| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
Plant Physiology 133:1051-1071 (2003) © 2003 American Society of Plant Biologists Genome-Wide Characterization of the Lignification Toolbox in Arabidopsis1,[w]Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, Technologiepark 927, B–9052 Gent, Belgium
Lignin, one of the most abundant terrestrial biopolymers, is indispensable for plant structure and defense. With the availability of the full genome sequence, large collections of insertion mutants, and functional genomics tools, Arabidopsis constitutes an excellent model system to profoundly unravel the monolignol biosynthetic pathway. In a genome-wide bioinformatics survey of the Arabidopsis genome, 34 candidate genes were annotated that encode genes homologous to the 10 presently known enzymes of the monolignol biosynthesis pathway, nine of which have not been described before. By combining evolutionary analysis of these 10 gene families with in silico promoter analysis and expression data (from a reverse transcription-polymerase chain reaction analysis on an extensive tissue panel, mining of expressed sequence tags from publicly available resources, and assembling expression data from literature), 12 genes could be pinpointed as the most likely candidates for a role in vascular lignification. Furthermore, a possible novel link was detected between the presence of the AC regulatory promoter element and the biosynthesis of G lignin during vascular development. Together, these data describe the full complement of monolignol biosynthesis genes in Arabidopsis, provide a unified nomenclature, and serve as a basis for further functional studies.
Lignin is an aromatic heteropolymer that is mainly present in secondary thickened plant cells, where it provides rigidity and impermeability to the cell walls. In addition, lignin deposition may be induced upon wounding and infection to protect plant tissues against invading pathogens. Lignin is composed of different phenylpropanoids, predominantly the monolignols p-coumaryl, coniferyl, and sinapyl alcohols that differ in their degree of methoxylation (Fig. 1). When these monolignols are incorporated into lignin, they are called p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units, respectively. In addition to the three monolignols, other phenylpropanoids, such as hydroxycinnamyl aldehydes, hydroxycinnamyl acetates, hydroxycinnamyl p-hydroxybenzoates, hydroxycinnamyl p-coumarates, and hydroxycinnamate esters, are also present in the polymer (Ralph et al., 2001  ; Boerjan et al., 2003). Considerable variation exists in lignin composition between taxa, cell types, and developmental and environmental conditions.
Over the last decade, there has been a tremendous effort in cloning new genes involved in the monolignol biosynthetic pathway and in tackling the enzyme kinetics of the corresponding proteins and the role these enzymes play in controlling the amount and composition of lignin to be deposited in the cell wall (Anterola and Lewis, 2002
Although enzymatic assays and transgenic plants have contributed extensively to our understanding of the in vivo role of the enzymes, the role of individual gene family members has been more difficult to tackle, a limitation that can only be overcome in plant species such as Arabidopsis, for which the genome sequence and efficient reverse genetics tools are available (Arabidopsis Genome Initiative [AGI], 2000 As a first step toward studying the role of individual family members, we have undertaken a bioinformatics approach to identify, in Arabidopsis, all the gene family members of all monolignol biosynthesis genes known today. In many cases, only a subset of a given gene family, mostly obtained by homology-based gene isolation, has been characterized in the past. As a consequence, more distant family members might not have been discovered when, for example, primers were designed on only a few members of the family. This has led to an important bias in the range of sequence data available in public databases. Here, we have used sensitive computational methods to delineate, in Arabidopsis, all members of the gene families currently known to be involved in monolignol biosynthesis. The integration of expression studies and promoter sequence analyses of the individual family members with phylogenetic analysis of the family has allowed us to select 12 genes as the most likely candidates to be involved in the developmental lignification in vascular tissues. Importantly, the promoter comparisons revealed a possible link between G lignin biosynthesis and the presence of the AC element that is correlated with a strong xylem expression. Together, these data describe the full complement of monolignol biosynthesis genes in Arabidopsis, introduce a unifying nomenclature for all genes of the pathway (Table I), and serve as a basis for further functional studies.
A semiautomatic structural annotation and a phylogeny-based classification were performed using prediction results, experimental data, and information from homologous sequences (see "Materials and Methods"). A total of 34 candidate monolignol biosynthesis genes were annotated, of which nine had, to our knowledge, never been described before (Table I). In addition, 27 closely related superfamily members ("likes") were identified in this process (Table I). To get a first insight into whether all these genes are indeed expressed and, more importantly, whether their expression pattern correlates with developmental lignification, their expression was analyzed in a set of tissues and for six developmental stages of inflorescence stem known to contain a high portion of lignifying cells. These data were compared with previous expression data from Arabidopsis and with information extracted from public databases of expressed sequence tag (EST). In addition, putative promoter elements, which drive expression during lignification, in pathogen and wound responses, and after induction by stress-related hormones, and potential subcellular localization signals were identified (due to size limitations, tables compiling all these data are available as supplemental data and at http://www.psb.ugent.be/bioinformatics/lignin/and are indexed by an "s" throughout the manuscript).
PAL (E.C. 4.3.1.5) is the first enzyme of the general phenylpropanoid pathway and catalyzes the nonoxidative deamination of Phe to trans-cinnamic acid and NH3 (Fig. 1). PAL mediates the influx from primary metabolism into the phenylpropanoid pathway and becomes rate limiting when its activity is reduced below a threshold of 20% to 25% in transgenic tobacco (Nicotiana tabacum; Bate et al., 1994 ; Sewalt et al., 1997).
By using a thorough semiautomated annotation method, four genes encoding PAL proteins were detected in the Arabidopsis genome, three of which have been described previously (Ohl et al., 1990
PAL1 and PAL2 are not only structurally very similar, but they also share common promoter elements and a similar expression pattern (supplemental Table Is). mRNAs from both genes are most abundant in roots and stems, where the expression increases during the later stages of development (Fig. 3; supplemental Table Is; Wanner et al., 1995). Analysis of the fusion between the AtPAL1 promoter and
In addition, and in accordance with the expression pattern, the promoters of PAL1 and PAL2 contain well-conserved AC elements that specify vascular expression of phenylpropanoid genes (supplemental Table Is; Ohl et al., 1990 In conclusion, all PAL genes are expressed in the inflorescence stem, a tissue with a high portion of lignifying cells. However, the presence of an AC element qualifies PAL1 and PAL2 as the most likely candidates to be involved in monolignol biosynthesis in the vascular lignifying cells. In accordance, the corresponding mutants show defects in lignin formation (A. Rohde and W. Boerjan, unpublished data).
C4H (E.C. 1.14.13.11) controls the conversion of cinnamate into p-coumarate (Fig. 1). C4H (CYP73A5) belongs to the cytochrome P450-dependent monooxygenases, like the two other hydroxylases in the pathway (C3H, F5H). So far, only one C4H gene has been described in Arabidopsis (Bell-Lelong et al., 1997 ; Mizutani et al., 1997; Urban et al., 1997). Although multiple family members have been detected in other plants (Betz et al., 2001, and refs. therein), we could not find any evidence for additional CYP73 genes in Arabidopsis. Phylogenetic analysis shows that two classes of C4H genes exist in plants (Fig. 4; Nedelkina et al., 1999; Betz et al., 2001). Furthermore, the tree topology indicates that the origin of these two classes has predated the divergence of gymnosperms and angiosperms, suggesting that class II members must have existed at some time in the evolution for most plant lineages. The Arabidopsis C4H gene belongs to class I; a class II homolog was most probably lost during the evolution of this species.
C4H is expressed in all tissues and upon exposure to light, wounding, and fungal infection (supplemental Table IIs; Bell-Lelong et al., 1997
By TargetP (Emanuelsson et al., 2000
4CL (E.C. 6.2.1.12) catalyzes the formation of CoA esters of p-coumaric acid, caffeic acid, ferulic acid, 5-hydroxyferulic acid, and sinapic acid (Fig. 1; Lee et al., 1997 ; Hu et al., 1998). The plethora of additional potential substrates may explain why there are many 4CL isoenzymes in most plants. In addition to the different substrate specificities, the genes typically have a distinct spatio-temporal expression pattern (Lewis and Yamamoto, 1990; Hu et al., 1998; Harding et al., 2002).
