research article cellular localization and biochemical characterization...

Research ArticleCellular Localization and Biochemical Characterization ofa Chimeric Fluorescent Protein Fusion of Arabidopsis CelluloseSynthase-Like A2 Inserted into Golgi Membrane

Monica De Caroli Marcello S Lenucci Gian-Pietro Di Sansebastiano Michela TunnoAnna Montefusco Giuseppe Dalessandro and Gabriella Piro

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali Universita del Salento (DiSTeBA) Provinciale Lecce-Monteroni73100 Lecce Italy

Correspondence should be addressed to Gabriella Piro gabriellapirounisalentoit

Received 29 August 2013 Accepted 27 October 2013 Published 14 January 2014

Academic Editors L Costa D Sarkar B Vyskot and C R Wilson

Copyright copy 2014 Monica De Caroli et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Cellulose synthase-like (Csl) genes are believed to encode enzymes for the synthesis of cell wallmatrix polysaccharidesThe subfamilyof CslA is putatively involved in the biosynthesis of 120573-mannans Here we report a study on the cellular localization and the enzymeactivity of an Arabidopsis CslA family member AtCslA2 We show that the fluorescent protein fusion AtCslA2-GFP transientlyexpressed in tobacco leaf protoplasts is synthesized in the ER and it accumulates in the Golgi stacksThe chimera is inserted in theGolgimembrane and is functional sincemembrane preparations obtained by transformedprotoplasts carry out the in vitro synthesisof a 14C-mannan starting from GDP-d-[U-14C]mannose as substrate The enzyme specific activity is increased by approximately38 in the transformed protoplasts with respect to wild-type Preliminary tests with proteinase K biochemical data and TMdomain predictions suggest that the catalytic site of AtCslA2 faces the Golgi lumen

1 Introduction

Cellulose hemicelluloses and pectins are themain structuralpolysaccharides of the plant cell wall Glycosyltransferases(Gts) are the enzymes involved in the synthesis of thesepolysaccharides and other glycans Plasmamembrane rosettecomplexes are the Gts involved in the synthesis of cellu-lose [1] whereas Gts located in the Golgi apparatus areresponsible for the pectin and hemicellulose synthesis [2ndash4] Cellulose synthase (CesA) genes were first identified incotton fibers [5] Later in addition to CesA genes Ara-bidopsis genome sequencing revealed a large gene familywith homology to CesA [6] named cellulose synthase-like(Csl)

Two classes of Gts are mainly involved in cell wallpolysaccharide biosynthesis the multimembrane spanningand the type II transmembrane (TM) Gts the latter con-sisting of a short N-terminal domain on the cytoplasmicside of the membrane a single TM domain a stem of

variable length and a large globular C-region containing thecatalytic domain Multi-membrane spanning Gts (celluloseand callose synthases) are typically associated with theplasma membrane while both Gts classes type II and multi-membrane spanning have been found in Golgi stacks wherethey are likely involved in the biosynthesis of complex 120573-glycan polymers [7ndash9] The Golgi multi-membrane spannedCsl proteins have several TM domains close to the N-terminus followed by a large hydrophilic loop containing thepredicted active site motif DDDQxxRW highly conservedin CesA and Csl family members a second hydrophilicloop and a variable number of TM domains beside theC-terminus are also present [6 10 11] Depending on thenumber of N-terminus TM helices the catalytic domain mayface the cytoplasmic (even) or the luminal (odd) side of Golgimembranes [7 12] The first type like CesA uses substratedonor NDP-sugars of the cytosolic pool the second type usesNDP-sugars transported into the Golgi by specific carrierproteins [13 14]

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 792420 7 pageshttpdxdoiorg1011552014792420

2 The Scientific World Journal

Until now little evidence for the functional role of Cslgenes has been collected Among the six groups of the Cslgene family identified only few members of CslA and CslChave been functionally characterized suggesting their respec-tive involvement in (gluco)mannan [15 16] and xyloglucansynthesis [17] A role in the synthesis of mixed 120573-glucan hasbeen proposed for members of CslG and CslF groups [18 19]

In this study we constructed a fluorescent fusion of anArabidopsis CslA family member AtCslA2-GFP to identifyits final localization in a heterologous system represented bytobacco leaf protoplasts The chimera was localized in theGolgi stacks in a functional organization since an increase inmannan synthesis was quantified in transformed protoplastsCombining confocal observation biochemical data andTM domain predictions the topologic organisation of thechimera in the Golgi membrane is discussed

2 Material and Methods

21 Plasmid Construction The cDNA coding for AtCslA2was purchased from The Arabidopsis Information Resource(TAIR Ohio State University) The cDNA sequence wasamplified with primers Man01 (51015840-ggagcccggatccatggacggtg-31015840) and Man02 (51015840-caactgctagctcgggacataagtccc-31015840) tointroduce respectively BamHI and NheI restriction sitesThe BamHINheI fragment was introduced into a GFP-containing vector PGIP2-GFP [20] to construct AtCslA2-GFP The construct was confirmed by sequencing (PRIMMsrl Milan Italy)

22 Protoplast Preparation Transformation and Drug Treat-ments Tobacco protoplasts were prepared from the leavesof Nicotiana tabacum cv SR1 plants and transiently trans-formed as described by [21] Equivalent quantities (20120583g) ofplasmids were used for the colocalization experiments Thefluorescent pattern of over 1200 cells was analysed BrefeldinA (10 120583gmL Sigma-Aldrich) or cycloheximide (30 120583gmLSigma-Aldrich) was added into the incubation medium for2 h before confocal analyses

