Inositol methyl tranferase from a halophytic wild rice,Porteresia coarctata Roxb. (Tateoka): regulation of pinitolsynthesis under abiotic stress

SONALI SENGUPTA, BARUNAVA PATRA, SUDIPTA RAY & ARUN LAHIRI MAJUMDER

Plant Molecular and Cellular Genetics, Bose Institute (Centenary Building), P1/12, C.I.T. Scheme VIIM, Kolkata 700054,India

ABSTRACT

Methylated inositol D-pinitol (3-O-methyl-D-chiro-inositol) accumulates in a number of plants naturally or inresponse to stress. Here, we present evidence for accumu-lation and salt-enhanced synthesis of pinitol in Porteresiacoarctata, a halophytic wild rice, in contrast to its absencein domesticated rice. A cDNA for Porteresia coarctatainositol methyl transferase 1 (PcIMT1), coding for theinositol methyl transferase implicated in the synthesis ofpinitol has been cloned from P. coarctata, bacterially over-expressed and shown to be functional in vitro. In silicoanalysis confirms the absence of an IMT1 homolog inOryza genome, and PcIMT1 is identified as phylogeneti-cally remotely related to the methyl transferase gene familyin rice. Both transcript and proteomic analysis show theup-regulation of PcIMT1 expression following exposureto salinity. Coordinated expression of L-myo-inositol1-phosphate synthase (PcINO1) gene along with PcIMT1indicates that in P. coarctata, accumulation of pinitol viainositol is a stress-regulated pathway. The presence ofpinitol synthesizing protein/gene in a wild halophytic rice isremarkable, although its exact role in salt tolerance of P.coarctata cannot be currently ascertained. The enhancedsynthesis of pinitol in Porteresia under stress may be one ofthe adaptive features employed by the plant in addition toits known salt-exclusion mechanism.

Key-word: inositol methyl transferase 1.

Abbreviations: 2-D PAGE, two-dimensional polyacryla-mide gel electrophoresis; BAP, benzyl amino purine; ECL,enhanced chemiluminescence; EST, expressed sequencetags; HRP, horseradish peroxidase; IAA, indole acetic acid;IBA, indole butyric acid; IEF, iso electric focussing;MALDI-TOF, matrix-assisted laser desorption ionizationtime-of-flight; NAA, napthalene acetic acid; PcAct1, Porter-esia coarctata Actin1; PcIMT1, Porteresia coarctata inositolmethyl transferase 1; PcINO1, Porteresia coarctata L-myo-inositol-1-phosphate synthase; PMSF, phenylmethylsulpho-

nyl fluoride; RT-PCR, reverse transcriptase PCR; TBS-T,Tris buffer saline-Tween 20; WPM, woody plant medium.

INTRODUCTION

Accumulation of polyols, either straight-chain metabolitessuch as mannitol and sorbitol, or cyclic polyols such asmyo-inositol and its methylated derivatives such as pinitol(Loewus & Dickinson 1982), is correlated with tolerance todrought and/or salinity (Vernon & Bohnert 1992).This phe-nomenon is observed in many species including bacteria,yeasts, marine algae, higher plants and animals. Becausemany naturally salt- or drought-tolerant plants accumulatesuch compounds when stressed, these metabolites are con-sidered as compatible solutes that act by providing osmoticadjustment and by lowering the osmotic potential, thusincreasing the capacity to retain water (LeRudulier &Bouillard 1983). Other mechanisms by which these metabo-lites might enhance tolerance to stress are still not wellunderstood. Some of these metabolites could act as scaven-gers of extremely toxic hydroxyl radicals, short-lived activeoxygen species that have no known enzymatic systemsfor breakdown (Smirnoff & Cumbes 1989; Smirnoff 1993;Asada 1994; Shen, Jensen & Bohnert 1997a,b). They arealso known for their protective function in maintaining thehydration sphere of proteins under water-stress conditions(Galinski 1993; Papageorgiou & Murata 1995).

Figure 1 depicts the pathway for synthesis of inositol andits methylation to pinitol with the probable regulation sitesof the pathway. Myo-inositol, the precursor of pinitol, issynthesized through an evolutionary conserved enzymeL-myo-inositol 1-phosphate synthase (MIPS; EC 5.5.1.4)coded by the INO1 gene (Majumder et al. 2003). The enzy-matic product of MIPS is specifically dephosphorylated by aMg++-dependent L-myo-inositol 1-phosphate phosphatase(EC 3.1.3.25) to form free inositol. Inositol is methylated topinitol by the inositol methyl transferase (EC 2.1.1.4) codedby an IMT1 gene in an S-adenosyl methionine (SAM)-dependent reaction, and is closely linked to the activatedmethyl cycle described in Fig. 1. To date, the IMT1 gene hasbeen isolated only from the halophytic ice plant, Mesem-bryanthemum crystallinum, where salt-induced transcrip-tional induction of the IMT1 is coordinated with the INO1gene for pinitol biosynthesis under salt stress (Ishitani et al.

Correspondence: A. L. Majumder. Fax: +91 33 2355 3886; e-mail:lahiri@bic.boseinst.ernet.in

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1996). Two distinct steps for regulation of the pathway canbe visualized, namely the relative availability of inositol forthe IMT1 reaction over other competing pathways of utili-zation, and the availability of SAM, indirectly regulatedby methionine production through homocysteine methyltransferase (EC 2.1.1.14). Several of these steps arethought to play important role in stress perception andamelioration.

In addition to the presence of an active IMT1 function,D-pinitol accumulation under saline environment dependson the availability of the required pool of myo-inositolunder such condition. We have previously demonstratedthat the halophytic wild rice Porteresia coarctata harbours aunique salt-tolerant MIPS coded by PcINO1 (Majee et al.2004; Ghosh Dastidar et al. 2006).As this particular enzymeis able to generate myo-inositol even at high salt concen-tration, the inositol pool in the plant is well maintainedduring salinity. It was intriguing to investigate whether,under salt stress, this pool of inositol in Porteresia may beconverted to pinitol. Several halophytic and non-halophyticspecies are reported to contain pinitol as the major solublecarbohydrate in their leaves (Bieleski 1994) although theIMT1 gene coding for the key enzyme for such conversionwas hitherto reported and characterized only from the fac-ultative halophyte, M. crystallinum (Vernon & Bohnert1992).

In the present communication, we report the isolation,cloning and characterization of a cDNA for inositol methyltransferase coding IMT1 gene from P. coarctata, a

monocotyledonous wild halophytic rice plant. The cDNAwas cloned from an in vitro propagated plant system.The cDNA clone was bacterially expressed, purified to nearhomogeneity, and the identity was confirmed by immuno-detection as well as by the production of the enzymaticreaction product, pinitol. The presence of the active IMT1protein in the plants and its increased synthesis during saltstress was supported by analysis of soluble sugar content.The relative transcript content data analysed by RNA gelblot and semi-quantitative RT-PCR support this observa-tion. A proteomic analysis to quantify the two gene prod-ucts further strengthens this finding. The co-ordinateregulation patterns of the two genes, PcINO1 and PcIMT1,under different abiotic stresses have also been studied toacquire some idea about their interplay during salt stress inthis system.