We detected four 4CL and nine 4CL-like genes in the Arabidopsis genome. Phylogenetic analysis of the predicted proteins, together with characterized 4CL proteins and luciferases, acetate, and fatty acid CoA-ligases (other adenylate-forming enzymes; data not shown), shows that 4CL proteins fall into two classes (Fig. 5; Ehlting et al., 1999
Our expression analysis showed that 4CL genes are expressed in almost all investigated tissues, with 4CL4 having the most restricted expression (Fig. 3). The latter observation is supported by the smallest number of ESTs found for 4CL4 among the 4CL genes (supplemental Table IIIs). 4CL1 and 4CL2 are expressed throughout inflorescence stem development and expression increases during the later stages (supplemental Table IIIs; Lee et al., 1995
In conclusion, 4CL1 and 4CL2 are the best candidates for a function in monolignol biosynthesis during developmental lignification, as suggested previously by Ehlting et al. (1999
HCT belongs to a large family of acyltransferases that are involved in the biosynthesis of diverse secondary metabolites. Only recently, the first HCT has been purified from tobacco stems, and the corresponding gene was cloned (Hoffmann et al., 2003 ). In tobacco, HCT catalyzes the conversion of p-coumaroyl-CoA and caffeoyl-CoA to the corresponding shikimate or quinate esters (Fig. 1). These shikimate and quinate esters, themselves being important intermediates in the phenylpropanoid pathway, have been shown recently to be good substrates for C3H (Kühnl et al., 1987; Schoch et al., 2001; Franke et al., 2002a, 2002b; Nair et al., 2002). Moreover, HCT catalyzes also the reverse transesterification (Hoffmann et al., 2003). Therefore, HCT might play a critical role up- and downstream of C3H. For the Arabidopsis HCT homolog, a biochemical activity similar to that of the tobacco HCT has been shown (Hoffmann et al., 2003). Here, one HCT gene was detected in the Arabidopsis genome (Fig. 6). Because only two homologs were characterized and the family is apparently well conserved (approximately 60% identity between monocot and dicot members; data not shown), no more distantly related genes were included.
The expression analysis shows that HCT is expressed in all tissues investigated but strongly in the inflorescence stem (Fig. 3; supplemental Table IVs). The promoter contains an AC element. The high and ubiquitous expression is confirmed by the second highest number of ESTs found for the 10 gene families analyzed (supplemental Table IVs). Interestingly, the combined presence of an H and a G box was observed, as for PAL4 and F5H2, suggesting transcriptional regulation by the pathway intermediate p-coumaric acid (Loake et al., 1992
C3H was originally named after its suspected function in C3-hydroxylation of p-coumaric acid, but recently, CYP98A3 (C3H1) was shown to preferentially convert the shikimate and quinate esters of p-coumaric acid into the corresponding caffeic acid conjugates, whereas p-coumaric acid and p-coumaroyl-CoA were not substrates of this enzyme (Fig. 1; Schoch et al., 2001 ; Franke et al., 2002b; Nair et al., 2002). We detected three C3H genes in the Arabidopsis genome, which all belong to the CYP98 class of the P450 enzymes. Only a few proteins of this class could be found from other species for phylogenetic analysis (Fig. 7). Arabidopsis C3H1 clusters with all known C3Hs of other species, whereas C3H2 and C3H3 (CYP98A8 and CYP98A9, respectively) probably constitute a different class that diverged before the gymnosperm-angiosperm split (Fig. 7).
The expression analysis shows that C3H1 is expressed in all tissues, an observation that is supported by ESTs from various tissues (supplemental table Vs). Previous studies detected the highest expression in the vascular tissues of stem and root (supplemental table Vs; Schoch et al., 2001
Analysis of the N terminus by TargetP predicts the C3H1 protein to contain an ER-targeting peptide, but it overlaps, as for C4H, with the membrane anchor region of P450 enzymes. The C3H1 protein has previously been localized in the membrane fraction in yeast (Franke et al., 2002b
CCoAOMT (E.C. 2.1.1.104) catalyzes the methylation of caffeoyl-CoA to feruloyl-CoA (in vitro and in vivo) and 5-hydroxyferuloyl-CoA to sinapoyl-CoA (at least in vitro) and is, together with COMT, responsible for the methylation of the monolignol precursors (Fig. 1; Ye et al., 1994 ; Zhong et al., 1998; Pinçon et al., 2001).
Seven putative members of the CCoAOMT gene family were detected in the Arabidopsis genome (Fig. 8). Plant CCoAOMT genes fall into two classes: Class I contains the Arabidopsis CCoAOMT1 gene together with the majority of experimentally characterized CCoAOMT genes (e.g. Zhong et al., 1998
CCoAOMT1 is expressed in all tissues investigated and has by far the highest number of ESTs (Fig. 3; supplemental Table VIs). Moreover, the CCoAOMT1 gene has two AC elements in its promoter. CCoAOMT1 is highly expressed in the basal portion of the inflorescence as compared with the apical portion (Goujon et al., 2003
CCoAOMT genes of other species were shown to be responsive to pathogens or elicitors (e.g. Pakusch et al., 1991 Based on its clustering in class I, its expression characteristics and level, and the presence of two AC elements in its promoter, CCoAOMT1 is the main candidate gene to be involved in the monolignol pathway during developmental lignification.
CCR (E.C.1.2.1.44) catalyzes the conversion of cinnamoyl-CoA esters to their respective cinnamaldehydes and is the first enzyme of the monolignol-specific part of the lignin biosynthetic pathway (Fig. 1). The two previously described CCR genes and five new CCR-like genes were found (Fig. 9; Jones et al., 2001 ; Lauvergeat et al., 2001).
CCR1 is highly expressed in all tissues examined, whereas CCR2 is in all tissues but flowers, siliques, and the earliest stage of inflorescence stem development (Fig. 3). Although CCR2 was hardly detected in stem by RNA gel blots (Lauvergeat et al., 2001
In conclusion, CCR1 and CCR2 are expressed during both developmental lignification and pathogen response, as documented by our expression analysis and ESTs (Fig. 3; supplemental Table VIIs). The role of CCR1 in lignification has clearly been established through the irx4 (irregular xylem) mutant characterization (Jones et al., 2001
F5H, also called coniferaldehyde 5-hydroxylase, is a cytochrome P450-dependent monooxygenase (CYP84) that is required for the production of syringyl lignin because it is responsible for the 5-hydroxylation of coniferaldehyde and/or coniferyl alcohol (Fig. 1; Humphreys et al., 1999 ; Li et al., 2000; Humphreys and Chapple, 2002).
The Arabidopsis genome harbors two F5H homologs, both belonging to the CYP84 family of the P450 monooxygenases. F5H1 (CYP84A1) has been characterized in Arabidopsis, Liquidambar styraciflua, and Brassica napus (Meyer et al., 1996
Our expression analysis revealed F5H1 expression in all tissues and an increasing expression during inflorescence stem development (Fig. 3), in accordance with results of earlier studies (supplemental Table VIIIs; Meyer et al., 1998
In the promoter analysis, for both genes an H box was found and for F5H2 a G box was also found, suggesting that both genes may be inducible and that F5H2 may be regulated by p-coumarate (Loake et al., 1992
COMT (E.C. 2.1.1.68) was originally postulated to be a bifunctional enzyme methylating caffeic acid and 5-hydroxyferulic acid. However, in vitro and transgenic studies revealed that the predominant role of COMT is the methylation of 5-hydroxyconiferaldehyde and/or 5-hydroxyconiferyl alcohol to sinapaldehyde and/or sinapyl alcohol, respectively (Fig. 1; Osakabe et al., 1999 ; Li et al., 2000; Chen et al., 2001; Guo et al., 2001; Parvathi et al., 2001; Goujon et al., 2003).
We detected only one COMT gene in the Arabidopsis genome. Furthermore, 13 proteins similar to COMT were detected that clustered in-between the functionally characterized COMT clade and the cluster containing the hydroxycinnamic acid/hydroxycinnamoyl-CoA ester O-methyltransferase protein (AEOMT; Li et al., 1997
Our RT-PCR data show that COMT is expressed in all tissues investigated, and the numerous ESTs point toward a generally high and ubiquitous expression (Fig. 3; supplemental Table IXs). Ninety-nine COMT ESTs, with a fifth being stress related, is almost twice the number found for any other gene in this analysis (supplemental Table IXs). COMT expression is particularly high in the inflorescence stem, with an increase during the later stages of development (Fig. 3; supplemental Table IXs). Correspondingly, COMT::GUS expression occurs in xylem, differentiating fibers, and mature phloem (Goujon et al., 2003
Interestingly, the COMT protein might be myristoylated. The N-terminal MGSTAETQLTPVQVTDDE sequence was identified as a "twilight zone" myristoylation signal, which corresponds both with truly myristoylated proteins and with false positives (Maurer-Stroh et al., 2002
CAD (E.C. 1.1.1.195) catalyzes the last step in monolignol biosynthesis, i.e. the reduction of cinnamyl aldehydes into their corresponding alcohols (Fig. 1). CAD reduces various aldehydes, present in different cell types or during different stages of development. Besides the function in developmentally regulated lignification, a number of CAD genes have been characterized for their response to plant pathogens (Kiedrowski et al., 1992 ).
Here, nine putative CAD genes were detected in the Arabidopsis genome (Table I; Tavares et al., 2000
Class II CADs (CAD3, CAD4, and CAD5) cluster with a number of alcohol dehydrogenases with diverse substrate preferences, such as the poplar (Populus tremuloides) sinapyl alcohol dehydrogenase (Li et al., 2001
Class III CADs (CAD1, CAD7, and CAD8) cluster in a group with an alcohol dehydrogenase from alfalfa (Medicago sativa), which is able to catalyze the reduction of cinnamaldehyde, sinapaldehyde, and coniferaldehyde, but also several aliphatic aldehydes and various substituted benzaldehydes (Brill et al., 1999
All CAD genes, except CAD2, CAD4, and CAD5, are expressed in all stages of inflorescence stem development (Fig. 3). Moreover, CAD2 and CAD6 are expressed in the inflorescence stem close to the bundle and interfascicular cambium, as revealed by promoter::GUS constructs (Sibout et al., 2003
The promoter analysis revealed that CAD6 from class I and CAD5 from class II contain AC elements (supplemental Table Xs). In addition, an A box was detected in the CAD6 promoter. The fact that only one gene in the pathway contains both an AC element and an A box casts doubt on the previous assumption that an A box works in conjunction with AC elements (Logemann et al., 1995
Based on the fact that they cluster with other well-characterized "true" CAD genes in the phylogenetic tree, CAD2 and CAD6 are the most likely candidates for the monolignol pathway in Arabidopsis. Of these two, only CAD6 has an AC element. Moreover, only the CAD6 gene mutant, but not that of CAD2, showed altered lignin content and structure (Sibout et al., 2003
Toward the Core Monolignol Biosynthesis Gene Set for Developmental Lignification
Lignification is a process that occurs predominantly in cells of the vascular tissue, found in almost all organs, but most abundantly in stems and roots. A strong expression of monolignol biosynthesis genes in stems and roots is documented in numerous publications (supplemental Tables Is–Xs, and refs. therein). Possibly, lignification cDNAs are relatively highly represented in root libraries because of the absence of other very abundant processes, such as photosynthesis, or, as could be concluded from AtC4H::GUS analysis (Nair et al., 2002
All 34 genes, annotated from the Arabidopsis genome sequence for their potential involvement in monolignol biosynthesis, are expressed at some stage of inflorescence stem development, a tissue with a prominent portion of lignifying cells (Table I and supplemental Tables Is–Xs; Dharmawardhana et al., 1992
A constitutive expression in the inflorescence stem (Fig. 3) and the phylogenetic classification in groups with functionally characterized proteins of other species were used as the first two criteria to delineate those family members that are the most likely to be involved in monolignol biosynthesis during developmental lignification (Table II). These criteria are fulfilled for 14 genes: PAL1, PAL2, PAL3, PAL4, C4H, 4CL1, 4CL2, HCT, C3H1, CCoAOMT1, CCR1, F5H1, COMT, and CAD6. Of these 14, eight genes have been already certified for their involvement in monolignol biosynthesis through the characterization of the corresponding mutants: PAL1 (pal1), PAL2 (pal2), C4H (ref3), C3H1 (ref8), CCR1 (irx4), F5H1 (fah1), COMT (comt1), and CAD6 (cad-D; Chapple et al., 1992
AC elements, originally identified in the promoters of the parsley PAL1 gene, the bean PAL2 and PAL3 genes, and the parsley 4CL1 gene (Cramer et al., 1989
Given the importance of AC elements in specifying vascular expression, the presence of an AC element in the promoters of the 34 annotated genes has been examined. In the past, most AC elements were identified by consensus sequences built from both experimentally verified AC elements and AC elements detected by sequence similarity. Often on top of such a consensus, a number of mismatches were allowed. Moreover, AC elements were often subdivided into ACI and ACII boxes, despite the fact that they align perfectly and were shown to be functionally redundant with respect to vascular expression (see supplemental data; Hatton et al., 1995
By searching all 29,787 Arabidopsis genes predicted with EuGene (Schiex et al., 2001
Seven gene families have at least one family member with an AC element in their promoter (Table II). Genes with an AC element do not simply correspond with genes that are highly expressed as estimated from the number of ESTs (Table II). Rather, AC elements coincide with those gene family members that were assigned to be involved in developmental lignification based on expression and phylogeny (see above): of these 14 genes, nine contain an AC element (Table II). Thus, within their respective gene families, the following genes are extra-qualified for playing a role in developmental lignification in vascular tissues: PAL1, PAL2, 4CL1, 4CL2, HCT, C3H1, CCoAOMT1, CCR1, and CAD6. CAD5 has an AC element but did not cluster with the true CAD clade in the phylogenetic tree (Fig. 12). In contrast, no AC elements were found in the gene families C4H, F5H, and COMT. Of these three gene families, C4H and COMT are single genes that, contrary to multigene families, may have acquired a more relaxed promoter organization compatible with expression in a broader range of cells and conditions. Maybe these genes contain more degenerated AC elements that were not picked up under the stringent search parameters used. The F5H family consists of two genes that are not functionally redundant because F5H2 fails to compensate for the loss of F5H1 in the fah1 mutant (Meyer et al., 1998
A tantalizing alternative hypothesis starts out from the notion that all AC element-containing monolignol biosynthesis genes code for enzymes acting in the G branch of the pathway (Fig. 1; Table II). None of the 14 other promoter elements analyzed, including stress- and elicitor-responsive elements, could be linked in a similarly meaningful way to particular groups of genes (supplemental Tables Is–Xs), underscoring how important the presence of AC elements may be for a common regulation of G-branch genes. A separate regulation of S-branch genes is a valid option to explain why the latter lack AC elements, given the spatio-temporal differences in deposition of S and G lignin (Dharmawardhana et al., 1992
If this scenario were true, AC elements correlate with a strong expression of G-lignin genes. Furthermore, COMT and F5H would have been recruited specifically into the S branch during the evolution of angiosperms because no S lignin is made in gymnosperms. As a consequence, a putatively S-specific alcohol dehydrogenase, as identified in poplar (Li et al., 2001
Growing evidence suggests that cytochrome P450 enzymes provide membrane anchors in the ER for assembling multienzyme complexes involved in metabolic channeling within the phenylpropanoid pathway (Wagner and Hrazdina, 1984
Among the three P450 enzyme families of the pathway (C4H, C3H, and F5H), C4H, C3H1, F5H1, and F5H2 have a well-conserved membrane-anchoring region, in agreement with their proposed localization in the ER membrane (Ro et al., 2001
In addition, CCoAOMT3 contains also a putative ER-targeting signal. Besides membrane association, this could also imply a vacuolar or extracellular localization of this enzyme. Sinapoyl-Glc:malate sinapoyltransferase (SMT) and sinapoyl-Glc:choline sinapoyltransferase, involved in modification of sinapoyl-Glc, have been identified as proteins with an ER-targeting peptide (Lehfeldt et al., 2000
Finally, a putative myristoylation site was detected in COMT, possibly involved in membrane anchoring. In agreement with this finding, a fraction of COMT from alfalfa stem was shown to be associated with the microsomal membranes, and channeling by COMT and F5H was suggested from coniferaldehyde to sinapaldehyde in the S branch of the monolignol pathway (Guo et al., 2002
The number of candidate monolignol biosynthesis genes found in the Arabidopsis genome varies greatly among the gene family studied, ranging from single genes to large gene families. A complex history of gene duplications has caused the expansion and diversification of the respective gene families. Interestingly, the polyploidy event, estimated to have occurred 24 to 86 million years ago, that marks the evolutionary history of Arabidopsis (Simillion et al., 2002 In conclusion, the genome-wide analysis of monolignol biosynthesis genes, as presented here, provides the foundation of the next steps in unraveling the monolignol pathway. The combination of reverse genetics with transcript and metabolite profiling analyses of the respective mutants will profoundly enlarge our understanding of this pathway and its relation with plant development.