23 Laser Scanning Confocal Microscopy Protoplasts tran-siently expressing fluorescent constructs were observed by alaser scanning confocal microscope (LSM 710 Zeiss) in theirculture medium at different times after transformation Todetect GFP fluorescence a 488 nm argon ion laser line wasused and the emission was recorded with 505ndash530 nm filterset RFPwas detectedwith a 560ndash615 nmfilter set afterHendashNelaser excitation at 543 nm while chlorophyll epifluorescencewas detected with the filter set for tetramethylrhodamineisothiocyanate (TRITCgt650 nm) and eliminatedThe powerof each laser line the gain and the offset were identicalfor each experiment so that the images were comparableAppropriate controls were performed to exclude the possibil-ity of crosstalk between the two fluorochromes before imageacquisition

24 Protein Extraction Immunoblotting Analyses Cell Lysisand Phase Separation Extracellular and intracellular protein

fraction cell lysis phase separation and immunoblot analy-ses were performed as previously described [20] Protoplastswere harvested by gentle centrifugation at 65timesg withoutbreak after dilution of the incubation medium with twovolumes of W5 The medium was concentrated by filtrationon Centricon Plus 20 (Amicon) to obtain the extracellularprotein fraction (OUT) The protoplasts were suspendedin homogenization buffer (40mM Hepes-NaOH buffer pH75 10mM imidazole 1mM benzamidine 5mM 6-amino-hexanoic acid 10mM dithiothreitol and 1mM phenyl-methylsulfonyl fluoride) and sonicated The homogenatewas centrifuged at 800timesg for 10min at 4∘C to removecell wall debris and the supernatant was precipitated with80 acetone at minus20∘C (three times) to obtain the proteinintracellular fraction (IN) that includes endomembrane com-partments and organelles Protein concentration was deter-mined according to [22] Identical amounts of total proteinfrom each subcellular preparation were subjected to SDS-PAGE electrophoresis [23] and immunoblotting analysesAfter electrophoresis protein samples were transferred to anitrocellulose membrane (Hybond-C extra GE Healthcare)and blocked with 5 skim-milk powder in TBS (20mMTris-HCl pH 75 500mM NaCl 5 wv milk powder) for2 h Anti-GFP (1 5000 vv) (Molecular Probes) was usedin TBS + 1 skimmed-milk powder Each protein fractionwas visualized after ECL (ECL western blotting analysissystem GE Healthcare) chemodetection Cell lysis and phaseseparation were carried out as described by [24] modified by[20] To test AtCslA2 solubility in Triton X-114 AtCslA2-GFPtransformed protoplasts were freeze-thawed twice in 10mMTris-buffer pH 75 containing 015M NaCl and 1mM EDTAand centrifuged at 800timesg for 10minThe 800timesg supernatantwas adjusted to 15mL with Tris-buffer (pH 75) and TritonX-114 was added into a final concentration of 1 vv

25 Enzyme Assay To assay mannan synthase activity pro-toplasts were suspended and sonicated in 01M sodium-phosphate buffer (pH 72) containing 10mM MgCl

2 1 mM

dithiothreitol 04M sucrose and 1 bovine serum albuminThe resulting homogenate was centrifuged at 800timesg for10min at 2∘C The 800timesg supernatant was further cen-trifuged at 100000timesg for 60min at 2∘C to obtain a pelletconsisting of membranous material (total membrane frac-tion) This pellet was suspended in 05mL of 01M sodium-phosphate buffer (pH 72) as described above homogenizedand immediately stored in Eppendorf tubes under liquidnitrogen until use Mannan synthase assays were performedas described by [25] Standard reaction was carried out ina final volume of 50120583L containing 020 nmol of GDP-d-[U-14C]mannose (approximately 1500 Bq) 20 nmol of GDP-d-mannose and 10 120583L of total membrane fraction in 01Msodium-phosphate buffer (pH72) Reactionswere performedat 27∘C for 30min and stopped by adding 500 120583L of 70ethanol Ethanol insolublemolecules including 14C-polymerwere precipitated by centrifugation at 10000timesg for 5minThepellet was rewashed five times with 70 ethanol in orderto remove unreacted radiolabelled precursors To verify thepresence of a 2-epimerase activity the incubation medium

The Scientific World Journal 3

depleted of polymers was paper-electrophoresed (pH 35)[26] and the elutedGDP-sugars were hydrolysed in 01MHCland paper-chromatographed (solvent A) When the effectof proteinase K and Triton X-100 was tested equal aliquotsof total membrane fraction were incubated with 400 120583gmLProteinase K with or without the addition of 1 Triton X-100for 30min at 25∘CThe reactionswere stopped by the additionof 500120583L of 200mM PMSF

26 Acid and Enzymic Hydrolysis Total acid hydrolysis ofthe 14C-polymer was carried out in 3 (ww) H

2SO4at

120∘C 103 kPa for 1 h The hydrolysates were neutralised bya bicarbonate form of amberlite IR-45 (OH) resin rotary-evaporated to dryness under reduced pressure and dissolvedin 30 120583L of H

2O and an aliquot used for paper chro-

matography Enzymic hydrolysis with 120573-mannanase fromBacillus subtilis or Penicillium wortmannin was performed asdescribed by [27]

