Recruiting & HR

Arabidopsis DREB2C functions as a transcriptional activator of HsfA3 during the heat stress response

Arabidopsis DREB2C functions as a transcriptional activator of HsfA3 during the heat stress response
of 7
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Related Documents
  12  Arabidopsis  DREB2C functions as a transcriptional activator of   HsfA 3 during 3  the heat stress response 4  Huan Chen a , Jung Eun Hwang a , Chan Ju Lim a , Dool Yi Kim b , Sang Yeol Lee a , Chae Oh Lim a, ⇑ 5  a Division of Applied Life Science, Graduate School of Gyeongsang National University, Plant Molecular Biology and Biotechnology Research Center, Jinju 660-701, Republic of Korea 6  b National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Republic of Korea 78 1 0 a r t i c l e i n f o 11  Article history: 12 Received 17 August 201013 Available online xxxx14  Keywords: 15 Gene regulation16 Heat shock factor17 Signal transduction18 Transgenic plant19 Transient assay20 2 1 a b s t r a c t 22 The dehydration-responsive element binding protein (DREB) family is important in regulating plant 23 responses to abiotic stresses.  DREB2C   is one of the  Arabidopsis  class 2  DREB s and is inducedby heat stress 24 (HS). Here, we present data concerning the interaction of   DREB2C   with  heat shock factor A3  ( HsfA3 ) in the 25 HS signal transduction cascade. RT-PCR showed that  HsfA3  is the most up-regulated gene among the 21 26  Arabidopsis Hsf  s in transgenic plants over-expressing  DREB2C  .  DREB2C   and  HsfA3  displayed similar tran- 27 scription patterns in response to HS and DREB2C specifically transactivated the DRE-dependent tran- 28 scription of   HsfA3  in  Arabidopsis  mesophyll protoplasts. Yeast one-hybrid assays and  in vitro 29 electrophoretic mobility shift assays further showed that DREB2C interacts with two DREs located in 30 the  HsfA3  promoter with a binding preference for the distal DRE2. Deletion mutants of DREB2C indicated 31 that transactivation activity was located in the C-terminal region. In addition, dual activator-reporter 32 assays showed that the induction of   heat shock protein  ( Hsp ) genes in transgenic plants could be attrib- 33 uted to the transcriptional activity of HsfA3. Taken together, these results indicate that DREB2C and 34 HsfA3 are key players in regulating the heat tolerance of   Arabidopsis . 35   2010 Published by Elsevier Inc. 3637 38  1. Introduction 39  The heat stress response (HSR) is characterized by a series of  40  reactions triggered by the exposure of living organisms to high 41  temperature stress, resulting in a set of physiological, biochemical 42  andmolecularchanges[1].AcommonfeatureoftheHSRisitsabil- 43  ity to confer resistance to ambient temperatures above optimal 44  growth conditions, a phenomenon known as thermotolerance [2]. 45  Emerging evidence suggests that redox and hormonal signals are 46  implicated in HSR  [3]. The accumulation of heat shock proteins 47  (Hsps) under the control of heat shock transcription factors (Hsfs) 48  is imperative in thermotolerance [4]. 49  In  Arabidopsis , two classes of dehydration-responsive element 50  (DRE)-binding proteins (DREBs) are present, encoded by  DREB1 51  and  DREB2  class genes, respectively [5].  DREB1 s are transcription- 52  ally induced by exposure to low temperatures and impart toler- 53  ance to drought, freezing and high-salinity in transgenic plants 54  [6,7].  DREB2 s were initially identified as drought and high-salinity 55  responsive genes [8,9], and were lately shown to be induced in re- 56  sponse to HS [10,11]. Overexpression of   DREB2C   and a constitu- 57  tively active form of   DREB2A  (DREB2A-CA), lacking a negative 58  regulatoryregion,activateddownstreamgenesandincreasedther- 59  motolerance in  Arabidopsis  plants. Additionally,  DREB2A  is induced 60 transiently within 1h of HS treatment.  