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Plant Physiology and Biochemistry 47 (2009) 796–806

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Plant Physiology and Biochemistry

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Research article

Roles of a membrane-bound caleosin and putative peroxygenase in bioticand abiotic stress responses in Arabidopsis

Mark Partridge, Denis J. Murphy*

Biotechnology Unit, Division of Biological Sciences, University of Glamorgan, Treforest, CF37 1DL, United Kingdom

a r t i c l e i n f o

Article history:Received 3 September 2008Accepted 27 April 2009Available online 9 May 2009

Keywords:Abiotic stressBiotic stressCaleosinOxylipinsPeroxygenaseLeptosphaeria maculansSalicylic acid

* Corresponding author. Tel.: þ44 1443 483 747; faE-mail address: dmurphy2@glam.ac.uk (D.J. MurpURL: http://people.glam.ac.uk/view/184

0981-9428/$ – see front matter � 2009 Elsevier Masdoi:10.1016/j.plaphy.2009.04.005

a b s t r a c t

We report here the localisation and properties of a new membrane-bound isoform of caleosin and itsputative role as a peroxygenase involved in oxylipin metabolism during biotic and abiotic stressresponses in Arabidopsis. Caleosins are a family of lipid-associated proteins that are ubiquitous in plantsand true fungi. Previous research has focused on lipid-body associated, seed-specific caleosins that haveperoxygenase activity. Here, we demonstrate that a separate membrane-bound constitutively expressedcaleosin isoform (Clo-3) is highly upregulated following exposure to abiotic stresses, such as salt anddrought, and to biotic stress such as pathogen infection. The Clo-3 protein binds one atom of calcium permolecule, is phosphorylated in response to stress, and has a similar peroxygenase activity to the seed-specific Clo-1 isoform. Clo-3 is present in microsomal and chloroplast envelope fractions and has a type Imembrane orientation with about 2 kDa of the C terminal exposed to the cytosol. Analysis of ArabidopsisABA and related mutant lines implies that Clo-3 is involved in the generation of oxidised fatty acids instress related signalling pathways involving both ABA and salicylic acid. We propose that Clo-3 is part ofan oxylipin pathway induced by multiple stresses and may also generate fatty acid derived anti-fungalcompounds for plant defence.

� 2009 Elsevier Masson SAS. All rights reserved.

1. Introduction Caleosin proteins are characterised by a single calcium binding

Caleosins comprise a group of related genes and their encodedproteins that are probably ubiquitous in multicellular plants, greenalgae, and the true fungi [30]. Caleosin-like genes are present in allhigher plant genomes sequenced to date, including Arabidopsis,rice, rapeseed, barley, soybean, maize, wheat, cotton and loblollypine. Very similar genes are found in the bryophytes Physcomitrellapatens and Selaginella tamariscina and in the two divergent algalspecies Auxenochlorella protothecoides and Chlamydomonas rein-hardtii. Intriguingly, caleosin-like genes are also present in a widerange of fungi, including many economically important plant andanimal pathogens, such as Aspergillus niger, Auxenochlorella oryzae,Auxenochlorella terreus, Auxenochlorella fumigatus, Auxenochlorellanidulans, Ustilago maydis, Magnaporthe grisea, Neurospora crassa,Chaetomium globosum and Coprinus cinereus as well as the mycor-rhizal symbiont, Laccaria bicolor. However, caleosin-like sequencesappear to be absent from the genomes of the yeasts, as well as fromanimal and prokaryotic genomes.

x: þ44 1443 482 285.hy).

son SAS. All rights reserved.

EF hand motif, a putative membrane bilayer spanning domain, plusseveral potential phosphorylation and haem-binding sites. Struc-tural studies with recombinant seed-specific caleosins indicate thatthe native proteins are able to bind calcium [8,48], phosphate [40]and haem [17]. Caleosins appear to be highly flexible proteins thatcan dramatically alter their secondary structures in response to thepolarity of the medium in which they are embedded [40]. Caleosinsare most frequently described in the literature as oil-body associ-ated proteins occurring in storage tissues, such as developing orgerminated seeds or caryopses [1,25,30,49] and in somatic embryos[7], and several caleosin isoforms are prominent components of theoil-body proteome [10,22]. However, we have previously noted thepresence of membrane associated caleosin isoforms in subcellularfractions from a range of non-seed tissues, including leaves, flowersand roots [18].

The genome of Arabidopsis thaliana contains six expressedcaleosin-like sequences for which corresponding ESTs can bedetected, plus one caleosin-like pseudogene [36]. Microarray andEST studies show that the oil-body associated caleosins, Clo-1 andClo-2, are among the most highly expressed genes in developingseeds [12,29]. These seed-specific caleosin isoforms may havespecialised roles in processes associated with oil-body formationand mobilisation [31,32,37]. Similarly, in barley, two caleosin-likegenes were mainly expressed in storage tissues and appeared to

M. Partridge, D.J. Murphy / Plant Physiology and Biochemistry 47 (2009) 796–806 797

have both oil-body and vesicle membrane locations [25]. A widerphysiological role for caleosins in seeds was recently suggested bythe demonstration that the Arabidopsis oil-body associated Clo-1protein has a calcium-dependent haem–oxygenase activity that isregulated by one or two conserved ferric-binding histidine residues[17]. A peroxygenase of this type is likely to be involved in theformation of epoxy hydroxy alcohols from fatty acid hydroperox-ides. These and other oxylipin metabolites are known to playprominent roles in plant responses to a range of biotic and abioticstresses, from drought tolerance to fungal infection. Similar oxy-lipins are also involved in various aspects of fungal spore devel-opment and probably serve in some fungi as anti-fungalcompounds that can deter the growth of competing fungal species[50].