27 Paper Chromatography Paper Electrophoresis andRadioactive Counting Procedure Descending paper chroma-tography was performed on Whatman no 1 paper anddeveloped with Solvent A ethyl acetate-pyridine-water(8 2 1 by vol) Paper electrophoresis was carried out onWhatman no 1 paper in acetic acid (8 vv) and formicacid (2 vv) buffer pH 20 5 kV for 45min to separateGDP-sugars and sugar phosphates from polysaccharidesand neutral material [21] To separate NDP-sugars paperelectrophoresis was carried out in pyridine 5 (vv) aceticacid buffer pH 35 5 kV for 30ndash45min [26] Detection of themarkers and radioactive counting procedure were performedas described by [25]

28 Statistical Analyses Thebiochemical data fromwild-typeand AtCslA2-GFP transformed tobacco leaf protoplasts werecompared using Studentrsquos 119905-test and values were expressedas means plusmn standard deviation of at least three independentreplicated experiments (119899 = 3) SigmaStat version 311 soft-ware (Systat Software Inc) was used for statistical analysis 119875value of less than 005 was considered to indicate statisticalsignificance

3 Results

31 Cellular Localization of AtCslA2 Expressed in Tobacco LeafProtoplasts A C-terminal fusion of the full-length codingregion of AtCslA2 to the green fluorescent protein (GFP)was constructed and transiently expressed in tobacco leafprotoplasts to investigate its subcellular localization Laserconfocal scanning microscopy revealed fluorescence in acortical reticulate network and some high mobile punctatestructures (Figure 1(a)) suggesting the chimera localizationin the ER and Golgi compartments

Treatment with Brefeldin A a lipophilic fungal toxinknown to induce disassembly of the Golgi stacks amongother effects [28] determined the aggregation of the punctatestructures (Figures 1(b) and 1(c)) and during time theredistribution of the fluorescence into the tubular and cortical

network (Figure 1(d))This suggested that AtCslA2-GFPmaybe localized in the Golgi stacks To further demonstrate thislocalization the chimera was coexpressed with ST52-mRFP atrans-Golgi stack marker [20 29] A complete colocalizationof the green and red fluorescence was observed (Figures1(e)ndash1(g)) evidencing that both ST52-mRFP and AtCslA2accumulated in the Golgi stacks The final localization ofthe chimera in the Golgi stacks was also confirmed bytreating AtCslA2-GFP transiently transformed protoplastswith the protein synthesis inhibitor cycloheximide After2 h of treatment the punctate structures were the only flu-orescent compartments evidenced (Figure 1(h)) Altogetherthese data demonstrate that GFP-tagged AtCslA2 protein iscotranslationally inserted into ER to further reach the Golgiapparatus which represents its stable localization

32 AtCslA2-GFP Is Integrated in the Golgi Membranes Thepresence of the chimera in the intracellular protein fractionincluding the endomembrane system was confirmed byimmunoblot analyses of protein extracted from the trans-formed protoplasts using an anti-GFP antibody (Figure 2(a))The protein band shows the expected molecular mass of885 kDa A very small amount of the chimera was detectedin the protein fraction of the medium The amino acid (aa)sequence analysis of the AtCslA2 cDNA product predicteda polypeptide of 534 aa showing four TM domains andtwo hydrophilic regions between the first and second andthe third and fourth TM domains (TMHMM server v 20httpwwwcbsdtudkservicesTMHMM) To ascertain theinsertion of the chimera into the lipid bilayer its solubil-ity in the nonionic detergent Triton X-114 was analysedAfter extraction and phase separation of the total AtCslA2transformed protoplast proteins the major percentage of thechimera was detected in the detergent phase confirming thatit was an integral membrane protein (Figure 2(b))

33 Biochemical Characterization of AtCslA2-GFP Expressedin Tobacco Leaf Protoplasts AtCslA2 is a member of Ara-bidopsis CslA gene family encoding for a putative mannansynthase To determine whether AtCslA2-GFP was func-tional in tobacco protoplasts we tested its enzymatic activityisolating the total membrane fraction from the homogenatesof wild-type and AtCslA2-GFP transiently transformed pro-toplasts and incubating them in the presence of GDP-d-[U-14C]mannose as substrate After 30min the radioactivityincorporated in the 70 ethanol precipitated product wasmeasured and the enzymatic activity associated with themembranes was calculated (Table 1) Radioactive polymerswere synthesized either from wild-type or transformed pro-toplasts 14C-polymers were immobile when electrophoresedin acetic acidformic acid buffer (pH 20) and were not elutedfrom Whatman no 1 paper after several washing either withH2O or with mixtures of chloroform-methanol (1 1 vv) and

chloroform-methanol-water (3 48 47 10 10 3 vv) Totalacid hydrolysis and 120573-mannanase treatment of the 14C-polymers released only mannose as radioactive sugar (paperchromatography solvent system A) No other radioactivesugars were detected when the incubation medium was


(a) (b) (c) (d)

(e) (f) (g) (h)

Figure 1 Subcellular localization of AtCslA2-GFP AtCslA2-GFP transiently expressed in tobacco leaf protoplasts labels ER and Golgi stacks(a) Brefeldin A determines the aggregation of the Golgi and the redistribution of the fluorescence in the ER (bndashd) AtCslA2-GFP colocaliseswith the Golgi marker ST52-mRFP (endashg) Golgi stacks are the only fluorescent compartment labelled after 2 h of cycloheximide treatment(h) Scale bar 20 120583m

IN OUT

97 kD

54 kD

(a)

DET AQ

97 kD

54 kD

(b)

