- Email: [email protected]
Isolation and characterisation of three cinnamyl alcohol dehydrogenase homologue cDNAs from perennial ryegrass (Lolium perenne L.)
J. Plant Physiol. 159. 653 – 660 (2002) Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp
Isolation and characterisation of three cinnamyl alcohol dehydrogenase homologue cDNAs from perennial ryegrass (Lolium perenne L.) Damian Lynch, Angela Lidgett, Russell McInnes, Helen Huxley, Elizabeth Jones, Natalia Mahoney, German Spangenberg* Plant Biotechnology Centre, Agriculture Victoria, Department of Natural Resources and Environment, and CRC for Molecular Plant Breeding, La Trobe University, Bundoora, Victoria 3086, Australia Received December 18, 2001 · Accepted February 12, 2002
Summary Three cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) homologue cDNAs (LpCAD1, LpCAD2 and LpCAD3) were isolated and characterised in perennial ryegrass (Lolium perenne). Sequence analysis revealed a partial LpCAD3 clone is highly homologous to typical monocot CAD cDNAs while full-length LpCAD1 and LpCAD2 cDNAs show lower homology to known CAD cDNAs. Northern hybridisation analysis revealed LpCAD1 and LpCAD3 transcripts in lignifying tissues while LpCAD2 was uniquely abundant in stem tissue. All three CAD cDNAs are upregulated in response to mechanical wounding stimulus. Southern hybridisation analysis suggested that LpCAD3 is a single copy gene within the Lolium genome while LpCAD1 and LpCAD2 each belong to a small gene family. Key words: CAD – cDNA – cinnamyl alcohol dehydrogenase – lignin – Lolium perenne – perennial ryegrass Abbreviations: ADH = alcohol dehydrogenase. – CAD = cinnamyl alcohol dehydrogenase. – CCR = cinnamoyl-CoA reductase. – cv. = cultivar. – DH = doubled haploid. – DIG = digoxigenin. – LG = linkage group. – LOD = log of odds. – MTD = Mannitol Dehydrogenase. – OMT = O-methyltransferase. – ORF = open reading frame. – RFLP = restriction fragment length polymorphism. – SAD = sinapyl alcohol dehydrogenase. – UTR = untranslated region
Introduction Lignin is an aromatic polymer deposited during the secondary thickening of plant cell walls. The role of lignin in vascular plants is to contribute to structural support, solute transport and plant defence (Iiyama et al. 1993). In grasses, the biosynthesis of monolignols is a multi-step process beginning with phenylalanine and tyrosine (Iiyama et al. 1993, Whetten 1998). These precursors are deaminated and hydroxylated in * E-mail corresponding author: [email protected]
the general phenylpropanoid biosynthetic pathway before entering the monolignol-specific branch [for reviews see Boudet (2000) and references therein]. The two reductive steps of the monolignol-specific pathway are catalysed by cinnamoylCoA reductase (CCR, EC 1.2.1.44) and cinnamyl alcohol dehydrogenase (CAD). Governing the last committed step of the pathway, CAD converts the hydroxycinnamaldehydes to their corresponding cinnamyl alcohols (monolignols). In angiosperms the three major monolignols are p-coumaryl, coniferyl and syringyl alcohols which are polymerised at the cell wall interface to form lignin. The varying combination of 0176-1617/02/159/06-653 $ 15.00/0
654
Damian Lynch et al.
monolignols incorporated into lignin is largely responsible for the heterogeneity displayed between plant species, tissues, developmental stage and sub-cellular location (Lewis and Yamamoto 1990). Typical CADs have been isolated from gymnosperms and angiosperms that display high sequence identity in key catalytic residues, however atypical CADs isolated exhibit sequence divergence (Brill et al. 1999). Being a final biosynthetic step, CAD may represent an ideal target to control lignin content and composition (Grima-Pettenati and Goffner 1999). Several observations suggest it is possible to alter lignin content and composition by manipulating CAD activity in planta. Decreased CAD activity has been implicated in the bm mutants of maize and sorghum and the cad n-1 mutant of loblolly pine (Pillonel et al. 1991, Mackay et al. 1997, Halpin et al. 1998), while CAD transgenic tobacco, poplar and lucerne (Halpin et al. 1994, Baucher et al. 1996, 1999) have displayed altered lignin profiles. Similar mutants or studies in forage or turf grasses have not been reported. The functional assessment of CAD function in planta via sense and antisense technology is dependent upon the availability of cDNA or gene sequences. We report the isolation and characterisation of three diverse CAD homologue cDNAs from perennial ryegrass as an initial step towards the molecular genetic dissection of the monolignol biosynthetic pathway.
Materials and Methods
seedlings (Heath et al. 1998). The library was screened with [32P] dCTP-labelled PCR generated probes. Excised and cloned cDNA inserts were obtained using the ExAssist helper phage with SOLR strain (Stratagene) as described by the manufacturer.
DNA sequence analysis High quality plasmid DNA (Maxi Kit, Qiagen) was extracted and sequenced by the dideoxy chain termination method (BigDyeTM version 2.0, ABI) on an ABI 373 automated sequencer. Protein and DNA comparisons were made using BLAST and GCG applications available through ANGIS (Australian National Genomic Informatiom Service). Phylogenetic analysis was performed using BioNavigator (http:// www.entigen.com).
Genomic Southern hybridisation analysis Perennial ryegrass genomic DNA of doubled haploid genotype DH297 (10 µg) was digested with DraI, BamHI, EcoRI, EcoRV, HindIII and XbaI, electrophoresed on a 1% agarose gel, capillary blotted and UV-fixed to a nylon membrane (Hybond-N, Amersham). Membranes were hybridised with DIG labelled full-length LpCAD1 (EcoRI/ApaI), LpCAD2 (EcoRI/XhoI) probes and a 730 bp LpCAD3 (EcoRI/ApaI) probe (Fig. 1). Hybridisation conditions were: 4 × SSC, 50 % formamide, 0.1 % N-Lauroyl-sarcosine, 0.02 % SDS, and 2 % blocking solution at 42 ˚C. Membranes were washed under high stringency: twice in 2 × SSC/0.1 % SDS for five minutes at room temperature, fifteen minutes in 0.2 × SSC/0.1 % SDS at 68 ˚C, then fifteen minutes in 0.1 × SSC/0.1% SDS at 68 ˚C.
Plant material Seeds of perennial ryegrass (Lolium perenne L.) cv. Ellet, tall fescue (Festuca arundinacea Schreb.) cv. Triumph and phalaris (Phalaris aquatica L.) cv. Holdfast were surface sterilised and germinated as previously described (Heath et al. 1998). Wounding experiments were performed with 10-day-old perennial ryegrass plants (cv. Ellet). Wounding was achieved by applying five compressions between 40grit sandpaper. Leaves and stems were harvested at 0, 6, 12, 24 and 48 hours post-wounding. The developmental expression of cDNAs was investigated using roots (3 – 5 day, 7–10 day, 6-week, 10-week), shoots (3 – 5 day, 7–10 day) seedlings (7–10 day), leaves (6-week, 10week) and stems (6-week, 10-week).
