JXB Advance Access originally published online on April 28, 2003
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Journal of Experimental Botany, Vol. 54, No. 387, pp. 1553-1564,
June 1, 2003
© 2003 Oxford University Press
In situ localization associates biologically active plant natriuretic peptide immuno-analogues with conductive tissue and stomata
Received 13 March 2003; Accepted 18 March 2003
1 School of Biological and Chemical Sciences, Deakin University, Geelong, Victoria, 3217, Australia
2 Department of Biotechnology, University of the Western Cape, Private Bag X17, Bellville 7535, South Africa
3 Department of Pharmaceutical Biology and Pharmacology, Victorian College of Pharmacy, Monash University, 381 Royal Parade, Parkville, Melbourne, Victoria 3052, Australia
4 Present address: Faculty of Biology, Gadjah Mada University, Yogyakarta, Indonesia 55281.
5 To whom correspondence should be addressed. Fax: +27 21 959 2266. E-mail:cgehring{at}uwc.ac.za
Abbreviations: ANP, atrial natriuretic peptide; PNP, plant natriuretic peptides.
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Abstract |
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Plant natriuretic peptide immuno-analogues (irPNP) have previously been shown to affect a number of biological processes including stomatal guard cell movements, ion fluxes and osmoticum-dependent water transport. Tissue printing and immunofluorescent labelling techniques have been used here to study the tissue and cellular localization of irPNP in ivy (Hedera helix L.) and potato (Solanum tuberosum L.). Polyclonal antibodies active against human atrial natriuretic peptide (anti-hANP) and antibodies against irPNP from potato (anti-StPNP) were used for immunolabelling. Tissue prints revealed that immunoreactants are concentrated in vascular tissues of leaves, petioles and stems. Phloem-associated cells, xylem cells and parenchymatic xylem cells showed the strongest immunoreaction. Immunofluorescent microscopy with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG supported this finding and, furthermore, revealed strong labelling to stomatal guard cells and the adjacent apoplastic space as well. Biologically active immunoreactants were also detected in xylem exudates of a soft South African perennial forest sage (Plectranthus ciliatus E. Mey ex Benth.) thus strengthening the evidence for a systemic role of the protein. In summary, in situ cellular localization is consistent with physiological responses elicited by irPNPs reported previously and is indicative of a systemic role in plant homeostasis.
Key words: Conductive tissue, expansins, Hedera helix L., homeostasis, immuno-analogues, plant natriuretic peptides, Plectranthus ciliatus E. Mey ex Benth., Solanum tuberosum L., tissue printing.
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Introduction |
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Over the last decade it has become apparent that plants also contain peptidic signalling molecules that play vital roles in cell-to-cell communication (Matsubayashi et al., 2001; Lindsey et al., 2002). Systemin (Pearce et al., 1991) was the first plant peptide shown to have a role in plant signalling where it functions in the systemic wound response. Subsequently, several other peptide systems have been identified. These include ENOD40 with a role in root nodulation (van de Sande et al., 1996), phytosulphokines which function in cell division (Matsubayashi and Sakagami, 1996), CLAVATA3 which is involved in shoot meristem organization (Fletcher et al., 1999), S-locus cysteine rich (SCR) proteins that act in self-incompatibility (Schofer et al., 1999) and RALF which arrests root growth and development (Pearce et al., 2001). Other protein and peptide families have been identified that are antimicrobial (Garcia-Olmedo et al., 2001) and yet others such as POLARIS have been reported to influence plant growth (Casson et al., 2002; Lindsey et al., 2002). This growing number of functional peptide families has led to a reassessment of the role of peptide hormones, as well as further exploration to discover novel peptide families involved in plant signalling.
The immunoaffinity purification of novel, biologically active proteins from ivy (Hedera helix) and potato (Solanum tuberosum) with antibodies directed against vertebrate atrial natriuretic peptide (ANP) (Billington et al., 1997; Maryani et al., 2001) have previously been reported and these proteins termed immunoreactant plant natriuretic peptides (irPNPs) (Gehring, 1999). Subsequently, N- and C-terminal sequences of an irPNP from Solanum tuberosum (StPNP) were obtained (Maryani et al., 2001) which, in turn, led to the identification and isolation of two Arabidopsis thaliana orthologues (AtPNP-A and AtPNP-B) (Ludidi et al., 2002). The AtPNP-A and AtPNP-B encoded proteins show similarity to CjBAp12, a functionally undefined protein from citrus that is induced in response to blight infection (Ceccardi et al., 1998). CjBAp12 shows some sequence similarity to domains found in the cell wall loosening expansins, but has tested negative for cell wall loosening activity (Ceccardi et al., 1998). Subsequent in silico analysis has established the evolutionary and functional relationships of irPNP-like molecules within the superfamily of expansins, pollen allergens and distantly related molecules such as endoglucanases and shown that irPNP-like molecules are related to expansins and fall into two closely related groups; one includes CjBAp12 and the other AtPNP-A (Li et al., 2002; Ludidi et al., 2002).
