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Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment0140-7791Blackwell Publishing Ltd 2005? 2005 29?303313 Original Article Differential synthesis of acid phosphatase isozymes G. G. Bozzo et al.

Plant, Cell and Environment (2006) 29, 303–313

Differential synthesis of phosphate-starvation inducible purple acid phosphatase isozymes in tomato (Lycopersicon esculentum) suspension cells and seedlings GALE G. BOZZO1, EVELYN L. DUNN2 & WILLIAM C. PLAXTON1,2 1

Departments of Biology and 2Biochemistry, Queen’s University, Kingston, Ontario K7L 3N6, Canada

ABSTRACT This study compares the influence of phosphate (Pi) deprivation on the coordinate synthesis of the principle Pistarvation inducible (PSI) acid phosphatase (AP) isozymes in a suspension cell culture with the homologous in planta system. Tomato suspension cells express three PSI purple AP isozymes: a heterodimeric intracellular AP (IAP) composed of 63 and 57 kDa subunits, and two monomeric secreted APs (SAPs) (84 kDa SAP1 and 57 kDa SAP2) localized in the culture media. Immunoblots probed with rabbit antibodies raised against purified SAP1 or IAP indicated the immunological distinctiveness of SAP1 relative to IAP and SAP2. Time-course studies of cells and seedlings undergoing a transition from Pi sufficiency to Pi deficiency revealed a close relationship between total IAP or SAP activity and relative amounts of antigenic IAP or SAP polypeptides. Upregulation of the pre-existing IAP in 6-day-old Pi-deficient (–Pi) suspension cells coincided with a 20-fold reduction in intracellular free Pi levels, which occurred 2 d prior to initial accumulation of SAP1 and SAP2 in the culture media. Similarly, root-specific SAP synthesis in –Pi seedlings occurred at least 7 d following IAP induction in roots or shoots. Preferential sequestration of limiting Pi to the leaves of the –Pi seedlings was suggested by the delayed induction of leaf versus root IAP. Our results confirm recent transcript profiling studies suggesting that PSI proteins are subject to both temporal and tissue-specific syntheses in- Pi plants. Key-words: biochemical adaptation, nutrient limitation, phosphate starvation, stress acclimation

INTRODUCTION Plants acclimatize to phosphate (Pi) deficiency via the coordinated induction of large numbers of Pi-starvation inducible (PSI) genes in order to reprioritize internal Pi use and to maximize external Pi acquisition (Raghothama 1999; Hammond et al. 2003; Vance, Uhde-Stone & Allan 2003; Wu et al. 2003; Hammond, Broadley & White 2004; Plaxton Correspondence: William C. Plaxton. Fax: +1 613 533 6617; e-mail: [email protected] © 2006 Blackwell Publishing Ltd

2004; Ticconi & Abel 2004). The adaptive value of acclimating at the molecular level to Pi stress was recently estimated to extend the viability of Pi-deficient (–Pi) Brassica napus suspension cells by at least 3 weeks (Singh et al. 2003). The induction of intracellular acid phosphatase (IAP) and secreted acid phosphatase (SAP) (EC 3.1.3.2) isozymes is an important component of the plant Pi stress response. As PSI IAPs and SAPs appear to be ubiquitous in vascular plants, their activities have provided useful biochemical markers of plant Pi deficiency (Duff, Sarath & Plaxton 1994). The likely functions of IAPs are the scavenging and recycling of Pi from expendable intracellular organophosphate pools. This is accompanied by marked reduction in cytoplasmic P metabolites during extended Pi stress (Duff et al. 1989; Lauer, Blevins & Sierzputowska-Gracz 1989; Lee & Ratcliffe 1993; Plaxton 2004). SAPs belong to a group of secreted PSI phosphohydrolases that are induced to mobilize Pi from external organophosphates, known to comprise up to 80% of total soil P (Ticconi & Abel 2004). For example, the combined action of secreted PSI ribonucleases, phosphoesterases and acid phosphatases (APs) allows –Pi tomato cell cultures and seedlings to efficiently scavenge extracellular nucleic acids as their sole source of nutritional Pi (Bosse & Köck 1998; Ticconi & Abel 2004). High-affinity plasmalemma Pi transporters are also induced in response to Pi limitation and facilitate extracellular Pi uptake against a steep (up to 10 000-fold) concentration gradient (Raghothama 1999). The upregulation of AP activity in –Pi plants and yeast has been correlated with de novo AP synthesis in several systems including Brassica nigra and B. napus suspension cells (Duff, Plaxton & Lefebvre 1991; Carswell, Grant & Plaxton 1997), proteoid lupin roots (Miller et al. 2001) and Saccharomyces cerevisiae (Oshima, Ogawa & Harashima 1996). Molecular analysis of the yeast Pi starvation response indicates the synchronized control of genes encoding APs, plasma membrane Pi transporters and Pi sensor protein kinases, termed the pho regulon (Oshima et al. 1996). A similar Pi emergency rescue and sensing system has been proposed for plants and includes the induction of Pi scavenging, recycling, transport and metabolism-related genes (Raghothama 1999; Hammond et al. 2003; Vance et al. 2003; Wu et al. 2003; Hammond et al. 2004; Ticconi & Abel 2004). By contrast, resupply of Pi to –Pi plants leads to the rapid 303

