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First published online June 28, 2007; 10.1104/pp.107.098970 Plant Physiology 144:1986-1999 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Related Arabidopsis Serine Carboxypeptidase-Like Sinapoylglucose Acyltransferases Display Distinct But Overlapping Substrate Specificities1,[OA]Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907 (C.M.F., M.G.T., A.M.S., J.A.S., T.S., M.C.H., C.C.); and United States Dairy Forage Research Center, United States Department of Agriculture-Agricultural Research Service, Madison, Wisconsin 53706 (J.R.)
The Arabidopsis (Arabidopsis thaliana) genome encodes 51 proteins annotated as serine carboxypeptidase-like (SCPL) enzymes. Nineteen of these SCPL proteins are highly similar to one another, and represent a clade that appears to be unique to plants. Two of the most divergent proteins within this group have been characterized to date, sinapoyl-glucose (Glc):malate sinapoyltransferase and sinapoyl-Glc:choline sinapoyltransferase. The fact that two of the least related proteins within this clade are acyltransferases rather than true serine carboxypeptidases suggests that some or all of the remaining members of this group may have similar activities. The gene that encodes sinapoyl-Glc:malate sinapoyltransferase (sinapoyl-Glc accumulator1 [SNG1]: At2g22990) is one of five SCPL genes arranged in a cluster on chromosome 2. In this study, an analysis of deletion mutant lines lacking one or more genes in this SCPL gene cluster reveals that three of these genes also encode sinapoyl-Glc-dependent acyltransferases. At2g23000 encodes sinapoyl-Glc:anthocyanin acyltransferase, an enzyme that is required for the synthesis of the sinapoylated anthocyanins in Arabidopsis. At2g23010 encodes an enzyme capable of synthesizing 1,2-disinapoyl-Glc from two molecules of sinapoyl-Glc, an activity shared by SNG1 and At2g22980. Sequence analysis of these SCPL proteins reveals pairwise percent identities that range from 71% to 78%, suggesting that their differing specificities for acyl acceptor substrates are due to changes in a relatively small subset of amino acids. The study of these SCPL proteins provides an opportunity to examine enzyme structure-function relationships and may shed light on the role of evolution of hydroxycinnamate ester metabolism and the SCPL gene family in Arabidopsis and other flowering plants.
Plant secondary metabolites account for a great amount of the biochemical diversity that exists in nature. These compounds have been estimated to number between 100,000 to 200,000, with the majority of them yet to be studied in detail (Wink, 1988  ). As a class of molecules, plant secondary metabolites include compounds that possess many important properties: they function as antifeedents, phytoanticipins and phytoalexins, signaling molecules, UV protectants, and in a host of other physiologically important roles (Li et al., 1993; Wajant et al., 1994; Ku , 1995; Kliebenstein et al., 2005; Taylor and Grotewold, 2005). Aside from their functions within the plant kingdom, they have also proven to be invaluable to humanity, playing long-standing roles in medicine and agriculture. Secondary metabolites are therefore not only crucial to our understanding of plant physiology, but are of great importance to our own health and survival as well.
A common metabolic step in the production of numerous secondary metabolites involves the attachment of an acyl group via an activated donor molecule (Croteau and Hooper, 1978
The enzymes responsible for the final step in the synthesis of isobutyryl Glc esters in Solanum berthaultii and Lycopersicon pennellii, as well as sinapoylcholine and sinapoylmalate in Arabidopsis (sinapoyl-Glc:choline sinapoyltransferase [SCT] and sinapoyl-Glc:malate sinapoyltransferase [SMT], respectively) are Ser carboxypeptidase-like (SCPL) proteins (Lehfeldt et al., 2000 ; Li and Steffens, 2000; Shirley et al., 2001). As a class of enzymes, SCPL proteins bear the trademark Ser-Asp-His catalytic triad along with other sequence features that are characteristic of known Ser carboxypeptidases (SCPs) such as carboxypeptidase-Y from yeast (Saccharomyces cerevisiae). In this article we report the characterization of three additional Arabidopsis SCPL proteins that are sinapoyl-Glc-dependent sinapoyltransferases. The functions of these proteins were identified through the phenotypic characterization of fast neutron-induced sinapoyl-Glc accumulator1 (sng1) deletion alleles in which the deletions affect not only the SNG1 gene, but also one or more of the SCPL genes that flank it. We report here that At2g23000 encodes a protein that catalyzes the sinapoyl-Glc-dependent sinapoylation of anthocyanins in Arabidopsis, demonstrating that this activity is not dependent upon a BAHD acyltransferase as has been suggested previously (Luo et al., 2007). Furthermore, we show that the SCPL protein encoded by At2g23010 catalyzes the disproportionation of two molecules of sinapoy-Glc to generate 1,2-disinapoyl-Glc (Fig. 1) and an additional, as yet unidentified compound. We have therefore designated the SCPL proteins encoded by At2g23000 and At2g23010 as sinapoyl-Glc:anthocyanin sinapoyltransferase (SAT) and sinapoyl-Glc:sinapoyl-Glc sinapoyltransferase (SST), respectively. Finally, we show that both SMT and the protein encoded by At2g22980 are capable of catalyzing the formation of 1,2-disinapoyl-Glc, although this activity is clearly not the primary function of SMT.