Evidence has been obtained that suggests that these proteins have a function in modulating plant water and solute homeostasis (Gehring, 1999). Exogenous PNP stimulates stomatal opening (Billington et al., 1997; Maryani et al., 2001) and activates the H+ -ATPase (Maryani et al., 2001). Moreover, irPNP rapidly and specifically induces the transient elevation of cGMP levels in maize root stele tissue (Pharmawati et al., 1998) and stomatal guard cell protoplasts (Pharmawati et al., 2001). This finding may suggest the presence of plant PNP receptors that contain guanylate cyclase domains. Such domains have been identified in the receptors NPR-A and NPR-B receptors for ANP in vertebrates (Chinkers et al., 1989). Not only does irPNP influence water movement via opening stomata, it also enhances osmoticum-dependent volume changes in leaf mesophyll protoplasts (Maryani et al., 2001). The immuno-reactants (irPNP) directly or indirectly modulate ion net fluxes across plant membranes leading to a rapid (<60 s) net influx of H+ and a delayed (>20 min) net influx of K + and Na+ in maize stele (conductive tissue in roots) tissue (Pharmawati et al., 1999). These findings all point to irPNP having a role, albeit as yet undefined, in regulating water and solute movements. Although irPNP can be isolated from leaf tissue (Billington et al., 1997; Maryani et al., 2001), to date its localization within the leaf is not known, nor whether it occurs in other regions of the plant.
The present study was aimed at gaining further insight into the role of natriuretic peptides in Hedera helix L. by localizing NP immuno-analogues at the tissue level using tissue prints and tissue section immunolocalization. Antibodies against human ANP (anti-hANP) and potato irPNP (anti-StPNP) were used in this study. This approach provided evidence of distinct localizations and this evidence is discussed in relation to functional and structural assays.
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Materials
Rabbit anti-hANP polyclonal antibodies were purchased from Auspep (Parkville Victoria 3052, Australia), rabbit anti-tomato glutamine synthetase (anti-GS) polyclonal antibodies were kindly provided by Dr Alejandro Pérez-García (Departamento de Microbiología, Facultad de Ciencias, Universidad de Málaga, Malaga, Spain) and rabbit anti-potato plant natriuretic peptide polyclonal antibodies (anti-StPNP) were produced by the Institute of Medical and Veterinary Science, Adelaide (South Australia). Recombinant AtPNP-A was expressed in Saccharomyces cerevisiae Y294 using the Yep352 yeast expression vector under the control of the ADH-2p promoter. The recombinant protein was prepared by Dr Ganka Pironcheva (University of the Western Cape).
IrPNP extraction and biochemical analyses
IrPNP was extracted from ivy leaves. The leaves (25 g) were snap-frozen in liquid N2 and ground to a fine powder with a mortar and pestle. The powder was then taken up in 125 ml of extraction buffer (50 mM KCl, 1 mM EDTA, 10 mM TRIS-HCl; pH 7.4) to which was added an equal volume of methanol. Extraction was allowed to proceed for 60 min at 4 °C under continuous stirring. The extract was then filtered through two layers of muslin cloth and the filtrate placed on a rotary evaporator until all the solvent had evaporated. The resulting plant material was resuspended in 100 ml of extraction buffer. This crude protein extract was precipitated in fractions at saturations of 25, 50 and 80% in successive steps by the addition of finely ground ammonium sulphate. The precipitate was removed at each step by centrifugation at 10 000 rpm for 15 min at 4 °C. Resulting pellets of fractions were resuspended in 2 ml of extraction buffer and dialysed against two changes of extraction buffer at 4 °C, overnight using SnakeSkin Pleated Dialysis Tubing with molecular weight cut-off 3500 (Pierce, Rockford IL). Aliquots of the ammonium sulphate saturated fractions were separated by SDS-polyacrylamide (15%) gel electrophoresis using a Mini-Protean 3 system (BioRad, Hercules CA) and total protein detected by staining with SilverQuest Silver Staining Kit (Invitrogen, Paisley, UK). Westerns were performed with a Mini-Protean 3 Transfer System. The nitrocellulose membranes were developed using a 1/5000 dilution of rabbit anti-human ANP (anti-hANP) or rabbit anti-StPNP and a 1/10 000 dilution of Goat anti-Rabbit IgG (CALTAG, Burlingame, CA) in TTBS and immunoreactivity detected with the ECL Plus Western Blotting Detection System (Amersham-Pharmacia-Biotech, Little Chalfont, UK).
