Kazuchika Yamauchi

Kazuhiko Fukushima

Comptes Rendus Biologies (CRBIOL)

3/is-ected toin. Thesinapicin wasy stageyringylnthesis)biniercoupesue de leurtion den. L’acideénéral, lais,C. R. Biologies 327 (2004) 791–797http://france.elsevier.com/direct/CRASSPlant biology and pathology / Biologie et pathologie végétalesThe regulation from guaiacyl to syringyl lignin in thedifferentiating xylem ofRobinia pseudoacacia ✩Kazuchika Yamauchi, Kazuhiko Fukushima∗Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, JapanReceived 27 January 2004; accepted after revision 15 July 2004Available online 17 September 2004Presented by Michel ThellierAbstract13C- and deuterium (D)-labeled ferulic acid and sinapic acid ([8-13C, 3-OCD3]-ferulic acid and [8-13C, 3,5-OCD3]-sinapicacid) were administered to robinia (Robinia pseudoacacia L.) shoots. To estimate the distribution of the label from admintrated ferulic or sinapic acid, continuous 50-µm-thick tangential sections cut from the cambium of robinia were subjlignin chemical analysis by the DFRC method. Labeled ferulic acid was incorporated into guaiacyl and syringyl lignincorporation of labeled ferulic acid into syringyl units was observed only in the later stage of lignification. Labeledacid was incorporated into syringyl lignin in the early stage and the later stage of lignification. In general, syringyl ligndeposited in the later stage of cell wall lignification. Thus, the incorporation of sinapic acid to syringyl lignin in the earlof lignification was abnormal. Taken together, the aromatic ring-modifying reactions (the conversion from guaiacyl to smoiety, including the hydroxylation and methylation) were more important for the regulation of the sinapyl alcohol biosythan the reducing reactions (the reduction of acids to alcohols) in the differentiating xylem.To cite this article: K. Yamauchi,K. Fukushima, C. R. Biologies 327 (2004). 2004 Académie des sciences. Published by Elsevier SAS. All rights reserved.RésuméRégulation du passage de lignines de type quaiacyle vers des lignines de type syringile dans le xylème en voie dedifférenciation de Robinia pseudoacacia Des doses d’acides férulique et sinapique marqués au13C- et au deutérium (D([8-13C, 3-OCD3]-acide férulique et [8-13C, 3,5-OCD3]-acide sinapique) ont été administrés à de jeunes pousses de ro(Robinia pseudoacacia L.). Pour estimer la distribution du marquage de l’acide férulique ou sinapique administré, destangentielles sériées de 50 µm, obtenues à partir du cambium du robinier, ont été soumises à une analyse chimiqlignine par la méthode DFRC. L’acide férulique marqué s’est incorporé aux lignines guaiacyle et syringyle. L’incorporal’acide férulique marqué dans les unités syringyles a été seulement observée lors de la dernière étape de la lignificatiosinapique marqué s’est incorporé à la lignine syringyle lors des première et dernière étapes de la lignification. En g✩ This article is dedicated to a pioneer who has fixed his eyes upon thelignin structures and distributions from the new angle, thatmorphological aspect, differentiation of the cell or evolution of plant.* Corresponding author.E-mail address: kazu@agr.nagoya-u.ac.jp(K. Fukushima).1631-0691/$ – see front matter 2004 Académie des sciences. Published by Elsevier SAS. All rights reserved.doi:10.1016/j.crvi.2004.07.012792 K. Yamauchi, K. Fukushima / C. R. Biologies 327 (2004) 791–797l’acidections deylation)acides enlignine syringyle se dépose lors de la dernière étape de la lignification des parois cellulaires. Ainsi, l’incorporation desinapique à la lignine syringyle lors du premier stade de lignification est-elle anormale. Prises ensemble, les réamodification du noyau aromatique (la conversion de la fonction guaiacyle en syringyle, avec l’hydroxylation et la méthsont plus importantes pour la régulation de la biosynthèse de l’alcool sinapylique que les réactions de réduction (desalcools) dans le xylème en cours de différenciation.Pour citer cet article : K. Yamauchi, K. Fukushima, C. R. Biologies 327(2004). 