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. 2005 Jun;138(2):1136–1148. doi: 10.1104/pp.104.057950

An Expression and Bioinformatics Analysis of the Arabidopsis Serine Carboxypeptidase-Like Gene Family1,[w]

Christopher M Fraser 1, Lance W Rider 1,2, Clint Chapple 1,*
PMCID: PMC1150427  PMID: 15908604

Abstract

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 α/β 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; Mizutani et al., 1998; Jackson et al., 2001; Li et al., 2001b; Lim et al., 2001; Stracke et al., 2001; Aubourg et al., 2002; Jackson et al., 2002; Naur et al., 2003). SCPL proteins appear to represent a similar case of recruitment and diversification in plant lineages that has received limited attention to date.

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.

RESULTS

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). The predicted translation products of another two (At3g56540 and At5g22960) terminate shortly after the putative catalytic Ser, and thus lack the active site Asp and His residues. A search of the downstream regions of these genes revealed no additional exons, suggesting that they are pseudogenes. As a result, they were excluded from further consideration, bringing the apparent total of Arabidopsis SCPL genes to 50. Finally, inspection of the Arabidopsis genome identified a gene annotated as a hydroxynitrile lyase-like protein (At4g15100), which encodes an SCPL polypeptide that is 74% identical to the protein encoded by At1g11080. Combined, these results suggest that there are a total of 51 functional SCPL proteins in Arabidopsis.

The Majority of SCPL Gene Annotation Errors Involve Splice Site Predictions

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.

Table I.

Summary of annotation errors found in Arabidopsis SCPL genes and their encoded proteins

Dash indicates RIKEN cDNA sequences not available.

SCPL Protein Gene Accession No. RIKEN cDNA Accession Nos. Description of Annotation Error(s)
1 At5g36180 AY093044 Additional exon (14) predicted incorrectly
AY128925 22 bp at 3′ end of final exon (13) not predicted
5 At1g73290 – Incorrect splice phase between exons 12 and 13 (final exon)
8 At2g22990 AF361601 Incorrect splice phase between exons 12 and 13 (final exon)
AY035052
AY051060
AY143880
15 At3g12240 – Additional 114 bp at 3′ end of exon 13 predicted incorrectly
Entire final exon (14) not predicted
18 At1g33540 – 54 bp at 3′ end of final exon (11) not predicted
19 At5g09640 AY063833 156 bp at 5′ end of exon 9 not predicted
AY091309 5′ and 3′ ends, and reading frame of exon 11 predicted incorrectly
5′ and 3′ ends, and reading frame of exon 13 predicted incorrectly
Final exon (14) not predicted
20 At4g12910 AY136365 15 bp at 5′ end of exon 1 not predicted
BT000181 Exon 6 skipped0
22 At2g24000 – Additional 30 bp at 3′ end of exon 1 predicted incorrectly
23 At2g24010 – Start codon predicted incorrectly
Signal peptide not predicted
28 At2g35770 – Start codon predicted incorrectly
Signal peptide not predicted
29 At4g30810 AY050958 Exon 5 skipped
AY133774 6 bp at 5′ end of exon 6 not predicted
28 bp at 3′ end of exon 6 not predicted
17 bp at 5′ end of exon 7 not predicted
41 bp at 3′ end of exon 7 not predicted
Additional 14 bp at 5′ end of exon 8 predicted incorrectly
30 At4g15100 – Start codon predicted incorrectly
Signal peptide predicted incorrectly
32 At1g61130 – Additional 39 bp at 3′ end of exon 5 predicted incorrectly
43 At2g12480 BT004306 Additional 78 bp at 5′ end of exon 7 predicted incorrectly
45 At1g28110 AY059854 15 bp at 3′ end of exon 6 not predicted
BT008763
46 At2g33530 AY074317 21 bp at 3′ end of exon 6 not predicted
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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; Table II; Supplemental Fig. 1), and an additional three CPY active site residues are conserved in all but a few SCPL proteins. In CPY, all of these residues either bind the terminal carboxylate group of the enzyme's substrate or comprise the binding pockets for the substrate's terminal and penultimate amino acid residues, referred to as the S1′ and S1 sites, respectively. Interestingly, only one of the seven residues that contribute to the S1 site in CPY is conserved in the Arabidopsis SCPL proteins compared to four of twelve S1′ site residues. A further five conserved amino acids correspond to residues that are thought to orient S1 or S1′ site residues in CPY such that they make appropriate substrate contacts (Endrizzi et al., 1994). There are no amino acids that can be identified as putative substrate binding residues within the region that aligns with CPY between L178 and W312, despite the fact that this region contributes nine residues to the S1 and S1′ sites of CPY. Overall, this region has low amino acid identity among all Arabidopsis SCPL proteins and corresponds to the primarily α-helical region between amino acids 180 and 317 of CPY that has been identified as an ancient insertion in the α/β hydrolase fold family (Endrizzi et al., 1994). Six Cys residues appear in conserved positions in almost all of the sequences and align near the Cys that form disulfide bridges in CPY. Results from SignalP (Bendtsen et al., 2004) indicate that all of the 51 SCPL proteins contain signal peptides that are likely to target them to the endoplasmic reticulum (Supplemental Fig. 1), possibly for posttranslational modification and subsequent transport to the vacuole or secretion from the cell. Consistent with this observation, many potential glycosylation sites that fit the consensus sequence NX(S/T) are found within the SCPL protein sequences (Marshall, 1972). Further, an N-terminal sequence found in eight of the SCPL proteins aligns with the N-terminal propeptide in CPY (Endrizzi et al., 1994). Finally, over one-half of the SCPL proteins, including SCT, contain a region of low sequence identity between the fourth and fifth conserved Cys residues (columns 400–460, Supplemental Fig. 1), which aligns within the α-helical region between amino acids 180 and 317 of CPY mentioned previously. When these Arabidopsis SCPL proteins are aligned with carboxypeptidase II from wheat (Triticum aestivum; CPDW-II; GenBank accession no. A29639), a well-studied enzyme thought to be involved in the mobilization of protein reserves in seeds (Breddam et al., 1987), this variable region corresponds to a sequence that is proteolytically removed from the mature wheat protein (Breddam et al., 1987; Liao and Remington, 1990), suggesting that there may be few constraints on amino acid changes that occur within this region.