Like SCT and SMT, SST and SAT belong to a family of 51 SCPL proteins encoded by the Arabidopsis genome, and are members of a clade that includes 15 other closely related SCPL proteins (Fraser et al., 2005
sng1-5 and sng1-6 Harbor Deletions of an SCPL Gene Cluster on Chromosome 2
The SNG1 gene is one of five SCPL genes that are arranged in tandem on chromosome 2 (Fig. 2
). The proteins encoded by these SCPL genes are highly similar, with any two of them being between 71% and 78% identical (Fraser et al., 2005
Etiolated sng1-6 Seedlings Lack a Major Sinapate Ester Found in Etiolated Wild-Type, sng1-1, and sng1-5 Seedlings
The high sequence similarity shared by SMT and the SCPL proteins encoded by At2g22980, At2g23000, and At2g23010 suggested that one or more of the latter might also be sinapoyl-Glc-dependent sinapoyltransferases. To test this hypothesis, extracts from a variety of tissues from the sng1-5 and sng1-6 mutants were analyzed via HPLC to determine if any putative sinapate esters present in wild-type and sng1-1 plants were absent from one or both of the mutants. In these experiments, two compounds not previously identified in Arabidopsis with absorption spectra characteristic of sinapate esters were found to be present in wild-type, sng1-1, and sng1-5 seedling extracts, but were absent from sng1-6 extracts (Fig. 3A
). Given that At2g22980 and At2g23010 are the only two SCPL genes uniquely deleted from the sng1-6 mutant relative to the sng1-5 mutant, the absence of these compounds (hereafter referred to as compounds 1 and 2) in the sng1-6 seedlings suggested that either At2g22980 or At2g23010 or both are required for their synthesis. Subsequent complementation analyses (see below) confirmed that this is indeed the case. Preliminary liquid chromatography/mass spectrometry (MS) analysis suggested that both compounds were disinapoylated monosaccharides (data not shown), suggesting that one of these two compounds might be1,2-disinapoyl-Glc, which has been shown to accumulate in the cotyledons of radish (Raphanus sativus) seedlings (Strack et al., 1984
NMR Analysis Identifies Compound 2 as 1,2-Disinapoylglucose
NMR spectra of compound 2 not only established its identity as 1,2-disinapoyl-Glc but provided the full NMR data and its complete assignment. The key experiment was the long-range 13C–1H (HMBC) experiment, which delineated which sinapoyl unit was attached to the 1 versus 2 position (Fig. 4B
). Then, with the coupling networks from each unit revealed in the normal way by COSY and HMBC correlations, all of the protons from each sinapoyl unit (S1 and S2) could be unambiguously assigned, with the possible exception of the methoxyl protons. Short-range 13C–1H (HSQC) correlations unambiguously established the assignments for the protonated carbons (Fig. 4A) and HMBC spectra identified all of the nonprotonated carbons. As seen from Figure 4A, some of the protons are poorly resolved in the one-dimensional (1D) proton spectrum, but the proton data are readily extracted from the HSQC spectrum (as well as by COSY; data not shown). The proton and carbon data presented in Tables I
and II
(in two solvents, CD3OD, and 9:1 acetone d6:D2O) are therefore unambiguously authenticated, again, with the possible exception of the methoxyl protons. Data presented in Tables I and II include previously obtained data (Strack et al., 1984
At2g23010 Complements the sng1-6 Phenotype To test the hypothesis that At2g23010 is required for the synthesis of compound 1 and 1,2-disinapoyl-Glc, the sng1-6 mutant was transformed with pCC579, a vector containing the genomic upstream, downstream, and coding regions of the gene. HPLC analysis of extracts from etiolated, 6-d-old sng1-6/pCC579 seedlings showed that both compound 1 and 1,2-disinapoyl-Glc are present at wild-type levels in the transformed mutant seedlings, indicating that At2g23010 complements this aspect of the sng1-6 phenotype (Fig. 3A), and that this gene thus encodes SST. To determine if the protein encoded by At2g23010 is uniquely responsible for the synthesis of 1,2-disinapoyl-Glc in etiolated seedlings, we obtained a SALK Institute T-DNA line (SALK_133207) harboring an insertion in At2g23010. Homozygosity for the T-DNA insertion was initially determined by screening for kanamycin resistance and by PCR genotyping, and Southern analysis was then used to confirm these results (data not shown). Although compound 1 is absent from extracts of etiolated SALK_133207 seedlings, 1,2-disinapoyl-Glc is still accumulated in the mutants seedlings (Fig. 3B), albeit at lower levels than in the wild type. These data suggested that one or more of At2g22980, SNG1, or At2g23000 are capable of synthesizing 1,2-disinapoyl-Glc.