Figure 2 AtCslA2-GFP is insoluble in the detergent Triton X-114 Western blot analysis of protein fractions (intracellular IN incubationmedium OUT) obtained from AtCslA2-GFP transformed protoplasts (a) AtCslA2-GFP is Triton X-114 insoluble and is mainly recoveredin the detergent phase (DET) a small contamination of the chimera was present in the aqueous phase (Aq) (b) Bands were detected usinganti-GFP serum

analysed for the presence of a GDP-mannose 2-epimeraseactivity (data not shown) These results demonstrated thepresence of a 120573-mannosyl transferase activity in tobacco leafprotoplasts and an appreciable highly statistically significant(119875 = 0004) increase of this activity approximately 38in AtCslA2-GFP transformed protoplasts indicating that theinserted chimera functions properly in theGolgimembranes

Based on TM domain prediction (TMHMM server)AtCslA2 active site should face the Golgi lumen To ascertainthis hypothesis mannan synthase activity was tested in thepresence of proteinase K with or without Triton X-100 Theenzymatic activity was drastically reduced (119875 lt 0001 withrespect to untreated AtCslA2-GFP transformed protoplasts)when membranes were permeabilized with detergent duringproteinase K treatment (Table 1) The results indicate that thecatalytic site of AtCslA2 faces the lumen of the Golgi stacks

according to bioinformatic predictions A hypotheticalmodelof the polypeptide topology based on the locations of thepredicted TM domains is reported in Figure 3

4 Discussion

In order to define the intracellular localization of AtCslA2(putative mannan synthase encoded by a member of Ara-bidopsis CslA gene family) a fusionwithGFPwas constructed(AtCslA2-GFP) and transiently expressed in tobacco leafprotoplasts The fluorescent chimera obtained was retainedin the intracellular compartments labelling ER and Golgistacks The presence of the chimera in the ER was due toits synthesis and transit in this compartment In fact Golgistacks were the stable and final localization of the chimera as


Table 1 Mannan synthase activity in wild-type and AtCslA2-GFPtransformed tobacco protoplasts and effect of protease K with orwithout Triton X-100The reactionmixture contained the following020 nmol of GDP-d-[U-14C]mannose 20 nmol GDP-d-mannoseand 10 120583L (130120583g of protein) of total membrane fraction isolatedfrom wild-type and AtCslA2-GFP transformed tobacco protoplastsin 01M sodium-phosphate buffer pH 72 in a total volume of 50120583LReaction time at 27∘C was 30min Data are the means plusmn standarddeviation of three independent replicates (119899 = 3)

Protoplasts Bq Specific activity(nmolsdotminmg of protein)

Wild-type 39 plusmn 02 370 plusmn 25

AtCslA2-GFP transformed 54 plusmn 03 510 plusmn 32

+ Proteinase K 46 plusmn 03 438 plusmn 30

+ Triton X-100 22 plusmn 01 209 plusmn 16

+ Proteinase K + TritonX-100 08 plusmn 01 76 plusmn 5

GFP

C

N

Cytosol

Lumen

34

36856

389 404

426 509

531

Figure 3 Hypothetical model of membrane topology of AtCslA2-GFP Four transmembrane domains predicted by TMHMM serverare reported in red numbers mark location of the amino acids Twohydrophilic region are predicted the larger of which contains thepredicted active site (blue star) and faces the Golgi lumen

evidenced by a notable colocalization with the Golgi markerST52-mRFP (Figures 1(e)ndash1(g)) and the disappearance offluorescence from ER compartment in the presence of theprotein inhibitor cycloheximide (Figure 1(h)) Furthermoreas previously described for other Golgi resident proteinsBrefeldin A induced a redistribution of the chimera fromGolgi stacks to the ER (Figures 1(b)ndash1(d)) [29]

AtCslA2 is predicted to have four transmembrane (TM)domains (TMHMM server) and therefore it should be anintegral membrane protein we validated this predictiondemonstrating its insolubility in the non-ionic detergentTriton X-114 Bioinformatic tools predict the first TMdomainas very close to the N-terminus (see scheme in Figure 3)followed by a large hydrophilic region a second hydrophilicregion separates the third from the fourth TM domain Thelast TM domain is very close to the C-terminus where GFPwas fused AtCslA2 seems to have a membrane topologysimilar to that of CesA callose synttase (CalS) and the othermembers of Csl family All of them contain multiple TMdomains clustered in two regions with a large hydrophiliccentral loop containing the putative catalytic site [1 30]

The catalytic domain faces the cytoplasm in CesA and CalSfamilies [7 30] and may be located in either the cytosolor the Golgi lumen in Csl proteins [31] The ArabidopsisCslD2 [32] and CslC4 [33] are examples of Csl proteinswith a catalytic domain facing the cytoplasmic side of theGolgi showing a topologic organization similar to CesA Onthe contrary 120573-mannan synthases of guar seeds (CtManS)[15] and Arabidopsis (CslA9) [33] are predicted to have thecatalytic domain within Golgi cisternae The number andlocation of the predicted TM domains (1 TM helice close totheN-terminus) inAtCslA2 suggest that the large hydrophilicloop containing the putative catalytic domains likely faces theGolgi lumen A second hydrophilic region between the thirdand fourth TM domain is predicted for AtCslA2 as well asfor CtManS [15] This domain is reported to be much longerthan any corresponding inter-TM domains toward the C-terminus in CesA [34] and is predicted to be in the Golgilumen on the same side of the catalytic domain for bothCtManS and AtCslA2 A role in interacting with a galactosyltransferase that adds galactose onto amannan backbone hasbeen hypothised for this domain in CtManS [15]

