General information on AtFUT1 gene family

The following information was collected from TAIR, NCBI and Uniprot database (see Table 25 A and B). Predictions have been made using common Web Tools like TMHMM, TargetP and SignalP servers. AtFUT1 and its nine homologues are located on three different chromosomes but the lengths of both coding sequences and the corresponding proteins are similar. The presence of one transmembrane domain is predicted for each of the fucosyltransferase homologue, even two in the case of AtFuT10. At the notable exception of AtFUT1, AtFUT3 and AtFUT10, all fucosyltransferase candidates are predicted to be localised to the secretory pathway, indicated by strongest values for predicted signal peptides. AtFUT1 is predicted to the mitochondrion (with low reliability), which is clearly illustrating the limits inherent to this kind of predictions as the modification of xyloglucans by AtFUT1 occurs within the secretory pathway. No localisation could be predicted for AtFUT3 and AtFUT10.

Analysis of potential signal peptides showed predicted presence of signal anchors for AtFUT1 to AtFUT6. A signal peptide was predicted for AtfUT6, AtFUT8 and AtFUT10.

AtFUT7 and AtFUT10 were predicted as non-secretory proteins. Expression profile shows that all 10 candidate genes are expressed in several plant organs, and that expression is often limited to just one organ. AtFUT1 is mainly expressed in shoot apex, buds, sepals, stamen, roots, young siliques and seeds. AtFUT2 expression is limited to the stamen. AtFUT3 is mainly expressed in flowers, seeds and young siliques. AtFUT4 expression is limited to sepals and young rosette leaves as well as senescent leaves.

AtFUT5 is exclusively expressed in young roots. Both AtFUT6 and AtFUT7 are mainly expressed in young roots and senescent leaves. AtFUT8 is exclusively expressed in closed buds. AtFUT9 expression is limited to seeds stage 10. AtFUT10 is exclusively expressed in young roots (data from TAIR, illustrated by e-FP browser at TAIR website).

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Table 25: General information and predictions about all 10 fucosyltransferase candidates.  These informations concerns chromosomal localisation, size of genes and proteins, molecular weight, numbers of potential transmembrane helices as well as predicted  localisation according to analysis of potential signal peptides.  Source: TAIR; 1 ) NCBI; 2 ) TMHMM Sever v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) (Krogh et al., 2001); 3 )  TargetP 1.1 Server: predicted localisation based on presence of N‐terminal signal peptides. Abbreviations: cTP ‐ chloroplast transit peptide, mTP ‐ mitochondrial targeting  peptide, SP ‐ signal peptide, Loc – localisation (S – secretory pathway; M – Mitochondrion, C – Chloroplast, “‐“ ‐ any other location), RC‐ reliability class with values ranging from  1 to 5, where 1 indicates strongest prediction  (http://www.cbs.dtu.dk/services/TargetP/) (Emanuelsson et al., 2007); 4 ) SignalP 3.0 Server  (http://www.cbs.dtu.dk/services/SignalP/) (Emanuelsson et al., 2007) maximal cleavage site probability in brackets. 

3.7.1 Sequence homology between the ten members of AtFUT1 gene family

The amino acid sequences of the fucosyltransferase candidates were compared and analysed using web tool Multalin (http://multalin.toulouse.inra.fr/multalin/).

AtFuT1 MDQNSYRRRSSPIRTTTGGSKSVNFSELLQMKYLSSGTMKLTRTFTTCLIVFSVLVAFSMIFHQHPSDSNRIMGFAE---ARVLDAGVFPNVTNINSDKL 97 AtFuT2 ---MRITEILALFMVLVPVSLVIVAMFGYDQGNGFVQASRFITMEPNVTSSSDDSS---LVQRDQEQKDSVDMSL 69 AtFuT3 --MKRGKKNSDAGDRLTNSDTRTGSSELNAMMKPSLSSMKTMGLLLAVLMVASVMFSLSVVLRDPPSDDVIETEAASRVLQSRLHQAIESDGGLSEKKAQ 98 AtFuT4 ---MYHIFQISGEVIKGLGLKTKILITIVFSTLLILSVMLLSFSNNFNNKLFAAT---INDESETPGR--DRL 65 AtFuT5 ---MYQKFQISGKIVKTLGLKMKVLIAVSFGSLLFI----LSYSNNFNNKLLDAT---TKVDIKETEKPVDKL 63 AtFuT6 ---MKILLTLVFSGLLIWSVVLVSFSNDFNNQLLVAT---SNVS-RESETPRDRL 48 AtFuT7 ---MLLLLSFSNIFKHQLLGAT---INVGSKDSVKPRDRL 34 AtFuT8 ---MKVVITVVT-CLFLLSVMQLSFFNIFNYQLLDAT---TNGS-KDSRKSKDKL 47 AtFuT9 ---MIKLTIAIATCLVLCLVLLLPSSNISYRHKYDL---PTNGLNDSEQQSEKL 48 AtFuT10 --- 0

