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Keywords:

  • Triticum aestivum;
  • acid phosphatase;
  • glucose 1-phosphate;
  • inositol hexaphosphate;
  • phosphate;
  • phosphomonoesterase;
  • phosphorus;
  • phytase;
  • soil organic P;
  • wheat

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Wheat seedlings exhibited a differential ability to utilize P from a range of organic P substrates when grown in agar culture under sterile conditions. Plants showed limited ability to obtain P from inositol hexaphosphate (IHP), whereas other monoester substrates such as glucose 1-phosphate (G1P), were equivalent sources of P for plant growth as compared with inorganic phosphate (Pi). Poor utilization of IHP was exemplified by significantly lower rates of dry matter accumulation and reduced P content of tissues, which were generally not significantly different to control plants that were grown in the absence of added P. The inability of wheat seedlings to obtain P from IHP was not associated with poor substrate availability but was due to either insufficient root phytase activity or inappropriate localization of phytase within root tissues. Phytase activities of 4 and 24 mU g1 root fresh weight (FW) were determined for crude root extracts prepared from plants that were grown with either adequate P or under deficient conditions, respectively. Similar levels of phytase activity (approximately 12 mU g1 FW) were observed in assays using intact roots, although no secreted activity was detected. By comparison, a secreted acid phosphomonoesterase activity was observed, and activities of between 466 and 1029 mU phosphomonoesterase g1 root FW were measured for intact roots. On the basis of the differences in enzyme activity, and the observed differences in the ability of wheat seedlings to utilize G1P and IHP, it is evident that low intrinsic levels of phytase activity in wheat roots is a critical factor that limits the ability of wheat to obtain P from phytate when supplied in agar under non-limiting conditions. This hypothesis was further supported by the observation that the ability of wheat to obtain P from IHP was significantly improved when the seedlings were inoculated with a soil bacterium (Pseudomonas sp. strain CCAR59) that possesses phytase activity.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Plants meet their phosphorus (P) requirement by the uptake of phosphate anions from the soil solution. To be available to plants, organic forms of soil P (Po) must first be mineralized to release phosphate, a process which is mediated by phosphatase enzymes ( Bieleski & Ferguson 1983). Some 50–80% of the total P in soil occurs in organic forms ( Dalal 1977; Harrison 1987) and it is generally assumed that Po is an important potential source of P for plant nutrition. A range of Po compounds occur in soil, with inositol penta- and hexa-phosphates (i.e., soil phytate) constituting a major component ( Dalal 1977). Typically, around 25% of soil Po may occur as phytate ( Anderson 1980). However, despite its abundance in most soils, the contribution of phytate to the P nutrition of plants remains poorly understood. Previous studies, using a wide range of growth media, have indicated that phytate-P is either completely available ( Adams & Pate 1992) or of limited availability to plants ( Findenegg & Nelemans 1993; Hayes, Simpson & Richardson 2000). Poor availability of phytate in soils is a major limitation to its utilization by plant roots ( Martin 1973; Adams & Pate 1992). However, it has also been suggested that the ability of plants to use P from phytate in low P-fixing growth media may be limited by the rate of hydrolytic cleavage ( Findenegg & Nelemans 1993).

Phosphatases with various substrate specificities (e.g. phosphomono- and phosphodi-esterases) and a range of pH optima have been characterized in plant roots ( Tadano & Sakai 1991; Bosse & Kock 1998). More recently, phytases (i.e., phosphomonoesterases with high specific activity against phytate) have also been described in roots ( Hübel & Beck 1996; Li et al. 1997a , b; Asmar 1997; Hayes, Richardson & Simpson 1999). Observations of enhanced activities of root phosphatases and phytases in response to P deficiency, and higher levels of activity within the rhizosphere relative to the bulk soil, are perceived as evidence for the role of these enzymes in the P nutrition of plants.

