Phylogenetic Analysis of the UDP-glycosyltransferase Multigene Family of Arabidopsis thaliana*,

Yi Li, Sandie Baldauf, Eng-Kiat Lim, and Dianna J. BowlesDagger

From the Department of Biology, University of York, P.O. Box 373, York YO10 5DD, United Kingdom

Received for publication, August 16, 2000, and in revised form, September 25, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

A class of UDP-glycosyltransferases (UGTs) defined by the presence of a C-terminal consensus sequence is found throughout the plant and animal kingdoms. Whereas mammalian enzymes use UDP-glucuronic acid, the plant enzymes typically use UDP-glucose in the transfer reactions. A diverse array of aglycones can be glucosylated by these UGTs. In plants, the aglycones include plant hormones, secondary metabolites involved in stress and defense responses, and xenobiotics such as herbicides. Glycosylation is known to regulate many properties of the aglycones such as their bioactivity, their solubility, and their transport properties within the cell and throughout the plant. As a means of providing a framework to start to understand the substrate specificities and structure-function relationships of plant UGTs, we have now applied a molecular phylogenetic analysis to the multigene family of 99 UGT sequences in Arabidopsis. We have determined the overall organization and evolutionary relationships among individual members with a surprisingly high degree of confidence. Through constructing a composite phylogenetic tree that also includes all of the additional plant UGTs with known catalytic activities, we can start to predict both the evolutionary history and substrate specificities of new sequences as they are identified. The tree already suggests that while the activities of some subgroups of the UGT family are highly conserved among different plant species, others subgroups shift substrate specificity with relative ease.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Glycosyltransferases are found in all living organisms, catalyzing the transfer of a glycosyl moiety from an activated donor to an acceptor molecule, forming a glycosidic bond. These glycosyl transfer reactions have been highlighted as the most important biotransformation on earth, since in quantitative terms they account for the assembly and degradation of the bulk of biomass (1).

A unique signature motif has been identified in the amino acid sequence of many of these glycosyltransferases, leading to their classification into a single UDP-glycosyltransferase (UGT)1 superfamily (2). Of these, the mammalian UGTs using UDP-glucuronic acid have attracted considerable attention in pharmaceutical and clinical research due to their central role in the metabolism and detoxification of foreign chemicals such as carcinogens and hydrophobic drugs (3, 4).

Plant UGTs are involved in a parallel range of activities, the majority of which use UDP-glucose in the transfer reaction. These reactions are known to have a number of important consequences. First, compounds can be activated or inactivated by their conjugation to glucose. For example, glucose esters are high energy compounds that are known to act as biosynthetic intermediates for further reactions involving the aglycones (5). In contrast, many of the plant hormones are known to be inactivated following glucosylation (6, 7). Second, glucosylation alters the solubility of compounds by increasing their hydrophilic properties and providing access to active membrane transport systems that recognize the glucosides but not the aglycones (8).

As a consequence of these events, glucosylation plays a crucial role in the maintenance of cellular homeostasis in plants through regulating the level, activity, and location of key cellular metabolites. Despite this general importance and the likely large number of these enzymes, given the diversity of substrates, plant UGTs have not been studied systematically. Rather, individual UGTs have been purified on the basis of a particular catalytic activity (9-20). The disadvantage of this approach is that the relationships of different UGTs cannot be defined easily, and, in consequence, predictions of catalytic activities based on structure-function relatedness cannot be made.

Genome sequencing programs offer a new route into understanding multigene families both within a single species and across different species. In this study, we have used the data available from the Arabidopsis genome sequencing program to start to build a foundation for understanding the UGT multigene family. This analysis focuses on the phylogeny and evolution of UGTs and complements parallel investigations into substrate specificity using recombinant proteins corresponding to known UGT sequences (21, 22).


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Data Base Searching and Sequence Analysis for Coding Regions and Introns-- A UGT signature motif in a known tomato UGT (23) was used in a BLAST search of the GenBankTM data base (available on the World Wide Web). ORFs and intron positions of putative Arabidopsis UGT sequences were first identified using Map and Codepreference programs of Wisconsin Package version 10.0 (Genetic Computing Group). Intron positions were decided by visual scanning of likely exon boundaries for Arabidopsis intron splice site consensus sequences (available on the World Wide Web) and by analyses using the program NetGene2 (available on the World Wide Web). In all cases, both methods found identical results. In all 12 cases where cDNAs were available, the predicted intron boundaries were confirmed by sequencing.

