Transcript
Page 1: A role for plant natriuretic peptide immuno-analogues in NaCl- and drought-stress responses

A role for plant natriuretic peptide immuno-analogues in NaCl- and

drought-stress responses

Suhail Rafudeena, Gugu Gxabab, Gile Makgokea, G. Bradleya, Ganka Pironchevaa, Lincoln Raittb, Helen Irvingc and

Chris Gehringa,*

aDepartment of Biotechnology, University of the Western Cape, Private Bag X17, Bellville 7535, South AfricabDepartment of Botany, University of the Western Cape, Private Bag X17, Bellville 7535, South AfricacDepartment of Pharmaceutical Biology and Pharmacology, Victorian College of Pharmacy, Monash University, 381 Royal Parade,Parkville, Melbourne, Victoria 3052, Australia*Corresponding author, e-mail: [email protected]

Received 30 January 2003; revised 24 April 2003

Higher plants contain biologically active molecules that are

recognized by anti-human atrial natriuretic polypeptide rabbit

serum (anti-ANP). These molecules are termed immunoreact-ant plant natriuretic peptides (irPNPs) and have previously

been shown to be associated with conductive tissue and to

affect ion fluxes, protoplast volume regulation and stomatalguard cell responses. Herein an irPNP from the brassicaceus

weed Erucastrum strigosum is identified and it is demon-

strated that the relative amounts of irPNP expressed as a

percentage of total water : methanol (50 : 50) extracted pro-teins are increased when plants are exposed to 300mM NaCl.

Since 100 and 200mM NaCl reduce dry and fresh mass as

well as increase total tissue NaCl load, it is hypothesized that

irPNP up-regulation is a late and possibly adaptive response.IrPNP is also significantly up-regulated in Arabidopsisthaliana suspension culture cells in response to 150mM NaCl

and even more so in response to iso-osmolar amounts of sorbitol.Finally, a recombinant A. thaliana irPNP (AtPNP-A) pro-

motes net water-uptake into the protoplast and thus volume

increases. This response is dependent on de novo protein

synthesis and may suggest a complex and possibly regulatoryfunction for irPNP-like molecules in plant homeostasis.

Introduction

Sustaining water and solute homeostasis is a key require-ment for living systems and in vertebrates homeostasis is,in part, achieved by natriuretic peptides (NPs), a familyof peptide hormones (for review see Anand-Srivastavaand Trachte 1993, Kone 2001, Suzuki et al. 2001). Themolecular structures and physiological functions ofnatriuretic peptides (NPs) in animals are the subject ofa large and growing literature. Since natriuretic peptidesare critically involved in salt and water homeostasis inanimals it is not surprising to find that several peptidesof the NP family modulate cation movements (Kourieand Rive 1999). Such modulations include the inhibitionof the apical Na1 channels in the kidney medulla (Zeidel1993) and deactivation of Na1, K1-ATPases (Aperiaet al. 1994). ANPs have been shown to affect Na1/H1

antiporters (Petrov et al. 1994). It has also been reportedthat ANPs can promote K1 excretion (e.g. Martin et al.

1990) and in particular that ANPs increase a K1 con-ductance in rat glomerular mesangial cells (Cermak et al.1996) as well as facilitate a K1 current in atrial ventricu-lar papillary muscle (Kecskemeti et al. 1996). Further-more, Ca21-dependent K1 channels in mesangial cellsare activated by ANP as well as its putative secondmessenger cGMP (Stockand and Sansom 1996). Finally,an additional mechanism that links ANP to the main-tenance of water and salt homeostasis has been reportedin animal systems (Patil et al. 1997). This mechanismimplies a direct and stimulatory effect of ANP on waterchannels.

There is structural and functional evidence to suggestthat an immunologically related peptide hormone systemmay operate in plants (e.g. Vesely et al. 1993, Billingtonet al. 1997, Gehring 1999). First, a synthetic peptideidentical to the C-terminus (amino acids 99–126) of the

PHYSIOLOGIA PLANTARUM119: 554–562. 2003 Copyright# Physiologia Plantarum 2003

Printed in Denmark – all rights reserved

Abbreviations – NP, natriuretic peptide; ANP, atrial natriuretic peptide; irPNP, immunoreactant plant natriuretic peptide.