Annotation For each of the 10 enzymes of the monolignol biosynthetic pathway, the corresponding genes were annotated in four steps: (a) experimentally certified family members were collected from a variety of species, and a family-specific profile was created; (b) an Arabidopsis protein database was scanned with this profile; (c) true family members were selected; and (d) prediction on the selected genes was improved with information from different sources, such as cDNA and EST sequences and within-family sequence similarity.
More specifically, from a ClustalW protein alignment of experimentally certified family members from different plant species, a hidden Markov model-based profile was created using the HMMER package (Thompson et al., 1994
To delineate the gene family, several factors were taken into account. First, only HMMER hits with an E-value score below the default cutoff value (E = 10.0) were considered. Second, in most cases, a clear "drop" in the E-value score could be detected, indicating that sequences below this threshold did not fulfill the family model as well as did those above, thus providing a means to distinguish potential family members from false positives or—in the case of large superfamilies—genes of other subfamilies. This approach can potentially lead to wrong conclusions because of incomplete or biased sampling of the family. For this reason, a third method was applied, based on a phylogenetic analysis of the (super)family using the detected genes, close homologs, and more distantly related members of distinct, well-known (sub)families, retrieved from GenBank (Benson et al., 2003
For the family members selected through these three criteria, the automatic annotation was improved by using information from different sources. First, the public databases were searched using BLASTN (Altschul et al., 1997
For the annotation of the C4H, C3H, and F5H families, a substantial amount of information from the P450 databases (at http://www.biobase.dk/P450/p450.shtml and http://drnelson.utmem.edu/CytochromeP450.html) was used to improve the annotation. Prediction of myristoylation sites was done with the algorithm of Maurer-Stroh et al. (2002
The nonredundant protein database was scanned for homologous sequences using BLASTp (Altschul et al., 1997
Both strands of upstream regions (1,000 bp before the ATG codon or the distance between the previous gene and the ATG) and first and second introns of the genes were analyzed for regulatory elements with MatInspector (Quandt et al., 1995
A list of potentially interesting motifs was compiled on the basis of the following three criteria: the motif had to be (a) experimentally characterized, (b) implicated in transcriptional regulation of known genes in the monolignol biosynthesis pathway, and/or (c) involved in elicitor, wound, or pathogen response. The motifs (and their respective calculated random occurrences in the Arabidopsis genome) that passed these criteria were: for Arabidopsis, GCC box (1/73,000 bp), jasmonate- and ethylene-responsive element (1/1,239,000 bp), W box (1/2,300 bp; withdrawn from results because of its high random occurrence), and S box (1/24,000 bp), all responsive to elicitation, wounding, and pathogens (Rushton et al., 2002
Expression analysis was carried out in Arabidopsis ecotype Columbia plants. Seeds were surface sterilized and placed on Murashige and Skoog medium supplemented with 10 g L–1 Suc. After the seeds had undergone a cold treatment for homogenous germination (overnight at 4°C), they were exposed to 20°C, 50 µmol m–2 s–1 light intensity, and 70% relative humidity, under a 16-h-light/8-h-dark cycle. Fourteen days after germination, plants were transferred to soil and cultivated in a greenhouse. Conditions were as follows: 23°C, 50 µmol m–2 s–1 light intensity at plant level (MBFR/U 400 W incandescent lamps; Philips, Eindhoven, The Netherlands), 40% relative humidity, and a 16-h-light/8-h-dark cycle, without shielding from incident day light. Material was harvested from a number of plants (within brackets) and pooled: seedling leaves and roots of 14-d-old in vitro plants (n = 100); rosette leaves, flowers, and green siliques of 7-week-old plants (n = 50); and inflorescence stems at 1-, 3-, 5-, 10-, 15-, and 20 cm length (n = 20 for 1, 3, and 5 cm; n = 10 for all later stages). At 20 cm, the stems were fully grown.
Total RNA was extracted with a LiCl method according to Goormachtig et al. (1995
Data on size and nature of EST libraries were obtained from http://www.ncbi.nlm.nih.gov/UniLib/, http://www.ncbi.nlm.nih.gov/Entrez/, and additionally for the RIKEN Arabidopsis full-length cDNA clones from Seki et al. (2002
The authors would like to thank Vincent Thareau for the gene-specific primer design; Pierre Rouzé for helpful discussions; Gunnar von Heijne for help in the interpretation of the TargetP results; Clint Chapple, Lise Jouanin,Michael Walter, and Carine Serizet for sharing unpublished data; Vanessa Hostyn for excellent technical assistance; Stephane Rombauts for help with promoter analysis; and Martine de Cock for help in preparing the manuscript. Received May 6, 2003; returned for revision July 11, 2003; accepted August 18, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.026484.
During the review process, another study on lignification genes in Arabidopsis was published by Goujon et al. (Goujon T, Sibout R, Eudes A, MacKay J, Jouanin L (2003
1 This work was supported in part by the European Commission programs EDEN (grant no. QLK5–CT–2001–00443) and by the Fund for Scientific Research–Flanders (postdoctoral fellowship to A.R.).
[w] The online version of this article contains Web-only data.
2 These authors contributed equally to the paper. * Corresponding author; e-mail wout.boerjan{at}psb.ugent.be; fax 32–9–331–3809.
Achnine L, Rasmussen S, Blancaflor E, Dixon RA (2002) Metabolic channeling at the entry point into the phenylpropanoid pathway: physical association between L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase. In I El Hadrami, ed, Proceedings of the XXI International Conference on Polyphenols, Imprimerie El-Watania, Marrakech, Morocco, pp 7–8 AGI (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408: 796–815[CrossRef][Medline]
Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402
Anterola AM, Jeon J-H, Davin LB, Lewis NG (2002) Transcriptional control of monolignol biosynthesis in Pinus taeda: factors affecting monolignol ratios and carbon allocation in phenylpropanoid metabolism. J Biol Chem 277: 18272–18280 Anterola AM, Lewis NG (2002) Trends in lignin modification: a comprehensive analysis of the effects of genetic manipulations/mutations on lignification and vascular integrity. Phytochemistry 61: 221–294[CrossRef][Web of Science][Medline]
Bate NJ, Orr J, Ni W, Meromi A, Nadler-Hassar T, Doerner PW, Dixon RA, Lamb CJ, Elkind Y (1994) Quantitative relationship between phenylalanine ammonia-lyase levels and phenylpropanoid accumulation in transgenic tobacco identifies a rate-determining step in natural product synthesis. Proc Natl Acad Sci USA 91: 7608–7612 Bell-Lelong DA, Cusumano JC, Meyer K, Chapple C (1997) Cinnamate-4-hydroxylase expression in Arabidopsis. Plant Physiol 113: 729–738[Abstract]
Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Wheeler DL (2003) GenBank. Nucleic Acids Res 31: 23–27 Betz C, McCollum TG, Mayer RT (2001) Differential expression of two cinnamate 4-hydroxylase genes in "Valencia" orange (Citrus sinensis Osbeck). Plant Mol Biol 46: 741–748[CrossRef][Web of Science][Medline]
Blanc G, Hokamp K, Wolfe KH (2003) A recent polyploidy superimposed on older large-scale duplications in the Arabidopsis genome. Genome Res 13: 137–144 Boerjan W, Ralph J, Baucher M (2003) Lignin biosynthesis. Annu Rev Plant Biol 54: 519–546[CrossRef][Medline]
Borevitz JO, Xia Y, Blount J, Dixon RA, Lamb C (2000) Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. Plant Cell 12: 2383–2393 Bowers JE, Chapman BA, Rong J, Paterson AH (2003) Unravelling angiosperm genome evolution by phylogenetic analysis of chromosomal duplication events. Nature 422: 433–438[CrossRef][Medline] Brill EM, Abrahams S, Hayes CM, Jenkins CLD, Watson JM (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[CrossRef][Web of Science][Medline] Chapple C (1998) Molecular-genetic analysis of plant cytochrome P450-dependent monooxygenases. Annu Rev Plant Physiol Plant Mol Biol 49: 311–343[CrossRef][Web of Science] Chapple CCS, Shirley BW, Zook M, Hammerschmidt R, Somerville SC (1994) Secondary metabolism in Arabidopsis. In EM Meyerowitz, CR Somerville, eds, Arabidopsis, Cold Spring Harbor Monograph Series, Vol 27. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 989–1030
Chapple CCS, Vogt T, Ellis BE, Somerville CR (1992) An Arabidopsis mutant defective in the general phenylpropanoid pathway. Plant Cell 4: 1413–1424
Chen C, Meyermans H, Burggraeve B, De Rycke RM, Inoue K, De Vleesschauwer V, Steenackers M, Van Montagu MC, Engler GJ, Boerjan WA (2000) Cell-specific and conditional expression of caffeoyl-CoA O-methyltransferase in poplar. Plant Physiol 123: 853–867 Chen F, Kota P, Blount JW, Dixon RA (2001) Chemical syntheses of caffeoyl and 5-OH coniferyl aldehydes and alcohols and determination of lignin O-methyltransferase activities in dicot and monocot species. Phytochemistry 58: 1035–1042[Medline] Chen M, McClure JW (2000) Altered lignin composition in phenylalanine ammonia-lyase-inhibited radish seedlings: implications for seed-derived sinapoyl esters as lignin precursors. Phytochemistry 53: 365–370[Medline] Chen W, Chao G, Singh KB (1996) The promoter of a H2O2-inducible, Arabidopsis glutathione S-transferase gene contains closely linked OBF- and OBP1-binding sites. Plant J 10: 955–966[CrossRef][Web of Science][Medline] Cramer CL, Edwards K, Dron M, Liang X, Dildine SL, Bolwell GP, Dixon RA, Lamb CJ, Schuch W (1989) Phenylalanine ammonia-lyase gene organization and structure. Plant Mol Biol 12: 367–383 Cukovic D, Ehlting J, VanZiffle JA, Douglas CJ (2001) Structure and evolution of 4-coumarate:coenzyme A ligase (4CL) gene families. Biol Chem 382: 645–654[CrossRef][Web of Science][Medline] Czichi U, Kindl H (1977) Phenylalanine ammonia-lyase and cinnamic acid hydroxylase as assembled consecutive enzymes on microsomal membranes of cucumber cotyledons: co-operation and subcellular distribution. Planta 134: 133–143[CrossRef][Web of Science] Dharmawardhana DP, Ellis BE, Carlson JE (1992) Characterization of vascular lignification in Arabidopsis thaliana. Can J Bot 70: 2238–2244[CrossRef] Dixon RA, Chen F, Guo D, Parvathi K (2001) The biosynthesis of monolignols: a "metabolic grid", or independent pathways to guaiacyl and syringyl units? Phytochemistry 57: 1069–1084[CrossRef][Web of Science][Medline] Donaldson LA (2001) Lignification and lignin topochemistry: an ultrastructural view. Phytochemistry 57: 859–873[CrossRef][Web of Science][Medline]
Eddy SR (1998) Profile hidden Markov models. Bioinformatics 14: 755–763 Ehlting J, Büttner D, Wang Q, Douglas CJ, Somssich IE, Kombrink E (1999) Three 4-coumarate:coenzyme A ligases in Arabidopsis thaliana represent two evolutionarily divergent classes in angiosperms. Plant J 19: 9–20[CrossRef][Web of Science][Medline] Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300: 1005–1016[CrossRef][Web of Science][Medline] Ermolaeva MD, Wu M, Eisen JA, Salzberg SL (2003) The age of the Arabidopsis thaliana genome duplication. Plant Mol Biol 51: 859–866[CrossRef][Web of Science][Medline]
Florea L, Hartzell G, Zhang Z, Rubin GM, Miller W (1998) A computer program for aligning a cDNA sequence with a genomic DNA sequence. Genome Res 8: 967–974 Franke R, Hemm MR, Denault JW, Ruegger MO, Humphreys JM, Chapple C (2002a) Changes in secondary metabolism and deposition of an unusual lignin in the ref8 mutant of Arabidopsis. Plant J 30: 47–59[CrossRef][Web of Science][Medline] Franke R, Humphreys JM, Hemm MR, Denault JW, Ruegger MO, Cusumano JC, Chapple C (2002b) The Arabidopsis REF8 gene encodes the 3-hydroxylase of phenylpropanoid metabolism. Plant J 30: 33–45[CrossRef][Web of Science][Medline] Goormachtig S, Valerio-Lepiniec M, Szczyglowski K, Van Montagu M, Holsters M, de Bruijn FJ (1995) Use of differential display to identify novel Sesbania rostrata genes enhanced by Azorhizobium caulinodans infection. Mol Plant-Microbe Interact 8: 816–824[Web of Science][Medline] Goujon T, Sibout R, Pollet B, Maba B, Nussaume L, Bechtold N, Lu F, Ralph J, Mila I, Barrière Y et al. (2003) A new Arabidopsis thaliana mutant deficient in the expression of O-methyltransferase: I. Impact on lignins and on sinapoyl esters. Plant Mol Biol 51: 973–989[Medline] Grimmig B, Matern U (1997) Structure of the parsley caffeoyl-CoA O-methyltransferase gene, harbouring a novel elicitor responsive cis-acting element. Plant Mol Biol 33: 323–341[CrossRef][Web of Science][Medline] Guo D, Chen F, Dixon RA (2002) Monolignol biosynthesis in microsomal preparations from lignifying stems of alfalfa (Medicago sativa L.). Phytochemistry 61: 657–667[Medline] Guo D, Chen F, Wheeler J, Winder J, Selman S, Peterson M, Dixon RA (2001) Improvement of in-rumen digestibility of alfalfa forage by genetic manipulation of lignin O-methyltransferases. Transgenic Res 10: 457–464[CrossRef][Web of Science][Medline] Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41: 95–98