The irPNP, mainly present in the 50% ammonium sulphate fraction, was affinity purified on a POROS 20 AL anti-hANP affinity column using the BioCAD SPRINT system. The column was prepared as follows: Rabbit anti-hANP antibody was resuspended in coupling buffer (0.1 M HEPES, pH 7). An aliquot of the resuspended rabbit anti-hANP antibody (55 µl) was added to 0.8 g POROS 20 AL resin (PerSeptive Biosystems, Framingham, USA) in 5 ml coupling buffer and rotated at room temperature for 8 h. The resultant Schiff base was reduced by adding 5 mg NaBH4 ml–1 bed volume (11.5 mg) to the coupling solution and rotated for a further 2 h at room temperature. The coupling mixture was centrifuged at 13 000 rpm (Eppendorf bench-top centrifuge 5415D) for 1 min at room temperature and the supernatant discarded. Residual aldehydes were quenched by adding 1.5 ml 0.2 M TRIS buffer containing 11.5 mg NaBH4 to the pelleted resin and rotated at room temperature for 2 h. The POROS 20 AL anti-hANP affinity resin was packed into a POROS PEEK column (diameter: 4.6 mm; length: 100 mm) and washed with 10 column volumes of equilibration buffer (10 mM TRIS-HCl, pH 7.5). To purify irPNP, 1.5 ml of the fraction precipitated with 50% ammonium sulphate was applied to the POROS 20 AL anti-hANP affinity column at a rate of 20 ml min–1. The column was washed with two column volumes equilibration buffer and the bound protein eluted with five column volumes equilibration buffer containing 1 M NaCl before re-equilibrating the resin with 15 column volumes equilibration buffer. Aliquots (1 ml) were collected at a flow rate of 20 ml min–1. The column eluent was monitored at 280 nm and fractions containing protein were concentrated using a Speed Vac and resuspended in 50 µl of distilled water before being subjected to SDS-PAGE and western blot analysis.
Tissue printing
Tissue prints were obtained following the method detailed previously (Pérez-Garcia et al., 1998). Cross-sections of approximately 1–2 mm thickness were prepared using a clean double-edged razor blade to cut leaves, petioles (from the middle leaflet), and the main stems (approximately 10 mm from the shoot). The section surface was rinsed with distilled water and gently blotted with Kim-wipes for a few seconds to remove the excess liquid and then transferred to a nitrocellulose membrane. The blotted section was laid on the membrane, the upper surface covered with four layers of Kim-wipes before being firmly and evenly pressed for 20–30 s. The membrane was air-dried at room temperature and incubated in TBS (TRIS-buffered saline: 20 mM TRIS-HCl (pH 7.5) and 500 mM NaCl) for 10 min at room temperature. Blocking of endogenous peroxidases was achieved by incubation in TBS containing 1% [w/v] periodic acid for 30 min at room temperature followed by three washes in TBS and blocking with 3% [w/v] gelatine in TBS at room temperature for 1 h. After two 10 min washes in TTBS (Tween TRIS-buffered saline: 0.1% [v/v] Tween-20 in TBS), the membranes were incubated at room temperature overnight with either anti-hANP, anti-StPNP or anti-GS diluted at 1:500 or 1:1000 in TTBS containing 1% [w/v] gelatine. Nitrocellulose membranes were subsequently washed three times with TTBS for 10 min at room temperature and then incubated with goat anti-rabbit IgG serum conjugated to horseradish peroxidase (HRP, BioRad Life Science Research Product, CA, USA) at a 1:3000 dilution in 1% gelatine in TTBS for 1 h at room temperature. The membrane was washed twice with TTBS and once with TBS for 10 min each and the peroxidase activity was developed using HRP detection reagents containing 4-chloro-1-naphthol in diethylene glycol and hydrogen peroxide (BioRad Life Science Research Product, CA, USA) according to the manufacturer’s instructions. Antigen on the tissue prints was localized and photographed under incident and bright light with a digital camera (RT Slider (Diagnostic Instruments. Inc. USA)) connected to a microscope (Zeiss, Germany) and a computer with digital image analysis software (Image Pro Express). Tissue printed specimens were compared with either the fresh or de-paraffinized paraplast embedded tissue sections stained with 0.1% [w/v] toluidine blue O in phosphate buffer (pH 5.5) for 10 min to show the anatomical features of the organs.