Characterization and assignment of the specific transg-lycosylase activity to a glycosyltransferase have always beendifficult when either a biochemical or a genetic approachwas applied to the problem Heterologous expression hasprovided a valid method to determine the enzymatic activityof plant Gts Heterologous expression in Drosophila Schnei-der 2 (S2) cells of a group of Csl genes from Arabidopsisrice loblolly pine and Physcomitrella patens has allowed todetermine a 120573-mannan synthase activity for several membersof CslA gene family [16 35] Also a guar putative ManSgene was expressed in embryogenic soybean suspension-culture cells to ascertain its involvement in the synthesisof galactomannans [15] In the present study we transientlyexpressed AtCslA2-GFP fusion protein in tobacco leaf pro-toplasts and analysed its catalytic activity in the total mem-brane fraction in the presence of GDP-d-[U-14C]mannoseas substrate Membrane preparations from untransformedtobacco protoplasts showed an appreciable mannan syn-thase activity (370 nmolsdotminmg of proteins)which increasedby approximately 38 in the transformed cells Althoughgymnosperms contain higher amounts of (galacto)mannansandor (galacto)glucomannans with respect to angiospermsthe presence of mannans as structural and storage polysac-charides in plants and algae is well documented [36ndash39] Aconstitutive 120573-mannan synthase activity in tobacco leaf istherefore not surprising The increased 120573-mannan synthaseactivity in transformed protoplasts clearly indicated a properfolding and a correct function of the chimera into Golgimembranes despite the presence of GFP as fluorescenttag A mannan synthase activity has been reported forproteins encoded by CslA1 and CslA9 genes from Arabidopsisexpressed in Drosophila S2 cells with an enzymatic activityvaluable in the order of pmolemg of protein [35] Theheterologous system represented by tobacco protoplastsdespite the constitutive background seems to be moreefficient than animal cells since a specific activity in theorder of nmolmg of protein was related to AtCslA2-GFPoverexpression


The chemical characterization of the radiolabelled poly-meric material obtained from the in vitro incubation ofthe total membrane fractions isolated from both wild-typeand AtCslA2-GFP transformed protoplasts with GDP-d-[U-14C]mannose demonstrated that it consisted mainly ofpolysaccharide whose complete acid hydrolysis gave onlymannose as radioactive glycosyl residue No other sugarswere detected either in the polymer or in the reactionmixture indicating that GDP-mannose was not 2-epimerisedinto GDP-glucose This result agrees with previously datashowing the absence of a 2-epimerase activity in amembranesystem isolated from pea stem which leads to the synthesisof mannans using GDP-mannose as substrate [25] On thecontrary an epimerase activity was found in particulateenzymic preparation isolated from pine cambial and xylemcells which leads to the synthesis of glucomannans using onlyGDP-mannose as substrate [27] The presence of a GDP-mannose 2-epimerase activity in our system would allow usto easily determine the possible involvement of AtCslA2 inglucomannan biosynthesis by the presence of both d-[U-14C]mannose and d-[U-14C]glucose residues after completeacid hydrolysis of polysaccharides We did not test trans-formed protoplast membrane preparations in the presenceof both radioactive GDP-mannose and GDP-glucose andorUDP-galactose therefore we cannot exclude that AtCslA2gene may have a role in the synthesis of (galacto)mannansor (galacto)glucomannans A role in (galacto)mannan and(galacto)glucomannan synthesis has been evidenced for sev-eral CslA gene products from Arabidopsis guar and poplar[15 16 35 40ndash43]

5 Conclusions

The present study extends previous analyses on CslA geneproducts demonstrating that AtCslA2 brings about the syn-thesis of 120573-mannan The heterologous expression of thefluorescent chimera AtCslA2-GFP in tobacco leaf protoplastscan be considered an efficient experimental system thatallows either to follow the correct final localization of thechimera or to test its enzymatic activity The possibilityto overexpress gene products involved in the synthesis ofmannan glucomannan andor galactoglucomannan mayhelp to better understand how the encoded proteins operateto synthesize these polysaccharides This information mayhave relevant consequences for several technological aspectssuch as the production of biofuel and gums of high value

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by Italian grant from MinisterodellrsquoIstruzione dellrsquoUniversita e della Ricerca-Programmidi Rilevante Interesse Nazionale (MIUR-PRIN) 2009 Theauthors thank Dr R Y Tsien (Howard Hughes Medical

Institute and Department of Pharmacology California Uni-versity) for allowing the use of the RFPmarker Special thanksare due to ldquoRegione Pugliardquo for supporting the Project no14 ldquoReti di Laboratori Pubblici di Ricercardquo ldquoSELGErdquo throughwhich the Zeiss LSM710 confocal microscope was bought

References

[1] M S Doblin I Kurek D Jacob-Wilk and D P DelmerldquoCellulose biosynthesis in plants from genes to rosettesrdquo Plantand Cell Physiology vol 43 no 12 pp 1407ndash1420 2002

[2] D J Cosgrove ldquoGrowth of the plant cell wallrdquo Nature ReviewsMolecular Cell Biology vol 6 no 11 pp 850ndash861 2005

[3] A P S Sandhu G S Randhawa and K S Dhugga ldquoPlant cellwall matrix polysaccharide biosynthesisrdquo Molecular Plant vol2 no 5 pp 840ndash850 2009

[4] A H Liepman R Wightman N Geshi S R Turner and H VScheller ldquoArabidopsismdasha powerful model system for plant cellwall researchrdquo Plant Journal vol 61 no 6 pp 1107ndash1121 2010