DREB2B  showed similar ra- 61 pid induction to  DREB2A , but expression continues for 12h after 62 heat treatment, whereas  DREB2C   exhibited a slower rate of induc- 63 tion than either of the other genes [11,12], and the results sug- 64 gested that DREB2A and DREB2C may function as early and late 65 regulators, respectively, inducing expression of common target 66 genes under the HS conditions. Recent work established that DRE- 67 B2A activates the  HsfA3  promoter and subsequently regulates the 68 expression of   Hsp  genes involved in early phases of the HSR  69 [13,14]. Thus, the late expression of   HsfA3  should require DREB2C 70 expression under HSR. 71 Here, we used a number of   in vivo  and  in vitro  approaches to 72 demonstrate whether DREB2Ctransactivates DRE-dependent tran- 73 scription of   HsfA3  in late phases of the HSR. 74 2. Materials and methods 75  2.1. Plant materials and HS treatments 76  Arabidopsis thaliana  L. Heynh. ecotype Columbia (Col-0) was 77 used as wild-type plant. Plants were grown in soil or  in vitro  on 78 MS medium containing 1% sucrose and 0.25% Phyta-gel (pH 5.7), 79 under16hof100 l Es  1 m  1 lightand8hdark,at23  C.Generation 80 of   35S:DREB2C   transgenic lines and HS treatments were described 81 previously [12]. 0006-291X/$ - see front matter    2010 Published by Elsevier Inc.doi:10.1016/j.bbrc.2010.09.038 ⇑ Corresponding author. Fax: +82 55 759 9363. E-mail address: (C.O. Lim).Biochemical and Biophysical Research Communications xxx (2010) xxx–xxx Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: YBBRC 25629 No. of Pages 7, Model 5G20 September 2010 Please cite this article in press as: H. Chen et al.,  Arabidopsis  DREB2C functions as a transcriptional activator of   HsfA 3 during the heat stress response, Bio-chem. Biophys. Res. Commun. (2010), doi:10.1016/j.bbrc.2010.09.038  82  2.2. Reverse transcription (RT)-PCR 83  Total RNA was extracted from 7-day-old seedlings using the 84  TRIzol reagent (Invitrogen). Complementary DNA was synthesized 85  and RT-PCR analysis was performed as described previously [12] 86  usingthegene-specificprimersetsindicatedinSupplementaryTa- 87  ble 1. RT-PCR reactions were conducted using at least two biolog- 88  ical replicates. 89  2.3. Transactivation assay with Arabidopsis mesophyll protoplasts 90  To investigate the function of two putative binding sites ( DRE1 91  and DRE2 ) forDREB2Cinthe HsfA3  promoter,the DREB2C   cDNAse- 92  quenceswasfusedtotheFlag-tagsequence(F)andexpressedunder 93  the control of a  CaMV35S   promoter (35S) and the TMV translation 94  enhancingomegaelement( X )(Fig.1C).Toconstructreporterplas- 95  mids harboring mutations in either  DRE1  [  pHsfA3 ( m1 ) :GUS  ] or 96  DRE2  [  pHsfA3 ( m2 ) :GUS  ], a 1.1kb fragment of the  HsfA3  upstream 97  regulatory region was subjected to mutation using sequence-spe- 98  cificprimers(SupplementaryTable2)bymegaprimer-PCR [15]. 99  Transient transformation of   Arabidopsis  mesophyll protoplasts 100  wasperformedusingamethoddescribedpreviously[16].Typically, 101  300 l l of protoplast suspension (5  10 6 cells per milliliter) was 102 transiently co-transfected with 20 l g effector plasmid, 15 l g of  103 GUS reporter plasmid and 5 l g of   35S:LUC   control plasmid in a 104 15ml disposable conical tube and mixed with an equal volume of  105 40% PEG 4000 (Sigma–Fluka). GUS activity assays were performed 106 as previously described [17]. Luciferase assays were conducted 107 using the Luciferase Assay System (Promega) and measured on a 108 20/20 n Luminometer (Turner Biosystems). Promoter activity was 109 calculatedasunitsofGUSactivityperunitofluciferaseactivity. 