The endo-1,4-β-glucanase Korrigan exhibits functional conservation between gymnosperms and angiosperms and is required for proper cell wall formation in gymnosperms

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The endo-1,4-β-glucanase Korrigan exhibits functional conservation between gymnosperms and angiosperms and is required for proper cell wall formation in gymnosperms

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  The endo-1,4- b -glucanase  Korrigan  exhibits functionalconservation between gymnosperms and angiosperms and isrequired for proper cell wall formation in gymnosperms  Victoria J. Maloney  1 , A. Lacey Samuels 2 and Shawn D. Mansfield  1 1 Department of Wood Science, Faculty of Forestry, The University of British Columbia, 2424 Main Mall, Vancouver, BC, V6T 1Z4 Canada;  2 Department of Botany, The University of BritishColumbia, 6270 University Boulevard, Vancouver, BC, V6T 1Z4 Canada  Author for correspondence:  Shawn D. Mansfield Tel: +1 604 822 0196Email: shawn.mansfield@ubc.ca Received:  4 August 2011 Accepted:  29 October 2011New Phytologist   (2012)  193 : 1076–1087 doi : 10.1111/j.1469-8137.2011.03998.x Key words:  cell wall, cellulose,endoglucanase, trachieds, ultrastructure,vessels, xylem. Summary •  The evolution of compositional polymers and their complex arrangement and deposition inthe cell walls of terrestrial plants included the acquisition of key protein functions. •  A membrane-bound endoglucanase, termed  Korrigan  (KOR), has been shown to berequired for proper cellulose synthesis. To date, no extensive characterization of the gymno-sperm KOR has been undertaken. •  Characterization of the white spruce ( Picea glauca ) gene encoding KOR ( PgKOR ) showsconserved protein features such as polarized targeting signals and residues predicted to beessential for catalytic activity. The rescue of the  Arabidopsis thaliana kor1-1  mutant by theexpression of  PgKOR  suggests gene conservation, providing evidence for functional equiva-lence.Analysesofendogenous KOR expressioninwhitesprucerevealedthehighestexpressionin young developing tissues, which corresponds with primary cell wall development. Addition-ally,RNAinterferenceoftheendogenousgymnospermgenesubstantiallyreducedgrowthandstructuralglucosecontent,buthadnoeffectoncelluloseultrastructure. •  Partial functional conservation of KOR in gymnosperms suggests that its role in cell wallsynthesis dates back to 300 million yr ago (Mya), predating angiosperms, which arose130 Mya, and shows that proteins contributing to proper cellulose deposition are importantconserved features of vascular plants. Introduction Plant evolution has been characterized by distinctly defined adap-tive events, including the development of a stiff, upright stemthat permitted the expansion of the growth habit of plantsmigrating from an aquatic to a terrestrial ecosystem. What werethe key factors in the evolution of the cell wall of terrestrialplants? It is possible that the evolution of the proteins necessary for the formation of the critical cellulose–hemicellulose network of the cell wall was a key element in allowing plants to move fromaquatic to terrestrial environments.The arrangement of polysaccharides in both primary andsecondary cell walls is an important determinant for cell shape,growth and cell wall properties in vascular plants (Estevez  et al. ,2009; Siddhanta   et al. , 2009). This unique arrangement could bea key factor in cell wall evolution. The cellulose of nonvascularplants has a higher level of crystallinity and a larger surface area than higher plant cellulose (Ek   et al. , 1998; Stromme  et al. ,2002). In addition, highly specialized proteins such as endotrans-glycosylases and expansins are present in