2004 Académie des sciences. Published by Elsevier SAS. All rights reserved.Keywords: robinia; tracer experiment; DFRC method; syringyl lignin biosynthesis; lignificationMots-clés : robinier ; expérience de marquage ; méthode DFRC ; biosynthèse de la lignine syringyle ; lignificationv-,dydn-gylole-UVl-efore-inlyselerrysy-iaa-ouso Sidoli-n)s tonlyifyfer-ibleden-iaeinib-a-),etrytionnge-ianofftheAbbreviationsDFRC, Derivatization followed by reductive cleaage; GC-MS, gas spectrometry–mass spectrometer; Gguaiacyl; S, syringyl; SIM, selected ion monitoring.1. IntroductionLignin is one of the major components of wooplant cell walls, and it gives rigidity to plants anallows water conduction. Lignin molecules in agiosperms are composed of guaiacyl (G) and syrin(S) units, which are derived from coniferyl alcohand sinapyl alcohol, respectively[6]. A difference inthe ratio of G to S units in different morphological rgions of some angiosperms has been shown bymicroscopy[2,3,10], bromination-TEM-EDXA[13],and the chemical characterization of various tissuefractions[1,7]. In addition, the selective radio labeing of specific units in lignin and visualization of thlabeled units by microautoradiography is usefulstudying the formation and structure of lignin with rspect to its location in wood tissues[11,12,17]. Thesestudies demonstrated that G lignin is deposited main the early stage of xylem differentiation on veswalls and the compound middle lamella of the fibwall, while S lignin is deposited on fiber secondacell walls in the later stage of differentiation.Recent studies suggest that the precursor ofringyl lignin, sinapyl alcohol, is biosynthesized vferuloyl–CoA thioester, coniferaldehyde, and sinpaldehyde without forming sinapic acid[4,5,8,14].However, we previously demonstrated that exogenferulic acid was incorporated into G and S lignin, andexogenous sinapic acid was also incorporated intlignin in robinia[9,15]. This suggests that sinapic acis an intermediate of syringyl lignin. Sinapyl alcohis formed from ferulic acid via the aromatic ring modfying reaction (the hydroxylation and the methylatioand the reducing reactions (the reduction of acidalcohols), whereas it formed from sinapic acid ovia the reducing reductions. It is important to clarthe differences in the regions where sinapic andulic acid are incorporatedin differentiating xylem inorder to elucidate the enzymatic systems responsfor modifying the aromatic ring and reducing the sichain.In this study, we prepared different stage of cotinuous sections of newly formed xylem from robinfed labeled ferulic and sinapic acids, to reveal wherthese compounds are incorporated into S ligninthe differentiating xylem. We estimated the distrution of the labeled lignin by using the derivatiztion followed by reductive cleavage method (DFRCfollowed by gas chromatography–mass spectrom(GC-MS). The difference in thein situ incorporationof the labeled precursors is discussed in the regulaof sinapyl alcohol biosynthesis in the differentiatixylem.2. Material and methods2.1. Synthesis of labeled precursors[8-13C, 3-OCD3]-ferulic acid and [8-13C, 3,5-OCD3]-sinapic acid (Fig. 1) were synthesized as dscribed previously[9,15].2.2. Plant material and administration of precursorsThe upper parts of 2-year-old shoots of robin(Robinia pseudoacacia L.) trees that were growing othe campus of the Nagoya University were cutin July 2000. A small depression was made atK. Yamauchi, K. Fukushima / C. R. Biologies 327 (2004) 791–797 793mlis-ed inec-embyledds.er-sec-C-o-t:ec-l).ndicg)asedlo-o-oustedoreaneFig. 