110  2.4. Yeast one-hybrid assay 111 In yeast one-hybrid assay, the  pGAD424:DREB2C   and dual  HIS3- 112 LacZ   reporters under the control of four tandem repeats of wild- 113 type DRE   (4wDRE)or mutated DRE   (4mDRE)(SupplementaryTable 114 3) were transformed into yeast strain YM4271, as described previ- 115 ously [12]. Qualitative colony-lift filter assays and quantitative  b - 116 galactosidase activity analysis were performed according to the 117 manufacturer’s protocols (Clontech, Protocol No. PT3024-1). 118  2.5. Electrophoretic mobility shift assay (EMSA) 119 Two antiparallel oligonucleotides spanning the wild-type  DRE  120 (wDRE1; 5 0 -TTTGTCGGTT-3 0 , wDRE2; 5 0 -AATGTCGGTT-3 0 ) or Fig. 1.  Transcript levels of   Hsf   genes in  Arabidopsis  plants. (A) Expression profiles of 21  Arabidopsis Hsf  s in wild-type (WT) and  DREB2C  -over-expressing transgenic lines(  35S:DREB2C-a  and  35S:DREB2C-b ).  Actin2  and  Tubulin8  genes were used as loading controls. RT-PCR cycle for most genes was 35, and for  HsfA1d ,  HsfA2 ,  HsfA5 ,  HsfA9 ,  HsfC1 was40,respectively.TheexpectedsizesofampliconsareindicatedinSupplementaryTable1.(B)Expressionpatternsofselected Hsf  sfollowingexposuretoheatstress(37  C)for the times indicated in 7-day-old WT seedlings.  Actin2  was analyzed for control. RT-PCR cycle was 30. (C) DREB2C transactivates DRE-dependent expression of   HsfA3 . Theactivator construct contains the  CaMV35S   promoter and the TMV X element fused to  DREB2C   cDNA. A Flag-tag sequence (F) was added to the N terminus of   DREB2C  . NOS-T,nopalinesynthasegene3 0 -terminator.Thereporterconstructsaredrivenby1.1kbsequenceupstreamofthe HsfA3  transcriptionstartsite(+1)containingtwowild-type DRE  s(  pHsfA3:GUS  ),base-substituted DRE1 [  pHsfA3 ( m1 ) :GUS  ]and DRE2 [  pHsfA3 ( m2 ) :GUS  ],respectively.RelativeGUS/LUCactivitymeasurementsweredescribedinsection2.5.Barsrepresent the SD of five replicates of relative GUS/LUC activity assays. Ratios represent the fold-increase in activity of test constructs compared to the  pHsfA3:GUS   plasmidonly (normalized as 1).2  H. Chen et al./Biochemical and Biophysical Research Communications xxx (2010) xxx–xxx YBBRC 25629 No. of Pages 7, Model 5G20 September 2010 Please cite this article in press as: H. Chen et al.,  Arabidopsis  DREB2C functions as a transcriptional activator of   HsfA 3 during the heat stress response, Bio-chem. Biophys. Res. Commun. (2010), doi:10.1016/j.bbrc.2010.09.038  121  mutated  DRE   (mDRE1; 5 0 -TTTaaaaGTT-3 0 , mDRE2; 5 0 -AATaaaaGTT- 122  3 0 ) identified in the  HsfA3  promoter (Supplementary Table 3) were 123  annealed at a 1:1M ratio by heating at 70  C for 20min in 50mM 124  NaClandbyslowlycoolingdowntoroomtemperatureforaperiod 125  of 30min. The annealed oligonucleotides were labeled with  a 32 P- 126  dATP and were purified by MicroSpin TM G-25 columns (GE Health- 127  care).ThepurificationofrecombinantGST-DREB2C (P49–C176) protein 128  andEMSAwerecarriedoutasdescribedpreviously[12]. 129  2.6. Yeast monohybrid assays 130  To test the activator potential of DREB2C, monohybrid assays 131  wereperformedas describedpreviously[18]. Theentirecodingse- 132  quenceof  DREB2C  aswellassequencedeletionsweresubclonedinto 133  pGBT9  and transformed into yeast strain pJ69-4a (Clontech). The 134  gene-specificprimersusedinamplificationof  DREB2C   deletionser- 135  iesarelistedinSupplementaryTable4.Thequalitativecolony-liftfil- 136 terassaywithX-galandquantitative b -galactosidaseactivityassay 137 withONPG(o-nitrophenyl b - D -galactopyranoside;Sigma)wereper- 138 formedaccordingtothemanufacturer’sprotocols(Clontech). 