Immunolocalization of irPNP
Cut tissues were immediately immersed in a fixation solution (0.5% [v/v] glutaraldehyde and 3% [w/v] paraformaldehyde mixture in PBS (phosphate buffer saline: 135 mM NaCl, 2.7 mM KCl, 1.5 mM KH 2PO4, pH 7.2)) and infiltrated under vacuum for 30 min ( Dewitte et al., 1999). The tissue pieces were stored in the fixative for 2–5 h at 4 °C on a shaker with gentle agitation and then washed in PBS at 4 °C for 60 min with three changes of buffer. The tissue pieces were dehydrated in a graded series of ethanol in H2O (EtOH: 10%, 20%, 30%, 50%, 70%, and 85%) each for 30 min at room temperature. Tissue pieces were then further incubated twice in 95% and 100% EtOH and left in 100% EtOH overnight at 4 °C. The samples were further dehydrated in 100% EtOH for 30 min and transferred into a series of xylene in EtOH steps (Xylene: 25%, 50%, 75%) each for 30 min at room temperature and twice in 100% xylene. Xylene was replaced with a series of Paraplast Plus/xylene solutions (Paraplast Plus: 25%, 50%, 75%, 100%, 100%) at 58 °C for 30 min in a water bath followed by three 30 min vacuum infusions of pure liquid Paraplast Plus at 58 °C. After infiltration, tissue pieces were embedded in 100% Paraplast Plus. For immunostaining tissue sections (10 µm thickness) were cut with a sliding microtome (Reichert Jung, Germany) and attached to slides after floating on adhesive solution (0.1% [w/v] gelatin, 0.02% [w/v] chromium potassium sulphate in dH2 O). Paraffin was removed from the tissue using two 10 min xylene washes followed by a 10 min rinse in 100% EtOH for 10 min at room temperature. Mounted tissue sections were rehydrated in a graded EtOH series (95%, 70%, 50%, and 25%) at room temperature by repeated dipping until all streaks disappeared followed by two 5 min washes with TBSA solution at room temperature. Sections were incubated with primary antibodies (anti-StPNP) diluted 1:500 in TBSA. After three 5 min washes with TBSA, FITC-conjugated goat anti-rabbit IgG antibodies diluted 1:1000 with TBSA was applied at 37 °C for 1 h followed by two 5 min washes in TBSA with occasional gentle agitation. Sections were covered with mounting medium (Cytoseal 60, PST Pro Sci Tech, Australia) and observed under the fluorescence microscope.
Collection of xylem sap
Xylem sap was collected from the soft South African perennial forest sage (Plectranthus ciliatus E. Mey ex Benth.). P. ciliatus plants were grown in well-watered pots and xylem sap was collected as root pressure exudate following removal of the shoot. Stems were cut 10 mm above the soil level and the wound area was immediately swabbed to remove contaminants from cut cells. The sap was allowed to exude for 10 min and this exudate (
250 µl) was discarded. A further 1.5 ml of sap was collected in a glass tube placed over the cut stem and sealed with Parafilm. Then 1 ml of exudate per plant was concentrated and analysed for the presence of irPNP with an anti-hANP affinity column on the BioCAD.
Stomatal guard cell assay
In each experiment three Arabidopsis thaliana leaves were rinsed and submerged at 20–25 °C in stomatal assay solution (10 mM PIPES (pH 6.3), 50 mM KCl, 1 mM MgCl2, and 100 µM CaCl2) in microtitre plate wells and treated at 20–25 °C under incandescent light (430 nm at 35 W m–2) for 30 min. Pore widths of >20 stomata from three separate leaves for each treatment were measured microscopically using a calibrated ocular micrometer and the results are the mean ±SE of >60 stomata subjected to a one-way analysis of variance (ANOVA) to establish differences in treatments.