Although several of the oil-body associated caleosins have beencharacterised, the role(s) of the other four caleosin-like genes thatare expressed in the Arabidopsis genome remain to be determined.One clue to their possible function is that the first caleosin-likesequences were originally reported to be among the most highlyresponsive genes to treatments with abscisic acid (ABA) or osmoticstress in rice caryopses [9]. One of the major ABA-inducible anddesiccation stress related proteins in Arabidopsis, termed RD20[48,53], is now known to be a non-seed caleosin isoform, which wehave termed Clo-3. We therefore selected Clo-3 for further analysiswith regard to its localisation, regulation, activity and possible rolesin various form of stress response in plants. In this study, we reportthe regulation of Clo-3 following abiotic stresses, including salt,drought, cold and heat; biotic stress such as infection with thehemibiotrophic fungus, Leptosphaeria maculans; or treatment witha variety of phytohormones or analogues including abscisic acid(ABA), 2,6-dichloroisonicotinic acid (DCINA), jasmonic acid (JA) andsalicylic acid (SA). We also report on the subcellular localisationand membrane orientation of Clo-3, its capacity to bind calciumand phosphate, its activity as a peroxygenase, and its possible rolein plant oxylipin signalling pathways.

2. Methods

2.1. Plant growth

A. thaliana (Columbia ecotype), originally obtained from NASC(National Arabidopsis Stock Centre, Nottingham, UK) but propa-gated locally, was grown either in soil or hydroponically under glassat 22 �C day/15 �C night under long day conditions (16 h light/8 hdark cycles) to flowering stage. For growth in soil, seeds were sownon a 1:1:1 mixture of vermiculite, perlite and peat placed at 4 �C for4 days to break residual dormancy, and transferred to normalgrowth conditions. For hydroponic growth, the seeds were grownas outlined in Huttner & Bar-Zvi [20] using the growth medium ofGibeaut et al. [11]. Nutrient concentrations were 1.5 mM Ca(NO3)2,1.25 mM KNO3, 0.75 mM MgSO4, 0.5 mM KH2PO4, 70 mM Fe-diethylenetriamine pentaacetate, 50 mM KCl,50 mM H3BO3, 10 mMMnSO4, 2 mM ZnSO4, 1.5 mM CuSO4, and 0.075 mM ammoniummolybdate. The transgenic Arabidopsis thaliana line containing theNahG gene was obtained from the ABRC, Ohio State University,Columbus. The mutants npr1-1, npr1-2, aba1-1 and aba151 wereprovided by European Arabidopsis stock centre at NottinghamUniversity, UK. All mutant and transgenic lines were derived fromthe Columbia (Col-0) ecotype.

Potted plants subject to abscisic, and jasmonic acid treatmentswere sprayed with 200 mM of the respective hormone or a watercontrol once per day for the required incubation period at roomtemperature under white light. For salt treatment plants weregrown hydroponically and supplemented with 200 mM NaCl atroom temperature. For cold treatment, potted plants were

transferred to a 4 �C growth chamber under white light. Fordrought treatment, plants grown in potted soil were carefullyremoved and dehydrated on filter paper as described by Yama-guchi-Shinozaki & Shinozaki [53]. Roots, leaves, stems, flowers andsiliques were harvested after the appropriate stress incubationperiod and either snap frozen in liquid nitrogen and stored at�80 �C for subsequent RNA extraction or immediately used forprotein extraction.

2.2. Subcellular fractionations

All steps for homogenisation and centrifugation were performedat 4 �C. Plant tissues (leaves, stem, silique, root and flower) wereblended in an Ultra-Turrax� homogeniser or ground with a mortarand pestle in 100 mM Hepes (pH 7.5) buffer containing 0.4 Msucrose, 10 mM KCl, 1 mM EDTA, 2 mM DTT. Samples were filteredthrough double layered Miracloth and subsequently centrifuged at6000g for 2 min, yielding crude homogenates. Aliquots of 50 ml ofthe crude homogenate was removed for SDS-PAGE and theremaining homogenate was microfuged at 13,000g for 20 min.Aliquots of 50 ml of the resulting supernatant were removed forSDS-PAGE and stored at �20 �C. The remaining supernatant wascentrifuged at 125,000g for 60 min in an airfuge (Beckman andCoulter) after which the final soluble fraction was aspirated andstored at �20 �C. The remaining microsomal fraction was resus-pended in 10 ml of 50 mMTris/containing 10 mM CaCl2 and stored at4 �C for no longer than 1 h before proteolysis and running out on anSDS-PAGE. Protein concentrations were determined according toBradford [5].