AtFuT1 LGGL-LASGFDEDSCLSRYQSV-HYRKPSPYKPSSYLISKLRNYEKLHKRCGPGTESYKKALKQLDQEHID----GDGECKYVVWISFSG-LGNRILSLA 190 AtFuT2 LGGL-LVSGFKKESCLSRYQSY-LYRKASPYKPSLHLLSKLRAYEELHKRCGPGTRQYTNAERLLKQKQTG--EMESQGCKYVVWMSFSG-LGNRIISIA 164 AtFuT3 LGNINLVPSFDKESCLSRYEAS-LYRKESPFKQSSYLDYRLQRYEDLHRRCGPFTRSYNLTLDKLKSGDRS--DGEVSGCRYVIWLNSNGDLGNRMLSLA 195 AtFuT4 IGGL-LTADFDEGSCLSRYHKTFLYRKPSPYKPSEYLVSKLRSYEMLHKRCGPGTKAYKEATKHLSHDENYNASKSDGECRYVVWLADYG-LGNRLLTLA 163 AtFuT5 IGGL-LTADFDEGSCLSRYHKYFLYRKPSPYKPSEYLVSKLRSYEMLHKRCGPDTEYYKEAIEKLSRDD---ASESNGECRYIVWVAGYG-LGNRLLTLA 158 AtFuT6 IGGL-LTADFDEGSCLSRYQQSLLYRKASPYKPSEYLVSKLRSYEKLHKRCGPGTDAYKKATEILGHDDENYASKSVGECRYIVWVAVYG-LGNRILTLA 146 AtFuT7 LGGL-LTADFDEDSCLSRYQSS-LYRKPSPYRTSEYLISKLRNYEMLHKRCGPGTDAYKRATEKLGHDHENVGDSSDGECKYIVWVAVYG-LGNRILTLA 131 AtFuT8 LGGL-LTADFDEDSCLSRYESS-LYRKPSPYKPSRYLVSKLRSYEMLHKRCGPGTEAYKKATEILGHDDENHSTKSVGECRYIVWIAVYG-LGNRILTLA 144 AtFuT9 LGGL-LATGFEEKSCLSRYDQS--MSKPSPYKPSRHIVSKLRSYEMLHKRCGPGTKAYKRATKQLGHNELS---SSGDECRYVVWMPMFG-LGNRMLSLV 141 AtFuT10 ---MPSEYLVSELRSYEMLHKRCGPDTKAYKEATEKLSRDEYY-ASESNGECRYIVWLARDG-LGNRLITLA 67