304 G. G. Bozzo et al. repression of PSI genes (Miller et al. 2001; Müller et al. 2004) while simultaneously inducing proteases that appear specifically to target intra- and extracellular PSI proteins (Bozzo, Singh & Plaxton 2004b). Although several recent transcript profiling studies revealed the differential expression of various plant PSI genes in both a temporal and a tissue-specific fashion (Hammond et al. 2003, Wu et al. 2003; Hammond et al. 2004; Zimmermann et al. 2004), we are unaware of any corroborative biochemical evidence demonstrating that PSI proteins are themselves subject to temporal and tissuespecific synthesis in –Pi plants. Thanks to the pioneering work of Alan Goldstein and co-workers (Goldstein, Baertlein & McDaniel 1988), tomato suspension cells have become a useful model system for examining the biochemical and/or molecular responses of vascular plants to Pi limitation (Köck et al. 1998; Raghothama 1999; Ticconi & Abel 2004). These cultures represent a uniform system in which a homogeneous cell population is equally exposed to the environmental conditions prevalent in liquid culture. Three PSI APs that demonstrated distinctive physical and kinetic properties were recently purified and biochemically characterized from –Pi tomato suspension cells (Bozzo, Raghothama & Plaxton 2002, 2004a). Two are secreted monomeric SAPs (84 kDa SAP1 and 57 kDa SAP2), whereas the third is a 142 kDa heterodimeric IAP composed of a 1:1 ratio of 63 and 57 kDa subunits. All three tomato PSI AP isozymes are purple APs (PAPs) as they each demonstrated a violet colour in solution, and N-terminal and/or internal peptide amino acid sequence similarity to portions of that deduced for several putative or previously characterized plant PAPs (Bozzo et al. 2002, 2004a). PAPs represent a specific AP class containing a bimetallic active centre that endows them with a characteristic purple or pink colour in solution. In the Arabidopsis genome, 29 putative PAP genes have been identified, several of which appear to respond to Pi deficiency (Li et al. 2002). In the present study, AP activity assays and immunological studies were employed to gain insight into the coordinate synthesis of the three principal PSI tomato PAP isozymes of tomato cell cultures in both suspension cells and seedlings undergoing a transition from Pi sufficiency to Pi deficiency.

MATERIALS AND METHODS Plant material Heterotrophic tomato (Lycopersicon esculentum cv. Moneymaker) cell suspensions were routinely cultured in 100 mL of Murashige–Skoog (MS) medium (Caisson Laboratories, Rexburg, ID, USA) containing 3.5 mM Pi, as described previously (Bozzo et al. 2002, 2004a). This Pi concentration is the minimum required concentration to maintain these cells fully Pi-sufficient (+Pi) over 8 d in batch culture. Pi-deficiency treatments were initiated 7 d following subculture of the cells into the +Pi medium. A 100 mL +Pi cell suspension was centrifuged axenically at 4000 g for 12 min at 25 °C. The cells were washed with –Pi

MS medium and were used to inoculate four separate flasks that each contained 100 mL of +Pi or –Pi medium. At various times over a 14-d growth period, the cells and their surrounding culture media were harvested by filtration through Whatman 541 filter paper (Fisher Scientific, Nepean, Ontario, Canada) on a Büchner funnel. The cells and aliquots of the corresponding cell culture filtrates (CCF) were frozen in liquid nitrogen and stored at −80 °C. Tomato seeds (cv. Moneymaker) were obtained from Bountiful Gardens (Willits, CA, USA). Seed surface sterilization and imbibition were conducted as described previously (Murley, Theodorou & Plaxton 1998). The seeds were germinated on Whatman No. 1 filter paper soaked in sterile Milli-Q water (Millipore Canada Ltd., Etobicoke, Ontario, Canada) and then transferred to Magenta Boxes (nine seeds per box) on sterile agar-solidified MS media containing 0.7% (w/v) agar, 2% (w/v) sucrose and either 0 or 2.5 mM Pi. The seedlings were cultivated in growth chambers at 22 °C and 80% relative humidity with a 16:8 h light:dark regime. Light intensity was 300 µmol m−2 s−1. The seedlings were watered at 14 and 28 d with 2 mL of MS medium (lacking agar) as described above, and harvested periodically over a 40-d growth period. Intact seedlings were removed from the agar medium and rinsed with MilliQ water. Leaves, stems and roots were rapidly excised, frozen in liquid N2 and stored at −80 °C.

AP extraction and assays Clarified extracts were prepared as described previously (Bozzo et al. 2004a). Frozen cells/tissues were powdered in liquid N2 and homogenized (2:1, w/v) in an ice cold extraction buffer with a mortar and pestle containing a spatula tip of acid-washed sand. The extraction buffer was 50 mM potassium acetate (pH 5.6) containing 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), 5 mM thiourea, 1 mM 2,2′-dipyridyl disulfide and 1 mM phenylmethylsulfonyl fluoride. The homogenates were centrifuged for 10 min at 14 000 g with a Beckman GS-15R Centrifuge (Beckman Coulter Canada Inc., Mississauga, Ontario, Canada), and supernatants used for IAP assays. A quick-thawed CCF was used for SAP assays. For routine measurements of total SAP or IAP activity, the hydrolysis of phosphoenolpyruvate (PEP) to pyruvate was coupled to the lactate dehydrogenase reaction and assayed at 25 °C by continuously monitoring the oxidation of reduced NADH at 340 nm with a Spectromax 250 Microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). Standard AP assay conditions were 50 mM Na acetate (pH 5.3 and 5.1 for SAP and IAP assays, respectively) (Bozzo et al. 2002, 2004a), 5 mM PEP, 10 mM MgCl2, 0.2 mM NADH and 3 units mL−1 of desalted rabbit muscle lactate dehydrogenase. All assays were initiated by the addition of an enzyme preparation and corrected for any NADH oxidase activity by omitting PEP from the reaction mixture. AP activity in all the assays was proportional to the amount of added cell extract or CCF and remained linear with respect to time.

