to a lesser extent than observed for the recent addition of phosphate ( Table 1 ). <h1>Discussion</h1> <h2>Expression of phytase in tobacco allows the utilization of P from phytate</h2> Transformation of N. tabacum with the ex::phyA gene was confirmed by the presence of mRNA in the shoot tissue of all three ex::phyA lines. One of the transformed lines ( ex::phyA-3 ) showed reduced expression of phyA by Northern blot hybridization compared with the other lines, whereas the line which had insertions in at least three loci (ex::phyA-2) showed the highest expression. These levels of phyA expression were consistent with phytase activity in both shoot material and root exudates. Of significance was the fact that the phytase activity exuded to the rhizosphere was greatly increased in the transgenic lines compared with the undetectable levels exuded by control plants. The level of phytase activity exuded from the roots of tobacco, even for the low expressing line ( ex::phyA-3 ), was equivalent to the activity exuded by other phyA -expressing plants, including Arabidopsis , potato and subterranean clover ( Richardson et al ., 2001a ; Zimmermann et al ., 2003 ; George et al ., 2004 ). The enhanced exudation of phytase by transgenic N. tabacum conferred an ability to acquire P from phytate in sterile non-sorbing agar media. Transgenic line ex::phyA-3 , which had the lowest exuded phytase activity, accumulated more P than vector control plants when grown in agar supplied with soluble phytate as the only P source. The greater extracellular phytase activity produced by the other transgenic lines is assumed to be adequate to achieve, at least, a similar response to that observed for ex::phyA-3 , as was the case for subterranean clover expressing ex::phyA ( George et al ., 2004 ). Greater accumulation of P in the shoots of N. tabacum that express ex::phyA is consistent with previous observations for transgenic Arabidopsis and subterranean clover ( Mudge et al ., 2003 ; George et al ., 2004 ). This result confirms the inability of plants to acquire P from phytate without the expression of heterologous genes for the extracellular release of phytase. <h2>Improved growth and P nutrition of plants are not evident in unamended soils</h2> The response observed in agar, however, was compromised when the plants were grown in soil. The P nutrition of plants expressing ex::phyA was no better than that of the vector controls in unamended soil. This is consistent with observations made for soil-grown subterranean clover which also expressed ex::phyA ( George et al ., 2004 ). Nevertheless, when ex::phyA-3 plants were grown in soil that had received P fertilizer applications in the field, P nutrition was improved by 38% compared with vector control plants. To further understand the basis of this response, three ex::phyA tobacco lines were grown in two contrasting soils which had been amended with combinations of either phytate or phosphate and lime. Consistent with previous observations, none of the independent transgenic lines showed improved P nutrition in either of the two unamended soils, and all plants were restricted by P deficiency. This could be attributed to the low concentrations of plant-available inorganic P observed in both of these soils. Moreover, all plants achieved the same P nutrition despite the presence, in both soils, of a pool (> 23 µg/g) of organic P that could be hydrolysed by a nonspecific phytase preparation. The lack of response by transgenic plants in unamended soils suggests that the portion of phytate in the enzyme-labile fraction was either too small or not sufficiently available in soil to act as an effective substrate. Alternatively, as the crude enzyme preparation (Sigma phytase) used is known to have activity towards a range of organic P substrates other than phytate ( Hayes et al ., 2000a ), NaOH-Pphos may be equally available to all plant lines even though the control lines do not have appreciable extracellular phytase activity. <h2>Amendment of soils confers a P nutrition advantage to plants that express ex::phyA</h2> The availability of inorganic P in both soils and the P nutrition of all plants were improved by the addition of phytate. This suggests that a portion of the phytate was mineralized by microorganisms when the soils were incubated prior to planting ( Cosgrove, 1976 ; Richardson et al ., 2001b ). Alternatively, added phytate may compete with phosphate for sorption sites and thus increase P availability by displacing orthophosphate ( Anderson and Malcolm, 1974 ). Significantly, the addition of phytate to soils improved the P nutrition of transgenic plants that expressed ex::phyA to a greater extent than that of the controls. In Hall soil, transgenic ex::phyA lines accumulated 52% more P on average than the control lines. This response was coincident with significant increases in both organic P and the fraction of this pool that was amenable to hydrolysis by the nonspecific phytase. Such responses were less consistent in Tilba soil, where only the transgenic lines with the highest expression of phyA accumulated significantly more P (28%) than the control lines. Phytate addition to Tilba soil also had less of an impact on both the concentration of organic P and that which was hydrolysed by the nonspecific phytase. Interestingly, when phytate was added to both soils in conjunction with lime, the P nutrition of all plants was markedly reduced and the differences between transgenic and control plants, seen at natural soil pH, were not evident. However, resin-extractable P in the soils was increased to a lesser extent when phytate was added in the presence of lime ( Table 1 ), and the availability and/or mineralization of the added phytate may therefore have been reduced. Excess calcium addition in lime and the addition of phytate as Ca phytate may have precipitated the phytate in limed soils. The solubility of Ca phytate is reduced significantly at pH values above pH 7.0 ( Jackman and Black, 1951 ), and low substrate availability may therefore have contributed to the lack of response of the transgenic plants compared with controls under such conditions. As observed in the initial experiment, improved P nutrition of plants expressing ex::phyA was evident when grown in soils with added phosphate. In contrast with the situation with phytate addition, the most significant responses of the transgenic ex::phyA -expressing lines to the addition of phosphate were observed in limed soil. Under such conditions, the three lines accumulated, on average, 32% more P than controls in both soil types. In contrast, in phosphate-amended soils at natural pH, significant responses as a result of phyA expression were only seen in Hall soil and only for one of the three transgenic lines. This inconsistent response to phosphate-addition in acidic Hall soil is comparable with the observations made for subterranean clover under the same conditions, where only one of five transgenic lines showed a response in P nutrition ( George et al ., 2004 ). Moreover, the improved P nutrition of transgenic plants was only consistently observed in soils in which phosphate had been recently applied, as compared with long-term application of phosphate in the field. In Hall soil, the improved P nutrition of the phyA -expressing plants with recent phosphate applications, was coincident with a twofold increase in the pool of organic P that was hydrolysed by the nonspecific phytase. However, increases in this pool of phytase-labile P were not evident in Tilba soil, and therefore could not explain the improved P nutrition of transgenic ex::phyA -expressing plants after application of phosphate with lime. The consistent improvement in the P nutrition of all transgenic lines in limed soils after phosphate addition, compared with both control lines, is a significant observation and contrasts with the observations found when phytate was added with lime. Although it is difficult to determine the reason for this difference, it may be a consequence of a number of factors. Although the presence of excess Ca at high soil pH may reduce the availability of added