To test the hypothesis that one or more of the SCPL genes clustered near At2g23010 also have SST activity, two additional sng1-6 transgenic lines were generated: one harboring SNG1 (pCC398) and one harboring At2g22980 (pCC881) under the control of their native regulatory elements. Extracts from 6-d-old etiolated seedlings of each transgenic line contained 1,2-disinapoyl-Glc but not compound 1 (Fig. 3B), thus exhibiting a phenotype similar to that of the SALK At2g23010 knockout line. These data show that in addition to the At2g23010 protein, both of these enzymes have the capacity to synthesize 1,2-disinapoy-Glc.
In addition to the observed redundancy with respect to the genes missing from the sng1-6 mutants, results from enzyme assays suggest that another Arabidopsis protein, likely an SCPL enzyme, exhibits SST activity in vitro. We carried out enzyme assays using crude protein extracts from 6-d-old, etiolated wild-type and sng1-6 seedlings, and found roughly equivalent SST activity for both (data not shown). Given that at least five other clade 1 SCPL genes are expressed in seedlings (Fraser et al., 2005
Although At2g23010 is expressed in wild-type leaves (Fraser et al., 2005
In the course of investigating the bright trichomes1 (brt1) mutant, which is defective in a major sinapic acid:UDPG glucosyltransferase (Sinlapadech et al., 2007 ), we generated a brt1-1/sng1-1 double mutant. HPLC analysis of the double mutant showed that total sinapate ester content in the brt1-1/sng1-1 leaf extracts was not substantially different from that of the brt1-1 mutant; however, 1,2-disinapoyl-Glc levels were dramatically reduced compared to sng1-1 (Fig. 6
). Taken together, these data suggest that SMT and SST are probably expressed in the same cell types where they compete with one another for substrate. Furthermore, they suggest that BRT1 is the major UDP-Glc:sinapic acid glucosyltransferase (SGT) expressed in cells that synthesize 1,2-disinapoyl-Glc, and conversely, that 1,2-disinapoyl-Glc is synthesized in only a subset of cells that accumulate sinapoyl-Glc.
The sng1-5 and sng1-6 Mutants Lack the Sinapoylated Form of the Major Anthocyanin in Arabidopsis
The most abundant anthocyanin in Arabidopsis is a sinapoylated cyanidin derivative, 3-O-[6-O-(4-O-
At2g23000 Is Uniquely Responsible for Anthocyanin Sinapoylation in Vivo The data above do not exclude the possibility that SMT is also an SAT, and that SMT and At2g23000 are redundant in function, at least with respect to anthocyanin sinapoylation. Since a T-DNA insertional mutant for At2g23000 is not currently available, the sng1-5 mutant was transformed with pCC398, the aforementioned vector harboring the SNG1 gene, to determine the effect of a loss of At2g23000 function alone. Twelve independent transformed lines from the T2 generation were obtained and screened via HPLC. Levels of sinapoylmalate for each of transformed lines varied, but in no case were sinapoylated anthocyanins detected (Fig. 8 ). The presence of sinapoylmalate in these plants indicate that SMT is expressed and catalytically active, and the lack of sinapoylated anthocyanin demonstrates that the enzyme is incapable of acting as a redundant SAT in vivo.
SCPL proteins represent a class of proteins whose diversity of function has become apparent only recently. SCPL proteins have been designated as SCP like solely on the basis of their overall sequence similarity with respect to known SCPs but the enzymatic function of SCPL proteins implied by their annotation is entirely assumed. While the annotation is in some cases accurate, as with the Arabidopsis SCPL protein BRS1 (Zhou and Li, 2005 ), it is now clear that as a class, SCPL proteins are diverse in both the reactions they catalyze and the functions they serve. Indeed, the SCPL hydroxynitrile lyase from Sorghum bicolor (SbHNL; Wajant et al., 1994), the SCPL isobutyryl-Glc acyltransferases from L. pennellii (Li and Steffens, 2000), and the SCPL-SGAs from Arabidopsis and other members of the Brassicaceae all illustrate that SCPL proteins exhibit a range of catalytic activities (Lehfeldt et al., 2000; Shirley et al., 2001; Milkowski et al., 2004).