Genetic mapping using RFLPs RFLPs were detected with PCR-amplified [32P]-labelled probes of two distinct perennial ryegrass CAD homologues, LpCAD1 and LpCAD2, and the perennial ryegrass O-methyltransferase (OMT, EC 2.1.1.6) homologue, LpOMT1 (Heath et al. 1998), as previously described (Jones et al. 2002). RFLPs were mapped using 110 progeny individuals of the p150/112 perennial ryegrass reference population restricted with EcoRV (LpCAD1), EcoRI (LpCAD2) and DraI (LpOMT1). Genetic mapping data for p150/112 was obtained from the public database FoggDB (http://ukcrop.net/perl/ace/search/FoggDB) and full-length cDNAs LpCAD1, LpCAD2 and LpOMT1 were mapped within this marker framework using MAPMAKER 3.0 (Lander 1987).
Isolation of CAD sequences for cDNA library screening Oligonucleotide primers were designed to amino acid CAD domains (motifs CAGVTVYS and DVRYRFV) conserved between pine (AAB38774), lucerne (AAC35845), Arabidopsis (AAA99511), Aralia cordata, Eucalyptus botryoides and maize to create a generic hybridisation probe (HPCAD1) via PCR. A second specific hybridisation probe (HPCAD2) was generated by designing primers to a previously identified perennial ryegrass CAD (GenBank accession AF010290). Sequences encoding CAD were amplified from cDNA prepared from perennial ryegrass seedling total RNA and were cloned into pBluescript (Stratagene).
cDNA library screening A Uni-ZAPTM XR cDNA library (ZAP-cDNA Synthesis Kit, Stratagene) was constructed from whole 3 – 5-day-old etiolated perennial ryegrass
Northern hybridisation analysis Total RNA was isolated using Trizol (GibcoBRL) and 15 µg was electrophoresed on a 1.2 % agarose gel containing 6 % formamide, capillary blotted and UV-fixed to a nylon membrane (Hybond-N, Amersham). In order to verify equal loading of RNA, gels and membranes were either stained with ethidium bromide or 0.2 % methylene blue/ 0.3 mol/L sodium acetate respectively. 150 ng of [32P] dCTP labelled LpCAD1, LpCAD2 and LpCAD3 probes were used. Hybridisation conditions were: 4 × SSC, 50 % formamide, 0.5 % SDS, 5 × Denhardt’s solution, 5 % dextrane sulphate, 0.1 % herring sperm DNA, at 42 ˚C over-night. Membranes were washed under high stringency: three times in 2 × SSC/0.1 % SDS for ten minutes at 42 ˚C, three times in 0.2 × SSC/0.1 % SDS for ten minutes at 65 ˚C, and twice in 0.1 × SSC/ 0.1% SDS for ten minutes at 65 ˚C.
Cinnamyl alcohol dehydrogenase homologue cDNAs from Lolium
Results
655
216 bp 3′ UTR and a single 1086 bp ORF that encodes a protein of 361 amino acids (Fig. 1).
Isolation of perennial ryegrass CAD cDNA sequences Three CAD homologue cDNAs were isolated from a cDNA library constructed from RNA of 3 – 5-day-old etiolated perennial ryegrass seedlings (Heath et al. 1998). Two novel fulllength CAD cDNAs, LpCAD1 and LpCAD2, were isolated using a generic cDNA library hybridisation probe (HPCAD1). A third partial cDNA, LpCAD3, was isolated using a probe (HPCAD2) designed specifically to a known perennial ryegrass CAD sequence (AF010290) that was deposited in GenBank during the course of this study. Of the cDNA clones isolated, LpCAD1 is a 1325 bp cDNA with a 21 bp 5′ UTR, 74 bp 3′ UTR and a single 1230 bp ORF that encodes a protein of 409 amino acids. The coding region of LpCAD2 is a 1378 bp cDNA with a 101 bp 5′ UTR, 164 bp 3′ UTR and a single 1113 bp ORF that encodes a protein of 370 amino acids. LpCAD3 is a 780 bp fragment that is representative of a full-length 1382 bp cDNA with a 80 bp 5′ UTR, a
Analysis of LpCAD cDNA sequences Nucleotide sequence comparisons revealed that LpCAD1, LpCAD2 and LpCAD3 full-length cDNAs are only 56 % identical. Amino acid identity alignments (Table 1) showed that LpCAD3 is highly homologous to monocot CADs (99 – 85 %) with lower homology to dicot CADs. Both LpCAD1 and LpCAD2 show a lower degree of homology to CAD sequences (60 – 38 %). Phylogenetic analysis revealed LpCAD3 clustering with tall fescue, maize and sugarcane CADs while LpCAD1 and LpCAD2 group with atypical CADs from lucerne, Arabidopsis and Stylosanthes humilis (Fig. 2). Deduced amino acid sequence of perennial ryegrass CADs revealed functional protein domains retained amongst all CAD sequences, the Zn-1 and Zn-2 domains for catalytic and structural zinc ion binding and the NADPH binding do-
Figure 1. Partial restriction maps of CAD homologue cDNAs. LpCAD1 and LpCAD2 are full-length cDNAs while LpCAD3 is a partial cDNA clone identical to a full-length cDNA (AF010290). Full-length cDNAs were used as Southern and northern hybridisation probes.
Table 1. Amino acid identity (%) between selected plant cinnamyl alcohol dehydrogenases. LpCAD3 shows high identity to typical monocot CAD enzymes from tall fescue, maize and sugarcane, while LpCAD1 and LpCAD2 show lower homology. Details of each CAD sequences are given in Figure 2.
656
Damian Lynch et al. main (Fig. 3). The deduced protein sequences for LpCAD2 and LpCAD3 (370 and 361 amino acids, respectively) are similar to maize and sugarcane CADs (367 and 365 amino acids, respectively) while LpCAD1 is significantly larger, 409 amino acids.
Gene organisation and mapping Southern hybridisation analysis of DH perennial ryegrass genomic DNA using full length LpCAD1 and LpCAD2 cDNA probes revealed complex hybridisation patterns, suggesting that both are members of separate small gene families (Fig. 4). In contrast, LpCAD3 appeared to be a single copy gene in the perennial ryegrass genome. Both LpCAD1 and LpCAD2 were mapped within the framework of the perennial ryegrass p150/112 genetic linkage map as RFLPs. One LpCAD1 locus was polymorphic and mapped to perennial ryegrass LG2, while two LpCAD2 loci were polymorphic and mapped to LG2 and LG7 (Fig. 5). The LpCAD2 on LG7 mapped to the same overlapping region as LpCCR1 (McInnes et al. 2002) and LpOMT1 (this study).