Acid phosphomonoesterase activity has been shown to increase under P-deficiency in intact roots of wheat (Triticum aestivum L.) and in the rhizosphere of wheat seedlings grown under sterile and non-sterile conditions ( Ridge & Rovira 1971; McLachlan 1980). By contrast, phytase activity in wheat roots has not previously been reported and it is presently unclear whether root phytases are involved in the P utilization of plants supplied with phytate. A phytase has been purified and characterized from wheat bran ( Nagai & Funahashi 1962) and it has been shown that the enzyme is involved in the mobilization of phosphate from seed reserves of phytate during germination ( Barrier-Guillot et al. 1996 ). In this paper we characterize the acid phosphomonoesterase and phytase activities of wheat roots and investigate the utilization of a range of Po compounds, including inositol hexaphosphate, by wheat seedlings grown under sterile conditions. The objective of this work was to establish the role of root phytases in the P nutrition of wheat plants supplied with phytate.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Plant growth conditions

Plant material

Seeds of Triticum aestivum L. (cv. Genaro) were surface-sterilized by immersion in 95% ethanol for 1 min, then 4·0% sodium hypochlorite for 30 min, followed by 10 rinses in sterile, deionized water. Seeds were then transferred to yeast–mannitol medium ( Vincent 1970), containing 1·5% agar, and germinated overnight at 20 °C prior to transfer to agar slants. Germination plates (containing unused seeds) were incubated for a further 2–3 d to confirm seed sterility.

Sterile agar culture

Wheat plants were grown on agar slants in foil-capped tubes (150 mm × 25 mm diam.) sealed with a rubber ring, such that the roots were maintained under sterile conditions ( Fig. 1). Each tube contained 40 mL of agar medium (1·5% agar w/v, Difco Bacto; Difco Laboratories, Detroit, MI, USA) with macronutrients supplied as KNO3 (4 m M), Ca(NO3)2·4H2O (4 m M), NH4Cl (3 m M), MgSO4·7H2O (1·5 m M), Fe-Na EDTA (0·1 m M), and micronutrients as MnCl2·4H2O (46 μM), H3BO3 (23 μM), ZnCl2 (15 μM), CuSO4·5H2O (1·6 μM), Na2MoO4·2H2O (1·0 μM), and CoCl2·6H2O (1·0 μM). The media (pH 5·5) contained either no added P (NoP), P as Na2HPO4 (Pi) or P supplied as one of the following Po substrates: myo-inositol hexaphosphoric acid dodecasodium salt (IHP), D(–)3-phosphoglyceric acid tri-sodium salt (PGA), adenosine 3′:5′-cyclic monophosphate sodium salt (AMP), ribonucleic acid Type VI (RNA; all obtained from Sigma Chemical Co., St. Louis, MO, USA); α- D-glucose 1-phosphate disodium salt (G1P; Calbiochem – Novabiochem Corp., La Jolla, CA, USA); or adenosine-5′-triphosphate disodium salt (ATP; Boehringer Mannheim, Germany). Stock solutions of P (100 × the final concentration) were adjusted to pH 5·5 and sterilized by membrane filtration (Millex-HA, 0·45 μm, Millipore Corp., Bedford, MA, USA); prior to their addition to bulk solutions of autoclaved media which had been cooled to approximately 42 °C. The media were dispensed aseptically into individual sterile culture tubes, which were then sealed and solidified at an angle of approximately 10° to horizontal.

image

Figure 1. Cultural set-up for the growth of wheat seedlings on agar slants under sterile conditions.

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Three growth experiments were conducted: (I) plants were supplied with P at an equivalence of 1·0 m M (i.e., 1239 μg P seedling−1) as Pi, IHP, AMP, PGA, RNA, ATP or G1P; (II) plants were supplied with P at an equivalence of 0·8 m M P (991 μg P seedling−1) as Pi, IHP or G1P and root phosphomonoesterase and phytase activities were measured; and (III) plants were supplied with P at an equivalence of either 0·8 m M P (as either Pi or IHP) or at 4·8 m M P as IHP (i.e., 0·13 or 0·80 m M IHP, respectively), and were, or were not, inoculated with Pseudomonas sp. strain CCAR59, a soil bacterium isolated for its marked ability to dephosphorylate IHP ( Richardson & Hadobas 1997). Strain CCAR59 was grown overnight in 100 mL of half-strength tryptic soy broth (Difco Laboratories), centrifuged (8000 g, 10 min) and resuspended, then washed twice in the basal nutrient solution. The prepared bacterial inoculum was added to the cooled agar medium (approximately 42 °C) to provide approximately 107 bacteria per plant. For all three experiments, control treatments of plants that received no P were also included.