Intact chloroplasts were isolated from Arabidopsis using a chlo-roplast isolation kit (Sigma). Briefly, 15 g of 4-week old leaves werecut into small pieces and homogenised in an Ultra-Turrax� with40 ml of chloroplast isolation buffer (CIB) with 0.1% BSA. Themacerate was passed through a filter mesh and centrifuged for3 min at 200g to remove cell debris. The supernatant was trans-ferred to a fresh tube and chloroplasts sedimented by centrifugingfor 7 min at 1000g. The pellet was resuspended in 1 ml of CIB bygentle pipetting up and down. Intact chloroplasts were separatedfrom the broken chloroplasts by centrifugation on top of a 40%Percoll layer. The pellet was retained and resuspended in 1 ml of CIBwithout BSA. An aliquot was retained for loading on a gel. Thechloroplasts were lysed by freeze-thaw in liquid nitrogen. Toseparate the membrane material from soluble stromal proteins thesolution was centrifuged at 3000g for 10 min. A stromal supernatantwas collected and thylakoids resuspended in distilled water prior toloading onto protein gels. The stromal supernatant was centrifugedat 100,000g for 60 min to sediment the chloroplast envelope. Thestromal fraction was retained and the pellet dissolved in 100 ml ofdistilled water. For immunoblotting, 100 mg of each fraction wasloaded onto an SDS-PAGE gel.

2.3. Biochemical methods

2.3.1. Proteinase K digestionAliquots of intact microsomes from stressed and non-stressed

leaves (5 ml containing 200 mg protein) were incubated withincreasing quantities of proteinase K (50–3000 mg) dissolved in50 mM Tris–HCl (pH 8.0) in a total volume of 50 ml at 37 �C over-night. One sample was also treated with 2% v/v Triton X-100 tosolubilise the microsomal membranes. Proteolysis was terminatedby 1 mM phenylmethylsulfonylfluoride.

2.3.2. Calcium mobility shift assayAliquots of leaf total protein were incubated with increasing

concentrations of calcium chloride and Ethylene glycol-bis

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(2-aminoethylether)-N,N,N0,N0- tetraacetic acid (EGTA) (10–50mM) in a total volume of 40 ml at room temperature for 35 min.Samples were then loaded onto a gel with an appropriate volume ofsample buffer and run under standard conditions.

2.3.3. Phosphorylation assayDetection of phosphorylated proteins was by means of Phos-

tag� 300/460 phosphoprotein gel stain (Perkin Elmer). Briefly,75 mg of total protein samples were run on an SDS-PAGE gel understandard conditions. Appropriate molecular weights markers wereused for size estimation (Sigma). After electrophoresis, the gel wasfixed in 50% methanol/10% acetic acid and washed in distilledwater. The gel was incubated with Phos-tag concentrate at 1:100with stain buffer at room temperature for 90 min. The gel wasdestained with wash buffer, rinsed in distilled water and viewedwith a BioDoc-It� UV transilluminator imaging system (Bioimag-ing systems).

2.3.4. Peroxygenase assayPeroxygenase activity was measured as outlined in Blee & Durst

[3] and Ishimaru and Yamazaki [21]. Briefly, the oxidation of aniline(1 mM) to nitrosobenzene at 310 nm was followed in the presenceof microsomes from non-stressed and stressed leaves (0.5 mgprotein) in 0.1 M potassium phosphate buffer, pH 7.2, at 25 �C (finalvolume: 1 ml). The reaction was initiated by the addition of cumenehydroperoxide (1 mM).

2.4. RNA extraction and blotting

RNA isolation was based on the protocol of Prescott and Martin[38]. Samples were ground in liquid nitrogen in a cooled pestle andmortar and the resulting fine powder was transferred to 3 ml LETSextraction buffer (2 mM aurintricarboxylic acid, 150 mM LiCl, 5 mMEDTA, 50 mM Tris–Cl, pH 9.0, 5% SDS) in a 15 ml falcon tube.Following addition of an equal volume of water-saturated phe-nol:chloroform, the tube was vortexed and left on ice for 5 min withoccasional vortexing. The phases were separated by centrifugationat 1500g for 3 min and the aqueous phase was extracted again withphenol:chloroform and then with chloroform. To precipitate theRNA, 8 M LiCl was added to the aqueous phase to a final concen-tration of 2 M and the tubes stored at 4 �C overnight. The RNA wasrecovered by centrifugation and the pellet resuspended in 600 mlwater containing 0.1% diethyl pyrocarbonate (DEPC). The solutionwas transferred to microfuge tubes containing 200 ml 8 M LiCl andthe tubes were kept at �20 �C overnight. The RNA was re-pelletedand resuspended in 400 ml DEPC water and aliquoted into smallsamples and frozen at �80 �C. The RNA concentrations weredetermined using a spectrophotometer and its integrity demon-strated by running a sample on a native RNA gel.

Northern blot analysis was as described by Sambrook et al. [42].Total RNA samples were separated on 1% agarose gels containing 1%formaldehyde and MOPS buffer under standard conditions. Afterelectrophoresis, the RNA was blotted onto a positively chargednylon membrane (Roche) and hybridized with digoxigenin labelledClo-3 riboprobes. The blot was hybridized overnight with labelledprobe at 65 �C in DIG Easy Hyb (Roche) and then washed threetimes at 65 �C for 30 min in 2�SSC þ 0.1% SDS, 1�SSC þ 0.1% SDS,and in 0.5�SSC þ 0.1% SDS respectively. A full length cDNA clone ofthe Clo-3 gene was obtained from the Riken Plant Science Centre asa cDNA insert (clone # RAFL06-69-K02) cloned into a modifiedBluescript vector containing the T7 and T3 promoter sequences.The vector was transformed into E. coli JM109 competent cells fromPromega using manufacturer’s instructions. The RAFL plasmidcDNA was subsequently isolated from E. coli following the mini-prep DNA purification protocol from QIAGEN�. A linearised RAFL

cDNA fragment was prepared by digestion of plasmid DNA andpurified to 1 mg/ml. Riboprobes were synthesized and labelledwith digoxigenin from cDNA by the Roche in vitro transcriptionreaction kit using SP6 and T7 phage RNA polymerases according tomanufacturer’s instructions.