MATERIALS AND METHODS

In vitro clonal propagation of plants

Porteresia coarctata plants collected from Canning area inthe Sunderban mangroves of West Bengal were main-tained in earthen pots in the net house. From profuselygrowing mature plants, multinodal segments of ~10 cmwere collected as explants and were washed in 1%aqueous solution of Tween-20 (v/v). Eradication of theendogenous fungi was achieved by overnight incubation ina combination of fungicides Bavistin (0.1%) and SAAF

Figure 1. Synthesis of pinitol from inositol and the associated methyl cycle. Pinitol is synthesized by the enzyme inositol methyltransferase 1 (IMT1) using inositol as substrate and S-adenosyl methionine (SAM) as co-substrate. SAM serves as a secondary methyldonor in this reaction and is converted to S-adenosyl homocysteine (SAH). SAH is split into adenosine and homocysteine by adenosylhomocysteinase. Homocysteine is used to regenerate methionine and subsequently, SAM. This regeneration is mediated by methioninesynthase/homocysteine methyl transferase using N5-methyl tetrahydrofolate (N5-met-THF) as primary methyl donor generated fromphotorespiratory activity. The symbol indicates limiting points in the whole process.

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(0.1%). Surface sterilization with 70% ethanol, 0.1%sodium hypochlorite and 0.1% mercuric chloride was fol-lowed by in vitro culture of the multinodal segments inLloyd & McCown’s (1980) WPM with 3% sucrose(pH 5.8). The sprouting of new shoots from the nodal seg-ments was induced by supplementing the basal mediumwith an optimum concentration of 2.2 mm kinetin and5.5 mm BAP. Rooting of the shoots was induced by supple-menting the basal medium with an optimum concentrationof 1.0 mg L-1 NAA and 2.5 mg L-1 IBA, following a trans-fer to a regulator-free medium after 12–14 d. Cultures weremaintained in sterile 180 mL glass jars at 26 � 10 °C. Shootemergence was aided by an initial continuous illuminationprovided by white fluorescent tubes for 3 d, followed by a10:14 h light : dark period. A minimum of 10 replicateswere employed during standardization for each experi-mental hormone combination tried. All experiments wererepeated at least three times.

Stress treatment of plants

Eight-week-old plantlets were transferred to a half-strengthWPM and were acclimatized for 15 d. Growth was main-tained at 26 � 10 °C with 10 h of light/14 h of dark and~40% relative humidity. For abiotic stress treatments, theseplants were then transferred in the same medium contain-ing of 400 mm NaCl/10-6 m paraquat/75 mm abscisic acid.The plants were also subjected to 40 °C dry heat/4 °C coldtreatment. Leaf samples from ~12-cm-tall plantlets werecollected at 48 and 96 h of stress application.

Measurement of physiological parameters:fresh weight, chlorophyll concentration andphotosynthetic efficiency under stress

The procedure for chlorophyll determination was based onthe property of absorption of light by aqueous acetone(80%) extracts of chlorophyll (Mackinney 1941). Chloro-phyll concentration was determined in mg mg-1 fresh weightbasis. Transients of chlorophyll a fluorescence were mea-sured from stressed and unstressed plants using a portableHandy PEA equipment (Hansatech Instruments, Ltd.,Norfolk, UK). The excitation light intensity was 600 W m-2

from an array of six red light-emitting diodes with a peakwavelength at 650 nm focused on the leaf surface in theclips on a spot of 4 mm in diameter. From the measuredparameters, JIP test parameters were derived using theBiolyzer software (created by Ronald Maldonado Rod-riguez) to analyse the stepwise flow of energy through pho-tosystem II (PSII) at different levels.

Inositol/pinitol profiling by gaschromatography (GC)

Total soluble leaf sugar was isolated following the modifiedmethod of Bieleski & Redgwell (1977). For authenticidentification and estimation, the samples were TMS

(tri-methyl silyl)-derivatized with Tri-Sil-Z (Pierce, Rock-ford, IL, USA) and were run through gas liquid chromatog-raphy in a Chemito 1000 GC (Chemito Instruments Pvt.Ltd., Nashik, India) equipped with a flame ionization detec-tor. GC conditions were as follows: 3% SP-2100 stationaryphase (Supelco, Sigma-Aldrich Unit, St. Louis, MO, USA)supported on chromosorb-W (Sigma, St Louis, MO, USA)packed in a 1.8 mts(l) ¥ 2 mm (i.d.) glass column with N2

(flow rate, 31 mL min-1) as carrier gas, and oven tempera-ture programmed between 130 and 320 °C at 100 °C min-1.Quantification was made against similar runs with authenticsugar samples as standard. The pinitol standard was a kindgift from Prof. Hans J Bohnert, University of Illinois atUrbana-Champaign, Urbana, IL, USA.

RNA isolation and gel-blot analysis

Total RNA was isolated from the stress-treated anduntreated P. coarctata leaves using a modified pine-treemethod described by Chang, Puryear & Cairney (1993).Northern blotting was performed according to Sambrook,Fritsch & Maniatis (1989) from the salt-treated anduntreated plants using radiolabelled purified gel eluted M.crystallinum methyl transferase (McIMT1) ~1.1 kb frag-ment (~100 ng) as a probe.

Amplification of the desired cDNA fragmentand cloning

First-strand cDNA was prepared from ~5 mg of total RNAusing Stratagene’s ProSTAR First Strand RT-PCR Kit (LaJolla, CA, USA) according to the manufacturer’s instruc-tion. For PCR amplification, primers were designed basedon the sequence of McIMT1 mRNA (accession numberM87340). Primer sequences were as follows: forward primer– 5′CATATGACCACCTACACCA 3′, reverse primer –5′AAGCTTCTTCTTGTAGAGCT 3′. PCR reaction wascarried out in a Bio-Rad iCycler (Hercules, CA, USA) inthe PCR conditions mentioned: 94 °C for 3 min (1 cycle);denaturation, 94 °C for 30 s; annealing, 50 °C for 45 s; exten-sion, 72 °C for 90 s (28 cycles); and final extension, 72 °C for15 min.

The amplified ~1.1 kb PCR product was purified fromagarose gel and was cloned in pGEMT-Easy cloning vector(Promega, Madison, WI, USA) according to the manu-facturer’s instructions. The product was sequenced using96-well capillary DNA Sequencer (ABI Prism, AppliedBiosystems, Foster City, CA, USA) at the genomics andproteomics facility of the institute.

In silico analysis of the PcIMT1 sequence

Blastn analysis of the sequence obtained with the availabledatabase at the National Center for Biotechnology Infor-mation (NCBI) confirms the identity of the product. Thesequence was virtually translated, and probable proteinsequence was aligned with the IMT1 sequence of M.

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crystallinum as well as some of the other methyl trans-ferases using ClustalW or Multalin. The Oryza sativagenome database (RAP-DB 2008 update; build 3 assembly)was scanned with the sequence to investigate any probablematch in NCBI and TIGR (The Institute for GenomicResearch). Other available transcript/EST/genome assem-blages were also scanned to look for homologous sequencesamong other plant species (Supporting InformationTable S5). Domain architecture was predicted using theInterProscan tool from Expasy against PROSITE, Pfam,PRINTS and other family and domain databases. Themethyl transferase domain containing matching proteinsequences of O. sativa was aligned with PcIMT1, and phy-logenetic analysis was carried out in MEGA4 (Felsenstein1985; Saitou & Nei 1987; Tamura et al. 2007). The majoruniprotKB sequences used for this analysis are listed inSupporting Information Table S4.

Bacterial overexpression and purificationof PcIMT1

The full-length ~1.1 kb fragment of IMT1 cDNA of P. coarc-tata (PcIMT1) was digested out from pGEMT-Easy clonefollowing Nde1/HindIII digestion. The fragment was thencloned in pET15b(+) vector backbone carrying an N-terminal 6X histidine tag. PcIMT1-pET15b(+) clone wasexpressed in Escherichia coli BL21(DE3) PLys(S) strain.Twelve percent sodium dodecyl sulphate–polyacrylamidegel electrophoresis (SDS–PAGE) analysis was performedaccording to Laemmli (1970). The protein, predominantlyexpressed in pellet fraction, was solubilized (8 m urea, 0.5 mNaCl, 20 mm Tris-HCl, pH 8.0, 10 mm b-ME, 2 mm PMSF)and loaded onto a Qiagen Ni-NTA resin column (Valencia,CA, USA) under the same denaturing condition.The boundprotein fraction is eluted in 0–500 mm linear gradient ofimidazole. The purified fractions were then dialysedthrough a serial dilution of urea from 8 m to a urea-free50 mm Tris-Cl (pH 8.0) buffer in 5% glycerol for completeremoval of urea and imidazole. The dialysed fractions werechecked for homogeneity in 12% SDS–PAGE as describedby Laemmli (1970). The purified refolded enzyme wasassayed as described by Rammesmayer et al. (1995). Theproduct generated by enzyme reaction was analysed by GCand was identified using standard sugars.

