ORIGINAL ARTICLE

Targeted expression of L-myo- inositol 1-phosphate synthasefrom Porteresia coarctata (Roxb.) Tateoka confers multiplestress tolerance in transgenic crop plants

Lily Goswami & Sonali Sengupta & Sritama Mukherjee &

Sudipta Ray & Rajeswari Mukherjee & Arun Lahiri Majumder

Received: 8 January 2013 /Accepted: 8 April 2013# Society for Plant Biochemistry and Biotechnology 2013

Abstract Improving crop tolerance to osmotic stresses is alongstanding goal of agricultural biotechnology. In the presentwork the PcINO1 gene coding for a salt-tolerant L-myo-inositol-1-phosphate synthase (MIPS) from Porteresiacoarctata (Roxb.) Tateoka, a halophytic wild rice wasintrogressed into cultivated mustard, Brassica juncea varB85. The transgenic plants demonstrate increased toleranceto salinity and oxidative stress with elevated level of inositolin both roots and shoots. The yield and crop quality of trans-genic Brassica plants remain uncompromised and the plantswere able to stably grow, set seeds and germinate in salineenvironments. When targeted to seeds of Nicotiana, PcINO1was able to improve the seed survival rate under salinity anddehydration. Inositol and its derivatives regulate stressresponses in various ways, serving as compatible solutes orsignaling molecules. It is implicated that engineering inositolmetabolism may affect the plant metabolic network leading toa stress tolerant phenotype as enumerated here in transgeniccrop plants. How inositol itself or its derivatives affect the

overall metabolic pathways leading to a stress-tolerant pheno-type remains an intriguing question for future investigations.

Keywords Brassica . Lmyo-inositol-1 phosphate synthase .

Transgenic plants . Porteresia coarctata . Drought . Salinity

AbbreviationsCaMV35S 35S promoter from Cauliflower Mosaic VirusFID Flame ionization detectorGC Gas liquid chromatographyGMO Genetically modified organismMIPS L-myo-inositol-1-phosphate synthaseOsINO1 L-myo-inositol-1-phosphate synthase from

Oryza sativaPcINO1 L-myo-inositol-1-phosphate synthase from

Porteresia coarctataPEA Photosynthetic efficiency analyzerTLC Thin layer chromatographyTMS Tri methyl silyl

Introduction

The yield potential of a crop is largely limited by abiotic stress(Oerke et al. 1994). Such stress initiates several events in aplant system namely, perception of stress, activation of sig-naling pathways, cellular stress responses (activation of tran-scription factors and gene expression) followed by theactuation of stress phenotypes. Elite combination of stressresponses may help the plant to cope up with stress withoutcompromising the normal phenotype. Genetic engineeringoffers the unmatched capability to introduce beneficial genesfrom any origin, singly or sequentially to improve existingelite gene combinations, which further improves crop yieldand survival under adverse environmental conditions.

Electronic supplementary material The online version of this article(doi:10.1007/s13562-013-0217-7) contains supplementary material,which is available to authorized users.

L. Goswami : S. Sengupta : S. Mukherjee : S. Ray :R. Mukherjee :A. Lahiri Majumder (*)Division of Plant Biology, Bose Institute, Centenary Building,P-1/12, C I T Scheme VII M,Kolkata 700 054, Indiae-mail: lahiri@bic.boseinst.ernet.in

A. Lahiri Majumdere-mail: alahirimajumder@yahoo.com

Present Address:S. RayDepartment of Botany, University College of Science,Kolkata 700 019, India

J. Plant Biochem. Biotechnol.DOI 10.1007/s13562-013-0217-7

Plant responses to abiotic stress are dynamic, complex(Skirycz and Inze 2010; Cramer 2010) and often overlapping.At times, such abiotic stresses co-occur in nature resulting in acompound effect. Drought stress is often accompanied by hightemperature stress whereas salinity stress is again associatedwith drought stress. Hyperosmolarity is also associated withoxidative stress caused by excessive production of reactiveoxygen species (ROS). The combined effects of multiplestresses on plants are not specifically known in molecular orcellular level.

Many osmoprotectants enhance stress tolerance of theplants when expressed as transgenes (Bohnert and Jensen1996; Zhu 2001). Metabolic engineering of the pathwaysproducing various osmoprotectants/ osmolytes or sugars isthus a promising approach to produce stress- tolerant trans-genics (Abebe et al. 2003; Gage and Rathinasabapathi 1999;Garg et al. 2002; Kishor et al. 1995; Paul and Cockburn1989; Shen et al. 1997; Shen et al. 1999). Increase inproduction of antioxidants and compatible solutes such asproline, mannitol, sorbitol, trehalose, inositol and glycinebetaine (Nuccio et al. 1999) and alteration of glutathioneand ascorbate metabolism (Bartoli et al. 2000) has beenattempted in plants. The results showed variability in theimproved resistance and in some cases adverse phenotypiceffects were observed (Supplementary Table 1).

Myo-inositol derived from glucose-6-phosphate serves asa precursor to a number of metabolites with slow turnover inthe cell (Loewus and Murthy 2000). Based on the diversefunctions of inositol in plant metabolic pathways, it washypothesized that locale-specific expression of inositol willhave significant effects on plant reaction to stress. Inositolgenerates diversified lipid and sugar derivatives and providesa link between lipid and sugar signaling (Valluru and Ende2011). Inositol derivatives have significant dual functions assignals and key metabolites under salt stress. D-ononitol,pinitol, galactinol are inositol derivatives which can act ascompatible solutes or osmoprotectants; besides, inositol itselfcan function as an osmoprotectant being a sugar alcohol(Loewus and Murthy 2000). For these reasons, engineeringinositol metabolism for stress tolerance may have certainadvantages over other osmolytes with single mode of action(Sengupta et al. 2012) .

However, it is essential that production and supply ofinositol remains constant during salinity stress. To achievethat, the key enzyme for inositol synthesis in the cell, L-myo-inositol-1-phosphate synthase (MIPS; EC 5.5.1.4) mustremain active at high salinity. MIPS is an evolutionarilyconserved protein among kingdoms though it does not pos-sess an intrinsic salt tolerance in general, resulting in deple-tion of inositol pool during salt stress.