[5] D P Delmer ldquoCellulose biosynthesis exciting times for adifficult field of studyrdquo Annual Review of Plant Biology vol 50pp 245ndash276 1999

[6] T A Richmond and C R Somerville ldquoThe cellulose synthasesuperfamilyrdquoPlant Physiology vol 124 no 2 pp 495ndash498 2000

[7] M S Doblin C E Vegara S Read E Newbigin and A BacicldquoPlant cell wall biosynthesismaking the bricksrdquo inAnnual PlantReviews The Plant Cell Wall J K C Rose Ed Blackwell 2003

[8] W-R Scheible andM Pauly ldquoGlycosyltransferases and cell wallbiosynthesis novel players and insightsrdquo Current Opinion inPlant Biology vol 7 no 3 pp 285ndash295 2004

[9] A H Liepman and D M Cavalier ldquoThe cellulose synthase likeA and cellulose synthase like C families recent advances andfuture perspectivesrdquo Frontiers in Plant Science vol 3 no 109pp 1ndash7 2012

[10] I M Saxena R M Brown Jr M Fevre R A Geremiaand B Henrissat ldquoMultidomain architecture of 120573-glycosyltransferases Implications for mechanism of actionrdquo Journal ofBacteriology vol 177 no 6 pp 1419ndash1424 1995

[11] T A Richmond and C R Somerville ldquoIntegrative approachesto determining Csl functionrdquo Plant Molecular Biology vol 47no 1-2 pp 131ndash143 2001

[12] A Oikawa C H Lund Y Sakuragi and H V Scheller ldquoGolgi-localized enzyme complexes for plant cell wall biosynthesisrdquoTrends in Plant Science vol 18 no 1 pp 49ndash58 2013

[13] L Norambuena L Marchant P Berninsone C B HirschbergH Silva and A Orellana ldquoTransport of UDP-galactose inplants Identification and functional characterization ofAtUTr1an Arabidopsis thaliana UDP-galactoseUDP-glucose trans-porterrdquoThe Journal of Biological Chemistry vol 277 no 36 pp32923ndash32929 2002

[14] F Reyes and A Orellana ldquoGolgi transporters opening the gateto cell wall polysaccharide biosynthesisrdquo Current Opinion inPlant Biology vol 11 no 3 pp 244ndash251 2008

[15] K S Dhugga R Barreiro B Whitten et al ldquoGuar seed 120573-mannan synthase is a member of the cellulose synthase supergene familyrdquo Science vol 303 no 5656 pp 363ndash366 2004

[16] A H Liepman C G Wilkerson and K Keegstra ldquoExpressionof cellulose synthase-like (Csl) genes in insect cells reveals thatCslA family members encode mannan synthasesrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 102 no 6 pp 2221ndash2226 2005


[17] J-C Cocuron O Lerouxel G Drakakaki et al ldquoA gene fromthe cellulose synthase-like C family encodes a 120573-14 glucansynthaserdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 104 no 20 pp 8550ndash8555 2007

[18] R A Burton S M Wilson M Hrmova et al ldquoCellulosesynthase-like CslF genes mediate the synthesis of cell wall(1314)-120573-D-glucansrdquo Science vol 311 no 5769 pp 1940ndash19422006

[19] M S Doblin F A Pettolino S M Wilson et al ldquoA barley cel-lulose synthase-like CSLH gene mediates (1314)-120573-D- glucansynthesis in transgenic Arabidopsisrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 106 no14 pp 5996ndash6001 2009

[20] M De Caroli M S Lenucci G-P Di Sansebastiano GDalessandro G De Lorenzo and G Piro ldquoProtein trafficking tothe cell wall occurs through mechanisms distinguishable fromdefault sorting in tobaccordquo Plant Journal vol 65 no 2 pp 295ndash308 2011

[21] M R Leucci G-P Di Sansebastiano M Gigante G Dalessan-dro and G Piro ldquoSecretion marker proteins and cell-wallpolysaccharides move through different secretory pathwaysrdquoPlanta vol 225 no 4 pp 1001ndash1017 2007

[22] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[23] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[24] M P Lisanti I W Caras T Gilbert D Hanzel and ERodriguez-Boulan ldquoVectorial apical delivery and slow endo-cytosis of a glycolipid-anchored fusion protein in transfectedMDCK cellsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 87 no 19 pp 7419ndash74231990

[25] G Piro A Zuppa G Dalessandro and D H NorthcoteldquoGlucomannan synthesis in pea epicotyls the mannose andglucose transferasesrdquo Planta vol 190 no 2 pp 206ndash220 1993

[26] D Pacoda A Montefusco G Piro and G Dalessandro ldquoReac-tive oxygen species and nitric oxide affect cell wall metabolismin tobacco BY-2 cellsrdquo Journal of Plant Physiology vol 161 no10 pp 1143ndash1156 2004

[27] G Dalessandro G Piro and D H Northcote ldquoA membrane-bound enzyme complex synthesising glucan and glucomannanin pine tissuesrdquo Planta vol 175 no 1 pp 60ndash70 1988

[28] C Ritzenthaler A Nebenfuhr A Movafeghi et al ldquoReevalu-ation of the effects of brefeldin a on plant cells using tobaccobright yellow 2 cells expressing golgi-targeted green fluorescentprotein and copi antiserardquo Plant Cell vol 14 no 1 pp 237ndash2612002