Developmental and wound-induced expression of LpCADs
Figure 2. A phylogenetic tree constructed from amino acid sequences of plant CADs rooted against HADH (Horse Liver ADH, AAA30931). Amino acid sequences used for analysis and their respective accession numbers were: AcCAD (Aralia cordata, BAA03099), AgMTD (Apium graveolans, AAC15467), AtCAD (Arabidopsis thaliana AAA99511), AtEli3–1 (CAA48027), AtEli3 – 2 (CAA48026), AtCAD1 (AAK43875), AtCAD2 (AAK44076), AtCAD3 (AAL11561), AtCAD4 (AAK59426), EbCAD (Eucalyptus botryoides, BAA04046), EgCAD (E. gunni, CAA46585), EgbCAD (E. globulus, AAC07987), EgCAD1– 5 (CAA61275), FaCAD (Festuca arundinacea, AAK97809), FrCAD (Fragaria × ananassa, AAD10327), LpCAD1 (Lolium perenne, AF472591), LpCAD2 (AF472592), LpCAD3 (AAB 70908), McEli3 (Mesembryanthenum crystallium, AAB38503), MdCAD (Malus domestica, AAC06319), MsCAD1 (Medicago sativa, (AAC35846), MsCAD2 (AAC35845), NtCAD14 (Nicotiana tabacum, CAA44216), NtCAD19 (CAA44217), PaCAD (Picea abies, CAA51226), PcEli3 (Petroselinum crispum, CAA48028), PrCAD (Pinus radiata, AAB38774), PtCAD (Pinus taeda, CAA86072), PoptCAD (Populus tremuloides, AAF43140), PoptSAD (AAK58693), ShCAD1 (Stylosanthes humilis, AAA74882), ShCAD2 (AAA74883), SoCAD (Saccharum officinarum, CAA13177), ZeCAD (Zinnia elegans, BAA19487), ZmCAD (Zea mays, CAA06 687). Amino acid alignments were bootstrapped with Seqboot, subjected to maximum parsimony analysis using Protpars and a consensus tree created through Consense (Felenstein 1989). Fork numbers indicate bootstrap values where the number of times branch position to the right occurred out of 100 trees.
Northern hybridisation analysis of LpCAD1, LpCAD2 and LpCAD3 revealed three unique expression profiles, the level of expression varying with maturity and between organs (Fig. 6 a). The expression of LpCAD1 exhibited a constitutive profile with transcript particularly abundant in roots and mature stem. Similarly, LpCAD3 was highly expressed at all stages of root development and in mature stem tissue, but only lowly expressed in shoots and leaves. In contrast, LpCAD2 transcript was not detected in roots but accumulated heavily in mature shoot and especially stem tissue. Interspecific hybridisation was detected for LpCAD1 in phalaris and tall fescue but only in tall fescue for LpCAD2 and LpCAD3. Hybridisation analysis also revealed that LpCAD1, LpCAD2 and LpCAD3 were similarly upregulated in response to a mechanical wounding stimulus (Fig. 6 b). In all cases, transcript abundance increased in ten day old shoots within six hours of wounding. Levels of expression returned to normal between twenty four to forty eight hours post-wounding.
Discussion We report here for the first time, the isolation and analysis of three divergent CAD homologue cDNAs from perennial ryegrass. Expression analysis, protein and nucleotide alignments and phylogenetic comparisons disclose the divergent nature of these three CADs. LpCAD3 corresponds to a full-length cDNA (AF010290) that encodes a protein belonging to a conserved group of monocot CADs. Maize and sugarcane CADs have been implicated in conventional lignification (Halpin et al. 1998, Selman-Housein et al. 1999), suggesting that LpCAD3 may have a role in structural lignification class CAD genes. In contrast,
Cinnamyl alcohol dehydrogenase homologue cDNAs from Lolium
657
Figure 3. Amino acid sequence alignment of plant CADs. Shaded areas indicate conserved residues. Key functional domains highlighted are: the Zn-1 catalytic zinc binding domain (- - - -) and associated invariant residues (䊉), the Zn-2 structural zinc binding domain (*), the NADPH binding domain domain ( + + + ), residues for coenzyme binding specificity (䉬), and residues for active site specificity (bold and underlined) (Jornvall et al. 1987, McKie et al. 1993).
both LpCAD1 and LpCAD2 appear to be divergent defence related CAD cDNAs on the basis of their sequence and phylogeny. Although they retain critical amino acid domains implicated in CAD function, they display significant sequence divergence in residues implicated in substrate specificity (McKie et al. 1993, Lauvergeat et al. 1995). When compared with other known CAD enzymes, LpCAD1 and LpCAD2 group more closely to other atypical CADs that have been implicated in defence lignification or other biosynthetic roles (eg: Eli3s, SAD) (Kiedrowski et al. 1992, Williamson et al. 1995, Somssich et al. 1996, Logemann et al. 1997, Brill et al. 1999, Li et al. 2001). Furthermore, LpCAD1 exhibits an atypical 38 amino acid carboxy terminus extension. This long tail
domain contains no conventional targeting signal (Barpeled et al. 1996, Trelease et al. 1996). Southern hybridisation analysis and mapping data suggests the CADs represent three separate genes located within the ryegrass genome. Of interest is the location of three key lignification genes (LpOMT1, LpCCR1 and LpCAD2) in close proximity on perennial ryegrass LG7. A gene cluster of this nature could be indicative of a coordinated regulation of this complex pathway (Douglas 1996). Functional divergence of the perennial ryegrass CADs is supported by the different expression patterns. While all the CADs display a similar up-regulation in response to mechanical wounding, their developmental expression profiles sug-
658
Damian Lynch et al.
Figure 4. Southern hybridisation analysis of perennial ryegrass genomic DNA. DH DNA (10 µg) was digested with DraI (D), BamHI (B), EcoRI (EI), EcoRV (EV), HindIII (H), XbaI (X), none of which cut within the cDNAs, and the resulting membrane probed with a DIG-labelled LpCAD1, LpCAD2 and LpCAD3 cDNAs.
Figure 5. Map locations of LpCAD1 and LpCAD2 on the perennial ryegrass reference map. The most likely positions of CADs are indicated by arrows, with the adjacent bar indicating the span of possible map locations at LOD > 2.0. Map positions of LpOMT1, LpCCR1 and LpCAD2 in the same overlapping region are indicated on LG7.
Cinnamyl alcohol dehydrogenase homologue cDNAs from Lolium
659
Figure 6. Northern hybridisation analysis of CAD gene expression in perennial ryegrass. Membranes were probed with radiolabelled LpCAD1, LpCAD2 and LpCAD3 cDNAs. A. Roots (R1) from seedlings 3 – 5 days, roots (R2) from seedlings 7–10 days, roots (R3) from 6-week-old plants, and roots (R4) from 10-week-old plants. Shoots (Sh1) from seedlings 3 – 5 days, shoots (Sh2) from seedlings 7–10 days, leaves (L1) from 6-weekold plants, and leaves (L2) from 10-week-old plants. Stem (St1) from 6-week-old plants, stem (St2) from 10-week-old plants, whole plant seedling (P) of 11-day-old phalaris, and whole plant seedling (F) of 7-day-old tall fescue. B. Wounded and unwounded control ten day old shoots harvested at 0, 6, 12, 24, 48 hours post-wounding.
gest complex transcriptional regulation. LpCAD1 and LpCAD3 were expressed in most plant organs and were particularly abundant in roots, while LpCAD2 displayed transcript abundance in mature stem tissues. Tightly regulated expression, such as that displayed by LpCAD2, highlights
avenues for discovering novel promoter elements to facilitate specific directed gene expression studies. Studies in transgenic tobacco, poplar, and lucerne (Halpin et al. 1994, Baucher et al. 1996, 1999) have shown by manipulating CAD activity, via sense and antisense technologies,
660
Damian Lynch et al.
alterations in cell wall and lignification profiles are possible. As yet, no transgenic studies have investigated the possible role of the defence related or atypical CAD genes in lignification. Moreover, none of these approaches have accounted for the functional redundancy displayed for CAD in angiosperms (Whetten et al. 1998). The isolation and analysis of three distinctive CAD homologue cDNAs provides suitable targets for the molecular genetic dissection of CAD function in planta. This work will enhance our understanding of lignin biosynthesis, monomeric composition and properties in grasses and facilitate the production of pasture grasses with enhanced herbage quality and turf grasses with improved resilience. Acknowledgements. This work was supported by the Australian CRC for Molecular Plant Breeding, the Australian Dairy Research and Development Corporation and the Department of Natural Resources and Environment, Victoria, Australia.