2.5. Antibody preparation and purification

A 15-mer peptide was synthesised with a sequence corre-sponding to the first 15 residues of the N-terminal peptidedomain of Clo-3, i.e. MAGEAEALATTAPLA. The peptide was cross-linked to keyhole limpet haemocyanin and injected into NewZealand white rabbits. Rabbit antiserum was subsequently raisedto the peptide conjugate. Immunoglobulins were purified fromthe antiserum by precipitation with caprylic acid (octanoic acid)a dialysis step followed by ammonium sulphate precipitation anda second dialysis. Briefly, an equal volume of sodium acetate(120 mM, pH 4.0) was added to the antiserum and the final pHadjusted to 4.8. Caprylic acid was added dropwise with constantmixing to a final concentration of 0.17 M. The mixture wasincubated at room temperature for 5 min and was centrifuged at5000g for 30 min at 4 �C. The supernatant was retained anddialysed against PBS at 4 �C overnight with two changes. Thedialysed sample was then mixed with an equal volume of satu-rated ammonium sulphate solution ((NH4)2SO4) and storedovernight at 4 �C. The sample was centrifuged at 3000g for30 min at 4 �C and the pellet resuspended in two pellet volumesof PBS and dialysed as above. Western blotting confirmed thespecificity of the antibody for Arabidopsis Clo-3.

2.6. SDS-PAGE and western blotting

Proteins (75 mg) were separated on 12% SDS-PAGE gelsaccording to the method of Schagger and von Jagow [43]. SDS-PAGE standards with appropriate molecular weights were used assize markers (Sigma) and prestained markers were used for use inwestern blot (Sigma). After electrophoresis, proteins were trans-ferred to nitrocellulose membranes (Hybond C, Amersham, UK)blocked for 2 h at 4 �C in 1� Tris-buffered saline Tween-20 (TBST)containing 3% w/v non-fat milk powder. Primary antibody (Clo-3N-terminal) was diluted 1:1000 in 1� TBST. Blots were incubatedwith primary overnight at 4 �C followed by three 20 min washesin TBST and followed by incubation with alkaline phosphatase-conjugated goat anti-rabbit secondary antibody (diluted 1:30,000)for 1 h. The membranes were washed as above and alkalinephosphatase activity was visualized by BCIP/NBT colorimetricdetection.

3. Results

3.1. Genomic analysis of caleosins

The Arabidopsis genome contains seven caleosin-like sequenceslocated on all but one of its chromosomes as shown in Supplemen-tary data, Fig. A1. The tandemly arranged gene pair, Clo-4 & Clo-5,probably arose from a relatively recent gene endoduplication eventand these two genes are expressed at very low levels in most planttissues [36]. The Clo-7 locus is also the result of gene endoduplicationbut in this case the resulting larger than average caleosin transcript isspliced to produce a mature mRNA that encodes Clo-7, which isa 27 kDa extracellular protein found in the pollen coat ([27],Supplementary data, Fig. A2).

The expression profile of the Arabidopsis Clo-3 gene undervarious environmental conditions was followed using a gene-specific riboprobe. Unlike previously characterised caleosins, most

M. Partridge, D.J. Murphy / Plant Physiology and Biochemistry 47 (2009) 796–806 799

of which are seed-specific and associated with lipid storage, theClo-3 gene was constitutively expressed at low levels throughoutthe Arabidopsis plant, but was then highly upregulated in someorgans in response to various forms of abiotic stress or stressrelated hormones. We also followed the expression profile of theseed-specific Clo-3 gene and, as reported elsewhere [18,33], thisgene was specifically expressed in the seeds of maturing siliquesand was at very low levels in other organs (data not shown).Following the kinds of abiotic stress treatments described below,we found a slight but consistent seed-specific upregulation of Clo-1gene expression, particularly with salt stress, but the magnitude ofthis induction was always far lower than in the case of Clo-3 (datanot shown).

In Fig. 1A the relatively low level of Clo-3 expression in fivedifferent organs from control plants is contrasted with varyinglevels of enhanced expression in organs from stressed plants. Coldstress caused a small amount of upregulation, especially in flowers(whole inflorescences) and leaves. Salt stress led to the higheststimulation of Clo-3 expression and was particularly marked inleaves and flowers. Drought conditions led to modest but consis-tent upregulation in all organs examined. As expected, applicationof ABA led to a large induction of gene expression that was almostas marked as in salt treated plants and was similarly concentratedin leaves and flowers. However, the phytohormone JA caused onlyminor upregulation in all organs. In Fig. 1B, the time course ofinduction of Clo-3 gene expression is shown in the treatmentsassociated with the highest levels of upregulation. Although someincrease in mRNA abundance was seen after as little as 5 h, the mostsignificant increases occurred around 24 h, and in the case of ABAtreatments expression levels were still rising after 48 h. Qualita-tively similar time courses were observed with drought and SAtreatments but increases were lower than for ABA or salttreatments.