[29] C Saint-Jore-Dupas A Nebenfuhr A Boulaflous et al ldquoPlantN-glycan processing enzymes employ different targetingmech-anisms for their spatial arrangement along the secretory path-wayrdquo Plant Cell vol 18 no 11 pp 3182ndash3200 2006

[30] D P S Verma and Z Hong ldquoPlant callose synthase complexesrdquoPlant Molecular Biology vol 47 no 6 pp 693ndash701 2001

[31] O Lerouxel D M Cavalier A H Liepman and K KeegstraldquoBiosynthesis of plant cell wall polysaccharidesmdasha complexprocessrdquo Current Opinion in Plant Biology vol 9 no 6 pp 621ndash630 2006

[32] W Zeng and K Keegstra ldquoAtCSLD2 is an integral Golgimembrane protein with its N-terminus facing the cytosolrdquoPlanta vol 228 no 5 pp 823ndash838 2008

[33] J Davis F Brandizzi A H Liepman and K Keegstra ldquoAra-bidopsis mannan synthase CSLA9 and glucan synthase CSLC4have opposite orientations in the Golgi membranerdquo PlantJournal vol 64 no 6 pp 1028ndash1037 2010

[34] J R Pear Y Kawagoe W E Schreckengost D P Delmer andD M Stalker ldquoHigher plants contain homologs of the bacterialcelA genes encoding the catalytic subunit of cellulose synthaserdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 93 no 22 pp 12637ndash12642 1996

[35] A H Liepman C J Nairn W G T Willats I Soslashrensen AW Roberts and K Keegstra ldquoFunctional genomic analysissupports conservation of function among cellulose synthase-like a gene family members and suggests diverse roles ofmannans in plantsrdquo Plant Physiology vol 143 no 4 pp 1881ndash1893 2007

[36] D H Northcote ldquoChemistry of plant cell wallrdquo Annual Reviewof Plant Physiology vol 23 pp 113ndash132 1972

[37] A Bacic P J Harris and B A Stone ldquoStructure and functionof plant cell wallsrdquo in Biochemistry of Plants J Preiss Ed vol14 pp 297ndash371 Academic Press New York NY USA 1988

[38] H Meier and J S G Reid ldquoReserve polysaccharides other thanstarch in higher plantsrdquo in Encyclopedia of Plant Physiology FA Loewus and W Tanner Eds vol 13 pp 418ndash471 SpringerBerlin Germany 1982

[39] G Piro M Lenucci G Dalessandro et al ldquoUltrastructurechemical composition and biosynthesis of the cell wall inKoliella antarctica (Klebsormidiales Chlorophyta)rdquo EuropeanJournal of Phycology vol 35 no 4 pp 331ndash337 2000

[40] S Suzuki L Li Y-H Sun and V L Chiang ldquoThe cellulosesynthase gene superfamily and biochemical functions of xylem-specific cellulose synthase-like genes in Populus trichocarpardquoPlant Physiology vol 142 no 3 pp 1233ndash1245 2006

[41] S Ubeda-Tomas E Edvardsson C Eland et al ldquoGenomic-assisted identification of genes involved in secondary growthin Arabidopsis utilising transcript profiling of poplar wood-forming tissuesrdquo Physiologia Plantarum vol 129 no 2 pp 415ndash428 2007

[42] F Goubet C J Barton J C Mortimer et al ldquoCell wallglucomannan in Arabidopsis is synthesised by CSLA glycosyl-transferases and influences the progression of embryogenesisrdquoPlant Journal vol 60 no 3 pp 527ndash538 2009

[43] O O Obembe E Jacobsen R G F Visser and J-P VinckenldquoCellulose-hemicellulose networks as target for in planta mod-ification of the properties of natural fibresrdquo Biotechnology andMolecular Biology Review vol 1 no 3 pp 76ndash86 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Until now little evidence for the functional role of Cslgenes has been collected Among the six groups of the Cslgene family identified only few members of CslA and CslChave been functionally characterized suggesting their respec-tive involvement in (gluco)mannan [15 16] and xyloglucansynthesis [17] A role in the synthesis of mixed 120573-glucan hasbeen proposed for members of CslG and CslF groups [18 19]

In this study we constructed a fluorescent fusion of anArabidopsis CslA family member AtCslA2-GFP to identifyits final localization in a heterologous system represented bytobacco leaf protoplasts The chimera was localized in theGolgi stacks in a functional organization since an increase inmannan synthesis was quantified in transformed protoplastsCombining confocal observation biochemical data andTM domain predictions the topologic organisation of thechimera in the Golgi membrane is discussed

2 Material and Methods

21 Plasmid Construction The cDNA coding for AtCslA2was purchased from The Arabidopsis Information Resource(TAIR Ohio State University) The cDNA sequence wasamplified with primers Man01 (51015840-ggagcccggatccatggacggtg-31015840) and Man02 (51015840-caactgctagctcgggacataagtccc-31015840) tointroduce respectively BamHI and NheI restriction sitesThe BamHINheI fragment was introduced into a GFP-containing vector PGIP2-GFP [20] to construct AtCslA2-GFP The construct was confirmed by sequencing (PRIMMsrl Milan Italy)

22 Protoplast Preparation Transformation and Drug Treat-ments Tobacco protoplasts were prepared from the leavesof Nicotiana tabacum cv SR1 plants and transiently trans-formed as described by [21] Equivalent quantities (20120583g) ofplasmids were used for the colocalization experiments Thefluorescent pattern of over 1200 cells was analysed BrefeldinA (10 120583gmL Sigma-Aldrich) or cycloheximide (30 120583gmLSigma-Aldrich) was added into the incubation medium for2 h before confocal analyses