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Differential synthesis of acid phosphatase isozymes 305

Pi extraction and assays Frozen suspension cells or seedling tissues were powdered under liquid N2, ground in 10% (w/v) perchloric acid and centrifuged at 14 000 g for 10 min. Supernatants were neutralized with 5 M KOH/1 M triethanolamine. The neutralized extracts were centrifuged as above, and the supernatants were used for soluble Pi determinations. Total cellular Pi was determined on suspension cells that had been wet ashed by HNO3 as described previously (Cook, Daughton & Alexander 1978). This involved the digestion of cells (1:1, w/v) in a mixture of 750 mM perchloric acid, 1 M sulphuric acid and 10.5 M nitric acid in a vacuum oven at 200 °C for 60–80 min. Pi was assayed as described previously (Drueckes, Schinzel & Palm 1995). Free Pi and total Pi extracts (1– 40 µL) were combined with 200 µL of a freshly prepared solution of four parts 10% (w/v) ascorbate with one part 10 mM ammonium molybdate in 15 mM zinc acetate (pH 5.0). Samples were incubated for 20 min at 37 °C, and the A660 was determined with a Spectromax 250 Microplate spectrophotometer (Molecular Devices). A standard curve over the range of 1–133 nmol of Pi was constructed for each set of assays. Esterified Pi was determined as the difference between total and free Pi concentrations.

2002). Preliminary studies indicated that the anti-(tomato SAP1 or IAP) antibodies detected numerous glycoproteins. Therefore, the oxidation of antigenic glycosylated side chains was routinely performed by pretreatment of immunoblots with sodium-m-periodate (Laine 1988). The relative amount of immunoreactive polypeptides was determined by quantification of the antigenic bands on immunoblots (in terms of A663) using an LKB Ultroscan XL laser densitometer and GelScan software (Version 2.1) (Amersham Biosciences; Baie d’Urfe, Quebec, Canada). The derived A663 values were linear with respect to the amount of the immunoblotted extract. Immunological specificities were confirmed by performing immunoblots

Antibody production Purified SAP1 (120 µg) (Bozzo et al. 2002) was dialysed overnight against Pi-buffered saline, filtered through a 0.2 µm membrane and emulsified in RIBI adjuvant (1 mL total volume) (RIBI ImmunoChem Research, Hamilton, MT, USA). After pre-immune serum was collected, the SAP1 was injected (0.6 mL subcutaneously, 0.4 mL intramuscularly) into a 2 kg New Zealand rabbit. A secondary injection (70 µg) was administered subcutaneously after 28 d. At 10 d after the final injection, blood was collected by cardiac puncture. After incubation overnight at 4 °C, the clotted cells were removed by centrifugation at 1000 g for 10 min. The antiserum was frozen in liquid N2 and stored at −80 °C in 0.04% (w/v) NaN3. For immunoblotting, anti(tomato IAP)–immunoglobulin G (IgG) was obtained by affinity purification from the corresponding immune serum by using 50 µg of the purified –Pi tomato IAP’s α-(63 kDa) and β-(57 kDa) subunits (Bozzo et al. 2004a) separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto poly(vinylidene difluoride) (PVDF) membrane as described previously (Plaxton 1989).

Electrophoresis and immunoblotting SDS–PAGE, relative molecular mass (Mr) estimates, immunoblotting onto PVDF membranes (Immobilon transfer; 0.45 µm pore size; Millipore Canada Ltd, Etobicoke, Ontario, Canada) and visualization of antigenic polypeptides using an alkaline phosphatase-tagged secondary antibody were performed as described previously (Bozzo et al.

Figure 1. Schematic representation of the protocol used for the immunoblotting of root surface secreted acid phosphatases (SAPs) following in situ 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) SAP-activity staining. The diagram describes the following steps in chronological order: (1) Place the excised roots from the 35-dayold phosphate (Pi)-sufficient (+Pi) or Pi-deficient (–Pi) tomato seedlings onto a 10% polyacrylamide gel electrophoresis (PAGE) mini-gel between two 0.75 mm thick mini-gel spacers, then immediately pour the BCIP–agar mixture (3 mL) onto the root preparation. (2) Remove the plastic spacers and incubate the root–BCIP preparation in the dark for 16 h at 25 °C. (3) Electroblot the root preparation onto a poly(vinylidene difluoride) (PVDF) membrane at 100 V for 75 min. (4) The carbohydrate epitope abolishment and immunodetection of the Pi-starvation inducible (PSI) tomato SAP isozymes are performed as described in the Materials and methods.

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306 G. G. Bozzo et al. in which rabbit pre-immune serum was substituted for the corresponding anti-(tomato SAP1) immune serum or affinity-purified anti-(tomato IAP)–IgG.

Root surface AP activity staining and tissue printing Excised roots from 35-day-old +Pi and –Pi seedlings were placed on a precast 10% PAGE minigel (lacking SDS) (Fig. 1). To visualize root surface AP activity, the root preparations were incubated with a 5-bromo-4-chloro-3indolyl-phosphate (BCIP)–agar overlay solution [50 mM Na-acetate, pH 5.3; 10 mM MgCl2; 0.6% (w/v) agar and 0.1 mg mL−1 BCIP] for 16 h at 25 °C. The agar-fixed root/ gel preparation was subsequently electroblotted onto a PVDF membrane, after which immunoblotting was carried out by using IAP or SAP1 antibodies as outlined in Fig. 1. Immunological specificities were confirmed by probing parallel immunoblots of BCIP-stained intact roots with rabbit pre-immune serum.