3.2. Expression profile of the Clo-3 protein

Although gene expression profiling can give useful information,it is also important to correlate such data with analysis of proteinlevels wherever possible. In Fig. 2 the steady state levels of Clo-3protein are shown as determined by means of a specific antibodyprobe. This antibody was raised against a 15-mer peptide, namelyn-MAGEAEALATTAPLA-c, corresponding to a unique sequence atthe N terminal portion of Clo-3 that was not present in other Ara-bidopsis caleosins and did not correspond to any other sequences asverified by a BLAST search of public databases. This antibodyspecifically recognised the Clo-3 protein, which has an apparentmolecular mass of 27 kDa on SDS-PAGE but did not cross react withthe seed-specific Clo-1 isoform, which has an apparent molecularmass of 25 kDa on SDS-PAGE. We refer to the measurements of Clo-3 protein as steady state levels because, unlike Clo-1 which isassociated with storage oil bodies that have very low rates ofturnover during seed development, Clo-3 is present in cellularmembranes in non-storage tissues, such as leaves and roots, thatare constantly turning over.

As shown in Fig. 2, Clo-3 protein was detectable, albeit in verylow quantities in most organs of non-stressed plants and waspresent at especially high levels in leaves. Following stress orhormone treatments, Clo-3 protein levels responded in a broadlysimilar way to the gene expression profile shown in Fig. 1. Inparticular, the protein was highly upregulated after salt or ABAtreatments in leaves and flowers. Broadly similar results were alsofound in the time course of Clo-3 protein abundance, with theobvious difference that increases in level of translation productsalways lags behind that of gene transcription products. In this case,protein levels were still rising after 72 h.

3.3. Subcellular localisation, membrane orientationand accessibility

We have previously reported the dual localisation of seed-specific caleosins from Arabidopsis and Brassica, which can occureither on the surfaces of storage oil bodies or as integral membraneproteins in microsomes derived mainly from the ER [18]. In Fig. 3,the subcellular localisation of Clo-3 from control and salt stressedleaves is shown. As with some of the seed-specific caleosins, Clo-3was localised on microsomal membranes, although a significantband was also present in the 5000g pellet corresponding to thechloroplast fraction. The latter was investigated in more detail byisolating intact chloroplasts, which were then osmotically andmechanically lysed and separated into stroma, thylakoid andenvelope fractions. Control leaves had insufficient Clo-3 to givea detectable signal, but in salt stressed leaves it was clear thatplastidial membrane-bound Clo-3 was mainly located in theenvelope fraction.

In Fig. 4, the membrane orientation and accessibility of leafmicrosomal Clo-3 is elucidated using proteinase K and Triton X-100 treatments. The full length protein has a relative mass ofabout 27 kDa and, following solubilisation of microsomalmembrane by Triton X-100, the protein was completely degradedby low ratios of proteinase K. This protease cleaves at the carboxylside of aliphatic, aromatic or hydrophobic amino acid residues[24]. Such degradation can only occur if most or all of suchresidues are accessible for digestion by the protease. However, themicrosome-associated form of Clo-3 was protected fromproteinase K digestion, with the exception of a small fragment ofabout 2 kDa, which was removed by protease levels in excess of100 mg. The remainder of the Clo-3 protein was still detectablefollowing removal of this fragment, indicating that the cleavagehad occurred at the C-terminus and that the protein was thereforeorientated with its N-terminus facing into the microsomalvesicles.

3.4. Calcium and phosphate binding

Seed-specific caleosins have been reported to bind calcium andto be phosphorylated [8,17,40,48]. Like the seed-specific Clo-1isoform, the stress-induced Clo-3 isoform described in the presentstudy contains a single EF hand motif potentially able to binda single calcium atom. The Clo-3 protein sequence also containsseveral putative phosphorylation sites. In view of the response ofClo-3 to abiotic stress and its possible role in signalling, it wastherefore important to determine the calcium and phosphatebinding characteristics of this isoform. In Fig. 5A, the binding ofcalcium to Clo-3 from the leaves of untreated leaves is shown.Binding of one atom of calcium binding results in a mobilityincrease on SDS-PAGE equivalent to an apparent shift of 2 kDa inmolecular mass compared to identical polypeptides withoutbound calcium. Binding was observed over a range of calciumconcentrations from 10 to 50 mM and was effectively abolishedby incubation with EGTA. Native Clo-3 was always capable ofbinding calcium, whether it was present in untreated or stressedplants, although we observed that salt-stressed leaves containedelevated levels of Clo-3 compared to controls. The phosphoryla-tion status of Clo-3 from stressed and unstressed plants asrevealed by PhosphoTag assays is shown in Fig. 5B and C. InFig. 5B, control leaves showed very low levels of phosphorylation,whereas Clo-3 in salt or ABA stressed leaves was significantlyphosphorylated. Given that the expression of Clo-3 protein incontrol tissue was approximately 30% lower than that of saltstressed tissue, the control lanes of Fig. 5C were purposely over-loaded by a factor of three such that the intensity of

Fig. 1. Variation in Clo-3 gene expression in response to abiotic stresses and plant growth regulators. Panel A, Tissue specificity of response. Chilling stress was 4 �C, salt was250 mM NaCl, drought was withdrawal of watering (see Methods), ABA and JA were each applied at 200 mM. All treatments were for 24 h except drought which was for 12 h. Clo-3detection was by means of a specific cDNA probe RAFL06-69-K02 from RIKEN bioresources. Lane 1, leaf (mature); lane 2, stem; lane 3, silique (green); lane 4, flower (wholeinflorescences); lane 5, root. Panel B, Time course from 0 to 48 h of Clo-3 gene expression in leaves in response to treatments with either 200 mM ABA, 250 mM NaCl (salt), 4 �C(chilling), or 200 mM salicylic acid.