phytate as a result of precipitation (as discussed above), higher soil pH would also be expected to release phytate from adsorption sites in the soil ( Celi et al ., 2001 ), and the addition of phosphate anions may further displace phytate into solution ( Anderson and Malcolm, 1974 ). Alternatively added phosphate may be readily immobilized into soluble forms of phytate by soil microorganisms ( Cosgrove, 1976 ), although this possibility remains to be confirmed. Furthermore, George et al . (2005 ) have shown that the adsorption of phytase enzyme to the solid phase in these two soils is reduced markedly at neutral pH. More phytase remaining in soil solution at higher pH is likely to improve the capacity of the root-secreted enzyme to interact with its substrate. However, higher soil pH maybe less favourable for phytase activity, which is optimal at pH 5.5 ( Wyss et al ., 1999 ), and activity would be reduced by ∼50% at pH 6.5. Nevertheless, our results indicate that liming in conjunction with phosphate addition may potentially improve the availability of phytate in soil to plants that release extracellular phytase. The observations made here are important, not only because they were made using naturally formed soils, but also because they were verified using three independent transgenic lines against transgenic control plants. Comparisons made without such stringency have been shown to be unreliable ( George et al ., 2004 ), a point reiterated by the lone response of the low-expressing transgenic line ( ex::phyA-3 ) in field-fertilized Hall soil against only one control. <h2>Exudation of phytase from roots as a strategy to improve the P nutrition of plants</h2> Given that phytate is a relatively abundant form of phosphorus in most soils ( Turner et al ., 2002 ), it is intriguing that naturally occurring plants have not evolved an extracellular phytase for the direct utilization of P from phytate ( Hayes et al ., 2000b ). However, consistent with this paradox is the fact that, when plants that express a fungal phytase gene and exude phytase to the rhizosphere are grown in unamended soil, they do not achieve better P nutrition than controls. Limitations to the performance of such plants may include the poor availability of the substrate, a deactivation of the phytase activity when exuded to the rhizosphere or a compensatory effect of phytase-exuding rhizosphere microorganisms. Results from the present study suggest that transgenic plants that exude phytase to the rhizosphere can have an advantage over wild-type controls, and are able to accumulate more P from soil, under conditions in which phytate availability was putatively improved. This implies that the growth of transgenic plants which express phyA , in combination with amendments to soils which improve phytate availability, may directly improve the P nutrition of crops and also the efficiency with which applied phosphate fertilizers are utilized. Consequently, further research to understand the factors that govern the availability of phytate in soil is required. A number of agronomic and molecular approaches are possible and include: (i) appropriate soil management practices, such as the application of P fertilizer and lime as shown here; (ii) the use of animal manures with large phytate contents, particularly from non-ruminant animals, for field fertilization ( Eeckhout and De Paepe, 1994 ; Brinch-Pendersen et al ., 2002 ); and (iii) the enhancement of the rhizosphere concentration of organic anions, such as citrate, which have been shown to increase the phytase lability of organic P in soil extracts ( Hayes et al ., 2000a ). The latter may be achieved by rotation of phytase-exuding crop plants with organic anion-exuding break-crops, such as Lupinus albus ( Horst et al . 2001 ), or by genetic manipulation of plants for extracellular release of both phytase and organic anions ( de la Fuente et al ., 1997 ; Delhaize et al ., 2001 ; Sasaki et al ., 2004 ). In conclusion, our results indicate that the expression and extracellular release of phytase by plants confers a unique characteristic that enables them to utilize P from phytate. However, this advantage is poorly realized in unamended soil and is only apparent under circumstances in which the availability of phytate has been putatively increased. These results indicate that phytate availability in soil is a major limitation restricting the ability of plants to access this abundant form of soil organic P. Consequently, the development of approaches to increase the availability of phytate in soil are expected to confer distinct advantages to transgenic plants that release extracellular phytase. Such an integrated approach will improve the efficiency with which phosphate fertilizers are utilized in agricultural systems, and thus increase the economic viability and sustainability of P-limited agriculture, while also reducing the potential negative environmental impacts of excess P use. <h1>Experimental procedures</h1> <h2>Transformation of N. tabacum</h2> N. tabacum plants (var. W38) were transformed with a phytase gene ( phyA ) from A. niger using Agrobacterium -mediated transformation. The fungal phytase gene, under the control of the cauliflower mosaic virus (CaMV) 35S promoter and ocs terminator ( Figure 1A ), was modified for extracellular secretion by inclusion of an extracellular targeting sequence from the carrot extensin ( ex ) gene ( Richardson et al ., 2001a ). Primary transformant calli containing ex::phyA (T0 generation) were selected on kanamycin (50 µg/mL) and transformation was verified by Southern blot (not shown). Vector control plants (pPLEX502; Schünmann et al. , 2003 ) were also generated. Twelve independent lines of both ex::phyA and vector alone were initially selected by resistance of T0 plants to kanamycin. From these, three transformed lines containing ex::phyA insertions, both at a single locus ( ex::phyA-1 and ex::phyA-3 ) and with multiple insertions ( ex::phyA-2 ), and one vector control were selected and transferred to a fertilized potting mix for collection of T1 generation seed. Transformed ex::phyA and vector control plants for subsequent experimentation were selected from segregating T1 seedlings by screening for resistance to kanamycin (100 µg/mL) in nutrient agar ( Richardson et al ., 2001a ), and comparisons were made with wild-type plants which had been grown to the same stage in the absence of kanamycin. Verification of the transformation of T1 generation material was performed by Northern blot hybridization, using the Aspergillus phyA or tobacco 18S RNA genes as 32 P-labelled DNA probes ( Figure 1B ). The relative expression of phyA was quantified by comparing the hybridization intensity on sequential Northern blot hybridizations using FujiFilm™ Multigauge Software. The number of dark pixels (PSL) produced by phyA hybridization per 1000 dark pixels produced by the 18S ribosomal DNA hybridization within an equivalent area was compared using three replicate bands, each representing separate RNA preparations for each of the different plant lines ( Figure 1C ). The lowest expressing line was subsequently used in sterile agar containing phytate as the sole source of P to demonstrate an advantage in P nutrition of expressing phyA , based on the assumption that lines with greater expression of phyA will have at least an equal, if not greater, advantage ( George et al ., 2004 ). <h2>Determination of phytase activity in shoots and exudates</h2> The phytase activity of shoots was determined on the same plant material as used for Northern blots, which had been ground in liquid nitrogen and resuspended in five parts of homogenizing buffer [15 m m 2-( N -morpholino)ethanesulphonic acid (MES) pH 5.5, 3 m m ethylenediaminetetraacetic acid (EDTA), 5 m m cysteine]. The extracts was centrifuged (15 000 g , 15 min) and the supernatant was taken to represent soluble cell contents. Total protein was assayed by reaction with Bradford reagent ( Bradford, 1976 ). Extracellular phytase activities were determined on plants grown in 20 mL of sterile nutrient solution (4 m m KNO 3 , 4 m m Ca(NO 3 ) 2 ·4H 2 O, 1.5 m m MgSO 4 ·7H 2 O, 3 m m NH 4 Cl, 0.1 m m Fe-EDTA and micronutrients) containing 50 µ m P (Na 2 HPO 4 ) and 10 g/L sucrose. Plants were initially grown for 14 days, with constant swirling and light (∼200 µE/m 2 ) in a growth room at 21 °C, and were then transferred to a no-P, sucrose-free equivalent nutrient solution under the same conditions for a further 14 days. Phytase activities were determined in the root bathing solutions for four replicate samples. The dry weights of shoots and roots were determined from oven-dried (65 °C) material. Bathing solution volumes were weighed to account for transpirational losses and stored at 4 °C prior to enzyme assays. Phytase activities in both soluble shoot contents and exudates were determined using myo -inositol hexa kis phosphate (InsP6; Sigma-Aldrich Corp., St. Louis, MC), as described by Richardson et al . (2000 ). Briefly, aliquots of extracts or root exudates were made up to 300 µL with buffer (15 m m MES, 1 m m EDTA, pH 5.5) containing InsP6 (2 m m ) and incubated at 37 °C for 60 min. Reactions were terminated with equal volumes of 10% trichloroacetic acid (TCA) at either time zero or at the end of incubation. The concentrations of phosphate released during the assay were measured spectrophotometrically at 620 nm after reaction with malachite green ( Irving and McLaughlin, 1990 ). Enzyme activities were calculated from the difference in phosphate concentrations in supernatants between time zero and the end of incubation, and expressed as either the activity per milligram of protein (shoot activity) or the activity exuded per root dry weight per day (exudate activity). <h2>Growth and P uptake by transgenic plants grown in agar</h2> The ability of plants to use phytate as a source of P was determined by growth in laboratory media (1.2% Bacto™ agar; Becton Dickinson and Company, Sparks, MD) under sterile conditions. Individual plants were grown on nutrient agar slants supplied with all essential nutrients (4 m m KNO 3 , 4 m m Ca(NO 3 ) 2 ·4H 2 O, 1.5 m m MgSO 4 ·7H 2 O, 3 m m NH 4 Cl, 50 µ m Fe-EDTA, 30 n m H 3 BO 3 , 6 µ m CuSO 4 , 6 µ m MnSO 4 , 0.6 µ m ZnSO 4 , 42 n m NH 4 Mo 7 , 12 µ m Co 4 (NO 3 ) 2 ), except P, which was supplied either as InsP6 (phytate) at a concentration of 0.8 m m with respect to P or as orthophosphate supplied as Na 2 HPO 4 (0.8 m m P); otherwise, plants were grown in the absence of P ( Hayes et al ., 2000b ; Richardson et al ., 2001b ). Fourteen replicates of transgenic line ex::phyA-3 and a vector control line were selected on kanamycin, transplanted to the agar slants and grown for 28 days in a growth cabinet at 20 °C with a light intensity of 375 µE/m 2 and a 12 h light period. Shoots were harvested and oven dried (65 °C) for the measurement of dry weight. Shoot P content was determined by ashing (550 °C) the entire shoot and dissolving the ash in 100 parts (w/v) 1.8 N H 2 SO 4 . Total P was measured by reaction with malachite green ( Irving and McLaughlin, 1990 ). <h2>Soil characterization and amendments</h2> Two acidic topsoils (0–10 cm depth) from permanent pastures, which were low in available P, were used. Soil collected from the CSIRO experiment station in Hall, ACT, Australia (Hall soil) was an Alfisol (USDA Soil Taxonomy) with a moderate P sorption capacity and the following soil P characteristics: Colwell P, 10.4 µg P/g; total P, 316.5 µg P/g; total organic P, 67%. Soil collected from Tilba, NSW, Australia (Tilba soil) was a Spodosol (USDA Soil Taxonomy), had low P sorption capacity and the following soil P characteristics: Colwell P, 9.45 µg P/g; total P, 60.0 µg P/g; total organic P, 93%. An additional topsoil from the Hall site, which had received regular P fertilization in the field over 10 years as part of a long-term fertilizer trial, was also collected (field-fertilized Hall soil). Soils were air-dried, mixed and passed through a 2 mm sieve, and were either left unamended or amended with various combinations of lime or P, supplied as either KH 2 PO 4 or Ca phytate (Koch-Light Laboratories Ltd, Colnbrook, UK). Additions of phosphate or P as phytate were based on the amount of orthophosphate required to increase plant-available P (resin-extractable P) by ∼10-fold ( Table 1 ). As a result of the greater P sorption capacity of Hall soil, P addition was 200 mg P/kg soil (918.7 mg Ca phytate/kg soil or 878.9 mg KH 2 PO 4 /kg soil), whereas, in Tilba soil, 60 mg P/kg soil (275.6 mg Ca phytate/kg soil or 263.7 mg KH 2 PO 4 /kg soil) was added. Where required, soils were limed to increase soil pH to between 6.5 and 7.0, at a rate equivalent to 5 tonnes of lime per hectare (3.3 g CaCO 3 /kg soil). Soils were then brought up to 80% field capacity (0.18 or 0.22 mL H 2 O/g soil for Hall or Tilba soil, respectively) and incubated for 28 days prior to analysis and plant growth. Incubated soils were analysed for pH (1 : 5 w/v deionized H 2 O), anion exchange resin-extractable P (resin-P or plant-available P) ( Saggar et al ., 1990 ) and 0.1 m NaOH-extractable inorganic and organic P (NaOH-Pi or NaOH-Po) ( Tiessen and Moir, 1993 ). The anion exchange resin method of Saggar et al . (1990 ) was modified in that resins were charged with Na 2 HCO 3 and eluted with HCl. The component of the NaOH extracts that was amenable to enzyme hydrolysis (NaOH-Pphos) was also determined by incubation with excess activity of a commercially available crude phytase preparation from Aspergillus sp. (Sigma Phytase; Sigma-Aldrich Ltd, St. Louis, MO). This preparation of phytase has been shown to be active against InsP6 and also other monoester and diester forms of organic P ( Hayes et al ., 2000a ). For assays, 100 µL of NaOH extract was made up to 300 µL with buffer (15 m m MES, 1 m m EDTA, pH 5.5) and phytase at an activity of 0.5 nKat/mL, followed by incubation at 37 °C for 6 h to allow the reaction to run to completion. Reactions were terminated with equal volumes of 10% TCA at either time zero or at the end of incubation. The amounts of phosphate released during the assay were measured and expressed on a per soil basis as NaOH-Pphos. <h2>Growth and P uptake by transgenic plants in soil</h2> Five replicate pots containing 475 g (weight at 80% field capacity) of each soil treatment were sown with five tobacco seedlings for each plant line. Prior to planting, transgenic plants were germinated and grown for 14 days on agar containing 100 µg kanamycin/L. Wild-type plants were grown to the same stage on nutrient agar in the absence of kanamycin. Pots were thinned to three plants per pot after establishment, and maintained by weight at ∼80% field capacity during growth. All nutrients except P were supplied weekly by the addition of 5 mL of nutrient solution [3 m m NH 4 SO 4 , 2 m m KNO 3 , 1 m m MgSO 4 , 10 m m Ca(NO 3 ) 2 , 80 µ m Fe-EDTA and micronutrients (B, Cu, Mn, Zn, Mo and Co)]. Plants were grown in a randomized design in a glasshouse at between 14 and 22 °C with an approximate daylight length of 16 h. Shoots were harvested after 25 days of growth and the biomass was determined after oven drying at 65 °C. Shoot materials were milled and analysed for total P content after digestion with a sulphuric acid–hydrogen peroxide mix ( Heffernan, 1985 ). Preliminary soil growth studies using field-fertilized and unamended Hall soil ( Table 2 ) were performed in the same way as described above, except that only one transgenic line ( ex::phyA-3 ) was grown and plants were grown for 45 days. All environmental parameters were the same, except for a shorter day length of approximately 12 h. <h2>Data calculation and statistical analyses</h2> All data are presented as the mean of between three and 14 replicates and error bars represent one standard error of the mean. Significant differences were established using general analysis of variance ( anova ) and treatment means were compared by least significant difference (LSD) ( P = 0.05) (Genstat v5; Rothamsted Experiment Station, Hertfordshire, UK). All data were tested for normality prior to analysis and, where required, skewed data were transformed to natural log values (resin-P and NaOH-Pphos). All data are presented as measured except for the following: (i) exuded phytase activity (nKat/g root dry weight/day) was calculated by converting from activity per 14 days to activity per day, which assumes that the exudation rate is constant over the time period; and (ii) P accumulated in shoots was calculated by multiplying the shoot P concentration (µg P/mg) by the biomass (mg). For plants grown in soil ( Figure 4 ), data for shoot P accumulation are presented as they provide a more sensitive indicator than the shoot dry weight of the plant's ability to acquire P from the soils. Although the P content and shoot growth are generally correlated under P-deficient conditions, shoot growth does not respond when plants are near or above their critical P requirement (e.g. Figures 2 and 4 ; phosphate- and lime-amended Tilba soil). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Biotechnology Journal Wiley