Plant growth

A germinated wheat seed was transferred aseptically to each agar slant by carefully inserting the radicle through a small puncture in the foil cap. Seedlings were maintained under high humidity for 48 h prior to being transferred to a naturally-lit glasshouse (maximum photon irradiance of 1400 μmol m−2 s−1 and temperatures of 25 and 19 °C for day and night periods, respectively). Brown paper sleeves were placed around each tube to minimize the penetration of light to the roots ( Fig. 1). Ten (experiments I and III) or 20 (experiment II) replicates arranged in a completely randomized design were established for each P treatment and any tubes which became visibly contaminated with either bacteria or fungi were discarded.

Plant harvest

For experiments I and III, shoot material only from at least eight replicates for each P treatment was harvested from plants grown for 19 and 20 d, respectively. Excised shoots were oven-dried in preweighed borosilicate glass tubes at 75 °C for 48 h, for dry weight (DW) determinations. For experiment II, between 12 and 15 replicate plants for each P treatment were carefully removed from the agar after 13 d of growth, rinsed with sterile water and then equilibrated in 15 m M MES (2-[N-Morpholino]ethanesulfonic acid) (pH 5·5), 0·5 m M CaCl2 solution for 1 h. Acid phosphomonoesterase and phytase activities of the roots were then determined on subsets (four to five replicates) of plants as outlined below. Shoot and root fresh weights (FW) were measured for all samples and shoot and root dry weights and total P contents were measured for non-destructed samples.

The total P content of shoots and roots was determined on material ashed in a muffle furnace at 550 °C for 16 h. Ashed samples were dissolved in 0·9 M H2SO4 at approximately 10 mg DW mL−1 acid. Concentrations of P in the extracts were subsequently determined by the molybdate-blue method ( Murphy & Riley 1962).

Availability of P substrates in agar

The relative availabilities of Pi, IHP and G1P in agar were determined by extracting (end-over-end shaker; 30 r.p.m.) 20 mL plugs of agar containing 0·8 m M of the three P substrates with 20 mL of deionized water for 16 h. Extracts were then filtered through Whatman 42 paper. The Pi contents of the extracts were determined by the molybdate-blue method. Total P in the extracts were determined subsequent to autoclaving (121 °C; 120 kPa) 5 mL samples for 1 h in a total volume of 15 mL, containing 0·6 M H2SO4 and 0·5 g ammonium persulphate ( Schoenau & Huang 1991). Following autoclaving, samples were adjusted for volume loss.

Phosphomonoesterase and phytase assays

Enzyme activities were determined on (i) wheat root crude extracts, (ii) intact roots and (iii) in solutions external to the root. Phosphomonoesterase and phytase activities were expressed on either a mU g−1 root FW or a mU mg−1 protein basis, where 1 U is defined as the release of 1 μmol of Pi min−1 under the assay conditions described.

Enzyme activities in root extracts

Root material was ground in the presence of fine sand with a mortar and pestle at 4 °C, with 5 volumes (w/v) of 15 m M MES (pH 5·5), 0·5 m M CaCl2, 1 m M EDTA buffer. The extracts were then centrifuged for 10 min at 12 000 g (4 °C) to remove insoluble components. Enzyme assays were conducted by incubating 50 μL or 250 μL of extract (for phosphomonoesterase and phytase, respectively) in a total volume of 500 μL of 15 m M MES (pH 5·5), 0·5 m M CaCl2 buffer at 27 °C in the presence of either 10 m Mp-nitrophenyl phosphate (pNPP; phosphomonoesterase assays) or 2 m M IHP (filter sterilized, pH 5·5; phytase assays) ( Hayes et al. 1999 ). Phosphomonoesterase and phytase assays were conducted over 30 and 60 min, and reactions were terminated by the addition of equal volumes of 0·25 M NaOH and 10% TCA (trichloroacetic acid), respectively. Phosphomonoesterase activity was calculated from the release of p-nitrophenol (pNP), as determined spectrophotometrically at 412 nm relative to standard solutions. Phytase activity was calculated from the release of Pi in samples that were centrifuged (12 000 g, 10 min) and were determined using the molybdate-blue assay. The protein content of root extracts was measured using bovine serum albumin standards, according to Bradford (1976).

Enzyme activities of intact roots

Enzyme activities of intact roots were determined by incubating the roots of whole plants for 30 or 60 min (for phosphomonoesterase and phytase, respectively) at 27 °C in 4 mL of MES/Ca buffer, containing either 10 m MpNPP or 2 m M IHP. Following the incubation period, root material was rinsed in distilled water and washed in 0·9% NaCl for 30 min, prior to being dried and weighed. For phytase activity, a set of control plants were also incubated in the absence of IHP to account for P efflux from roots during the assay period. However, correction was made to account for possible P-uptake ( Asmar 1997).