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rat atrial natriuretic peptide (rANP) binds specifically toisolated leaf microsomes in vitro (Gehring et al. 1996)and leaf tissue in situ (Suwastika et al. 2000). Second,rANP promotes stomatal opening in a concentration-and conformation-dependent manner (Gehring et al.1996, Pharmawati et al. 1998a, 2001). Third, there areindications that this NP effect on stomatal guard cells isinfluenced by cGMP since it does not occur in the pres-ence of LY 83583, an inhibitor of particulate guanylatecyclase, but can be induced by the cell permeant cGMPanalogue 8-Br-cGMP (Pharmawati et al. 1998a, b, 2001).Fourth, and most importantly, we have isolated andpurified by immunoaffinity chromatography biologicallyactive plant natriuretic peptide immuno-analogues(irPNP) (Billington et al. 1997). We have since identifiedand isolated two Arabidopsis thaliana irPNP-encodinggenes termed AtPNP-A and AtPNP-B and describedthe domain organization of the encoded proteins (Ludidiet al. 2002). AtPNP-A encodes a protein of 13 998Daincluding a predicted signal peptide of 3949Da, AtPNP-B encodes a protein of 13 228Da including a signal pep-tide of 3201Da (Ludidi et al. 2002). Both proteins arenovel and have not been functionally characterized; how-ever, AtPNP-B has an orthologue in citrus that is asso-ciated with responses to citrus blight (Ceccardi et al.1998). Citrus blight leads to severe disturbances in hosthomeostasis and eventually to dieback and this responsemay be revealing.

Following on from these findings, we set out to testhow an increased environmental NaCl load would affectthe brassicaceous weed Erucastrum strigosum andexplore if irPNP levels correlate with the growthresponse under conditions of different NaCl loads. Wehave also tested the irPNP response to NaCl and sorbitolin an A. thaliana suspension culture system. The resultsare interpreted with a view to further defining the bio-logical role of irPNP-like molecules in plant homeostasis.

Materials and methods

Chemicals

Rabbit anti-ANP (1–28 human) antibody was purchasedfrom Peninsular Laboratories Inc. (Belmont, CA, USA)and the nutrient solution ‘Kompel’ is from ChemicultProducts Ltd. (Camps Bay, RSA).

Plant material and growth conditions

Erucastrum strigosum seeds were germinated directly inpure silica sand in pots (diameter: 15 cm) and wateredwith ‘Kompel’, a complete nutrient solution includingboth macro- and micronutrients in a greenhouse underseasonal light conditions. After the emergence of seed-lings, they were thinned out to leave two per pot. Theplants were grown in a random block design experimentand the blocks were replicated three times. Four final saltconcentrations (0, 100, 200 and 300mM NaCl) wereapplied once the plants were well established with step-

wise increases in salinity of 100mM per week until thehighest concentration was reached. The plants were har-vested after they had been subjected to the respectivefinal salt concentration for 1week. Fresh and dry mass,as well as mineral content was assessed and subjected toa multifactorial analysis. A Shapiro–Wilks test was per-formed to assess for non-normality, and orthogonalpolynomials were extracted from each factor. Arabidopsisthaliana cell suspensions were grown from fresh callussuspended in sterile MSMO (Murashigo and SkoogMinimum Organics) media (4.43 g MSMO, 30 g sucrose,50 ml kinetin (1mgml�1) and 500ml naphthylacetic acid(NAA, 1mgml�1) made up to a volume of 1 litre,pH 5.7). The A. thaliana cell suspension cultures weresubcultured by adding 10ml of a 1-week-old culture to90ml of fresh MSMO media. The subcultures weregrown for 72 h before the NaCl and sorbitol treatmentswere performed on respective 100ml aliquots of the cellsuspensions. The cell suspensions were then allowed togrow for a further 24h after which 50ml from each of thecell suspensions was centrifuged at 3000� g for 15min.The pellet and supernatants were stored separately at�20�C until analysed for irPNP content.

Ion measurements

On harvesting the E. strigosum plants, the roots wereseparated from the shoots. The shoots were weighted toobtain the fresh mass, then dried in an oven at 70�C toconstant dry mass. The oven-dried shoots were ground ina Wiley mill and acid digested with H2SO4 and H2O2.Cations (Ca21, Mg21, K1 and Na1) were assayed usingan UNICAM Solar M Series Atomic Absorption Spec-trophotometer (Cambridge, UK). The growth and iondata were then subject to variance analysis.