139  2.7. Dual activator-reporter assays 140 Effectorplasmidsusedinthedualactivator-reporterassaywere 141 constructed using DNA fragments containing the coding region of  142 DREB2C   or  HsfA3  (Supplementary Table 5), which was expressed 143 under the control of the  CaMV 35S   promoter and the TMV X  ele- 144 ment.For reporterplasmidconstruction,theputativepromoterse- 145 quences of   Hsp18.1-Cl ,  Hsp25.3 ,  Hsp26.5  and  RD29A  were amplified 146 by PCR from wild-type  Arabidopsis  genomic DNA using gene-spe- 147 cificprimers(SupplementaryTable5)andfusedin-frametothese- 148 quence encoding the  GUS   gene in a  pUC19 -derived plasmid [19]. 149 Transient transformation of   Arabidopsis  mesophyll protoplasts 150 was performed using a method described above section 2.3. Fig. 2.  DREB2Cbinds totwo  DRE   motifs inthe  HsfA3  promoter. (A) Schematic diagramof the  HsfA3  promoter containingthe  DRE  s. Two  DRE   motifs (proximal  DRE1  and distal DRE2 ), theputativeTATA-box, andtheputativetranscriptionstartsite(TSS;denotedbyarrow)are shown. Thenumbersindicatethe nucleotidepositionsrelativetoTSS(+1).Bait or probe sequences of the promoter containing either wild-type  DRE   (wDRE) or mutant  DRE   (mDRE) are shown. The mutant  DRE   core sequence variants are indicated bysmallletters.(B)TranscriptionalactivationviaDREB2Cinyeast.pAD:DREB2CdenotestheGAL4activationdomain(AD)fusedwiththeentireDREB2Ccodingregionunderthecontrol of a truncated  ADH1  promoter. LacZ and His reporters are expressed under the control of four tandem copies of wDRE (4wDRE) and mDRE (4mDRE). The growth of transformed yeast cells were examined on synthetic dropout mediumlacking uracil, histidine and leucine (SD-UHL) and supplemented with the indicated concentrations of 3-ATasaninhibitorof the HIS3  geneproduct. LacZactivitywasdeterminedbycolony-liftfilterandliquidcultureassays. Errorbars indicatetheSDoffivereplicates. (C) DRE  -binding activity of DREB2C was determined by EMSA. Radio labeled wDRE1 and wDRE2 were used as probes and unlabeled wDRE1, wDRE2, mDRE1 and mDRE2 fragmentwere employed as competitors.  , Probe without recombinant DREB2C; +, probe with recombinant DREB2C or GST. Filled triangles denote increasing amount of unlabeledcompetitors. H. Chen et al./Biochemical and Biophysical Research Communications xxx (2010) xxx–xxx  3 YBBRC 25629 No. of Pages 7, Model 5G20 September 2010 Please cite this article in press as: H. Chen et al.,  Arabidopsis  DREB2C functions as a transcriptional activator of   HsfA 3 during the heat stress response, Bio-chem. Biophys. Res. Commun. (2010), doi:10.1016/j.bbrc.2010.09.038  151  3. Results and discussion 152  3.1. Expression of Hsf genes in DREB2C-overexpressor  153  To determine whether the  DREB2C  -overexpressors affects 154  endogenous  Hsf   gene expression, we performed RT-PCR analysis 155  using the 21  Arabidopsis Hsf   family genes in wild-type and two 156  DREB2C  -overexpressor lines that were grown under the non- 157  stressed conditions. The underlying assumption of this approach 158  is that the constitutive expression of   DREB2C   in transgenic plants 159  activates putative target  Hsf   genes whose expression levels 160  would remain relatively low in wild-type plants under non- 161  stressed conditions.  HsfA3  was the most up-regulated  Hsf  162  (Fig. 1A) and in wild-type plants undergoing a HSR at 37  C 163  (Fig. 1B), expression of   HsfA3  started after 3h of exposure to ele- 164  vated temperature and continued to increase for 24h. A similar 165  pattern of expression was observed for  DREB2C  . In contrast, the 166  expression of   DREB2A  was rapidly and transiently induced by 167  HS and peaked within 1h. Several HS-responsive  Hsfs  also 168  peaked at 1h and then declined under HS conditions, most likely 169  reflecting the influence of DREB2A expression [14]. Hence, the 170  strong induction of   HsfA3  in  DREB2C  -overexpressors suggests 171  that DREB2C may regulate  HsfA3  expression, especially