Enzyme activities in external root solutions

The activities of phosphomonoesterase and phytase that were secreted into external-root solutions were determined for plants incubated for 4 h in 4 mL of MES/Ca buffer. The plants were removed prior to the addition of either pNPP or IHP substrate and the enzyme assays were conducted using 450 μL of the root incubation solutions.

Statistical analyses

The data were analysed by one-way analyses of variance and, where F ratios were significant (P < 0·05), treatment means were compared using either Fisher’s protected least significant difference (lsd) or unpaired t-tests (P = 0·05). Percentage data were arcsin-transformed prior to analysis. Coefficients of determination (r2) were derived from linear regression analyses.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Growth and P content of wheat plants supplied with organic P substrates (experiment I)

The dry weight and P content of wheat shoots supplied with a range of Po sources and grown under sterile conditions is shown in Table 1. With the exception of plants supplied with IHP, shoot dry weights were not significantly different (P < 0·05) for any of the P treatments relative to the Pi-fed controls. In contrast, the mean shoot dry weight of plants supplied with IHP was only 59% of that for the Pi controls and was the same as plants that were grown with no added P ( Table 1).

Table 1.  Dry weight and phosphorus accumulation of shoots from wheat seedlings grown for 19 d in sterile agar and supplied with various sources of P
Phosphorus sourceShoot dry weight (mg plant−1) P content (μg shoot−1) P concentration (% dry weight)
  1. For each phosphorus source, P was supplied at a rate equivalent to a Pi concentration of 1·0 m M. Within each column, values followed by a different letter are significantly different (P < 0·05).

Na2HPO4 (Pi) 68·0ab371·0ab0·55ab
adenosine 3′:5′-cyclic monophosphate (AMP) 59·1b224·7c0·38c
ribonucleic acid (RNA)70·3ab238·2c0·33c
α- D-glucose 1-phosphate (G1P) 68·1ab380·6ab0·57a
D(−)3-phosphoglyceric acid (PGA)67·1ab337·5b0·51b
adenosine-5′-triphosphate (ATP)73·1a406·7a0·56ab
myo-inositol hexaphosphoric acid (IHP) 40·6c 74·4d0·18d
no added P (NoP)34·9c 27·2e0·08e

The P content and concentration in shoots of plants supplied with P as the phosphate monoesters G1P, PGA and ATP were not significantly different (P < 0·05) to plants supplied with Pi ( Table 1). By contrast, lower shoot P was observed for plants that received P as either of the phosphate diesters RNA or AMP (P concentrations 0·33 and 0·38%, respectively) or as IHP (containing monoester phosphate linkages). Plants supplied with IHP were clearly P-deficient and had a mean shoot P concentration that was similar to that for plants that received no P ( Table 1). Shoots of plants supplied with IHP contained only 20% of the P in the Pi-fed controls, whereas the P content of shoots supplied with other P substrates ranged between 61 and 64% (diester phosphates) and 91 and 109% (monoester phosphates) relative to the Pi-fed control seedlings ( Table 1).

Utilization of organic P substrates and acid phosphomonoesterase and phytase activities of roots (experiment II)

Availability of P substrates in agar

The relative availabilities of Pi, G1P and IHP in agar were investigated by measuring P concentrations in water extracts prepared from agar plugs. No inorganic P was detected in the organic P treatments, whereas a 98% recovery of the expected phosphate concentration occurred in the Pi treatment ( Table 2). By contrast, the recovery of total P from the extracts containing organic P sources was 68 and 93% for IHP and G1P, respectively. Similarly, for the treated extracts the recovery of phosphate in the Pi treatment was 92% ( Table 2).

Table 2.  Phosphorus content of extracts prepared from agar media used for plant-growth analysis in experiment II
 Phosphate PTotal P
Treatment(μg P ml−1 extract)(% recovery)1(μg P ml−1 extract)(% P recovery)1
  1. Each value is the mean of three replicates and is shown ± the standard error.

  2. IHP, myo-inositol hexaphosphoric acid; G1P, α- D-glucose 1-phosphate.

  3. 1Relative to phosphate recovered from samples of each treatment prepared in distilled water only. Agar samples were extracted with an equal volume of water and, for P treatments, the initial P content of the agar was 24·8 μg P ml−1.