Protein analyses

IrPNP was extracted from E. strigosum leaves. Theleaves (1–10 g) were snap-frozen in liquid N2 and groundto a fine powder with a mortar and pestle. The powderwas then re-suspended in 50ml extraction buffer (50mMKCl, 1mM EDTA, 10mM Tris-HCl; pH7.4) to whichwas added an equal volume of methanol. Extraction wasallowed to proceed for 60min at 4�C under continuousstirring. The extract was then filtered through glass wooland the filtrate centrifuged at 15 000� g for 10min. Theresulting supernatant was freeze-dried and re-suspendedin 2ml of H2O.

IrPNP was extracted from A. thaliana cell suspensioncultures. Cells were harvested from the suspension cul-tures (100ml) by centrifugation at 3000� g in a Eppendorfbenchtop centrifuge. The cell pellets were frozen inliquid N2 and ground to a fine powder with mortar andpestle. The powder was re-suspended in 50ml extractionbuffer (50mM KCl, 1mM EDTA, 10mM Tris-HCl;pH 7.4) to which was added an equal volume of metha-nol. Extraction was allowed to proceed for 60min at 4�Cunder continuous stirring. The extract was then filtered

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through glass wool and the filtrate centrifuged at15 000� g for 10min. The resulting supernatant wasfreeze-dried and re-suspended in 2ml of H2O. The super-natant was dialysed against several changes of water,freeze-dried and taken up in 2ml of H2O.

Aliquots of the concentrated peptides were separatedby SDS-polyacrylamide (15%) gel electrophoresis usinga Mini-Protean 3 system (Bio-Rad, Hercules, CA, USA).Total protein was detected by silver staining (SilverQuest Silver staining Kit; Invitrogen, Carlsblad, CA,USA). Western blots were performed with a Mini-Protean 3 Transfer System and the irPNP detected usingrabbit anti-ANP (1–28 human) antibody and the ECLPlus Western Blotting Detection System (Amersham-Pharmacia-Biotech, Little Chalfont, UK).

The irPNP was affinity purified on a POROS 20 ALanti-ANP affinity column using the BIOCAD SPRINTsystem (Applied BioSystems, Foster City, CA, USA). Thecolumn was prepared as follows: Rabbit anti-ANP anti-body was re-suspended in coupling buffer (0.1M HEPES,pH7). An aliquot of the re-suspended rabbit anti-ANPantibody (55ml) was added to 0.8 g POROS 20 AL resin(PerSeptive Biosystems, Framingham, MA, USA) in 5mlcoupling buffer and rotated at room temperature for 8 h.The resultant Schiff’s base was reduced by adding 5mgNaBH4 per ml bed volume (11.5mg) to the couplingsolution and rotated for a further 2 h at room tempera-ture. The coupling mixture was centrifuged at 20 000 g for1min at room temperature and the supernatant discarded.Residual aldehydes were quenched by adding 1.5ml 0.2MTris buffer containing 11.5mg NaBH4 to the pelleted resinand rotated at room temperature for 2 h. The POROS 20AL anti-ANP affinity resin was packed into a POROSPEEK column (4.6mm [D]� 100mm [L], 1.7ml) andwashed with 10 column volumes of equilibration buffer(1mM Tris/HCl, pH7.5). To quantify the level of irPNPin the plant extracts, 50ml was applied to the POROS 20AL anti-ANP affinity column. The column was washedwith 24 column volumes (CV) equilibration buffer andthe bound protein eluted with 5CV equilibration buffercontaining 1MNaCl before re-equilibrating the resin with15CV equilibration buffer. Aliquots (1ml) were collectedat a flow rate of 20mlmin�1. The peaks were integratedand the area under each peak calculated using theBIOCAD chromatogram analysis software.

Purified irPNP protein was analysed on a MALDITOF mass spectrometer (Voyager-DE BiospectrometryWork Station; PerSeptive Biosystems) to determine themolecular mass of the isolated proteins. The MALDI-MS was fitted with a nitrogen UV laser (337 nm), and thematrix used was Sinapinic Acid (10mgml�1) with 50%acetonitrile, 3% trifluoroacetic acid (TFA) in de-ionizedwater as solvent.