Identification of Pseudogenes-- Sequence alignment indicated eight pseudogenes. Of these, five (71B4, 72D2, 89A1, 76E10, and 84B3)2 were found to contain a single nucleotide substitution, insertion, or deletion, therefore causing interruption of ORFs. The single base changes for 71B4, 72D2, and 84B3 were confirmed by sequencing of the cloned PCR amplification products from genomic DNA using Pfu DNA polymerase. 76E8 had two single nucleotide nonsense substitutions. 71B3 contained a 40-base pair deletion causing a frameshift and in-frame stop codon; 90A3 contained multiple nonsense substitutions, insertions, and deletions throughout.

PCR Amplification, Cloning, and Sequencing-- DNA sequences were amplified from the genomic DNA of Arabidopsis thaliana Columbia by PCR, using Pfu DNA polymerase (Stratagene) and gene-specific oligomers. PCR products were subcloned into restriction sites on the multiple cloning site of pBluescriptII (24). Nucleotide substitutions and insertion/deletions in the pseudogenes were confirmed by sequencing using the appropriate oligomers. Intron positions in 73B1, 73B2, 74D1, 74E2, 74F2, 76C5, 76E3, 78D1, 87A2, and 88A1 were also confirmed by sequencing their corresponding expressed sequence tags obtained from the Arabidopsis Biological Resource Center.

Sequence Alignment and Phylogenetic Analysis-- The amino acid sequences of the ORFs were initially aligned using the program ClustalX version 1.8 with default gap penalties (25). These alignments were then reconciled and further adjusted by eye to minimize insertion/deletion events. The conserved motifs were defined as alignable regions among all sequences. Smaller groups of more closely related sequences were then aligned separately by the same methods. Only regions of unambiguous alignments were used in the subsequent phylogenetic analyses.

Trees were constructed initially from amino acid sequences of nine conserved motifs present and alignable in all sequences, giving a total of 290-300 amino acid positions for all sequences. Distance analyses used the program Protdist of Phylip 3.5c with a PAM250 substitution matrix and trees calculated by neighbor-joining algorithms. Distance bootstrap analyses consisted of 1000 replicates using the same protocol. Parsimony analyses utilized PAUP* 4.0b2a. Shortest tree searches consisted of 1000-10,000 replicates of random sequence addition with tree bisection-reconnection branch swapping. Bootstrap analyses used 1000 replicates of a single round of random addition each.

Strongly supported subgroups (bootstrap percentage >90%) were then analyzed further to refine all of the subtrees. Analysis of 105 UGTs, including Arabidopsis and other plant species, used only the distance method.


    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The UGT Family of Arabidopsis Is Large and Diverse-- A signature sequence involved in the binding of the UDP moiety of the nucleotide sugar has been identified as a characteristic of UGT sequences from a range of prokaryotic and eukaryotic organisms (2, 26, 27). To gain insight into the size of the UGT family in the model plant Arabidopsis thaliana, a 44-amino acid sequence corresponding to this signature motif in a known UGT from tomato (23, 28) was used to screen the GenBankTM data base. To our surprise, 99 sequences containing the signature motif were identified. Since to date (March 1, 2000), 86.5% of the Arabidopsis genome is sequenced and publicly available, our data estimate that the total genome will contain ~120 UGTs. This indicates that UGTs constitute one of the largest multigene families in Arabidopsis.

Each of the putative UGT genes identified in this study has been newly classified using the standardized system recommended by the UGT Nomenclature Committee (2) (Table S1). Of the 99 UGT sequences identified, eight were found to be pseudogenes on the basis of interruptions to ORFs (71B3P, 71B4P, 72D2P, 76E8P, 76E10P, 84B3P, 89A1P, and 90A3P); three were only partial (76B1, 76C2, and 85A3). Detailed sequence analyses indicated 19 errors to current data base annotation (Table S2). This left 88 unique, complete genes, which were then subjected to further analysis. The deduced amino acid sequences of the putative UGTs ranged from 442 amino acids (79B7 and 79B8) to 507 amino acids (73D1).

While all of the UGT sequences identified contain the signature motif, the overall sequence similarity varies substantially between individual pairs of sequences, from 95% to lower than 30% identity. Despite this diversity, a total of nine conserved motifs are clearly present. Conserved motif 5 separates all of the UGTs into N-terminal and C-terminal regions. The N-terminal region is more highly variable than the C-terminal region. This observation supports the suggestion that the N-terminal region is likely to be involved in the recognition and binding of the highly diverse aglycone substrates, whereas the C-terminal region, including the signature sequence in motif 7, is involved in binding the nucleotide sugar substrates. The complete alignment of these unique sequences is provided in Fig. S1.