Raising antibody against McIMT1

For immunodetection of the expressed protein, polyclonalantibody was generated in rabbit against the alreadyreported IMT1 protein from M. crystallinum. The McIMT1cDNA clone was cloned into pET15b(+) expression vector,bacterially expressed, and purified in a similar manner asdescribed previously. The identity of recombinant proteinwas confirmed through immunodetection by mouse anti-His antibody.Anti-McIMT1 antibody was raised against thepurified recombinant IMT1 in rabbit, following the stan-dard procedure (Sambrook et al. 1989).

Immunodetection of the bacterially expressedprotein PcIMT1

For immunodetection, proteins were electroblotted onto anitrocellulose membrane, and the blots were probed withthe rabbit anti-McIMT1 antibody (1:1000) as well as themouse anti-His antibody (1:5000).The bound antibody pro-portion was detected by the luminescence caused by goatanti-rabbit and anti-mouse IgG HRP-conjugated second-ary antibody (1:10 000 dilution in TBS-T) respectivelydeveloped with a fluorescent ECL detection kit reagent(Amersham Pharmacia Biotech, Little Chalfont, Bucks,UK) according to the instruction of the manufacturer.Fluorescence was detected by exposing the thoroughlywashed membrane to Kodak [X-O(MAT)AR] film (Roch-ester, NY, USA).

Isolation of total leaf protein from salt-treatedand untreated Porteresia plants

The collected leaves were frozen immediately in liquidnitrogen after harvest and were ground to a fine powder inpre-cooled mortar and pestle along with 1% w/w frozenplant protease inhibitor cocktail (Sigma). To 1 g of groundtissue, 5 mL of chilled extraction buffer [50 mm Tris-HCl,pH 8.0, 50 mm EGTA (ethylene glycol tetra acetic acid),900 mm sucrose, 2% PVP (poly vinyl pyrrolidone), 2%PEG-6000, 20% Triton X-100, 4% NP-40 and 10 mm DTT(di-thio-threitol)] was added and transferred to a 30 mLfalcon tube, vortexed and ice-incubated by shaking for10 min. An equal volume of Tris-HCl-saturated phenol(pH 8.8) was added followed by sonication pulses. Thesample was centrifuged for 10 min at 5500 g and 4 °C. Theupper phenolic phase was recovered carefully, leaving theaqueous phase and insoluble material, which was backextracted with 3 mL Tris-buffered phenol similarly and wascombined with the first extraction. Four volumes of precipi-tation solution (0.1 m ammonium acetate in chilled metha-nol) was added to it and was left overnight at -20 °C after athorough mixing. Proteins were pelleted by centrifugation(10 min, 5500 g, 4 °C), and were washed three times withchilled precipitation solution and then 80% chilled acetonecontaining 10 mm DTT. The pellet was finally dried undervacuum.

Protein solubilization and quantification

The final pellet was resuspended in IEF buffer (7 m urea,2 m thiourea, 4% CHAPS, 10% Triton X-100, 20 mm DTT,1.6% ampholytes, pH 3–10) and was incubated at roomtemperature with constant shaking. Protein quantificationwas carried out following the modified procedure ofRamagli & Rodriguez (1985), based on acidification ofthe samples, and assaying them by Bradford assayreagent (BioRad). Serial dilutions of BSA (bovine serumalbumin) were used as standards.

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2-D PAGE analysis

Immobilized pH gradient (IPG) strips (17 cm) (linear,pH 3–10) from Bio-Rad were rehydrated at 20 °C with 0.75–1.0 mgof protein in 450 mL for 15–16 h. Focusing was carried out ina Bio-Rad Protean IEF Cell for a total of 35 000 volt hours.After focusing, the strips were equilibrated with reductionand alkylation buffers, and a second-dimension PAGE wascarried out onto a 12% (w/v) acrylamide gel. Gels werestained with Coomassie Brilliant Blue R-250, and imageswere digitized with a Bio-Rad Versa Doc imaging systemequipped with a 12-bit camera. Experimental molecularmass and pI were calculated from digitized 2-D PAGEimages using standard molecular mass–pI marker proteinsfrom BioRad. For immunodetection of the desired spots,two-dimensional gels were electroblotted and challengedwith anti-IMT1 and anti-INO1 antibody. Image analysis andquantitative analysis were carried out with PDQuest 8.0advanced software (BioRad), according to the manufactur-er’s instructions. Fold change was calculated as the normal-ized product quantity of stressed sample/normalizedproduct quantity in control sample. For each analysis, theanalysed data showed a high level of reproducibilitybetween normalized spot volumes of 3–5 replica gels and alow standard deviation from average.

Peptide mass fingerprinting (PMF) throughMALDI-TOF analysis of the spots

The gel piece containing the unknown protein was excisedfrom the gel, destained, reduced/alkylated and digestedusing the Montage In-Gel Digest Kit (Millipore, Billerica,MA, USA) following the kit’s instructions. Peptide sampleswere extracted and spotted onto a MALDI-TOF microtiterplate, and were covered with matrix CHCA (a-cyano-4-hydroxycinnamic acid; Sigma).

Analysis was performed using the Voyayer-DE PROBioSpectrometry workstation from Applied Biosystems(Foster City, CA, USA). Spectrum was obtained in the massrange of 500–4000 Da, and was calibrated using a calibra-tion mixture containing angiotensin I, Substance P, ACTH(1–17), ACTH (18–39) and somatostain (28). Peptidemasses of the unknown proteins were sent to two differentPMF databases, Mascot from Matrix Science (http://www.matrixscience.com), MS-Fit from Protein Prospector(http://prospector.ucsf.edu) and/or Aldente (http://www.expasy.ch/tools/aldente). Search parameters were asfollows: maximum allowed peptide mass error of 100 ppm,consideration of one incomplete cleavage per peptide, andat least four peptides identified.

Semi-quantitative RT-PCR

Semi-quantitative RT-PCR was performed to assay fortarget transcript amount for both PcIMT1 and PcINO1genes. A 500 bp part of rice Actin1 gene was used as theinternal control in the experiments. The protocol describedin Zhang et al. (1997) was followed for RT-PCR and

quantitation. Total RNA was reverse-transcribed, and PCRamplification was performed following the conditionsdescribed previously and was quantified for Actin1, INO1and IMT1 cDNA fragments. The end products were quan-tified in-gel using Quantity One software from BioRad.Theproduct quantity was normalized against Actin1 productsafter background subtraction. Fold change was calculatedas the normalized product quantity in stressed sample/normalized product quantity in control sample. Results inall cases are presented as the mean of three to five experi-ments with standard deviation.

RESULTS

Pinitol synthesis in clonally propagatedP. coarctata under salt stress

The plants grown in vitro in a NaCl-free medium up to10–12 cm were subjected to 400 mm NaCl stress, and theinositol and pinitol content of control and salt-treatedplants were determined by GC. In the no-salt condition, theplants showed a high inositol content in the leaves (up to~30 mmol g-1 fresh weight), whereas after 10 d incubation in400 mm NaCl, it declined to ~3.5 mmol g-1 fresh weight, con-comitant with the prominent increase of another polyolidentified as D-pinitol (O-methylated inositol) to about35–40 mmol g-1 fresh weight. A salt-sensitive variety (IR64)and a salt-tolerant variety (Pokkali) of domesticated ricetreated similarly were also analysed in search for a similarpresence of pinitol. In sharp contrast to the wild rice, theyproduced no pinitol under either stressed or unstressedcondition (Fig. 2a).

