Phylogenetic and Expression Analysis of the Glutamate...

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Mol. Biol. Evol. 19(7):1066–1082. 2002q 2002 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038

Phylogenetic and Expression Analysis of the Glutamate-Receptor–LikeGene Family in Arabidopsis thaliana

Joanna C. Chiu,* Eric D. Brenner,* Rob DeSalle,† Michael N. Nitabach,* Todd C. Holmes,*and Gloria M. Coruzzi**Department of Biology, New York University; and †Department of Entomology, American Museum of Natural History,New York

The ionotropic glutamate receptor (iGluR) gene family has been widely studied in animals and is determined to beimportant in excitatory neurotransmission and other neuronal processes. We have previously identified ionotropicglutamate receptor–like genes (GLRs) in Arabidopsis thaliana, an organism that lacks a nervous system. Upon thecompletion of the Arabidopsis genome sequencing project, a large family of GLR genes has been uncovered. Apreliminary phylogenetic analysis divides the AtGLR gene family into three clades and is used as the basis for therecently established nomenclature for the AtGLR gene family. We performed a phylogenetic analysis with extensiveannotations of the iGluR gene family, which includes all 20 Arabidopsis GLR genes, the entire iGluR family fromrat (except NR3), and two prokaryotic iGluRs, Synechocystis GluR0 and Anabaena GluR. Our analysis supportsthe division of the AtGLR gene family into three clades and identifies potential functionally important amino acidresidues that are conserved in both prokaryotic and eukaryotic iGluRs as well as those that are only conserved inAtGLRs. To begin to investigate whether the three AtGLR clades represent different functional classes, we performedthe first comprehensive mRNA expression analysis of the entire AtGLR gene family. On the basis of RT-PCR, allAtGLRs are expressed genes. The three AtGLR clades do not show distinct clade-specific organ expression patterns.All 20 AtGLR genes are expressed in the root. Among them, five of the nine clade-II genes are root-specific in 8-week-old Arabidopsis plants.

Introduction

Sequence homology screening has become an in-creasingly popular method for cloning genes of interest,drawing from the large amount of sequence data gen-erated from genome sequencing projects of various pro-karyotic and eukaryotic organisms. Using the Arabidop-sis thaliana genomic sequence and expressed sequencetag database, Lam et al. (1998) identified two comple-mentary DNA clones, GLR1.1 and GLR3.1 (a.k.a. GLR1and GLR2), and two genomic sequences, GLR2.1 andGLR3.4 (a.k.a. GLR3 and GLR4), that have sequencesimilarity with animal ionotropic glutamate receptor(iGluR) genes. In addition to primary sequence similar-ity, the Arabidopsis GLR (AtGLR) genes are also pre-dicted to be similar to animal iGluRs, in terms of sec-ondary structure, i.e., transmembrane topology, asshown by hydropathy plots (Lam et al. 1998). Genesthat have sequence similarity with the other family ofanimal glutamate receptors, the metabotropic glutamatereceptors, have not been found in Arabidopsis. However,Turano et al. (2001) suggested a possible evolutionarylink between AtGLRs and seven transmembrane G-pro-tein–linked receptors, which includes metabotropic glu-tamate receptors as well as GABA receptors.

Animal iGluR genes encode subunits for ligand-gated ion channels, which account for a major fraction

Abbreviations: AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; GlnH, glutamate-binding domain; iGluR, ionotropicglutamate receptor; KA, kainate; M, transmembrane domain; NMDA,N-methyl-D-aspartate; P, pore.

Key words: glutamate receptor, plant, Arabidopsis thaliana,mRNA expression, character mapping.

Address for correspondence and reprints: Gloria Coruzzi, Depart-ment of Biology, New York University, 100 Washington Sq. East, NewYork 10003. E-mail: [email protected].

of fast excitatory neurotransmission. They form a largegene family that can be divided into multiple classes (N-methyl-D-aspartate [NMDA], a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid [AMPA]-kainate[KA], and Delta) based on ligand selectivity and ionconductance properties (Sprengel and Seeburg 1995).NMDA iGluRs have been implicated in neurodegener-ative diseases and are believed to be involved in neu-ronal cell death for pathological conditions such as is-chemia (Rameau et al. 2000). AMPA and KA receptorsare often grouped together as non-NMDA iGluRs be-cause of ligand cross-reactivity and their similarities insequence as well as ion-conducting properties (Sutcliffe,Wo, and Oswald 1996). We will refer to AMPA and KAiGluRs as a single class in this paper for the reasonsmentioned above (AMPA-KA). Lastly, the delta iGluRsdo not appear to have functional ion channel and ligand-binding activity in their wild-type form (Araki et al.1993; Lomeli et al. 1993). However, the widely studied‘‘Lurcher’’ mutation in the d2 subunit results in a con-stitutively open ion channel with properties similar toAMPA-KA receptor channels and leads to neurodegen-eration in mice (Wollmuth et al. 2000). d2 receptors arefound to express predominantly in cerebellar Purkinjecells (Kohda, Wang, and Yuzaki 2000).