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immunodetected control and salt stressed Clo-3 were equivalent.The corresponding western blot shows equal abundance of Clo-3in each lane. Accordingly, the phosphoTag assay clearly shows theincreased phosphorylation status of Clo-3 in salt stressed leaf androot compared to controls regardless of the decreased totalprotein loading compared to controls.

3.5. Peroxygenase activity

The seed-specific caleosin isoform, Clo-1, has been demon-strated to have a histidine-dependent peroxygenase activity [17]but similar activities have not been previously reported for othercaleosin isoforms, most notably the stress-induced Clo-3, which we

Fig. 2. Steady state levels of Clo-3 protein in response to abiotic stresses and plant growth regulators. Panel A, Steady-state levels of Clo-3 protein in various tissues. Chilling stresswas 4 �C, salt was 250 mM NaCl, drought was withdrawal of watering (see Methods), ABA and JA were applied at 200 mM. All treatments were for 48 h; 50 mg protein was loaded ineach lane. Clo-3 protein detection was by means of a specific antibody raised against its N terminal portion. Lane 1, leaf; lane 2, stem; lane 3, silique; lane 4, flower; lane 5, root. PanelB, Time course from 0 to 48 h of steady-state levels of Clo-3 protein in leaves in response to treatments with either 200 mM ABA, 250 mM NaCl (salt), or 4 �C (chilling). 50 mg proteinwas loaded in each lane. Clo-3 protein detection was by means of a specific antibody raised against its N terminal portion.

Fig. 3. Subcellular localisation and membrane orientation of Clo-3 protein in leaves. Localisation of Clo-3 protein in subcellular fractions of leaves in the absence (Panel A) orpresence (Panel B) of 250 mM NaCl (salt). Lane 1 total homogenate; lane 25,000g pellet; lane 3,100,000g pellet; lane 4, 100,000g supernatant Localisation of Clo-3 protein insubchloroplast fractions of leaves in the absence (Panel C) or presence (Panel D) of 250 mM NaCl (salt). Lane 1 total homogenate; lane 2, intact chloroplast (5000g pellet); lane 3,stroma (100,000g supernatant); lane 4, thylakoids (100,000g pellet).

M. Partridge, D.J. Murphy / Plant Physiology and Biochemistry 47 (2009) 796–806 801

Fig. 4. Membrane orientation of Clo-3 as determined by proteinase K incubations with intact or Triton X-100 treated microsomes. Clo-3 protein was detected by immunoblotting asdescribed in Methods and each lane was loaded with 150 mg microsomal protein previously incubated � proteinase K for 16 h at room temperature. Lane 1, control microsomes; lane2, microsomes þ 2% Triton X-100 þ 50 mg proteinase K; lanes 3–9, contain respectively 100, 200, 400, 750, 1000, 2000 and 3000 mg proteinase K.

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show to be especially upregulated in stressed leaves. We examinedperoxygenase activity by following the oxidation of aniline tonitrosobenzene by microsomal fractions from leaves. The totalperoxygenase activities of microsomes (0.5 mg protein/assay) fromcontrol leaves in which caleosin protein levels were relatively lowwere in the range of 26 � 1 mM nitrosobenzene formed/min/mgprotein Table 1. In contrast, peroxygenase activities of microsomesfrom salt stressed leaves (250 mM NaCl � 48 h) in which caleosinprotein levels were significantly upregulated were in the range of30 � 2 mM nitrosobenzene formed/min/mg protein. Similar resultswere obtained from independently replicated experiments.Although in a total microsomal fraction peroxygenase activity couldresult from several enzymes, the modest, but consistent increaseafter salt stress followed a significant increase in Clo-3 proteinlevels (Fig. 5) and is indirect evidence consistent with the activity ofClo-3 as a peroxygenase in vivo.

3.6. Responses to SA and biotic stress

A common source of biotic stress for many Brassicaceae,including Arabidopsis and Brassica spp is the pathogen, L. maculans,anamorph Phoma lingam. This fungus initially elicits a biotrophicresponse in the host plant mediated by the salicylic acid pathway[46]. Later in the infection cycle, necrotic greyish lesions punctu-ated by pycnidia appear on leaves of younger plants, often pro-gressing to stem canker and death in adult plants [45]. Weinvestigated the role of Clo-3 in the initial biotrophic response byexamining protein levels 48 h after treatment with salicylic acid orits synthetic mimic, DCINA (Fig. 6). In both cases there wasa several-fold increase in Clo-3 protein as determined by immu-noblotting. The increase in Clo-3 protein levels was even moremarked during the later necrotrophic phase as determined 4 weeksafter initial inoculation with Phoma, indicating that Clo-3 inductionwas maintained throughout the infection cycle.