Synthesis of recombinant protein

Saccharomyces cerevisiae Y294 were cultured on eitherYPD medium: 1% yeast extract, 2% peptone, 2% glu-cose or on selective synthetic (SC) medium for yeast with

100mgml�1 ampicillin (Rose et al. 1990).The cells werecultured at 30�C on a rotary shaker at 150 r.p.m.

AtPNP-A was obtained by RT-PCR (Ludidi et al.2002) digested with HindIII and XhoI and ligated intopBluescriptSK (1/–) (Stratagene, North Torrey PinesRoad, La Jolla, CA, USA). The AtPNP-A gene wasthen digested from this plasmid with EcoRI and XhoIand ligated into the YEp352 plasmid (Stratagene) carry-ing the ADH2 promoter and terminator. DNA and pro-tein analyses were performed using the DNAMANversion 4.13. Saccharomyces cerevisiae Y294 transformedwith the YEp-AtPNP-A were cultured for 48 h in SCmedium with 100mgml�1 ampicillin at 30�C, then pelletedand ground with glass beads under liquid N2 in 0.05MTris-HCl (pH7.5) and 0.5M NaCl. The lysate was spunat 20 000 g for 60min at room temperature, then washedwith four volumes of 0.05M Tris-HCl (pH7.5), 0.5Murea, 0.5M NaCl and stirred in the same buffer for30min. This step was repeated twice. The protein wasconcentrated and desalted with an Ultrafree-CL Amiconcolumn (Millipore, Bedford, MA) prior to immuno-affinity purification (see above).

Protoplast preparation and cell volume measurement

Protoplast isolation from cell cultures of A. thaliana(100ml) were obtained as described previously(Pharmawati et al. 2001) with the exception of the sus-pension medium used which was 0.4M sorbitol. Theprotoplasts were incubated with the recombinant proteinfor 10min at room temperature, visualized under themicroscope with a calibrated micrometer and picturesof the protoplasts were taken. The volumes of morethan 50 randomly selected protoplasts of the controlsand the samples were calculated and the results analysedby an ANOVA and paired Students t-test.

Results

Effect of NaCl on the growth of Erucastrum strigosum

A step-wise increase (see methods) of the external NaClload was applied to E. strigosum plants in order toinduce different degrees of NaCl stress. Fresh and drymass values of Erucastrum shoots (leaves and stems)grown under standard greenhouse conditions (see methods)reveal the effect of additional NaCl to the growthmedium (Fig. 1). The step-wise increase of externalNaCl load significantly reduced shoot fresh mass. Thebiggest fresh mass reduction was reached at 300mMexternal NaCl. In contrast, a significant loss in drymass was only observed between 0 and 100mM externalNaCl. Further external increases in NaCl did not signifi-cantly impact on dry mass values.

Effect of NaCl on the ion levels in Erucastrum strigosum

The effects of external NaCl load on Na1, K1, Ca21 andMg21 in shoots are presented in Fig. 2A and B. Increases

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in Na1 were highly significant and steepest between0mM salt and the external concentration of 100mMNaCl (Fig. 2A). The largest Na1 load was observed at300mM. The shoot K1 load showed no significantchanges and possibly a slight downwards trend between0 and 100mM external NaCl (Fig. 2A).

Changes in external NaCl over the whole rangebetween 0 and 300mM were not reflected in shootCa21 content (Fig. 2B). In contrast, the shoot Mg21

content decreased significantly with external NaCl andthe steepest decrease was seen between 0 and 100mMNaCl (Fig. 2B).

Effect of NaCl on irPNP levels in Erucastrum strigosum

The effect of NaCl load on the relative abundance ofirPNPs in total liquid phase [water :methanol; 50% (v/v)]-extracted proteins in shoots and leaves was alsoassessed. Anti-ANP was subsequently coupled to aPOROS 20 matrix to affinity purify total liquid phase-extracted proteins (irPNPs). The elution profile of pro-teins extracted from unstressed plants (Fig. 3A) revealedthat the bulk of proteins was not retained (firstpeak;. 95%) and that at fraction 40 the baseline wasreached again. A subsequent increase in NaCl released thebound affinity-purified fractions that contain immunoreact-ant molecules (Fig. 3A). The SDS-Page gel (Fig. 3B) ofimmunoaffinity-purified irPNP from untreatedE. strigosumplants revealed that two species of molecules in the massrange between 6.5 and 14.3kDa were retained.