RESULTS Specificity of anti-(tomato PAP) antibodies The affinity-purified rabbit anti-(tomato IAP)–IgG readily cross-reacted with immunoblots of 20 ng of homogeneous IAP or SAP2 isolated from –Pi tomato cell cultures, but failed to detect 100 ng of the corresponding SAP1 isozyme (Fig. 2a, lanes 1–3). Similarly, the rabbit anti-(tomato SAP1) immune serum readily cross-reacted with 10 ng of purified SAP1, but not with 100 ng of homogeneous SAP2 or IAP (Fig. 2b, lanes 1–3). Immunoblots of cell extracts and CCF from 8-day-old –Pi tomato suspension cells indicated that: (1) the anti-(tomato IAP)–IgG was monospecific for the SAP2 and IAP polypeptides (Fig. 2a, lanes 4 and 5), whereas (2) the anti-(tomato SAP1) immune serum was monospecific for the SAP1 polypeptide and that SAP1 is restricted to the CCF of –Pi cells (Fig. 2b, lanes 4 and 5).

Influence of Pi nutrition on the growth and Pi content of tomato suspension cells L. esculentum suspension cells cultured for 14 d in the absence of Pi accumulated approximately 40% of the fresh weight (FW) of the corresponding +Pi cells (results not shown). Their reduced growth was correlated with depletion of exogenous Pi to undetectable levels within 1 d following subculture of the +Pi cells into the –Pi media (Fig. 3a). Pi in the CCF of +Pi cells steadily decreased from 3.5 mM at day 0 to undetectable levels by day 8 (Fig. 3a). Figure 3 also displays the intracellular free Pi and total P concentrations of the +Pi and –Pi cells. Decreases of 10-, 25- and 50-fold in the intracellular free Pi content occurred in the 6-, 11- and 14-day-old –Pi cells, respectively, whereas free Pi levels only decreased by about 2.5-fold, 14 d following subculture of the cells into the +Pi media (Fig. 3c). Likewise, total P and esterified –Pi levels were both approx-

Figure 2. Immunoblot analysis of the purified inorganic phosphate (Pi)-starvation inducible (PSI) purple acid phosphatase (PAP) isozymes, and the clarified extracts and cell culture filtrate (CCF) of Pi-deficient (–Pi) tomato suspension cells was performed using (a) a 1:50 dilution of the affinity-purified rabbit anti-[tomato intracellular acid phosphatase (IAP)]–immunoglobulin G (IgG), or (b) a 1:2000 dilution of the rabbit anti-[tomato secreted acid phosphatase1 (SAP1) immune serum. After carbohydrate epitope abolishment (Laine 1988), the immunoreactive polypeptides were visualized with an alkaline phosphatase-tagged secondary antibody and chromogenic detection (Bozzo et al. 2002, 2004a). (a) Lanes 1–3 contain 100, 20 and 20 ng of homogeneous SAP1, SAP2 and IAP, respectively. Lanes 4 and 5 respectively contain clarified CCF (1.5 µg) and cell extract proteins (10 µg) from 8-day-old –Pi tomato suspension cells. (b) Lanes 1–3 contain 10, 100 and 100 ng of homogeneous SAP1, SAP2 and IAP, respectively. Lanes 4 and 5 respectively contain clarified CCF (1.5 µg) and cell extract proteins (10 µg) and from 8-day-old –Pi tomato suspension cells. The indicated relative molecular mass (Mr) values denote the respective migration of various pre-stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) Mr standards.

© 2006 Blackwell Publishing Ltd, Plant, Cell and Environment, 29, 303–313

Differential synthesis of acid phosphatase isozymes 307 imately sixfold lower in the 14-day-old –Pi versus +Pi cells (Fig. 3b & d).

Influence of Pi nutrition on the synthesis of PSI PAP isozymes in tomato suspension cells Figure 4 shows the time-course activity profiles and immunoblots of PSI tomato IAP and SAP isozymes following subculture of +Pi suspension cells into +Pi or –Pi MS media. The affinity-purified anti-(tomato IAP)–IgG cross-reacted with the 63 kDa α- and 57 kDa β-subunits of the purified IAP in a 1:1 ratio (Fig. 4c). However, IAP’s α-subunit was

generally about twofold more immunoreactive than its βsubunit on the immunoblots of extracts from –Pi or +Pi cells sampled over the respective time courses (Fig. 4c). Levels of IAP activity and immunoreactive polypeptides remained relatively low and constant in the +Pi cells, but increased by about 2.5-fold, 6–11 d following subculture of the cells into –Pi media (Fig. 4a & c). The subsequently reduced IAP activity of the 14-day-old –Pi cells was correlated with a similar decrease in the relative amounts of IAP’s immunoreactive α- and β-subunits. SAP activity was initially detected in the 8-day-old –Pi tomato cells and became maximal by day 14 (Fig. 4b). CCF immunoblots probed with anti-(tomato SAP1) immune serum or affinity-purified anti-(tomato IAP)–IgG revealed the simultaneous induction of antigenic 84 kDa SAP1 and 57 kDa SAP2 polypeptides beginning 8 d after subculture of the tomato cells into the –Pi media (Fig. 4d & e). The results of Fig. 4e corroborate those of Fig. 2a, as well as our earlier report that the PSI IAP from tomato cell cultures is antigenically related to the 57 kDa SAP2 monomer (Bozzo et al. 2004a). The CCF immunoblots indicated a further approximate twofold increase in immunoreactive 84 kDa SAP1 and 57 kDa SAP2 polypeptides in the 11- to 14-dayold –Pi cells (Fig. 4d & e). Although SAP1 polypeptides remained undetectable on immunoblots of CCF harvested from the +Pi cells, relatively small amounts of SAP activity and SAP2 polypeptides appeared in CCF harvested from the 8- to 14-day-old +Pi cells that had been subcultured into MS media containing 3.5 mM Pi at day 0 (Fig. 4b & e).