Because animal iGluRs have been studied exten-sively in mammalian CNS (Sprengel and Seeburg 1995),the discovery of their putative homologs in Arabidopsis,an organism without a nervous system, was surprising(Lam et al. 1998). By performing an initial phylogeneticanalysis of animal iGluRs and four Arabidopsis GLRgenes, Chiu et al. (1999) determined that the divergenceof animal iGluRs and Arabidopsis GLRs preceded thedivergence of animal iGluR classes (AMPA-KA,NMDA, Delta). This preliminary comparative phyloge-netic analysis of plant GLRs and animal iGluRs sug-

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Table 1GenBank Accession Numbers and Nomenclature for the AtGLR Gene Family

PREVIOUS NAME

CDNA

Full Length Splice Variants

GENOMIC

BAC Protein ID

I . . . . . AtGLR1.1AtGLR1.2AtGLR1.3AtGLR1.4

AtGLR1a AF079998AY072064b

AY072066b

AY072065b

AY072067b

AC016829AB020745.1AB020745.2AC009853

AAF26802.1BAA96960.1BAA96961.2AAF02156.1

II. . . . . AtGLR2.1AtGLR2.2AtGLR2.3AtGLR2.4AtGLR2.5AtGLR2.6

AtGLR3a

AY072068bAF007271AC007266.1AC007266.2AL031004AL360314.1AL360314.2

AAB61068.1AAD26895.1AAD26894.1CAA19752.1CAB96656.1CAB96653.1

AtGLR2.7AtGLR2.8AtGLR2.9

Glur9AY072069b

AJ311495AC005315.1AC005315.2AC005315.3

AAC33239.1AAC33237.1AAC33236.1

III . . . . AtGLR3.1AtGLR3.2AtGLR3.3AtGLR3.4AtGLR3.5AtGLR3.6AtGLR3.7

AtGLR2a, ACL1c

AtGluR2d

AtGLR4a, GLUR3e

GLR6

GLR5

AF079999AF159498

AF167355AF170494

AF210701

AF038557

AY072070b

AC002329AL022604AC025815AC000098AC005700.1AL133452AC005700.2

AAF63223.1CAA18740.1AAG51316.1AAB71458.1AAC69939.1CAB63012.1AAC69938.1

a Lam et al. 1998.b Current paper.c ACL1 corresponds to the splice variant AF038557.d Kim et al. 2001.e GLUR3 is deposited into GenBank as AF167355.

gested that signaling by amino acids might be a primi-tive mechanism that existed before the divergence ofplants and animals. This hypothesis was subsequentlyverified with the identification of the first prokaryoticfunctional ionotropic glutamate receptor, GluR0, in Sy-nechocystis (cyanobacteria) (Chen et al. 1999).

It is currently unknown whether Arabidopsis GLRs,like animal iGluRs and Synechocystis GluR0, are ca-pable of forming functional ion channels. Glutamate-gated calcium fluxes have been observed in Arabidopsisroots (Dennison and Spalding 2000), but links of thisactivity to specific AtGLR genes have not yet been es-tablished. Despite the fact that the biochemical proper-ties of GLR proteins remain an open question, theAtGLR genes have already been implicated in processes,such as light signal transduction (Lam et al. 1998; Bren-ner et al. 2000) and calcium homeostasis (Kim et al.2001) in plants. We therefore expect that AtGLRs willbe involved in many different signaling and physiolog-ical processes in plants, especially because a large fam-ily of 20 AtGLR genes has been uncovered with thecompletion of the Arabidopsis genome sequencing pro-ject (AGI 2000). A nomenclature has recently been es-tablished for the AtGLR gene family (Lacombe et al.2001). The nomenclature is based on a preliminary par-simony analysis that divides the AtGLR gene family intothree clades.