Subsequent mass spectroscopic analyses of a proteinextract from NaCl-stressed E. strigosum post-affinitycolumn, showed a mass of 11.685 kDa (Fig. 3C) whereasthe smaller peak (11.8907 kDa) observed in the massspectroscopy trace was an internal standard applied con-

currently with the sample. We have subsequently clonedthe E. strigosum AtPNP-A orthologue (Fig. 3D). Thededuced amino acid sequence of the E. strigosum ortho-logue revealed that only a single amino acid was replaced(isoleucine to valine) and that the molecule also containsa putative signal peptide that was typical for excretedproteins and peptides. The sequence (accession numberAAM18791), including the putative signal peptide, con-tained 126 amino acids and had a predicted mass of14.016 kDa, whereas the protein after removal of thesignal peptide had only 104 amino acids and a predictedmass of 11.676 kDa. The latter corresponded very closelyto that obtained from the mass spectroscopy trace(11.685 kDa, Fig. 3C) indicating that the immunoreact-ant isolated from the NaCl-stressed E. strigosum does

Fig. 1. NaCl-dependent changes in plant mass wet and dry mass.Wet (——) and dry (- - - -) mass changes in response to stepwiseincreased (see methods) NaCl treatments of Erucastrum strigosumshoots. There was no evidence against normality (P. 0.05).Student’s t-least significant differences (LSD) were calculated at a5% level to compare treatment means. Data points with differentletters are significantly different from each other.

Fig. 2. Total Na1 and K1 content (A) and total Ca21 and Mg21

content (B) are expressed in mg g�1 dry mass. Plant material wasdried to constant dry mass, ground, acid digested and assayed byatomic absorption spectrophotometry. Ion data were subject tovariance analysis. Identical letters signify no significant differences(P, 0.01) between points, while different letters denote significantdifferences between points.

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not contain the signal peptide. The same immunoaffinitycolumn was also subsequently used to determine andquantify the proportion of irPNP molecules in the totalliquid-extracted proteins from samples after differentNaCl exposure. The peaks obtained in the elution pro-files were integrated and the area under the peaks calcu-

lated using the BIOCAD chromatogram analysissoftware. The ratio for the sample with no added NaClwas designated the value of 100%. Increasing the exter-nal NaCl challenges of the E. strigosum led to insignifi-cantly decreased percentages of irPNP at 100 and200mM NaCl. However, a significant (P, 0.05) increasewas observed at 300mM NaCl (Fig. 4). Each bar repre-sents the mean of three determinations and the sametrend was observed in three independent series of experi-ments. The affinity column result was further validatedwith a western blot of protein extracted from salt-challenged plants and eluted from the affinity column(Fig. 4; inset). Since the dry weight was decreasing inresponse to NaCl treatment (Fig. 1), the observedincrease in irPNP can be attributed to either specificresistance to protein degradation or increased synthesisin response to the biotic stress at 300mM NaCl.

NaCl and osmotic stress induce irPNP increases in

Arabidopsis thaliana

When different NaCl treatments were applied to A. thalianasuspension culture cells it was observed that the additionof 50mM NaCl did not impair growth whereas thehigher concentrations tested, exerted a marked effect(Fig. 5A). At 100mM NaCl growth was slowed downwhereas at 150mM NaCl growth was arrested. It wasalso observed that the cell suspension cultures treatedwith 50mM NaCl were identical to the control whereasthe cultures treated with 100mM NaCl were slightlybleached and those with 150mM NaCl were totallybleached (unpublished observation). Determination ofthe proportion of irPNP molecules in the total liquid-extracted proteins from the cell pellet, 24 h after treat-ment, revealed a slight reduction at 50mM NaCl and a