We have previously reported isolation and characteriza-tion of an INO1 gene (termed PcINO1) from a halophyticwild rice, Porteresia coarctata (Roxb.) Tateoka coding for a

unique MIPS enzyme that remains active in vitro upto500 mM of NaCl. The homologues of PcINO1, such asOsINO1 from the cultivated rice Oryza sativa and BjINO1from Brassica juncea code for salt-sensitive MIPS proteins.Sequence analysis of PcINO1 displayed a unique 37 aminoacid stretch that was established to be responsible for con-ferring an in vitro salt tolerance trait (Majee et al. 2004;GhoshDastidar et al. 2006). The PcINO1 gene has beenshowed to confer salt tolerance when introgressed into modelplants and other evolutionary diverse organisms from bacteriato crop plants (Das Chatterjee et al. 2006).

In the present investigation, introgression of PcINO1 inBrassica juncea was studied to judge its potential to raise agenetically modified (GM) food crop. Indian mustard orbrown mustard Brassica juncea (L.) Czern. & Coss.(Commonly called rai) is one of the most widely cultivatedoilseed crop in the Indian sub-continent. Brassica trans-genics have earlier been characterized with respect to herbi-cide tolerance (Mehra et al. 2000), resistance to pathogens(Mondal et al. 2007) and seed oil content (Sharma et al.2008). In the present study we report the development of asalt-tolerant Brassica juncea line by transformation with asingle PcINO1 gene and characterization of the transformedplants for their ability to sustain long periods of salt stress,protection against Reactive Oxygen Species (ROS) andmaintenance of crop quality as well reproductive behavior .The transgenics obtained showed significantly unaltered foodvalue and no allergenicity was predicted for the introducedprotein conforming to international consensus that the concen-trations of key toxic, anti-nutritional and allergenic com-pounds in the Genetically Modified Organism (GMO)should be within the range found in the parental variety(Anon 2002a, b, c; Schauzu 2000). In addition, we alsoshowed that localized expression of PcINO1 in seeds of amodel plant, tobacco, protects the seeds from water stress aswell as combined salt and water stress during seed maturationand germination. We propose that depending on thelocale of expression, introgression of PcINO1 can offermultiple stress tolerance to a crop plant without anyharmful alteration in its crop value and without the probableeffect of a foreign gene insertion.

Materials and methods

Allergenicity testing of PcINO1 sequence

The amino acid sequences of the introduced proteinswere screened for homology with known allergens athttp://AllergenOnline.com/. The Food and AgriculturalOrganization/ World Health Organization, FAO/WHOexpert panel and Codex Alimentarius Commission(CAC 2003a, b) suggest that an identity greater than

J. Plant Biochem. Biotechnol.

35 % over 80 or more amino acids should be used as aguideline to suggest or predict allergenicity (FAO/WHO2001). Proteins showing significant matches(>50 %) toknown allergens may carry potential clinical risks andshould be further confirmed by serum testing, skin pricktesting or even food challenge (Aalberse 2000).

Vector construction and plant transformations

OsINO1 and PcINO1 cDNA sequences (~1.5 kb; fromOryza sativa and Porteresia coarctata respectively), codingfor respective MIPS proteins were cloned into XbaI andKpnI sites of the plant expression vector pCAMBIA 1301.The 0.3 kb NOS terminator was cloned into EcoRI and SacIsite of pCAMBIA 1301. The CAMV35S promoter, 0.8 kb inlength, was cloned at Hind III and XbaI site ofpCAMBIA1301 containing NOS terminator. To make aseed-specific promoter-driven construct, Napin, a potentseed specific promoter was cloned at PstI and SalI sites ofpCAMBIA 1301. pCAMBIA 1301 with OsINO1/PcINO1sequences at XbaI and KpnI site and functioning undereither CAMV35S or Napin promoter and NOS terminatorwere used for plant transformation. All constructs weretransformed into Agrobacterium tumefaciens LBA4404 ina freeze-thaw method (Nishiguchi et al. 1987). TransformedA. tumefaciens strain LBA4404 were grown overnight in15 ml of AB medium (Chilton et al. 1974) supplementedwith appropriate antibiotics. The cells were collected bycentrifugation and suspended in liquid MS medium to obtainan OD600 of 0.5. These cells were then used for infection ofhypocotyl explants ofBrassica following themethod of Donnaand Barfield (1991) with few modifications. For tobacco trans-formation, matured leaf discs were taken as explants, essen-tially following the method as in Horsch et al. (1985).

Molecular analysis of the transgenic lines

Plant genomic DNAwas isolated by modified CTAB method(Chen and Ronald 1999). Transformants were confirmed byPCR using hptII gene specific primers, 5′ ATG AAA AAGCCT GAA CTC ACC GCG 3′ forward and 5′CTATTT CTTTGC CCT CGG ACG AGT 3′ reverse primers. Introgressionof PcINO1 was confirmed using PcINO1 specific primers ,bp526 CTC TCC CTG GCA TCT ATG ATC C bp547 andbp956 CCG GTT TTT TTT TTT GGT TTG CCC bp933,designed from a region of nucleotide present in PcINO1 genebut absent in OsINO1 gene, amplifying a region of 430 bp inPcINO1 gene only. Southern blot analysis was performed toconfirm the integration of the PcINO1 gene according tostandard protocol. The genomic DNA was digested withEcoRI/ HindIII . PcINO1 specific 430 bp (526–933) andOsINO1 and PcINO1 common 600 bp (1–600) regions wereused as probes radiolabelled with α32P dCTP.

Maintenance and generation advancement of the transgeniclines

The transgenic plants were maintained in green house(Temperature: 25±1 °C, Relative humidity: 50±10 %,Photoperiod: 14-h/10-h light/dark) and allowed to set seeds.Seed germination was accompanied by segregation analysisthrough hygromycin resistance. The transgenic Brassicaplants were advanced upto T4 generation while the trans-genic tobacco lines were advanced upto T2 generation , withmolecular analysis performed in each generation.

Stress treatment of plants in soil and physiological analysis

Plants were grown in soil and watered with 300 mM NaClsolution for 30 days every alternate day and the growth wasmonitored followed by different studies.

Photosynthetic performance of plants was measured byusing chlorophyll a fluorescence values measured using aPhotosynthetic Efficiency Analyzer (Hansatech InstrumentsLtd, King’s Lynn, UK). A single flash of light intensity of3,000 μmol / m2 / s was used dark adoption. Chlorophyll afluorescence transient was analyzed as described in Strasseret al. (1995) .