In this paper, we confirm the identification of 20GLR genes in Arabidopsis and report the characteriza-tion of five new full-length AtGLR cDNA sequences. Weexamine the genealogical relationships of the iGluRfamily and identify residues that are invariant (‘‘invari-ant’’ carries the meaning of ‘‘unvaried’’ rather than ‘‘in-

variable’’ in this paper) in both prokaryotic and eukary-otic iGluRs by performing a parsimony analysis withextensive annotations using the amino acid sequences ofa complete family of animal iGluRs, all 20 ArabidopsisGLRs, and two prokaryotic cyanobacterial iGluRs, Sy-nechocystis GluR0 and Anabaena GluR. SynechocystisGluR0 encodes the first identified functional prokaryoticiGluR channel subunit (Chen et al. 1999). On the otherhand, functional studies on the Anabaena GluR have notbeen published yet. This study demonstrates that phy-logenetic analysis can be a powerful tool to guide func-tional studies of large gene families as well as to un-derstand evolutionary relationships. We also examinemRNA expression patterns of all 20 GLR genes in dif-ferent organs and determine cell-type–specific expres-sion patterns for a gene from each of the three clades.This analysis has allowed us to investigate whether thephylogenetic division of the AtGLR gene family hasfunctional implications by testing the hypothesis ofclade-specific gene expression patterns.

Materials and MethodsObtaining Sequences for Phylogenetic Analysis

The complete family of 20 Arabidopsis GLR genesidentified upon the completion of the Arabidopsis ge-nome sequencing project is listed in table 1. These 20AtGLR genes were identified by BLAST search (Al-tschul et al. 1997) using AtGLR1.1 cDNA sequence(AF079998) as the query sequence. The AtGLR se-quences used in our parsimony analysis include a com-bination of full-length complementary DNA sequencesthat were available at the time of the analysis (GLR1.1,

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Table 2GenBank Accession Numbers and Abbreviations for Two Prokaryotic GlutamateReceptors, Rat Glutamate Receptors, and Bacterial Periplasmic Amino Acid–BindingProteins Examined in this Study

Gene Species Accession NumberAbbreviation Used

(if different from name)

Bacterial periplasmic amino acid binding proteinglnH . . . . . . . .glnP . . . . . . . .glnP . . . . . . . .

Escherichia coliArcharoglobus fulgidusSalmonella typhimurium

X14180AE001090U73111

ecoliglnhafgglnpsalglnp

Prokaryotic iGluRGluR0 . . . . . .GluR. . . . . . . .

Synechocystis PCC6803Anabaena PCC7120

D90909AP003591

SynGluR0AnaGluR

Rat iGluRGluRA . . . . . .GluRB . . . . . .GluRC . . . . . .GluRD . . . . . .GluR5 . . . . . .GluR6 . . . . . .GluR7 . . . . . .KA-1 . . . . . . .KA-2 . . . . . . .Delta 1 . . . . . .Delta 2 . . . . . .NMDAR1 . . .NR2A. . . . . . .NR2B. . . . . . .NR2C. . . . . . .NR2D. . . . . . .

Rattus norvegicusR. norvegicusR. norvegicusR. norvegicusR. norvegicusR. norvegicusR. norvegicusR. norvegicusR. norvegicusR. norvegicusR. norvegicusR. norvegicusR. norvegicusR. norvegicusR. norvegicusR. norvegicus

X17184X54655M36420M36421M83560Z11715M83552U08257Z11581Z17238Z17239X63255M91561M91562M91563L31612

RatK1RatK2RatGluRCRatGluRDRatGluR5-1RatGluR6RatGluR7RKA1RKA2RDELTA1RDELTA2RNMDAR1RNR2ARNR2BRNR2CRNR2D

GLR1.2, GLR1.4, GLR2.2, GLR2.7, GLR3.1, GLR3.2,GLR3.4, GLR3.5, and GLR3.7) and predicted cDNA se-quences from genomic sequence analysis for the re-maining genes. Whereas the full-length cDNA sequenc-es for GLR1.1, GLR3.1, GLR3.2, GLR3.5, and GLR3.7were obtained from GenBank, the full-length cDNAclones for GLR1.2, GLR1.4, GLR2.2, and GLR2.7 werenewly constructed for this analysis by RT-PCR (see Ma-terials and Methods—RT-PCR section), and the full-length cDNA clone for GLR3.4 was obtained throughcDNA screening. The remaining 10 AtGLR sequencesused in our analysis (GLR1.3, GLR2.1, GLR2.3, GLR2.4,GLR2.5, GLR2.6, GLR2.8, GLR2.9, GLR3.3, andGLR3.6) were predicted from genomic sequences. In ourpreliminary alignment, cDNA sequences predicted byGenBank were used for all AtGLR genes for whichcDNA clones were not available. Upon inspection of thepreliminary alignment, conserved iGluR exon sequencesin some of the predicted AtGLR genes were regarded asintron sequences by the gene prediction program usedby GenBank and were spliced out. We therefore editedthe predicted cDNA sequence from GenBank for theAtGLR genes in question using alignment and existingand newly constructed AtGLR cDNAs as guidelines.

The sequences for the two prokaryotic iGluRs (Sy-nechocystis GluR0 and Anabaena GluR) and the ratiGluR genes used in this study were obtained fromGenBank (table 2). Bacterial periplasmic amino acid–binding protein sequences from Escherichia, Salmonel-la, and an archaebacteria were included in this analysisas outgroups (table 2), as in our previous analysis (Chiuet al. 1999).