Fig. 3. Isolation of irPNP and BIOCad immunoaffinity purificationof irPNP from untreated shoot tissue extracts (A). The arrow(!)indicates the release of bound immunoreactant protein withincreasing ionic strength. The solid line is the protein trace and thebroken line represents the NaCl concentration. Inset: Silver stainedSDS gel of protein marker (M) and total extracted protein (TP).SDS-Page gel of immunoaffinity-purified irPNP from untreatedErucastrum strigosum shoots (B). Mass spectroscopic trace of irPNPfrom Erucastrum strigosum shoots treated with 300mM NaCl (C).Amino acid sequence of the Erucastrum strigosum irPNP-Aorthologue (D). Open arrow (

)

) indicates the C-terminal end ofthe putative signal peptide, the solid arrow (!) shows the aminoacid substitution (I!V) and the underlined sequence is the domainthat shows similarity to vertebrate natriuretic peptides.

Fig. 4. Relative amount of irPNP expressed as percentage of totalprotein. The 100% level is defined as the percentage of irPNP intotal extracted protein from untreated samples. The bars representthe standard deviations of triplicate measurements. Different lettersrepresents a significant change (P, 0.01). Inset: Western blot ofproteins from unstressed plants (C), plant exposed to 100mM NaCl(1) and (2) 300mM NaCl, probed with anti-ANP.

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slight elevation at 100mM NaCl (Fig. 5B). At 150mMNaCl the relative irPNP levels were at their highest(Fig. 5B). There was no significant difference betweencontrol and 50mM NaCl (P. 0.01) and between controland 100mM NaCl (P. 0.01), but there was a significantincrease above control at 150mM NaCl treatment(P, 0.01).

When osmotic stresses were applied with sorbitolrather than NaCl, cell growth was not affected at100mM sorbitol and only slightly reduced at 200 and300mM sorbitol (Fig. 6A). Determination of irPNPmolecules accumulated after 24 h. (Fig. 6B) showed nosignificant difference above control for the 100mM

(P. 0.01) and 200mM (P. 0.01) sorbitol treatment,but a significant increase at the 300mM sorbitol(P, 0.01). At this concentration, 2.88% of total proteinwas immunoreactant.

Recombinant AtPNP-A causes protoplast swelling

In order to test physiological responses to irPNP-A-likemolecules, AtPNP-A was cloned and expressed in a yeastvector. Purification on the anti-ANP affinity columndemonstrated that the recombinant AtPNP-A is a bonafide natriuretic peptide immuno-analogue (Fig. 7A; seeinset). Since osmotic stress induced an irPNP

Fig. 5. Effect of NaCl onArabidopsis thaliana cells insuspension culture (A) and irPNPexpressed as percentage of totalprotein (B). The bars represent thestandard deviation of triplicatemeasurements and differentletters represent a significantchange (P, 0.01). Cell suspensioncultures were grown for 72 hbefore treatment with differentconcentrations of NaCl. The cellsuspension cultures were grownfor a further 24 h beforeharvesting and irPNP extractionand analysis. Protein extraction ofcell pellets, obtained aftercentrifugation, was performedusing the extraction proceduredetailed in the methods. Theextracted protein was freeze-driedand re-suspended in 2ml dH2O.Aliquots (500ml) were analysed onthe BioCAD using an anti-ANP-affinity column.

Fig. 6. Effect of sorbitol onArabidopsis thaliana cells insuspension culture (A) and irPNPexpressed as percentage of totalprotein (B). Cell suspensioncultures were grown for 72 hbefore treatment with variousconcentration of sorbitol. The cellsuspension cultures were grownfor a further 24 h beforeharvesting for irPNP analysis.Protein extraction of cell pelletsobtained after centrifugation wasperformed using the methanolextraction procedure described inthe methods. The extractedprotein was freeze-dried and re-suspended in 2ml dH2O. Aliquots(500ml) were analysed on theBioCAD using an anti-ANP-affinity column (see methods).The bars with different lettersrepresent significantly (P, 0.01)different values.

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up-regulation, we were interested to investigate the effectof this molecule in H2O transport. To this end we haveisolated protoplasts in 400mM sorbitol and suspendedthem in 400mM sorbitol. The result shows (Fig. 7B) thatsignificant and concentration-dependent increases inaverage cell volume result from this treatment. Moreover(Fig. 7C), the increase in response to 50 ng recom-binant proteinml�1 was significantly reduced when thecells had been pre-incubated for 30min with the proteinsynthesis inhibitor cycloheximide (50 ngml�1). Thisimplies that de novo protein synthesis was required forthis AtPNP-A-dependent reaction to occur.