higher plants thatmodify the complex cross-linking of cellulose with hemicellulosesduring growth and morphogenesis (Cosgrove, 2005). It is thiscross-linking that permits the cell wall to be strong yet flexible.Some of these proteins act on the hemicellulose–cellulose net-work in the cell wall, while other proteins act during cellulosedeposition.One particular group of enzymes, the endoglucanases(E.C.3.2.1.4), have the capacity to hydrolyse the  b -1,4 linkage of the cellulose chain, and have been shown to be more active onamorphous cellulose than on crystalline cellulose (Carrard  et al. ,2000). A membrane-bound endoglucanase called Korrigan(KOR) has been shown to be required for synthesis of theordered, load-bearing cellulose–hemicellulose network (Nicol et al. , 1998; Sato  et al. , 2001). KOR was srcinally isolated in a mutant  Arabidopsis thaliana   plant ( kor1-1 ) that showed pro-nounced architectural alterations in the primary cell wall whengrown in the absence of light (Nicol  et al. , 1998). It was furthershown that  KOR   possesses a single N-terminal membrane-spanning domain and therefore presumably acts at the plasma membrane–cell wall interface (Nicol  et al. , 1998). Additional KOR   mutations ( kor1-2  ) have been isolated and shown to causethe formation of aberrant cell plates, incomplete cell walls, and Research 1076  New Phytologist   (2012)  193 : 1076–1087 www.newphytologist.com   2011 The Authors New Phytologist     2011 New Phytologist Trust  multinucleated cells, leading to abnormal seedling morphology (Zuo  et al. , 2000). In addition, the identification of irregularxylem mutants of   KOR  , such as  irregular xylem 2   (Szyjanowicz et al.,  2004), indicates that  KOR   is required for normal xylemvessel development. Related membrane-bound endoglucanaseshavebeenidentifiedintomato( Lycopersiconesculentum  ;Brummell et al. , 1997a,b), oilseed rape ( Brasica napus  ; Molhoj  et al. ,2001a,b), rice ( Oryza  ; Zhou  et al. , 2006), and loblolly pine( Pinus taeda  ; Nairn  et al. , 2008). A number of studies examining the function of thesemembrane-bound endoglucanases have been undertaken in anattempt to elucidate the role(s) these enzymes may play withrespect to cell wall remodelling, and more generally the overallphysiology of plants. While none of these studies has been able toreveal the mechanism of KOR function, the results from thesestudies do indicate that plants, regardless of species, possess oneparticular membrane-bound endoglucanase that appears to havesimilar functionality (Brummell  et al. , 1997a,b; Lane  et al. ,2001; Molhoj  et al. , 2001a, 2002; Master  et al. , 2004; Robert et al. , 2005; Bhandari  et al. , 2006; Takahashi  et al. , 2009;Maloney & Mansfield, 2010). In 2002, Molhoj  et al.  classifiedthese proteins as class IX glycosyl hydrolases.Given the irregular xylem phenotype in  Arabidopsis thaliana  , itis interesting that expression of sequences related to the  A. thaliana KOR   (  AtKOR  ) was correlated with cellulose deposi-tion in secondary xylem of   Populus spp. , suggesting a role for KOR in wood formation (Sterky   et al. , 1998; Hertzberg   et al. , 2001;Bhandari  et al. , 2006). Hybrid poplar endoglucanase activity wasexperimentally demonstrated (Rudsander  et al. , 2003; Master et al. , 2004). Within the angiosperms,  KOR   from hybrid aspen( Populus tremula   L.  ·  tremuloides   Michx.;  PttKOR  ) comple-mentedthe  A. thaliana   mutantsandoverexpression ofthe PttKOR  in  A. thaliana   led to lower cellulose crystallinity (Takahashi  et al. ,2009). When  KOR   expression was suppressed by RNA inter-ference (RNAi) in hybrid poplar ( Populus alba   ·  grandidentata; PaxgKOR  ), there was less cellulose present but it was more crystal-line, while levels of the hemicellulose xylan increased (Maloney &Mansfield, 2010). In addition, the irregular xylem phenotypeshown in  A. thaliana kor   mutants was also observed for poplarsecondaryxylem vessels (Maloney & Mansfield, 2010). Although it has been speculated that  KOR   predates the splitbetween angiosperms and gymnosperms (Molhoj  et al. , 2002;Nairn  et al. , 2008), to date no detailed characterization of a gymnosperm  KOR   has been undertaken. Should functional con-servation of these membrane-bound endoglucanases in the gym-nosperms exist, it would suggest that their role in vascular plantevolution dates back to 300 million yr ago (Mya), predating angiosperms, which arose 130 Mya. Here we report on theisolation and characterization of a membrane-bound endo-glucanase gene (denoted  PgKOR  ) from white spruce ( Picea glau- ca  ). The rescue of the  A. thaliana kor1-1  irregular xylem anddwarf phenotype by the expression of   PgKOR   provides evidencefor functional equivalence of the gymnosperm gene in the angio-sperm. Analyses of endogenous  KOR   expression in white sprucerevealed the highest expression in young developing tissues,which corresponds with primary cell wall development. Additionally, decreased expression of   PgKOR   using RNAi-mediated suppression in white spruce trees resulted in substan-tially reduced growth and structural glucose content, but had noeffect on cellulose ultrastructure. The lack of an effect on thecellulose ultrastructure suggests that, while PgKOR can partially replace the function of AtKOR, there still might be somefunctional divergence after the split of gymnosperms andangiosperms. Materials and Methods PgKOR  isolation and construct development RNA was extracted from the green leader portion of   Picea glauca  (Moench) Voss line 653 stem tissue. To identify and character-ize the white spruce homologue of the  Arabidopsis thaliana   (L.)Heynh.  KOR   gene, the initial sequence was obtained by blasting against a spruce expressed sequence tag (EST) library (http://www.treenomix.ca), from which a set of contigs were selectedbased on their 73% sequence similarity to the  AtKOR   gene. A large portion of the gene was isolated with a forward primer(PtrKORFw) designed using the already characterized  Populus tremuloides KOR   ( PtrKOR  ; GenBank AY535003), which has83% similarity with the  AtKOR   sequence, and a reverse primer(PgKORESTRv) designed using the EST screen. In order forany amplification to occur, the forward primer had to bedesigned within the  PtrKOR   open reading frame (ORF), and wewere therefore unable to amplify the entire  PgKOR   ORF. Inorder to obtain the remainder of the ORF, 5 ¢  RLM RACE(RNA Ligase Mediated Rapid Amplification of cDNA Ends; Ambion, Austin, TX, USA) was employed to complete the5 ¢  sequence of the gene. Following sequence verification, oligo-nucleotides (PgKORgateFw and PgKORgateRv) were designedto generate a PCR product from cDNA for direct cloning intothe pENTR    ⁄   D-TOPO vector (Invitrogen, Carlsbad, CA, USA).The clone was then sequenced to verify the correct insertion of the  PgKOR   gene, and Gateway technology (Invitrogen) was usedto insert the gene into the 35S::hRLUC::attR destination vector(Subramanian  et al. , 2004). The resulting binary plasmid,35S::hRLUC::PgKOR, was transformed into  Agrobacterium tumefaciens   strain GV3101 and used for the complementation of the  kor1-1  mutant.The  PgKOR   genomic sequence was PCR-amplified using geno-mic DNA from the green leader portion of   P. glauca   653 stemtissue and four sets of oligonucleotides designed within the PgKOR   ORF (PgKORgen1FW&RV, PgKORgen2FW&RV,PgKOEgen3FW&RV and PgKORgen4FW&RV). The sequencing results for the four PCR