Phylogenetic Analysis

Full-length amino acid sequences were aligned byCLUSTAL X using three separate sets of alignment pa-rameters (gap-to-change 5 10, 15, and 20; gap exten-sion cost 5 1; amino acid substitution matrix 5 Blosum30). The three values for gap-to-change ratio were ar-bitrarily chosen as reasonable starting points for exam-ining the effect of gap-to-change cost on final alignmentand hence on tree topology. A gap extension cost of 1is conservative and will produce reasonable alignmentswithout excessive hypotheses of gap insertions. Thesealignments were examined for alignment-ambiguous re-gions, i.e., columns of amino acids that change fromalignment-cost to alignment-cost (Gatesy, DeSalle, andWheeler 1993). These regions of alignment instabilitywere culled from the matrix to ensure validity of theanalysis. Before parsimony analysis, the matrix was alsogap-coded as described in Chiu et al. (1999). In brief,we chose to gap-code the regions in our alignmentwhere long stretches of positions showed gaps, so thatthese regions are not weighted excessively in our phy-logenetic analysis. Our gap coding was implemented asunordered. Indel events, which created gaps, oftentimesspanned several amino acid positions, and such regionswere recoded as single multistate characters on the basisof their lengths (DeSalle and Brower 1997). The treat-ment of bacterial amino acid–binding protein sequencespresented another data-coding issue. There are two re-gions in these bacterial genes that show similarity witheukaryotic iGluRs (Paas 1998). These two regions cor-respond to the glutamate-binding domains in iGluRs and


FIG. 1.—Arabidopsis GLR genes group into three distinct clades.Phylogenetic tree generated from parsimony analysis of amino acidsequences of rat iGluRs, AtGLRs, and two prokaryotic cyanobacterialiGluRs using three bacterial periplasmic amino acid–binding proteinsequences as outgroups. Our search resulted in two equally parsimo-nious trees. The bootstrap consensus tree is shown here.

are used to root the present parsimony analysis as wellas our previous analysis (Chiu et al. 1999). All othercharacters in the glutamate receptor sequences that arenot in these two regions and are absent in the bacterialsequences were coded as missing in the outgroups.

In order to strengthen and confirm our analysis, wealso performed a second parsimony analysis where weprocessed the data matrix using elision (Wheeler, Ga-tesy, and DeSalle 1995) instead of a combination ofculling and gap-coding. As in the first parsimony anal-ysis, we used CLUSTAL X to align the sequences usingdifferent sets of alignment parameters (gap-to-change 510, 15, and 20; gap extension cost 5 1; amino acidsubstitution matrix 5 Blosum 30). We then combinedthe three matrices to generate a larger matrix, which weused for parsimony analysis.

We used PAUP* (phylogenetic analysis using par-simony; Swofford 1998) to infer phylogeny. Ten ran-dom-addition heuristic searches with TBR branch swap-ping were implemented, and all characters were equallyweighted in our analyses. We did not place a limit onthe number of trees saved in each search. When morethan one equally parsimonious tree was obtained, wegenerated strict consensus trees to represent our phylo-genetic hypothesis. To measure the robustness of allnodes in our parsimony analysis, bootstrap analysis (100replicates) was performed using PAUP*. Ten random-addition heuristic searches with TBR branch swappingwere used for each replicate of the bootstrap analysis.

Computation of Percent Identity Values

We used CLUSTAL X to generate the percent iden-tity values between different iGluRs when comparingthe entire amino acid sequences. The default parameters(gap-to-change cost 5 10; gap extension cost 5 1; ami-no acid substitution matrix 5 Blosum 30) were used forthe alignment, and percent identity values were gener-ated along with the alignment. We then compiled thesevalues to generate—(1) the percent identity values be-tween genes within the same rat iGluR classes or AtGLRclades (AMPA and KA iGluRs are grouped together asa single class), (2) the percent identity values betweengenes that are in different rat iGluR classes or AtGLRclades, and (3) the percent identity values between ratand Arabidopsis iGluR genes. The percent identity val-ues for the glutamate-binding domains as well as thepore (P) and surrounding transmembrane regions (M1and M2) were generated using the same method. Thesequence boundaries for the functional domains in ratiGluR genes used in this study follow the boundariesthat were previously established by structural and func-tional studies (Hollmann and Heinemann 1994; Stern-Bach et al. 1994). The sequence boundaries of trans-membrane domains in AtGLR genes were predicted bythe software PHD version 1996.1 (Rost 1996). The com-puter predictions generally coincided with the AtGLRtransmembrane domain boundaries predicted when us-ing the alignment of the AtGLR genes and rat iGluRgenes as guidelines. In cases where the two predictionsdid not match exactly, they only differed by one or two

amino acids. The sequence boundaries for the gluta-mate-binding domains were predicted using the align-ment of AtGLRs and rat iGluRs as guidelines becauseprograms specializing in predicting glutamate-bindingdomains are not available. Amino acid residues numbers406–527 and 628–737 were used for the alignment forthe glutamate-binding domains, and amino acid residuesnumbers 543–626 were used for the alignment for theP and surrounding transmembrane regions. The aminoacid numbering follows the sequence of AtGLR1.1(AF079998). The compiled percent identity values werethen mapped onto a schematized version of a tree gen-erated from our parsimony analysis.