Considering that the genes encoding SMT, SAT, SST, and the At2g22980 protein are arranged in a cluster, their divergence in substrate and product specificity serves as an excellent example of how gene duplication can contribute to metabolic diversity, and illustrates a means by which secondary metabolism in plants can evolve and diversify (Table III). Genes of secondary metabolism arranged in tandem often encode proteins that are similar in metabolic roles, substrate specificity, or catalytic activity (Frey et al., 1997 The evolution of the SCPL-SGAs is likely to have taken place by an initial shift in catalytic activity from hydrolytic cleavage exhibited by SCPs to acyltransferase activity, followed by divergence of substrate specificity. The initial shift in catalytic activity may also represent the point of divergence in the function of the ancestral SCP from involvement in primary cellular processes (e.g. protein processing and degradation), providing the opportunity for members of the ancestral gene family to assume a role in plant secondary metabolism. Although a distinct function for At2g22980 has not yet been identified, and the principle role for SMT is clearly in sinapoylmalate biosynthesis, the partial redundancy of these proteins with SST indicates that the evolution of these genes has not yet resulted in enzymes with completely distinct acyl acceptor specificities. In contrast, the ability of SMT, SAT, and SST to function uniquely in the biosynthesis of sinapoylmalate, sinapoylated anthocyanins, and compound 1, respectively, appears to be shared by no other members of the protein family encoded by this gene cluster. In summary, this SCPL-SGA gene cluster appears to provide a glimpse of the gene evolution underlying plant secondary metabolism.
It is interesting to note that the SCPL-SGA gene cluster on chromosome II is one of seven tandem clusters found within the Arabidopsis SCPL gene family, with the three largest clusters all encoding clade IA SCPL proteins (Fraser et al., 2005
Arabidopsis and other members of the Brassicaceae synthesize a wide array of sinapate esters in addition to those discussed here (Baumert et al., 2005
Sinapoylated anthocyanins have been found in many different members of the Brassicaceae, including cabbage (Brassica capitata; Hrazdina et al., 1977
The metabolic route to sinapoylation still remains to be determined. While it is possible that SAT exhibits substrate specificity toward a single anthocyanin such as 3-O-(6-O-p-coumaroyl-2-O-
Substrate Specificity of the Arabidopsis SCPL-SGAs May Be Due to a Small Subset of Amino Acid Residues SST, SAT, and the At2g22980 protein represent the latest additions to the class of SCPL-SGAs whose activities have been fully or partially characterized. As such, they not only expand the importance of this class of enzymes with respect to plant secondary metabolism, but they also provide an opportunity to study the relationship between the evolution of sequence and function. For example, the fact that SMT, SST, SAT, the protein encoded by At2g22980, and even SCT are relatively similar to one another would suggest that the relatively small number of amino acids by which these proteins differ is responsible for the differing substrate specificities exhibited by the Arabidopsis SCPL-SGAs, and that the most important amino acids affecting substrate specificity are likely to be an even smaller subset of residues clustered near the enzymes' active sites. In conclusion, the SCPL-SGAs represent a growing class of enzymes important to plant secondary metabolism. Our research and the research of others suggests that in the plant kingdom, a significant number of SCPL proteins have been recruited to serve as Glc acyltransferases, utilizing 1-O-sinapoyl-Glc as an activated acyl donor molecule. The results of this article further suggest that the study of the SCPL-SGA gene cluster provides an opportunity to elucidate the relationship between gene sequence, enzyme function, and the evolution of metabolism.