3.7. Clo-3 induction in SA and ABA pathway mutants ofArabidopsis

The above data show that Clo-3 was responsive to both the ABAand the SA induction pathways that operate in various abiotic andbiotic stress responses in plants. In order to study the involvementof Clo-3 in additional stress related signalling pathways, weobtained and tested a variety of mutant lines as follows. The aba1-1and aba1-5 deficient mutants have a block in the epoxidation ofzeaxanthin resulting in drastically reduced levels of ABA. In bothcases, these mutants showed a definite, if slightly reduced induc-tion of Clo-3 in response to salt stress compared to WT plants(Fig. 7). Salicylic acid or DCINA can modulate gene expression eithervia an induced pathogenesis related (PR) pathway or via a PR-independent pathway. To look at the latter process, we used the

npr1-1 mutant, which contains a point mutation in an IkB-likesignal-transduction component acting in the PR-independentpathway downstream of SA. This mutant showed reduced butmeasurable induction of Clo-3 by SA, indicating that Clo-3 couldalso be activated via a PR-independent pathway. The ability ofDCINA to substitute for SA as an inducer of Clo-3 was confirmedusing plants containing the NahG mutant, where SA-mediatedinduction of PR-1, PR-2, or PR-5 genes is blocked by ectopicexpression of an SA hydroxylase.

4. Discussion

The seemingly ubiquitous presence of caleosin-like sequences inthe genomes of all green plants (Chlorobionta/Viridiplantae),including non-spermatophytes such as mosses and single-celledalgae, indicates that these proteins have roles in addition to thosepreviously characterised in relation to seed development andgermination [17,37]. We show here that at least one caleosin–per-oxygenase isoform in Arabidopsis is strongly induced in non-seedtissues by a variety of environmental stresses in a mannersuggestive of its role in oxylipin signalling pathways, includingthose regulated by mediators like ABA and SA. Our observedresponse of the Arabidopsis Clo-3 gene to ABA induction is consis-tent with the presence of several consensus ABA-responsiveelement (ABRE)-like sequences (from �155 to �152 and �199 to�195) in its promoter region. The widespread occurrence of verysimilar caleosin-like sequences in all true-fungal genomes analysedto date is also consistent with the central role of oxylipin signallingpathways in fungal spore development and release [50] andpossibly in defence against other fungal species. However, giventhat the Opisthokonta (animals and fungi) are now generallyregarded as a monophyletic group that is descended froma common single-flagellate Protist ancestor [47], this raises a diffi-cult question about the wider origin and fate of caleosins. It isconceivable that there was a common Protist ancestor of Chlor-obionta and Opisthokonta that had caleosin gene(s) but this begsthe question of what happened to these genes in animals andorganisms such as yeast where they now appear to be completelyabsent.

The Arabidopsis Clo-3 protein was associated with microsomaland chloroplast envelope fractions where it exhibited a type Imembrane orientation [44] with about 2 kDa of its C terminalfacing the cytosol. Given that the protein sequence contains justone predicted transmembrane domain and that the N terminal wasprotected from proteolysis even at very high ratios of proteinase K/caleosin, this suggests that about 9 kDa of the N terminal regionfaces the lumen (see Supplementary data, Fig. D). This is consistentwith the observation that the seed-specific Clo-1 isoform wasembedded in storage oil bodies with about 2 kDa of its C terminalfacing the cytosol [17]. We were unable to determine whether the

Fig. 5. Calcium binding and phosphorylation status of Clo-3 protein in leaves and roots under various physiological conditions. Panel A, mobility shift of Clo-3 from non-stressed andstressed leaves in the presence of calcium: non-stressed: lane 1, no treatment; lane 2,10 mM Ca2þ; lane 3,10 mM Ca2þþ 30 mM EGTA. Salt stressed: lane 1, no treatment; lane 2,10 mMCa2þ; lane 3, 30 mM EGTA; lane 4,10 mM Ca2þþ 30 mM EGTA; lane 5,100 mM Ca2þþ 30 mM EGTA; lane 6,100 mM Ca2þþ 30 mM EGTA then a further 30 mM EGTA treatment. Panel B,phosphorylation status of Clo-3 protein as assayed by Phos-tools� (PerkinElmer); treatments were 200 mM ABA or 250 mM NaCl (salt): lane 1, control non-stressed leaf; lane 2, saltstressed leaf; lane 3, leaf þ ABA; lane 4, control non-stressed root; lane 5, salt stressed root; lane 6, root þ ABA. Each lane was loaded with 150 mg of total protein. Panel C, phos-phorylation status of Clo-3 protein as assayed by Phos-tools� (PerkinElmer); salt treatments were with 250 mM NaCl: lane 1, control non-stressed leaf; lane 2, salt stressed leaf; lane 3,control non-stressed root; lane 4, salt stressed root. The control lanes were loaded with 225 mg of total protein whereas the salt stressed lanes were loaded with 75 mg of total protein.The corresponding western blot below which serves as a protein loading control, shows equivalent abundance of Clo-3 in control against stressed samples.

M. Partridge, D.J. Murphy / Plant Physiology and Biochemistry 47 (2009) 796–806 803

microsomal Clo-3 was derived from endoplasmic reticulum, tono-plast or plasmalemma membranes, or indeed any combination ofthese, but this suggests that one or more of these membranesystems may be sites of oxylipin metabolism in plants.

Interestingly, an analysis of the Arabidopsis vacuolar proteomeisolated from leaf protoplasts has shown that Clo-3 is present onthe tonoplast membrane [6]. Plastid envelopes are also a well-known location for enzymes of oxylipin metabolism [28], although

Table 1Peroxygenase activity in microsomes from control and salt-stressed leaves. (seeMethods for assay details, 0.5 mg protein used per assay).