AtFuT1 SVFLYALLTDRVLLVDRGKDMDDLFCEPFLGMSWLLPLDFPMTDQ--FDGLNQESSRCYGYMVKNQVIDTEG----TLSHLYLHLVHDYGDHDKMFFCEG 284 AtFuT2 SVFLYAMLTDRVLLVEGGEQFADLFCEPFLDTTWLLPKDFTLASQ--FSGFGQNSAHCHGDMLKRKLINESS--VSSLSHLYLHLAHDYNEHDKMFFCEE 260 AtFuT3 SAFLYALLTNRFLLVELGVDMADLFCEPFPNTTWFLPPEFPLNSH---FNEQ---SLLR----NSGN---PMVAYRHVVRDSSDQQKLFFCED 275 AtFuT4 SVFLYALLTDRIILVDNRKDIGDLLCEPFPGTSWLLPLDFPLMKY--ADGYHKGYSRCYGTMLENHSINSTS----FPPHLYMHNLHDSRDSDKMFFCQK 257 AtFuT5 SVFLYALLTERIILVDNRKDVSDLLCEPFPGTSWLLPLDFPMLNYTYAWGYNKEYPRCYGTMSEKHSINSTS----IPPHLYMHNLHDSRDSDKLFVCQK 254 AtFuT6 SVFLYALLTERVVLVDQSKDISDLFCEPFPGTSWLLPLEFPLMKQ--IDGYNKGYSRCYGTMLNNQAINSTL----IPPHLYLHILHDSRDNDKMFFCQK 240 AtFuT7 SVFLYALLTERIILVDQRKDISDLFCEPFPGTSWLLPLDFPLMGQ--IDSFNREYSHCYGTMLKNHTINSTT----IPSHLYLHLLHDYRDQDKMFFCQK 225 AtFuT8 SLFLYALLTDRIMLVDQRTDISDLFCEPFPGTSWLLPLDFPLTDQ--LDSFNKESPRCYGTMLKNHAINSTTTESIIPSYLCLYLIHDYDDYDKMFFCES 242 AtFuT9 SVFLYALLTDRVMLVDQRNDITDLFCEPFPETSWLLPLDFPLNDQ--LDSFNREHSRCYGTMLKNHGINSTS---IIPSHLYLDIFHDSRDHDKKFFCEE 236 AtFuT10 SVFLYAILTERIILVDNRKDVSDLLCEPFPGTSWLLPLDFPMLNYTYAYGYNKEYPRCYGTMLENHAINSTS----IPPHLYLHNIHDSRDSDKLFFCQK 163 AtFuT1 DQTFIGKVPWLIVKTDNYFVPSLWLIPGFDDELNKLFPQKATVFHHLGRYLFHPTNQVWGLVTRYYEAYLSHADEKIGIQVRVFDEDPGPFQHVMDQISS 384 AtFuT2 DQNLLKNVPWLIMRTNNFFAPSLFLISSFEEELGMMFPEKGTVFHHLGRYLFHPSNQVWGLITRYYQAYLAKADERIGLQIRVFDEKSGVSPRVTKQIIS 360 AtFuT3 SQVLLEETPWLILKADSFFLPSLFSVSSFKQELQMLFPEKDTAFHFLSQYLFHPTNVVWGLITRYYNAYLAKADQRIGIYIGVSESGNEQFQHLIDQILA 375 AtFuT4 DQSLIDKVPWLIFRANVYFVPSLWFNPTFQTELTKLFPQKETVFHHLGRYLFHPKNQVWDIVTKYYHDHLSKADERLGIQIRVFRDQGGYYQHVMDQVIS 357 AtFuT5 DQSLIDKVPWLIVQANVYFVPSLWFNPTFQTELVKLFPQKETVFHHLARYLFHPTNEVWDMVTDYYHAHLSKADERLGIQIRVFGKPDGRFKHVIDQVIS 354 AtFuT6 DQSLVDKVPWLIVKANVYFVPSLWLNPTFQTELMKLFPQKEAVFHHLARYLFHPTNQVWGLITRSYNAYLSRADETLGIQIRVFSDRAGYFQHVMDQVVA 340 AtFuT7 DQSLVDKVPWLVVKSNLYFIPSLWLNPSFQTELIKLFPQKDTVFYHLARYLFHPTNQVWGMVTRSYNAYLSRADEILGIQVRVFSRQTKYFQHVMDQIVA 325 AtFuT8 DQILIRQVPWLVFNSNLYFIPSLWLIPSFQSELSKLFPQKETVFHHLARYLFHPTNQVWGMITRSYNGYLSRADERLGIQVRVFSKPAGYFQHVMDQILA 342 AtFuT9 DQAFLDKVTWLVVKSNLYFVPSLWMIPSFQTKLIKLFPQKETVFHHLARYLFHPTNQVWGMVTRSYNAYLSRADERLGIQVRVFSKPVGYFQHVMDQIL- 335 AtFuT10 DQSFIDKVPWLIIQTNAYFVPSLWLNPTFQTKLVKLFPQKETVFHHLARYLFHPTNEVWDMVTKYYDAHLSNADERLGIQIRVFGKPSGYFKHVMDQVVA 263 AtFuT1 CTQKEKLLPEVDTLVERSRH--VNTPKHKAVLVTSLNAGYAENLKSMYWEYPTSTGEIIGVHQPSQEGYQQTEKKMHNGKALAEMYLLSLTDNLVTS-AW 481 AtFuT2 CVQNENLLPRLSKGEEQYKQPSEEELKLKSVLVTSLTTGYFEILKTMYWENPTVTRDVIGIHQPSHEGHQQTEKLMHNRKAWAEMYLLSLTDKLVIS-AW 459 AtFuT3 CGTRHKLLPEVDKQRNLPSS-QVLNRKSKAVFISSSSPGYFKSIRDVYWENPTVMGEIISVHKPSYKDYQKTPRNMESKRAWAEIYLLSCSDALVVTGLW 474 AtFuT4 CTQREKLLPELATQEESKVN-ISNIPKSKAVLVTSLSPEYSKKLENMFSERANMTGEIIKVYQPSGERYQQTDKKVHDQKALAEMYLLSLTDNIVAS-SR 455 AtFuT5 CTQREKLLPEFATPEESKVN-ISKTPKLKSVLVASLYPEFSGNLTNMFSKRPSSTGEIVEVYQPSGERVQQTDKKSHDQKALAEMYLLSLTDNIVTS-AR 452 AtFuT6 CTRRENLLPEPAAQEEPKVN-ISRSQKLKAVLVTSLYPEYSETLKNMYWERPSSTGEIIEVYQPSGERVQQTDKKLHDQKALAEMYLLSLTDKIVTS-AR 438 AtFuT7 CTQREKLLPEFAAQEEAQVTNTSNPSKLKAVLVTSLNPEYSNNLKKMYWEHPTTTGDIVEVYQPSRERFQQTDKKLHDQKALAEMYLLSLTDKLVTS-AL 424 AtFuT8 CTQREKLLPEVFVLETQVTN-TSRSSKLKAVLVTSLYPEYSEILRQMYWKGPSSTGEIIQIYQPSQEIYQQTDNKLHDQKALAEIYLLSLTDYIVTS-DS 440 AtFuT9 ---YSDHLKNMFLEQASSTGETIEVYQPSGEKIQQTDKKLHDQKALAEIYLLSLTDELVTS-TR 395 AtFuT10 CTQREKLLPEFE--EESKVN-ISKPPKLKVVLVASLYPEYSVNLTNMFLARPSSTGEIIEVYQPSAERVQQTDKKSHDQKALAEMYLLSLTDNIVTS-GW 359 AtFuT1 STFGYVAQGLGGLKPWILYRPENRTTPDPSCGRAMSMEPCFHSPPFYDCKAK-TGIDTGTLVPHVRHCEDISW-GLKLV--- 558