Fresh weight and length of root and shoots of the trans-genic plant has been measured and additionally, ratio offresh leaves to dry leaves has been noted, using the follow-ing formula:

The Ratio was calculated by

Number of fresh leaves � number of dried leaves

Total number of leaves freshþ dried leavesð Þ

Recovery of the plants from the salt shock

The plants were grown in salt for 10 days and then thegrowth of either the excised shoot bud or the whole plantletwas monitored in non-saline conditions.

Oxidative stress tolerance of the transgenic plants

Oxidative stress was generated in vivo by the herbicide,paraquat (methyl viologen; 1, 1′-dimethyl-4,4′bipyridiniumdichloride), a known oxidative stress inducer, which acts viathe electron transport chain to produce intracellular superoxideanion. Leaf disc pieces of the transgenic and wt plants weredipped into aqueous solutions of paraquat (0–4 μM). The leafdiscs were preincubated in dark for 22 h at 25 °C and thenplaced under moderate light intensity for 2 h and again incu-bated in the dark for 24 h at 25 °C (Slooten et al. 1995).

The paraquat-dependant oxygen radical reduction of po-tential yield of photochemistry (Fv/Fm) was measured byPhotosynthetic efficiency analyzer (PEA) and Hansatech

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Biolyzer HP3 software. The chlorophyll content was alsodetermined after paraquat treatment (Arnon 1949).

Comparison of lipid profile of the transgenic seedswith control seeds

Total lipids were extracted from seeds by using n-hexane andmethanol:chloroform (2:1,v/v). The samples were thendissolved in anhydrous methanol containing concentrated sul-furic acid (1 %, v/v) for obtaining methyl esters of fatty acids(Christie 1982). Fatty acid methyl esters were then purifiedby TLC using a solvent system of n-hexane-diethyl ether(90:10, v/v). Methyl esters were recovered by extractingthe bands in a mini column in chloroform. GLC of thefatty acid methyl esters obtained by TLC was done on aChemito 1,000 instrument by Chemito-Toshniwal, equippedwith a flame ionization detector (FID) and quantified byWinchrom software.

Estimation of leaf inositol content

Leaf inositol content was estimated from the plants after a10-day salt shock following isolation of the soluble sugarsby the method of Bieleski and Redgwell (1977). GC wasdone in a Chemito 1,000 GC equipped with flame ionizationdetector. Samples were TMS-derivatized (Low et al. 1994)with Tri-Sil- Z (Pierce) and were run through 3 % SP-2100stationary phase (Supelco) supported on chromosorb—W(Sigma) packed in a 1.8mts(l)×2 mm (i.d) glass column withN2 (flow rate 31 ml/ min) as carrier gas and oven temperatureprogrammed between 130 °C to 320 °C @ 100 °C/min.Quantification was made against similar runs with myo-inositol from Sigma-Aldrich(St Louis, MO) as standard.

Desiccation tolerance of the transgenic seeds of Nicotiana

Seeds were surface sterilized and then spread in MSmedia supplemented with different amounts of mannitolor PEG −6000. After 10 days dark incubation, seeds weretransferred to light at 23±1 °C and allowed to grow foranother 10 days. The germination frequency was studied fromthe experiments. The segregation of the T1 seeds had beentaken into account and deducted from experimental results.

Results

Determining the allergenicity scores of OsINO1and PcINO1 proteins

Introduction of a new ‘foreign’ protein in food by geneticengineering can cause allergic reactions. Potential allergenicityof a foreign protein is tested using several approaches, including

bioinformatics, in vitro digestibility of the protein, and bindingto antisera of allergic patients (Stiekema and Nap 2004; FAOand WHO 2003). The sequence of OsINO1 and PcINO1 wascompared with different allergen sequences available in thedatabase (http://AllergenOnline.com/). The nearest match forPcINO1 was a negligible 25 % with Mite allergen Lepd7precursor and for OsINO1, a 42.857 % identity with profilinin Apium graveolens (Celery) was found. With further 80merSliding Window Search, neither protein showed any matchwith known allergens. Such bioinformatic analysis, however,should be viewed as a prediction only pending actual dem-onstration of non-allergenicity of the material by appropriateexperimentation. Hence, PcINO1 and OsINO1 can be con-sidered tentatively as non-allergens to be used for raisingtransgenic food crops.

Generation of transgenic Brassica juncea var. B85overexpressing OsINO1 and PcINO1

Brassica juncea var. B85 hypocotyl explants were transformedwith binary OsINO1 and PcINO1 plant transformation con-structs (genes under CaMV35S promoter and NOS terminator,Fig. 1a; i).B.juncea- CaMV35s-OsINO1 (BjOsINO1, 192 lines)

�Fig. 1 Salt tolerance in the BjPcINO1, BjOsINO1 and wt plants. a i.Vector construct used for transformation where OsINO1/PcINO1werecloned under CaMV35S promoter and NOS terminator. LB: Left Border,RB: Right Border, hpt: Hygromycin phosphotransferase gene, 35SP:CaMV35S promoter, OsINO1: OsINO1 gene, PcINO1: PcINO1 gene,GUS: β -Glucuronidase Reporter gene, NOS: Nopaline synthase termi-nator sequence. ii. PCR analysis of (1)BjPcINO1, (2)BjOsINO1 (3) wtwith (ii) common primers amplifying a region of 1.5 kb (iii), hptII primersamplifying a region of ~800bp (iv) PcINO1 specific primers ampli-fying a 430 bp fragment only in PcINO1. M1 is marker. b (i):Southern blot analysis of the transgenic lines and wt plants with a PcINO1specific 430bp probe and EcoRI digested genomic DNA. Lane 1: Positivecontrol (PcINO1 in pGEMT-EASY), 2wt, 3: BjOsINO1 (negative control,Rb2, T2), 4: BjPcINO1 (Pb11,T1), 5: BjPcINO1 Pb4 (T2), 6: BjPcINO1Pb2 (T2). (ii) Southern blot analysis for determining the copy number ofgenes introgressed using same probe and HindIII digested genomic DNA.Lane1: wt, 2: BjOsINO1 (negative control, Rb2;T2), 3: BjPcINO1(Pb11;T0 generation), 4: BjPcINO1 (Pb4,T0), 5: BjPcINO1 (Pb11,T1), 6:BjPcINO1 (Pb2; T2 generation); 7 : BjPcINO1 (Pb4 ,T2), 8: BjOsINO1(negative control,Rb19 ;T2) , 9: BjPcINO1 transformed line Pb9. Lane 7shows two copies of the transgene. (iii). Southern blot analysis of thetransgenic for determination of the copy number of the gene introgressed.A 600bp region of PcINO1 digested with BamHI , common to bothPcINO1 andOsINO1was used as the probe . Genomic DNAwas digestedwith HindIII . Lane1: PcINO1 in pCAMBIA 1301 (Positive Control), 4, 5and 6: BjOsINO1 (lines Rb2.1, Rb2.2, Rb2.3 respectively;T4), 7, 8 and 9:BjPcINO1 (lines Pb11.1, Pb11.2, Pb11.3 respectively,T4), 11: wt. c Wt,BjOsINO1 (Rb19, T2) and BjPcINO1 (Pb4, T2) plants photographed after300 mMNaCl stress treatment in soil for 30 days. d Reproductive growthof BjOsINO1 (Rb19, T2) and BjPcINO1 transformed (Pb4, T2) plants aftera 20 days recovery from salt stress. The wt plants did not survive the salttreatment. e Fv/Fm ratio of 10 days salt treated plants. f Radar plotpresentation of different parameters of the sub steps of photosynthesis ofthewt (red),BjOsINO1 (blue) andBjPcINO1 (green) transformed plants inunstressed condition. For details see experimental procedures. g Same as funder salinity stress (100 mM NaCl for 10 days)