1. Precursors labeled with stable isotopes. (A) Ferulicacid-[8-13C, 3-OCD3]. (B) Sinapic acid-[8-13C, 3, 5-OCD3].top of the remaining stem and was filled with 30of a 2-mM aqueous solution of each precursor dsolved in a phosphate buffer (pH. 7.01, 25◦C). After9 days, each shoot was harvested and was soak80% ethanol. And then 50-µm-thick tangential stions were cut from the surface of the bark-free xylwith a sliding microtome. All section was treatedethanol-benzene (1:2) for 8 h to remove the labeprecursor and low molecular phenolic compounThe analyses of lignin by the DFRC method were pformed using these sections, as shown inF g. 2 anddescribed previously[15].2.3. Analysis of ligninTo degrade and detect the labeled lignin, eachtion (approximately 1 mg) was applied to the DFRmethod[16] followed by GC-MS, with some modification as followed; AcBr stock solution: acetyl brmide/acetic acid (1:4, v/v); Acidic reduction solvendioxane/acetic acid/water (5:4:1, v/v/v). To wood stion was added the AcBr stock solution (0.5 mThe mixture was gently stirred at 50◦C for 3 h. Thesolvent was completely removed by blowing dowof N2 gas. The residue was dissolved in the acireduction solvent (0.5 ml). Zinc powder (10 mwas added to a well-stirred solution. Stirring wcontinued for 30 min. To the mixture were adddichloromethane (1 ml), a saturated ammonium chride solution (1 ml), and an internal standard (dcosane in dichloromethane). The pH of the aquephase was adjusted to less than 3 by adding diluHCl. The aqueous phase was extracted twice mwith dichloromethane. The combined dichloromethfractions were dried over anhydrous Na2SO4, andFig. 2. Administration of labeled precursors and sample preparation.794 K. Yamauchi, K. Fukushima / C. R. Biologies 327 (2004) 791–797rgllco-C-aa0 totCon-a-eseionideartheea-ionllra-thells,llch)rsorkseas-svi--ednitseof)evaporated in vacuo. The rsidue was acetylated fo40 min in 1.5 ml of dichloromethane containin0.2 ml of acetic anhydride and 0.2 ml of pyridine. Avolatile components were removed completely byevaporation with ethanol in vacuo.Acetylated DFRC products were analyzed by GMS. Mass spectra were recorded at 70 eV withGCMS-OP2010 (Shimadzu, Kyoto, Japan), withfused silica capillary column (DB-1, 30 m× 0.32 mmi.d.). The sample (1 µl) was injected at 220◦C. Thetemperature was programmed to increase from 15280◦C at 20◦C min−1 and, after 5 min, maintained a280◦C. The carrier gas was helium. Labeled DFRproducts were determined by using selected ion mitoring (SIM), according to the previous method[15].3. Results and discussionFig. 3shows the distribution of the DFRC degradtion products (GDFRC, SDFRC) in the robinia given la-beled ferulic acid. DFRC method only produces thproducts from 8-O-4′ structures in lignin[17]. Com-pared with non-labeled shoots, the lignin compositwas not affected by the administration of ferulic ac(data not shown). The low yields of the sections nthe cambium indicated that these tissues were ininitial stage of lignification. G lignin began to formin the early stage of cell-wall lignification, and thproportion of S lignin increased gradually as lignifiction proceeded. This indicates that lignin compositis clearly regulated by the differentiation of cell wain robinia. Previous studies using microautoradiogphy demonstrated that G lignin was deposited inearly stage of xylem differentiation on vessel wafollowed by the deposition of S lignin on the fiber cewall, in magnolia, lilac, and beech[12,17]. Our resultsfor robinia were similar to theirs.Fig. 4 shows the partial mass spectra of eaDFRC product (GDFRC and SDFRC) in controls (A)and robinia fed with labeled ferulic (B) or sinapic (Cacid. Each figure shows where the labeled precuwas incorporated into lignin. The molecular ion peafor GDFRC and SDFRC monomers are atm/z 264 and294, respectively. In general, aromatic acetatesily lose a ketene group (m 42). Therefore, the masspectra of GDFRC and SDFRC have base peaks (m) atm/z 222 and 252, respectively. As described preously [15], when fed with [8-13C, 3-OCD3]-ferulicacid, mass peaks were detected atm/z 226 (222+ 4;m + 4) in GDFRC and atm/z 256 (252+ 4; m + 4) inSDFRC (Fig. 4B). When fed with [8-13C, 3,5-OCD3]-sinapic acid, a mass peak was detected atm/z 259(252+ 7; m + 7) in SDFRC (Fig. 4C). Examination ofthe areas of them + 4, m + 7, andm peaks on