NoP 0·21 ± 0·01 0·62 ± 0·42
Pi13·37 ± 0·2798·2 ± 1·613·71 ± 0·4092·1 ± 3·2
IHP 0·17 ± 0·01 0·0 ± 0·0 8·31 ± 0·3667·8 ± 4·0
G1P 0·22 ± 0·00 0·1 ± 0·011·44 ± 0·6493·3 ± 4·5
Plant growth and P uptake

The growth and P uptake by wheat seedlings supplied with Pi, G1P or IHP, or grown with no added P, is shown in Fig. 2. Consistent with the results from experiment I ( Table 1), highest plant dry weights were observed in the Pi and G1P treatments (63·7 and 69·8 mg plant−1, respectively), which were significantly greater (P < 0·05) than for plants supplied with either IHP or grown in the absence of P (46·9 and 44·0 mg plant−1, respectively; Fig. 2a). These differences in dry matter accumulation were associated with the growth of shoots only. No differences were observed for root dry weights across the various P treatments. As a consequence, lower shoot-to-root ratios (1·5 to 1·6) were observed for the plants grown with IHP or no added P, compared to plants supplied with Pi or G1P (ratios of 2·5).

image

Figure 2. Dry weight (a) and total P content (b) of shoots (upper panels) and roots (lower panels) for wheat seedlings grown for 13 d in sterile agar supplied with P as either Na2HPO4 (Pi), α- D-glucose 1-phosphate (G1P), or myo-inositol hexaphosphoric acid (IHP), or grown in the absence of added P (NoP). For the phosphorus treatments, P was supplied at a rate equivalent to a Pi concentration of 0·8 m M. The values in parentheses show the shoot-to-root ratios. For each panel, and for shoots and roots separately, columns designated with a different letter are significantly different (P < 0·05).

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The mean total P content of plants grown with Pi was 596·3 μg P plant−1 ( Fig. 2b), of which approximately 80 μg of P was estimated to be derived from seed reserves. Thus, under the growth conditions used, plants used 52% of the total amount of P supplied as Pi. Similarly, for seedlings supplied with G1P, total P content was 477·3 μg P plant−1, and although this was significantly less (P < 0·05) than the Pi-fed plants, the seedlings still obtained approximately 40% of the total P supplied in the agar, which allowed for maximum dry weight accumulation ( Fig. 2). The P content of seedlings grown in the absence of added P was 89·5 μg P plant−1. Plants supplied with IHP were similarly P deficient (116 μg P plant−1) and accessed less than 4% of the P from the agar. Plants grown with IHP and without added P were P deficient, as indicated by shoot P concentrations of 0·25 and 0·21%, compared with 0·69 and 0·93% in the GIP and Pi treatments, respectively. Under P deficiency, the proportion of total P retained in the roots was higher relative to shoots, as indicated by shoot-to-root ratios for P distribution of between 2·1 and 2·3, compared to more than 4·0 for P-sufficient plants.

Acid phosphomonoesterase and phytase activities of roots

The activities of phosphomonoesterase and phytase were determined for wheat roots that were supplied with Pi, G1P or IHP, or grown in the absence of P (experiment II; Fig. 2). When intact roots were assayed, acid phosphomonoesterase activity was increased significantly, by ~2-fold, in plants that were P-deficient (i.e., supplied with either no P or IHP; Table 3). By comparison, the phosphomonoesterase activity of plants supplied with G1P was equivalent to that observed for the Pi-fed plants. In contrast to the response observed for intact roots, comparable levels of phosphomonoesterase activity were observed in soluble extracts prepared from ground roots from plants of each of the four P treatments when expressed on a FW basis. However, a significant (P < 0·05) increase was observed for the P-deficient treatments (i.e., NoP and IHP) when activities were determined relative to root protein content ( Table 3). Consistent with these observations, significant negative correlations (P < 0·05) were observed between phosphomonoesterase activity of intact roots and both shoot and root P concentrations (r2 = 0·639 and r2 = 0·675, respectively), whilst the phosphomonoesterase activity of root extracts, expressed on either fresh weight or protein content basis, was poorly correlated with shoot P concentration.