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and B.juncea- CaMV35s-PcINO1 (BjPcINO1, 253 lines) wereobtained with regeneration frequencies of 11.8 % and 13.2 %,respectively. Introgression of the genes was confirmed by PCRanalysis for hygromycin marker (hptII) and candidate gene(INO1). Positive transformants were maintained in green-house, allowed to set seeds by selfing, and advanced up tothe T4 generation.

Stringent molecular screenings for the transgenics wereperformed during generation advancement. PCR was carriedout for hptII and OsINO1/PcINO1 for Brassica transgenics(Fig. 1a, ii-iv). Southern blot hybridization of the transformedBrassica (Fig. 1b) plants was performed using probes specific

for genes introgressed. The copy number of the inserted genewas also determined in Brassica transgenics in T2 and T4

generations (Fig. 1b). All but oneBjPcINO1 lines carry a singlecopy of the transgene. Segregation tests performed duringgeneration advancement showed 3 Hyg R: 1Hyg S segregationratio indicating single copy insertion (data not shown).

Survival of the B.juncea-CaMV35s-PcINO1/OsINO1in saline soil

Performance of the plants in saline soil is the closest simulationof the conditions the plants would encounter in a field trial.

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One-month-old T2 plants were transferred to soil after 2 weeksof hardening in soil-rite mix. The growth of the plants wasstudied after being watered with 300 mM NaCl solution everyalternate day for 30 days. Thewt andBjOsINO1 lines exhibitedan inferior growth compared to that of BjPcINO1 lines(Fig. 1c, d). The BjOsINO1 lines survived high NaCl in soilbut growth was stunted and the plants were unable to enter thereproductive stage whereas the BjPcINO1 lines survived thesalt- stress and continued to grow and flower normally afterinitial wilting (Fig. 1c). When the same plants were allowed torecover from stress after a month by resuming normalwatering, wt plants did not recover from salt shock, but thestunted BjOsINO1 lines resumed growth to some extent,initiated flowering after 10–15 days and set seeds of normalvigor. The BjPcINO1 lines performed normally (Fig. 1d).

Photosynthetic efficiency of the transgenic Brassica linesupon exposure to salinity

During abiotic stresses including drought, salinity or oxidativestress, a reduction in photosynthesis accompanied by produc-tion of Reactive Oxygen Species (ROS) is observed. Photo-oxidative damage is a major contributor to poor agronomicperformance in salinity. The effect of salinity stress wasassessed by determining the quantum yield of PS II (Fv/Fm)photochemistry by using in vivo chlorophyll fluorescencetechniques (Strasser et al. 1995). 10 days of treatment in100–400 mM NaCl lowered Fv/Fm (the quantum yield ofPS II photochemistry) to 60 % and 50 % in 300–400 mMNaCl in wt and BjOsINO1 lines respectively. BjPcINO1 linesexhibited negligible drop (5–8 % in 300 mM and 400 mMNaCl, Fig. 1e).

Other photosynthetic parameters analyzed are representedin Radar plots (Strasser et al. 1995). From the multiparametriccomparison (relative to the corresponding value of control)of photosynthesis, it was observed that performance indicesvalues like PI (CS0) (Performance index per cross sectionat minimum fluorescence), PI (CSm) (Performance indexper cross section at maximum fluorescence) and PI (ABS)(Performance Index at equal absorbance) show comparablevalues for wt, BjOsINO1 and BjPcINO1 lines under un-stressed condition (Fig. 1f). However, the same parameterswere strikingly higher in the BjPcINO1 lines compared tothe BjOsINO1 and wt plants under salinity stress (Fig. 1g).Other parameters like ABS / CSm (Absorbance / Reactioncemtre at maximum fluorescence) and TR0/CSm (Trapping /Reaction center at maximum fluorescence), ET0/ CSm(Electron transported / Reaction center at maximum fluores-cence), RC/CSm (Density of reaction centers which remainopen at Fm) in NaCl also showed similar trends.

The physiological effects of stress upon transgenic plantshave been quantified in terms of shoot and root fresh weightand root length. After 10 days of growth in different

concentrations of salt, the leaves of wt and BjOsINO1 plantswere found to turn yellow from 200 mM of NaCl onwards.BjPcINO1 transformed plants were observed to survivebetter in comparison. The growth of roots showed signifi-cant difference in all the three cases. BjPcINO1 plantsexhibited much denser and new root growth in differentconcentrations of salt in comparison to others.

Since Brassica plants grow in an axillary growth mode,to compare the survival and tolerance to salt, the ratio offresh leaves and dried leaves becomes an important factor.The ratio of fresh vs. dried leaves were calculated after10 days of incubation in different concentrations of NaCland it was found that throughout the range of differentconcentrations of NaCl used, BjPcINO1 showed larger num-ber of fresh leaves than that of dried leaves in comparison tocontrol and OsINO1 transformed plants.