Influence of Pi nutrition on the growth and synthesis of the PSI PAP isozymes in the tomato seedlings

Figure 3. Time course of (a) extracellular [cell culture filtrate (CCF)], (b) intracellular total, (c) intracellular free and (d) intracellular esterified inorganic phosphate (Pi) levels of the tomato suspension cells cultured in Murashige–Skoog (MS) media containing 3.5 mM Pi [Pi-sufficient (+Pi); ] or 0 mM Pi [Pideficient (–Pi); ]. ‘Esterified [Pi]’ represents the difference between the respective levels of total versus free Pi. All values represent the means ± standard error of the mean (SEM) of n = 3 separate flasks. FW, fresh weight.

The L. esculentum seedlings cultivated in the absence of Pi began to display symptoms of Pi starvation (e.g. decreased growth and anthocyanin accumulation) by approximately 11 d post imbibition (Fig. 5a). By 24 d, the –Pi seedlings had accumulated about 65% of the FW of the +Pi seedlings (Fig. 5a; results not shown). A progressive threefold increase in the root:shoot FW ratio of the –Pi seedlings occurred by day 40 (Fig. 6a). In contrast, the root:shoot FW ratio of the +Pi seedlings was not altered over their 40-daygrowth period (Fig. 6a). The Pi deprivation resulted in considerably lower levels of Pi in the various tissues. By day 30, intracellular Pi levels of leaves, stems and roots of the –Pi seedlings were, respectively, 19-, 19- and 50-fold lower than the corresponding Pi levels of the +Pi seedlings (Fig. 7). The AP activity of clarified tissue extracts from the +Pi seedlings remained relatively low and unchanged over their 40-day growth period (Fig. 6b–d). By contrast, the AP activity in root extracts from the –Pi seedlings was almost twofold that of the +Pi seedlings by day 11 (Fig. 6d). However, in leaves of the –Pi seedlings, enhanced AP activity was not apparent until day 29 (Fig. 6b). Maximal (≈ sixfold) induction of leaf AP activity occurred by approximately day 40, relative to the maximal threefold increase in the AP activity

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Figure 4. Acid phosphatase (AP) activities and immunological detection of inorganic phosphate (Pi)-starvation inducible (PSI) purple AP (PAP) isozymes in clarified extracts and cell culture filtrate (CCF) of the tomato suspension cells cultured in Murashige–Skoog (MS) media containing 3.5 mM Pi [Pi-sufficient (+Pi); ] or 0 mM Pi [Pi-deficient (–Pi); ]. (a & b) Time course of intracellular AP (IAP) and secreted AP (SAP) activities, respectively, present in the clarified cell extracts (a) and CCF (b). All values represent the means ± standard error of the mean (SEM) of n = 3 separate flasks. (c–e) Immunoblot analyses of PSI tomato PAP isozymes. Purified IAP (c) (50 ng), SAP1 (d) (20 ng), SAP2 (e) (20 ng) and clarified cell-extract proteins (c) (5 µg lane−1) or CCF proteins (d & e) 1.4 µg lane−1 were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and blotted as described in the Materials and methods. Immunoblots were probed with a 1:50 dilution of the affinity-purified anti-(tomato IAP)–immunoglobulin G (IgG) (c & e) or with a 1:2000 dilution of the anti(tomato SAP1) immune serum (d). Relative amounts of the antigenic polypeptides were estimated via laser densitometry. Lanes labelled ‘+’ and ‘–’ denote clarified cell extracts or CCF from the +Pi and –Pi cultures, respectively.

that occurred by days 29 to 32 in roots and stems of the –Pi seedlings (Fig. 6b–d). No anti-(tomato SAP1) immune serum immunoreactive polypeptides were detected following immunoblotting of stem or leaf extracts of the +Pi or –Pi seedlings (Fig. 6e and results not shown). However, an immunoreactive 84 kDa polypeptide that co-migrated with homogeneous SAP1 was observed on an immunoblot of root extracts from 18- to 40-day-old –Pi seedlings that were probed with the anti(tomato SAP1) immune serum (Fig. 6e). From days 18 to 35, the relative amount of the antigenic SAP1 polypeptide on root extract immunoblots was estimated by densitometry to have increased by about 2.5-fold. The apparent rootspecific induction of SAP1 in the 18-day-old –Pi seedlings followed the initial appearance at day 11 of the anti(tomato IAP)–IgG immunoreactive 63 and 57 kDa polypeptides that co-migrated with the α- and β-subunits of the homogeneous IAP from the –Pi tomato suspension cells (Fig. 6f). Densitometric analyses indicated that the anti-(tomato IAP)–IgG uniformly cross-reacted with the immunoreactive 63 and 57 kDa root polypeptides in an approximate 1:2 ratio, and that a maximal twofold increase in relative amounts of these polypeptides occurred in 23-day-old versus 11-day-old –Pi roots (Fig. 6f and results not shown). Immunoblots of stem extracts probed with anti-(tomato IAP)–IgG indicated that: (1) an approximate