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Purification and characterization of irPNPs
When liquid-phase-isolated, water-soluble, ivy-leaf proteins are affinity-purified on an anti-human ANP column a single elution peak results (Fig. 1A). This peak is released with increasing ionic strength at a conductivity >25 mS (1 mS=15 mM NaCl). The SDS-PAGE resolution of immunoreactants from ivy (HhPNP) show two proteins of similar molecular mass (>13 kDa) and a protein of smaller mass (<11 kDa) (Fig. 1C). Such a column was also used to immunoaffinity purify proteins with close to identical molecular masses isolated from potato (StPNP). The StPNP peptides were used to generate polyclonal rabbit anti-StPNP antisera (prepared by the Institute of Medical and Veterinary Science, South Australia) that was used later for tissue printing and fluorescent in situ labelling. To test the efficacy of the StPNP antisera an immunoaffinity column was prepared with the anti-StPNP antibody. Water-soluble ivy-leaf proteins were loaded on to the column and one peak (Fig. 1B) containing two ivy proteins both close to 13 kDa (Fig. 1C) was eluted. The smaller protein was not detected in proteins purified with anti-StPNP antibody. Most importantly, a recombinant Arabidopsis thaliana irPNP (AtPNP-A) was made and successfully purified with the anti-StPNP column (Fig. 1C) and thus established that AtPNP-A contains an epitope recognized by the anti-StPNP antibody. A western dot blot shows that potato crude extract reacts strongly when probed with anti-StPNP (Fig. 2A). Not surprisingly, such a reaction is also seen when potato extract that has been immunoaffinity purified with anti-hANP (giving StPNP) is probed. When the anti-serum is preincubated with recombinant AtPNP-A this effect is abolished. AtPNP-A has thus competed for the binding site in the crude protein extract and the StPNP sample. In order to find out if this epitope is a key to biological activity, a functional assay was used. The guard cell aperture assay (Fig. 2B) shows a significant promotion of opening caused by the crude protein extract and imunnoaffinity purified potato extract (StPNP). In controls, where the crude protein extract or StPNP were preincubated with anti-hANP antisera, the stomatal apertures did not significantly differ from untreated controls.
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Localization of irPNP in conductive tissue
In longitudinal sections anti-hANP immunoreactivity was detected in the conductive tissue of potato stems (Fig. 3). The cell that contained immunoreactivity (Fig. 3A) was identified as a part of the conductive tissue, most likely phloem or xylem parenchyma. Nearby is a mature xylem vessel that shows prominent signs of secondary lignification with the diagnostic autofluorescence. Two strongly fluorescent signals are seen in Fig. 3B (see arrows) which occur in the area likely to represent the perforation plates of sieve cells. Interestingly, this signal is only seen on one end of the cells and this may indicate that the peptide has been blocked and accumulated due to plug formation in the perforation zone. The background fluorescence (Fig. 3C) in the control, in the presence of pre-absorbed primary antibody, is relatively slight as no prominent and highly fluorescent regions are evident. To test the antiserum further, a western blot on a potato leaf extract was performed with anti-hANP as a probe (Fig. 3D). The two bands resulting from the presence or absence of the signal peptide, respectively, are not seen when the antiserum is preincubated with recombinant AtPNP-A. The result is thus consistent with the in situ result (Fig. 3A–C). The alignment of AtPNP-A used to preincubate the antibody and human ANP (Fig. 3E) indicates the area of the recombinant protein that shares the greatest degree of similarity and most likely contains the epitope.
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Localization of irPNP in ivy using tissue prints
In order to determine the cellular location of irPNPs, tissue prints were made and probed with anti-hANP and anti-StPNP. The anatomical features of a transverse section through an ivy leaf midrib are presented in Fig. 4A. There are three types of mesophyll (parenchyma) cells present enveloped by epidermal cells. One or two layers of palisade parenchyma cells occur immediately under the upper epidermis while spongy parenchyma occur in the leaf lamina. A circular vascular bundle embedded in densely packed parenchyma cells forms the midrib. Xylem cells are located in the centre and inner layer of the vascular bundle and are surrounded by phloem tissue containing phloem cells and parenchyma. The immediate neighbouring transverse sections were used for tissue prints. Probing with tomato anti-glutamine synthetase (anti-GS) resulted in a fairly uniform labelling of the vascular tissues with similar signal strength in the xylem and phloem areas (Fig. 4B). Labelling was detected in the mesophyll, particularly in the midrib area below the central vascular bundle, and also in epidermal regions (Fig. 4B), similar to that reported previously (Pérez-Garcia et al., 1998). Incubation with anti-hANP polyclonal antibodies yielded a more contrasting signal distribution with a strong immunoreaction in association with vascular tissue and conceivably the strongest labelling in the area of the phloem (Fig. 4C). The area circumscribing the outer phloem region (bordering the mespophyll layer) was detected most clearly with the anti-StPNP antisera (Fig. 4 D). In addition, signal was evident in the midrib parenchyma, some spongy parenchyma and the epidermal regions. Since several antibodies were used, two further control experiments were conducted. Firstly, a control with no primary antibody was performed to determine if the secondary antibody bound to any particular regions. Secondly, a control with preimmune sera for the anti-StPNP antisera was made. No signal was observed in either case (data not shown) indicating that the signals observed were due to the primary antibodies.