The shoot and root fresh weights also serve as an impor-tant dataset in physiological property assessment. We havepreviously shown the beneficial effect of inositoloverproduction during salt stress on plant biomass. Itwas shown that increased NaCl in media exerts delete-rious effect on the fresh weight of BjOsINO1 and wtplants, whereas the effect is less pronounced inBjPcINO1 plants and they can also recover from salinitystress better than the others (Das Chatterjee et al. 2006;Fig. 1). Although these results may not entirely reflect theperformance of the transgenic plants in the field , at this stagethese provide a close approximate to the same. Furtherexperiments for evaluation of their field performance arecontemplated.

BjPcINO1 lines are considerably adept to copewith fluctuating salinity, and recover from salt shock

We have previously shown that the BjPcINO1 lines displayeda better tolerance to high salinity than BjOsINO1 or wt plantsin their first generation (Das Chatterjee et al. 2006). In thisstudy, we further tested the association of salinity tolerance ofBjPcINO1 with the product quality as a pre-requisite to large-scale production and suitability of the lines for release of thetransgenic plants for field trial as potential GM crop.

Plants are often exposed to fluctuating salinity and hencethey need to adjust to increasing salinity as well as to rapidlyreturn to the normal state once salt is removed. The BjPcINO1and BjOsINO1 lines were analyzed for their ability to recoverfrom salt stress. For whole plants, upto 100 mM of salt wasendured by the wild type (wt) , BjOsINO1 as well asBjPcINO1 lines and all of them recovered well. At 200 mMNaCl,wt andBjOsINO1 lines were inferior toBjPcINO1 lines,although all of them recovered the normal growth. At300 mM, wt and BjOsINO1 could not recover normal growthphenotype in contrast to the BjPcINO1 lines that recoveredand gave out new shoots in 21 days (Fig. 2a).

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Green shoot buds from salt-stressed plantlets (100, 200,300, or 400 mM NaCl for 10 days) were excised and allowedto recover and give rise to new plantlets in non-saline mediumas described in experimental procedures. Starting 200 mMNaCl concentration, the wt and BjOsINO1 shoot bud lost theirability to recover growth. The shoot buds from BjPcINO1lines, recovered to their full growth within 30 days, givingrise to a normal rooted plantlet (Fig. 2b). In 100 mMNaCl, theBjOsINO1 lines recovered to some extent, but growth patternwas inferior compared to BjPcINO1 lines (Fig. 2b).

These results show that in addition to superior salt en-durance, BjPcINO1 plants also attain a better survivalchance in dynamic groundwater salinity, compared to wtand BjOsINO1 lines, and this property is independent ofgrowth stages of the plant.

Inositol content and root/shoot allocation in the transgenicplants

A direct correlation between free inositol content and salinitytolerance in the PcINO1 transgenic plants was observed ear-lier (Majee et al. 2004; Das Chatterjee et al. 2006). Estimationof total inositol content in the roots and shoots of the exper-imental systems in T0 and T2 generations are summarized inTable 1. BjPcINO1 lines showed an increase in total shootinositol content compared to the wt and BjOsINO1 lines. Theanalysis of total inositol content was extended to differentconcentrations of salt ranging 100 to 300 mM NaCl.Interestingly, with increasing concentration of NaCl in themedia, the roots had a significant increase in inositol content,the highest being in the BjPcINO1 transformed plants in300 mM NaCl. Moreover, with the increase of total inositol

content in the root system, shoot inositol content graduallydeclines. Nelson and Jensen (1995) proposed, that a myo-inositol dependent leaf-to-root inositol transport mechanismis operative interconnected with sodium uptake. Understressed conditions, increased levels of leaf myo-inositol inBjPcINO1 might be transported to the roots, where it couldhelp in Na+ sequestration in vacuoles as suggested by Nelsonand Jensen (1995).

Crop value: Nutritional quality of mustard oil obtainedfrom transgenic Brassica seeds remains essentiallyuncompromised

Being an oilseed crop, oil obtained fromBrassica is of primaryeconomic importance. Mustard varieties commonly grown inIndia are characterized by high (30–51%) erucic acid and low(13–23%) oleic acid content. Oil high in oleic acid has demandin commercial food-service applications due to a long shelf lifeand cholesterol-reducing properties. Both linoleic andlinolenic acids are essential fatty acids but less than 3%linolenic acid is preferred for oil stability (Kaushik andAgnihotri 2003). High oleic, moderate linoleic and lowlinolenic acid contents in the oil are considered beneficial forhuman consumption. The oilseed quality of the transgenicplants in their third generation was analyzed by a lipid profil-ing (Table 2). Seeds of BjOsINO1 and BjPcINO1 lines show ahigher oleic acid and linoleic acid content as compared to thecontrol plants. Erucic acid however, is higher in one of theBjPcINO1 lines although generally lesser in both BjPcINOand BjOsINO1 plants. Taken together, the seed oil showsmarginal differences compared with the control plants, and issuitable for human consumption.

Fig. 2 Study of recovery of thetransgenic and wt plants understerile conditions a Recovery oftransformed B.juncea plantsfollowing 10 days of salttreatment. Plants werephotographed after 21 days ofrecovery. b Growth of excisedshoot bud from wt, BjOsINO1and BjPcINO1; after 30 daysrecovery from 10 days of saltstress

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PcINO1 introgression additionally imparts oxidative stresstolerance to transgenic plants

High salt concentrations affect the integrity of cellularmembranes, the activities of various enzymes, nutrientacquisition, and photosynthesis in part by generatingreactive oxygen species (ROS). Osmolytes are known todetoxify plants by scavenging ROS. To determine ifinositol overproduction results in stress-generated ROSscavenging, the BjPcINO1 lines were treated with paraquat(Methylviologen, MV), a potent herbicide. Paraquat reducesoxygen to superoxides, which are free radicals that affectchloroplast activity (Takahashi and Asada 1988). The reducedoxygen species formed by re-oxidation of reduced paraquatmight propagate to the reaction centers of PSII resulting in theloss of photosynthetic efficiency (Fv/Fm) (Slooten et al. 1995)and out of the chloroplasts to the cell membrane resulting in anincrease of ion leakage.

