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

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<ul><li><p>A role for plant natriuretic peptide immuno-analogues in NaCl- anddrought-stress responses</p><p>Suhail Rafudeena, Gugu Gxabab, Gile Makgokea, G. Bradleya, Ganka Pironchevaa, Lincoln Raittb, Helen Irvingc andChris Gehringa,*</p><p>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: cgehring@uwc.ac.za</p><p>Received 30 January 2003; revised 24 April 2003</p><p>Higher plants contain biologically active molecules that arerecognized by anti-human atrial natriuretic polypeptide rabbitserum (anti-ANP). These molecules are termed immunoreact-ant plant natriuretic peptides (irPNPs) and have previouslybeen shown to be associated with conductive tissue and toaffect ion fluxes, protoplast volume regulation and stomatalguard cell responses. Herein an irPNP from the brassicaceusweed Erucastrum strigosum is identified and it is demon-strated that the relative amounts of irPNP expressed as apercentage of total water : methanol (50 : 50) extracted pro-teins are increased when plants are exposed to 300mM NaCl.</p><p>Since 100 and 200mM NaCl reduce dry and fresh mass aswell as increase total tissue NaCl load, it is hypothesized thatirPNP up-regulation is a late and possibly adaptive response.IrPNP is also significantly up-regulated in Arabidopsisthaliana suspension culture cells in response to 150mM NaCland 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 volumeincreases. This response is dependent on de novo proteinsynthesis and may suggest a complex and possibly regulatoryfunction for irPNP-like molecules in plant homeostasis.</p><p>Introduction</p><p>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</p><p>antiporters (Petrov et al. 1994). It has also been reportedthat ANPs can promote K1 excretion (e.g. Martin et al.</p><p>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 suggest</p><p>that 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 99126) of the</p><p>PHYSIOLOGIA PLANTARUM119: 554562. 2003 Copyright# Physiologia Plantarum 2003Printed in Denmark all rights reserved</p><p>Abbreviations NP, natriuretic peptide; ANP, atrial natriuretic peptide; irPNP, immunoreactant plant natriuretic peptide.</p><p>554 Physiol. Plant. 119, 2003</p></li><li><p>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 test</p><p>how 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.</p><p>Materials and methods</p><p>Chemicals</p><p>Rabbit anti-ANP (128 human) antibody was purchasedfrom Peninsular Laboratories Inc. (Belmont, CA, USA)and the nutrient solution Kompel is from ChemicultProducts Ltd. (Camps Bay, RSA).</p><p>Plant material and growth conditions</p><p>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-</p><p>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 ShapiroWilks 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 (1mgml1) and 500ml naphthylacetic acid(NAA, 1mgml1) 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 at20C until analysed for irPNP content.</p><p>Ion measurements</p><p>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 70C 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.</p><p>Protein analyses</p><p>IrPNP was extracted from E. strigosum leaves. Theleaves (110 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 4C 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 suspension</p><p>cultures. 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 4Cunder continuous stirring. The extract was then filtered</p><p>Physiol. Plant. 119, 2003 555</p></li><li><p>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 separated</p><p>by 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 (128 human) antibody and the ECLPlus Western Blotting Detection System (Amersham-Pharmacia-Biotech, Little Chalfont, UK).The irPNP was affinity purified on a POROS 20 AL</p><p>anti-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 Schiffs 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 20mlmin1. The peaks were integratedand the area under each peak calculated using theBIOCAD chromatogram analysis software.Purified irPNP protein was analysed on a MALDI</p><p>TOF 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 (10mgml1) with 50%acetonitrile, 3% trifluoroacetic acid (TFA) in de-ionizedwater as solvent.</p><p>Synthesis of recombinant protein</p><p>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</p><p>100mgml1 ampicillin (Rose et al. 1990).The cells werecultured at 30C on a rotary shaker at 150 r.p.m.AtPNP-A was obtained by RT-PCR (Ludidi et al.</p><p>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 100mgml1 ampicillin at 30C, 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).</p><p>Protoplast preparation and cell volume measurement</p><p>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.</p><p>Results</p><p>Effect of NaCl on the growth of Erucastrum strigosum</p><p>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.</p><p>Effect of NaCl on the ion levels in Erucastrum strigosum</p><p>The effects of exter...</p></li></ul>

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