Harding SA, Leshkevich J, Chiang VL, Tsai C-J (2002) Differential substrate inhibition couples kinetically distinct 4-coumarate:coenzyme A ligases with spatially distinct metabolic roles in quaking aspen. Plant Physiol 128: 428–438 Hatton D, Sablowski R, Yung M-H, Smith C, Schuch W, Bevan M (1995) Two classes of cis sequences contribute to tissue-specific expression of a PAL2 promoter in transgenic tobacco. Plant J 7: 859–876[CrossRef][Web of Science][Medline] Hauffe KD, Lee SP, Subramaniam R, Douglas CJ (1993) Combinatorial interactions between positive and negative cis-acting elements control spatial patterns of 4CL1 expression in transgenic tobacco. Plant J 4: 235–253[CrossRef][Web of Science][Medline]
Hauffe KD, Paszkowski U, Schulze-Lefert P, Hahlbrock K, Dangl JL, Douglas CJ (1991) A parsley 4CL1 promoter fragment specifies complex expression patterns in transgenic tobacco. Plant Cell 3: 435–443
Hoffmann L, Maury S, Martz F, Geoffroy P, Legrand M (2003) Purification, cloning and properties of an acyltransferase controlling shikimate and quinate ester intermediates in phenylpropanoid metabolism. J Biol Chem 278: 95–103
Hu W-J, Kawaoka A, Tsai C-J, Lung J, Osakabe K, Ebinuma H, Chiang VL (1998) Compartmentalized expression of two structurally and functionally distinct 4-coumarate:CoA ligase genes in aspen (Populus tremuloides). Proc Natl Acad Sci USA 95: 5407–5412 Humphreys JM, Chapple C (2002) Rewriting the lignin roadmap. Curr Opin Plant Biol 5: 224–229[CrossRef][Web of Science][Medline]
Humphreys JM, Hemm MR, Chapple C (1999) New routes for lignin biosynthesis defined by biochemical characterization of recombinant ferulate 5-hydroxylase, a multifunctional cytochrome P450-dependent monooxygenase. Proc Natl Acad Sci USA 96: 10045–10050 Jin H, Cominelli E, Bailey P, Parr A, Mehrtens F, Jones J, Tonelli C, Weisshaar B, Martin C (2000) Transcriptional repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis. EMBO J 19: 6150–6161[CrossRef][Web of Science][Medline] Jones L, Ennos AR, Turner SR (2001) Cloning and characterization of irregular xylem4 (irx4): a severely lignin-deficient mutant of Arabidopsis. Plant J 26: 205–216[CrossRef][Web of Science][Medline] Kiedrowski S, Kawalleck P, Hahlbrock K, Somssich IE, Dangl JL (1992) Rapid activation of a novel plant defense gene is strictly dependent on the Arabidopsis RPM1 disease resistance locus. EMBO J 11: 4677–4684[Web of Science][Medline]
Krawczyk S, Thurow C, Niggeweg R, Gatz C (2002) Analysis of the spacing between the two palindromes of activation sequence-1 with respect to binding to different TGA factors and transcriptional activation potential. Nucleic Acids Res 30: 775–781 Kühnl T, Koch U, Heller W, Wellmann E (1987) Chlorogenic acid biosynthesis: characterization of a light-induced microsomal 5-O-(4-coumaroyl)-D-quinate/shikimate 3'-hydroxylase from carrot (Daucus carota L.) cell suspension cultures. Arch Biochem Biophys 258: 226–232[CrossRef][Web of Science][Medline] Lacombe E, Van Doorsselaere J, Boerjan W, Boudet AM, Grima-Pettenati J (2000) Characterization of cis-elements required for vascular expression of the Cinnamoyl CoA Reductase gene and for protein-DNA complex formation. Plant J 23: 663–676[CrossRef][Web of Science][Medline] Lauvergeat V, Lacomme C, Lacombe E, Lasserre E, Roby D, Grima-Pettenati J (2001) Two cinnamoyl-CoA reductase (CCR) genes from Arabidopsis thaliana are differentially expressed during development and in response to infection with pathogenic bacteria. Phytochemistry 57: 1187–1195[CrossRef][Web of Science][Medline] Lee D, Ellard M, Wanner LA, Davis KR, Douglas CJ (1995) The Arabidopsis thaliana 4-coumarate:CoA ligase (4CL) gene: stress and developmentally regulated expression and nucleotide sequence of its cDNA. Plant Mol Biol 28: 871–884[CrossRef][Web of Science][Medline] Lee D, Meyer K, Chapple C, Douglas CJ (1997) Antisense suppression of 4-coumarate:coenzyme A ligase activity in Arabidopsis leads to altered lignin subunit composition. Plant Cell 9: 1985–1998[Abstract]
Lehfeldt C, Shirley AM, Meyer K, Ruegger MO, Cusumano JC, Viitanen PV, Strack D, Chapple C (2000) Cloning of the SNG1 gene of Arabidopsis reveals a role for a serine carboxypeptidase-like protein as an acyltransferase in secondary metabolism. Plant Cell 12: 1295–1306
Lescot M, Déhais P, Moreau Y, Van de Peer Y, Rouzé P, Rombauts S (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res 30: 325–327 Lewis NG, Yamamoto E (1990) Lignin: occurrence, biogenesis, and biodegradation. Annu Rev Plant Physiol Plant Mol Biol 41: 455–496[CrossRef][Web of Science][Medline] Leyva A, Jarillo JA, Salinas J, Martinez-Zapater JM (1995) Low temperature induces the accumulation of phenylalanine ammonia-lyase and chalcone synthase mRNAs of Arabidopsis thaliana in a light-dependent manner. Plant Physiol 108: 39–46[Abstract]
Leyva A, Liang X, Pintor-Toro JA, Dixon RA, Lamb CJ (1992) cis-Element combinations determine phenylalanine ammonia-lyase gene tissue-specific expression patterns. Plant Cell 4: 263–271
Li L, Cheng XF, Leshkevich J, Umezawa T, Harding SA, Chiang VL (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 Li L, Osakabe Y, Joshi CP, Chiang VL (1999) Secondary xylem-specific expression of caffeoyl-coenzyme A 3-O-methyltransferase plays an important role in the methylation pathway associated with lignin biosynthesis in loblolly pine. Plant Mol Biol 40: 555–565[CrossRef][Web of Science][Medline]
Li L, Popko JL, Umezawa T, Chiang VL (2000) 5-Hydroxyconiferyl aldehyde modulates enzymatic methylation for syringyl monolignol formation, a new view of monolignol biosynthesis in angiosperms. J Biol Chem 275: 6537–6545
Li L, Popko JL, Zhang X-H, Osakabe K, Tsai C-J, Joshi CP, Chiang VL (1997) A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine. Proc Natl Acad Sci USA 94: 5461–5466
Lindermayr C, Fliegmann J, Ebel J (2003) Deletion of a single amino acid residue from different 4-coumarate-CoA ligases from soybean results in the generation of new substrate specificities. J Biol Chem 278: 2781–2786 Lindsay WP, McAlister FM, Zhu Q, He X-Z, Dröge-Laser W, Hedrick S, Doerner P, Lamb C, Dixon RA (2002) KAP-2, a protein that binds to the H-box in a bean chalcone synthase promoter, is a novel plant transcription factor with sequence identity to the large subunit of human Ku autoantigen. Plant Mol Biol 49: 503–514[CrossRef][Web of Science][Medline]