at later 172  stages of HS. 173  3.2. DREB2C directs the DRE-dependent expression of HsfA3 174 AstwoconservedDREmotifs(DRE1andDRE2)arelocatedinthe 175 HsfA3  promoter (Fig. 1C), the ability of DREB2C to regulate  HsfA3 176 transcriptionviathesemotifswasevaluatedbytransientco-expres- 177 sionassaysin  Arabidopsis mesophyllprotoplasts.AsshowninFig.1C, 178 the  pHsfA3:GUS  reportershowedadramaticincreaseinGUSactivity 179 (28.5-fold)whenco-expressedwith  35S  X :DREB2C   comparedtothe 180 reporter alone, indicating that DREB2C strongly transactivates the 181 transcriptionof  HsfA3 .Inaddition,co-expressionofDREB2Cresulted 182 in 32.1-fold and 24.2-fold activation of   pHsfA3 ( m1 ) :GUS   and 183  pHsfA3 ( m2 ) :GUS   reporters, respectively, revealing that each DRE is 184 strongly transactivated by DREB2C. Of particular interest, the sum 185 of the activities observed for  pHsfA3 ( m1 ) :GUS   (8.02) and 186  pHsfA3 ( m2 ) :GUS   (21.82) was approximately equivalent to that of  187  pHsfA3:GUS   (28.5)inthepresenceofDREB2C.Therefore,thesedata 188 suggest that DREB2C is a robust transactivator that is capable of  189 activatingDRE-dependenttranscriptionof  HsfA3  inplantcells. 190  3.3. DREB2C preferentially binds to the distal DRE2 located in the 191 HsfA3 promoter  192 To investigate the ability of DREB2C to physically bind to the 193 two  DRE  s contained within the  HsfA3  promoter (Fig. 2A), we Fig. 3.  Activation domain analysis and dual activator-reporter assay of DREB2C. (A) Activation domain analysis of DREB2C using yeast monohybrid assays. Full-lengthDREB2CanddeletionvariantswerefusedtotheGAL4DNA-bindingdomain(GAL4-DBD)andexpressedinyeast. NLS,nuclearlocalizationsignal(graybox);AP2/ERF,AP2/ERFDNA-binding domain (dotted box); AD, activation domain (striped box). The solid box denotes amino acid residues from 231 to 260. Transformed yeast were grown insyntheticdextrosemediumsupplementedwith(+His)orlackinghistidine(  His). Qualitativeassay(X-gal)andquantitative b -galactosidaseassaywereusedtomeasureLacZactivity. Bars indicate the SDof five replicates for quantitative  b -galactosidase assays. (B) Transcript levels of   Hsps  in  DREB2C  -over-expressing transgenic lines (  35S:DREB2C-a and  35S:DREB2C-b ). RT-PCR was used to estimate transcript abundance in 10-day-old plants grown under unstressed conditions.  Actin2  and  Tubulin8  were used asconstitutively expressed controls. (C) Dual activator-reporter assays in  Arabidopsis  mesophyll protoplasts. Assays were performed in protoplasts by co-transformation of effectorconstructs(  35S  X :HsfA3  and  35S  X :DREB2C  )withreporterconstructscontainingthepromoterfragmentsofselected Hsps  (  pHsp18.1-CI:GUS  ,  pHsp25.3-P:GUS  ,  pHsp26.5-P  ( r  ) :GUS  ) or  RD29A  (  pRD29A:GUS  ), fused to GUS. Effector constructs contain HsfA3 or DREB2C under control of the  CaMV35S   promoter and TMV X enhancer or, in the case of   pHsfA3:HsfA3 , under control of its own promoter. The resulting GUS/LUC activities are presented with error bars representing the SD of five independent replicates.4  H. Chen et al./Biochemical and Biophysical Research Communications xxx (2010) xxx–xxx YBBRC 25629 No. of Pages 7, Model 5G20 September 2010 Please cite this article in press as: H. Chen et al.,  Arabidopsis  DREB2C functions as a transcriptional activator of   HsfA 3 during the heat stress response, Bio-chem. Biophys. Res. Commun. (2010), doi:10.1016/j.bbrc.2010.09.038  194  carried out yeast one-hybrid (Y1H) experiments and EMSAs. In 195  Y1H assays, we monitored the ability of DREB2C prey construct 196  totransactivatebaitreporterconstructsharboringeitherwild-type 197  DRE1  (wDRE1; proximal  DRE   relative to translational start site) or 198  DRE2  (wDRE2; distal  DRE   relative to translational start