References Barpeled M, Bassham D, Raikhel N (1996) Transport of proteins in eukaryotic cells: more questions ahead. Plant Mol Biol 32: 223 – 249 Baucher M, Chabbert B, Pilate G, Van Doorsselaere J, Tollier M, Petitconil M, Cornu D, Monties B, Van Montagu M, Inze D, Jouanin L, Boerjan W (1996) Red xylem and higher lignin extractability by down-regulating a cinnamyl alcohol dehydrogenase in poplar. Plant Physiol 112: 1479–1490 Baucher M, Bernard-Vailhe M, Chabbert B, Besle J, Opsomer C, Van Montagu M, Botterman J (1999) Down-regulation of cinnamyl alcohol dehydrogenase in transgenic alfalfa (Medicago sativa L.) and the effect on lignin composition and digestibility. Plant Mol Biol 39: 437– 447 Boudet AM (2000) Lignins and lignification: Selected issues. Plant Physiol Biochem 38: 81– 96 Brill E, Abrahams S, Hayes C, Jenkins C, Watson J (1999) Molecular characterisation and expression of a wound-inducible cDNA encoding a novel cinnamyl-alcohol dehydrogenase enzyme in lucerne (Medicago sativa L.). Plant Mol Biol 41: 279 – 291 Douglas C (1996) Phenylpropanoid metabolism and lignin biosynthesis: from weeds to trees. Trends Plant Sci 1: 171–177 Felenstein J (1989) PHYLIP – Phylogeny Interference Package (Version 3.2). Cladistics 5: 164–166 Grima-Pettenati J, Goffner D (1999) Lignin genetic engineering revisited. Plant Sci 145: 51– 65 Halpin C, Knight M, Foxon G, Campbell M, Chabbert B, Tollier M, Schuch W (1994) Manipulation of lignin quality by downregulation of cinnamyl alcohol dehydrogenase. Plant J 6: 339 – 350 Halpin C, Holt K, Chojecki J, Oliver D, Chabbert B, Monties B, Edwards K, Barakate A, Foxon GA (1998) Brown-midrib maize (bm1) – a mutation affecting the cinnamyl alcohol dehydrogenase gene. Plant J 14: 545 – 553 Heath R, Huxley H, Stone B, Spangenberg G (1998) cDNA cloning and differential expression of three caffeic acid O-methyltransferase homologues from perennial ryegrass (Lolium perenne). J Plant Physiol 153: 649 – 657 Iiyama K, Lam T, Meikle P, Ng K, Rhodes D, Stone B (1993) Cell wall biosynthesis and its regulation. In: Jung H, Buxton D, Hatfield R, Ralph J (eds) Forage Cell Wall Structure and Digestibility. American Society of Agronomy, Madison, WI, pp 621– 683
Jones E, Mahoney N, Hayward M, Armstead I, Jones J, Humphreys M, Kishida T, Yamada T, Balfourier F, Charmet G, Forster J (2002) An enhanced molecular marker-based genetic map of perennial ryegrass (Lolium perenne L.) reveals comparative relationships with other Poaceae genomes. Genome 45: 282 – 295 Jornvall H, Persson B, Jeffery J (1987) Characteristics of alcohol/polyol dehydrogenases. The zinc-containing long-chain alcohol dehydrogenases. Eur J Biochem 167: 195 – 201 Kiedrowski S, Kawalleck P, Hahlbrock K, Somssich I, Dangl J (1992) Rapid activation of a novel plant defense gene is strictly dependent on the Arabidopsis RPM1 disease resistance locus. EMBO J 11: 4677– 4684 Lander E, Green P, Abrahamson J, Barlow A, Daly MJ, Lincoln SE, Newburg L (1987) MAPMAKER: an interactive computer package for constructing primary linkage maps of experimental and natural populations. Genomics 1: 174–181 Lauvergeat V, Kennedy K, Feuillet C, McKie JH, Gorrichon L, Baltas M, Boudet AM, Grima-Pettenati J, Douglas KT (1995) Site-directed mutagenesis of a serine residue in cinnamyl alcohol dehydrogenase, a plant NADPH-dependent dehydrogenase, affects the specificity for the coenzyme. Biochemistry 34: 12426–12434 Lewis N, Yamamoto E (1990) Lignin: occurrence, biogenesis and biodegradation. Annu Rev Plant Phys 41: 455 – 496 Li L, Cheng X, Leshkevich J, Umezawa T, Harding S, Chiang V (2001) The last step of syringyl monolignol biosynthesis in angiosperms is regulated by a novel gene encoding sinapyl alcohol dehydrogenase. Plant Cell 13: 1567–1585 Logemann E, Reinold S, Somssich IE, Hahlbrock K (1997) A novel type of pathogen defense-related cinnamyl alcohol dehydrogenase. Biol Chem 378: 909 – 913 Mackay JJ, Omalley DM, Presnell T, Booker FL, Campbell MM, Whetten RW, Sederoff RR (1997) Inheritance, gene expression, and lignin characterization in a mutant pine deficient in cinnamyl alcohol dehydrogenase. Proc Natl Acad Sci USA 94: 8255 – 8260 McInnes R, Lidgett A, Lynch D, Huxley H, Jones E, Mahoney N, Spangenberg G (2002) Isolation and characterisation of a cinnamoylCoA reductase gene from perennial ryegrass (Lolium perenne L.). J Plant Physiol: in press McKie JH, Jaouhari R, Douglas KT, Goffner D, Feuillet C, Grima-Pettenati J, Boudet AM, Baltas M, Gorrichon L (1993) A molecular model for cinnamyl alcohol dehydrogenase, a plant aromatic alcohol dehydrogenase involved in lignification. Biochim Biophys Acta 1202: 61– 69 Pillonel C, Mulder MM, Boon JJ, Forster B, Binder A (1991) Involvement of cinnamyl-alcohol dehydrogenase in the control of lignin formation in Sorghum bicolor L. Moench. Planta 185: 538 – 544 Selman-Housein G, Lopez MA, Hernandez D, Civardi L, Miranda F, Rigau J, Puigdomenech P (1999) Molecular cloning of cDNAs coding for three sugarcane enzymes involved in lignification. Plant Sci 143: 163–171 Somssich IE, Wernert P, Kiedrowski S, Hahlbrock K (1996) Arabidopsis thaliana defense-related protein Eli3 is an aromatic alcoholNADP + oxidoreductase. Proc Natl Acad Sci USA 93: 14199–14203 Trelease RN, Lee MS, Banjoko A, Bunkelmann J (1996) C-terminal polypeptides are necessary and sufficient for in vivo targeting of transiently-expressed proteins to peroxisomes in suspension-cultured plant cells. Protoplasma 195: 156–167 Whetten RW, MacKay J, Sederoff R (1998) Recent advances in understanding lignin biosynthesis. Annu Rev Plant Phys 49: 585 – 609 Williamson JD, Stoop JMH, Massel MO, Conkling MA, Pharr DM (1995) Sequence analysis of a mannitol dehydrogenase cDNA from plants reveals a function for the pathogenesis-related protein Eli3. Proc Natl Acad Sci USA 92: 7148–7152
Isolation and characterisation of three cinnamyl alcohol dehydrogenase homologue cDNAs from perennial ryegrass (Lolium perenne L.) Damian Lynch, Angela Lidgett, Russell McInnes, Helen Huxley, Elizabeth Jones, Natalia Mahoney, German Spangenberg* Plant Biotechnology Centre, Agriculture Victoria, Department of Natural Resources and Environment, and CRC for Molecular Plant Breeding, La Trobe University, Bundoora, Victoria 3086, Australia Received December 18, 2001 · Accepted February 12, 2002