Expression of a fungal phytase gene in Nicotiana tabacum improves phosphorus nutrition of plants grown in amended soils

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10.1111/j.1467-7652.2004.00116.x
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Abstract

<h1>Introduction</h1> Phosphorus (P) is an essential plant nutrient which limits agricultural production on a global scale. Inorganic P fertilizers are a scarce resource in much of the world and, where they are applied to promote crop yields, the majority of the added P is transformed into inorganic and organic forms which are of limited availability to plants ( Sanyal and De Datta, 1991 ). Only 10%−20% of fertilizer P is available for utilization by crops in the first year after application ( Holford, 1997 ). For this reason, it is paramount that P use efficiency by agricultural plants is improved. More efficient use of fertilizer P is also required to avoid adverse environmental effects as a result of P contamination of the wider environment ( Tunney et al ., 1997 ). Moreover, at current rates of consumption, known reserves of high-grade rock phosphate are predicted to be exhausted before the end of this century ( Runge-Metzger, 1995 ). Plants have developed a range of mechanisms to enhance their ability to acquire P from soil ( Marschner, 1995 ; Vance et al ., 2003 ). The most important are root morphological characteristics ( Lynch, 1995 ; Gahoonia and Nielsen, 1997 ; Ge et al ., 2000 ). However, mycorrhizal symbioses ( Smith and Read, 1997 ) and the production of root exudates, such as organic anions ( Ryan et al ., 2001 ) and phosphatase enzymes, may also enhance P acquisition ( Tadano et al ., 1993 ; Li et al ., 1997 ; George et al ., 2002 ). For P to be available to plants, it needs to be present as orthophosphate in soil solution. Therefore, the exudation of phosphatases which catalyse the hydrolysis of organic P, is a potentially important way for plants to enhance P availability, particularly as a large proportion of soil P (up to 80%) occurs in organic forms ( Richardson et al ., 2004 ). Phytases, enzymes that hydrolyse derivatives of inositol penta- and hexa kis -phosphates (phytate), are of particular interest because phytate constitutes up to 50% of the total organic P in soil ( Anderson, 1980 ; Turner et al ., 2002 ). Paradoxically, many plants have limited capacity to obtain P directly from phytate when grown under controlled conditions ( Hayes et al ., 2000b ; Richardson et al ., 2001b ). This is because the availability of phytate in soil is low (due to sorption and precipitation reactions; Anderson and Malcolm, 1974 ; Shang et al ., 1992 ; Celi et al ., 2001 ) and because the capacity of plants to exude phytase to the rhizosphere ( Hayes et al ., 1999 ; Richardson et al ., 2000 ) is limited. In low P-sorbing growth media (agar or sand), the ability of plants to use P from phytate is improved when they are inoculated with soil microorganisms which possess phytase activity, or when purified phytase is added to the medium ( Findenegg and Nelemans, 1993 ; Hayes et al ., 2000b ; Richardson et al ., 2001b ; Idriss et al ., 2002 ). Transgenic Arabidopsis thaliana , subterranean clover ( Trifolium subterraneum L.) and potato ( Solanum tuberosum L.), that express a phytase gene ( phyA ) from Aspergillus niger , release extracellular phytase and are able to utilize P supplied as phytate in agar ( Richardson et al ., 2001a ; Mudge et al ., 2003 ; Zimmermann et al ., 2003 ; George et al ., 2004 ). Improved growth and P nutrition of plants that express phytase have not been demonstrated for soil-grown plants. In this paper, the growth and P nutrition of transgenic lines of tobacco ( Nicotiana tabacum L.) expressing phyA were assessed in two P-deficient Australian soils which potentially contained endogenous phytate ( Williams and Anderson, 1968 ; Hayes et al ., 2000a ). Although growth and P accumulation by transgenic tobacco were not increased in unamended soils, P nutrition was improved in soils which had been amended with various combinations of phytate, phosphate and lime. <h1>Results</h1> <h2>Expression of ex::phyA in N. tabacum and utilization of P from phytate</h2> Tobacco plants were transformed with a constitutively expressed phyA gene from A. niger containing a 5′ signal peptide ( ex ) from carrot extensin for extracellular secretion of phytase ( Figure 1A ). Three of 12 independently transformed lines generated were tested, and the expression of ex::phyA in shoots was verified using Northern blot analysis. phyA mRNA was present in the shoot tissue of all transgenic lines ( ex::phyA-1 to ex::phyA-3 ) and absent in plants transformed with the vector alone (vector control) and in wild-type plants ( Figure 1B ). The amount of mRNA in shoots varied ( P < 0.05) between the transgenic lines. A line with transgene insertions at multiple loci ( ex::phyA-2 ) had 2.9-fold greater relative expression of phyA than line ex::phyA-1 , and 6.3-fold greater relative expression of phyA than line ex::phyA-3 ( Figure 1C ). Shoot phytase activity was increased by 2.0–4.8-fold ( P < 0.001) in transgenic plants that expressed the chimeric ex::phyA gene. Most importantly, all ex::phyA lines showed substantially greater extracellular phytase activities than controls ( P < 0.001), which had no detectable extracellular activity. The relative level of gene expression of the transgenic lines was apparent in the extracellular phytase activity, in that the line ( ex::phyA-3 ) which showed the lowest expression of phyA mRNA also had 2.8-fold lower extracellular phytase activity ( P < 0.05) ( Figure 1 ). The effectiveness of the expression of ex::phyA by plants was demonstrated by comparing the shoot biomass and P nutrition of the lowest expressing transgenic line ( ex::phyA-3 ) with vector control plants when grown in sterile agar supplied with P solely as phytate ( Figure 2 ). Control treatments included plants grown without P and those supplied with P as orthophosphate. Line ex::phyA -3 showed similar shoot biomass to the vector controls in all P treatments ( Figure 2A ). However, when P was provided as phytate, plants that expressed ex::phyA-3 showed a 1.8-fold greater shoot biomass than when no P was added ( P < 0.05), but no difference was observed for the vector control. Despite this, P nutrition was improved by expression of the phytase gene ( Figure 2B ). When plants were supplied with P as phytate, the ex::phyA-3 line accumulated fourfold more P than vector control plants ( P < 0.05) and 29-fold more P than plants grown in the absence of phosphorus ( P < 0.05). However, ex::phyA plants supplied with phytate did not accumulate as much P as equivalent plants supplied with the same amount of P as orthophosphate ( P < 0.05). <h2>Growth and P nutrition in soil of plants that release extracellular phytase</h2> Transgenic line ex::phyA-3 was also grown in an Alfisol soil which had been collected from plots (both unamended and fertilized) in a field fertilizer trial (Hall soil, Table 1 ). The shoot biomass and P nutrition of ex::phyA-3 plants were compared with those of the vector control after 45 days of growth. In soil taken from an unfertilized plot, the growth of both plant lines