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Cross-sections and tissue prints of an ivy petiole are seen in Fig. 4E to H. The ivy petiole contains several bicollateral vascular bundles that occur in distinct and separate strands concentrically arranged (Fig. 4E) as detailed previously (Esau, 1965). The phloem surrounds the bundle, but primarily occurs on the periphery of the petiole (Fig. 4F). Parenchyma rays occur throughout the xylem and phloem cells. In the petiole, immunoreactivity to anti-hANP (Fig. 4G) is restricted to the vascular bundle regions, particularly the periphery which corresponds to phloem cells, and the epidermis (Fig. 4G). In turn, the anti-StPNP probed tissue print (Fig. 4H) showed a strongly pronounced localization in the vasculature. The most intense signal is apparent in the periphery of the bundle cells that corresponds to the phloem and phloem parenchyma.
The vascular bundles are arranged in collateral (phloem towards the periphery) circle in the ivy stem (Fig. 5A, B). In tissue prints from ivy stem, the anti-GS signal is evenly distributed in the sub-epidermal mesophyll and absent from the vascular tissue and the pith (not shown). Anti-hANP (Fig. 5C) and anti-StPNP (Fig. 5D) labelling is most prominent in the regions of the conductive tissue and the epidermis. Control prints with pre-absorbed primary antibody treatment showed no signal, suggesting specific responses to the antibodies (not shown). The hexarch vascular bundle is located in the centre of the ivy root, the xylem in the middle encircled by phloem cells with limited secondary structure evident (Fig. 5E). Strong immunolabelling is obtained with anti-StPNP and is associated with the entire phloem region and, possibly, adjacent parts of the cortex but not the xylem (Fig. 5F).
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Tissue localization of irPNP using immunofluorescence
Tissue localization of irPNP was also examined using immunofluorescence on embedded sections of ivy tissue that were labelled with anti-StPNP antibodies followed by FITC-conjugated goat anti-rabbit IgG. Control treatments were made where the sections were only exposed to the fluorescently labelled secondary antibody in the absence of primary antisera (Fig. 6A, C, E) or with hANP pre-absorbed immune serum (not shown). The stomatal ledge wall material is strongly fluorescent in the presence of StPNP antiserum and guard cells themselves show distinct labelling (Fig. 6B). This labelling is associated with the vacuolar membrane rather than occurring throughout the cytoplasm or the cell wall. Labelling of vascular bundles within the leaf was also evident, particularly the cytoplasm-rich bundle sheath cells (Fig. 6D). Within the vascular bundle of the stem, both phloem and xylem regions stain positively (Fig. 6F). The signal in the xylem is strongly associated with wall material or small cells that could be parenchyma rays while cytoplasmic material may also be labelled in the phloem regions.
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Detection of irPNP in xylem exudate
The soft South African perennial forest sage (Plectranthus ciliatus E. Mey ex Benth.) (Codd, 1975) affords relatively easy access to xylem sap and was thus chosen as an experimental system to test if irPNP-like molecules are indeed present in conductive tissue. One ml of exudate per plant was collected, concentrated and analysed with an anti-hANP affinity column on the BioCAD. The result (Fig. 7A) shows the presence of immunoreactant protein. This protein is likely to have been synthesized in the root and is xylem mobile. To test if Plectranthus xylem exudate contained NP activity, it was tested in a stomatal guard cell assay (Fig. 7B) using Arabidopsis thaliana leaves. Stomatal aperture increased when 30 µl xylem exudates (10 µg total protein) was added to 270 µl incubation medium. To test if this effect was due to irPNP, the xylem exudate was run over the anti-hANP column (Fig. 7A) and the unbound proteins of the flow-through were tested. Importantly, no increase in stomatal aperture was observed in leaves exposed to the flow-through material. This indicates that the effect resulting from the complete xylem exudate is due to immunoreactant proteins. Testing of the very small amounts (<50 µg ml–1) of affinity purified proteins proved very difficult, since they are eluted with NaCl and required extensive desalting which makes subsequent recovery and quantification unreliable. As a positive control recombinant AtPNP-A was used at a concentration of 0.1 µg protein ml–1 medium that evoked a similar level of stomatal opening to xylem exudate (Fig. 7B).