Summary Three cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) homologue cDNAs (LpCAD1, LpCAD2 and LpCAD3) were isolated and characterised in perennial ryegrass (Lolium perenne). Sequence analysis revealed a partial LpCAD3 clone is highly homologous to typical monocot CAD cDNAs while full-length LpCAD1 and LpCAD2 cDNAs show lower homology to known CAD cDNAs. Northern hybridisation analysis revealed LpCAD1 and LpCAD3 transcripts in lignifying tissues while LpCAD2 was uniquely abundant in stem tissue. All three CAD cDNAs are upregulated in response to mechanical wounding stimulus. Southern hybridisation analysis suggested that LpCAD3 is a single copy gene within the Lolium genome while LpCAD1 and LpCAD2 each belong to a small gene family. Key words: CAD – cDNA – cinnamyl alcohol dehydrogenase – lignin – Lolium perenne – perennial ryegrass Abbreviations: ADH = alcohol dehydrogenase. – CAD = cinnamyl alcohol dehydrogenase. – CCR = cinnamoyl-CoA reductase. – cv. = cultivar. – DH = doubled haploid. – DIG = digoxigenin. – LG = linkage group. – LOD = log of odds. – MTD = Mannitol Dehydrogenase. – OMT = O-methyltransferase. – ORF = open reading frame. – RFLP = restriction fragment length polymorphism. – SAD = sinapyl alcohol dehydrogenase. – UTR = untranslated region
Introduction Lignin is an aromatic polymer deposited during the secondary thickening of plant cell walls. The role of lignin in vascular plants is to contribute to structural support, solute transport and plant defence (Iiyama et al. 1993). In grasses, the biosynthesis of monolignols is a multi-step process beginning with phenylalanine and tyrosine (Iiyama et al. 1993, Whetten 1998). These precursors are deaminated and hydroxylated in * E-mail corresponding author: [email protected]
the general phenylpropanoid biosynthetic pathway before entering the monolignol-specific branch [for reviews see Boudet (2000) and references therein]. The two reductive steps of the monolignol-specific pathway are catalysed by cinnamoylCoA reductase (CCR, EC 1.2.1.44) and cinnamyl alcohol dehydrogenase (CAD). Governing the last committed step of the pathway, CAD converts the hydroxycinnamaldehydes to their corresponding cinnamyl alcohols (monolignols). In angiosperms the three major monolignols are p-coumaryl, coniferyl and syringyl alcohols which are polymerised at the cell wall interface to form lignin. The varying combination of 0176-1617/02/159/06-653 $ 15.00/0
654
Damian Lynch et al.
monolignols incorporated into lignin is largely responsible for the heterogeneity displayed between plant species, tissues, developmental stage and sub-cellular location (Lewis and Yamamoto 1990). Typical CADs have been isolated from gymnosperms and angiosperms that display high sequence identity in key catalytic residues, however atypical CADs isolated exhibit sequence divergence (Brill et al. 1999). Being a final biosynthetic step, CAD may represent an ideal target to control lignin content and composition (Grima-Pettenati and Goffner 1999). Several observations suggest it is possible to alter lignin content and composition by manipulating CAD activity in planta. Decreased CAD activity has been implicated in the bm mutants of maize and sorghum and the cad n-1 mutant of loblolly pine (Pillonel et al. 1991, Mackay et al. 1997, Halpin et al. 1998), while CAD transgenic tobacco, poplar and lucerne (Halpin et al. 1994, Baucher et al. 1996, 1999) have displayed altered lignin profiles. Similar mutants or studies in forage or turf grasses have not been reported. The functional assessment of CAD function in planta via sense and antisense technology is dependent upon the availability of cDNA or gene sequences. We report the isolation and characterisation of three diverse CAD homologue cDNAs from perennial ryegrass as an initial step towards the molecular genetic dissection of the monolignol biosynthetic pathway.
Materials and Methods
seedlings (Heath et al. 1998). The library was screened with [32P] dCTP-labelled PCR generated probes. Excised and cloned cDNA inserts were obtained using the ExAssist helper phage with SOLR strain (Stratagene) as described by the manufacturer.
DNA sequence analysis High quality plasmid DNA (Maxi Kit, Qiagen) was extracted and sequenced by the dideoxy chain termination method (BigDyeTM version 2.0, ABI) on an ABI 373 automated sequencer. Protein and DNA comparisons were made using BLAST and GCG applications available through ANGIS (Australian National Genomic Informatiom Service). Phylogenetic analysis was performed using BioNavigator (http:// www.entigen.com).
Genomic Southern hybridisation analysis Perennial ryegrass genomic DNA of doubled haploid genotype DH297 (10 µg) was digested with DraI, BamHI, EcoRI, EcoRV, HindIII and XbaI, electrophoresed on a 1% agarose gel, capillary blotted and UV-fixed to a nylon membrane (Hybond-N, Amersham). Membranes were hybridised with DIG labelled full-length LpCAD1 (EcoRI/ApaI), LpCAD2 (EcoRI/XhoI) probes and a 730 bp LpCAD3 (EcoRI/ApaI) probe (Fig. 1). Hybridisation conditions were: 4 × SSC, 50 % formamide, 0.1 % N-Lauroyl-sarcosine, 0.02 % SDS, and 2 % blocking solution at 42 ˚C. Membranes were washed under high stringency: twice in 2 × SSC/0.1 % SDS for five minutes at room temperature, fifteen minutes in 0.2 × SSC/0.1 % SDS at 68 ˚C, then fifteen minutes in 0.1 × SSC/0.1% SDS at 68 ˚C.
Plant material Seeds of perennial ryegrass (Lolium perenne L.) cv. Ellet, tall fescue (Festuca arundinacea Schreb.) cv. Triumph and phalaris (Phalaris aquatica L.) cv. Holdfast were surface sterilised and germinated as previously described (Heath et al. 1998). Wounding experiments were performed with 10-day-old perennial ryegrass plants (cv. Ellet). Wounding was achieved by applying five compressions between 40grit sandpaper. Leaves and stems were harvested at 0, 6, 12, 24 and 48 hours post-wounding. The developmental expression of cDNAs was investigated using roots (3 – 5 day, 7–10 day, 6-week, 10-week), shoots (3 – 5 day, 7–10 day) seedlings (7–10 day), leaves (6-week, 10week) and stems (6-week, 10-week).