All of the UGTs identified as transferring glucuronic acid to hydrophobic substrates in mammalian cells are membrane-bound enzymes (29) localized in the endoplasmic reticulum with their catalytic sites facing the lumen. Two functional motifs in mammalian UGTs are thought to be important to the topology of proteins within the cell. First, a distinct hydrophobic C-terminal halt sequence anchors the enzymes to the membrane region (30-34). Second, the mammalian UGTs also contain the N-terminal signal sequence cleaved on cotranslational segregation into the endoplasmic reticulum (29, 35). However, neither motif was identified in our analyses of Arabidopsis UGTs using programs TopPred2 (available on the World Wide Web), SignalP (available on the World Wide Web), and Psort (available on the World Wide Web) (data not shown), supporting the general assumption that the plant UGTs are cytoplasmic enzymes.

Phylogeny of the UGT Superfamily-- An alignment of nine conserved motifs, encompassing ~60% of the total deduced protein sequence of all Arabidopsis UGTs, were analyzed by neighbor joining and parsimony with statistical confidence measured by bootstrap analysis. Both sets of analyses define 12 major groups, each with a bootstrap support greater than 90% in distance analysis (Fig. 1).



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Fig. 1.   Phylogenetic analysis of the Arabidopsis UGT superfamily shows 12 distinct groups. A, the tree shown was derived by neighbor-joining distance analysis of nine conservative motifs described in Fig. S1. Bootstrap values over 50% are indicated above the nodes, with the number on the left for the neighbor joining and right for parsimony. Bootstrap values in brackets were derived by analysis excluding the two long-branch sequences 78D1 and 83A1. Triangles indicate subtrees that were subjected to further refinement. The lengths of triangles are drawn roughly proportional to the overall branch length of the whole group. B, relationships among sequences within triangles were further refined using additional amino acid positions shared within each group. Hypothetical intron gains and losses are indicated by diamonds followed by intron number (see Fig. 3). Postulated intron gains are indicated by filled diamonds, intron losses by unfilled diamonds, and the questionable intron loss by striped diamonds.

Relationships of some individual UGTs in well supported subgroups could not be strongly resolved due to the very high similarity among closely related sequences in the nine conserved motifs (Fig. 1A). These subgroups were analyzed to better resolve fine level relationships using larger data sets with additional alignable residues (Fig. 1B).

The 12 well defined major groups of UGTs suggest that there were at one time 12 ancestral genes. However, the branching order among these 12 ancestral genes is not clearly resolved by these analyses. One possible source of artifact is the presence of two sequences (83A1 and 78D1) with long unique terminal branches, suggesting accelerated evolutionary rates. Such long branches tend to distort phylogenetic analyses by reducing apparent bootstrap support for nearby clades (36). Therefore, we reanalyzed the data with these two sequences removed. These analyses give strong statistical confidence that five of the ancestral genes, corresponding to groups G, H, J, K, and L, are likely to share a more recent common origin (Fig. 1A, 84/70% bootstrap). Within these groups, G and H are more likely to be closely related (Fig. 1A, 78/57% bootstrap).

The phylogenetic tree described in this study has not been rooted (Fig. 1). Several UGT sequences of mammalian and bacterial origin were used as outgroups in an attempt to define the earliest branch in the Arabidopsis UGT tree. However, no single branch was consistently or strongly identified (data not shown). This is most probably due to the remoteness of the outgroups used and the fact that only motif 7 is clearly alignable among all these sequences, so only ~60 amino acid positions could be used for the analyses (data not shown).

Chromosomal Organization of UGT Genes-- When the nearest recombinant inbred markers of the bacterial artificial chromosome clones (Table S1) were used as indications of their chromosomal positions, the UGT genes were found to be scattered throughout the chromosomes of the Arabidopsis genome, which is similar to the Arabidopsis R2R3-MYB gene family (37). Another distinctive feature of the Arabidopsis UGT gene family is the clustering of sequences into groups with sizes ranging from two to seven genes per group (Fig. 2). The genes in any particular cluster often show a high degree of sequence similarity among themselves, also reflecting their closeness in the terminal subgroups of the phylogenetic tree (Fig. 1).



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Fig. 2.   Deduced chromosomal positions of Arabidopsis UGT genes. The positions are given according to the nearest recombinant inbred markers. Genetic distances in centimorgans (cM) are according to the Lister and Dean recombinant inbred map.