Character Mapping

We imported the processed data matrix used forparsimony analysis and the consensus tree obtainedfrom parsimony analysis into MacClade version 4.0(Maddison WP and Maddison DR 1992) for character-mapping analysis. We identified conserved residues atdifferent nodes by using the trace character function inMacClade.

For mapping RT-PCR expression data on the par-simony tree, we converted AtGLR mRNA expression inleaves, roots, flowers, and siliques as detected by RT-PCR into binary format (0 5 undetected and 1 5 de-tected) and appended the four additional characters tothe matrix used to generate the parsimony tree shownin figure 1. The corresponding characters for the rat andcyanobacteria genes are coded as missing. We used thisnew matrix, with four additional characters, to generatea second parsimony tree. The resulting phylogenetic tree

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is identical to the one shown in figure 1. We then usedthe trace-character function in MacClade to map eachof the four expression characters onto the phylogenetictree.

Examining AtGLR Expression by RT-PCR

First-strand synthesis reactions using ThermoscriptRT (Invitrogen) were performed on mRNA (200 ng)generated from leaves, roots, flowers, and siliques, re-spectively (Ambion PolyA Pure mRNA isolation kit).Leaf and root tissues were collected from 8-week-oldArabidopsis (Columbia Col-0 ecotype) plants grown inshort days (8 h light-16 h dark) and hydroponic condi-tions (1 mM KH2PO4, 1 mM MgSO4, 0.1 mM NaFe-EDTA, 50 mM KCl, 30 mM H3BO3, 5 mM MnSO4, 1mM ZnSO4, 1 mM CuSO4, 0.1 mM (NH4)6Mo7O24, 0.5mM KNO3, and 0.25 mM Ca(NO3)2). Flowers and si-liques were collected from mature Arabidopsis plants (8-week-old Columbia Col-0 ecotype) grown under long-day condition (16 h light-8 h dark). To examine the ex-pression patterns of all 20 AtGLR genes, 20 sets of spe-cific primers corresponding to the distinct AtGLR genes(sequences for specific primers are available from au-thors upon request) were designed. The specificity of theprimers was tested by using them to amplify Arabidop-sis genomic DNA in which all AtGLR genes are presentat the same copy number. The resulting PCR productswere then sequenced to determine if the expected prod-ucts were amplified. The 20 primer sets were used toperform PCR using the cDNA produced from the firststrand synthesis reactions as templates (Takara Ex-Taqpolymerase from Panvera). All 20 sets of primers weredesigned to span intron sequences, thus allowing us toaccount for signals resulting from genomic DNA con-tamination. Primers that amplify TUB5 (accession num-ber M84702) from Arabidopsis (59-CTCGCAACAATACATCTCACTCACC-39 and 59-AAGACACCCAAAATAAATGGAAACTTC-39) were used as control primersto ensure uniform amplification and were added to eachPCR reaction together with the AtGLR gene-specificprimers. All primers were used at a concentration of 40ng/ml. Thermocycling time and temperature were as fol-lows: 948C for 5 min, followed by 30 cycles of 948Cfor 30 s, 558C for 30 s, 728C for 1 min, and a finalextension period of 728C for 10 min. Because of thedifference in primer efficiencies, we cannot compare theexpression levels between different AtGLR genes. Wetherefore assess the relative difference in expression lev-els in different organs for each AtGLR gene, respective-ly. It is important to note that the RT-PCR results pre-sented here are not truly quantitative because of theproblem of saturation (30 amplification cycles used).However, we confirmed the relative expression level ofeach AtGLR gene in the four different organ types byperforming a second set of PCR reactions using 20 am-plification cycles. In addition, the same experimentswere repeated using 40 amplification cycles to addressthe problem of inefficient amplification in cases wherePCR products were not detected.