Loake GJ, Faktor O, Lamb CJ, Dixon RA (1992) Combination of H-box [CCTACC(N)7CT] and G-box [CACGTG] cis elements is necessary for feed-forward stimulation of a chalcone synthase promoter by the phenylpropanoid-pathway intermediate p-coumaric acid. Proc Natl Acad Sci USA 89: 9230–9234
Logemann E, Parniske M, Hahlbrock K (1995) Modes of expression and common structural features of the complete phenylalanine ammonialyase gene family in parsley. Proc Natl Acad Sci USA 92: 5905–5909 Logemann E, Reinold S, Somssich IE, Hahlbrock K (1997) A novel type of pathogen defense-related cinnamyl alcohol dehydrogenase. Biol Chem 378: 909–913[Web of Science][Medline] Lois R, Dietrich A, Hahlbrock K, Schulz W (1989) A phenylalanine ammonia-lyase gene from parsley: structure, regulation and identification of elicitor and light responsive cis-acting elements. EMBO J 8: 1641–1648[Web of Science][Medline]
Lu SX, Hrabak EM (2002) An Arabidopsis calcium-dependent protein kinase is associated with the endoplasmic reticulum. Plant Physiol 128: 1008–1021
Lukashin AV, Borodovsky M (1998) GeneMark.hmm: new solutions for gene finding. Nucleic Acids Res 26: 1107–1115 Maurer-Stroh S, Eisenhaber B, Eisenhaber F (2002) N-terminal N-myristoylation of proteins: prediction of substrate proteins from amino acid sequence. J Mol Biol 317: 541–557[CrossRef][Web of Science][Medline]
Maury S, Geoffroy P, Legrand M (1999) Tobacco O-methyltransferases involved in phenylpropanoid metabolism: the different caffeoyl-coenzyme A/5-hydroxyferuloyl-coenzyme A 3/5-O-methyltransferase and caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase classes have distinct substrate specificities and expression patterns. Plant Physiol 121: 215–223 Maxwell CA, Harrison MJ, Dixon RA (1993) Molecular characterization and expression of alfalfa isoliquiritigenin 2'-O-methyltransferase, an enzyme specifically involved in the biosynthesis of an inducer of Rhizobium meliloti nodulation genes. Plant J 4: 971–981[CrossRef][Medline]
Meyer K, Cusumano JC, Somerville C, Chapple CCS (1996) Ferulate-5-hydroxylase from Arabidopsis thaliana defines a new family of cytochrome P450-dependent monooxygenases. Proc Natl Acad Sci USA 93: 6869–6874
Meyer K, Shirley AM, Cusumano JC, Bell-Lelong DA, Chapple C (1998) Lignin monomer composition is determined by the expression of a cytochrome P450-dependent monooxygenase in Arabidopsis. Proc Natl Acad Sci USA 95: 6619–6623
Meyermans H, Morreel K, Lapierre C, Pollet B, De Bruyn A, Busson R, Herdewijn P, Devreese B, Van Beeumen J, Marita JM et al. (2000) Modification in lignin and accumulation of phenolic glucosides in poplar xylem upon down-regulation of caffeoyl-coenzyme A O-methyltransferase, an enzyme involved in lignin biosynthesis. J Biol Chem 275: 36899–36909 Mizutani M, Ohta S, Sato R (1997) Isolation of a cDNA and a genomic clone encoding cinnamate 4-hydroxylase from Arabidopsis and its expression manner in planta. Plant Physiol 113: 755–763[Abstract]
Nair RB, Joy RW IV, Kurylo E, Shi X, Schnaider J, Datla RSS, Keller WA, Selvaraj G (2000) Identification of a CYP84 family of cytochrome P450-dependent mono-oxygenase genes in Brassica napus and perturbation of their expression for engineering sinapine reduction in the seeds. Plant Physiol 123: 1623–1634
Nair RB, Xia Q, Kartha CJ, Kurylo E, Hirji RN, Datla R, Selvaraj G (2002) Arabidopsis CYP98A3 mediating aromatic 3-hydroxylation: developmental regulation of the gene, and expression in yeast. Plant Physiol 130: 210–220 Nambara E, McCourt P (1999) Protein farnesylation: a greasy tale. Curr Opin Plant Biol 2: 388–392[CrossRef][Web of Science][Medline] Nedelkina S, Jupe SC, Blee KA, Schalk M, Werck-Reichert D, Bolwell GP (1999) Novel characteristics and regulation of a divergent cinnamate 4-hydroxylase (CYP73A15) from French bean: engineering expression in yeast. Plant Mol Biol 39: 1079–1090[CrossRef][Web of Science][Medline] Neustaedter D, Lee SP, Douglas CJ (1999) A novel parsley 4CL cis-element is required for developmentally regulated expression and protein-DNA complex formation. Plant J 18: 77–88[CrossRef][Web of Science][Medline]
Ohl S, Hedrick SA, Chory J, Lamb CJ (1990) Functional properties of a phenylalanine ammonia-lyase promoter from Arabidopsis. Plant Cell 2: 837–848
Osakabe K, Tsao CC, Li L, Popko JL, Umezawa T, Carraway DT, Smeltzer RH, Joshi CP, Chiang VL (1999) Coniferyl aldehyde 5-hydroxylation and methylation direct syringyl lignin biosynthesis in angiosperms. Proc Natl Acad Sci USA 96: 8955–8960
Pakusch A-E, Matern U, Schiltz E (1991) Elicitor-inducible caffeoyl-coenzyme A 3-O-methyltransferase from Petroselinum crispum cell suspensions: purification, partial sequence, and antigenicity. Plant Physiol 95: 137–143 Parvathi K, Chen F, Guo D, Blount DW, Dixon RA (2001) Substrate preferences of O-methyltransferases in alfalfa suggest new pathways for 3-O-methylation of monolignols. Plant J 25: 193–202[CrossRef][Web of Science][Medline] Pellegrini L, Geoffroy P, Fritig B, Legrand M (1993) Molecular cloning and expression of a new class of ortho-diphenol-O-methyltransferases induced in tobacco (Nicotiana tabacum L.) leaves by infection or elicitor treatment. Plant Physiol 103: 509–517[Abstract] Pinçon G, Maury S, Hoffmann L, Geoffroy P, Lapierre C, Pollet B, Legrand M (2001) Repression of O-methyltransferase genes in transgenic tobacco affects lignin synthesis and plant growth. Phytochemistry 57: 1167–1176[CrossRef][Web of Science][Medline] Pontier D, Balagué C, Bezombes-Marion I, Tronchet M, Deslandes L, Roby D (2001) Identification of a novel pathogen-responsive element in the promoter of the tobacco gene HSR203J, a molecular marker of the hypersensitive response. Plant J 26: 495–507[CrossRef][Web of Science][Medline]
Quandt K, Frech K, Karas H, Wingender E, Werner T (1995) MtaInd and MatInspector: new fast and versatile tools for detection of consensus matches in nucleotide sequence data. Nucleic Acids Res 23: 4878–4884 Raes J, Van de Peer Y (1999) ForCon: a software tool for the conversion of sequence alignments. EMBnet.news 6; http://www.ebi.ac.uk/embnet.news/vol6_1 Raes J, Vandepoele K, Saeys Y, Simillion C, Van de Peer Y (2003) Investigating ancient duplication events in the Arabidopsis genome. J Struct Funct Genom 3: 117–129 Ralph J, Lapierre C, Marita JM, Kim H, Lu F, Hatfield RD, Ralph S, Chapple C, Franke R, Hemm MR et al. (2001) Elucidation of new structures in lignins of CAD- and COMT-deficient plants by NMR. Phytochemistry 57: 993–1003[CrossRef][Web of Science][Medline] Randall SK, Crowell DN (1999) Protein isoprenylation in plants. Crit Rev Biochem Mol Biol 34: 325–338[CrossRef][Web of Science][Medline]
Rasmussen S, Dixon RA (1999) Transgene-mediated and elicitor-induced perturbation of metabolic channeling at the entry point into the phenylpropanoid pathway. Plant Cell 11: 1537–1551
Ro DK, Mah N, Ellis BE, Douglas CJ (2001) Functional characterization and subcellular localization of poplar (Populus trichocarpa x Populus deltoides) cinnamate 4-hydroxylase. Plant Physiol 126: 317–329