From similar samples as analysed previously, immunob-lotting of the total leaf protein with anti-McIMT1 showedpresence of a ~40 kDa immunoreactive protein in P. coarc-tata in both stressed and unstressed conditions, whereas inrice, this protein was undetectable. Probing with anti-OsINO1 antibody shows a ~60 kDa major band in both riceand Porteresia.

It appears that the INO1 protein content remained unal-tered in Porteresia under both stressed and unstressed con-ditions, whereas in both salt-sensitive and salt-tolerant ricevarieties, the amount of INO1 protein declined under salttreatment (Fig. 2b), commensurate with the known salt tol-erance and salt sensitivity of the corresponding PcINO1and OsINO1 enzyme proteins responsible for synthesis ofinositol (Majee et al. 2004).

Following detection of increased production of pinitol(Fig. 2a) and an immunodetectable presumptive IMT1protein (Fig. 2b) under salt treatment in Porteresia, it wasthought pertinent to look for any transcriptional inductionof the IMT1 protein in Porteresia. For this purpose,northern blotting was performed with total leaf RNA ofP. coarctata under stressed (400 mm NaCl) and unstressedconditions using the IMT1 cDNA from M. crystallinum as aprobe. RNA gel-blot analysis confirmed the presence of anIMT1 transcript, which showed a considerable increaseupon salt exposure (Fig. 2c).

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Isolation of PcIMT1 cDNA clone fromP. coarctata and in silico comparisonwith other methyl transferases from Oryza

Polyol analysis and the presence of IMT1 specific protein/transcript under salt stress presuppose that P. coarctata hasan IMT1 gene encoded by the genome of this wild rice,although the domesticated rice, O. sativa, may not have suchsequence. Using primers designed on the basis of thealready reported cDNA sequence of IMT1 from ice plant,M. crystallinum (GenBank accession number M87340;Vernon & Bohnert 1992), we obtained a PCR product of~1095 base pairs by RT-PCR of RNA isolated from NaCl-treated P. coarctata (Fig. 3a). Nucleotide sequencing revealsthat the cDNA obtained shows ~99% sequence similaritywith the reported ice plant IMT1 sequences. Multalin analy-sis of the virtually translated protein product (365-amino-acid long) shows only two amino acid deviation from theMcIMT1; G in place of D16, and I237 is replaced by V(Fig. 3b). It also has the O-methyl transferase 2 domain,SAM-binding motifs and the dimerization domain commonamong the plant methyl transferases. In addition, thesequence exhibits an N-glycosylation site at AA8 position(NYTQ) and an 18-amino-acid-long transmembranedomain from AA21 to AA38 (LAGLAVILANAAAFPMII)(Fig. 3c). The PcIMT1 sequence has been submitted toGenBank under the accession number EU240449 (NCBI).

However, similar RT-PCR procedure with the cultivatedindica variety of O. sativa failed to generate any such pre-sumptive IMT1 PCR product using McIMT1 based primers.The Oryza genome database (RAP-DB 2008 update, build3 assembly) was searched thereafter as well and no ORFfor IMT1 was found, which confirms the absence of IMT1gene sequences in domesticated rice in conformity withthe results in Fig. 2a,b. We analysed all the availablemethyl transferase 2 domain harbouring EST or putativesequences of Oryza from the database and aligned them

pairwise or in multiple in ClustalW. IMT1 sequences do notproduce significant match with any member of Oryzamethyl transferase family. Using the ClustalW-derivedalignment, a phylogenetic tree of all Oryza methyl trans-ferases, along with PcIMT1, was drawn in MEGA4 soft-ware. The tree shows PcIMT1 as an outgroup and to benearer to the unknown ancestor than to the other methyltransferases (Fig. 3d). It appears that PcIMT1 is an ancientand remotely related sequence in comparison with theOryza methyl transferases. The uniprotKB entries used foralignment is listed in Supporting Information Table S4.

An in silico analysis to search for possible IMT1sequences in other plant species was attempted. Databasesearch for related sequences from any other plant speciesshowed that, except for the M. crystallinum IMT1 sequence,the sequence does not yield a considerably high match withany annotated sequence to date. However, the sequenceis similar at C-terminal end with caffeic acid-3-O-methyltransferase coding transcripts from several plants (i.e. in theO-methyl transferase 2 domain, SAM-binding motifs anddimerization domain portions common among the plantmethyl transferases). An overview of some database searchresults is included in Supporting Information Table S5, indi-cating the database searched (with versions); the nearestmatches obtained from such database and the mRNA cov-erage in the obtained matches.

Bacterial expression, immunodetection andassay of the recombinant PcIMT1 protein

The cDNA for the putative IMT1 from P. coarctata(PcIMT1) was bacterially expressed as a ~40 kDa 6XHis-tagged protein product predominantly present in inclusionbody (Fig. 4a).The identity of the expressed PcIMT1 proteinwas confirmed by immunodetection with anti-McIMT1antibody, which showed a single immunoreactive band(Fig. 4b). The expressed PcIMT1 was solubilized from the

Figure 2. (a) Inositol and pinitol content present in total leaf sugar of Porteresia coarctata, Oryza sativa Pokkali variety (ST; salt tolerant)and IR64 variety (SS; salt sensitive) after 400 mm NaCl stress application and compared with control. (b) Western blot of same samples withanti-McIMT1 and anti-OsINO1 antibody. Total protein (15 mg) was loaded per lane. Purified protein samples (OsINO1 and McIMT1) wereused as marker. (c) RNA gel blot for PcIMT transcript from P. coarctata leaves. The upper panel shows the RNA blot using McIMT cDNAfragment as a probe. Equal loading of RNA was checked by ethidium bromide staining as shown in the lower pannel.

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pellet fractions and was purified as described in the methods.The IMT1 protein was detected from the various fractionsthrough SDS–PAGE and immunoblotting by both anti-McIMT1 and anti-His antibody (Fig. 4c,d). Further, thebacterially expressed and purified PcIMT1 was assayed forinositol methyl transferase activity, and pinitol productionin vitro by the complete system was confirmed by GC analy-sis (Fig. 4e,f). These experiments demonstrated that theexpressed PcIMT1 gene product is enzymatically functional.

Analysis of coordinated INO1 and IMT1expression during salt stress in Porteresia

An inverse relationship between inositol and pinitolcontent (Fig. 2a) and demonstration of increased IMT1 spe-cific protein/transcript in salt-stressed Porteresia (Fig. 2b,c)are suggestive of a salt-induced up-regulation of the IMT1gene in this system. Because pinitol is a product ofenzymatic methylation of inositol catalysed by the IMT1

Figure 3. (a) PCR amplification of PcIMT1 from cDNA of Porteresia coarctata. cDNA obtained from total leaf RNA of unstressedplantlets (lane 1) and 400 mm NaCl-treated plantlets (lane 2) amplified with McIMT1 cDNA-specific primers. Lane M shows 500 bp DNAladder. The amount of cDNA used in each reaction mix loaded per lane corresponds to 80 ng of total RNA. (b) Comparison of deducedamino acid sequence of PcIMT1 and McIMT1. Arrows indicate the sequence variations. (c) Diagrammatic representation of domainarchitecture of PcIMT1. NG, N-glycosylation domain; TM, transmembrane region; DMD, dimerization domain; OMT, O-methyltransferase domain; AA, amino acid. (d) Evolutionary relationships of 41 representative Oryza sativa methyl transferases with P. coarctatainositol methyl transferase 1. The evolutionary history was inferred using the neighbour-joining method. The optimal tree with the sum ofbranch length = 8.81382920 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstraptest (500 replicates) is shown next to the branches. The tree is drawn to scale. Branch lengths represent the evolutionary distancescomputed using the Poisson correction method and are in the units of the number of amino acid substitutions per site (PAM). Allpositions containing gaps and missing data were eliminated from the data set (complete deletion option). Phylogenetic analyses wereconducted in MEGA4.