We analyzed the PCR products using gel electro-phoresis followed by NIH image version 1.62. Thegraphic representation of the RT-PCR data is generatedby normalizing AtGLR expression against TUB5 expres-sion. To construct each AtGLR graph, we measured theintegrated density values of the TUB5 band and theAtGLR band for each of the four organ lanes on thedigitalized picture of the agarose gel (samples are from30 PCR cycles) using NIH image. The integrated densityvalues of the four AtGLR bands were then normalizedusing the integrated density values of the four TUB5bands as control. The normalized integrated density val-ues of the four AtGLR bands were then used to calculatethe relative intensity values shown on the graph. Thehighest of the four intensity values was regarded as 1,and the others were calculated and represented as a frac-tion of the highest value.

The above RT-PCR protocol was also used to ob-tain full-length cDNA clones for GLR1.2, GLR1.4,GLR2.2, and GLR2.7. However, instead of using thegene-specific primers used to examine expression pat-terns, primers that were predicted from genomic se-quence analysis to represent the beginning and end ofeach full-length cDNA sequence were used in the PCRamplification reactions.

Examining AtGLR Expression in Transgenic Plants

Representative AtGLR genes from each of the threeGLR clades were examined for cell-type expression pat-terns using a reporter gene system. Transcriptional fu-sions were made between the promoters of GLR1.1,GLR2.1 or GLR3.1, and the reporter gene b-glucuroni-dase (GUS) (Jefferson 1989). AtGLR promoter regionswere amplified by PCR from Columbia (Col-0) genomicDNA using Ex-Taq polymerase (Panvera). A 1,713-bpregion upstream of the start codon of GLR1.1 was am-plified at the 59 end with the primer JC99 (59-CGTCGAAGCTTATAAGAAACG-39) and at the 39 end withthe primer JC101 (59-CATATCTACTTGTGCCATGG-39). The amplified fragment has a HindIII site at the 59end and an NcoI site at the 39 end so that GUS is trans-lated at the presumptive ATG start site for GLR1.1. A1,553-bp region upstream of the start codon of GLR2.1was amplified at the 59 end with the primer EBP027 (59-GTCGACGATCAAAGGTTATGTCGCTAAAGGAG-39) and at the 39 end with the primer EBP017 (59-GTGGATCCTACTTAGCCGAAAAAGAATGAAACTTG-39). This introduced a SalI restriction site at the 59 endand an NcoI site at the 39 end. The NcoI site facilitatedthe translation of GUS at the presumptive start ATG sitefor GLR2.1. A 1,780-bp upstream region from GLR3.1was amplified at the 59 end with the primer EBP4 (59-TGAAGCTTCGTTCACTAATTGGAGTGCAT-39) andat the 39 end with EBP1 (59-ATGACCATGGAGCTTAACATTGAACAACAAAAAGAG-39). This introduced aHindIII restriction site at the 59 end and an NcoI site atthe 39 end so that GUS is translated at the presumptivestart ATG site for GLR3.1. All fragments were cut at theintroduced restriction sites and cloned into pTZGUS(Ngai, Tsai, and Coruzzi 1997). The promoter::GUS fu-


sions were excised with EcoRI and HindIII for GLR1.1and GLR3.1 and with EcoRI and SalI for GLR2.1 andindependently subcloned into the binary Agrobacteriumvector pBI101 (Jefferson, Kavanaugh, and Bevan 1987)at those respective sites. Binary constructs were trans-formed into Agrobacterium strain GV3101, and Arabi-dopsis plants (Columbia Col-0 ecotype) were transformed(Clough and Bent 1998). At least 20 independent trans-genic lines for each construct were selected for analysis.Transgenic Arabidopsis seedlings were germinated onMS media containing 1% sucrose and grown in 16 hlight-8 h dark day-night cycles. GUS staining was per-formed according to the method of Jefferson, Kavanaugh,and Bevan (1987).

ResultsArabidopsis GLR Gene Family: Genomic and cDNAClone Identification

We have identified a total of 20 distinct open read-ing frames that represent glutamate-receptor–like genesin Arabidopsis (also listed in Lacombe et al. 2001) byusing AtGLR1.1 (AF079998) as the query sequence toperform a BLAST search (Altschul et al. 1997) againstthe completed A. thaliana genome (AGI 2000). Table 1lists the 20 AtGLR genes using the recently establishednomenclature for glutamate receptor genes in Arabidop-sis (Lacombe et al. 2001). Here we report the charac-terization of the corresponding full-length complemen-tary DNA clones for five new GLR cDNAs, GLR1.2,GLR1.4, GLR2.2, GLR2.7, and GLR3.4, isolated fromcDNA libraries or by RT-PCR. Together with the full-length cDNA clones that we have previously isolated,GLR1.1 and GLR3.1 (Lam et al. 1998), and the full-length cDNA clones that were isolated by other groups,GLR2.8, GLR3.2, GLR3.5, and GLR3.7, a total of 11AtGLR genes are currently found to have correspondingcDNAs (table 1). Although cDNAs have not been iso-lated for the other nine members, Northern and RT-PCRanalyses presented herein indicate that all AtGLR genesare expressed (see RT-PCR section).