Table 3.  Acid phosphomonoesterase activities of root extracts, intact roots and in external root solutions from 13-day-old wheat seedlings
 Acid phosphomonoesterase activity
 Root extractsIntact rootsExternal-root solutions
Treatment(mU mg−1 protein)(mU g−1 root FW)(mU g−1 root FW)(mU h−1 g−1 root FW)
  1. Within each column, values followed by a different letter are significantly different (P < 0·05).

  2. IHP, myo-inositol hexaphosphoric acid; G1P, α- D-glucose 1-phosphate.

Pi330·5b469·8a 466·7b21·5a
G1P322·8b550·9a 540·8b24·5a
IHP458·8ab485·0a 931·1a31·1a
NoP552·0a509·7a1029·7a46·7a

Phosphomonoesterase activity that was excreted from roots (i.e., in the external solutions in which roots were incubated) were also higher for P-deficient plants, although these differences were not statistically significant ( Table 3). In particular, large coefficients of variation were evident for the NoP and IHP treatments (70 and 72%, respectively, compared to approximately 30% for P-sufficient plants). However, across the various P treatments, the mean phosphomonoesterase activity released per hour (per gram of root FW) appeared to be proportional (3·3 to 4·6%) to the total acid phosphomonoesterase activity of intact roots ( Table 3).

The phytase activity in soluble extracts from wheat roots ( Table 4) was substantially less than acid phosphomonoesterase activity. Unlike phosphomonoesterase activity, phytase activity was increased markedly in response to P deficiency (by up to approximately six- and nine-fold in the IHP and NoP treatments, respectively), when expressed on either a root fresh weight or protein content basis ( Table 4). Significant negative correlations (P < 0·05) between the phytase activity of root extracts and shoot P concentrations (r2 = 0·656 to 0·749) were evident.

Table 4.  Phytase activities of root extracts from 13-day-old wheat seedlings
 Phytase activity of root extracts
Treatment(mU mg−1 protein)(% of acid phosphatase)(mU g−1 root FW)(% of acid phosphatase)
  1. Within each column, values followed by a different letter are significantly different (P < 0·05).

  2. IHP, myo-inositol hexaphosphoric acid; G1P, α- D-glucose 1-phosphate.

Pi 3·0c0·9 4·4c0·9
G1P 3·6c1·1 6·2c1·1
IHP16·7b3·617·6b3·6
NoP26·7a4·823·9a4·7

Notably, phytase activities in root extracts constituted around 1% of the total acid phosphomonoesterase activity for plants that were P-sufficient, but were increased to approximately 4–5% in P-deficient plants ( Table 4). Analogous to the measurements of phosphomonoesterase activity, attempts were also made to measure the phytase activity of intact roots. For plants grown in the absence of added P (NoP treatment), a phytase activity of 12·1 ± 4·0 mU g−1 root FW was determined, which represents approximately 50% of the activity that was observed in root extracts ( Table 4). Comparable measurements of phytase activity for intact roots from Pi-fed plants could not be determined, as these estimates were clearly confounded by P efflux from the roots during the assay period, for which appropriate corrections could not be made. By contrast, efflux of P from plants in the NoP treatment was negligible (data not shown).

No phytase activity (i.e., < 0·3 mU g−1 FW) could be detected as a secreted enzyme in solutions that were collected from the roots of plants grown in any of the P treatments (including Pi-fed and NoP controls).

Utilization of myo-inositol hexaphosphoric acid by wheat plants when inoculated with a bacterium able to hydrolyse phytate (experiment III)

The growth of wheat seedlings supplied with P as IHP and inoculated with Pseudomonas sp. strain CCAR59 is shown in Fig. 3. Under sterile conditions, the mean shoot dry weights of plants that received IHP, at either of two levels (0·8 or 4·8 m M with respect to P concentration), were significantly lower (P < 0·05) than Pi-fed plants and were only marginally greater than plants grown without added P ( Fig. 3a). Poor utilization of P from IHP was also indicated by shoot P contents of 97·9 and 145·0 μg (0·8 and 4·8 m M IHP-P, P treatments, respectively) as compared to 502·6 and 28·4 μg P for the Pi-fed and NoP control treatments, respectively. The concentration of P in shoots was increased when IHP was supplied at the higher level, but remained significantly lower (P < 0·05) than shoot P concentrations observed for plants grown in the presence of Pi ( Fig. 3b).

image

Figure 3. Effect of bacterial inoculation on (a) dry weight accumulation and (b) P concentration of shoots from wheat seedlings grown for 20 d in agar culture. Plants were grown either under sterile conditions [solid bars (–)] or were inoculated with Pseudomonas sp. strain CCAR59 [hatched bars (+)]. Plants were supplied with P as either Na2HPO4 (Pi), or myo-inositol hexaphosphoric acid (IHP), or were grown in the absence of added P (NoP). For the IHP treatments, P was supplied at a rate equivalent to a Pi concentration of either 0·8 m M or 4·8 m M. For each panel, columns designated with a different letter are significantly different (P < 0·05).