Paraquat-mediated damage in the treated leaf segmentsfollowed by an exposure to light was measured by thereduction of maximum photosystem II (PSII) efficiency(Fv/Fm) in the dark-adapted leaf discs (see experimentalprocedures). With increasing concentration of MV, therewas a gradual decrease of Fv/Fm in wt and BjOsINO1 whileBjPcINO1 lines maintained high Fv/Fm values even inhigher concentrations of paraquat (Fig. 3a).

The total chlorophyll content of the leaves after paraquattreatment showed a sharp drop in the wt and BjOsINO1 lines.BjPcINO1 lines, however maintained constant chlorophyllcontent upto 2 μM followed by decrease at 3 μM MV. At4 μMMV, the BjPcINO1 lines displayed a drop of chlorophyllcontent of 28% as compared to 50% and 64% for control andBjOsINO1, respectively (Fig. 3b). It is possible that inositol orsome of the inositol- derivatives, on overproduction protectthe photosynthetic machinery from oxidative stress and leadto better survival.

Table 1 Comparison of totalinositol content of the wt , trans-genic BjOsINO1 (line Rb2) andBjPcINO1 (line Pb4) Brassicaplants from T0 and T2 generations

NaCl concentration T0 Generation T2 Generation

Shoots Roots Shoots Roots

Control (wt) 0 mM 5.19 – 3.25 1.9

100 mM 3.46 – 3.6 10.6

300 mM 1.31 – 4.9 –

OsINO1 (Rb2) 0 mM 5.73 – 6.7 3.2

100 mM 4.91 – 9.4 12.7

300 mM 2.35 – 9.5 17.2

PcINO1 (Pb4) 0 mM 6.86 – 5.9 5.76

100 mM 9.25 – 14.73 14.82

300 mM 5.54 – 13.09 42.63

Table 2 Comparison of the lipidprofile of three independentPcINO1 lines (Pb11,Pb2 andPb4), two OsINO1 lines (Rb2and Rb19) in their third genera-tion and Control plants. Majorfatty acids in Brassica includedErucic acid (22:1), Oleic acid(18:1), Linoleic acid (18:2ω3)and Palmitic acid (16:0)

Lipid profiles in seeds of representative lines of transgenic and control plants

Components Control Pb11 Pb2 Pb4 Rb2 Rb19

16:0 (Palmitic acid) 6.02 14.14 9.52 5.54 12.19 7.20

16:1(Palmitoleic acid) – 0.27 – – 0.16 0.20

18:0 (Stearic acid) 1.54 1.39 1.25 0.80 1.94 1.27

18:1(Oleic acid) 14.46 13.92 18.75 13.77 13.04 21.23

18:2ω6 1.71 1.26 0.74 2.34 3.72 0.51

18:2ω3 (Linoleic acid) 18.99 26.13 25.84 21.76 22.41 24.37

18:3ω3 (α-Linolenic acid) 8.19 6.35 5.40 3.8 2.37 9.76

20:0 (Arachidonic acid) 0.74 0.45 0.46 0.42 0.81 0.32

20:1 7.45 5.72 5.67 5.86 5.95 5.66

20:2 0.68 0.49 0.24 0.17 0.50 0.35

22:0 0.74 1.10 1.10 0.96 2.33 0.53

22:1(Erucic acid) 34.55 22.81 27.38 40.98 17.54 24.66

22:2 0.97 1.36 0.61 0.76 0.77 0.60

24:0 0.73 1.16 1.14 1.16 0.90 1.05

J. Plant Biochem. Biotechnol.

Seed specific expression of PcINO1 increases germinationpotential of seeds under stress

During seed setting, L-myo-inositol-1-phosphate is convertedto inositol hexaphosphate (Phytic acid), which is then hydro-lyzed to phosphate and myo-inositol during germination.Seed-specific overproduction of inositol during germinationmay thus increase germination potential of the seeds and suchproperties would be beneficial for a GM crop to ensure max-imum seed survival under conditions of drought stress. To testthis hypothesis in a model plant species, both OsINO1 andPcINO1were cloned into expression vectors under the controlof Napin , a seed–specific promoter and introgressed inNicotiana tabaccum (Fig. 4a).

Generation of transgenic Nicotiana tabaccum overexpressingOsINO1 and PcINO1 under seed specific promoter

Around 50 PCR-positive Nicotiana lines were obtained forboth OsINO1 and PcINO1. Expression of the genes wasdriven by Napin, a strong seed-specific promoter (Jako et al.2001; Iwabuchi et al. 2003; Mietkiewska et al. 2004; Mönkeet al. 2004). The independent lines of N.tabacum-Napin-OsINO1 and N.tabacum-Napin-PcINO1 were maintained ingreenhouse and selfed for two generations to obtain T2 seeds.

PCR was carried out for hptII and OsINO1/PcINO1for Nicotiana transgenics, (Fig. 4b, i,ii,iii). Southern blot

hybridization of the transformed plants was performedusing probes specific for genes introgressed (Fig. 4c).Segregation tests performed during generation advancementshowed 3 Hyg R: 1Hyg S segregation ratio indicating singlecopy insertion (data not shown).

The lines obtained are henceforth referred to asNtNOsINO1(Nicotiana tabaccum Napin OsINO1), NtNPcINO1 (Nicotianatabaccum Napin PcINO1 and NtCPcINO1 (Nicotianatabaccum CaMV35s OsINO1). Seeds from the transgenicplants were allowed to germinate in presence of NaCl, mannitolor polyethylene glycol to impose high salt and dehydrationbarrier during germination.

Seed-specific expression of PcINO1 demonstrates highergermination rate under salinity stress

Seed-specific expression of PcINO1 accompanied by in-creased inositol production should help germination in asaline environment. Seeds of wt, NtNOsINO1, NtNPcINO1and NtCPcINO1 plants were germinated in presence ofdifferent concentrations of NaCl.Wt, and NtNOsINO1 seedsshow poor germination frequency, small seedling size withrolled up leaves starting from 50 mM NaCl (Fig. 5a).NtCPcINO1 seeds show fair germination frequency upto100 mM NaCl. NtNPcINO1 seeds demonstrated the highestgermination frequency (50%) in 100 mM NaCl (Fig. 5b)and maximum seedling growth in terms of root and shootgrowth (Fig. 5c and d).