fragments were then assembled to obtainthe entire  PgKOR   genomic DNA sequence.The PgKOR-RNAi construct was built using two oligonucleo-tides, PgRNAiFW and PgRNAiRV, with the addition of either5 ¢  Bam  HI and 3 ¢  Cla  I (sense) or 5 ¢  Xho  I and 3 ¢  Kpn  I (antisense)restrictions sites. These oligonucleotides were used to amplify a 400-base pair fragment of the  PgKOR   coding region from cDNA.The fragments were then digested with the appropriate restrictionenzymes and ligated into the pKANNIBAL (Helliwell & New Phytologist  Research  1077   2011 The Authors New Phytologist     2011 New Phytologist Trust New Phytologist   (2012)  193 : 1076–1087 www.newphytologist.com   Waterhouse, 2003) cloning vector. Finally, the  Not  I fragmentfrom pKANNIBAL containing the hairpin RNA (hpRNA) cas-settes was subcloned into the binary vector pART27 (Gleave,1992) and used for plant transformations. Phylogenetic analyses  Amino acid sequence alignment for PgKOR with all of theknown  A. thaliana   glycosyl hydrolase family 9 proteins and allthe other known plant membrane-bound endoglucanases wasperformed with C LUSTAL  W in the B IO E DIT  program (Hall, 1999).The evolutionary history was inferred using the neighbour- joining method (Saitou & Nei, 1987). The optimal tree with thesum of branch length = 7.72127000 is shown in Fig. 2. The per-centage of replicate trees in which the associated taxa clusteredtogether in the bootstrap test (1000 replicates) is shown next tothe branches (Felsenstein, 1985). The tree is drawn to scale, withbranch lengths given in the same units as those of the evolution-ary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the JTT matrix-based method(Jones  et al. , 1992) and are given in units of the number of aminoacid substitutions per site. The analysis involved 49 amino acidsequences and all positions containing gaps and missing data wereeliminated. There were a total of 253 positions in the final data set. The final tree is rooted to an  Oryza sativa   endoglucanse(GenBank #BAG94612), which is not a class IX member. Evolu-tionary analyses were conducted in  MEGA  5 (Tamura   et al. , 2011). Plant strains and growth conditions TheT-DNAinsertionmutant  A. thaliana  line kor1-1 (Nicol et al. ,1998) was acquired from the Arabidopsis Biological ResourceCenter (Columbus, OH, USA). The T-DNA insertion of   kor1-1 carries a gene that results in resistance to kanamycin. Seeds weresterilizedandgerminatedatroomtemperature ina16 hlight:8 hdark cycle on half-strength Murashige and Skoog (MS) medium(Murashige & Skoog, 1962) containing 50 mg l ) 1 kanamycinsulfate (Sigma, St Louis, MO, USA) and no sugar. Plantlets weretransferred to soil after the first four primary leaves had emerged,andgrowthuntil maturitywasallowed tocontinueunder thesameconditions. The F 1  progeny from the  kor1-1  line were comparedwiththeWassilewskija(WS)ecotype,thebackgroundfor kor1-1 . White spruce somatic embryo tissue (Pg653) was acquiredfrom Dr K. Klimaszewska (CFS, Quebec, Canada). Embryoswere matured according to Klimaszewska   et al.  (2001). Plantletswere grown for 18 months, and then destructively harvested. Plant transformation kor1-1  plants were transformed by floral dip using   A. tumefaciens  carrying the  PgKOR   construct. The harvested seeds were selectedon MS medium containing 50 mg ml ) 1 kanamycin and 85 mg ml ) 1 PESTANAL (glufosinate ammonium; Sigma). Plantletsresistant to both selection agents were transferred to soil andplaced in a growth chamber and grown under the same condi-tions described previously. Pg653 somatic embryo tissue wastransformed with the  PgKOR-RNAi   construct according toKlimaszewska   et al.  (2001). Genomic DNA extraction and screening Genomic DNA was extracted from either the green leaderportion of   P. glauca   653 stem tissue or single leaves taken fromyoung soil-grown  A. thaliana   plants using a modified hexadecyl-trimethylammonium bromide (CTAB) extraction method(Rogers & Bendich, 1994). Briefly, tissue was placed in micro-centrifuge tubes and ground to a powder with liquid nitrogen.Then 1 ml of CTAB extraction buffer (2% (w    ⁄   v) CTAB (Sigma),100 mM Tris-HCl, pH 8.0, 1.4 M NaCl, 20 mM EDTA, 1%(w    ⁄   v) polyvinylprylidone, and 0.2% (v    ⁄   v) 2-mercaptoethanol)was added to each tube, and the tube was incubated at 65  C for60 min. An equal volume of chloroform was added, and the tubewas vortexed and then centrifuged in a microcentrifuge for10 min. Genomic DNA was precipitated from the aqueous phaseby the addition of 1 volume of isopropanol, incubation at ) 20  Cfor 10 min, and centrifugation for 5 min. Genomic DNA was re-suspended in RNase buffer (25 mM Tris-HCl, pH 7.5, 10 mMEDTA, and 100 mg ml ) 1 RNase A) and incubated at 37  C for30 min. Two volumes of ethanol were added, and the genomicDNA was recovered by centrifugation. Finally, the DNA was re-suspended in 50  l l of EB buffer (10 mM Tris-HCl, pH 8.0, and1 mM EDTA), quantified at A260, and stored at 4  C. PCR wasperformed on this DNA to determine the genotype (homozygousor heterozygous for the T-DNA insertion) of the plants with thegenomic primers (RP and LP) specifically designed to amplify genomic regions flanking the T-DNA insertion, as well as theT-DNA specific TAG7 primer, according to Nicol  et al.  (1998).The presence or absence of the  PgKOR   construct was determinedwith the primers hLUC3 ¢  Fw and PgKORgate Rv (for all primersequences, see Supporting Information Table S1). RNA extraction and real-time PCR Total RNA was extracted from  c.  500 mg of frozen ground8-wk-old whole  A. thaliana   plants using TRIzol reagent (Sigma)according to the manufacturer’s instructions. Total RNA wasisolated from white spruce tissues according to Kolosova   et al. (2004). RNA yield was measured by absorption at 260 nm, and10  l g was treated with DNAase (TURBO DNA-free; Ambion).Then 1  l g of the resulting DNA-free RNA was evaluated on a 1%Tris-acetate EDTA agarose gel in order to determine quality.Equal quantities of RNA (1  l g) were used for the synthesis of cDNA with SuperScript II reverse transcriptase (Invitrogen) and(dT)16 primers, according to the manufacturer’s instructions.Samples were run in triplicate with Platinum SYBR Green qPCR Master mix (Invitrogen) on an Mx3000p real-time PCR system(Stratagene, La Jolla, CA, USA). The real-time PCR analysisof the  A. thaliana   lines was performed using the primersPgKORRTFw and PgKORRTRv or AtKORRTFw and AtKORRT3 ¢ UTRRv, while the endogenous  PgKOR   expression inthe wild type and the PgKOR-RNAi lines was performed using the primers PgKORRNAiRTFw and PgKORRNAiRTRv. After 1078  Research New Phytologist   2011 The Authors New Phytologist     2011 New Phytologist Trust New Phytologist   (2012)  193 : 1076–1087 www.newphytologist.com  analysis of the dissociation curves to ensure single band amplifica-tion, critical threshold (Ct) values were quantified in triplicate andtranscript abundances were determined based on changes inCt values relative to elongation initiation factor 5A (Gutierrez et al. , 2008; primers AtEIF5AFw and AtEIF5ARv) for  A. thaliana  and actin (primers PgActinFw and PgActinRv) for white spruceusing the following equation:  D Ct  ¼  2 ð CtPgKOR  ;  AtKOR or PgKORRNAi     CtPgActin or AtEIF  5  A  Þ . Conditions for all PCR reactions were as follows: 95  C for 10 min, followed by 40 cyclesof 95  C for 30 s, 55  C for 1 min, and 72  C for 30 s. Structural carbohydrate analyses Ten-wk-old  A. thaliana   