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The relative abundance of irPNP in leaves and xylem sap differs (Table 1). Two observations can be made. Firstly, the irPNP content in leaf extracts is less than 1% of the total protein, while it forms 8% of xylem exudate protein. Secondly, while the amount of total protein in the xylem is reduced in the fraction collected between 2 h and 6 h, compared with the fraction collected between 10 min and 2 h the percentage of irPNP is greatly increased in the second fraction. In the sample collected between 2 h and 6 h, 25% of the collected protein is immunoreactant. This observation suggests that irPNP synthesis and/or release into the xylem is certainly not down-regulated and is quite possibly up-regulated as a result of the massive homeostatic disturbance caused by the removal of the shoot. Saturation of the column can be excluded as a source of interference since it is known from the experiments with the column as the tool for the purification of recombinant AtPNP-A that protein concentrations up to 200 µg ml –1 work very well.
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IrPNPs were initially isolated through the use of immunoaffinity to an antibody directed against ANP, a vertebrate peptide signalling molecule with a role in water and solute homeostasis (Billington et al., 1997; Gehring, 1999). Natriuretic peptides, ANPs and irPNPs have been shown to affect water and solute transport (Suwastika and Gehring, 1998; Pharmawati et al., 1999, 2001; Maryani et al., 2000) across plant cell membranes. Furthermore, water transport has been shown to be affected by ANP in vertebrate systems (Wolfensberger et al., 1994; Han et al., 1998) and both ANP and irPNP modulate osmoticum-dependent water transport (Maryani et al., 2001) in protoplasts. The physiological data thus suggests a role for PNP in homeostasis and, in particular, in solute transport. Since such a role may require systemically mobile molecules and distinct spatial localizations it was decided to make use of NP-specific antibodies to identify NPs in situ applying both tissue printing and embedded tissue immunolocalization. These methods have been successfully applied to localize cellular and extracellular proteins in a number of species (Pérez-Garcia et al., 1998; Bravo et al., 1999; Dewitte and Van Onckelen, 2001) and plant hormones, such as abscisic acid (Pastor et al., 1999), cytokinins (Kärkönen and Simola, 1999; Dewitte and Van Onckelen, 2001) and IAA (Shanfa, 2000). The detection of plant proteins and hormones in tissues by immunolocalization thus provides a useful tool to study the distribution of potential signal molecules.
Tissue print immunolocalization was used here to determine in situ where epitopes recognized by anti-hANP and anti-StPNP are within the plant. Anti-StPNP was made by raising antibodies against anti-hANP immunoaffinity purified potato proteins. Anti-hANP antibody recognizes two proteins of >11.5 kDa, and it was observed that this signal is substantially stronger under non-denaturing conditions (data not shown). While both antibodies show the strongest association with the vascular bundle, there is a markedly stronger signal with the anti-plant antibody and this is possibly due to the fact that the latter was raised against a plant protein. The association with the vasculature typically obtained with antibodies against natriuretic peptide-like molecules contrasts with the different hybridization pattern of the anti-GS antibody. This contrast further confirms that antibody based localizations are specific. One apparent discrepancy of signal positioning is the xylem labelling that is not prominent in the tissue prints, but pronounced in the fluorescent images. It appears most likely that the reason for this apparent inconsistency is in the mode of the sample preparation. Tissue printing includes procedures that are likely to, at least partly, remove aqueous and non-bound molecules prior to printing, thus the content of the conductive tissue is likely to be soaked off. In situ fluorescence microscopy, in turn, is done after sample preparation that is conducive to a high degree of topological stability, thus the (frozen and fixed) content of the conductive tissue is much more likely to remain in situ. Further support for the xylem localization comes from the experiments performed with extracted xylem sap of the soft South African perennial forest sage (Plectranthus ciliatus E. Mey ex Benth.). Plectranthus was chosen for the easy accessibility of xylem sap and the choice of this experimental system is further justified by the fact that irPNP-like molecules are highly conserved in all higher plant species tested to date (Ludidi et al., 2002). The Plectranthus experiments also suggest that irPNPs are made in the root (but not just) and are transported to the shoot. In this context, three findings are noteworthy. Firstly, the relative amount of irPNPs expressed as a percentage of total proteins is more than 10 times higher in the xylem than in leaf tissue extracts. Since irPNPs are not likely to be synthesized in the xylem, it follows that cells in the root release the molecules into the conductive tissue where it constitutes a significant part of the total protein content. Secondly, irPNPs extracted from the xylem are affecting stomatal aperture. This finding is significant as it is the first report of a xylem-mobile peptidic molecule synthesized in roots that affects a physiological process in stomata. Thirdly, the relative amount of irPNPs is significantly augmented (Table 1) in a situation of homeostatic disturbance. This observation could indicate that irPNPs are part of a stress response. Thus cellular and functional data add support to the hypothesis of a systemic role for irPNPs.
