Isolation And Characterization Of A Glutamate Decarboxylase (gad) Gene And Their Differential Expression In Response To Abiotic Stresses From Panax Ginseng C. A. Meyer

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  Isolation and characterization of a  Glutamate decarboxylase (GAD) gene and their differential expression in responseto abiotic stresses from  Panax ginseng  C. A. Meyer Jung-Hye Lee  • Yu-Jin Kim  • Dae-Young Jeong  • Gayathri Sathiyaraj  • Rama Krishna Pulla  • Ju-Sun Shim  • Jun-Gyo In  • Deok-Chun Yang Received: 10 September 2009/Accepted: 18 November 2009/Published online: 5 December 2009   Springer Science+Business Media B.V. 2009 Abstract  Glutamate decarboxylase (GAD) catalyzes theconversion of   L -glutamate to  c -aminobutyric acid (GABA).A full-length cDNA encoding GAD (designated as PgGAD ) was isolated and characterized from the root of  Panax ginseng  C. A. Meyer. The length cDNA of   PgGAD was 1881 bp and contained a 1491 bp open reading frame(ORF) encoding a glutamate decarboxylase protein of 496amino acids, possessing a Ser-X-X-Lys active site, whichbelongs to the GAD group. The deduced amino acidsequence of the  PgGAD  was classified in the plant GADfamily and has 76–85% high similarity with other plants aslike petunia,  Arabidopsis , tomato. Secondary structure of  PgGAD  was predicted by using SOPMA software program.Southern blot analysis of genomic DNA suggests that,there is more than one copy of the  PgGAD  gene. The organspecific gene expression pattern also studied in  P. ginseng seedlings, in which the stem showed elevated expressionthan root, leaf, bud and rhizomes. Along with this, we alsoconfirmed the gene expression of   PgGAD  under variousabiotic stresses like temperature stress, osmotic stress,anoxia, oxidative stress, and mechanical damage. Temporalanalysis of gene expression except exposure of oxidativestress revealed an enhanced expression after each stresses.The enzyme activity of PgGAD was stimulated to 2-foldunder cold stress. Keywords  Abiotic stresses    Characterization   Glutamate decarboxylase    Panax ginseng Abbreviations CaM CalmodulinGAD Glutamate decarboxylasecDNA Complementary DNAGABA  c -amino butyric acidEST Expressed sequence tagORF Open reading frameRT-PCR Reverse transcriptase-PCRTLC Thin layer chromatographyNCC Ninhydrin-colored complexPLP Pyridoxal 5 0 -Phosphate Introduction c -amino butyric acid (GABA) is a ubiquitous non-proteinamino acid which functions as a major inhibitory neuro-transmitter of the central nervous system [1, 2] and has been proved to be effective for lowering the blood pressureof experimental animals [3, 4]. GABA activates the cere- bral blood flow, accelerates metabolic functions, increasesthe amount of O 2  supply, and clinically as a medicine toameliorate sequel of stroke and cerebral artery sclerosis [5].In plants and in animals, GABA is mainly metabolized viaa short pathway composed of three enzymes, called theGABA shunt because it by passes two steps of the tricar-boxylic acid (TCA) cycle. The pathway is composed of thecytosolic enzyme glutamate decarboxylase (GAD), themitochondrial enzymes GABA transaminase (GABA-T)and succinic semialdehyde dehydrogenase (SSADH) [6]. Electronic supplementary material  The online version of thisarticle (doi:10.1007/s11033-009-9937-0) contains supplementarymaterial, which is available to authorized users.J.-H. Lee    Y.-J. Kim    D.-Y. Jeong    G. Sathiyaraj   R. K. Pulla    J.-S. Shim    J.-G. In    D.