Seed specific expression of PcINO1 augment desiccationtolerance during germination

There was a marked difference in the germination frequencyamong the wt, NtNOsINO1, NtNPcINO1 and NtCPcINO1seeds from 50mMmannitol onwards (Fig. 6a) which becomesmost prominent in 150 mM concentration. The percentage ofseed germination (Fig. 6b) in wt seeds reduces to 48% in150 mM and 28% in 200 mM; in NtNOsINO1 to 50% in150 mM and 38% in 200 mM while in NtNPcINO1 thereduction in germination percentage is negligible. 91% seedsgerminate in 150 mM mannitol and 81% germinate in200 mM mannitol. However, for all the seeds, the bestseedling growth is observed in unstressed condition. Instressed condition, the root and shoot length of the seed-lings were significantly reduced in wt and NtNOsINO1seedlings compared to that of NtNPcINO1 seedlings(Fig. 6c, d). A higher tolerance to water stressed condi-tions of the NtNPcINO1 seeds are thus indicated, althoughthe normal germination and growth are still compromisedto some extent.

Similar experiments for desiccation tolerance wereperformed by addition of PEG 6000 in the growth mediumin following concentrations- 0%, 5%, 10%, 15% and 20%

Fig. 3 Oxidative stress tolerance in PcINO1 transformed B.juncea plantsaComparison of Fv/Fm ratio of the leaves of wt,BjOsINO1 andBjPcINO1,treated with different concentrations of paraquat. b Chlorophyll content ofthe paraquat treated leaves

J. Plant Biochem. Biotechnol.

(corresponding osmotic pressures; 0MPa, −0.05 MPa, −0.15MPa, −0.30MPa and −0.49MPa; Lagerwerff et al. 1961). TheNtNPcINO1 seeds exhibited a fair germination frequency atupto 10% of PEG 6000, while the wt and NtNOsINO1 seedsfailed to show any germination in high PEG percentages(Fig. 7a, b). NtCPcINO1 seeds exhibited germination at upto5% PEG. Taken together; NtNPcINO1 seeds can toleratehigher negative osmotic pressures, compared to that of wt,NtNOsINO1 and even NtCPcINO1. This superior perfor-mance was also demonstrated in the growth of young seed-lings when root and shoot lengths were compared (Fig. 7c, d).

Cumulative effect of salt and dehydration on germinatingtransgenic seeds

The above seeds were further evaluated based on their germi-nation potential in presence of both drought and salinity stress.In a germination experiment involving wt, NtNOsINO1,NtNPcINO1, NtCPcINO1 seeds in media containing 5% PEGand 50 mM NaCl together; only the NtNPcINO1seeds germi-nated. However, the compound stresses caused compromisedphenotypes in NtNPcINO1 (Fig. 8a).

Role of exogenous inositol in desiccation tolerancein the transgenic seeds

Adding inositol exogenously in the media validated thedefinitive role of inositol in germination in a dehydrated

environment. Wt seeds were germinated in growth mediumcontaining 5% PEG-6000 in absence or presence of exoge-nous inositol (2uM to 7uM). In absence of inositol, wt seedsfail to germinate. However, when supplemented with exoge-nous inositol, the seeds were found to germinate well in 5%PEG −6000 (Fig. 8b). The dosage difference was not evidentin inositol-aided germination.

Discussion

Engineering inositol metabolism in plants to achieve stresstolerance indicates certain uniqueness over engineering path-ways for overproduction of dead-end metabolites like glycine-betaine or proline (Sengupta et al. 2012). Inositol occupies acentral position in cellular metabolism and can be used in cellsto generate (i) phosphatidylinositol (PtdIns) and its derivatives;(ii) inositol polyphosphates (InsPs) (iii) compatible solutes suchas pinitol, galactinol, raffinose-family oligosaccharides (RFOs)and (iv) cell wall polysaccharides. These compounds partici-pate in several functions including signal transduction, mem-brane trafficking, mRNA export, stress tolerance and cellularphosphorus storage (Loewus and Murthy 2000; Valluru andEnde 2011). Monophosphorylated isomers of PtdIns (PtdIns3P,PtdIns4P, PtdIns5P) have been implicated in stress signalingpathways, especially in hyperosmotic stress, salt stress anddehydration stress. Phosphatidylinositols act as membranestructural lipids as well as signaling molecules. Several stress

Fig. 4 Analysis of the Nicotiana tabacum transformed with PcINO1and OsINO1under Napin (NtNPcINO1 and NtNOsINO1) a Vectorconstruct used for Nicotiana transformation, where OsINO1/PcINO1were cloned under Napin promoter and NOS terminator. LB:Left Border, RB: Right Border, hpt: Hygromycin phosphotransferasegene, 35SP: CaMV35S promoter, OsINO1: OsINO1 gene, PcINO1:PcINO1 gene, GUS: β -Glucuronidase Reporter gene, NOS: Nopalinesynthase terminator sequence. b PCR analysis of the (1) wt, (2)

NtNOsINO1 and (3) NtNPcINO1: with (i) primers amplifying a sharedstretch of 1.5 kb, (ii)with hptII specific primers (ii) with PcINO1specific primers amplifying a 430 bp fragment. M1 is marker. cSouthern blot analysis of the transgenic lines. PcINO1 specific 430bpregion was used as the probe and the genomic DNA as well as thecontrol plasmid was digested with EcoRI . Lane1: Positive control(PcINO1 gene cloned in pGEMT-EASY vector), 2: wt, 3: NtNOsINO1(negative control), 4, 5NtN PcINO1, independent lines

J. Plant Biochem. Biotechnol.

and non-stress related pathways regulated by PtdInsP isoformsand associated enzymes (kinases and phosphatases) function inparallel to coordinate regulation of growth and stress responsesin plants. Recent evidence also indicates their possible role inmodulating chromatin structure: PtdIns5P is reported to beinvolved in an epigenetic mechanism by binding to a chromatinmodifier in Arabidopsis, ATX1 (Alvarez-Venegas andAvramova 2005). Increasing levels of PtdIns5P is associatedwith hyperosmotic stress, salt stress and dehydration (Meijer etal. 1999; Ndamukong et al. 2010; Pical et al. 1999). Inositolserves as a source of compatible compounds. It can be meth-ylated to pinitol and also can accept a galactosyl group to formgalactinol, the precursor in raffinose series oligosachharide(RFO) synthesis. Inositol, pinitol, and RFOs all are potentosmoprotectants enhancing stress tolerance in plants (Nelsonand Jensen 1995; Taji et al. 2002). Galactinol and RFOs arealso emerging as antioxidants and putative signaling com-pounds (Chaouch and Noctor 2010; Cho et al. 2008; Lee etal. 2008). In essence, inositol derivatives have complex ways toregulate stress response in plants, and are still a focus of activeresearch (Valluru and Ende 2011).