23 Laser Scanning Confocal Microscopy Protoplasts tran-siently expressing fluorescent constructs were observed by alaser scanning confocal microscope (LSM 710 Zeiss) in theirculture medium at different times after transformation Todetect GFP fluorescence a 488 nm argon ion laser line wasused and the emission was recorded with 505ndash530 nm filterset RFPwas detectedwith a 560ndash615 nmfilter set afterHendashNelaser excitation at 543 nm while chlorophyll epifluorescencewas detected with the filter set for tetramethylrhodamineisothiocyanate (TRITCgt650 nm) and eliminatedThe powerof each laser line the gain and the offset were identicalfor each experiment so that the images were comparableAppropriate controls were performed to exclude the possibil-ity of crosstalk between the two fluorochromes before imageacquisition

24 Protein Extraction Immunoblotting Analyses Cell Lysisand Phase Separation Extracellular and intracellular protein

fraction cell lysis phase separation and immunoblot analy-ses were performed as previously described [20] Protoplastswere harvested by gentle centrifugation at 65timesg withoutbreak after dilution of the incubation medium with twovolumes of W5 The medium was concentrated by filtrationon Centricon Plus 20 (Amicon) to obtain the extracellularprotein fraction (OUT) The protoplasts were suspendedin homogenization buffer (40mM Hepes-NaOH buffer pH75 10mM imidazole 1mM benzamidine 5mM 6-amino-hexanoic acid 10mM dithiothreitol and 1mM phenyl-methylsulfonyl fluoride) and sonicated The homogenatewas centrifuged at 800timesg for 10min at 4∘C to removecell wall debris and the supernatant was precipitated with80 acetone at minus20∘C (three times) to obtain the proteinintracellular fraction (IN) that includes endomembrane com-partments and organelles Protein concentration was deter-mined according to [22] Identical amounts of total proteinfrom each subcellular preparation were subjected to SDS-PAGE electrophoresis [23] and immunoblotting analysesAfter electrophoresis protein samples were transferred to anitrocellulose membrane (Hybond-C extra GE Healthcare)and blocked with 5 skim-milk powder in TBS (20mMTris-HCl pH 75 500mM NaCl 5 wv milk powder) for2 h Anti-GFP (1 5000 vv) (Molecular Probes) was usedin TBS + 1 skimmed-milk powder Each protein fractionwas visualized after ECL (ECL western blotting analysissystem GE Healthcare) chemodetection Cell lysis and phaseseparation were carried out as described by [24] modified by[20] To test AtCslA2 solubility in Triton X-114 AtCslA2-GFPtransformed protoplasts were freeze-thawed twice in 10mMTris-buffer pH 75 containing 015M NaCl and 1mM EDTAand centrifuged at 800timesg for 10minThe 800timesg supernatantwas adjusted to 15mL with Tris-buffer (pH 75) and TritonX-114 was added into a final concentration of 1 vv

25 Enzyme Assay To assay mannan synthase activity pro-toplasts were suspended and sonicated in 01M sodium-phosphate buffer (pH 72) containing 10mM MgCl

2 1 mM

dithiothreitol 04M sucrose and 1 bovine serum albuminThe resulting homogenate was centrifuged at 800timesg for10min at 2∘C The 800timesg supernatant was further cen-trifuged at 100000timesg for 60min at 2∘C to obtain a pelletconsisting of membranous material (total membrane frac-tion) This pellet was suspended in 05mL of 01M sodium-phosphate buffer (pH 72) as described above homogenizedand immediately stored in Eppendorf tubes under liquidnitrogen until use Mannan synthase assays were performedas described by [25] Standard reaction was carried out ina final volume of 50120583L containing 020 nmol of GDP-d-[U-14C]mannose (approximately 1500 Bq) 20 nmol of GDP-d-mannose and 10 120583L of total membrane fraction in 01Msodium-phosphate buffer (pH72) Reactionswere performedat 27∘C for 30min and stopped by adding 500 120583L of 70ethanol Ethanol insolublemolecules including 14C-polymerwere precipitated by centrifugation at 10000timesg for 5minThepellet was rewashed five times with 70 ethanol in orderto remove unreacted radiolabelled precursors To verify thepresence of a 2-epimerase activity the incubation medium




2SO4at






3 Results








(a) (b) (c) (d)

(e) (f) (g) (h)


IN OUT

97 kD

54 kD

(a)

DET AQ

97 kD

54 kD

(b)





4 Discussion










GFP

C

N

Cytosol

Lumen

34

36856

389 404

426 509

531








5 Conclusions




Acknowledgments



References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




2SO4at






3 Results








(a) (b) (c) (d)

(e) (f) (g) (h)


IN OUT

97 kD

54 kD

(a)

DET AQ

97 kD

54 kD

(b)





4 Discussion










GFP

C

N

Cytosol

Lumen

34

36856

389 404

426 509

531








5 Conclusions




Acknowledgments



References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b) (c) (d)

(e) (f) (g) (h)


IN OUT

97 kD

54 kD

(a)

DET AQ

97 kD

54 kD

(b)





4 Discussion










GFP

C

N

Cytosol

Lumen

34

36856

389 404

426 509

531








5 Conclusions




Acknowledgments



References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









GFP

C

N

Cytosol

Lumen

34

36856

389 404

426 509

531








5 Conclusions




Acknowledgments



References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



5 Conclusions




Acknowledgments



References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article cellular localization and biochemical characterization...

Documents