Proc. Natl. Acad. Sci. USA Vol. 84, pp. 7542-7546, November 1987 Botany Molecular cloning of complementary DNA encoding the lignin-forming peroxidase from tobacco: Molecular analysis and tissue-specific expression L. MARK LAGRIMINI*t, WILLIAM BURKHART, MARY MOYER, AND STEVEN ROTHSTEIN Ciba-Geigy Agricultural Biotechnology Unit, P.O. Box 12257, Research Triangle Park, NC 27705 Communicated by Mary-Dell Chilton, June 29, 1987 ABSTRACT Plant peroxidases play a major role in lignin The moderately anionic peroxidase isozymes (pI, 4.5-6.5) formation and wound healing and are believed to be involved from tobacco are highly expressed in wounded stem tissue in auxin catabolism and defense to pathogen attack. The (6). These isozymes have previously been localized to the cell function of the anionic peroxidase isozymes is best understood walls, and they have a moderate activity toward lignin in tobacco. These isozymes catalyze the formation of the lignin precursors. They may serve a function in wound healing or polymer and form rigid cross-links between lignin, cellulose, suberization by forming a water-tight barrier over the wound and extensin in the secondary plant cell wall. We report the (3). purification of the anionic peroxidase isozymes from tobacco The function of the anionic peroxidase isozymes (pI, and their partial amino acid sequence. An oligonucleotide 3.5-4.0) is understood best. This isozyme group is also cell probe deduced from the amino acid sequence was used to wall associated and has a high activity for the polymerization screen a tobacco leafcDNA library and a 1200-base-pair cDNA of cinnamyl alcohols in vitro (11). They function in lignifica- clone was isolated and sequenced in its entirety. The predicted tion and the cross-linking ofextensin monomers and feruloyl- amino acid sequence revealed a 22-amino acid signal peptide ated polysaccharides (12). Since the formation of the sec- and a 302-amino acid mature protein (Mr, 32,311). The amino ondary cell wall prohibits cell expansion, the activity and acid sequence was compared to that of the cationic peroxidases perhaps the de novo synthesis of the anionic isozymes are from horseradish and turnip and was found to be 52% and 46% likely regulated by auxin levels (13). homologous, respectively. By RNA blot analysis, the messenger We have begun to apply the tools of molecular biology to for the tobacco isozyme was found to be abundant in stem tissue better understand how peroxidases function in growth and while expressed at very low levels in leaf and root tissue. Four development and what role they may play in the plant's distinguishable copies of the gene were found on genomic DNA ability to recover from environmental stress, wounding, and blots. The gene copy number may reflect the allotetraploid infection. Our approach entails the isolation of molecular nature of Nicotiana tabacum. probes to monitor the expression of the various peroxidase isozymes. In this paper, we report the cloning and sequence The peroxidases (donor: hydrogen-peroxide oxidoreductase, of a cDNA encoding one ofthe two anionic peroxidases from EC 1.11.1.7) have been studied extensively in a wide variety A ofhigher plants [for a review, see Gaspar et al. (1)]. They play tobacco. cDNA probe was used to observe the levels of an integral role in secondary cell wall biosynthesis by the expression of the anionic peroxidase genes in several plant polymerization of cinnamyl alcohols into lignin (2) and by tissues. The tobacco genome was also probed to determine forming rigid cross-links between cellulose, pectin, hydroxy- the gene copy number and to characterize the nucleotide proline-rich glycoproteins, and lignin (2). Peroxidases are homology between peroxidase isozyme classes. In addition, also critical in wound healing, in which they in part form a the predicted amino acid sequence of the anionic peroxidase water-tight barrier over the wound by the deposition of of tobacco was compared to that of other plant peroxidases.$ polymeric aliphatic and aromatic compounds (3). Peroxi- dases may also have a role in auxin catabolism (4) and defense MATERIALS AND METHODS of the plant against pathogen attack (5, 6). Most higher plants possess a number of different peroxi- Plant Materials and Growth Conditions. Nicotiana tabac- dase isozymes whose pattern of expression is tissue specific, um (Coker 176), Nicotiana sylvestris, and Nicotiana tomen- developmentally regulated, and influenced by environmental tosiformis plants were grown from seed in the greenhouse factors (6). In the tobacco plant there are at least 12 with 14-hr daily light periods. distinguishable isozymes, which fall into three subgroups; the Anionic Peroxidase Purification and Protein Sequencing. anionic, the moderately anionic, and the cationic. Each group The anionic peroxidase isozymes were purified from tobacco is thought to serve a different function in the cell. leaves as described by Mader (14). Trypsin or cyanogen The cationic