Velocity (nM min�1 mg�1 protein)

Control 26 � 1Stressed 30 � 2

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a classic peroxygenase activity was not detected in the in vitro studyof Blee & Joyard [4]. Our data therefore suggest that Clo-3 occurs inmultiple membrane locations in leaf cells, which is consistent withits involvement in responses to several divergent forms of bioticand abiotic stress.

Fig. 6. Effects of biotic stress and salicylic acid related treatments on morphology and CloBrassica napus leaves after each treatment (as described below) Panel B, Clo-3 protein levels ileaf þ 200 mM salicylic acid; lane 5, leaf control; lane 6, leaf after 4 weeks of Phoma infect

Fig. 7. Induction of Clo-3 in ABA- and SA-related and in pathogenesis related pathway mutahas a mutation in the PR-independent pathway downstream of SA; NahG plants are blocke

The role of lipid peroxygenases in the generation of oxylipinderivatives of fatty acids has been established in seed tissues froma range of plant species [14,15]. The seed oil-body caleosin isoform,Clo-1, which is very similar in its amino acid sequence to Clo-3 (65%identity, 78% conservative substitution, see Supplementary data,Fig. B), has been shown to be a calcium binding haem–oxygenasewith peroxygenase activity [17]. In the present study, leaf micro-somal extracts enriched in Clo-3 also had peroxygenase activityand, like the seed isoform, it can be assumed that the single EF-handed Clo-3 also protein binds one calcium atom per molecule.The peroxygenase cascade is an alternative route for metabolism offatty acid hydroperoxides that leads to formation of epoxy andhydroxy derivatives of linolenic acid. This pathway is known to be

-3 protein levels in leaves of Arabidopsis and Brassica napus. Panel A, morphology ofn: lane 1, seed control; lane 2, leaf control þwater; lane 3, leaf þ 200 mM DCINA; lane 4,ion. Leaves in lanes 2–4 were incubated for 48 h; lanes 5–6 for 4 weeks.

nts of Arabidopsis. WT, wild type; ABA1-5 and ABA1-1 are ABA deficient mutants; NPR1d in SA-mediated induction of three PR genes.

M. Partridge, D.J. Murphy / Plant Physiology and Biochemistry 47 (2009) 796–806 805

part of the general defence response against fungal infection in thevegetative tissues of plants such as tomato or rice [23,34,35,52]. Inseed tissues, peroxygenases appear to have a role in the generationof anti-fungal metabolites such as hydroxy–epoxy fatty acids[16,39]. We show here that in Arabidopsis, Clo-3 was stronglyinduced in leaves following infection by the Phoma pathogen, Lmaculans. This implies that one role of Clo-3 may be as a perox-ygenase involved in producing hydroxy fatty acid derivatives thatcould have anti-fungal properties.

These observations are consistent with screening studies usingBrassica napus plants which have shown that a range of oxylipins,including hydroxy and hydroperoxy fatty acids, had varying degreesof anti-fungal activity to such pathogens as L maculans, Alternariabrassicae and Sclerotinia sclerotiorum [13,48,53]. Similarly to ourobservations with Clo-3 from Arabidopsis, the highly similar barleyBCI-4 gene was induced by SA, DCINA but only very slightly by JA [2].This barley caleosin-like gene is so highly upregulated that it is nowused as a marker in studies of chemically induced resistance, or CIR[19]. More recently, the same barley caleosin was shown to beupregulated following attack by the aphid species, Rhopalosiphumpadi and Metopolophium dirhodum [41]. In this case, the barleycaleosin was strongly upregulated in response to aphid herbivory inresistant plant lines but was not detectable in susceptible lines,indicating that elements of this particular oxylipin pathway may notbe operating in susceptible plants, possibly due to a lesion in anupstream signalling component. Our observations (Fig. 7) that Clo-3could be induced in ABA mutants and in npr1-1 mutants suggest thatat least some elements of oxylipin metabolism can respond both tohormone-sensitive and hormone-insensitive pathways and toinduced pathogenesis related (PR) and PR-independent pathways(see Supplementary data, Fig. C). The importance of these and othersignalling pathways in regulating responses to biotic stress in theArabidopsis relative, Boechera divaricarpa, was recently shown byVogel et al. [51]. Indeed, one of the herbivory-related genesdiscovered in this study was a homolog of Clo-3.

We demonstrate here that Clo-3 is responsive to a range ofenvironmental stresses, especially in leaves and roots, and can beupregulated by multiple signalling pathways. This is the first reportof a peroxygenase involvement in abiotic stress responses. Wefound that Clo-3 was especially highly induced in response to salt ordehydration stresses. The caleosin isoforms Clo-4 and Clo-5 areexpressed at very low levels in non-stressed vegetative tissues andalso barely respond to stresses such as salt or desiccation (seeSupplementary data, Fig. E). These two caleosins may thereforehave other, hitherto unrecognised, roles in plants. The more generalrole of caleosins in water stress has recently been reinforced by theobservation that a Clo-3-like gene was upregulated as part of thedesiccation tolerance response of the non-seed plant S. tamariscina[26]. Further work in our lab is now focused on the analysis of Clo-3knockout lines created by RNA interference technology.

Acknowledgments

This work was partially funded by a ‘Future People’ studentshipawarded to MP by the University of Glamorgan.

Appendix. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.plaphy.2009.04.005

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