Table II.

Conservation of amino acids in Arabidopsis SCPL proteins that align with residues making substrate contacts in the active site of CPY from yeast

In CPY, these residues are either involved in catalysis, binding of the C-terminal carboxylate of the substrate, or comprise the binding sites for R-groups of the C-terminal or penultimate amino acid residues of the substrate (S1′ and S1, respectively).

Residuea Conserved Columnb Function in CPY
N51 Only in Clades IB and II 168 Carboxylate binding
G52 Yes 169 Carboxylate binding
G53 Yes 170 S1′
C56 Yes, except A in SCPL protein 51 173 S1′
S57 Yes, except T in SCPL protein 18 174 S1′
T60 No 177 S1′
F64 No 182 S1′
E65 Yes, except A or Q in 3 SCPL proteins 183 S1′
E145 No, N,D, or Q in 22 SCPL proteins 275 Carboxylate binding
S146 Yes 276 Catalytic
Y147 Yes 277 S1
L178 No 322 S1
Y185 No 329 S1
Y188 No 332 S1
N254 No 402 S1′
Y256 No 447 S1′
Y269 No 460 S1′
L272 No 463 S1′
S297 No 492 S1′
W312 No 507 S1
D338 Yes 533 Catalytic
I340 No 535 S1
C341 No 536 S1
H397 Yes 594 Catalytic
M398 No 595 S1′
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a

Designated by amino acid and position in CPY.

b

Refers to column position in Supplemental Figure 2.

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; Shirley et al., 2001).

Figure 1.

Figure 1.

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Unrooted trees of SCPs and the SCPL protein family. A, Phylogenetic tree of the 51 Arabidopsis SCPL proteins and CPY from yeast. SCPL proteins are arbitrarily numbered proceeding counterclockwise. The GenBank gene accession numbers for each corresponding SCPL protein are shown on the right. B, Chromosomal positions of the Arabidopsis SCPL genes (pseudogenes are designated as P1–3). C, Phylogenetic tree of Arabidopsis, rice, fungal, and animal SCPL proteins. The GenBank identification numbers are provided on the right. Rice SCPL proteins 1 to 18 (not labeled) fall into Clades I and II. Tree nodes with bootstrap values above 90% are indicated in both A and C with black dots.