2:1 ratio of 63:57 kDa IAP polypeptides in this tissue and (2) that the initial induction of these IAP polypeptides in the stem tissue occurred around day 11 in the –Pi seedlings. In contrast to roots and stems, leaves of the –Pi seedlings appeared to induce mainly the 63 kDa IAP polypeptide (Fig. 6f). Densitometry indicated an approximate sixfold increase in the amount of the immunoreactive 63 kDa polypeptide in the 35-day-old versus 18-day-old –Pi leaves (Fig. 6f and results not shown). By contrast, immunologically detectable polypeptides corresponding to the subunits of the PSI tomato IAP were either barely visible or not detected on immunoblots of leaf extracts from +Pi seedlings that had been probed with the anti-(tomato IAP)–IgG (Fig. 6f). A BCIP-staining procedure was used to visualize potential SAP activity on the surface of the intact roots from the –Pi tomato seedlings (Fig. 1). BCIP is a soluble and colourless AP substrate, but upon AP hydrolysis it is visualized as an insoluble blue product (Goldstein et al. 1988). Tomato seedlings cultivated for 35 d under –Pi conditions exhibited blue-stained roots following their incubation with the BCIP–agar overlay (Fig. 5b), indicating SAP activity. Immunoblots of the intact roots probed with either anti(tomato SAP1) immune serum or anti-(tomato IAP)–IgG indicated the presence of immunoreactive polypeptides coincident with AP-activity staining using BCIP (Fig. 5b).

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Figure 5. (a) Tomato seedlings grown on the Murashige–Skoog (MS)–solidified agar supplemented with 0 mM [Pi-deficient (–Pi)] or 2.5 mM Pi [Pi-sufficient (+Pi)] were harvested at 11 d (shown on the left) and 24 d (shown on the right) post imbibition. (b) Localization of root surface secreted acid phosphatase (SAP) activity and protein. Thirty-five-day-old +Pi and –Pi tomato seedlings were harvested, and excised root material was incubated with 5-bromo-4chloro-3-indolyl-phosphate (BCIP) [a colourless acid phosphatase (AP) substrate]. Root surface SAP activity is visualized as blue in colour, indicating BCIP hydrolysis. Following in situ SAP-activity staining, immunoblots of the intact root preparations were probed with a 1:2000 dilution of the rabbit anti-(tomato SAP1) immune serum or with a 1:50 dilution of the affinity-purified anti-[tomato intracellular AP (IAP)]– immunoglobulin G (IgG). The antigenic polypeptides were visualized with an alkaline phosphatase-tagged secondary antibody (Bozzo et al. 2002).

By contrast, no root surface AP activity or PSI PAP antibody cross-reactivity was detected on the roots of the 35-day-old +Pi control seedlings.

DISCUSSION Few reports are available concerning the immunological relatedness of different APs within and between plant species. However, an anti-(B. nigra suspension cell PSI IAP)– IgG cross-reacted with the corresponding PSI SAP, as well as APs from diverse plant tissues (Duff et al. 1991). Thus, it was concluded that plant APs are immunologically closely related, implying divergence from a common ancestral

form. By contrast, our immunoblot results demonstrate that: (1) the tomato IAP antibodies readily cross-reacted with the 63 kDa α- and 57 kDa β-subunits of purified IAP and the 57 kDa SAP2 but not with purified SAP1 (Fig. 2a), whereas (2) tomato SAP1 antibodies readily detected the 84 kDa SAP1 but not SAP2 or either IAP subunit (Fig. 2b). Cloning of cDNAs encoding the three PSI tomato PAP isozymes will help to establish their genetic, evolutionary and structural relatedness. Interestingly, the N-terminal and tryptic peptide amino acid sequences of the 57 kDa IAP βsubunit that were obtained by Bozzo et al. (2004a) precisely correspond to portions of the deduced amino acid sequence of the tomato fruit cDNA [GenInfo Identifier (GI):

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Figure 6. Root : shoot fresh weight (FW) ratio (a), acid phosphatase (AP) activities (b–d) and immunological detection (e & f) of inorganic phosphate (Pi)-starvation inducible (PSI) purple AP (PAP) isozymes in the clarified extracts of various tissues of the tomato seedlings cultivated for up to 40 d on the agar-solidified Murashige–Skoog (MS) media containing 2.5 m M Pi [Pi-sufficient (+Pi); ] or 0 mM Pi [Pideficient (–Pi); ]. All values plotted in panels a–d represent the mean of duplicate determination with maximum variance < 15% of the mean value. (e & f) Immunoblot analyses of PSI tomato PAP isozymes. Purified secreted acid phosphatase1 (SAP1) (e) (20 ng), intracellular acid phosphatase (IAP) (f) (50 ng) and clarified extract proteins (6 µg lane−1) were resolved by sodium dodecyl sulfate (SDS)– polyacrylamide gel electrophoresis (PAGE) and blotted as described in the Materials and methods. Immunoblots were probed with a 1:2000 dilution of the rabbit anti-(tomato SAP1) immune serum (e) or with a 1:50 dilution of the affinity-purified anti-(tomato IAP)– immunoglobulin G (IgG) (f). Lanes labelled ‘+’ and ‘–’ denote extracts from the tissues of +Pi and –Pi seedlings, respectively.