Genetic mapping using RFLPs RFLPs were detected with PCR-amplified [32P]-labelled probes of two distinct perennial ryegrass CAD homologues, LpCAD1 and LpCAD2, and the perennial ryegrass O-methyltransferase (OMT, EC 2.1.1.6) homologue, LpOMT1 (Heath et al. 1998), as previously described (Jones et al. 2002). RFLPs were mapped using 110 progeny individuals of the p150/112 perennial ryegrass reference population restricted with EcoRV (LpCAD1), EcoRI (LpCAD2) and DraI (LpOMT1). Genetic mapping data for p150/112 was obtained from the public database FoggDB (http://ukcrop.net/perl/ace/search/FoggDB) and full-length cDNAs LpCAD1, LpCAD2 and LpOMT1 were mapped within this marker framework using MAPMAKER 3.0 (Lander 1987).
Isolation of CAD sequences for cDNA library screening Oligonucleotide primers were designed to amino acid CAD domains (motifs CAGVTVYS and DVRYRFV) conserved between pine (AAB38774), lucerne (AAC35845), Arabidopsis (AAA99511), Aralia cordata, Eucalyptus botryoides and maize to create a generic hybridisation probe (HPCAD1) via PCR. A second specific hybridisation probe (HPCAD2) was generated by designing primers to a previously identified perennial ryegrass CAD (GenBank accession AF010290). Sequences encoding CAD were amplified from cDNA prepared from perennial ryegrass seedling total RNA and were cloned into pBluescript (Stratagene).
cDNA library screening A Uni-ZAPTM XR cDNA library (ZAP-cDNA Synthesis Kit, Stratagene) was constructed from whole 3 – 5-day-old etiolated perennial ryegrass
Northern hybridisation analysis Total RNA was isolated using Trizol (GibcoBRL) and 15 µg was electrophoresed on a 1.2 % agarose gel containing 6 % formamide, capillary blotted and UV-fixed to a nylon membrane (Hybond-N, Amersham). In order to verify equal loading of RNA, gels and membranes were either stained with ethidium bromide or 0.2 % methylene blue/ 0.3 mol/L sodium acetate respectively. 150 ng of [32P] dCTP labelled LpCAD1, LpCAD2 and LpCAD3 probes were used. Hybridisation conditions were: 4 × SSC, 50 % formamide, 0.5 % SDS, 5 × Denhardt’s solution, 5 % dextrane sulphate, 0.1 % herring sperm DNA, at 42 ˚C over-night. Membranes were washed under high stringency: three times in 2 × SSC/0.1 % SDS for ten minutes at 42 ˚C, three times in 0.2 × SSC/0.1 % SDS for ten minutes at 65 ˚C, and twice in 0.1 × SSC/ 0.1% SDS for ten minutes at 65 ˚C.
Cinnamyl alcohol dehydrogenase homologue cDNAs from Lolium
Results
655
216 bp 3′ UTR and a single 1086 bp ORF that encodes a protein of 361 amino acids (Fig. 1).
Isolation of perennial ryegrass CAD cDNA sequences Three CAD homologue cDNAs were isolated from a cDNA library constructed from RNA of 3 – 5-day-old etiolated perennial ryegrass seedlings (Heath et al. 1998). Two novel fulllength CAD cDNAs, LpCAD1 and LpCAD2, were isolated using a generic cDNA library hybridisation probe (HPCAD1). A third partial cDNA, LpCAD3, was isolated using a probe (HPCAD2) designed specifically to a known perennial ryegrass CAD sequence (AF010290) that was deposited in GenBank during the course of this study. Of the cDNA clones isolated, LpCAD1 is a 1325 bp cDNA with a 21 bp 5′ UTR, 74 bp 3′ UTR and a single 1230 bp ORF that encodes a protein of 409 amino acids. The coding region of LpCAD2 is a 1378 bp cDNA with a 101 bp 5′ UTR, 164 bp 3′ UTR and a single 1113 bp ORF that encodes a protein of 370 amino acids. LpCAD3 is a 780 bp fragment that is representative of a full-length 1382 bp cDNA with a 80 bp 5′ UTR, a
Analysis of LpCAD cDNA sequences Nucleotide sequence comparisons revealed that LpCAD1, LpCAD2 and LpCAD3 full-length cDNAs are only 56 % identical. Amino acid identity alignments (Table 1) showed that LpCAD3 is highly homologous to monocot CADs (99 – 85 %) with lower homology to dicot CADs. Both LpCAD1 and LpCAD2 show a lower degree of homology to CAD sequences (60 – 38 %). Phylogenetic analysis revealed LpCAD3 clustering with tall fescue, maize and sugarcane CADs while LpCAD1 and LpCAD2 group with atypical CADs from lucerne, Arabidopsis and Stylosanthes humilis (Fig. 2). Deduced amino acid sequence of perennial ryegrass CADs revealed functional protein domains retained amongst all CAD sequences, the Zn-1 and Zn-2 domains for catalytic and structural zinc ion binding and the NADPH binding do-
Figure 1. Partial restriction maps of CAD homologue cDNAs. LpCAD1 and LpCAD2 are full-length cDNAs while LpCAD3 is a partial cDNA clone identical to a full-length cDNA (AF010290). Full-length cDNAs were used as Southern and northern hybridisation probes.
Table 1. Amino acid identity (%) between selected plant cinnamyl alcohol dehydrogenases. LpCAD3 shows high identity to typical monocot CAD enzymes from tall fescue, maize and sugarcane, while LpCAD1 and LpCAD2 show lower homology. Details of each CAD sequences are given in Figure 2.
656
Damian Lynch et al. main (Fig. 3). The deduced protein sequences for LpCAD2 and LpCAD3 (370 and 361 amino acids, respectively) are similar to maize and sugarcane CADs (367 and 365 amino acids, respectively) while LpCAD1 is significantly larger, 409 amino acids.
Gene organisation and mapping Southern hybridisation analysis of DH perennial ryegrass genomic DNA using full length LpCAD1 and LpCAD2 cDNA probes revealed complex hybridisation patterns, suggesting that both are members of separate small gene families (Fig. 4). In contrast, LpCAD3 appeared to be a single copy gene in the perennial ryegrass genome. Both LpCAD1 and LpCAD2 were mapped within the framework of the perennial ryegrass p150/112 genetic linkage map as RFLPs. One LpCAD1 locus was polymorphic and mapped to perennial ryegrass LG2, while two LpCAD2 loci were polymorphic and mapped to LG2 and LG7 (Fig. 5). The LpCAD2 on LG7 mapped to the same overlapping region as LpCCR1 (McInnes et al. 2002) and LpOMT1 (this study).