stems and 18-month-old glasshouse-grown white spruce stems were ground in a Wiley mill to pass a 0.4-mm screen (40 mesh) and Soxhlet-extracted overnight in hotacetone to remove extractives. Lignin and carbohydrate contentswere determined with a modified Klason (Coleman  et al. , 2009),in which extracted ground stem tissue (50 mg) was treated with3 ml of 72% H 2 SO 4  and stirred every 10 min for 2 h. Sampleswere then diluted with 112 ml of deionized water and autoclavedfor 1 h at 121  C. The acid-insoluble lignin fraction was deter-mined gravimetrically by filtration through a pre-weighedmedium coarseness sintered-glass crucible, while the acid-solublelignin component was determined spectrophotometrically by absorbance at 205 nm. Carbohydrate contents were determinedby using anion exchange high-performance liquid chroma-tography (Dx-600; Dionex, Sunnyvale, CA, USA) equipped withan ion exchange PA1 (Dionex) column, a pulsed amperometricdetector with a gold electrode, and a SpectraAS3500 auto injector(Spectra-Physics, Santa Clara, CA, USA). Cellulose characterization Microfibril angle estimates were generated by X-ray diffraction.The 002 diffraction spectra from five individual 18-month-oldwhite spruce trees from each of the transgenic lines and 10-wk-old  A. thaliana   stems were screened for  T   value distribution and sym-metry on a Bruker D8 discover X-ray diffraction unit equippedwithanareaarraydetector(generalareadetectordiffractionsystem(GADDS)). Wide-angle diffraction was used in the transmissionmode, and the measurements were performed with CuK  a 1 radia-tion ( k  = 1.54 A ˚). The X-ray source was fitted with a 0.5-mmcollimator, and the scattered photon was collected by a GADDSdetector.BoththeX-raysourceanddetectorweresettotheta = 0  .The degree of cellulose crystallinity was determined for whitespruce and  A. thaliana   stems as described previously (Mansfield et al. , 1997; Coleman  et al. , 2009) using the same X-ray para-meters as for microfibril angle (MFA) determination, with theexception of the source theta which was set at 17  . SignificantdifferencesfromthewildtypeweredeterminedusingaStudent t  -test. Cross-sectional staining and microscopy  A 1-cm segment was cut from the base of 18-month-old whitespruce stems and submerged in dH 2 O at room temperature for1 d. Samples were then radially cut into 20- l m cross-sectionsusing a Leica SM2000r hand sliding microtome (Leica Microsys-tems, Wetzlar, Germany) and again stored in dH 2 O until needed.Sections were treated either with 0.01% calcofluor white in1  ·  PBS for 3 min, and washed three times for 5 min each in1  ·  PBS to remove excess stain (Falconer & Seagull, 1985), orwith 10% phloroglucinol with the addition of concentrated HCL.Four-wk-old  A. thaliana   stems were also cut 1 cm above thebase and further hand-sectioned into  c.  500-nm-thick cross-sections. Sections were then stained in 0.05% toluidine blue stainfor 5 min. Excess stain was washed away with dH 2 O. All sectionswere mounted onto glass slides and examined with a Leica DRMmicroscope (Leica Microsystems) fitted with epifluorescenceoptics. Photographs were taken with a QICAM camera (QImaging,Surrey, Canada) and O PEN L  AB  software (PerkinElmer Inc., Waltham, MA, USA). Results Sequence analyses of PgKOR PgKOR   encodes a putative 65-kD protein with 617 amino acids(GenBank JF343550). A comparison of the  AtKOR  ,  PgKOR   and PtrKOR   genomic sequence structures shows that each gene con-sists of the same number of exons and introns, but the sizes of theintrons vary between species (Fig. 1a). A comparison of theprotein sequence with that of seven other putative KOR homo-logues from other plant species (Fig. 1b) demonstrates the strong overall similarity