peroxidase isozymes (pI, 8.1-11) efficiently bromide cleavage was carried out using standard procedures catalyze the synthesis ofH202 from NADH and H20 (7), and (15). Peptides were isolated by reverse-phase HPLC, and the have recently been localized to the central vacuole (8). These isolated peptides were sequenced on an Applied Biosystems isozymes have also been shown to possess an indoleacetic (Foster City, CA) model 470A gas-phase protein sequencer. acid oxidase activity in the absence of H202 (9). There has NH2-terminal sequence analysis was obtained by first de- been speculation that these isozymes regulate auxin levels, blocking with pyroglutamate amino peptidase (15). form ethylene from 1-aminocyclopropane-1-carboxylic acid (10), and provide H202 for other peroxidase isozymes (7). *To whom reprint requests should be addressed. Their actual function in the plant is as yet unclear. tPresent address: Department of Horticulture, Ohio State Univer- sity, Columbus, OH 43210. MThe sequence reported in this paper is being deposited in the The publication costs of this article were defrayed in part by page charge EMBL/GenBank data base (Bolt, Beranek, and Newman Labora- payment. This article must therefore be hereby marked "advertisement" tories, Cambridge, MA, and Eur. Mol. Biol. Lab., Heidelberg) in accordance with 18 U.S.C. §1734 solely to indicate this fact. (accession no. J02979). 7542 Botany: Lagrimini et al. Proc. Natl. Acad. Sci. USA 84 (1987) 7543 RNA Isolation and cDNA Library Construction. Poly(A)+ A. NH2-terminus QLSATFYDTTCPNVTSIVRGVMD RNA was isolated from either fresh leaf, root, or stem tissue B. Tryptic 1) GVMDQR (16). Leaf tissue (10 g) or pith tissue (50 g) was homogenized Peptides 2) GMDLTDVALSGAHTFG with a Brinkmann Polytron in equal volumes of grinding 3) LGNISPLTGTNGQ buffer (50 mM Tris HCl, pH 8.0/4% sodium p-aminosalicylic acid/1% sodium naphthalene-1,5-disulfonic acid) and water- CNBr 1) MIKLGNISPLTG saturated phenol. After separating the phases by centrifuga- Peptides 2) MIPQFTNKG tion, the aqueous phase was extracted again with phenol/ CHCl3, and then with CHCl3 alone. The aqueous phase was C. Met Ile Pro Gln Phe Thr Asn Lyg Gly then made 0.5 M LiCl/1 mM EDTA/0.1% Na2DodSO4. Up mRNA AUG AUA CCA CAA UUU ACA AAC AAA GGA-3' to 0.5 g of oligo(dT) cellulose was added and shaken for 10 U G G C G U G G min. The slurry was poured into a small glass column and C C C C poly(A)+ RNA was collected (17). Double-stranded cDNA U U U copies were made from leaf RNA as described poly(A)+ by 26 mer TAC TAT GGI GTT AAA TGI TTG TTT CC -5' Lapeyre and Amalric (18), ligated into Xgtll arms, and probe A C G A C packaged in vitro into phage particles (Vector, Burlingame, G CA). Oligonucleotide Screening. Mixed oligonucleotide probes FIG. 1. Protein sequence data and the synthetic oligonucleotide incorporating deoxyinosines were 5' 32P-end-labeled to a probe used in the cDNA cloning. (A) The NH2-terminal sequence of specific activity of 1 x 106 cpm/pmol with [-32P]ATP (>5000 the anionic tobacco peroxidase after deblocking with pyroglutanlate Ci/mmol; 1 Ci = 37 GBq; New England Nuclear) and T4 amino peptidase. (B) Tryptic and CNBr peptide sequences. (C) Partial amino acid sequence of CNBrpeptide fragment 2, followed by polynucleotide kinase (17). This was used to probe nitrocel- the deduced mRNA sequence, and the synthetic oligonucleotide lulose filter replicas ofthe tobacco leaf cDNA library (19, 20). sequence from the noncoding strand used as the probe. Amino acids Subcloning and DNA Sequencing. Restriction fragments are identified by the single-letter code. were isolated from the cDNA insert and were also subcloned into the appropriately digested vector for DNA sequencing. DNA sequencing was carried out by the dideoxy-chain the proteins with pyroglutamate amino peptidase (15), which termination method on alkali-denatured plasmid templates removes pyrrolidone carboxylic acid, the NH2-terminal se- (21, 22). quence was determined (Fig. 1). It should be noted that even DNA and RNA Filter Hybridizations. High molecular though NH2-terminal sequencing was carried out on an weight DNA was isolated from tobacco leaves as described equivalent mixture of two anionic peroxidases, they revealed by Bendich et al. (23). DNA (5 ,&g) was cleaved with various a unique sequence. This is most likely due to the similarity of restriction enzymes and subjected to agarose gel electropho- the two isozymes, although one cannot eliminate the possi- resis. The DNA was then transferred to nitrocellulose (24) bility that only one polypeptide was deblocked. and hybridized with nick-translated cDNA clones (25). The amino acid sequence of CNBr peptide 2 was used to Poly(A)+ RNA (1 ptg) from leaf, root, and stem tissue was deduce the corresponding nucleotide sequence. A DNA subjected to formaldehyde-agarose gel electrophoresis (26), probe consisting of a mixture of 48 oligonucleotides each 26 and transferred to nitrocellulose filters (27).