-C. Yang ( & )Korean Ginseng Center for Most Valuable Products and GinsengGenetic Resource Bank, Kyung Hee University, Seocheon,Giheung-gu, Yongin-si, Gyunggi-do 449-701, Republic of Koreae-mail: [email protected]  1 3 Mol Biol Rep (2010) 37:3455–3463DOI 10.1007/s11033-009-9937-0  The regulation of this conserved metabolic pathway seemsto have particular characteristics in plants. GABA alsorapidly produced in response to biotic and abiotic stresses[7]. GABA contents in both mature and germinated brownrice increased by a high-pressure and anaerobic stresses,respectively [8, 9]. And also in tea, leaves treated with anaerobic stress showed high level of GABA (2.5–7 times)than in control [10]. GABA in wheat seedling accumulatedin response to osmotic stress [11]. For these reasons,GABA-enriched foods come into the spotlight as thefunctional food material.Glutamate decarboxylase (GAD) catalyzes the decar-boxylation of glutamate to CO 2  and  c -amino butyric acid(GABA). GAD has been found in bacteria [12], animals[13], and plants [14]. Ueno [15] reported that basic structure of plant GAD is similar to that of   E. coli , but the structuralanalysis has revealed that an extra segment is attached to theC-terminal segment of some plant GADs. Unlike in animalsor microorganisms, a unique feature of plant GAD is thepresence of a calmodulin (CaM)-binding domain near theC-terminus [16, 17]. Several environmental factors can influence plants to increase the intracellular Ca 2 ? concen-trations, and as the H ? concentration increases, the Ca 2 ? that is joined with CaM activates GAD thereby increasingthe intracellular GABA concentration [18, 19]. In plants, GAD genes have been studied in petunia [16],Arabidopsis [20], tomato [21], rice [22], tobacco [23], fava bean [24]. Baum et al. [19] induced the entire  GAD  geneand the mutant  GAD  gene which was removed the CaM-binding site from petunia in transgenic tobacco plants toexamine the role of CaM in the expression of GADactivity. In both of all cases, GABA level was increasedand glutamate level was decreased, in tobacco, the mutant GAD  gene removed the CaM-binding site had a consider-able change when it was compared to the wild type plant,which has entire  GAD  gene. These results suggest thatplant GAD wields great influence upon metabolism,growth and development.This study was aimed to confirm the existence of a PgGAD  in  Panax ginseng  C. A. Meyer, to determine thebase sequence of the gene, also predict the amino acidsequence, and to determine the  PgGAD  involvement inexpression mediating stress responses. So, we isolated a PgGAD  cDNA from  P. ginseng  and analyzed expressionprofiles of   PgGAD  under various stress conditions. Materials and methods RNA purification and construction of cDNA libraryFor the construction of cDNA library, total RNA wasisolated from 14-year-old  Panax ginseng  plants usingaqueous phenol extraction procedure [25]. Poly(A) ? RNAwas purified with a Poly(A) Trac mRNA isolation system(Promega, USA). Then cDNA was synthesized from itusing cDNA library construction Kit (Clontech, PT3000-1,USA), according to the manufacturer’s instructions. Frac-tions containing cDNA greater than 500 bp were recoveredand this library was amplified to yield a final titer of 2  9  10 9 pfu ml - 1 . Individual colonies were propagatedand saved at  - 70  C until further use.Nucleotide sequencing and sequence analysisThe pBlue script vector was excised from the Uni-ZAP XRlibrary and used as templates for sequence analysis. The 5 0 ends of randomly selected cDNA inserts were sequencedby an automatic DNA sequencer (ABI prism 3700).Nucleotide and amino acid sequence analyses were per-formed using DNASIS program (Hitachi Software Engi-neering, Brisbane, CA). Primary amino acid sequences of GAD from different srcin were obtained using SWISS-PROT data bank software supported at the