Developmental and wound-induced expression of LpCADs
Figure 2. A phylogenetic tree constructed from amino acid sequences of plant CADs rooted against HADH (Horse Liver ADH, AAA30931). Amino acid sequences used for analysis and their respective accession numbers were: AcCAD (Aralia cordata, BAA03099), AgMTD (Apium graveolans, AAC15467), AtCAD (Arabidopsis thaliana AAA99511), AtEli3–1 (CAA48027), AtEli3 – 2 (CAA48026), AtCAD1 (AAK43875), AtCAD2 (AAK44076), AtCAD3 (AAL11561), AtCAD4 (AAK59426), EbCAD (Eucalyptus botryoides, BAA04046), EgCAD (E. gunni, CAA46585), EgbCAD (E. globulus, AAC07987), EgCAD1– 5 (CAA61275), FaCAD (Festuca arundinacea, AAK97809), FrCAD (Fragaria × ananassa, AAD10327), LpCAD1 (Lolium perenne, AF472591), LpCAD2 (AF472592), LpCAD3 (AAB 70908), McEli3 (Mesembryanthenum crystallium, AAB38503), MdCAD (Malus domestica, AAC06319), MsCAD1 (Medicago sativa, (AAC35846), MsCAD2 (AAC35845), NtCAD14 (Nicotiana tabacum, CAA44216), NtCAD19 (CAA44217), PaCAD (Picea abies, CAA51226), PcEli3 (Petroselinum crispum, CAA48028), PrCAD (Pinus radiata, AAB38774), PtCAD (Pinus taeda, CAA86072), PoptCAD (Populus tremuloides, AAF43140), PoptSAD (AAK58693), ShCAD1 (Stylosanthes humilis, AAA74882), ShCAD2 (AAA74883), SoCAD (Saccharum officinarum, CAA13177), ZeCAD (Zinnia elegans, BAA19487), ZmCAD (Zea mays, CAA06 687). Amino acid alignments were bootstrapped with Seqboot, subjected to maximum parsimony analysis using Protpars and a consensus tree created through Consense (Felenstein 1989). Fork numbers indicate bootstrap values where the number of times branch position to the right occurred out of 100 trees.
Northern hybridisation analysis of LpCAD1, LpCAD2 and LpCAD3 revealed three unique expression profiles, the level of expression varying with maturity and between organs (Fig. 6 a). The expression of LpCAD1 exhibited a constitutive profile with transcript particularly abundant in roots and mature stem. Similarly, LpCAD3 was highly expressed at all stages of root development and in mature stem tissue, but only lowly expressed in shoots and leaves. In contrast, LpCAD2 transcript was not detected in roots but accumulated heavily in mature shoot and especially stem tissue. Interspecific hybridisation was detected for LpCAD1 in phalaris and tall fescue but only in tall fescue for LpCAD2 and LpCAD3. Hybridisation analysis also revealed that LpCAD1, LpCAD2 and LpCAD3 were similarly upregulated in response to a mechanical wounding stimulus (Fig. 6 b). In all cases, transcript abundance increased in ten day old shoots within six hours of wounding. Levels of expression returned to normal between twenty four to forty eight hours post-wounding.
Discussion We report here for the first time, the isolation and analysis of three divergent CAD homologue cDNAs from perennial ryegrass. Expression analysis, protein and nucleotide alignments and phylogenetic comparisons disclose the divergent nature of these three CADs. LpCAD3 corresponds to a full-length cDNA (AF010290) that encodes a protein belonging to a conserved group of monocot CADs. Maize and sugarcane CADs have been implicated in conventional lignification (Halpin et al. 1998, Selman-Housein et al. 1999), suggesting that LpCAD3 may have a role in structural lignification class CAD genes. In contrast,
Cinnamyl alcohol dehydrogenase homologue cDNAs from Lolium
657
Figure 3. Amino acid sequence alignment of plant CADs. Shaded areas indicate conserved residues. Key functional domains highlighted are: the Zn-1 catalytic zinc binding domain (- - - -) and associated invariant residues (䊉), the Zn-2 structural zinc binding domain (*), the NADPH binding domain domain ( + + + ), residues for coenzyme binding specificity (䉬), and residues for active site specificity (bold and underlined) (Jornvall et al. 1987, McKie et al. 1993).
both LpCAD1 and LpCAD2 appear to be divergent defence related CAD cDNAs on the basis of their sequence and phylogeny. Although they retain critical amino acid domains implicated in CAD function, they display significant sequence divergence in residues implicated in substrate specificity (McKie et al. 1993, Lauvergeat et al. 1995). When compared with other known CAD enzymes, LpCAD1 and LpCAD2 group more closely to other atypical CADs that have been implicated in defence lignification or other biosynthetic roles (eg: Eli3s, SAD) (Kiedrowski et al. 1992, Williamson et al. 1995, Somssich et al. 1996, Logemann et al. 1997, Brill et al. 1999, Li et al. 2001). Furthermore, LpCAD1 exhibits an atypical 38 amino acid carboxy terminus extension. This long tail
domain contains no conventional targeting signal (Barpeled et al. 1996, Trelease et al. 1996). Southern hybridisation analysis and mapping data suggests the CADs represent three separate genes located within the ryegrass genome. Of interest is the location of three key lignification genes (LpOMT1, LpCCR1 and LpCAD2) in close proximity on perennial ryegrass LG7. A gene cluster of this nature could be indicative of a coordinated regulation of this complex pathway (Douglas 1996). Functional divergence of the perennial ryegrass CADs is supported by the different expression patterns. While all the CADs display a similar up-regulation in response to mechanical wounding, their developmental expression profiles sug-
658
Damian Lynch et al.
Figure 4. Southern hybridisation analysis of perennial ryegrass genomic DNA. DH DNA (10 µg) was digested with DraI (D), BamHI (B), EcoRI (EI), EcoRV (EV), HindIII (H), XbaI (X), none of which cut within the cDNAs, and the resulting membrane probed with a DIG-labelled LpCAD1, LpCAD2 and LpCAD3 cDNAs.
Figure 5. Map locations of LpCAD1 and LpCAD2 on the perennial ryegrass reference map. The most likely positions of CADs are indicated by arrows, with the adjacent bar indicating the span of possible map locations at LOD > 2.0. Map positions of LpOMT1, LpCCR1 and LpCAD2 in the same overlapping region are indicated on LG7.
Cinnamyl alcohol dehydrogenase homologue cDNAs from Lolium
659
Figure 6. Northern hybridisation analysis of CAD gene expression in perennial ryegrass. Membranes were probed with radiolabelled LpCAD1, LpCAD2 and LpCAD3 cDNAs. A. Roots (R1) from seedlings 3 – 5 days, roots (R2) from seedlings 7–10 days, roots (R3) from 6-week-old plants, and roots (R4) from 10-week-old plants. Shoots (Sh1) from seedlings 3 – 5 days, shoots (Sh2) from seedlings 7–10 days, leaves (L1) from 6-weekold plants, and leaves (L2) from 10-week-old plants. Stem (St1) from 6-week-old plants, stem (St2) from 10-week-old plants, whole plant seedling (P) of 11-day-old phalaris, and whole plant seedling (F) of 7-day-old tall fescue. B. Wounded and unwounded control ten day old shoots harvested at 0, 6, 12, 24, 48 hours post-wounding.
gest complex transcriptional regulation. LpCAD1 and LpCAD3 were expressed in most plant organs and were particularly abundant in roots, while LpCAD2 displayed transcript abundance in mature stem tissues. Tightly regulated expression, such as that displayed by LpCAD2, highlights
avenues for discovering novel promoter elements to facilitate specific directed gene expression studies. Studies in transgenic tobacco, poplar, and lucerne (Halpin et al. 1994, Baucher et al. 1996, 1999) have shown by manipulating CAD activity, via sense and antisense technologies,
660
Damian Lynch et al.
alterations in cell wall and lignification profiles are possible. As yet, no transgenic studies have investigated the possible role of the defence related or atypical CAD genes in lignification. Moreover, none of these approaches have accounted for the functional redundancy displayed for CAD in angiosperms (Whetten et al. 1998). The isolation and analysis of three distinctive CAD homologue cDNAs provides suitable targets for the molecular genetic dissection of CAD function in planta. This work will enhance our understanding of lignin biosynthesis, monomeric composition and properties in grasses and facilitate the production of pasture grasses with enhanced herbage quality and turf grasses with improved resilience. Acknowledgements. This work was supported by the Australian CRC for Molecular Plant Breeding, the Australian Dairy Research and Development Corporation and the Department of Natural Resources and Environment, Victoria, Australia.