was restricted and no differences in biomass or P nutrition were evident between lines ( Table 2 ). The growth and P nutrition of all plants were greatly enhanced, by between 10- and 19-fold, when plants were grown in soil that had received P fertilizer in the field. Significantly, the shoot biomass in the phyA -expressing line ( ex::phyA-3 ) was increased by 38% when compared with the control plants grown in the soil collected from fertilized plots ( P < 0.05) ( Table 2 ; Figure 3 ). P accumulation by transgenic plants expressing ex::phyA was also significantly improved by 34% compared with control plants in this soil ( P < 0.05). Further experiments were undertaken to validate the growth and P nutrition response observed for the ex::phyA-3 line of transgenic tobacco in field-fertilized Hall soil. Three independently transformed lines ( ex::phyA-1 to ex::phyA-3 ) were grown in two soils, the Hall soil (an Alfisol) and Tilba soil (a Spodosol), with moderate and low P sorption characteristics, respectively. Soils were either left unamended or supplemented with P as either calcium phytate (Ca phytate) or orthophosphate, with or without the addition of lime to increase the soil pH. P accumulation in shoots of the transgenic lines was compared with that in vector control and wild-type plants. Liming resulted in a pH increase in both soils ( P < 0.05) ( Table 1 ), and generally reduced the growth and P nutrition of all plants (Figure 4). With the addition of both phosphate and phytate, P availability increased ( P < 0.05), as measured by resin extraction, whilst NaOH-extractable inorganic and organic P pools were also greater. P availability was increased to a greater extent ( P < 0.05) with the addition of orthophosphate than with the addition of the same amount of P as Ca phytate ( Table 1 ). When grown in unamended soils, plant growth was severely restricted and all plants accumulated only small amounts of P. This was coincident with relatively low concentrations of resin-extractable P ( Table 1 ). In both the Tilba and Hall soils, no significant differences in shoot P accumulation were evident for the ex::phyA plant lines compared with the vector control or wild-type plants ( Figure 4A ). This was despite the presence of a pool of soil P that was amenable to hydrolysis by a crude phytase preparation (NaOH-Pphos; Table 1 ). Liming the unfertilized soils had little effect on the availability of soil P pools and did not affect the P nutrition of plant lines in the Hall soil, but caused a significant decrease in P accumulation in plants grown in the Tilba soil. <h2>Growth and P nutrition of plants in soils amended with phytate and lime</h2> When grown in soil supplemented with phytate, transgenic plants that expressed ex::phyA accumulated more P in shoots than did control plants ( Figure 4B ). In Hall soil, the shoot P content of ex::phyA plants was 52% greater than that in control plants and, on average, the plants accumulated 90 µg more P ( Figure 4B ). However, this difference in P content did not result in dry weights that were significantly different between the plant lines (mean shoot dry weight of 85.4 mg/plant across all lines; data not shown). In Tilba soil amended with phytate, only the higher expressing transgenic lines ( ex::phyA-1 and ex::phyA-2 ) accumulated more P (28% or 121 µg P per plant; Figure 4B ) than either of the controls and, in this case, differences in shoot dry weight were significant ( P < 0.05; mean shoot weight of 100.4 mg/plant for the two transgenic lines compared with 76.4 mg/plant for the controls). Soils supplemented with phytate had greater resin P ( P < 0.05) and more organic P extracted by NaOH. Moreover, amendment of both soils with phytate increased, by between 13% and 23%, the amount of NaOH-extractable organic P that was amenable to hydrolysis by a crude phytase preparation ( P < 0.05) ( Table 1 ). In contrast with plant responses in acidic soils, when phytate was added to soils that had been limed, P accumulation by transgenic ex::phyA plant lines was not significantly different from that of the controls, and the P nutrition of all plants was significantly less than in unlimed soils. The addition of phytate to limed soils also increased resin-extractable P, but to a lesser extent than in acidic soils. Unlike under acidic conditions, phytate addition did not increase the NaOH-extractable organic P and did not consistently increase the portion which was amenable to hydrolysis by crude phytase ( Table 1 ). These observations are consistent with the reduced P accumulation by all plants in limed soil after the addition of phytate ( P < 0.001), compared with the unlimed equivalent. <h2>Growth and P nutrition of plants in soils amended with phosphate and lime</h2> Shoot P accumulation was greatest in soils amended with phosphate, with all plants accumulating between three- and 22-fold more P than when the same amount of P was added as Ca phytate ( P < 0.05) ( Figure 4C ). This was coincident with increased ( P < 0.05) resin-extractable P concentrations in both soils. When plants were grown in Hall soil that had received a recent application of phosphate, transgenic ex::phyA -expressing plants accumulated, on average, 228 µg more P in shoots than did the control lines ( P < 0.05). There was no such response in phosphate-fertilized Tilba soil. However, in the presence of lime and phosphate, large increases in P accumulation by plants that expressed phyA were observed relative to the controls in both soils. Under these conditions, ex::phyA plant lines accumulated 597 and 347 µg more P than control plants in Tilba and Hall soils, respectively ( P < 0.001). This was equivalent to a 32% increase in P accumulation in both soils ( Figure 4C ) and, for the Hall soil, resulted in significantly greater shoot dry weights for the transgenic lines that expressed ex::phyA ( P < 0.05) (mean shoot dry weight of 319.3 mg/plant for the ex::phyA transgenic lines compared with 220.9 mg/plant for the controls). Although the addition of phosphate to Hall soil had little effect on the amount of organic P extracted by NaOH, the proportion of this organic P which was hydrolysed by a nonspecific phytase preparation was increased by between 54% and 100%. Comparable increases were not evident in the Tilba soil, although the amount of organic P extracted by NaOH increased, but this was only significant in unlimed soil. When grown in field-fertilized Hall soil, the P nutrition of all plants was improved in relation to equivalent plants grown in unamended soil (cf. Figure 4A and 4D ). This was again coincident with greater resin-extractable P ( P < 0.05). As previously observed ( Table 2 ), the ex::phyA-3 line accumulated significantly more P (35% more) than the vector control plants. However, this transgenic line was not different from the wild-type, and the two other ex::phyA lines showed no improvement in P nutrition compared with either of the controls. The field application of P to Hall soil increased the amount of NaOH-extractable inorganic and organic P ( P < 0.05) and increased the component of organic P that was hydrolysed by the crude phytase preparation, but to a lesser extent than observed for the recent addition of phosphate ( Table 1 ). <h1>Discussion</h1> <h2>Expression of phytase in tobacco allows the utilization of P from phytate</h2> Transformation of N. tabacum with the ex::phyA gene was confirmed by the presence of mRNA in the shoot tissue of all three ex::phyA lines. One of the transformed lines ( ex::phyA-3 ) showed reduced expression of phyA by Northern blot hybridization compared with the other lines, whereas the line which had insertions in at least three loci (ex::phyA-2) showed the highest expression. These levels of phyA expression were consistent with phytase activity in both shoot material and root exudates. Of significance was the fact that the phytase activity exuded to the rhizosphere was greatly increased in the transgenic lines compared with the undetectable levels exuded by control plants. The level of phytase activity exuded from the roots of tobacco, even for the low expressing