AtFuT2 STFGYVAQGLGGLRAWILYKQENQTNPNPPCGRAMSPDPCFHAPPYYDCKAK-KGTDTGNVVPHVRHCEDISW-GLKLVDNF---- 539 AtFuT3 SSLVEVAHGLGGLKPWVLNKAENGTAHEPYCVKARSIEPCSQATLFHGCKD--- 525 AtFuT4 STFGYVAYSLGGLKPWLLYLPNDNKAPDPPCVRSTSMEPCFLTPPTHGCEPDAWGTESGKVVPFVRYCEDIW--GLKLFDEL---- 535 AtFuT5 STFGYVSYSLGGLKPWLLYQPTNFTTPNPPCVRSKSMEPCYLTPPSHGCEAD-WGTNSGKILPFVRHCEDLIYGGLKLYDEF---- 533 AtFuT6 STFGYVAHSLGGLKPWLLYQPTGPTAPDPPCIQSTSMDPCHLTPPSHGCEPE-WGTNSGKVVPFVRHCEDRGNDGLKLFDEL---- 519 AtFuT7 STFGYVAQGLGGLKPWILYTPKKFKSPNPPCGRVISMEPCFLTPPVHGCEAK-KGINTAKIVPFVRHCEDLRHYGLKLVDDTKNEL 509 AtFuT8 STFGYVAQGLGGLKPWILYKPKNHTAPEPPCVRAVSMEPCFLRAPLYGCQAK---KVNITPFVMYCEDRIT-GLKLVDSN---- 516 AtFuT9 STFGYVAQGLGGLKPWILYEPRDKKTPNPPCVRAMSMEPCFLRAPLHGCQAK---TIKIPPFVRICEDWKT-GLKLVDVSDEL- 474 AtFuT10 STFGYVSYSLGGLKPWLLYQPVNFTTPNPPCVRSKSMEPCYHTPPSHGCEAD-WGTNSGKILPFVRHCEDMMYGGLKLYDDF---- 440