References Barpeled M, Bassham D, Raikhel N (1996) Transport of proteins in eukaryotic cells: more questions ahead. Plant Mol Biol 32: 223 – 249 Baucher M, Chabbert B, Pilate G, Van Doorsselaere J, Tollier M, Petitconil M, Cornu D, Monties B, Van Montagu M, Inze D, Jouanin L, Boerjan W (1996) Red xylem and higher lignin extractability by down-regulating a cinnamyl alcohol dehydrogenase in poplar. Plant Physiol 112: 1479–1490 Baucher M, Bernard-Vailhe M, Chabbert B, Besle J, Opsomer C, Van Montagu M, Botterman J (1999) Down-regulation of cinnamyl alcohol dehydrogenase in transgenic alfalfa (Medicago sativa L.) and the effect on lignin composition and digestibility. Plant Mol Biol 39: 437– 447 Boudet AM (2000) Lignins and lignification: Selected issues. Plant Physiol Biochem 38: 81– 96 Brill E, Abrahams S, Hayes C, Jenkins C, Watson J (1999) Molecular characterisation and expression of a wound-inducible cDNA encoding a novel cinnamyl-alcohol dehydrogenase enzyme in lucerne (Medicago sativa L.). Plant Mol Biol 41: 279 – 291 Douglas C (1996) Phenylpropanoid metabolism and lignin biosynthesis: from weeds to trees. Trends Plant Sci 1: 171–177 Felenstein J (1989) PHYLIP – Phylogeny Interference Package (Version 3.2). Cladistics 5: 164–166 Grima-Pettenati J, Goffner D (1999) Lignin genetic engineering revisited. Plant Sci 145: 51– 65 Halpin C, Knight M, Foxon G, Campbell M, Chabbert B, Tollier M, Schuch W (1994) Manipulation of lignin quality by downregulation of cinnamyl alcohol dehydrogenase. Plant J 6: 339 – 350 Halpin C, Holt K, Chojecki J, Oliver D, Chabbert B, Monties B, Edwards K, Barakate A, Foxon GA (1998) Brown-midrib maize (bm1) – a mutation affecting the cinnamyl alcohol dehydrogenase gene. Plant J 14: 545 – 553 Heath R, Huxley H, Stone B, Spangenberg G (1998) cDNA cloning and differential expression of three caffeic acid O-methyltransferase homologues from perennial ryegrass (Lolium perenne). J Plant Physiol 153: 649 – 657 Iiyama K, Lam T, Meikle P, Ng K, Rhodes D, Stone B (1993) Cell wall biosynthesis and its regulation. In: Jung H, Buxton D, Hatfield R, Ralph J (eds) Forage Cell Wall Structure and Digestibility. American Society of Agronomy, Madison, WI, pp 621– 683
Jones E, Mahoney N, Hayward M, Armstead I, Jones J, Humphreys M, Kishida T, Yamada T, Balfourier F, Charmet G, Forster J (2002) An enhanced molecular marker-based genetic map of perennial ryegrass (Lolium perenne L.) reveals comparative relationships with other Poaceae genomes. Genome 45: 282 – 295 Jornvall H, Persson B, Jeffery J (1987) Characteristics of alcohol/polyol dehydrogenases. The zinc-containing long-chain alcohol dehydrogenases. Eur J Biochem 167: 195 – 201 Kiedrowski S, Kawalleck P, Hahlbrock K, Somssich I, Dangl J (1992) Rapid activation of a novel plant defense gene is strictly dependent on the Arabidopsis RPM1 disease resistance locus. EMBO J 11: 4677– 4684 Lander E, Green P, Abrahamson J, Barlow A, Daly MJ, Lincoln SE, Newburg L (1987) MAPMAKER: an interactive computer package for constructing primary linkage maps of experimental and natural populations. Genomics 1: 174–181 Lauvergeat V, Kennedy K, Feuillet C, McKie JH, Gorrichon L, Baltas M, Boudet AM, Grima-Pettenati J, Douglas KT (1995) Site-directed mutagenesis of a serine residue in cinnamyl alcohol dehydrogenase, a plant NADPH-dependent dehydrogenase, affects the specificity for the coenzyme. Biochemistry 34: 12426–12434 Lewis N, Yamamoto E (1990) Lignin: occurrence, biogenesis and biodegradation. Annu Rev Plant Phys 41: 455 – 496 Li L, Cheng X, Leshkevich J, Umezawa T, Harding S, Chiang V (2001) The last step of syringyl monolignol biosynthesis in angiosperms is regulated by a novel gene encoding sinapyl alcohol dehydrogenase. Plant Cell 13: 1567–1585 Logemann E, Reinold S, Somssich IE, Hahlbrock K (1997) A novel type of pathogen defense-related cinnamyl alcohol dehydrogenase. Biol Chem 378: 909 – 913 Mackay JJ, Omalley DM, Presnell T, Booker FL, Campbell MM, Whetten RW, Sederoff RR (1997) Inheritance, gene expression, and lignin characterization in a mutant pine deficient in cinnamyl alcohol dehydrogenase. Proc Natl Acad Sci USA 94: 8255 – 8260 McInnes R, Lidgett A, Lynch D, Huxley H, Jones E, Mahoney N, Spangenberg G (2002) Isolation and characterisation of a cinnamoylCoA reductase gene from perennial ryegrass (Lolium perenne L.). J Plant Physiol: in press McKie JH, Jaouhari R, Douglas KT, Goffner D, Feuillet C, Grima-Pettenati J, Boudet AM, Baltas M, Gorrichon L (1993) A molecular model for cinnamyl alcohol dehydrogenase, a plant aromatic alcohol dehydrogenase involved in lignification. Biochim Biophys Acta 1202: 61– 69 Pillonel C, Mulder MM, Boon JJ, Forster B, Binder A (1991) Involvement of cinnamyl-alcohol dehydrogenase in the control of lignin formation in Sorghum bicolor L. Moench. Planta 185: 538 – 544 Selman-Housein G, Lopez MA, Hernandez D, Civardi L, Miranda F, Rigau J, Puigdomenech P (1999) Molecular cloning of cDNAs coding for three sugarcane enzymes involved in lignification. Plant Sci 143: 163–171 Somssich IE, Wernert P, Kiedrowski S, Hahlbrock K (1996) Arabidopsis thaliana defense-related protein Eli3 is an aromatic alcoholNADP + oxidoreductase. Proc Natl Acad Sci USA 93: 14199–14203 Trelease RN, Lee MS, Banjoko A, Bunkelmann J (1996) C-terminal polypeptides are necessary and sufficient for in vivo targeting of transiently-expressed proteins to peroxisomes in suspension-cultured plant cells. Protoplasma 195: 156–167 Whetten RW, MacKay J, Sederoff R (1998) Recent advances in understanding lignin biosynthesis. Annu Rev Plant Phys 49: 585 – 609 Williamson JD, Stoop JMH, Massel MO, Conkling MA, Pharr DM (1995) Sequence analysis of a mannitol dehydrogenase cDNA from plants reveals a function for the pathogenesis-related protein Eli3. Proc Natl Acad Sci USA 92: 7148–7152