line ( ex::phyA-3 ), was equivalent to the activity exuded by other phyA -expressing plants, including Arabidopsis , potato and subterranean clover ( Richardson et al ., 2001a ; Zimmermann et al ., 2003 ; George et al ., 2004 ). The enhanced exudation of phytase by transgenic N. tabacum conferred an ability to acquire P from phytate in sterile non-sorbing agar media. Transgenic line ex::phyA-3 , which had the lowest exuded phytase activity, accumulated more P than vector control plants when grown in agar supplied with soluble phytate as the only P source. The greater extracellular phytase activity produced by the other transgenic lines is assumed to be adequate to achieve, at least, a similar response to that observed for ex::phyA-3 , as was the case for subterranean clover expressing ex::phyA ( George et al ., 2004 ). Greater accumulation of P in the shoots of N. tabacum that express ex::phyA is consistent with previous observations for transgenic Arabidopsis and subterranean clover ( Mudge et al ., 2003 ; George et al ., 2004 ). This result confirms the inability of plants to acquire P from phytate without the expression of heterologous genes for the extracellular release of phytase. <h2>Improved growth and P nutrition of plants are not evident in unamended soils</h2> The response observed in agar, however, was compromised when the plants were grown in soil. The P nutrition of plants expressing ex::phyA was no better than that of the vector controls in unamended soil. This is consistent with observations made for soil-grown subterranean clover which also expressed ex::phyA ( George et al ., 2004 ). Nevertheless, when ex::phyA-3 plants were grown in soil that had received P fertilizer applications in the field, P nutrition was improved by 38% compared with vector control plants. To further understand the basis of this response, three ex::phyA tobacco lines were grown in two contrasting soils which had been amended with combinations of either phytate or phosphate and lime. Consistent with previous observations, none of the independent transgenic lines showed improved P nutrition in either of the two unamended soils, and all plants were restricted by P deficiency. This could be attributed to the low concentrations of plant-available inorganic P observed in both of these soils. Moreover, all plants achieved the same P nutrition despite the presence, in both soils, of a pool (> 23 µg/g) of organic P that could be hydrolysed by a nonspecific phytase preparation. The lack of response by transgenic plants in unamended soils suggests that the portion of phytate in the enzyme-labile fraction was either too small or not sufficiently available in soil to act as an effective substrate. Alternatively, as the crude enzyme preparation (Sigma phytase) used is known to have activity towards a range of organic P substrates other than phytate ( Hayes et al ., 2000a ), NaOH-Pphos may be equally available to all plant lines even though the control lines do not have appreciable extracellular phytase activity. <h2>Amendment of soils confers a P nutrition advantage to plants that express ex::phyA</h2> The availability of inorganic P in both soils and the P nutrition of all plants were improved by the addition of phytate. This suggests that a portion of the phytate was mineralized by microorganisms when the soils were incubated prior to planting ( Cosgrove, 1976 ; Richardson et al ., 2001b ). Alternatively, added phytate may compete with phosphate for sorption sites and thus increase P availability by displacing orthophosphate ( Anderson and Malcolm, 1974 ). Significantly, the addition of phytate to soils improved the P nutrition of transgenic plants that expressed ex::phyA to a greater extent than that of the controls. In Hall soil, transgenic ex::phyA lines accumulated 52% more P on average than the control lines. This response was coincident with significant increases in both organic P and the fraction of this pool that was amenable to hydrolysis by the nonspecific phytase. Such responses were less consistent in Tilba soil, where only the transgenic lines with the highest expression of phyA accumulated significantly more P (28%) than the control lines. Phytate addition to Tilba soil also had less of an impact on both the concentration of organic P and that which was hydrolysed by the nonspecific phytase. Interestingly, when phytate was added to both soils in conjunction with lime, the P nutrition of all plants was markedly reduced and the differences between transgenic and control plants, seen at natural soil pH, were not evident. However, resin-extractable P in the soils was increased to a lesser extent when phytate was added in the presence of lime ( Table 1 ), and the availability and/or mineralization of the added phytate may therefore have been reduced. Excess calcium addition in lime and the addition of phytate as Ca phytate may have precipitated the phytate in limed soils. The solubility of Ca phytate is reduced significantly at pH values above pH 7.0 ( Jackman and Black, 1951 ), and low substrate availability may therefore have contributed to the lack of response of the transgenic plants compared with controls under such conditions. As observed in the initial experiment, improved P nutrition of plants expressing ex::phyA was evident when grown in soils with added phosphate. In contrast with the situation with phytate addition, the most significant responses of the transgenic ex::phyA -expressing lines to the addition of phosphate were observed in limed soil. Under such conditions, the three lines accumulated, on average, 32% more P than controls in both soil types. In contrast, in phosphate-amended soils at natural pH, significant responses as a result of phyA expression were only seen in Hall soil and only for one of the three transgenic lines. This inconsistent response to phosphate-addition in acidic Hall soil is comparable with the observations made for subterranean clover under the same conditions, where only one of five transgenic lines showed a response in P nutrition ( George et al ., 2004 ). Moreover, the improved P nutrition of transgenic plants was only consistently observed in soils in which phosphate had been recently applied, as compared with long-term application of phosphate in the field. In Hall soil, the improved P nutrition of the phyA -expressing plants with recent phosphate applications, was coincident with a twofold increase in the pool of organic P that was hydrolysed by the nonspecific phytase. However, increases in this pool of phytase-labile P were not evident in Tilba soil, and therefore could not explain the improved P nutrition of transgenic ex::phyA -expressing plants after application of phosphate with lime. The consistent improvement in the P nutrition of all transgenic lines in limed soils after phosphate addition, compared with both control lines, is a significant observation and contrasts with the observations found when phytate was added with lime. Although it is difficult to determine the reason for this difference, it may be a consequence of a number of factors. Although the presence of excess Ca at high soil pH may reduce the availability of added phytate as a result of precipitation (as discussed above), higher soil pH would also be expected to release phytate from adsorption sites in the soil ( Celi et al ., 2001 ), and the addition of phosphate anions may further displace phytate into solution ( Anderson and Malcolm, 1974 ). Alternatively added phosphate may be readily immobilized into soluble forms of phytate by soil microorganisms ( Cosgrove, 1976 ), although this possibility remains to be confirmed. Furthermore, George et al . (2005 ) have shown that the adsorption of phytase enzyme to the solid phase in these two soils is reduced markedly at neutral pH. More phytase remaining in soil solution at higher pH is likely to improve the capacity of the root-secreted enzyme to interact with its substrate. However, higher soil pH maybe less favourable for phytase activity, which is optimal at pH 5.5 ( Wyss et al ., 1999 ), and activity would be reduced by ∼50% at pH 6.5. Nevertheless, our results