server from theNCBI (National Center for Biotechnology Information):http://www.ncbi.nlm.nih.gov/ , and also from the KEGGdata bank (Kyoto Encyclopedia of Genes and Genomes):http://www.genome.ad.jp/kegg/ . The functional classifica-tion of EST clone was done based on the results obtainedby comparising the non-redundant protein database of GenBank using the blastx algorithm. EST clone wasannotated manually by following the Munich InformationCenter for Protein Sequences (MIPS) role categorization[26]. We used ClustalX with default gap penalties to performmultiple alignment of GAD isolated in ginseng and pre-viously registered in other species [27]. Based on this alignment, a phylogenetic tree was constructed accordingto the neighbor-joining method, using the MEGA3 pro-grams [28]. Bootstrap analysis with 1,000 replicates was also conducted in order to obtain confidence levels for thebranches [29]. A search of homologous motifs in  PgGAD was carried out on the MEME (Multiple Em for Motif Elicitation; http://meme.sdsc.edu/meme/meme.html) withthe motif length set at 6–80, motif sites 2–200, e-value \ 1e - 10, and maxium number of motif 30 [30]. Secondary structure analysis molecular modeling for  PgGAD  wasperformed by SOPMA program [31], and the protein properties are estimated using ProtParam [32]. Plant materials and growth conditions P. ginseng  cv. ‘‘Yun-poong’’ seeds were immersed in 70%(v/v) ethanol for 1 min, surface-sterilized with 2% (v/v)sodium hypochlorite for 15 min, rinsed five times withsterilized distilled water. Inner zygotic embryos were 3456 Mol Biol Rep (2010) 37:3455–3463  1 3  dissected out and were placed on MS basal medium [33]. Itwas supplemented with 3% sucrose, and 0.7% agarose andadjusted the pH 5.7 to before autoclaving the medium.Zygotic embryos were cultured at 25  ±  2  C for 3 dayswith 16 h of light daily. Cultured plantlets were planted inglass bottles containing a 70 ml MS medium containing10 mg/l gibberellic acid and 3% sucrose and 0.7% agaroseunder the same conditions. After 3 weeks, ginseng planetswere used for the various stress treatments and nucleic acidextractions. For analysis of gene expression in differentorgans and enzyme assay, 1-year-old ginseng plants werecollected from leaves, roots, stems, rhizomes, buds andplantlets.Southern blot analysisGenomic DNA was isolated from young leaves of   P. gin-seng  using a GeneAll plant SV mini kit (GeneAll Bio-technology, Seoul, Korea) in accordance with themanufacturer’s recommendations, and then total DNA waspurified by ethanol precipitation. Approximately 10  l g of DNAs were digested with  Hin dIII,  Eco RI. The resultingfragments were fractionated by electrophoresis through a1.3% agarose gel with 25 ng DIG-labeled DNA MolecularWeight Marker III (Roche Applied Science, Mannheim,Germany). DIG-labeled DNA probe was hybridized at37  C for 16 h. Following hybridization, the blot waswashed twice for 5 min in 2  9  SSC containing 0.1% SDSat room temperature and was washed twice for 15 min in0.5  9  SSC containing 0.1% SDS at 68  C. The immuno-logical detection of the DIG-labeled probe was performedwith 1:10000 anti DIG-AP. The blot was exposed to X-rayfilm (AGFA, Germany) with CSPD.Abiotic stress treatmentFor real-time RT PCR analysis, ginseng plants were treatedwith various stresses in about 3 weeks after zygotic embryogermination. For the temperature stress treatment, theplanlets were exposed to temperatures of 4  C and 37  C for72 h. For treatment with NaCl (100 mM) and H 2 O 2 (10 mM),  P. ginseng  samples were incubated for 72 h inmedia containing each chemical at 25  C. Anoxic condi-tions were generated by submerging whole potted plantsfor the