Figure 4. (a) Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis of bacterially overexpressedrecombinant PcIMT1 protein from cell extract and (b) the corresponding immunoblot using anti-McIMT1 antibody. Lane1, purified6xHis-McIMT1; lane 2, supernatant from uninduced cell extract; lane 3, pellet from uninduced cell extract; lane 4, supernatant frominduced cell extract; lane 5, pellet from induced cell extract showing ~41 kDa overexpressed protein; lane 6, molecular weight marker.SDS–PAGE and the corresponding immunoblot of purified and dialysed fractions of 6xHis-PcIMT1 with anti-His (c) and anti-McIMT1(d) antibody. Lane 1, molecular weight marker; lanes 2–8, fractions eluted in 50–200 mm discontinuous gradient of Imidazole; lane 9,purified His-tagged McIMT1; lane 10, pulled PcIMT1 fractions. (e, f) Generation of methylated myo-inositol by recombinant PcIMT1. Gaschromatography (GC) traces of carbohydrates extracted from assay reactions are shown. Vertical axis shows the voltage applied.Retention times are plotted in the horizontal axis as well as indicated in the above peaks detected in all cases. (e) Control assay reactioncontaining myo-inositol and S-adenosyl methionine (SAM), without PcIMT1 enzyme after 60 min incubation. The position of myo-inositolis indicated (INS). Inset shows the GC trace for myo-inositol standard with exact retention time. (f) Post-assay reaction with PcIMT1enzyme after 60 min incubation shows generation of a new peak with retention time same as D-pinitol (PIN). Retention time of thestandard pinitol is shown in the inset.

Stress regulation of pinitol synthesis in wild rice 1449

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 1442–1459

function, a coordinated functioning of both the PcINO1and PcIMT1 genes is expected to be operative during saltstress in Porteresia for synthesis of pinitol.

To follow the expression of both PcINO1 and PcIMT1genes at protein level, a proteomic study was performed.The total leaf protein of 400 mm NaCl-treated anduntreated Porteresia plants was isolated and run in a 2-DPAGE to obtain a well-separated leaf proteomic profile ofP. coarctata, where each spot presented one expressedprotein in the experimental state and reflected their

quantitative pattern as well. From the respective gels, spotsimmunoreactive to anti-McIMT1 and anti-OsINO1 anti-body were detected (Fig. 5).The pI and molecular weight ofdetected spots match to those of the purified McIMT1 andOsINO1 proteins.

Further confirmation of the identity of these proteins wasobtained through PMF of the immunoreactive spots byMALDI-TOF analysis. The spot immunoreactive to anti-OsINO1 antibody was confirmed to be PcINO1 protein(Fig. 6a). However, for the spot immunoreactive to

Figure 5. Expression analysis on proteome profile through immunodetection. Data are average of three replicates � standard deviationfrom the mean. (a) 2-D-PAGE profiles of total leaf protein of Porteresia coarctata with or without NaCl stress, purified recombinantMcIMT1 and OsINO1 (left to right). In the total protein profiles, an equal protein amount (~170 mg) was loaded. Corresponding Westernblots with anti-McIMT1 (b) and anti-OsINO1 (c) antibody. (d) In-gel comparison of relative transcript level of PcIMT1 and PcINO1 inthe presence or absence of salt stress analysed by semi-quantitative RT-PCR. The product obtained from equal RNA amount is loaded.Product quantity is normalized with PcActin1 amplification. (e) Up-regulation of PcIMT1 and PcINO1 protein and transcript in terms offold change under 400 mm NaCl stress.

1450 S. Sengupta et al.

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Figure 6. Confirmation of spot identity through MALDI-TOF. (a) Mass spectrometric identification of the spot immunodetected withanti-OsINO1 antibody showing the closest match in database and confirmed as PcINO1. (b) The spot immunodetected with anti-McIMT1antibody shows no hit in database (spectrum I), although when matched with the spectrum of bacterially expressed McIMT1 and PcIMT1protein (spectrum II), the mass list gives 99% identity.

Stress regulation of pinitol synthesis in wild rice 1451

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 1442–1459

anti-McIMT1 antibody, no hit to a known protein was foundpresumably because of the absence of any mass data for anIMT1 protein in database. Hence, we compared the massspectra of this protein with the bacterially expressedMcIMT1 and PcIMT1 proteins. The m/z spectra matchedexactly with the mass spectra of the bacterially expressedpurified McIMT1/PcIMT1 protein with a ~99% identity inmass list, thus confirming its identity as IMT1 protein(Fig. 6b).

Indication of a coordinated functioning of PcINO1 andPcIMT1 genes during salt stress came from results oftheir transcript analysis through semi-quantitative RT-PCR.Both the transcripts were found to be up-regulated tovariable extent under 400 mm salt treatment, and theup-regulation of the PcIMT1 was several-fold higher thanthe PcINO1 (Fig. 5d,e; Supporting Information Table S2).The quantitative analysis of the protein spots obtainedin Fig. 5a–c in terms of spot volume indicates that whilePcIMT1 is up-regulated ~3-fold in 400 mm salt stress, thePcINO1 shows a moderate salt-induced uplift of expressionunder such condition (Fig. 5e; Table S3). Enhanced produc-tion of PcIMT1 enzyme can utilize the increased inositolpool because of up-regulated salt-tolerant PcINO1 functionto build up a stress-induced pinitol pool in Porteresia as isreflected in the inositol/pinitol content data (Fig. 2a). Suchcoordinated functioning of the INO1 and IMT1 hasearlier been demonstrated in the halophyte M. crystallinum(Ishitani et al. 1996).

Influence of different abiotic stresses ongrowth parameters of P. coarctata plantlets

Because salt stress remains as one of the most complexabiotic stresses that result in a number of secondarystresses, we employed four different stresses apart from400 mm NaCl, namely 10-6 m paraquat (Yun et al. 2000; Kojiet al. 2004), 75 mm ABA (Robertson et al. 1987; Johnson-Flanagan et al. 1991), dry heat (40 °C) and cold (4 °C) on2-month-old plantlets of P. coarctata, and monitored severalrelevant physiological parameters (Fig. 7). The detaileddose-dependent growth response of the plants in NaCl-containing medium (0–400 mm) is represented in Support-ing Information Fig. S1. For the other stresses, thetreatments were non-equivalent, and the performance ofthe plants under all such stresses is summarized in Fig. 7.There was an obvious bleaching of the shoot portions of theplantlets under paraquat and heat stress, indicating a loss ofchlorophyll as supported by the estimated chlorophyllcontent. Fresh weights of the plants were vividly affected inheat stress only. Paraquat and cold does not reduce orenhance the fresh weight of the plants within 96 h. It isconcluded from the results that 40 °C heat reduces growthperformances of the plants considerably and is ratherstressful. Interestingly, salinity did not affect the freshweight or chlorophyll content of the plants. It did not affectthe normal growth rate as well when we monitored thefresh weight at 48 and 96 h (Fig. 7a), neither did ABA.Photosynthetic efficiency of the plantlets was also measured

