Proc. Natl. Acad. Sci. USA Vol. 95, pp. 12803–12808, October 1998 Biochemistry NMR characterization of altered lignins extracted from tobacco plants down-regulated for lignification enzymes cinnamyl- alcohol dehydrogenase and cinnamoyl-CoA reductase (monolignolygenetic modificationyantisense RNAyconiferyl alcoholyferuloyl-CoA) JOHN RALPH*†‡,RONALD D. HATFIELD*, JOE¨L PIQUEMAL§,NABILA YAHIAOUI§,MICHEL PEAN¶, i CATHERINE LAPIERRE , AND ALAIN M. BOUDET§ *United States Dairy Forage Research Center, United States Department of Agriculture, Agricultural Research Service, Madison, WI 53706-1108; †Department of Forestry, University of Wisconsin, Madison, WI 53706-1598; §Unite´Mixte de Recherche, Centre National de la Recherche Scientifique 5546, Centre de Biologie et Physiologie Ve´ge´tale, Universite´Paul Sabatier Baˆt.4R1, 118 route de Narbonne, F-31062 Toulouse Cedex, France; ¶Commissariat a`l’Energie Atomique, De´partementd’Ecophysiologie Ve´ge´tale et Microbiologie, Cadarache, Baˆt.177, F-13108 Saint Paul lez Durance Cedex, France; and iInstitut National de la Recherche Agronomique, Laboratoire de Chimie Biologique, F-78850 Thiverval-Grignon, France Communicated by Ron Sederoff, North Carolina State University, Raleigh, NC, July 29, 1998 (received for review May 13, 1998) OHO OS OH OH ABSTRACT Homologous antisense constructs were CoA used to down-regulate tobacco cinnamyl-alcohol dehydro- CCL' CCR' CAD' p-Coumaryl genase (CAD; EC 1.1.1.195) and cinnamoyl-CoA reductase alcohol (CCR; EC 1.2.1.44) activities in the lignin monomer biosyn- OH OH OH OH 1) HYD1' 1) HYD2' 1) HYD3' 1) HYD4' thetic pathway. CCR converts activated cinnamic acids 2) OMT1' 2) OMT2' 2) OMT3' 2) OMT4' OHO OS OH OH (hydroxycinnamoyl–SCoAs) to cinnamaldehydes; cinnama- CoA CCR CAD ldehydes are then reduced to cinnamyl alcohols by CAD. The CCL Coniferyl transformations caused the incorporation of nontraditional X X alcohol Lignin OMe OMe OMe OMe components into the extractable tobacco lignins, as evi- OH OH OH OH 1) HYD1 1) HYD2 1) HYD3 1) HYD4 denced by NMR. Isolated lignin of antisense-CAD tobacco 2) OMT1 2) OMT2 2) OMT3 2) OMT4 OHO OS OH OH contained fewer coniferyl and sinapyl alcohol-derived units CoA Sinapyl that were compensated for by elevated levels of benzalde- CCL" CCR" CAD" alcohol hydes and cinnamaldehydes. Products from radical coupling MeO OMe MeO OMe MeO OMe MeO OMe of cinnamaldehydes, particularly sinapaldehyde, which were OH OH OH OH barely discernible in normal tobacco, were major compo- nents of the antisense-CAD tobacco lignin. Lignin content FIG. 1. Lignin biosynthetic pathway, simplified schematic. Not all was reduced in antisense-CCR tobacco, which displayed a of the pathways or enzymes are necessarily known or distinct. CAD is a generic term (cinnamyl-alcohol dehydrogenase) for the enzyme class markedly reduced vigor. That lignin contained fewer co- that catalyzes the final reduction to hydroxycinnamyl alcohols—CAD9 niferyl alcohol-derived units and significant levels of tyra- may or may not be different from CAD and CAD0. Similarly for CCR mine ferulate. Tyramine ferulate is a sink for the anticipated (cinnamoyl-CoA reductase). build-up of feruloyl–SCoA, and may be up-regulated in response to a deficit of coniferyl alcohol. Although it is not units. The structural observations begin to reveal the pro- yet clear whether the modified lignins are true structural cesses by which substantial lignin levels are maintained in components of the cell wall, the findings provide further some plants with severely depressed activity of lignifica- indications of the metabolic plasticity of plant lignification. tion enzymes. The CAD-deficient mutant appeared able to An ability to produce lignin from alternative monomers utilize other phenols as substrates for radical coupling would open new avenues for manipulation of lignin by genetic biotechnologies. reactions to produce polymers that may function similarly to normal lignin (15, 16). Whether this altered lignin is a true structural component of the cell wall remains to be deter- Lignins are phenolic polymers essential for mechanical sup- mined. port, defense, and water transport in vascular terrestrial plants To explore other plants with enzyme down-regulation, we (1–3), but they are a major obstacle to efficient utilization of plants for paper making or animal feed. A recent approach have selected tobacco plants transformed by CAD and cin- toward improved utilization has been the down-regulation of namoyl-CoA reductase (CCR; EC 1.2.1.44) antisense con- enzymes involved in the lignin monomer biosynthetic pathway structs. Tobacco is an excellent model plant with comprehen- (Fig. 1). Antisense technologies allow selective targeting of sive data on the genes of the lignification pathway; some