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Fig. 3.   Distribution of introns among 37 UGT genes of Arabidopsis. The introns are mapped and numbered to the alignment of their amino acid sequences. The nine conserved motifs are shown as shadow rectangles and aligned. Rectangles with broken lines represent regions with a variable number of amino acid residues. Inverted triangles indicate positions of introns that are found or predicted in the corresponding genes. Phase 0 introns are indicated by open inverted triangles; phase 1 introns by solid inverted triangles and phase 2 introns by shaded inverted triangles.

The presence of clusters of UGT sequences on individual chromosomes implies that they have evolved from gene duplications of more recent unequal recombination events. There are also clear examples of transpositional gene duplications in the UGT family. For example, in the 73B subfamily, a gene cluster comprising 73B1-73B3 is located on chromosome IV with a second cluster comprising 73B4 and 73B5 on chromosome II. Therefore, it is likely that ancestors of these genes were the result of a transpositional gene duplication event. Such features can also be found in the Arabidopsis multigene family encoding the detoxifying enzymes, glutathione S-transferases (38).

Intron Gain and Loss in the UGT Multigene Family-- Our study revealed that 37 in 88 UGT genes contained introns, with all but three of these genes containing only a single intron (Figs. 1 and 3). Comparing intron positions with sequence relationships predicted by phylogenetic analysis (Fig. 1), a minimum of nine independent intron insertion events appear to have happened in the course of UGT evolution. The most widespread, and therefore probably the oldest intron, is intron 2, which is found in all of the 23 UGT sequences in groups F-K (Fig. 1). All other introns are found only within a single restricted subgroup of closely related sequences or in only a single gene. This suggests a general pattern of intron gain during evolution of the UGT gene family.

A single clear case of recent intron loss is seen in the subfamily of closely related genes 73B1-73B5 in group D. Four of these genes contain intron 7, yet in the closely related pair of sequences 73B2 and 73B3 (85% sequence identity), only 73B2 contains the intron. This implies that an intron loss event has occurred along the line leading to 73B3, after the gene duplication event that gave rise to 73B2 and 73B3. Similarly, the absence of intron 2 in group L may also have arisen from an intron loss that occurred in the duplication event separating group L from groups I, J, and K. However, the relationship between group L and groups F-K is not resolved; therefore, this intron loss event is uncertain.

Thus, analysis of the evolution of the UGT multigene family provides evidence for both intron gain and intron loss and is thereby strongly consistent with the "intron-late" theory of intron evolution (39-42).

Structure-Function Relatedness of the UGT Family-- Numerous sequences containing the UGT signature motif have been identified from a wide range of plant species. Of these, 14 of the corresponding gene products have been characterized biochemically (Table I) (9-20, 43), and the functions of three additional proteins have been defined conclusively by genetic analysis (44-47). Therefore, we aligned these 17 additional sequences with 88 Arabidopsis sequences and constructed the phylogenetic trees. The trees were based on the nine conserved motifs by superimposing the predicted position of these 17 new sequences onto the tree in Fig. 1 based on the results of the 105 sequence analyses (Fig. 4). The results reveal a number of interesting relationships.


                              
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Table I
Substrate specificity of UGT enzymes from other plant species
The UGT designation allocated by the UGT nomenclature subcommittee is given in the first column; synonyms used by other authors are shown in the second column. GenBankTM database accession numbers are in the fourth column, and references are in the fifth column. Major aglycone substrates, nucleotide-sugar substrate, and major products of the enzymatic reactions are summarized in columns 6, 7, and 8 respectively. trans-CA: trans-cinnamic acid; BA, benzoic acid; IAA, indole-3-acetic acid; SA, salicylic acid.



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Fig. 4.   A composite tree of plant UGT sequences indicates the evolutionary relationships. Branch positions of 17 additional UGTs from plant species other than Arabidopsis were determined by neighbor-joining analysis of nine conserved motifs together with all 88 Arabidopsis UGT sequences. The relative positions of these 17 sequences (Table I) were then superimposed onto the tree shown in Fig. 1 as indicated by broken lines. Major groups identified in Fig. 1 are indicated in brackets.

The composite tree (Fig. 4) suggests the possible presence of an additional ancient UGT family distinct from the 12 groups we have so far identified in Arabidopsis (Fig. 1). This additional group includes two sequences encoding enzymes that glycosylate the plant hormone, zeatin (19, 20). While one of these UGTs uses UDP-glucose as the sugar donor, the other with identical aglycone specificity uses UDP-xylose. The two sequences are 87% identical, implying that they have arisen from a common ancestor with one of the pair subsequently shifting its substrate specificity.