among these proteins (Libertini  et al. , 2004).Overall, PgKOR is most similar to the KOR from loblolly pine(PtaKOR1; Nairn  et al. , 2008), sharing > 94% sequence iden-tity. A comparison of all known  KOR   genes revealed conservedpolarized targeting signals, predicted glycosylation sites, and resi-dues essential for catalytic activity; however, the predicted trans-membrane domains vary substantially, with only 40% sequencesimilarity between PgKOR and AtKOR (Fig. 1b).The  A. thaliana   genome contains  c.  25 genes that encodeendoglucanases that belong to glycosyl hydrolase family 9 (GH9)proteins. These 25 genes separate into at least nine different classesaccordingtoMolhoj et al. (2002),whocomparedtherelationshipsbetweenthe25endoglucanases in  A.thaliana   andtheir indentifiedhomologues in other plant species. Class IX appears tobe the mostunique class in that members of this class do not contain an endo-plasmic reticulum import sequence and are not secreted directly into the apoplast, but rather contain sequences that encode anN-terminal membrane-anchoring domain (Brummell  et al. ,1997b). In a phylogenetic comparison of PgKOR with all of theknown  A. thaliana   glycosyl transferase family 9 proteins and theclass IX proteins from other plant species, PgKOR clusters closestto the class IX proteins that are known to contain sequences thatencodeanN-terminalmembrane-anchoringdomain(Fig. 2). Complementation of the  A. thaliana kor1-1  mutant Having established phylogenetic relatedness of the  PgKOR   geneand the  KOR   from  A. thaliana  , we hypothesized that if KOR was New Phytologist  Research  1079   2011 The Authors New Phytologist     2011 New Phytologist Trust New Phytologist   (2012)  193 : 1076–1087 www.newphytologist.com  functionally conserved in gymnosperms and angiosperms, thenthe  PgKOR   gene would rescue the dwarf phenotype of   A. thaliana kor   mutants.  Arabidopsis thaliana   plants homozygousfor the T-DNA insertion  kor1-1  mutation (Nicol  et al. , 1998)were transformed with either the  PgKOR   gene or the endogenous  AtKOR   gene under the control of the cauliflower mosaic virus(CaMV) 35S promoter. None of the transformants displayed theelongation-deficient phenotype typical of the  kor1-1  mutant. TheT2 progeny, obtained after selfing, were again able to grow onPESTANAL selection media, and had a wild-type growth pheno-type, as shown in Fig. 3(a) for the positive control  AtKOR   lineand two representative  PgKOR   lines called PgKOR4 andPgKOR5. PCR-amplification of the T-DNA insertion (Fig. 3b)and real-time quantitative PCR analyses for the presence of theendogenous  AtKOR   transcript (Fig. 4a) confirmed that the repre-sentative lines were homozygous for the T-DNA insertion and Fig. 1  Molecular characterization of the  Korrigan  ( KOR )gene. (a) Schematic map of the gene structures of the AtKOR ,  PgKOR  and  PtrKOR  genes. Triangles indicate thelocation and size of the introns. (b) Alignment of eightdifferent plant membrane-bound endo-1,4- b -glucanaseprotein sequences. Amino acids shaded in black are thesame and those in grey are similar. Sequence shading:polarized targeting signals, purple; predicted transmem-brane domains, green; predicted glycosylation sites, red;residues essential for catalytic activity, blue. PgKOR,  Picea glauca ; PtaKOR1,  Pinus taeda ; AtKOR,  Arabidopsisthaliana ; Pa · gKOR,  Populus alba  ·  grandidentata ;LeCel3,  Lycopersicon esculentum ; OsCel9A,  Oryza sativa ; BnCel16,  Brassica napus ; PtrKOR,  Populustrichocarpa. 1080  Research New Phytologist   2011 The Authors New Phytologist     2011 New Phytologist Trust New Phytologist   (2012)  193 : 1076–1087 www.newphytologist.com
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