Discussion

Erucastrum strigosum is a fast-growing brassicaceus weedthat we use as a model plant for some physiological andecophysiological studies. Contrary to the closely related

A. thaliana, E. strigosum lends itself to growth in puresilica sand in pots and a single plant yields enoughbio-mass for both protein analyses including affinity puri-fication, RNA extractions and multiple ion determinations.Here the effect of step-wise increases of the externalNaCl load were applied in order to induce differentdegrees of NaCl stress. Fresh and dry mass values ofE. strigosum shoots reflect the effect of added NaCl tothe growth medium (Fig. 1). Decreases in fresh massparallels increasing external salt concentrations up tothe highest concentration (300mM), whereas dry massonly decreased significantly up to 100mM. The gradualdecrease in fresh weight suggests water loss as a result ofhigh exterior NaCl and the pattern of dry mass loss mayindicate that the initial major metabolic disturbanceoccurs at or below 100mM NaCl.

There is a significant and continual increase in shootNa1 content with the increase in external Na1 right up

Fig. 7. Expression and isolation ofAtPNP-A (A) and effect ofrecombinant AtPNP-A onprotoplast volume (B). Theasterisk (*) in the elution profileindicates the release of boundimmunoreactant protein withincreasing ionic strength. The solidline is the protein trace and thebroken line represents the NaClconcentration. Inset (left): Proteinfrom induced (Ind) and non-induced yeast (C) resolved on asilver-stained SDS gel. Inset(right): Silver-stained SDS-Page gelof immunoaffinity-purified AtPNP-A. The bars in (B) represent meanA. thaliana protoplast volumes inresponse to increasing amounts ofAtPNP-A, the bars in (C) comparemean protoplast volumes in controland treated protoplasts (50ngAtPNP-Aml�1) in the presence orabsence of cycloheximide(50ngml�1). Open bars representprotoplasts treated withoutcycloheximide and filled barsrepresent protoplasts treated withcycloheximide.

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to 300mM (Fig. 2A). Magnesium shows an initialdecrease (Fig. 2B) whereas Ca21 and K1 levels do notchange significantly demonstrating that K1 loss to com-pensate for increased Na1 loading does not occur. It isnoteworthy that neither Na1 concentrations nor dry andfresh mass parameters significantly change above300mM external NaCl while at this NaCl concentrationthe highest relative irPNP concentration is reached. Theincrease in irPNP at 300mM could be explained by threemechanisms. First, irPNP resists ubiquitin-dependent orproteolytic degradation, second, the molecule is in acompartment that is protected from degradation (e.g.the conductive tissue) or third, an increased synthesis inresponse to the biotic stress occurs.

The results presented in Fig. 3 may point to stressinduced processing of irPNPs. Two protein species, thefull length protein and the protein minus the signal pep-tide are present in unstressed plants (Fig. 3B) whereasunder stress conditions, only the shorter sequence is pres-ent (Fig. 3C). Since the role of the signal peptide is toensure export into the cytosol, the findings reported hereare both compatible with the domain architecture ofirPNPs (Ludidi et al. 2002) and their postulated bio-logical role as extracellular and mobile signalling mole-cules (Maryani et al. 2003).

In a further series of experiments the responses toNaCl and osmo-equivalents of sorbitol on growthwere investigated in an A. thaliana suspension culturesystem. This system is well characterized in terms ofgrowth parameters and allows for monitoring rapidresponses of synchronized cell populations. Theresponse to NaCl at 100mM NaCl which leads to areduction of growth does not cause a relative increasein irPNP levels whereas at 150mM NaCl growth isarrested and the irPNP levels are significantly elevated.This pattern may suggest an irPNP response to counter-act increasing osmotic stress rather than to modulateNaCl transport or compartmentalization. Such aninterpretation is supported by the observed responseto sorbitol in which only the concentration of 300mM(the osmo-equivalent of 150mM NaCl) leads toincreased irPNP levels.