selected ion-monitoring (SIM) chromatograms allowus to estimate the amount of labeled lignin subuin each section.Fig. 5 shows the distribution of thyields of labeled DFRC products in each sectionrobinia fed with labeled ferulic (A) or sinapic (BFig. 3. Distribution of the DFRC products (GDFRC, SDFRC) in the robinia given labeled ferulic acid.K. Yamauchi, K. Fukushima / C. R. Biologies 327 (2004) 791–797 795Fig. 4. Partial mass spectra of each DFRC product in robinia. (A) Control, no feeding; (B) fed with labeled ferulic acid; (C) fed with labeledsinapic acid.00ednacid. When fed with [8-13C, 3-OCD3]-ferulic acid,labeled GDFRC was located 250–350 and 550–7µm from the cambium. The distribution of labelGDFRC increased gradually as cell-wall lignificatioproceeded, and the labeled SDFRC was located 450–600 µm from the cambium. When fed with [8-13C,3,5-OCD3]-sinapic acid, labeled SDFRC was located150–250 and 450–700 µm from the cambium. Little796 K. Yamauchi, K. Fukushima / C. R. Biologies 327 (2004) 791–797(A)(B)Fig. 5. Distribution of the yield of labeled DFRC products. (A) Labeled GDFRC (m + 4) and SDFRC (m + 4) in robinia fed with labeled ferulicacid. (B) Labeled GDFRC (m + 4) and SDFRC (m + 7) in robinia fed with labeled sinapic acid.nton-ofithLa-intheera-geulicn-rylof the label from sinapic acid was incorporated intoGDFRC in any section.Labeled ferulic acid has begun to incorporate iG lignin in the early stages of cell-wall lignificatioand the incorporations increased as cell-wall lignification proceeded. And the labeled ferulic acid is alsoincorporated into S lignin during the late stagelignification. These localizations corresponded wthe heterogeneous deposition of lignin moieties.beled sinapic acid was incorporated into S ligninthe early and late stages of lignification. However,deposition of S lignin occurred mainly in the latstage of lignification. Thus, the incorporation of lbeled sinapic acid into syringyl lignin in the early stawould be abnormal conversion.These results indicated that the exogenous feracid was incorporated in the normal lignin biosythetic process, including the regulation of conifeK. Yamauchi, K. Fukushima / C. R. Biologies 327 (2004) 791–797 797ge-licod-a-idspyl-ionurr--nlyro-x-cylre-tods16)in-n.-y-)nlz-lpy,llofSA,ar-in,n,ro-r,for-r-of.forion-cad.-sy-l–n-ylondnk01)engupsku-nge97)and sinapyl alcohol biosynthesis, whereas the exonous sinapic acid was not. The conversion of feruacid to sinapyl alcohol goes via the aromatic ring mifying reaction (the hydroxylation and the methyltion) and the reducing reactions (the reduction of acto alcohols). The conversion of sinapic acid to sinaalcohol goes only via the reducing reactions (the reduction of acids to alcohols). Therefore, the regulatof the syringyl lignin biosynthetic process would occin the aromatic ring modifying reaction (the convesion from guaiacyl to syringyl moiety) in the differentiating xylem.4. ConclusionsIn robinia, sinapyl alcohols are generated maiin the later stage in the differentiating xylem. For theregulation of the sinapyl alcohol biosynthesis, the amatic ring-modifying reactions including the hydroylation and methylation (the conversion from guaiato syringyl moiety) are more important than theducing reactions (the reduction from sinapic acidsinapyl alcohol).AcknowledgementThis study was partly supported by Grant-in-Aifor Scientific Research (Nos. 14360097 and 152550from JSPS and for Bio Design Program from the Mistry of Agriculture, Forestry, and Fisheries of JapaReferences[1] T. Higuchi, Lignin biochemistry: biosynthesis and biodegradation, Wood Sci. 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The regulation from guaiacyl to syringyl lignin in the differentiating xylem of <i>Robinia pseudoacacia</i>

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The regulation from guaiacyl to syringyl lignin in the differentiating xylem of <i>Robinia pseudoacacia</i>

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