What sort of molecules are irPNPs? IrPNP from Solanum tuberosum (StPNP) has previously been immunoaffinity purified and partially sequenced and two closely related molecules identified in Arabidopsis thaliana (AtPNP-A and AtPNP-B) (Ludidi et al., 2002). Both related peptides share a significant degree of sequence similarity with CjBAp12, a recently discovered blight-induced protein of unknown function from rough lemon (Citrus jambhiri) (Ceccardi et al., 1998). Furthermore, recombinant AtPNP-A (13 kDa) has been expressed in a yeast expression system and shown that the recombinant peptide is indeed recognized by both anti-hANP and anti-StPNP (G Pironcheva and CA Gehring, unpublished observation). IrPNP-like molecules, much like expansins, contain a N-terminal signal peptide (Ludidi et al., 2002) that directs the molecules to the extracellular space and thus predisposes the molecule for a systemic role. Furthermore, an alignment of AtPNP-A with vertebrate ANPs (Ludidi et al., 2002) shows that the common epitope is not in the signal peptide, but the N-terminus of the cleaved peptide/protein. Consequently, anti-hANP will recognize both the unprocessed (14 kDa) as well as the processed (11.5 kDa) AtPNP-A like molecule. Using RT-PCR it was established that ivy also contains an AtPNP-A like molecule that is >95% identical at the amino acid level with the Arabidopsis molecule (Accession number: AY243474) and that this, in turn, can account for the larger of the two proteins seen in Fig. 1C. AtPNP-B does not contain similarity to the vertebrate epitope and is thus not purified with anti-hANP. The third and smallest peptide is either a different molecule or a further processed product of an AtPNP-A-like molecule that still contains the epitope, but has a truncated C-terminus. The second option is currently favoured since it was observed that stress, such as salinity, leads to an increase in the amount of the smallest processed molecule (G Bradley and CA Gehring; unpublished observation), thus suggesting a further protein-processing step as part of the biological response. Incidentally, protein processing is also an essential part of the synthesis of biologically active ANP (Gehring, 1999; Takei, 2001).
IrPNPs and CjBAp12 are probably not just structural homologues, but may also have some common physiological functions. Since CjBAp12 is induced by citrus blight which leads eventually to die-back, it is conceivable that CjBAp12 is in fact induced in response to homeostatic disturbances leading to the eventual death of the host. If so CjBAp12 may, at least in part, enable the host to counteract blight-induced disturbances of cation and water transport. It is noteworthy that the CjBAp12 message is present in roots but not leaves while the protein is present in xylem sap and leaves (Ceccardi et al., 1998). This must mean that the message in leaves has an extremely short half-life or more likely, that the protein is transported, presumably in the xylem, to the leaves. Such a conclusion is entirely compatible with the in situ localization reported here. However, the immunoreactant protein has previously been isolated from blight-unaffected tissue, suggesting that AtPNP-A is present in plants that are not challenged by pathogens. Similarly, when a RT-PCR approach was used to isolate the Arabidopsis orthologue (AtPNP-A), the template RNA was extracted from non-infected and non-stressed leaf tissue, thus supporting a more general physiological role (Ludidi et al., 2002). This does not preclude a stress-induced up-regulation.
Taken together, molecular organization and known physiological activities of PNP are compatible with the observed in situ localization in the conductive tissue and the association with stomata. Recombinant AtPNP-A is currently being tested with a view to characterizing the biological role of this novel family of molecules further.
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This work was supported by South African National Research Foundation (NRF) grants to GB and CAG and Australian Research Council grants to CAG and DMC. MMM was in receipt of an AusAid doctoral scholarship and MVM holds a NRF doctoral fellowship.
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