Figure 34: Sequence alignment of the ten fucosyltransferase amino acid sequences of A. thaliana from AtFuT1  gene family. 

Regions with high sequence similarities between 90 and 100 % are given in black. Regions with sequence  similarities between 50 and 90 % (5 to 8 genes) are given in dark grey. Gene‐abbreviations: AtFUT1 ‐  At2g03220; 

AtFUT2 – At2g03210; AtFUT3 – At1g74420; AtFUT4 – At2g15390; AtFUT5 – At2g15370; AtFUT6 – At1g14080; 

AtFUT7 – AT1g14070; AtFUT8 – AT1g14100; AtFUT9 – At1g14110; AtFUT10 – At2g15350.  

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3.7.2 Cloning of fucosyltransferase genes for expression in plants

In order to determine substrate specificity by enzymatic activity assays on different substrates, the coding sequences of all 10 fucosyltransferase genes (with exception of AtFUT9) were cloned and transiently expressed in N. benthamiana

With exception of AtFUT2, all fucosyltransferases genes were cloned into plant binary expression vector p21GT which led to creation of a C-terminal His-tag-fusion protein. AtFUT2 was cloned into vector ppT8 which led to the production of a C-terminal myc-tag-fusion protein. With exception of AtFUt5, AtFUT8 and AtFUT10, template cDNA was isolated from Col-0 leaves. Expression profile presented by e-FP browser at TAIR website indicated expression exclusively in young roots for AtFUT5 and AtFUT10, and expression in young buds for AtFUT8. Therefore, RNA was isolated from roots of three weeks old plants cultivated on MSS-agar plates for amplification of AtFUT5 and AtFUT10, as well as from young buds for amplification of AtFUT8. All amplified fragments were subcloned and sequenced with vector- and insert-specific primers given in Table 8, cloned into the plant expression vectors and sequenced again before transformation into A. tumefaciens.

3.7.3 Enzymatic assays with recombinant fucosyltransferases

In order to determine potential α1,2-fucosyltransferase activity on RG-II, AtFUT2 to AtFUT10 proteins were transiently expressed in N. benthamiana leaves and analysed by enzymatic activity assays. AtFUT1 which is active on xyloglucans and an empty vector control were included as controls for the assay. All expressed proteins were mixed with either unfucosylated tamarind xyloglucan or unfucosylated RG-II chain B.

The activity assay on AtFUT1 with tamarind xyloglucan showed significant activity in the transfer of L-fucose on XXLG to give XXFG. A mass of about 1390 could be detected which corresponds to XXFG, whereas the mass of 1244 corresponds to XXLG. About 43.3 % of XXFG could be detected, whereas the empty vector control showed no conversion at all and stayed at the level of the substrate XXLG (Figure 35).

None of the other AtFUT candidates was able to fucosylated either xyloglucan or RG- II structures.

  Figure 35: Fucosyltransferase 1 activity assay.  

High activity was observed with AtFUT1 which was transiently expressed in N. benthamiana leaves. The empty  vector p21 was included as a negative control. Substrate was tamarind xyloglucan. With AtFUT1 as the enzyme, a  conversion of unfucosylated XXLG into fucosylated XXFG of about 43.4 % could be detected. No XXFG could be  detected in the empty vector control p21.  

3.8 Study of knock-out or k nock-down plants affected in genes