In order to progress with the characterization of theplant natriuretic peptide system and its biological func-tions and mechanisms of action, we have produced arecombinant A. thaliana (AtPNP-A) in a yeast expres-sion system. First, the fact that the recombinant cansuccessfully be affinity-purified on an anti-ANP columnestablishes AtPNP-A as a true plant immunoanalogue ofANPs. Second, the recombinant is biologically activeand leads to concentration-dependent increases in proto-plast volume. Such volume increases in turn must becaused by net H2O uptake. We argue that this net uptakemay not be a direct response to net cation uptake mainlybecause we have previously shown that in Zea mays steletissue net K1 uptake in response to irPNP occurs with asignificant delay (. 20min) (Pharmawati et al. 1999) andduring this lag phase protoplast expansion has alreadyoccurred. If the two experimental systems, the maize stele

and A. thaliana protoplasts, are indeed comparable, ourinterpretation would be that K1 net uptake is in fact aresponse to net H2O uptake rather than the cause for it.The observed delayed K1 uptake could restore the cyto-solic concentration that was lowered as a result of volumeincreases. We also demonstrate that the swelling responseis significantly reduced when protein synthesis is inhibitedby cycloheximide and this argues for a mechanism thatrequires rapid de novo protein synthesis. Although it isnot clear at present what specific proteins are synthesizedin response to AtPNP-A, it is conceivable that a rapidproduction of compatible solutes could account for theobserved effects and such a production might require denovo synthesis of enzymes. However, a more precise pic-ture will emerge with the results of proteomics-basedexperiments that are currently being undertaken.

We have previously reported several direct or indirecteffects of both ANP and irPNP on stomatal guard cells(e.g. Gehring 1999, Pharmawati et al. 2001), ion trans-port (Pharmawati et al. 1999, Maryani et al. 2000) andosmoticum-dependent volume regulation (Maryani et al.2001). These observations, supported by biochemicalbinding assays (Gehring et al. 1996, Suwastika et al.2000) and in situ localization (Maryani et al. 2003)have led to the conclusion that plants specifically recog-nize biologically active molecules that share an epitopewith the highly conserved vertebrate ANPs. In addition,similarities in biological responses to ANP and irPNP inplants (e.g. Gehring 1999) have implied that irPNP-likemolecules might have a role in plant homeostasis muchlike ANPs have in vertebrates. Such a role, we arguemight necessitates a change in irPNP levels in responseto changes in water and salt balance.

Recently, progress has been made in the elucidation ofthe molecular structure of irPNP-like molecules. InA. thaliana two irPNP encoding genes (AtPNP-A andAtPNP-B) have been identified and isolated (Ludidiet al. 2002). AtPNP-A and -B are small proteins of lessthan 130 amino acids and are related to expansins.AtPNP-A and -B fall in two groups. Members of bothgroups share sequence homology with expansins but donot contain the tryptophan and tyrosine-rich C-terminalputative polysaccharide-binding domain that is typical ofexpansins or bacterial cellulases and hemicellulases(Ludidi et al. 2002). We have argued that both irPNP-like molecules and expansins have evolved from ancestralglucanase-like molecules that hydrolysed the cellwall. We have speculated that the absence of such apolysaccharide-binding domain would increase the mobilityof these molecules. Importantly, we have previouslydemonstrated that irPNPs act on protoplasts (Maryaniet al. 2001), that is plant cells without cell walls as well asmicrosomes, indicating that these novel proteins specifi-cally interact with the plasma membrane. In addition, wehave shown association of irPNP immunoreactivity withconductive tissue in situ and have extracted biologicallyactive immunoreactants from the xylem, thus supportingthe hypothesis of a systemic role of irPNP-like molecules(Maryani et al. 2003).

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In summary, the concept of increased mobilitytogether with the previously reported effects of irPNPson ion and solute transport (Pharmawati et al. 1999,Maryani et al. 2001) and the demonstrated up-regulationin response to NaCl and osmoticum stress suggests thatirPNP-like molecules have a systemic function in planthomeostasis and abiotic stress responses in particular.The exact nature of the function and the biochemicalmechanisms await further elucidation.

Acknowledgements – This work was supported by South AfricanNational Research Foundation (NRF) grants to L.R. and C.A.G.M.S.R. receives a Claude Leon Harris Foundation postdoctoralfellowship.

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