indicate that liming in conjunction with phosphate addition may potentially improve the availability of phytate in soil to plants that release extracellular phytase. The observations made here are important, not only because they were made using naturally formed soils, but also because they were verified using three independent transgenic lines against transgenic control plants. Comparisons made without such stringency have been shown to be unreliable ( George et al ., 2004 ), a point reiterated by the lone response of the low-expressing transgenic line ( ex::phyA-3 ) in field-fertilized Hall soil against only one control. <h2>Exudation of phytase from roots as a strategy to improve the P nutrition of plants</h2> Given that phytate is a relatively abundant form of phosphorus in most soils ( Turner et al ., 2002 ), it is intriguing that naturally occurring plants have not evolved an extracellular phytase for the direct utilization of P from phytate ( Hayes et al ., 2000b ). However, consistent with this paradox is the fact that, when plants that express a fungal phytase gene and exude phytase to the rhizosphere are grown in unamended soil, they do not achieve better P nutrition than controls. Limitations to the performance of such plants may include the poor availability of the substrate, a deactivation of the phytase activity when exuded to the rhizosphere or a compensatory effect of phytase-exuding rhizosphere microorganisms. Results from the present study suggest that transgenic plants that exude phytase to the rhizosphere can have an advantage over wild-type controls, and are able to accumulate more P from soil, under conditions in which phytate availability was putatively improved. This implies that the growth of transgenic plants which express phyA , in combination with amendments to soils which improve phytate availability, may directly improve the P nutrition of crops and also the efficiency with which applied phosphate fertilizers are utilized. Consequently, further research to understand the factors that govern the availability of phytate in soil is required. A number of agronomic and molecular approaches are possible and include: (i) appropriate soil management practices, such as the application of P fertilizer and lime as shown here; (ii) the use of animal manures with large phytate contents, particularly from non-ruminant animals, for field fertilization ( Eeckhout and De Paepe, 1994 ; Brinch-Pendersen et al ., 2002 ); and (iii) the enhancement of the rhizosphere concentration of organic anions, such as citrate, which have been shown to increase the phytase lability of organic P in soil extracts ( Hayes et al ., 2000a ). The latter may be achieved by rotation of phytase-exuding crop plants with organic anion-exuding break-crops, such as Lupinus albus ( Horst et al . 2001 ), or by genetic manipulation of plants for extracellular release of both phytase and organic anions ( de la Fuente et al ., 1997 ; Delhaize et al ., 2001 ; Sasaki et al ., 2004 ). In conclusion, our results indicate that the expression and extracellular release of phytase by plants confers a unique characteristic that enables them to utilize P from phytate. However, this advantage is poorly realized in unamended soil and is only apparent under circumstances in which the availability of phytate has been putatively increased. These results indicate that phytate availability in soil is a major limitation restricting the ability of plants to access this abundant form of soil organic P. Consequently, the development of approaches to increase the availability of phytate in soil are expected to confer distinct advantages to transgenic plants that release extracellular phytase. Such an integrated approach will improve the efficiency with which phosphate fertilizers are utilized in agricultural systems, and thus increase the economic viability and sustainability of P-limited agriculture, while also reducing the potential negative environmental impacts of excess P use. <h1>Experimental procedures</h1> <h2>Transformation of N. tabacum</h2> N. tabacum plants (var. W38) were transformed with a phytase gene ( phyA ) from A. niger using Agrobacterium -mediated transformation. The fungal phytase gene, under the control of the cauliflower mosaic virus (CaMV) 35S promoter and ocs terminator ( Figure 1A ), was modified for extracellular secretion by inclusion of an extracellular targeting sequence from the carrot extensin ( ex ) gene ( Richardson et al ., 2001a ). Primary transformant calli containing ex::phyA (T0 generation) were selected on kanamycin (50 µg/mL) and transformation was verified by Southern blot (not shown). Vector control plants (pPLEX502; Schünmann et al. , 2003 ) were also generated. Twelve independent lines of both ex::phyA and vector alone were initially selected by resistance of T0 plants to kanamycin. From these, three transformed lines containing ex::phyA insertions, both at a single locus ( ex::phyA-1 and ex::phyA-3 ) and with multiple insertions ( ex::phyA-2 ), and one vector control were selected and transferred to a fertilized potting mix for collection of T1 generation seed. Transformed ex::phyA and vector control plants for subsequent experimentation were selected from segregating T1 seedlings by screening for resistance to kanamycin (100 µg/mL) in nutrient agar ( Richardson et al ., 2001a ), and comparisons were made with wild-type plants which had been grown to the same stage in the absence of kanamycin. Verification of the transformation of T1 generation material was performed by Northern blot hybridization, using the Aspergillus phyA or tobacco 18S RNA genes as 32 P-labelled DNA probes ( Figure 1B ). The relative expression of phyA was quantified by comparing the hybridization intensity on sequential Northern blot hybridizations using FujiFilm™ Multigauge Software. The number of dark pixels (PSL) produced by phyA hybridization per 1000 dark pixels produced by the 18S ribosomal DNA hybridization within an equivalent area was compared using three replicate bands, each representing separate RNA preparations for each of the different plant lines ( Figure 1C ). The lowest expressing line was subsequently used in sterile agar containing phytate as the sole source of P to demonstrate an advantage in P nutrition of expressing phyA , based on the assumption that lines with greater expression of phyA will have at least an equal, if not greater, advantage ( George et al ., 2004 ). <h2>Determination of phytase activity in shoots and exudates</h2> The phytase activity of shoots was determined on the same plant material as used for Northern blots, which had been ground in liquid nitrogen and resuspended in five parts of homogenizing buffer [15 m m 2-( N -morpholino)ethanesulphonic acid (MES) pH 5.5, 3 m m ethylenediaminetetraacetic acid (EDTA), 5 m m cysteine]. The extracts was centrifuged (15 000 g , 15 min) and the supernatant was taken to represent soluble cell contents. Total protein was assayed by reaction with Bradford reagent ( Bradford, 1976 ). Extracellular phytase activities were determined on plants grown in 20 mL of sterile nutrient solution (4 m m KNO 3 , 4 m m Ca(NO 3 ) 2 ·4H 2 O, 1.5 m m MgSO 4 ·7H 2 O, 3 m m NH 4 Cl, 0.1 m m Fe-EDTA and micronutrients) containing 50 µ m P (Na 2 HPO 4 ) and 10 g/L sucrose. Plants were initially grown for 14 days, with constant swirling and light (∼200 µE/m 2 ) in a growth room at 21 °C, and were then transferred to a no-P, sucrose-free equivalent nutrient solution under the same conditions for a further 14 days. Phytase activities were determined in the root bathing solutions for four replicate samples. The dry weights of shoots and roots were determined from oven-dried (65 °C) material. Bathing solution volumes were weighed to account for transpirational losses and stored at 4 °C prior to enzyme assays. Phytase activities in both soluble shoot contents and exudates were determined using myo -inositol hexa kis phosphate (InsP6; Sigma-Aldrich Corp., St. Louis, MC), as described by Richardson et al . (2000 ). Briefly, aliquots of extracts or root exudates were made up to 300 µL with buffer (15 m m MES, 1 m m EDTA, pH 5.5) containing InsP6 (2 m m ) and incubated at 37 °C for 60 min. Reactions were terminated with equal volumes of 10% trichloroacetic acid (TCA) at either time zero or at the end of incubation. The concentrations of phosphate released during the assay were measured spectrophotometrically at 620 nm after reaction with malachite green ( Irving and McLaughlin, 1990 ). Enzyme activities were calculated from the difference in phosphate concentrations in supernatants between time zero and the end of incubation, and expressed as either the activity per milligram of protein (shoot activity) or the activity exuded per root dry weight per day (exudate activity). <h2>Growth and P uptake by transgenic plants grown in agar</h2> The ability of plants to use phytate as a source of P was determined by growth in laboratory media (1.2% Bacto™ agar; Becton Dickinson and Company, Sparks, MD) under sterile conditions. Individual plants were grown on nutrient agar slants supplied with all essential nutrients (4 m m KNO 3 , 4 m m Ca(NO 3 ) 2 ·4H 2 O, 1.5 m m MgSO 4 ·7H 2 O, 3 m m NH 4 Cl, 50 µ m Fe-EDTA, 30 n m H 3 BO 3 , 6 µ m CuSO 4 , 6 µ m MnSO 4 , 0.6 µ m ZnSO 4 , 42 n m NH 4 Mo 7 , 12 µ m Co 4 (NO 3 ) 2 ), except P, which was supplied either as InsP6 (phytate) at a concentration of 0.8 m m with respect to P or as orthophosphate supplied as Na 2 HPO 4 (0.8 m m P); otherwise, plants were grown in the absence of P ( Hayes et al ., 2000b ; Richardson et al ., 2001b ). Fourteen replicates of transgenic line ex::phyA-3 and a vector control line were selected on kanamycin, transplanted to the agar slants and grown for 28 days in a growth cabinet at 20 °C with a light intensity of 375 µE/m 2 and a 12 h light period. Shoots were harvested and oven dried (65 °C) for the measurement of dry weight. Shoot P content was determined by ashing (550 °C) the entire shoot and dissolving the ash in 100 parts (w/v) 1.8 N H 2 SO 4 . Total P was measured by reaction with malachite green ( Irving and McLaughlin, 1990 ). <h2>Soil characterization and amendments</h2> Two acidic topsoils (0–10 cm depth) from permanent pastures, which were low in available P, were used. Soil collected from the CSIRO experiment station in Hall, ACT, Australia (Hall soil) was an Alfisol (USDA Soil Taxonomy) with a moderate P sorption capacity and the following soil P characteristics: Colwell P, 10.4 µg P/g; total P, 316.5 µg P/g; total organic P, 67%. Soil collected from Tilba, NSW, Australia (Tilba soil) was a Spodosol (USDA Soil Taxonomy), had low P sorption capacity and the following soil P characteristics: Colwell P, 9.45 µg P/g; total P, 60.0 µg P/g; total organic P, 93%. An additional topsoil from the Hall site, which had received regular P fertilization in the field over 10 years as part of a long-term fertilizer trial, was also collected (field-fertilized Hall soil). Soils were air-dried, mixed and passed through a 2 mm sieve, and were either left unamended or amended with various combinations of lime or P, supplied as either KH 2 PO 4 or Ca phytate (Koch-Light Laboratories Ltd, Colnbrook, UK). Additions of phosphate or P as phytate were based on the amount of orthophosphate required to increase plant-available P (resin-extractable P) by ∼10-fold ( Table 1 ). As a result of the greater P sorption capacity of Hall soil, P addition was 200 mg P/kg soil (918.7 mg Ca phytate/kg soil or 878.9 mg KH 2 PO 4 /kg soil), whereas, in Tilba soil, 60 mg P/kg soil (275.6 mg Ca phytate/kg soil or 263.7 mg KH 2 PO 4 /kg soil) was added. Where required, soils were limed to increase soil pH to between 6.5 and 7.0, at a rate equivalent to 5 tonnes of lime per hectare (3.3 g CaCO 3 /kg soil). Soils were then brought up to 80% field capacity (0.18 or 0.22 mL H 2 O/g soil for Hall or Tilba soil, respectively) and incubated for 28 days prior to analysis and plant growth. Incubated soils were analysed for pH (1 : 5 w/v deionized H 2 O), anion exchange resin-extractable P (resin-P or plant-available P) ( Saggar et al ., 1990 ) and 0.1 m NaOH-extractable inorganic and organic P (NaOH-Pi or NaOH-Po) ( Tiessen and Moir, 1993 ). The anion exchange resin method of Saggar et al . (1990 ) was modified in that resins were charged with Na 2 HCO 3 and eluted with HCl. The component of the NaOH extracts that was amenable to enzyme hydrolysis (NaOH-Pphos) was also determined by incubation with excess activity of a commercially available crude phytase preparation from Aspergillus sp. (Sigma Phytase; Sigma-Aldrich Ltd, St. Louis, MO). This preparation of phytase has been shown to be active against InsP6 and also other monoester and diester forms of organic P ( Hayes et al ., 2000a ). For assays, 100 µL of NaOH extract was made up to 300 µL with buffer (15 m m MES, 1 m m EDTA, pH 5.5) and phytase at an activity of 0.5 nKat/mL, followed by incubation at 37 °C for 6 h to allow the reaction to run to completion. Reactions were terminated with equal volumes of 10% TCA at either time zero or at the end of incubation. The amounts of phosphate released during the assay were measured and expressed on a per soil basis as NaOH-Pphos. <h2>Growth and P uptake by transgenic plants in soil</h2> Five replicate pots containing 475 g (weight at 80% field capacity) of each soil treatment were sown with five tobacco seedlings for each plant line. Prior to planting, transgenic plants were germinated and grown for 14 days on agar containing 100 µg kanamycin/L. Wild-type plants were grown to the same stage on nutrient agar in the absence of kanamycin. Pots were thinned to three plants per pot after establishment, and maintained by weight at ∼80% field capacity during growth. All nutrients except P were supplied weekly by the addition of 5 mL of nutrient solution [3 m m NH 4 SO 4 , 2 m m KNO 3 , 1 m m MgSO 4 , 10 m m Ca(NO 3 ) 2 , 80 µ m Fe-EDTA and micronutrients (B, Cu, Mn, Zn, Mo and Co)]. Plants were grown in a randomized design in a glasshouse at between 14 and 22 °C with an approximate daylight length of 16 h. Shoots were harvested after 25 days of growth and the biomass was determined after oven drying at 65 °C. Shoot materials were milled and analysed for total P content after digestion with a sulphuric acid–hydrogen peroxide mix ( Heffernan, 1985 ). Preliminary soil growth studies using field-fertilized and unamended Hall soil ( Table 2 ) were performed in the same way as described above, except that only one transgenic line ( ex::phyA-3 ) was grown and plants were grown for 45 days. All environmental parameters were the same, except for a shorter day length of approximately 12 h. <h2>Data calculation and statistical analyses</h2> All data are presented as the mean of between three and 14 replicates and error bars represent one standard error of the mean. Significant differences were established using general analysis of variance ( anova ) and treatment means were compared by least significant difference (LSD) ( P = 0.05) (Genstat v5; Rothamsted Experiment Station, Hertfordshire, UK). All data were tested for normality prior to analysis and, where required, skewed data were transformed to natural log values (resin-P and NaOH-Pphos). All data are presented as measured except for the following: (i) exuded phytase activity (nKat/g root dry weight/day) was calculated by converting from activity per 14 days to activity per day, which assumes that the exudation rate is constant over the time period; and (ii) P accumulated in shoots was calculated by multiplying the shoot P concentration (µg P/mg) by the biomass (mg). For plants grown in soil ( Figure 4 ), data for shoot P accumulation are presented as they provide a more sensitive indicator than the shoot dry weight of the plant's ability to acquire P from the soils. Although the P content and shoot growth are generally correlated under P-deficient conditions, shoot growth does not respond when plants are near or above their critical P requirement (e.g. Figures 2 and 4 ; phosphate- and lime-amended Tilba soil).

Journal

Plant Biotechnology Journal – Wiley

Published: Jan 1, 2005

Keywords: ; ; ; ; ;

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