through chlorophyll fluorescence, which revealed interest-ing features. Excess excitation energy that is produced as aresult of several abiotic stresses almost always upshots in aprofound imbalance between the energy absorbed throughthe light-harvesting complex and the amount of energy thatcan be dissipated or transduced by photosystems in theform of heat or irradiance. This imbalance in energy trans-fer through PSII is detectable through the JIP test. In caseof the stressed plantlets, we observed a phenomenologicalflux of a particular dark-adapted leaf sample, which gives usinformation about the stepwise flow of energy on the levelof the excited cross-section (CS) for energy absorptionmaxima (ABS/CSm), energy trapping maxima (TR0/CSm),energy dissipation (DI0/CSm) and electron transport (ET0/CSm) (Fig. 7d). The density of active PSII reaction centresper CS was also estimated (Fig. 7d), along with the mea-surement of maximum quantum yield of primary photo-chemistry (FV/FM), which in this terminology is equal to theefficiency by which an absorbed photon will be trapped bythe PSII (Fig. 7c). The observed results suggest that salinitydoes not hamper PSII activity in Porteresia, neither hindersnormal growth nor degrades chlorophyll. Paraquat and heatreduce the quantum yield of the plants considerably. Inter-estingly, paraquat and heat also reduce the chlorophyllcontent; hence, the inefficient energy flow could occurbecause of the loss of chlorophyll. Reaction centres are alsoblocked to a significant percentage in these two stresses.Although the study of a more detailed effect of stresses wasnot undertaken in this work, the results obtained pointed atPorteresia’s inherent ability to fight stresses, like salinity,hyperoxidation and chilling, on a short-term basis. Further-more, it is suggestive of the presence of unique factors thatkeep the plant functioning in a multitude of abiotic stressesof which osmolytes could play an important role.

Regulation of pinitol synthesis pathway inP. coarctata under different abiotic stresses

To dissect out the regulation of pinitol synthesis under suchdifferent stress regime, transcript and protein analysesfor PcINO1 and PcIMT1 were carried out. Total RNAand protein were isolated from Porteresia leaves at 48and 96 h of stress treatment. Semi-quantitative RT-PCRand proteomic analysis were performed for the PcINO1and PcIMT1 transcripts (Fig. 8a; Supporting InformationTable S2) and the corresponding proteins through 2-DPAGE (Fig. 8b; Supporting Information Table S1). A fairlyuniform correlation between the transcript and the corre-sponding protein for the two genes were noticed underall stress conditions applied. The results show that PcIMT1is highly up-regulated in high salinity, oxidative stress(paraquat) and ABA. Cold stress does not alter thetranscript/protein quantity appreciably, whereas heatreduces the transcript quantity drastically. PcINO1,however, shows maximum up-regulation on application ofABA, oxidative stress and cold, moderately rises in highsalinity, and heat reduces it to some extent. Clearly, theresults support that in Porteresia, salt stress up-regulates

1452 S. Sengupta et al.

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 1442–1459

synthesis of pinitol, and inositol keeps a fair balance with it,harmonizing the need of the substrate for pinitol produc-tion. Moreover, as ABA increases the transcription of bothgenes, the strategy of salt tolerance through pinitol synthe-sis as employed in Porteresia may be an ABA-dependentone.

DISCUSSION

O-Methylated inositol or pinitol is a sugar alcohol that hasbeen known to be associated with salinity and other stressesin a diverse set of plant species including maritime pine(Nguyen & Lamant 1988), several legumes (Gorham,

ABS/CSm

TR0/CSm

DI0/CSm

1

1

zerom-496

ET0/CSm

ABS/CSm

TR0/CSm

DI0/CSm

ET0/CSm

ABS/CSm

TR0/CSm

DI0/CSm

ET0/CSm

ABS/CSm

TR0/CSm

DI0/CSm

ET0/CSm

ABS/CSm

TR0/CSm

DI0/CSm

ET0/CSm

ABS/CSm

TR0/CSm

DI0/CSm

ET0/CSm

LMScale 3123

LM Scale

LM Scale

ssg 22.1-365

3

ssg 22.1-367

3439

3439

LM Scale6

ssg 22.1-370

3439

LM Scale7

ssg 22.1-371

3439

LM Scale5

ssg 22.1-369

3439

Figure 7. Photosynthesis and growth parameters of the plants in multiple stresses as indicated. (a) Fresh weight of the plants measuredunder multiple stresses (at 48 and 96 h time points). (b) Chlorophyll content of the plants measured under multiple stresses (at 48 and96 h time points). (c) Quantum yield maxima (FV/FM) of the plants measured under multiple stresses (at 48 and 96 h time points).(d) Phenomenological yield models per excited cross-section under different stresses; yellow triangle, absorption maxima per excitedcross-section (ABS/CSm); blue pipe, electron transport per excited cross-section (ET0/CSm); green triangle, trapped energy per excitedcross-section (TR0/CSm); red block, dissipation maxima per excited cross-section (DI0/CSm); empty circles, active reaction centres; filledcircles, inactive reaction centres.

Stress regulation of pinitol synthesis in wild rice 1453

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 1442–1459

Figure 8. (a) Semi-quantitative RT-PCR analysis from Porteresia coarctata under different stress conditions. Several abiotic stresses, asindicated, were applied to P. coarctata plantlets, and the sample was collected after 48 and 96 h. Expression was checked in transcript levelby semi-quantitative RT-PCR. The amount of cDNA loaded in each lane was prepared from ~60 ng total RNA. Products were analysedfrom 15–30 cycles, and 25 cycles were found to be optima of the PCR reaction, after which the reaction entered into plateau phase. Thus,the product from 25 cycles were analysed quantitatively in Quantity One software, as an average of five replicates, after normalizationagainst PcAct. (i–iii) In-gel profile of PcIMT1, PcINO1 and PcAct1 transcript quantity after 25 cycles. (iv) Fold change in transcriptquantity after 48 h; (v) fold change in transcript quantity after 96 h. (b) Comparison of PcIMT1/PcINO1 protein level on 2-D PAGEunder different stress conditions (pI range, 3–10; strip length, 7 cm). Spots identified to be immunoreactive with anti-McIMT1/anti-OsINO1 antibodies were picked up from the profile and were analysed with PDQuest software. Data shown are average of fivereplicates with standard deviation. (i) In-gel changes in protein content after 48 h; (ii) corresponding fold change in protein quantity.

1454 S. Sengupta et al.

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 1442–1459

Hughes & Wyn Jones 1981), halophytic mangroves (Popp1984) and in the ice plant M. crystallinum (Vernon &Bohnert 1992). The exact physiological role of sugar alco-hols during osmotic stress is a matter of conjecture as yetand is not fully understood. There are two major assump-tions; one suggests that accumulation of sugar alcohols incytosol balances high osmotic potential of the vacuolesresulting from selective sequestration of ions away from thecytosol (Liu & Hellebust 1976; Pollard & Wyn Jones 1979).Compounds that serve this way are called osmolytes, andare non-reactive and non-disruptive to enzyme structureand activity even in high concentration (Liu & Hellebust1976; Gorham et al. 1981). In addition, as they accumulateto high intracellular concentration, they are generally meta-bolically inert. Pinitol exhibits such criteria and has alreadybeen claimed to serve as an osmolyte in several plants andstudied at greater details in ice plant.

The second purpose served by sugar alcohols in stress-tolerant organisms is macromolecular osmoprotection(Schobert 1977). In vitro studies indicate that these com-pounds may have hydrogen-bonding characteristics thatallow them to protect macromolecules from the adverseeffects of excessive water loss, high temperature andincreased ionic strength in the surrounding medium(Bieleski 1982), presumably by tightly associating withproteins and membrane components to replace waterlost during desiccation (Schobert 1977).