Ruegger M, Meyer K, Cusumano JC, Chapple C (1999) Regulation of ferulate-5-hydroxylase expression in Arabidopsis in the context of sinapate ester biosynthesis. Plant Physiol 119: 101–110
Rushton PJ, Reinstädler A, Lipka V, Lippok B, Somssich IE (2002) Synthetic plant promoters containing defined regulatory elements provide novel insights into pathogen- and wound-induced signaling. Plant Cell 14: 749–762
Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajandream M-A, Barrell B (2000) Artemis: sequence visualization and annotation. Bioinformatics 16: 944–945
Sablowski RWM, Baulcombe DC, Bevan M (1995) Expression of a flower-specific Myb protein in leaf cells using a viral vector causes ectopic activation of a target promoter. Proc Natl Acad Sci USA 92: 6901–6905 Schiex T, Moisan A, Rouzé P (2001) EUGÈNE: an eukaryotic gene finder that combines several sources of evidence. Lect Notes Comput Sci 2066: 111–125[CrossRef]
Schmidt HA, Strimmer K, Vingron M, von Haeseler A (2002) TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics 18: 502–504
Schneider K, Hovel K, Witzel K, Hamberger B, Schomburg D, Kombrink E, Stuible HP (2003) The substrate specificity-determining amino acid code of 4-coumarate:CoA ligase. Proc Natl Acad Sci USA 100: 8601–8606
Schoch G, Goepfert S, Morant M, Hehn A, Meyer D, Ullmann P, Werck-Reichhart D (2001) CYP98A3 from Arabidopsis thaliana is a 3'-hydroxylase of phenolic esters, a missing link in the phenylpropanoid pathway. J Biol Chem 276: 36566–36574 Séguin A, Laible G, Leyva A, Dixon RA, Lamb CJ (1997) Characterization of a gene encoding a DNA-binding protein that interacts in vitro with vascular specific cis elements of the phenylalanine ammonia-lyase promoter. Plant Mol Biol 35: 281–291[CrossRef][Web of Science][Medline] Seki H, Ichinose Y, Kato H, Shiraishi T, Yamada T (1996) Analysis of cis-regulatory elements involved in the activation of a member of chalcone synthase gene family (PsChs1) in pea. Plant Mol Biol 31: 479–491[CrossRef][Web of Science][Medline]
Seki M, Narusaka M, Kamiya A, Ishida J, Satou M, Sakurai T, Nakajima M, Enju A, Akiyama K, Oono Y et al. (2002) Functional annotation of a full-length Arabidopsis cDNA collection. Science 296: 141–145 Sewalt VJH, Ni W, Blount JW, Jung HG, Masoud SA, Howles PA, Lamb C, Dixon RA (1997) Reduced lignin content and altered lignin composition in transgenic tobacco down-regulated in expression of L-phenylalanine ammonia-lyase or cinnamate 4-hydroxylase. Plant Physiol 115: 41–50[Abstract]
Shah J, Klessig DF (1996) Identification of a salicylic acid-responsive element in the promoter of the tobacco pathogenesis-related Shirley AM, McMichael CM, Chapple C (2001) The sng2 mutant of Arabidopsis is defective in the gene encoding the serine carboxypeptidase-like protein sinapoylglucose:choline sinapoyltransferase. Plant J 28: 83–94[CrossRef][Web of Science][Medline]
Sibout R, Eudes A, Pollet B, Goujon T, Mila I, Granier F, Seguin A, Lapierre C, Jouanin L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol dehydrogenases in Arabidopsis: isolation and characterization of the corresponding mutants. Plant Physiol 132: 848–860
Simillion C, Vandepoele K, Van Montagu M, Zabeau M, Van de Peer Y (2002) The hidden duplication past of Arabidopsis thaliana. Proc Natl Acad Sci USA 99: 13627–13632
Somssich IE, Wernert P, Kiedrowski S, Hahlbrock K (1996) Arabidopsis thaliana defense-related protein ELI3 is an aromatic alcohol:NADP+ oxidoreductase. Proc Natl Acad Sci USA 93: 14199–14203 Strack D, Sharma V (1985) Vacuolar localization of the enzymatic synthesis of hydroxycinnamic acid esters of malic acid in protoplasts from Raphanus sativus leaves. Physiol Plant 65: 45–50
Sugimoto K, Takeda S, Hirochika H (2000) MYB-related transcription factor NtMYB2 induced by wounding and elicitors is a regulator of the tobacco retrotransposon Tto1 and defense-related genes. Plant Cell 12: 2511–2527
Takeshita N, Fujiwara H, Mimura H, Fitchen JH, Yamada Y, Sato F (1995) Molecular cloning and characterization of S-adenosyl-L-methionine: scoulerine-9-O-methyltransferase from cultured cells of Coptis japonica. Plant Cell Physiol 36: 29–36
Tamagnone L, Merida A, Parr A, Mackay S, Culianez-Macia FA, Roberts K, Martin C (1998) The AmMYB308 and AmMYB330 transcription factors from Antirrhinum regulate phenylpropanoid and lignin biosynthesis in transgenic tobacco. Plant Cell 10: 135–154 Tavares R, Aubourg S, Lecharny A, Kreis M (2000) Organization and structural evolution of four multigene families in Arabidopsis thaliana: AtLCAD, AtLGT, AtMYST and AtHD-GL2. Plant Mol Biol 42: 703–717[CrossRef][Web of Science][Medline] Terashima N, Fukushima K, Tsuchiya S (1986) Heterogeneity in formation of lignin: VII. An autoradiographic study on the formation of guaiacyl and syringyl lignin in poplar. J Wood Chem Technol 6: 495–504 Thompson GA Jr, Okuyama H (2000) Lipid-linked proteins in plants. Prog Lipid Res 39: 19–39[CrossRef][Web of Science][Medline]
Thompson JD, Higgins DG, Gibson TJ (1994) ClustalW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673–4680
Urban P, Mignotte C, Kazmaier M, Delorme F, Pompon D (1997) Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-cytochrome P450 reductases with P450 CYP73A5. J Biol Chem 272: 19176–19186
Van de Peer Y, De Wachter R (1994) TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput Appl Biosci 10: 569–570 Vernon DM, Bohnert HJ (1992) A novel methyl transferase induced by osmotic stress in the facultative halophyte Mesembryanthemum crystallinum. EMBO J 11: 2077–2085[Web of Science][Medline]
Wagner GJ, Hrazdina G (1984) Endoplasmic reticulum as a site of phenylpropanoid and flavonoid metabolism in Hippeastrum. Plant Physiol 74: 901–906 Wanner LA, Mittal S, Davis KR (1993) Recognition of the avirulence gene avrB from Pseudomonas syringae pv. glycinea by Arabidopsis thaliana. Mol Plant-Microbe Interact 6: 582–591[Web of Science][Medline]
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 Winkel-Shirley B (1999) Evidence for enzyme complexes in the phenylpropanoid and flavonoid pathways. Physiol Plant 107: 142–149[CrossRef] Ye Z-H, Kneusel RE, Matern U, Varner JE (1994) An alternative methylation pathway in lignin biosynthesis in Zinnia. Plant Cell 6: 1427–1439[Abstract] Yu LM, Lamb CJ, Dixon RA (1993) Purification and biochemical characterization of proteins which bind to the H-box cis-element implicated in transcriptional activation of plant defense genes. Plant J 3: 805–816[Web of Science][Medline] Zhang X-H, Chinnappa CC (1997) Molecular characterization of a cDNA encoding caffeoyl-coenzyme A 3-O-methyltransferase of Stellaria longipes. J Biosci 22: 161–175
Zhong R, Morrison WH III, Negrel J, Ye Z-H (1998) Dual methylation pathways in lignin biosynthesis. Plant Cell 10: 2033–2046 This article has been cited by other articles:
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ASPB Publications | PLANT PHYSIOLOGY® | THE PLANT CELL | |
|---|---|---|---|
TOP
ABSTRACT
RESULTS
-glucuronidase (GUS) revealed that the expression is located in the vascular tissues (Ohl et al., 1990