Plant Material and Growth Conditions
Arabidopsis (Arabidopsis thaliana) plants were grown under a 16 h light/8 h dark photoperiod in soilless potting mix (Redi-Earth potting mixture, Scotts-Sierra Horticultural Products) at 23°C. For seedling plant material to be used in the analysis of sinapate ester accumulation, seeds were surface sterilized for 10 min in a 2:1 mixture of 0.1% Triton X-100 and household bleach. Seeds were rinsed thoroughly with sterile water and planted on modified Murashige and Skoog medium (Murashige and Skoog, 1962
Sinapate esters were extracted from fresh plant tissue in 50% (v/v) methanol. Samples were incubated at 65°C for 30 min, centrifuged at 12,000g for 2 min, and analyzed by HPLC on a Microsorb-MV C18 column (Varian) using a gradient from 10% acetonitrile in 1.5% acetic acid to 35% acetonitrile in 1.5% acetic acid at a flow rate of 1 mL min–1. Sinapate esters were detected by their UV A335. Anthocyanins were extracted from fresh plant tissue in 4:46:50 acetic acid:water:methanol. Samples were incubated at 65°C for 30 min, centrifuged at 12,000g for 2 min, and analyzed by HPLC on an end-capped C18 column (Waters) using a gradient from 10% acetonitrile in 10% formic acid to 20% acetonitrile in 10% formic acid flow rate of 1 mL min–1. Anthocyanins were detected by their UV A525. Anthocyanin fractions corresponding to each peak were collected and analyzed by MALDI-TOF MS.
For the identification of the sinapate esters not found in the sng1-6 mutant, sng1-1 seedlings were grown on moistened filter paper for 7 d in complete darkness and were extracted in 50% methanol. This extract was concentrated in vacuo, and analyzed using HPLC as described above. The pooled fractions containing 1,2-disinapoyl-Glc were again reduced in vacuo to 2 mL and analyzed by NMR.
A subsample (approximately 1 mg) of the putative 1(E),2(E)-di-O-sinapoyl-
To construct a plant transformation vector containing At2g23010 (pCC579) a cosmid carrying the corresponding genomic sequence (pCC301) was digested with NsiI to liberate a 7,500 bp fragment that contained the genomic coding sequence for At2g23010 as well as 2,200 bp upstream and 1,628 bp downstream of the At2g23010 open reading frame. This fragment was subsequently subcloned into the compatible PstI site of the binary vector, pCAMBIA 2300, generating pCC579. To construct the plasmid containing At2g22980 a cosmid carrying the corresponding genomic sequence (pCC305) was digested with HindIII and XbaI to liberate a 5,865 bp fragment that contained the genomic coding sequence of At2g22980, 1,066 bp upstream and 741 bp downstream of the At2g22980 open reading frame. The plasmid containing At2g22990 (SNG1) was constructed as described elsewhere (Lehfeldt et al., 2000
Constructs for plant transformation were introduced into Agrobacterium tumefaciens C58 pGV3850 (Zambryski et al., 1983
Samples recovered after HPLC were spotted on a stainless steel MALDI plate with an equal volume of 5 mg/mL
The authors would like to thank the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants and the Arabidopsis Biological Resource Center for the distribution of these materials. Received March 6, 2007; accepted June 22, 2007; published June 28, 2007.
1 This work was supported by the National Science Foundation (grant no. 0091419 to C.C.) and a graduate fellowship from Purdue University (to C.M.F.). J.R. was supported in part by funding through the Department of Energy Biosciences program (grant no. DE–AI02–00ER15067). NMR experiments on the Bruker DMX-500 cryoprobe system were carried out at the National Magnetic Resonance Facility at Madison with support from the National Institutes of Health Biomedical Technology Program (grant no. RR02301) and additional equipment funding from the University of Wisconsin National Science Foundation Academic Infrastructure Program (grant no. BIR–9214394), National Institutes of Health Shared Instrumentation Program (grant nos. RR02781 and RR08438), National Science Foundation Biological Instrumentation Program (grant no. DMB–8415048), and the U.S. Department of Agriculture. A portion of the mass spectrometry analysis was conducted at the Purdue University Metabolic Profiling Facility, supported by the National Science Foundation (grant no. DBI–0421102). This is journal number 2007–18146 from the Purdue University Agricultural Experiment Station. 
2 Present address: Department of Chemistry, Purdue University, West Lafayette, IN 47907.
3 Present address: BASF Plant Science L.L.C., 26 Davis Drive, Research Triangle Park, NC 27709. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Clint Chapple (chapple{at}purdue.edu).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.098970 * Corresponding author; e-mail chapple{at}purdue.edu; fax 765–494–7897.
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ABSTRACT
RESULTS
-D-xylopyranosyl-
= 3.31 ppm; our data differ by approximately 0.04 ppm from Strack's. **, Assignments may be interchanged. s, Singlet; d, doublet; dd, doublet of doublets; m, multiplet.
-cyano-4-hydroxycinnamic acid in 50% acetonitrile/0.1% trifluoroacetic acid and allowed to air dry. Mass spectra were acquired with an Applied Biosystems 4700 MALDI TOF/TOF instrument operating in positive reflectron mode. The instrument was initially calibrated with a series of peptide standards to a mass accuracy better than 50 ppm. 