duration of the experiment. For mechanicalwounding stress, healthy leaves and stems of plantlets werewounded with a sterile scalpel. All treatments were carriedout on MS media [33] with or without the treatmentsolution (NaCl and H 2 O 2 ). For GAD assay analysis, 1 yearginseng plants were exposed to temperatures of 4  C duringfor 1, 2, 3, 4, 5 and 7 days. The stressed plant materialsfrom all completed treatments were immediately frozen inliquid nitrogen and stored at  - 70  C until required.Organ specific expressionTotalRNAwasextractedfromdifferenttissuesof1-year-old P. ginseng  (buds, leaves, stems, roots, rhizomes, and plant-lets) to study organ-specific expression using the RNeasymini kit (Qiagen, Valencia, CA, USA). RNA samples werequantified spectrophotometrically.Quantitative real-time PCR analysisThe gene expression of   PgGAD  was studied using quan-titative real-time PCR (RT-PCR). For RT-PCR, 200 ng of total RNA was used as a template for reverse transcriptionusing oligo (dT) primer (0.2 mM) (INTRON Biotechnol-ogy, Inc., South Korea) for 10 min at 65  C. The reactionmixture was then incubated with AMV Reverse Trans-criptase (10 U/  l l) (INTRON Biotechnology, Inc., SouthKorea) for 60 min at 42  C. The reaction was inactivated byheating the mixture at 94  C for 5 min. The real-time RT-PCR was conducted with SYBR Green SensiMix  Plus  kit(Quantace, Watford, United Kingdom) and carried outusing gene-specific primers. Three-step RT-PCR procedurewas performed in all experiments using to the manufac-turer’s instruction. Real-time reactions were accomplishedin a 10  l l final volume containing 3  l l aliquot of the firststand cDNA, 1  l l each of specific primers (10 pmol) forcoding region of   PgGAD  (forward, 5 0 -CAA CAC ATAACC GTG CTT CG-3 0 ; reverse, 5 0 -ACC TCC TCG GCAGTT TTC TT-3 0 ), and 5  l l of SensiMix  Plus  SYBR. As acontrol, the primers specific to the  P. ginseng  actin genewere used (forward, 5 0 -GAA CGG GAA ATT GTT CGAGA-3 0 ; reverse, 5 0 -GCA GAT TCC ATT CCG ATC AT-3 0 ).The reaction incubated as follows: an initial denaturationfor 10 min at 95  C, 40 amplification cycles [10 s at 95  C(denaturation), 10 s at 60  C (annealing), and 20 s at 72  C(polymerization)]. Actin gene was used in the same con-ditions as the internal control to normalize each sample forvariations in the amounts of RNA used. Relative quantityof the  PgGAD  expression level was performed using Rotor-Gene 6000 real-time rotary analyzer (Corbett Life Science,Sydney, Australia), and calculated using the comparativecycle threshold (C T ) method according to the manufac-turers’ instructions for data normalization.Analysis of GAD activityGAD activity was carried out by the modified methoddescribed by Oh et al. [34] and Sethi [35]. The ground ginseng samples (1 g) were thawed in 50 mM bis-Tris-HCl(pH 7.0) buffer containing 2 mM ethylenediaminete-traacetic acid (EDTA), 2 mM  b -mercaptoethanol, 0.1 mMPLP, 1 mM phenylmethylsulphonyl fluoride (PMSF), 2%(w/v) polyvinylpyrrolidone (PVPP), 10% (v/v) glycerol. Mol Biol Rep (2010) 37:3455–3463 3457  1 3  The homogenate was centrifuged at 11,000 rpm for 15 minat 4  C and the supernatant was collected. The total proteinconcentration was determined according to the method of Bradford [36] using a Coomassie brilliant blue reagent (Bio-Rad Lab., Richmond, USA) with bovine serumalbumin (BSA) as the standard.The GAD assay was performed using modified colori-metric method [35]. Samples were incubated at 37  C for15 min with reaction mixture containing, 0.5 mM sodiumacetate (pH 5.0), 0.3 M sodium chloride and 25  l M sodiumglutamate; the reaction was stopped by cooling it on ice for15 min, and analyzed via thin layer chromatography (TLC)with  N  -Propanol-water(1:1)solvent.Afterdevelopment,theTLC plate was dried and sprayed with 0.2% ninhydrinsolution. The NCC bands of the substrate and enzyme-sub-strate and GABA were separated from TLC plate, andextracted with 6 ml of extraction solution containing 75%ethanoland0.05%cupricsulfate,byheatingtoboiling.Aftercooling, the absorption readings were taken by spectro-photometer (Ultraspec 2100 pro, Amersham Biosciences,Buckinghamshire,England) at575 nm. The standardcurveswere prepared by plotting the NCC absorption readings of substrate and enzyme-substrate from the incubated reactionmixtureagainstsubstrateconcentration.Acontrolcurvewasprepared from substrate obtained from the reaction mixtureagainst substrate concentration at 25  C. Results Cloning and sequence analysis of a  PgGAD  cDNAAn expressed sequence tags (ESTs) analysis of a cDNAlibrary prepared with the RNA obtained from seedling of   P.ginseng  was screened using two probes generated via PCR.A cDNA encoding a glutamate decarboxylase (GAD),designated  PgGAD  was isolated and sequenced. The, PgGAD  is 1881 bp in length and it has an open readingframe (ORF) of 1491 bp nucleotide (For detail see sup-plementary Fig. 1). This ORF of   PgGAD  encodes 496amino acids, beginning at the initiation codon ATG(Position 182) and ending at the stop codon TGA (Position1672) of the cDNA. The predicted molecular weight andisoelectric point of the protein is 56.06 kDa and 5.69,respectively. In the deduced amino acid sequence of  PgGAD , the total number of negatively charged residues(Asp  ?  Glu) was amounted to 67 while the total number of positively charged residues (Arg  ?  Lys) was 59.Homology analysisHomology analysis was done by BLAST search of nucle-otide and protein databases using nucleotide sequence andamino acid sequence of   P. ginseng  GAD. The deducedamino acid sequence of   PgGAD  showed high homologywith  Petunia x hybrid   [16],  Arabidopsis thaliana  [17],  Lycopersicon esculentum  [21] and other species from NCBI and KEGG data bank was performed using theClustal method. The  PgGAD  shared high homology withthe  P. hybrida , two  A.thaliana  (GAD1, GAD2), and  L. esculentum  having 85%, 84%, 81%, and 76% of sequence identity, respectively (For detail see supplemen-tary Fig. 2). However,  PgGAD  shares lower than 50%degrees of identity to GAD of microorganisms and ani-mals. A phylogenetic analysis of   GAD  from plants,microorganism and animal has been carried out using theClustal method and it indicates plant GADs and microor-ganism GADs are closely related to each other and formone subgroup; whereas, animal GADs are distant from thissubgroup (For detail see supplementary Fig. 3). Furtheranalysis revealed 30 motifs of a repeated amino acidsequence in the studied proteins (For colored picture seesupplementary Fig. 4). Motif characteristic of GAD genewas shown in Fig. 1.  PgGAD  contains a set of 14 motifswith other plants as like  P. hybrida ,  A.thaliana , therefore 1,2, 4, 5, 7, 9, 12, 13, 15, 18, 19, 20, 25, and 28 set of motif.In comparison with GAD of   L. esculentum ,  PgGAD  con-tains a set of 12 motifs without region 18 and 25. Bacteriaspecies contain 16 motifs (1, 2, 4, 5, 7, 12, 13, 15, 19, 20,21, 22, 24, 26, 27, and 30) and animal species contain 13motifs (1, 2, 3, 6, 8, 10, 11, 13, 14, 15, 16, 17, and 23).Secondary structure analysis of   PgGAD The secondary structure analysis revealed that  PgGAD consists of 224  a -helix and 35  b -turns jointed by 67extended strands, and 168 random coils (For detail seesupplementary Fig. 5). This result is also highly similar tothe secondary structure of GADs of   P. hybrida , whichcontains 225  a -helix, 30  b -turns jointed by 67 Extendedstrands, and 172 random coils;  A.thaliana  (GAD1), whichcontains 187  a -helix, 32  b -turns jointed by 84 extendedstrands, and 191 random coils;  A. thaliana  (GAD2), whichcontains 216  a -helix, 30  b -turns jointed by 71 extendedstrands, and 177 random coils;  L. esculentum , which con-tains 216  a -helix, 30  b -turns jointed by 71 extendedstrands, and 177 random coils.Southern blot analysis of   PgGAD In order to estimate the copy number of GAD gene in Panax ginseng , we performed a southern blot analysisusing ginseng genomic DNA digested with 2 differentenzymes  Eco RI and  Hin dIII .  The digests showed one band(1.38 kbp) in  Eco RI restriction digest lane and two bands(3.4 and 3.2 kbp) in the  Hin dIII restriction digest lane (For 3458 Mol Biol Rep (2010) 37:3455–3463  1 3  detail see supplementary Fig. 6), respectively. Theseresults confirmed that, there are more than one copy of the PgGAD  exit in  P. ginseng .Expression patterns of   PgGAD  in different tissuesThetissue-specific PgGAD mRNAabundancewasanalyzedfrom different tissues like leaves, roots, stems, rhizomes andbuds of   P. ginseng . Relative gene expression levels werenormalized by measuring actin gene and expressed relativetobuds.All PgGAD swerefoundindetectableamountsinthetissues examined (Fig. 2). Highest expression of   PgGAD was found in stems (5.78-fold higher than buds;  P \ 0.05).Inleaves, PgGAD wassignificantlyincreasedwith4.45-foldhigher than buds ( P \ 0.05). Regarding tissue-specificexpression in roots and rhizomes,  PgGAD  was increasedwith 2.32 and 2.74 fold higher than buds  P \ 0.05),respectively. The expression of   PgGAD  is high in this order,stem [ leaves [ rhizomes [ roots [ buds.Expression levels of   PgGAD  in responseto different stressesTo investigate whether the expression of the  PgGAD  genesare stress-induced, total RNA were extracted from seed-lings under chilling and heating, NaCl and H 2 O 2  treat-ments, anoxia, and wounding stresses. The expressionpatterns of   PgGAD  at different time points after treatmentswere also analyzed using real-time PCR. Data were nor-malized to the actin gene, and further expressed relative tothe untreated (control 0 h). When ginseng seedlings wereexposed to chilling stresses at 4  C, the  PgGAD  mRNAlevel was slightly induced at 1 h and 4 h, with 2.81 and2.74-fold higher than the control plants (Fig. 3a). Theexpression level then rapidly peaked at 8 h with 5.94-foldinduction. The expression of   PgGAD  decreased to 2.81-fold and 2.4-fold at 24 h and 48 h, respectively, and waskept exponential level at 72 h (2.06-fold). Figure 3b showsthe gene expression pattern of   PgGAD  by incubating the Fig. 1  Comparison of the motifs for  Panax ginseng  GAD with otherspecies;  Petunia x hybrida  (Q07346)  , Arabidopsis thaliana  (GAD1,Q42521; GAD2, Q42472),  Lycopersicon esculentum  (P54767), Escherichia coli  K-12 (P69908),  Escherichia coli  O157:H7 (P58228), Shigella flexneri  (Q83PR1),  Listeria innocua  (Q928R9),  Lactococcuslactis  (Q9CG20),  Listeria monocytogenes  (Q9F5P3),  Saccharomycescerevisiae  (Q04792)  , Homo sapiens  (GAD1, Q99259; GAD2,Q05329)  , Rattus norvegicus  (GAD1, P18088; GAD2, Q05683),  Musmusculus  (GAD1, P48318; GAD2, P48320). Each motif is repre-sented by numbers in the different colored box. The grey linesrepresent the non conserved sequences Fig. 2  Relative expression levels of   PgGAD  in tissues. Expressionvalues were normalized to actin gene of the  P. ginseng . Data areexpressed as the ratio (calculated using 2 - ( D Ct) ) of target mRNA toactin of   P. ginseng  mRNAMol Biol Rep (2010) 37:3455–3463 3459  1 3