site). Yeast 199  co-transformed with both bait and prey constructs grew in selec- 200  tive media and containing 20mM 3-AT, and also induced LacZ 201  activity (Fig. 2B). Conversely, yeast harboring the mutated  DRE1 202  (mDRE1) and  DRE2  (mDRE2) bait constructs and co-transformed 203  with DREB2C prey were incapable of growing on same selective 204  media and showed no LacZ activity. These results reveal that 205  DREB2C specifically interacted with wild-type DREs but not with 206  mutated versions of these sequences. Interestingly, wDRE2- 207  containing reporters were more abundantly induced compared to 208  wDRE1-containing reporters, suggesting that DREB2C preferen- 209  tially interacts with wDRE2 in yeast cells. 210  In complementary EMSA experiments, DNA–protein binding 211  complexes were observed when labeled probes consisting of  212  wDRE1 and wDRE2 sequence were incubated with recombinant 213  purified DREB2C protein (Fig. 2C). Competitive assays demon- 214 strated that addition of unlabeled wDRE1 and wDRE2 effectively 215 reduced DREB2C binding to the wild-type DREs, but addition of  216 unlabeled mDRE1 and mDRE2 sequences did not. These results 217 further demonstrate DREB2C directly binds to wDRE1 and wDRE2 218 in vitro . The signal generated by binding of DREB2C to wDRE2 was 219 stronger than that with wDRE1, providing additional evidences 220 that DREB2C binds more efficiently to wDRE2 than to wDRE1. 221  3.4. Mutational analysis of DREB2C transactivation activity 222 The C-terminal region of DREB2C is rich in acidic amino acids 223 and is hypothesized to contain a transcriptional activation domain 224 [20]. To identify domains required for transcriptional activity, we 225 carried out yeast monohybrid assays [18]. As shown in Fig. 3A, 226 the GAL4-DBD control vector and a series of effector constructs 227 containing various amino acid domains of DREB2C (001-170, 228 048-170, and 001-200) did not activate the expression of reporter 229 construct. The effector construct expressing amino acids 1–230 230 weakly stimulated the expression of the reporter in comparison 231 to the effector expressing amino acids 1–200. As predicted, the Fig. 4.  DREB2C   overexpression results in growth stage-specific thermotolerance. (A) Enhanced vegetative thermotolerance of   DREB2C  -over-expressing transgenic lines(  35S:DREB2C-a  and  35S:DREB2C-b ). Three-day-old seedlings were treated at 45  C for 70 (middle panel) or 90min (lower panel) and photographs were taken after 7days of recoveryat 23  C, asillustrated. (B) DREB2C  -over-expressing transgenic lines reducedseedgerminationunder heat stress. Imbibedseeds weretreatedat 54  Cfor 60(middlepanel)or70min(lowerpanel)andphotographsweretakenafter7daysofrecoveryat23  C,asillustrated.(C)Enhancedvegetativethermotoleranceof  HsfA3 -over-expressingtransgenic lines (  35S:HsfA3-S12  and  35S:HsfA3-S20 ). Five-day-old seedlings were treated at 45  C for 70min and photographs were taken after 7days of recovery at 23  C, asillustrated. (D)  HsfA3 -over-expressing transgenic lines reduced seed germination under heat stress. Imbibed seeds were treated at 54  C for 70min and photographs weretaken after 7days of recovery at 23  C, as illustrated. All experiments (A, B, C, D) were performed in triplicate using 32 imbibed seeds per test. H. Chen et al./Biochemical and Biophysical Research Communications xxx (2010) xxx–xxx  5 YBBRC 25629 No. of Pages 7, Model 5G20 September 2010 Please cite this article in press as: H. Chen et al.,  Arabidopsis  DREB2C functions as a transcriptional activator of   HsfA 3 during the heat stress response, Bio-chem. Biophys. Res. Commun. (2010), doi:10.1016/j.bbrc.2010.09.038
Similar documents
View more...
Related Search
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks