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First published online May 20, 2005; 10.1104/pp.104.057950 Plant Physiology 138:1136-1148 (2005) © 2005 American Society of Plant Biologists An Expression and Bioinformatics Analysis of the Arabidopsis Serine Carboxypeptidase-Like Gene Family1,[w]Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907
The Arabidopsis (Arabidopsis thaliana) genome encodes a family of 51 proteins that are homologous to known serine carboxypeptidases. Based on their sequences, these serine carboxypeptidase-like (SCPL) proteins can be divided into several major clades. The first group consists of 21 proteins which, despite the function implied by their annotation, includes two that have been shown to function as acyltransferases in plant secondary metabolism: sinapoylglucose:malate sinapoyltransferase and sinapoylglucose:choline sinapoyltransferase. A second group comprises 25 SCPL proteins whose biochemical functions have not been clearly defined. Genes encoding representatives from both of these clades can be found in many plants, but have not yet been identified in other phyla. In contrast, the remaining SCPL proteins include five members that are similar to serine carboxypeptidases from a variety of organisms, including fungi and animals. Reverse transcription PCR results suggest that some SCPL genes are expressed in a highly tissue-specific fashion, whereas others are transcribed in a wide range of tissue types. Taken together, these data suggest that the Arabidopsis SCPL gene family encodes a diverse group of enzymes whose functions are likely to extend beyond protein degradation and processing to include activities such as the production of secondary metabolites.
Serine carboxypeptidases (SCPs) are members of the  / hydrolase family of proteins, which make use of a Ser-Asp-His catalytic triad to cleave the carboxyterminal peptide bonds of their protein or peptide substrates (Hayashi et al., 1973 , 1975; Bech and Breddam, 1989; Liao and Remington, 1990; Liao et al., 1992; Ollis et al., 1992). Proteins that contain this catalytic triad and are otherwise homologous to SCPs have been found in a variety of organisms (Doi et al., 1980; Kim and Hayashi, 1983; Breddam, 1986; Baulcombe et al., 1987; Galjart et al., 1988; Bradley, 1992; Degan et al., 1994; Endrizzi et al., 1994; Wajant et al., 1994; Jones et al., 1996; Li and Steffens, 2000). Many of these serine carboxypeptidase-like (SCPL) proteins have been annotated as peptidases based only on this shared sequence similarity; however, studies have shown that some of them function not as peptidases, but as acyltransferases and lyases (Wajant et al., 1994; Lehfeldt et al., 2000; Li and Steffens, 2000; Shirley et al., 2001). For example, several SCPL proteins have been shown to catalyze the production of plant secondary metabolites involved in herbivory defense and UV protection (Wajant et al., 1994; Lehfeldt et al., 2000; Li and Steffens, 2000), including the hydroxynitrile lyase from Sorghum bicolor (SbHNL) necessary for cyanogenesis, and the acyltransferases in wild tomato (Lycopersicon pennellii) that catalyze the formation 2,3,4-isobutyryl-Glc (Wajant et al., 1994; Lehfeldt et al., 2000). Two enzymes in Arabidopsis (Arabidopsis thaliana) responsible for sinapate ester biosynthesis are also SCPL proteins. Sinapoylglucose:malate sinapoyltransferase (SMT) catalyzes the formation of sinapoylmalate, a UV-protectant in seedlings (Landry et al., 1995; Lehfeldt et al., 2000) and sinapoylglucose:choline sinapoyltransferase (SCT) is required for the formation of sinapoylcholine in Arabidopsis seeds (Shirley et al., 2001).
Plants accumulate a broad array of secondary compounds and appear to have recruited many classes of proteins to serve in their synthesis and regulation. In the course of evolutionary history, the genes that encode these proteins have diversified to coordinate these metabolic pathways unique to plants. Examples that have been particularly well studied include the Cyt P450-dependent monooxygenases, MYB transcription factors, terpene synthases, and UDP-glucosyltransferases (Meyer et al., 1996 In this paper, we present a bioinformatics-based examination of the Arabidopsis SCPL genes and their encoded proteins. Inspection of the 51 SCPL genes demonstrated that many of them required reannotation. A phylogenetic analysis of the corresponding corrected protein sequences suggested that Arabidopsis SCPL proteins fall into several distinct classes. Extending this analysis to include SCPL proteins from other organisms reveals a division between plant-specific SCPL proteins and those SCPL proteins common to a broader spectrum of organisms. Together with an expression profile for the entire SCPL gene family generated using reverse transcription (RT)-PCR, these analyses may help to assign roles to members of the SCPL gene family that are as yet of unknown function.
The Arabidopsis Genome Encodes 51 SCPL Proteins
Annotation of the Arabidopsis genome has identified 53 putative SCPL genes. One of these (At2g22960) was previously identified as a pseudogene because the ATPase-like sequences upstream of the first exon suggest that this SCPL gene lacks a promoter (Lehfeldt et al., 2000
Based on a comparison of each RIKEN Arabidopsis full-length (RAFL) cDNA with the corresponding genomic sequence in GenBank, we found that eight SCPL genes are incorrectly annotated. The majority of the errors are located at splice junctions, resulting in incorrect exon lengths, the exclusion of entire exons, and the inclusion of portions of introns in the predicted mRNAs (Table I). Eight likely annotation errors in genes for which no cDNAs are currently available were identified as well, based on comparisons between the genes in question and other, closely related SCPL genes. For example, the predicted splice sites joining the penultimate and final exon of At1g73290 are in a slightly different position and alternate phase from the highly conserved exon splice sites of closely related Arabidopsis SCPL genes. Since inspection of the sequence of At1g73290 revealed alternate 5' and 3' intron splice sites in the same phase and position as the sites in these related genes, its annotation was revised. Similarly, the predicted protein sequence for At3g12240 contains an aberrant carboxyterminal region. Examination of the corresponding genomic sequence revealed that the 3' end of the penultimate exon of the gene was not identified correctly during annotation, resulting in the inferred translation continuing through the final intron until a stop codon was reached. Translation of the 3' untranslated region (UTR) revealed the presence of an additional exon virtually identical to the final exon of a closely related and physically linked SCPL protein (At3g12230); thus, the annotation of At3g12240 was updated accordingly. Conversely, exon 13 of gene At1g33540 appeared to be truncated when compared to those of related genes, and translation of the 5' end of the subsequent intron revealed a sequence similar to those found in other closely related SCPL genes. These findings suggested that the 3' end of the exon had been incorrectly predicted, and thus, a portion of the correct inferred sequence was missing from the database. A similar exon/intron boundary prediction error resulted in the extension of the 3' end of the first exon of SCPL gene At2g24000 (Table I). Finally, the inferred translation products of At2g24010, At2g35770, and At4g15100 appeared to be truncated at the N termini, and selection of an upstream in-frame start codon yielded an amino acid sequence more closely in agreement with other SCPL proteins. All subsequent analyses described here were performed using these 16 corrected sequences.
Arabidopsis SCPL Proteins Exhibit Conserved Sequence Motifs
In addition to the Ser-Asp-His catalytic triad, 15 amino acid residues are conserved in carboxypeptidase Y (CPY) and all Arabidopsis SCPL proteins. Of these, three align with residues that have been shown to be involved in substrate binding in CPY from yeast (Saccharomyces cerevisiae; Endrizzi et al., 1994
Most Arabidopsis SCPL Proteins Can Be Grouped into Two Major Clades
Phylogenetic analysis of the 51 Arabidopsis SCPL proteins showed that the majority of sequences cluster into two major clades, although there are five additional members that are less closely related (Fig. 1A). For the sake of convenience and brevity in subsequent discussion, these proteins have been numbered counterclockwise as they are presented in Figure 1, beginning from the top right of Clade I. Clade I can be further subdivided into Clades IA (SCPL proteins 1 through 19) and IB (SCPL proteins 20 and 21) based upon the degree of amino acid identity shared between these two subgroups. The SCPL proteins of Clade IA include the known acyltransferases SMT (SCPL protein 8) and SCT (SCPL protein 19; Fig. 1A; Lehfeldt et al., 2000
Several subclades of Clade IA are associated with clusters of SCPL genes that are tandemly arranged in the genome. For example, SCPL proteins 2 to 6 are between 79% and 86% identical, and their corresponding genes are located together at the bottom of chromosome I (Fig. 1B). Interestingly, SCPL gene 1 is located on chromosome 5, but its encoded protein is 96% identical to that of SCPL gene 2 on chromosome 1 (Fig. 2). The mRNA sequences of these genes are 97% identical, nine of their 12 introns are greater than 74% identical (e.g. intron 10 of SCPL gene 1 is 97% identical to intron 10 of SCPL gene 2), and their 5' and 3' UTRs are highly similar. Furthermore, this identity extends through the region downstream of the two genes to include a portion of the gene that follows SCPL gene 2 (At1g72290) duplicated in the region downstream of SCPL gene 1. Taken together, these data suggest that SCPL gene 1 represents a duplication of SCPL gene 2 onto chromosome 5. Another SCPL gene cluster includes genes 8 through 13 on chromosome II. This group includes SNG1 (SCPL gene 8), which encodes SMT (Fig. 1A). Given that proteins encoded by the genes flanking SNG1 are over 70% identical to SMT (Fig. 2), it is tempting to speculate that these enzymes may share some functional characteristics.
The pairwise percent identity between any two Clade IA SCPL proteins ranges from 51% to 96%, averaging 66% overall (Fig. 2). The exon/intron splicing patterns of the Clade IA SCPL genes are also alike, with the phase, number, and location of splice sites being identical for each SCPL gene with the exception of only two sites (Supplemental Fig. 1). Clade II SCPL proteins are more varied in sequence, with any two proteins being 41% identical on average (Fig. 2). In contrast to the 261 residues uniquely conserved among 75% of Clade IA members, only 56 amino acids meet the same criteria for Clade II proteins. The exon/intron splicing patterns of Clade II genes also show more variability, with fewer splice sites of the same phase and location conserved among them (Supplemental Fig. 1). SCPL proteins 20 and 21 (Clade IB) are between 32% and 38% identical to Clade IA SCPL proteins and 20% to 32% identical to Clade II members (Fig. 2), but among those shared residues are a high percentage of the residues conserved only among the Clade IA proteins (Supplemental Fig. 1). Further, all but two of the consensus intron/exon splice sites in the Clade IA genes are conserved in position and phase in SCPL genes 20 and 21. These observations suggest that they are more closely related to SCPL proteins 1 to 19 than the others, leading us to designate them as Clade IB (Fig. 1A).
To determine whether the clade structure observed for Arabidopsis SCPL proteins reflected the diversity and relationships between similar proteins in other plants, we included other plant SCPL proteins in a separate sequence analysis. In this analysis, the sequences of the isobutyryl-Glc acyltransferase from wild tomato and three similar enzymes from wild potato (GenBank accession nos. AF248647, AF006078, AF006079, and AF006080) were grouped into Clade IA. The pairwise identities between these individual proteins and most Clade I proteins range from 38% to 46%. Similarly, a wound/jasmonate-inducible SCPL protein from Lycopersicon esculentum (accession no. AF242849) is highly similar to SCPL proteins 20 and 21 from Arabidopsis with pairwise identity values of 62% and 66%, respectively (Moura et al., 2001
Several relatively well-studied SCPs and SCPL proteins from other plant species fell into Clade II when included in the phylogenetic analysis of the Arabidopsis SCPL proteins. CPDW-II (Breddam et al., 1987
Extending the analysis of SCPL proteins to sequences from organisms outside the plant kingdom revealed that homologs of SCPL proteins 47 to 51 are found in diverse eukaryotic lineages (Fig. 1C) and cluster into an additional four clades. Arabidopsis SCPL proteins 47 to 49 are most similar to CPY and an SCPL protein from Schizosaccharomyces pombe (YBR139W) in Clade IV (Fig. 1B; Endrizzi et al., 1994
To provide a foundation for determining the function of the Arabidopsis SCPL proteins, RT-PCR analyses were carried out using RNA extractions from eight different tissue types. These experiments revealed a diversity of expression patterns for Arabidopsis SCPL genes, even among members of the five SCPL gene clusters. For example, each member of the cluster on chromosome 1 (SCPL genes 2–6) is expressed in roots and seedlings, but SCPL gene 5 is also expressed in siliques, and SCPL gene 4 is expressed in all tissues surveyed (Fig. 3). The shared expression pattern in seedlings and roots exhibited by SCPL gene 1 on chromosome 5 is consistent with its recent duplication from SCPL gene 2 on chromosome 1.
The members of each of the other two Clade I gene clusters are also differentially expressed (Fig. 3). Consistent with previous findings (Lehfeldt et al., 2000 ), SCPL gene 8 (SNG1) is expressed in all tissue types. On the other hand, expression of SCPL gene 9 is below detectable limits in roots and stems, SCPL gene 10 shows positive results only for senescent leaves, and the PCR results for SCPL gene 11 suggest that it is highly expressed in leaves. The SCPL gene cluster on chromosome 3 likewise includes a gene expressed primarily in roots (SCPL gene 15), one expressed in all leaf stages examined (SCPL gene 16), and one expressed primarily in siliques (SCPL gene 17). The Clade II gene cluster on chromosome 3 containing SCPL genes 36, 37, and 39 exhibits the most uniformity of expression, with each gene expressed in roots, seedlings, flowers, and siliques; however, 36 is more limited in its expression than the other two. Thirteen SCPL genes appear to be expressed chiefly in single tissue types: two in senescent leaves (genes 7 and 10), four in roots (genes 3, 6, 15, and 18), two in siliques (genes 17 and 19), four in flowers (genes 11, 32, 36, and 41), and one in seedlings (gene 41; Fig. 3). Twenty-three of the 51 SCPL genes appear to be expressed in both roots and seedlings, an observation that may reflect the fact that the intact seedlings used for mRNA preparation included root tissue. In contrast, SCPL gene 41 is one of three genes expressed in seedlings but not in roots, the other two being 9 and 17. The data for these genes suggest that the actual site of their expression is either the hypocotyls or cotyledons.
Expression in all but one or two tissue types appears to be a characteristic of several SCPL genes. For instance, SCPL gene 20 is expressed in all tissues except young leaves, and 25 is not expressed in senescent leaves. In contrast to the consistently positive PCR results for the majority of SCPL genes, several of them yielded only negative PCR results: 13, 22, 23, 30, 42, and 43 (Fig. 3). Although we cannot exclude the possibility that some of these reactions did not yield product due to technical issues associated with primer design, it is likely that some or all of these genes are expressed only at low levels or in a highly tissue-specific manner, or only in response to abiotic or biotic stimuli that were not studied here. Finally, our expression data were in very good agreement with both Massively Parallel Signature Sequencing (MPSS) expression data (http://mpss.udel.edu/at/java.html; Brenner et al., 2000
Many Arabidopsis SCPL Proteins Are Misannotated
Although the advent of completely sequenced and annotated genomes has become an indispensable resource for the scientific community, independent verification of gene annotation is required to ensure database reliability. Within the 51 annotated genes analyzed in this study, most of the predicted splice sites appear to have been identified correctly. Given that the likelihood of a splice site annotation error within a gene is directly dependent upon the number of introns it contains, even a low error rate can still be problematic when studying genes with multiple introns. For example, misannotations were found in almost one-third of the SCPL genes examined, with many of the sequences containing more than one error. Moreover, many of the errors are substantial, resulting in large deletions, insertions, and truncations. Further, we identified several annotated SCPL genes that are almost certainly pseudogenes and an additional SCPL gene not annotated as such. These data demonstrate that careful reexamination of sequence annotation is necessary prior to detailed molecular and phylogenetic analyses, and that studies completed in the absence of this type of analysis are likely to contain a substantial number of errors. Errors of this type can be found in a previous analysis of the Arabidopsis SCPL protein family, in addition to three protein sequence duplications: SMT, SCT, and At1g73310 (Milkowski and Strack, 2004
Some of the common sequence characteristics of the Arabidopsis SCPL proteins may provide preliminary insights into structural features required for activity and/or subcellular localization. For example, the presence of the Ser-Asp-His triad in every SCPL protein suggests that some or all of them may employ a similar catalytic mechanism. Indeed, inhibition studies conducted with SMT and SCT indicate that the Ser residue is required for the catalytic activity of both enzymes (Lehfeldt et al., 2000
Two observations suggest that the six Cys residues present in almost all of the Arabidopsis SCPL proteins may be important in maintaining the tertiary structure of the functional enzymes. First, the six residues align with Cys in CPY that are known to form three disulfide bonds that are likely to contribute to the stability of the protein. Second, two of these residues are situated on either side of a region that is highly variable among many SCPL family members. This variable region aligns with a sequence in CPDW-II that is similarly bordered by disulfide bond-forming Cys and is proteolytically excised from the mature enzyme (Breddam et al., 1987
The identification of signal peptides via SignalP in 49 of the 51 SCPL proteins is consistent with experimental work showing that CPY and SMT are transported to the vacuole (Marshall, 1972
RT-PCR reveals that members of the SCPL gene family are expressed in all major tissue types examined: seedlings, roots, stems, leaves, flowers, and siliques. Thus, it seems likely that SCPL genes and their encoded proteins have diversified in Arabidopsis to function differently both in terms of their activities and their tissue-specific expression. Alternatively, some groups of SCPL proteins may have maintained the same metabolic roles, with different members serving the same function in different tissues. In any case, the diversification of SCPL gene expression patterns appears to have continued until fairly recently, as indicated by the distinct regulation exhibited by SCPL genes found in four of the six chromosomal clusters. Our RT-PCR results may also serve as a guide for identifying the functions of Arabidopsis SCPL proteins. These results will be particularly important in experiments aimed at identifying metabolic pathways blocked in T-DNA insertional mutants. For example, in both the sng1 and sng2 mutants, the activated Glc ester substrate for the SCPL protein in question accumulates in a tissue-specific manner. If phenotypes of this kind can be expected to occur in other SCPL acyltransferase-deficient mutants, SCPL gene expression patterns can be used to direct metabolic profiling experiments, and the identity of the Glc ester accumulated will help to identify the pathway in which the enzyme is involved.
Finally, some reactions that might be catalyzed by SCPL proteins are known to occur in specific tissues. As a result, our RT-PCR gene expression survey may help to identify candidate genes for these enzymes. For example, sinapoylcholinesterase activity is found only in germinating seedlings (Strack et al., 1980
Our analyses of Arabidopsis SCPL proteins and related sequences from other organisms suggest that these proteins can be divided into six clades. Of these, Clades I and II appear to be plant specific, based upon the presence of highly similar proteins identified in an assortment of plants and the absence of closely related coding sequences in the genomes of insects, yeast, and animals that were examined. Consistent with this hypothesis, a preliminary phylogenetic analysis of three annotated SCPL proteins from the initial draft of the Chlamydomonas reinhardtii genome (http://genome.jgi-psf.org/chlre2/chlre2.home.html) suggested that they fall into Clades I, II, and VI. These findings suggest that at some point in the evolutionary history of the plant lineage, an ancestral SCPL protein may have acquired an enzymatic activity that added to the catalytic repertoire of plants. This seems like a particularly likely explanation for Clade IA proteins given that SMT, SCT, and the SCPL isobutyryl acyltransferases are grouped within this clade. Indeed, considering that SMT and SCT are both acyltransferases but are among the most distantly related of Clade IA proteins, it seems likely that some or all of the as-yet-uncharacterized Clade IA proteins are also acyltransferases.
Our grouping of SCPL proteins 20 and 21 with proteins 1 to 19 to form Clade I is based primarily on sequence characteristics not reflected by pairwise percent identities. Whereas there are only slightly higher shared identities between SCPL proteins 20, 21, and the proteins in Clade IA versus the members of Clades II to VI (Fig. 2), the patterns of amino acid and splice site conservation sets 20 and 21 apart more clearly from Clades II to VI. The similarity of SCPL proteins 20 and 21 to the wound/jasmonate-inducible Ser carboxypeptidase from tomato suggest that they may be important to defense responses in Arabidopsis. As in tomatoes, jasmonate acts as a signaling molecule in Arabidopsis in the defense against insects, pathogens, and abiotic stress (McConn et al., 1997
Aside from the wound-inducible SCP from tomato, all of the other known plant SCPs included in our analysis fall into Clade II. This suggests that Arabidopsis SCPL proteins 22 to 46 act as genuine carboxypeptidases, although some members may be esterases or lyases (Degan et al., 1994
Our understanding of the reactions catalyzed by plant SCPL proteins is limited at best; however, some reactions likely to be catalyzed by SCPL proteins can be identified in the biochemical literature. For example, it has been shown that the most abundant anthocyanin in wild carrot (Daucus carota) is a sinapoylated cyanidin glycoside (Harborne et al., 1983
Another potential role for Clade IA SCPL proteins is in the acylation of glucosinolates. Glucosinolates are amino acid-derived sulfated thioglucosides that are characteristic of the members of the Brassicaceae, including Arabidopsis (Wittstock and Halkier, 2002
Finally, Glc ester transesterification reactions are found in a number of other pathways leading to plant secondary metabolites, including chlorogenic acid in sweet potato and gallotannins in oak (Gross, 1983
All of the SCPL proteins and known SCPs outside of the plant kingdom fall into Clades III to VI. The similarity of Arabidopsis SCPL proteins 47 to 51 to fungal and animal SCPs suggests that these proteins may perform similar or identical functions in these divergent organisms and may thus be true orthologs. In fact, the analysis of Arabidopsis SCPL proteins within the context of rice, animal, and fungal SCPL proteins suggests that Clade III is specific to animals, Clade IV is specific to only plants and fungi, and Clades V and VI represent proteins that serve a function conserved among all eukaryotic organisms. We have not included two SCPL proteins from S. pombe and one from yeast in Clade IV because they are only distantly related to the members of this clade, and the relevant branch points are not supported by high bootstrap values. Further analysis of fungal SCPL proteins may reveal that these proteins define additional SCPL clades that are not characteristic of plants and animals.
Although the biological role of the majority of Arabidopsis SCPL proteins remains to be determined, our research and the research of others suggests that SCPL proteins are likely to function in a broad range of biochemical pathways including those involved in secondary metabolite biosynthesis. SCPL proteins may therefore be important to normal plant growth and development, as well as to the synthesis of compounds that protect plants against pathogens and UV light and for resistance to natural and man-made xenobiotics. Regardless of the actual enzymatic function, RT-PCR results indicate that few Arabidopsis SCPL genes are expressed predominantly in tissues undergoing extensive protein breakdown such as cotyledons and senescent leaves, suggesting that previous assumptions about the roles of these proteins being confined solely to protein turnover may be in error.
Search of On-Line Sequence Databases
To identify all SCPL and SCP sequences in the Arabidopsis (Arabidopsis thaliana) genome and in other fully or partially sequenced genomes, nonredundant genomic, cDNA, and protein sequences with the keywords "Ser carboxypeptidase" in their definition fields were obtained from GenBank (Benson et al., 2000
Two different approaches were taken to evaluate the annotation of the Arabidopsis SCPL genes. For each of the 20 SCPL genes with one or more available RAFL cDNAs, the predicted open reading frame was aligned with the RAFL cDNA sequence to determine whether differences existed between the two. In cases where these sequences were not identical, the genomic sequence was then examined to determine how this discrepancy may have arisen, such as by the erroneous addition, deletion, extension, or truncation of exons. In these cases, the RAFL cDNA was adopted as the revised SCPL coding sequence. To evaluate the annotation of SCPL genes for which RAFL cDNA sequences were not available, their inferred amino acid sequences were aligned together with those of the RAFL cDNAs described above to identify likely sites of misannotation.
All alignments were performed using the default parameters in AlignX, a part of the Vector NTI program suite 9.0.0, which uses ClustalW as a base alignment algorithm (Thompson et al., 1994
RNA extractions were performed as described previously (Lehfeldt et al., 2000
The authors are grateful to J. Lohmann, M. Schmid, and D. Weigel, MPI for Developmental Biology, Tuebingen, Germany, for release of their microarray dataset prior to publication. Received December 9, 2004; returned for revision February 3, 2005; accepted February 11, 2005.
1 This work was supported by the National Science Foundation (grant to C.C.) and by Purdue University (graduate fellowships to C.M.F.). This is journal paper number 2005–17562 from the Purdue University Agricultural Experiment Station. 
2 Present address: Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109.
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.057950. * Corresponding author; e-mail chapple{at}purdue.edu; fax 765–496–7213.
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