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.

Figure 2.

Figure 2.

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Pairwise identity matrix for the Arabidopsis SCPL proteins. Percent identity values are shown for each pairwise alignment of the 51 Arabidopsis SCPL proteins coded on the gray scale shown at bottom. In the top right half of the matrix, the proteins are arranged by their numbered tree positions given in Figure 1. Bars next to the numbers on the top horizontal and right axes indicate clade divisions as designated in Figure 1. In the bottom left half of the matrix, the proteins are arranged in order of their chromosomal positions, from the top of chromosome 1 to the bottom of chromosome 5. The solid bars next to the SCPL protein numbers on the left and lower horizontal axes designate each of the five chromosomes, and the dashed bars designate groups of clustered SCPL genes.

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).

Arabidopsis SCPL Proteins Have Homologues in Other Plants

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) is 58% and 60% identical with respect to SCPL proteins 26 and 27. The hydroxynitrile lyase from Sorghum bicolor (accession no. AJ421152; Wajant et al., 1994) is also 45% identical to proteins 26 and 27. Further, SCPL protein 40 is 50% identical to an SCP from barley grain (CP-MII; accession no. AAB31589) and 57% identical to a gibberellin-induced SCP from pea (CP-A; accession no. AJ251969; Degan et al., 1994; Jones et al., 1996). Finally, among 26 nonredundant SCPL genes annotated in the rice (Oryza sativa) genome, 12 of the encoded proteins group among the Clade II Arabidopsis SCPL proteins and six cluster into Clade I (Fig. 1C).

Animal and Fungal SCPL Proteins Help to Define Four Additional SCPL Clades

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). SCPL protein 50 is encoded by an intronless gene and lacks many of the conserved Cys residues found in the majority of other Arabidopsis SCPL proteins (Supplemental Fig. 1). It clusters into Clade V with five SCPL proteins from rice, four of which are encoded by genes with no introns, and the other by a gene with a single intron. Also grouped into Clade V is an SCPL protein from Drosophila (lacking introns) and SCPs from human and zebrafish (Fig. 1C). As with Clade V, Clade VI includes SCPL proteins from both plants and animals including Arabidopsis, human, Drosophila, zebrafish, and Caenorhabditis elegans. In fact, Arabidopsis SCPL protein 51 and rice SCPL protein 26 (accession no. AAR87281) are each 41% identical to the human retinoid-inducible SCP (Chen et al., 2001) but only 21% to 25% identical to any other plant SCPL proteins outside of Clade VI. Clade III stands apart from the other clades in that it contains exclusively animal SCPL proteins, including the human SCP lysosomal protective protein also known as cathepsin A (Galjart et al., 1988; Fig. 1C). Finally, it is noteworthy that a query of the 240 sequenced genomes of prokaryotes available through BLASTP using the amino acid sequence of SMT, retinoid-inducible Ser carboxypeptidase, and CPY yielded only a single protein (NP_902682) with an E value lower than 1e-7. Since even this protein did not appear to include the catalytic Ser, Asp, and His residues, as determined by alignment, it would appear that Ser carboxypeptidase-like proteins are not commonly found in prokaryotes.

Arabidopsis SCPL Genes Exhibit Tissue-Specific Expression Patterns

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.

Figure 3.

Figure 3.

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RT-PCR analysis of Arabidopsis SCPL gene expression. RNA was extracted from the tissues indicated and used for RT-PCR to evaluate SCPL gene expression. The Arabidopsis SCPL genes are identified according to the numbering scheme used in Figure 1. Genes that are tandemly arranged in the genome are boxed, with the dotted box surrounding gene 1 reflecting its close sequence identity with SCPL gene 2. Results for SCPL genes 1 to 21 and 22 to 46 are grouped together because they represent Clades I and II, respectively. Results for the ACT2/8 positive control are given on bottom left.

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) and microarray data (http://www.weigelworld.org/resources/microarray/AtGenExpress/) made available to the Arabidopsis research community.

DISCUSSION

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).

Arabidopsis SCPL Proteins Have Structural Features in Common

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; Shirley et al., 2001), and the kinetic analysis of SCT indicates that a fundamental alteration of the catalytic mechanism is not required to bring about transacylation activity (Shirley and Chapple, 2003). Thus, it may be that the majority of SCPL enzymes, including both acyltransferases and hydrolases, utilize the Ser-Asp-His triad in catalysis, although our alignment suggests that with regard to other residues involved in substrate binding, these enzymes have diverged substantially over evolutionary time.

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; Liao and Remington, 1990). Since this posttranslational modification results in the production of a heterodimeric protein molecule, the disulfides that cross-link the monomers are probably important to prevent their dissociation.

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; Stevens et al., 1982; Sharma and Strack, 1985; Valls et al., 1987; Hause et al., 2002). The SignalP results are also supported by the fact that the signal peptide sequence removed from the mature form of SMT (Lehfeldt et al., 2000) is accurately predicted by the algorithm. An additional N-terminal feature found in eight of the SCPL proteins is the presence of a variable-length peptide sequence that aligns with the propeptide of CPY (Ramos et al., 1994). These peptides could serve a chaperone-like role similar to that of the CPY propeptide (Winther and Sorensen, 1991). Consistent with the presence of the N-terminal signal peptides in SCPL proteins are the many putative glycosylation sites found in most of their sequences. These data suggest that many SCPL proteins may be posttranslationally modified as they are processed through the endoplasmic reticulum and Golgi. Finally, and as mentioned above, the inferred translations of many SCPL proteins contain a variable region known to be removed in CPDW-II (Breddam et al., 1987; Liao and Remington., 1990). Protein gel blots of SCT expressed in planta and in yeast suggest that this region is also removed during the maturation of this SCPL acyltransferase (Shirley and Chapple, 2003). Taken together, these data suggest a pattern of subcellular targeting and posttranslational processing that may be required to produce functional enzymes.

SCPL Gene Expression Profiles May Help to Reveal the Function of Their Encoded Proteins

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) where it functions to hydrolyze sinapoylcholine, liberating choline and sinapate for the biosynthesis of membrane lipids and UV-protective sinapoylmalate, respectively (Strack, 1981). Among the 51 SCPL genes of Arabidopsis, the expression of only one (SCPL 44, At1g43780) is seedling specific, making it an excellent candidate for the SCE (sinapoylcholinesterase) gene.

SCPL Proteins in Clades I and II Appear to Represent Plant-Specific Proteins

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; Thomma et al., 1999; Overmyer et al., 2000; Rao et al., 2000). SCPL proteins 20 and 21 might therefore play a role similar to their tomato homolog, with the diverse expression of SCPL gene 20 making its encoded protein a particularly good candidate for involvement in plant defense.

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; Wajant et al., 1994; Jones et al., 1996; Moura et al., 2001). Although its true substrate and the nature of its catalytic activity remain unknown, the finding that SCPL protein 24 is involved in brassinosteroid signaling suggest that Clade II members may play important roles in plant growth and development (Li et al., 2001a). Whatever the exact function of the Clade II SCPL proteins may be, the fact that they do not appear to be encoded within the genomes of animals and fungi suggests that these proteins have become specialized to perform functions that are likely to be unique to plants.

Candidate Reactions for SCPL Acyltransferases Can Be Found throughout Plant Metabolism

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; Glässgen et al., 1992). The enzyme that catalyzes the sinapoylation of this anthocyanin uses sinapoyl-Glc as the activated sinapate donor (Glässgen and Seitz, 1992; Nakayama et al., 2003; Suzuki et al., 2004). As in Daucus, the major anthocyanin in Arabidopsis is acylated with a sinapoyl moiety (Bloor and Abrahams, 2002), which may be added by a sinapoyl-Glc-utilizing SCPL protein. Further, since isolated Daucus vacuoles actively accumulate the sinapoylated anthocyanin, but not the nonacylated form, the sinapate moiety may function as a vacuolar uptake or retention tag (Hopp and Seitz, 1987). Alternatively, the sinapate moiety may be required for, or be the site of, glutathione derivatization by a glutathione S-transferase analogous to that encoded by the maize Bronze2 gene (Marrs et al., 1995). Since anthocyanins are accumulated in Arabidopsis at high levels in senescing leaves, SCPL genes 7 and 10 are good candidates for genes encoding this putative anthocyanin sinapoyltransferase because they both belong to Clade 1A and are expressed at high levels in the appropriate tissue.