Parsimony Analysis of iGluRs from Prokaryotes andEukaryotes Defines Three AtGLR Clades

Previously, a phylogenetic analysis of the iGluRgene family was performed using animal iGluRs and thefour AtGLR genes (GLR1.1, GLR2.1, GLR3.1, andGLR3.4) that were available at the time (Chiu et al.1999). This initial analysis showed that the divergenceof animal iGluRs and AtGLRs preceded the divergenceof animal iGluR classes (AMPA-KA, NMDA, and Del-ta). The completion of the Arabidopsis genome sequenc-ing project led to the identification of 16 additionalAtGLR genes. We therefore performed a new, morecomprehensive phylogenetic analysis using all the 20AtGLR gene family members of the model plant Ara-bidopsis (table 1) and all known iGluR genes of a singleanimal, rat (except NR3; table 2). In addition, two pro-karyotic iGluRs, Synechocystis GluR0 (Chen et al. 1999)and Anabaena GluR (table 2), were also included in thisphylogenetic analysis to examine where they fit into the

evolutionary history, relative to the complete plant andanimal glutamate receptor gene families.

Visual inspection of the alignment of all Arabidop-sis, rat, and two prokaryotic glutamate receptor genesreveals a high degree of similarity in most of the iGluRfunctional domains. These include the two glutamate-binding domains (GlnH1 and 2), transmembrane domainM1, the P, and transmembrane domain M2. On the otherhand, most of the sequences before GlnH1 and the re-gions after GlnH2, which includes the last transmem-brane domain M3 (absent in Synechocystis GluR0 andAnabaena GluR), show limited similarity. These regionsof low conservation and ambiguous alignment wereculled (Gatesy, DeSalle, and Wheeler 1993) accordinglyduring matrix processing before the first parsimonyanalysis. Although these highly variable regions may notbe absolutely required for protein activity, they maycontain domains with modulatory functions, e.g., regu-latory kinase-binding sites (Nitabach et al. 2001).

Parsimony analysis of the iGluR gene family usingbacterial sequences as outgroups gave two equally par-simonious trees of 4,235 steps, with a consistency indexof 0.616 and a retention index of 0.730. Figure 1 showsthe consensus parsimony tree with bootstrap values. Asshown in our previous analysis (Chiu et al. 1999), thedivergence of animal iGluR and AtGLR precedes thedivergence of animal iGluR classes (AMPA-KA,NMDA, and Delta). This phylogenetic inference is sup-ported by in vivo data showing that glutamate-gatedcalcium fluxes in Arabidopsis roots, which may belinked to AtGLR gene products, cannot be inducedwhen L-glutamate is replaced by animal iGluR class-specific ligands, such as AMPA or NMDA (Dennisonand Spalding 2000). The two prokaryotic iGluRs, Sy-nechocystis GluR0 and Anabaena GluR, are sister taxa,and they fall outside the multicellular clade. The well-characterized rat iGluR genes belonging to different an-imal iGluR classes all reside within their appropriateclades, with the exception of RNMDAR1. Instead offorming a monophyletic clade with the other fourNMDA iGluRs (RNR2A to 2D), it groups with the restof the animal iGluRs with a weak bootstrap value of54% (fig. 1). It is important to note that althoughRNMDAR1 has indeed diverged greatly from the otherfour NMDA iGluRs, its placement outside the cladeconsisting of the other four NMDA genes is only weaklysupported. In fact, in a similar analysis, where all butthe Anabaena GluR sequence is included, RNMDAR1forms a monophyletic clade with the other four NMDAgenes (bootstrap value 5 62%, data not shown). In gen-eral, the animal iGluR clades resulting from this parsi-mony analysis coincide with the classes established infunctional studies. Like animal iGluRs, the AtGLR genefamily also separates into clades (Clades I, II, and III)with strong node support (Bootstrap values 5 95%–100%).

The division of the AtGLR gene family into threeclades is also demonstrated by the comparison of aminoacid percent identity values between genes that are inthe same AtGLR clade (40%–57%) and genes that arefrom different AtGLR clades (21%–33%; fig. 2a). As

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FIG. 2.—Amino acid percent identity divides AtGLR genes into three clades. Amino acid percent identity values between different glutamatereceptor genes generated using: a, full-length amino acid sequences; b, sequences for the two glutamate-binding domains (amino acid residuenumbers 406–527 and 628–737); c, sequences for the P and the surrounding M1 and M2 transmembrane domains (amino acid residue numbers543–626). Amino acid numbering follows sequence of AtGLR1.1 (GenBank accession number AF079998).