The remaining 15 UGT genes from plant species other than Arabidopsis all cluster within the 12 groups identified by this study (Fig. 4). Interestingly, three of these UGTs clustered in group F are known to be involved in the 3-O-glucosylation of anthocyanidins, implying that the Arabidopsis 78D1 gene products may have a related specificity. Since this cluster contains sequences from maize, gentian, and grapevine (12, 10, 45), the biochemical function of the product from the common ancestral gene may have evolved prior to the division of monocots and dicots. Although a fourth UGT, from petunia, is also present in the lower branches of the same cluster, the enzyme is known not to glycosylate anthocyanidins (16).

The 3-O-glucosides of anthocyanidins can be further modified either by the addition of rhamnose to the glucose moiety or by glucosylation of other hydroxyl groups. The petunia UGTs transferring rhamnose to the glucose ring of anthocyanins (46, 47) are located in group A of the composite phylogenetic tree (Fig. 4), whereas an example of a UGT from perilla (15) transferring glucose to the 5-O position is in group L. This suggests that the enzymes involved in these further modifications of anthocyanins have evolved independently. Similarly, while the biosynthesis of glucosinolates and cyanogenic glucosides share common intermediates in the form of oximes and also share the involvement of UGTs (17, 43, 48, 49), there is no close evolutionary relationship between these UGTs. The enzyme involved in glucosinolate formation (43) is found within group L, whereas that involved in cyanogenic glucoside formation (17) is found within Group G.

In parallel studies to this phylogenetic analysis, we have screened representatives from each of the major groups of the Arabidopsis multigene family for their ability to glucosylate cinnamic acids. The results have shown that within 36 family members that have been analyzed, the ability to form glucose esters only resides within group L (21). This group contains the maize iaglu gene product (9) and the equivalent enzyme of Arabidopsis, 84B1, that forms the glucose ester of indole-3-acetic acid (22). Group L also contains the recently described tobacco gene product forming the glucose ester of benzoic acid and salicylic acid (18). These data suggest that the formation of glucose esters may have evolved from a deep ancestral branch recognizing carboxyl groups of the aglycones. From data currently available, biochemical analyses indicate that two UGTs from petunia and Brassica napus (15, 43) that are also located within group L form glucosides. However, phylogenetic analysis of their sequences shows that they are of relatively recent origin compared with the UGTs involved in glucose ester formation. This is further supported by the comparatively recent evolution of the glucosinolate synthesis pathway, which is restricted only to plants in some families in the order of Capparalle (49).

In conclusion, this study indicates that in the Arabidopsis genome, the number of sequences containing the signature characteristic of UGTs is extremely high. The phylogenetic analysis, defining 12 distinct evolutionary groups, provides a useful new foundation for understanding the structure-function relatedness of the UGT family members and a new framework for exploring and modifying their substrate specificities.


    ACKNOWLEDGEMENTS

We thank the Arabidopsis Biological Resource Center for providing all expressed sequence tag clones. We also thank Dr. Joe Ross for critical reading of the manuscript and helpful discussions.


    Note Added in Proof

An additional 18 complete UGT sequences have been identified in Arabidopsis genome data base subsequent to the submission of this manuscript. These do not change the composition of the groups defined in this manuscript, and they add an additional two groups to the tree.


    FOOTNOTES

* Financial support of this work was provided by Biotechnology and Biological Sciences Research Council grant 87/97 8855 to D. J. B.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplementary material including additional details of the Arabidopsis UGTs analyzed in this study (Table S1), corrections to the data base annotations of UGT sequences (Table S2), and the complete amino acid sequence alignment of 88 Arabidopsis UGTs (Fig. S1).

Dagger To whom correspondence should be addressed. Tel.: 44-1904-434334; Fax: 44-1904-434336; E-mail: djb32@york.ac.uk.

Published, JBC Papers in Press, October 20, 2000, DOI 10.1074/jbc.M007447200

2 In the UGT nomenclature system, a gene name includes (a) UGT, defining a putative UDP-dependent glycosyltransferase; (b) an Arabic number from 71 to 100, which designates a UGT family of plant origin, the same number being used for sequences with >45% sequence identity; (c) a letter, representing a subfamily whose members share >60% identity; and (d) a number, corresponding to the individual gene. The letter "P" after the gene number is used to denote a pseudogene. For clarity, the prefix UGT is omitted throughout this paper.


    ABBREVIATIONS

The abbreviations used are: UGT, UDP-glycosyltransferase; ORF, open reading frame; PCR, polymerase chain reaction.


    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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