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The growth of wheat seedlings supplied with IHP was improved considerably when the seedlings were inoculated with strain CCAR59 ( Fig. 3). Under inoculated conditions, the shoot dry weights of plants supplied with IHP were equivalent to those observed for Pi-fed plants ( Fig. 3a). Similarly, bacterial inoculation resulted in tissue P contents that were between 3·3- and 3·6-fold higher (354·5 to 483·7 μg P shoot−1) than those observed for the corresponding IHP treatments grown under sterile conditions. As a consequence, the shoot tissue P concentrations of plants that were both inoculated and supplied with IHP were either equivalent to, or where the IHP was supplied at the higher concentration, were significantly higher (P < 0·05) than, the P concentrations of plants that received phosphate (Pi-fed; Fig. 3b). By comparison, inoculation of control plants grown with phosphate or in the absence of added P (NoP treatment) did not result in increased shoot dry weight, shoot P content or tissue P concentration ( Fig. 3).

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

To be available to plants, phosphate from Po substrates must first be hydrolysed by phosphatases. The observations from the present work, where seedlings were grown in agar under sterile conditions, indicate that with the exception of IHP, wheat roots possessed sufficient phosphomonoesterase activity to meet plant P requirements from a range of monoester P substrates. When supplied at an equivalent rate of P (i.e., 1·0 m M), G1P, PGA and ATP were equal sources of P to Pi in terms of both dry weight accumulation and plant P uptake. By contrast, plants were less able to obtain P from AMP and RNA (diester P substrates), but still achieved maximum shoot dry weight.

For intact wheat roots, the activity of acid phosphomonoesterase (measured using pNPP) was around 500 mU g−1 root FW for plants that were P-sufficient, and increased approximately two-fold under P deficiency. Assuming an average root fresh weight of approximately 100 mg for seedlings during 13 d of growth ( Fig. 2), these levels of enzyme activity would be sufficient to liberate 2–4 mg Pi day−1 from a non-limiting supply of monoester P, against which the measured phosphomonoesterase was assumed to have a similar specific activity. By comparison, the total P uptake by the plants during 13 d of growth was only approximately 0·5 mg when supplied with Pi ( Fig. 2). The potential for liberation of P from organic sources by root phosphomonoesterases is therefore well in excess of the plants’ requirement for P. This suggestion is consistent with observations in the present study of the ability of plants to use organic P substrates such as G1P ( Fig. 2), and with reports published elsewhere ( Barrett-Lennard, Dracup & Greenway 1993; Hayes et al. 2000 ).

By contrast, wheat seedlings had only limited ability to obtain P from IHP. The growth and P uptake by plants supplied with IHP was significantly reduced and was comparable to plants grown in the absence of added P. Despite this, phytase activity was detectable both in crude extracts prepared from ground roots and for intact roots. Phytases are a class of phosphomonoesterase that show a high specific activity against phytate (derivatives of IHP), usually in addition to activity against other Po substrates ( Nagai & Funahashi 1962; Konietzny, Greiner & Jany 1995). For wheat plants that were grown with sufficient P (i.e., Pi or G1P), the phytase activity of the extracts represented approximately 1% of the total acid phosphomonoesterase activity. However, and in contrast to total phosphomonoesterase activity, phytase activity in root extracts increased six-fold in plants that were P-deficient, and accounted for 4–5% of the total phosphomonoesterase activity in these plants. For intact roots, the phytase activity of P-deficient plants accounted for approximately 1% of the total phosphomonoesterase activity (approximately 12 mU phytase compared to 1030 mU total acid phosphomonoesterase g−1 root FW). However, the estimate of the phytase activity for intact roots may be an underestimate of the actual activity, as no allowance was made for possible P uptake by the plant during the 60 min assay period. Phytase activity was not detected in solutions external to the root, which is in contrast to observations for total acid phosphomonoesterase, where approximately 5% was secreted from the roots over a 4 h period. Assuming that a comparable proportion of phytase was released, activities of around 0·6 mU g−1 root FW would be expected, which is only marginally above the sensitivity of the assays described (i.e., approximately 0·3 mU g−1 root FW).