47105718] that was recently submitted and shown to display significant similarity to known plant PAPs (http:// www.ncbi.nlm.nih.gov). The reduced biomass accumulation of –Pi tomato cell cultures and seedlings (Fig. 5a and results not shown), and enhanced root : shoot FW ratio of the –Pi seedlings (Fig. 6a) were correlated with pronounced reductions in intracellular free Pi contents (Figs 3c & 7). The magnitude

of Pi decline upon Pi starvation corroborates a previous study of –Pi tomato seedlings (Bosse & Köck 1998), as well as observations from other plant systems including B. nigra suspension cells and seedlings (Duff et al. 1989; Theodorou & Plaxton 1994), soybean leaves (Lauer et al. 1989), and maize roots and leaves (Loughman, Ratcliffe & Southon 1989; Lee, Ratcliffe & Southon 1990; Lee & Ratcliffe 1993). During early plant Pi starvation responses, cytoplasmic Pi

© 2006 Blackwell Publishing Ltd, Plant, Cell and Environment, 29, 303–313

[Pi] (mmol•g–1 FW)

Differential synthesis of acid phosphatase isozymes 311

14.0 12.0 10.0 8.0 6.0 4.0

+Pi seedlings –Pi seedlings

0.8 0.6 0.4 0.2 0.0

Leaves

Stems

Roots

Figure 7. Intracellular inorganic phosphate (Pi) concentration of leaves, stems and roots from the 30-day-old tomato seedlings grown in a medium containing 2.5 mM [Pi-sufficient (+Pi)] or 0 mM Pi [Pi-deficient (–Pi)]. All values represent the means ± standard error of the mean (SEM) of n = 3 separate extracts. FW, fresh weight.

is maintained at the expense of the non-metabolic vacuolar Pi reserve (Rebeille et al. 1983; Mimura, Sakano & Shimmen 1996). Prolonged Pi starvation eventually depletes vacuolar Pi stores, resulting in marked decreases in cytoplasmic free Pi, nucleoside-P and other P-metabolite pools (Lauer et al. 1989; Loughman et al. 1989; Lee & Ratcliffe 1993; Plaxton 2004). Considering the extremely low levels of intracellular free Pi respectively present in the 14- and 30-day-old –Pi tomato suspension cells and seedling tissues (Figs 3c & 7), one would expect both vacuolar and cytoplasmic Pi pools to have been almost completely exhausted. By day 30, the intracellular Pi levels of leaves/stems and roots of the –Pi seedlings were, respectively, about 20- and 50fold lower than the corresponding levels in the +Pi seedlings (Fig. 7). Similarly, the –Pi suspension cells displayed approximate 20- and 50-fold reductions in their intracellular free Pi levels by days 8 and 14, respectively, whereas a 2.5-fold reduction in intracellular free Pi occurred by day 14 in +Pi cells (likely because of the depletion of media Pi from 3.5 mM at day 0 to undetectable by day 8) (Fig. 3c). The latter results substantiate Pi nutrition studies of B. nigra and B. napus suspension cells reporting that the standard 1.25 mM Pi concentration of MS nutrient media is insufficient to maintain batch-cultured plant suspension cells fully +Pi over their typical 7–14 d culture period (Lefebvre et al. 1990; Carswell et al. 1997). In tomato suspension cells and seedlings experiencing transition from Pi sufficiency to deficiency, the total amount of antigenic IAP or SAP proteins correlated well with total IAP or SAP activity (Figs 4 & 6). Tomato seedlings remobilize Pi from stored phytic acid over their first 8–10 d of germination, and thus –Pi seedlings do not display morphological or biochemical symptoms of Pi stress until about 10– 12 d after sowing (Bosse & Köck 1998) (Figs 5a & 6). Likewise, there was a marked (> 5 d) lag between the elimination of exogenous Pi, and the de novo synthesis of PSI PAP isozymes by the –Pi tomato cell cultures (Figs 3 & 4). This delay was likely coincident with the depletion of the vacu-

olar Pi pool (Rebeille et al. 1983) and points to an intracellular mechanism of Pi sensing. This is substantiated by: (1) the rapid induction of PSI genes following incubation of cultured tomato cells in a +Pi medium supplemented with D-mannose and other compounds known to sequester intracellular Pi into organic compounds (Köck et al. 1998), and (2) the results of Miller et al. (2001), who demonstrated that the gene encoding a PSI SAP of proteoid lupin roots was responsive to internal Pi concentrations. Purified PSI IAP from–Pi tomato cells exists as a 142 kDa heterodimer composed of an equivalent ratio of α- (63 kDa) and β- (57 kDa) subunits (Bozzo et al. 2004a). Immunoblots of clarified extracts probed with affinitypurified anti-IAP–IgG indicated that +Pi and –Pi tomato cells and seedlings contain varying ratios of these antigenic IAP polypeptides, ranging from an approximate 2:1 α- : β-subunit ratio in suspension cells and stems, to 1:2 in roots and up to 6:1 in leaves (Figs 4c & 5f). These results are reminiscent of the PSI plasma inorganic pyrophosphate (PPi)-dependent phosphofructokinase of B. nigra suspension cells and seedlings that displayed varying ratios of its regulatory α- (66 kDa) and catalytic β- (60 kDa) subunits depending upon the Pi nutritional status and tissue being investigated (Theodorou et al. 1992; Theodorou & Plaxton 1994). Structure–function analyses, coupled with the genetic manipulation of the relevant genes in planta, will help to delineate the relative contribution of the IAP’s α- and β-subunits to intracellular Pi recycling in –Pi tomato. That enhanced IAP synthesis plays an important in vivo role recycling Pi from intracellular P metabolites in the 6to 11-day-old –Pi tomato cells is supported by the concomitant twofold reduction in the levels of the intracellular esterified –Pi over the same period (Figs 3d & 4a,c). In addition, purified PSI IAP from –Pi tomato suspension cells displayed potent allosteric (mixed competitive) inhibition by Pi (Ki = 1.6 mM) (Bozzo et al. 2004a). The intracellular Pi concentration of the 0- to 4-day-old +Pi cells (≈ 2.0–4.5 mM, assuming 1 g FW ≈ 1 mL) (Fig. 3c) should exert significant IAP inhibition in vivo. Conversely, the subsequent > 20-fold reductions in the intracellular Pi levels of the 6- to 11-day-old –Pi cells are expected to relieve the PSI tomato IAP from any Pi inhibition. Between days 11 and 14, the IAP activity and concentration of the –Pi suspension cells decreased to that of +Pi cells (Fig. 4a & c). This could be due to IAP-mediated depletion of intracellular P metabolites to a minimal level necessary to sustain cellular viability. It is notable that the large reduction in intracellular Pi content of the –Pi tomato cells was associated with the temporal induction of IAP (day 6) and SAP (day 8) activities and corresponding antigenic IAP and SAP1/SAP2 polypeptides, respectively, that are present in clarified cell extracts and CCF (Fig. 4). Similarly, SAP1 polypeptide induction occurred at least 7 d following the induction of IAP protein in roots of the –Pi seedlings (Fig. 6e & f). It will be of interest to determine if the temporal induction of IAP versus SAP1/SAP2 isozymes in tomato cell cultures