The present work shows that overexpression and main-tenance of an unabated pool of inositol in transgenic plantsimparts a substantial tolerance to salt and oxidative stress.Seed specific overexpression of inositol imparts enhanceddehydration tolerance and improved germination under stress.However, at this stage it was not possible to determine all themechanisms involved in this process. Overproduction ofosmolytes in transgenic plants (Trehalose, Mannitol,Ectoine, Glycine betaine, Sorbitol, D-ononitol, Pinitol andProline; supplementary Table 1) imparts stress tolerance inmost cases. However it appears from the present work thatinositol might not work primarily or solely as an osmolyte. Innaturally accumulating plants, osmolyte concentrations typi-cally lie between 5 and 50 μmolg-1 fresh weight (~6–60 mM)(Rhodes and Hanson 1993; Pilon-Smits et al. 1995) and mayreach 200 mM when stressed (Rhodes and Samaras 1994).The PcINO1 transformed Brassica plants produced a netbalance of 9–15 μmolg-1 fresh weight of inositol in the pres-ence of 100 mM NaCl. Unless the titer of the cyclitol indifferent cellular locales is much higher than the estimatednet amount, it is difficult to assume a compatible solute

Fig. 5 Germination oftransformed N.tabacum seedsin the presence of mannitola Germination of wt,NtNOsINO1, NtNPcINO1 andNtCPcINO1 seeds in differentconcentrations of mannitolb Germination frequency of thetransgenic and wt seeds indifferent concentrations ofmannitol. The error barsrepresent mean standarddeviation. c Root length andd shoot length of wt,NtNOsINO1, NtNPcINO1 andNtCPcINO1 seedlings grownin different concentrations ofmannitol. The error barsrepresent mean standarddeviation

J. Plant Biochem. Biotechnol.

function for inositol in stress tolerance. We propose thatunabated pool of inositol and its derivatives, in stress condi-tions, help in revitalizing the cellular system and maintainnormal growth. As demonstrated in this work, the role ofinositol in scavenging ROS can be one such mechanism, ascan be the activation of different signaling pathways throughPtdIns, RFOs, galactinol and ascorbic acid.

The overall performance of the BjPcINO1 plantsscore to be satisfactory for a potential salt tolerantGM crop to be released for a field trial. Growth ofthe plants in saline environment has been normal andresults in about 3-fold better survival than wt inprolonged salinity stress. The plants were able to enterreproductive phase and set seeds both under stress andwhile recovering from stress. The seed and pod qualityand quantity under normal conditions were comparableto wt plants and when exposed to stress, they exhibitsuperior quality to that of wt. There were no deleteriousaccumulations of unwanted metabolites in the seed oil.Moreover, being a beneficial food supplement, accumulationof inositol improves food quality in contrast to foreignproteins that may alter food value.

PcINO1 transformed Brassica plants show enhancedtolerance to oxidative stress and physiological drought

as an added advantage. The transformation events gaverise to a high number of positive lines which are ad-vanced to T4 generation and passed through segregationanalysis and screening every generation. The overall as-sessment of the BjPcINO1 lines satisfy the criteria ofbeing an important GM food crop and are thus suitablefor preliminary field trials.

The seed specific expression of inositol in tobacco seedsenhance germination rate under dehydration stress and sa-linity stress, single or compounded. Inositol derivatives,especially phytic acid plays an important role in seed mat-uration and germination. During seed maturation, thedehydration-generated iron cations are chelated by phytate,to save the seeds from stress. During germination, phytate isgenerally dephosphorylated to inositol; used in the produc-tion of galactinol and RFOs that play several roles in ROShomeostasis. Seed-targeted inositol overexpression thuspresents a better survival rate in salinity affected soilprovided excess inositol does not result in imbalancednutritional quality of the seeds. Further studies will benecessary to dissect the mechanisms by which inositoloverproduction imparts salinity tolerance and how itslocale-specific expression correlates with the ameliorationof different abiotic stresses.

Fig. 6 Germination oftransformed N.tabacum seedsin the presence of PEG-6000a Germination of wt,NtNOsINO1, NtNPcINO1 andNtCPcINO1 seeds in differentconcentrations of PEG-6000.b Germination frequency of thetransgenic and wt seeds indifferent concentrations ofPEG-6000. The error barsrepresent mean standarddeviation. c Root length andd shoot length of wt,NtNOsINO1, NtNPcINO1 andNtCPcINO1 seedlings grown indifferent concentrations ofPEG-6000. The error barsrepresent mean standarddeviation

J. Plant Biochem. Biotechnol.

Fig. 7 Salinity Germination oftransformed N. tabacum seedsin the presence of NaCla Germination of wt,NtNOsINO1, NtNPcINO1 andNtCPcINO1 seeds in differentconcentrations of NaCl.b Germination frequency of thetransgenic and wt seeds indifferent concentrations ofNaCl. The error bars representmean standard deviation. c Rootlength and d shoot length of wt,NtNOsINO1, NtNPcINO1 andNtCPcINO1 seedlings grown indifferent concentrations ofNaCl. The error bars representmean standard deviation

Fig. 8 a Effect of combinedsalinity and drought ongermination of transformedN.tabacum seeds in the presenceof NaCl. Germination of the wt,NtNOsINO1, NtNPcINO1seedsin the presence of 50 mM NaCland 5%PEG. b Exogenousinositol can help in germinationunder drought. Wt seeds weregerminated in the presence of 5%PEG −6000 with or withoutexogenous inositol supplementin the media

J. Plant Biochem. Biotechnol.

Acknowledgments The work has been supported by research grants(to ALM) from the Department of Biotechnology (DBT), Govt. of India.LG, SR and SM were Senior Research Fellowship Awardees from theCouncil of Scientific & Industrial Research and RM is a ResearchAssociate of DBT. SSG is a Staff Scientist in a DBT supported project.Napin promoter was a kind gift from Dr. I.B. Maiti from TobaccoResearch & Development Center, University of Kentucky.

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