Although it was evident that wheat roots did exhibit phytase activity, it was clear that seedlings grown with IHP remained P-deficient. It was shown that the inability of plants to utilize phytate was not associated with poor substrate availability, as a recovery of IHP of at least 68% was obtained from the agar and this was not substantially different to that observed for G1P or Pi (approximately 90% recovery). Rather, the utilization of P from IHP by wheat roots appeared to be limited by root phytase activity. Wheat seedlings either possessed insufficient levels of phytase activity, or had phytase with an affinity for IHP that was too low for efficient hydrolysis of the substrate when supplied at a concentration of approximately 0·15 m M. Nagai & Funahashi (1962) have measured a Km (Michaelis-Menten constant) of 0·57 m M IHP for purified wheat bran phytase. The ineffectiveness of endogenous wheat root phytases was further highlighted by the observation that inoculation of seedlings with Pseudomonas sp. strain CCAR59 significantly enhanced the availability of IHP, such that the growth and P content of these plants was equivalent to that observed for the Pi-fed controls ( Fig. 3). The effectiveness of supplementing wheat plants with bacterial phytase may have been due to either increased levels of phytase in the growth medium, or to the introduction of an enzyme with a higher affinity for IHP, or to a combination of both effects. The phytase characterized from a Pseudomonas sp. by Irving & Cosgrove (1971) was shown to have a Km for phytate of 16·3 μM, and is low in comparison to reported Km values for a range of plant phytases (e.g., Nagai & Funahashi 1962; Konietzny et al. 1995 ; Hayes et al. 1999 ).

Furthermore, it was evident from the present experiments that phytase was not secreted from the roots and it is therefore apparent that the phytase activity in wheat roots is located in tissues that are inappropriate for the utilization of phytate from external sources. Using a cytochemical technique, Hübel & Beck (1996) showed that phytase in maize roots is predominantly located in the root endodermis, whereas non-specific acid phosphatases are evident throughout the entire root, including the root cortex and in epidermal layers. Similarly, using in situ hybridizations and immunolocalizations, Maugenest et al. (1999) have shown that phytase mRNA and phytase protein are specifically located within the endodermis and pericycle of maize, although they were also detected in the rhizodermis. On the basis of these observations it has been suggested that the phytases in maize roots are involved predominantly in the internal remobilization of phytates, which may act as important transient storage compounds for P. In contrast, Li et al. (1997b) have measured secreted phytase activity for roots from a wide range of plant species, indicating that phytases may be important for the utilization of phytate external to the roots by some species. However, the measured levels of secreted phytase activity (obtained by a collection procedure using dialysis tubing) were comparable to total acid phosphatase activity determined by the same procedure. This is not consistent with the results of the present study for wheat seedlings, where the phytase activity of both crude root extracts and intact roots was very low compared to total acid phosphomonoesterase activity.

In the present experiments we also showed that the utilization of P derived from IHP by wheat seedlings in sterile agar was significantly enhanced when a soil bacterium with phytase activity was introduced. In soil environments, plants may similarly be reliant on rhizosphere microorganisms to increase nutrient availability ( Richardson 1994; Tarafdar & Marschner 1995). For example, Tarafdar & Jungk (1987) showed that for wheat plants grown in soil, a significant zone of Po depletion occurred around the root and that this region coincided with higher levels of phosphatase activity and substantially larger populations of both bacteria and fungi ( Tarafdar & Jungk 1987). However, it has also been shown that the accessibility of phytate to enzyme hydrolysis in soil environments is limited by poor substrate availability and that this is a major factor that limits the ability of plants to utilize P from phytate ( Adams & Pate 1992; Martin 1973; Tarafdar & Claassen 1988). The results of the present study using wheat, together with observations reported elsewhere for various plant species ( Barrett-Lennard et al. 1993 ; Hayes et al. 2000 ; Findenegg & Nelemans 1993), indicate that low intrinsic levels of phytase activity in plant roots is also an important factor that limits the ability of plants to obtain P from phytate.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Support for this project was provided by Australian woolgrowers and the Australian Government through the Australian Wool Research and Promotion Organization (The Woolmark Company).

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
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