© 2006 Blackwell Publishing Ltd, Plant, Cell and Environment, 29, 303–313

312 G. G. Bozzo et al. and seedlings is dependent upon critical thresholds of Pi depletion during the transition from Pi sufficiency to deficiency. A similar temporal-synthesis pattern was previously documented for a pair of PSI root SAP electrophoretic isoforms over a 28-day time course of Pi deprivation in white clover seedlings (Zhang & McManus 2000). A recent transcript profiling study also revealed the temporal and tissue-specific expression of a pair of putative PSI potato PAPs (Zimmermann et al. 2004). Likewise, genome-wide expression studies of –Pi Arabidopsis seedlings implicated a variety of PSI genes that display short-term expression, whereas prolonged Pi deficiency elicited a switch to the expression of alternate PSI genes (Hammond et al. 2003, 2004; Wu et al. 2003). The marked delay in IAP induction in leaves and stems relative to roots of the –Pi tomato seedlings agrees with reports that –Pi plants preferentially accumulate limiting Pi in leaves at the expense of roots (Theodorou & Plaxton 1994; Mimura et al. 1996). This has been hypothesized to represent an adaptive strategy to sequester limiting Pi to the leaves to maintain optimal rates of photosynthesis for as long as possible in –Pi plants, and is in accord with our observation that free Pi levels of leaves of the 30-day-old – Pi tomato seedlings were at least threefold greater than those of the corresponding roots (Fig. 7). Of further interest was the apparent tissue-specific synthesis of SAP1 in roots of –Pi tomato seedlings (Figs 5b & 6e). No AP-activity staining or anti-(tomato IAP or SAP1)– IgG immunoreactive material was detected on the surface of intact roots harvested from the 30-day-old +Pi seedlings (Fig. 5b). By contrast, in situ localization of root surface SAP activity and immunoreactive SAP proteins confirmed SAP1 and probable SAP2 secretion by roots of 30-day-old –Pi tomato seedlings (Fig. 5b). The possible accumulation of the 57 kDa SAP2 in stems or leaves of the –Pi seedlings cannot be excluded because it cannot be distinguished from IAP’s 57 kDa β-subunit on the basis of immunogenicity or mobility upon SDS–PAGE (Bozzo et al. 2004a). Nevertheless, it is apparent that SAP-activity staining of the root surface of –Pi tomato seedlings was coincident with the accumulation of the same two SAP isozymes that were initially purified from the CCF of –Pi tomato suspension cells (Bozzo et al. 2002). Root-specific PAP synthesis has been described at the transcript level for –Pi potato and white lupin seedlings (Wasaki et al. 2003; Zimmermann et al. 2004). Similarly, transcript profiling of –Pi Arabidopsis seedlings indicated that numerous PSI genes exhibited contrasting expression in leaves versus roots (Wu et al. 2003), lending support to the idea that different plant organs exhibit distinct Pi starvation response strategies. Root surface and CCF synthesis of PSI SAP1 and SAP2 that respectively occurs in –Pi tomato seedlings and cell cultures are proposed to function with additional PSI phosphohydrolases (Bosse & Köck 1998; Ticconi & Abel 2004) to mobilize Pi from extracellular organophosphates that are released into the culture media or apoplast by damaged or dying cells, or are otherwise present in the rhizosphere of the –Pi seedlings.

Conclusions The activity and synthesis profile of each PSI PAP isozyme in tomato cell culture were correlated with a significant reduction in intracellular, but not extracellular, Pi. The temporal synthesis of IAP, relative to SAP1 and SAP2 during Pi stress is suggestive of a differential mechanism of transcriptional, translational and/or proteolytic control. The analysis of the whole tomato plant response to Pi deficiency demonstrated the temporal and tissue-specific synthesis of the same three tomato PSI PAP isozymes first identified in the –Pi suspension cells. This provides additional evidence that suspension cell cultures represent a valuable model system for further investigations of the biochemical and molecular adaptations of vascular plants to suboptimal Pi nutrition.

ACKNOWLEDGMENTS This work was supported by research and equipment grants from the Natural Sciences and Engineering Research Council of Canada.

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