Adherents of the compatible solute hypothesis have longconsidered methylation to be a critical factor affecting thestabilizing properties of osmolytes that may reduce thecharge density and confer partial hydrophobicities toorganic osmolytes, as well as increase the size of the hydra-tion shell. Methylated sugar alcohols, like pinitol, can carryout either osmoregulatory or osmoprotective roles, depend-ing on the level to which they accumulate. It is difficult torecognize the relative importance of the overlapping rolesof sugar alcohols in vivo. The stress-induced increase inlevels of a limited number of such metabolites in a diverseset of organisms spanning all of the biological kingdomsargues in favour of the proposal that accumulation of com-patible solutes/osmoprotectants is an evolutionarily ancienttrait (Hare, Cress & Staden 1998). However, higher planttaxa show preferential accumulation of some compatiblesolutes over the others, which may be a reflection of theavailability of its precursors under adverse conditions. Onemay also hypothesize that differences in the solute typeaccumulated by various species may be correlated with thetaxonomic/ecological state of the plant.

In P. coarctata leaves, pinitol accumulates under salinitystress to a level of 30 mmol g-1 fresh weight, which is com-parable to the Nicotiana tabaccum transgenics overexpress-ing McIMT1 (Sheveleva et al. 1997). In the unstressedcondition, free inositol shows an accumulation of up to30 mmol g-1, and pinitol accumulates to a concentration of3.5 mmol g-1 (Fig. 2a). With application of stress, there is anincrease in transcript and protein levels of both PcINO1and PcIMT1. As PcINO1 is an intrinsically salt-tolerantenzyme, it can still produce the substrate for IMT1 in vivo

under salt environment, which seems to be rapidly con-verted to pinitol, concomitant with a reduction in free inosi-tol content. The stress-enhanced accumulation of pinitol inthe plant fits the requirements for an osmoprotectant func-tion, if not an osmolyte (Bohnert, Nelson & Jensen 1995;Ishitani et al. 1996).

The plant O-methyl transferases show high substratespecificity. IMT1 uses inositol as substrate, and SAM asco-substrate. The ado-met-dependent enzymes exhibitcommon signature motifs for SAM and S-adenosylhomocysteine (SAH) binding. SAH is the product remain-ing after the methyl transfer from SAM, and is a potentcompetitive inhibitor of SAM-dependent methyl trans-ferase reactions (Fig. 1). Thus, the methyl transfer rate onthe IMT1 is limited by three factors, namely availability offree inositol pool, availability of functional IMT1 enzymeand the intracellular ratios of SAM to SAH.

Our results show that PcIMT1 enzyme activity is notlimited in vivo by the substrate pool, as PcINO1 is active andcan synthesize free inositol even under stress (Majee et al.2004;Das-Chatterjee et al. 2006;Ghosh Dastidar et al. 2006).Although the intracellular ratio of SAM to SAH in Porter-esia under stress is not experimentally measured, based onearlier work ofVernon & Bohnert (1992) that SAH acts as aninhibitor of McIMT1 in vitro, and because the sequences ofPcIMT1 and McIMT1 are identical with exceptions of twoamino acid substitutions, it is possible that the activity ofPcIMT1 will also depend on the cellular turnover of SAH.SAH is hydrolysed to adenosine and homocysteine in cell bythe activity of SAH hydrolase. Homocysteine is recycled tomethionine by homocysteine methyl transferase with themethyl group derived from N5-tetrahydrofolate (THFA),one of the tetrahydrofolate compounds originating from C1

pool (Fig. 1).Salinity or dehydration stress-induced stomatalclosure is known to bring a reduction in internal CO2 levels.This results in elevated photorespiratory activity. Photosyn-thetic carbon oxidation (PCO) cycle activates the produc-tion of substantial amounts of glycine, serine, glutamate andN5-methylenetetrahydrofolate (MeTHF) (Hare et al. 1998).Operation of this activated methylation cycle (Fig. 1) bal-ances the SAM/SAH ratio in plants.Elevated PCO cycle andsubsequent activation of SAM production can compete withSAH production in cell. As a result of salt-enhancedfunctional IMT1 and INO1 expression and presumablyenhanced SAM production, increased pinitol synthesisunder stress may be operational in the plant.Salinity stress isknown to activate the methyl cycle with several instanceslike salt-induced transcript of a SAM synthase in tomato(Espertero, Pintor-Toro & Pardo et al. 1994) and threelight- and salt-responsive cytosolic SAM-dependentN-methylation reactions involved in choline synthesis inspinach (Weretilnyk et al. 1995). Support to this hypothesisalso comes from the findings of Sheveleva et al. (1997),whichproposed that methyl groups from photorespiration maycontribute to ononitol production in transgenic tobaccoexpressing the McIMT1. During salt stress, net CO2 fixationrates in these plants were less inhibited than in the wild type,and plants producing high amount of ononitol give better

Stress regulation of pinitol synthesis in wild rice 1455

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 1442–1459

Tab

le1.

Som

epl

ant

spec

ies

know

nto

prod

uce

pini

toli

nre

spon

seto

stre

ss

Spec

ies

Fam

ilySt

ress

/con

diti

ons

asso

ciat

edw

ith

elev

ated

pini

tolp

rodu

ctio

nN

ativ

eha

bita

tA

ccum

ulat

ion

leve

lof

pini

tola

sst

ated

inth

ere

fere

nces

Ref

eren

ces

Gly

cine

max

Leg

umin

osae

Hig

h-te

mpe

ratu

reda

mag

e,bu

tno

tlo

w-t

empe

ratu

rein

juri

esH

igh

nitr

ogen

;hum

idso

il30

mg

g-1dr

yw

eigh

t(n

odul

es)

Guo

&O

oste

rhui

s(1

995)

Sim

mon

dsia

chin

ensi

sC

aryo

phyl

lace

aeD

roug

ht,h

igh

salin

ity

Des

ert

and

mou

ntai

nsu

b-sl

ope

13m

gg-1

dry

wei

ght

(lea

fbl

ades

)N

elso

n&

Bar

tels

(199

8)A

ster

trip

oliu

mA

ster

acea

eIn

crea

sed

dehy

drat

ion

orsa

linit

yst

ress

Coa

stal

rock

ycl

iffs

36m

olm

-3G

orha

met

al.(

1981

)

Hon

keny

a(=

Are

nari

a)pe

ploi

des

Car

yoph

ylla

ceae

Incr

ease

dde

hydr

atio

nor

salin

ity

stre

ssSt

ony

coas

talr

idge

s15

.5m

olm

-3G

orha

met

al.(

1981

)

Mac

ropt

ilium

atro

purp

ureu

mL

egum

inos

aeIn

crea

sed

dehy

drat

ion

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linit

yst

ress

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ific

isla

nds,

natu

raliz

edto

aw

ide

rang

eof

salin

eso

il–

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ham

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.(19

81)

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umin

osae

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ease

dde

hydr

atio

nor

salin

ity

stre

ssW

ater

logg

edst

eam

bank

s,sa

line

and

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line

soil

–B

iele

ski(

1994

)

Med

icag

osa

tiva

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umin

osae

Incr

ease

dde

hydr

atio

nor

salin

ity

stre

ssD

rypl

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,tol

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tto

drou

ght

and

tem

pera

ture

fluct

uati

on22

348

nmol

g-1fr

esh

wei

ght

(nod

ules

)B

iele

ski(

1994

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uspi

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dehy

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yst

ress

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ntai

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1994

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llinu

mA

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ceae

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ease

dde

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atio

nor

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stre

ssSa

ndy/

rock

yco

asta

lare

as10

0m

olm

-3(c

hlor

opla

sts)

Bie

lesk

i(19

94)

Dis

phym

aau

stra

leA

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ease

dde

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nor

salin

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stre

ssC

oast

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es–

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aeIn

crea

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dehy

drat

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orsa

linit

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fore

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ycl

iffs

3.9

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g-1fr

esh

wei

ght

(lea

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iele

ski(

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agon

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igyn

aA

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stre

ssM

argi

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grow

son

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nes

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iffs

1.93

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ght

(gam

etop

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gula

ria

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sh4.