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). These compounds are substrates for myrosinases, which convert the various glucosinolates into an array of toxic and volatile substances that are important in plant-insect interactions (Rask et al., 2000). The enzymes that catalyze the core reactions of glucosinolate biosynthesis have been identified (Wittstock and Halkier, 2002); however, a number of glucosinolates are further modified by additions of other substituents that include sinapoyl and benzoyl groups (Linscheid et al., 1980; Reichelt et al., 2002; Brown et al., 2003). This acylation may have an impact on glucosinolate turnover and plant-herbivore interactions because they are not readily degradable targets for myrosinases (Taipalensuu et al., 1997). The presence of benzoylated glucosinolates in Arabidopsis has already been demonstrated, and sinapoylated glucosinolates have been detected in Arabidopsis seed extracts (Graser et al., 2001; Reichelt et al., 2002). Sinapoyl-Glc and benzoyl-Glc are potential donor molecules in the acylation reaction, making Clade IA SCPL enzymes good candidates for the catalysts involved.

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; Villegas and Kojima, 1986). Similarly, indole-3-acetic acid (IAA)-Glc synthesized by indole-3-acetic acid glucosyltransferase serves as an activated form of IAA, which is used in a transesterification reaction to form IAA-inositol (Michalczuk and Bandurski, 1982; Jackson et al., 2001, 2002; Ljung et al., 2002). This latter reaction is analogous to sinapoylmalate biosynthesis and preliminary indications suggest that it may be catalyzed by an SCPL enzyme (Kowalczyk et al., 2003).

SCPL Proteins in Clades III to VI May Function in Pathways Conserved among Animals, Plants, and Fungi

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.

CONCLUSION

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.

MATERIALS AND METHODS

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). To identify any SCPL genes that had not been annotated as such, we used a subset of Arabidopsis SCPL amino acid sequences to again query the genome databases using PSIBlast (Altschul et al., 1997). Once identified, sequences were downloaded directly into a Vector NTI database (Informax/Invitrogen, Carlsbad, CA) using the on-line retrieval tool. GenBank identification numbers for all sequences included in our analyses are provided in Figure 1, with the exception of CPY (GenBank accession no. 576359).

Reannotation of SCPL Genes

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.

Phylogenetic and Sequence Analyses

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). Alignments were exported to the PHYLIP software package (Felsenstein, 1985) and used to generate unrooted neighbor joining trees and bootstrap values, also using the default parameters. AlignX was also used to generate pairwise identity values for SCPL protein sequences. SignalP (http://www.cbs.dtu.dk/services/SignalP; Bendtsen et al., 2004) was used with the default parameters to identify signal peptides. Tree graphics were generated using the TreeView program (Page, 1996).

RT-PCR

RNA extractions were performed as described previously (Lehfeldt et al., 2000). RT reactions were carried out using Promega ImProm-II (Promega, Madison, WI) reverse transcriptase for each tissue type following the protocol provided by the manufacturer. Four-fold dilutions of cDNA were used for each PCR reaction. Oligonucleotides were chosen to minimize the likelihood of nonspecific annealing by designing the forward primer within regions that were most divergent between any subset of closely related SCPL proteins (Supplemental Table I). Each reverse primer was designed against the similarly diverse 3′ UTR of each gene. BLASTN (Altschul et al., 1990) was then used to search the Arabidopsis genome with each primer pair to ensure a low risk of nonspecific PCR amplification. For all but one gene, the region to be amplified included at least one intron in the genomic sequence, such that amplicons from contaminating genomic DNA could be distinguished from those derived from cDNA. Each PCR reaction was run with an annealing temperature at the lower primer Tm for each primer pair. A primer pair common to actin genes 2 and 8 (An et al., 1996) was used for positive control PCR reactions.

Supplementary Material

Supplemental Data
plntphys_pp.104.057950_index.html (1,005B, html)

Acknowledgments

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.

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.

[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.

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