data single enzymes in the pathway and transgenic plants have been have been published on lignins from CCR (8) and CAD obtained with altered lignin content andyor lignin structure down-regulated tobacco plants (9, 12, 13, 17). For this study, (4–14). uniformly 13C-enriched lignins were isolated from down- Recently, a lignin isolated from a pine mutant deficient in regulated CAD and CCR tobacco plants and compared with cinnamyl-alcohol dehydrogenase (CAD; EC 1.1.1.195) was control plants through the application of NMR techniques to characterized (15). This lignin had significantly lower co- niferyl-alcohol-derived units, compensated for by elevated Abbreviations: CAD, cinnamyl-alcohol dehydrogenase; CCR, cin- aldehyde levels, and major levels of dihydroconiferyl alcohol namoyl-CoA reductase; HMQC, heteronuclear multiple quantum coherence; HSQC, heteronuclear single quantum coherence; TOCSY, The publication costs of this article were defrayed in part by page charge total correlation spectroscopy; HMBC, heteronuclear multiple bond correlation; G, guaiacyl; S, syringyl. payment. This article must therefore be hereby marked ‘‘advertisement’’ in A Commentary on this article begins on page 12742. accordance with 18 U.S.C. §1734 solely to indicate this fact. ‡To whom reprint requests should be addressed at: U.S. Dairy Forage © 1998 by The National Academy of Sciences 0027-8424y98y9512803-6$2.00y0 Research Center, USDA-ARS, 1925 Linden Drive West, Madison, PNAS is available online at www.pnas.org. WI 53706-1108. e-mail: [email protected]. 12803 Downloaded by guest on September 27, 2021 12804 Biochemistry: Ralph et al. Proc. Natl. Acad. Sci. USA 95 (1998) examine how plants respond to the down-regulation of specific iments, the lignins were extracted with methylene chloride to enzymes. remove possible low molecular weight components. This wash- ing caused neither substantial material loss nor the reduction MATERIALS AND METHODS of the aldehyde or tyramine ferulate signals, which were therefore included in the polymer. Acetylation of small Transgenic Tobacco Plants (Nicotiana tabacum L. Cv. Sam- amounts of each of the lignins was by means of acetic anhy- sun). Antisense CAD plants. We used seeds resulting from dride and pyridine. The acetylated lignins were extracted into self-pollination of primary transformant T37 (17) carrying a freshly distilled ethyl acetate and washed with aqueous EDTA 1-kb CAD cDNA in antisense orientation, associated to the to remove trace metal contaminants prior to NMR study. 35S cauliflower mosaic virus (CaMV) promoter and the 39 NMR. NMR experiments were performed at 360 MHz on a terminator of the nopaline synthase. Bruker AMX-360 using a narrow-bore probe with conven- Antisense CCR plants. We used seeds resulting from a test tional coil geometry and without gradients, or at 750 MHz on cross on primary transformant B3 (8) carrying a 1.3-kb CCR a Bruker DMX-750 using a narrow-bore (5 mm) triple reso- cDNA in antisense orientation, associated to the 35S CaMV nance probe (1H, 13C, 15N; the 15N was not used) optimized for promoter and the 39 terminator of the nopaline synthase. inverse experiments and with three-axis gradients. Experi- Both CAD and CCR antisense constructs also carried the ments used were standard Bruker implementations of tradi- neomycin phosphotransferase gene, which confers resistance tional or gradient-selected versions of inverse (1H-detected) to kanamycin. heteronuclear multiple quantum coherence (HMQC), hetero- Growth and Labeling Conditions. Seeds were sterilized for nuclear single quantum coherence (HSQC), HMQC-total 5 min in 10% sodium hypochlorite and rinsed in sterile water. correlation spectroscopy (TOCSY), HSQC-TOCSY, and het- After 15 days in vitro [lightydark regime of 16 h, 20–30 eronuclear multiple bond correlation (HMBC) experiments. mEzm22zsec21, 27°C; 1 E (einstein) 5 1 mol of photons] on a TOCSY experiments used a 100-ms spin lock period; HMBC solid MS (Murashige and Skoog) medium supplemented with experiments used a 100-ms long-range coupling delay. kanamycin (500 mgyliter) to select the seedlings harboring the transgenes, resistant plantlets were transferred to vermiculite RESULTS AND DISCUSSION and grown in a culture room (same culture conditions) for 26 days. Six-week-old plants (10 expanded leaves, stem height Definition of Isolated Tobacco Lignin. All following com- 7–10 cm) were transferred to Cadarache for labeling in a C23A ments refer to an extracted ‘‘lignin’’ that is not necessarily chamber (18). This strictly closed module (750 liters) derived representative of lignin in situ. Indeed, it has been suggested from glove boxes used for nuclear manipulation, allows accu- (49) that the isolated lignins [in particular from the mutant rate regulation