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nace

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drat

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idto

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il35

.76

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(lea

ves)

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i(19

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abili

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lapa

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nace

aeIn

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sed

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idac

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soil

1.66

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i(19

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ders

tore

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dsu

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6.6

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(lea

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lesk

i(19

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ussy

lves

tris

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e(5

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kym

ount

ain

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1456 S. Sengupta et al.

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 1442–1459

stress tolerance. In line with these, salt-subtracted cDNAlibrary of P. coarctata shows an up-regulated transcriptwith a high sequence similarity to O. sativa and Zeamays homocysteine S-methyltransferase (NCBI accessionnumber CO805234, our own unpublished data), which is anindication of the enhanced SAM production in P. coarctataunder salt stress.

The transcript and protein content of PcIMT1 was sub-stantially up-regulated in the presence of salt. Several otherstresses were applied on the plant, and the IMT1 transcriptor protein seems to be highly up-regulated in salinity, oxi-dative stress (paraquat) and ABA.When compared with thetranscription level of PcINO1, it seems clear that inositol,the precursor of pinitol, is also synthesized in accordanceand is being utilized too. When we measured plant growthand photosynthesis under similar stress conditions, itappeared that this high amount of pinitol is phenomeno-logically correlated with substantial protection of photosys-tem. In heat or oxidative stress, the PcIMT1 transcript wasreduced, and photosynthesis was also hampered, resultingin bleaching and even occasional wilting of the plant.However, this is only a correlative observation, and a directconnection of pinitol synthesis to the salt tolerance of theplant is yet to be established. P. coarctata leaves show uni-cellular salt hairs that are epidermal outgrowths and highlyvacuolated. The mechanisms of retention of sequesteredsalt in salt hairs are yet unknown, and so is the homeostasispathways in such hair cells. Flowers et al. (1990) and ourown unpublished data show that continued salinity stressresults in increase of Na+ ion concentration to ~7-fold, evenafter washing off the salt hairs and the salt excreted bythem. Such high NaCl concentration still needs homeostasisin the cell and protection of macromolecular structures andphotosystem, which can be achieved in part throughincreased production of inositol/pinitol. At induced methylcycle operating, this situation could be favoured over freeinositol accumulation as observed in ice plant as well as inwild rice.

To date, all the plants known to produce pinitol aredicots, except Carex extensa, the long bract sedge fromCyperaceae, which occurs in salt marshes (Gorham et al.1981). With the first report of isolation of a salt-up-regulated monocot IMT1 cDNA from the halophytic wildrice, we present evidence suggesting that pinitol productionpathway is up-regulated in Porteresia as a response of highsalinity experienced by the plant as in its native habitat. Ithas been shown in the halotolerant green algae, Dunaliella,that in the presence of a very high NaCl concentration(~3 m), it does not only survive, but also responds byenhancement of photosynthetic CO2 assimilation and bydiversion of carbon and energy resources for synthesis ofglycerol, the osmotic element in Dunaliella (Liska et al.2004). Salinity-induced high PCO cycle drives the accumu-lation of methyl group donors in cell (Hare et al. 1998),which might stimulate methylation of inositol as observedin Porteresia. Stress-induced pinitol synthesis is thecommon phenomena observed in several plant species, assummarized in Table 1.The absence of an IMT1 sequence in

cultivated rice proves interesting in this regard where theenzyme OsINO1 is inherently salt sensitive and cannotprovide the plant an uninterrupted inositol precursor poolunder salt stress, in contrast to PcINO1, which is active inthe presence of salt (Majee et al. 2004; Das-Chatterjee et al.2006; Ghosh Dastidar et al. 2006). The different nativehabitat could be correlated to such discrimination betweenthe metabolic pathway of the two species, namely O.sativa and P. coarctata, despite their close phylogeneticrelationship. P. coarctata is considered to have a moreancient origin than domesticated rice (Ge et al. 1999). Ourstudy also suggests that IMT1 sequence may be moreancient than the other methyl transferases present inO. sativa. The presence of a salinity-enhanced IMT1 in P.coarctata indicates that this more ancient plant needed thedead-end pinitol synthesis pathway in its native habitat(corresponding to 500–650 mm NaCl, seawater NaCl con-centration), which is absent in domesticated rice growing innon-saline soil (Fig. 3d). The search for more homologs ofIMT1 from other salt-tolerant monocots native to saltyenvironment and additional transgenic approaches involv-ing the total inositol methylation pathway would elucidatethe exact role of pinitol in the salt tolerance of P. coarctata,the halophytic wild rice.

ACKNOWLEDGMENTS

Thanks are due to Prof. Hans J. Bohnert, University ofIllinois, USA, for the gift of McIMT1 clone. This work hasbeen supported by research grants (to ALM) from theDepartment of Biotechnology, Government of India. S.S.G.is a Senior Research Fellowship Awardee of the Council ofScientific and Industrial Research, Government of India.

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Received 9 April 2008; received in revised form 20 June 2008;accepted for publication 27 June 2008

SUPPORTING INFORMATION

Additional Supporting Information may be found in theonline version of this article:

Figure S1. Dose and time-dependent growth-response ofPorteresia coarctata under NaCl treatment. (a) Change inleaf biomass of Porteresia coarctata under 0, 100, 200 and400 mm NaCl. Data taken in 24 h interval up to 96 h. Thebiomass is calculated as leaf dry weight in mg mm-2 leafarea and the change in biomass with time is represented inpercentage with respect to control (0 h is the control forevery treatment). The data are average of three randomreplicates. Vertical error bars represent standard deviationfrom mean. (b) Percent increase in plant fresh weight under0, 100, 200 and 400 mm NaCl. Data taken in 24 h interval upto 96 h. Increase in fresh weight (mg) with time is repre-sented in percentage with respect to control (0 h is the

control for every treatment). The data are average of threerandom replicates. Vertical error bars represent standarddeviation from mean. (c) Chlorophyll content of the plantsunder 0, 100, 200 and 400 mm NaCl. Data taken in 24 hinterval up to 96 h. The data are average of three randomreplicates. Vertical error bars represent standard deviationfrom mean. (d) Efficiency of open PSII centres (Fv/Fm) ofPorteresia coarctata under 0, 100, 200 and 400 mm NaCl.Data taken in 24 h interval up to 96 h. The data are averageof three random replicates. Vertical error bars representstandard deviation from mean.Table S1. Mean spot volume in 2-D PAGE, fold changeand the standard deviation among experimental replicatesunder different stresses. For each experimental conditionfive biological replicates were analysed.Table S2. Mean product quantity in reverse transcriptasePCR, fold change and the standard deviation amongexperimental replicates under different stresses. For eachexperimental condition five biological replicates wereanalysed. *Mean normalized product quantity = volume oftarget cDNA/volume of PcActin1 cDNA.Table S3. Quantification of bands in western blot. Data areaverage of three replicates.Table S4. Thirty-four PIRSF entries (UniProtKBsequences) for rice O-Methyl Transferases (OMT) family 2.Table S5. Some representative database search details forPcIMT1 cDNA sequence across available genomic/cDNA/EST assemblages. Some of the matches were putativelyannotated as various partial clones of methyl transferaseswith sequence similarity to PcIMT1 cDNA. High match wasobtained from Mesembryanthemum crystallinum cDNA/ESTs which is reported as inositol methyl transferasecDNA.

Please note: Blackwell Publishing are not responsible forthe content or functionality of any supporting materialssupplied by the authors. Any queries (other than missingmaterial) should be directed to the corresponding authorfor the article.

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