shown by the amino acid percent identity values gen-erated by comparing entire proteins, there is a clearboundary between AtGLR clades based on sequenceidentity. As a test case, amino acid percent identity val-ues between different rat iGluRs also support the treetopology established in our parsimony analysis, whichagrees with the established animal iGluR classes. Thereis a clear separation between animal iGluR classes basedon amino acid percent identity values, with the excep-tion of the NMDA class. The amino acid percent iden-tity is relatively low (19%–38%) within the NMDAclass because NMDAR1 has diverged greatly from theother four NMDA genes (NR2A-D). When the percentidentity values generated for the genes within theNMDA class are excluded, the percent identity valuesbetween genes that are within the same animal iGluRclass are relatively high (29%–56%), whereas that be-tween genes from different animal iGluR classes are rel-atively low (10%–26%).

We then repeated the analysis using amino acid res-idues for important functional domains (see Materialsand Methods for boundaries of functional domains).When the amino acid percent identities were generatedusing only the ligand-binding domains (GlnH1 and 2)instead of the entire protein, the percent identity valuesincrease overall, but the boundary between AtGLRclades remains clear-cut (fig. 2b). The same holds truefor the animal iGluR classes as long as the percent iden-tity values generated for the genes within the NMDAclass are excluded. On the other hand, when the P andsurrounding regions (M1, P, M2) were used to generateamino acid percent identities, the clear boundary be-tween AtGLR clades disappeared (fig. 2c). Using onlythe P and surrounding regions, the amino acid percentidentities between genes that are in the same AtGLRclade (41%–92%) now overlap with the amino acid per-cent identities between AtGLR genes from differentclades (35%–68%), which might suggest an overlap inion selectivity, if AtGLR proteins are indeed ion channelsubunits. Interestingly, for rat iGluRs, the amino acidpercent identities for the P and surrounding regions pro-vide an observable boundary between distinct iGluRclasses with different ion selectivity (as long as the per-cent identity values generated for the genes within theNMDA class are excluded).

In addition to establishing the three clades ofAtGLR genes, our phylogenetic analysis also showedthat clades I and II are sisters to each other (bootstrapvalue 5 58%). To confirm the results of our analysis,we performed a second parsimony analysis using elision(Wheeler, Gatesy, and DeSalle 1995) as an alternativemethod to process the matrix, instead of a combinationof culling and gap-coding. The topology of the resultingparsimony tree is essentially identical to the one dis-cussed above (data not shown).

Defining Invariant Amino Acid Residues for the iGluRGene Family

With the inclusion of all 20 AtGLR gene familymembers and the two prokaryotic iGluRs (SynechocystisGluR0 and Anabaena GluR) in our analysis, we can nowidentify potential functionally important iGluR aminoacid residues that are absolutely conserved before thedivergence of plants and animals and those that are con-served even before the divergence of prokaryotes andeukaryotes. These iGluR invariant residues are present-ed in figure 3a. The present analysis is more compre-hensive than a previous one (Chiu et al. 1999) becausethe sequences for the entire AtGLR gene family (16more genes) and two prokaryotic iGluRs are now in-cluded. Another difference between this and our previ-ous analysis is that animal iGluR sequences included inthis analysis are limited to the sequences of a singleanimal, rat. In our previous analysis, animal iGluR se-quences from other species, such as Drosophila melan-ogaster, Caenorhabditis elegans, pigeon, and fish wereincluded. The exclusion of non-rat animal iGluR se-quences has allowed us to directly compare the entireiGluR gene family of a single plant (Arabidopsis) anda single animal (rat). Such an analysis could potentiallyincrease the number of conserved residues, but this doesnot appear to be the case. The only invariant residueidentified in this analysis as a result of excluding non-rat animal iGluR sequences is G701 in GlnH2 (fig. 3b;amino acid residue numbering is based on sequence ofAtGLR1.1), which is found in all animal iGluRs, exceptDrosophila GluR2 (M73271, data not shown). G701 isalso replaced by proline and asparagine in SynechocystisGluR0 and Anabaena GluR, respectively, and is there-


FIG. 3.—Identification of residues invariant to animal, plant, and cyanobacterial iGluRs. Diagrams showing the functional domains of theglutamate receptor protein and the locations of invariant amino acid residues between (a) rat iGluRs, AtGLRs, and cyanobacterial iGluRs; (b)rat iGluRs and AtGLRs; (c) all AtGLRs. The boxes representing the functional domains are not drawn to scale and do not reflect sequencelength. Invariant residues between rat iGluRs, AtGLRs, and cyanobacterial iGluRs are shown in black in all three panels. Invariant residuesbetween rat iGluRs and AtGLRs that are absent in cyanobacterial iGluRs are shown in red in panels b and c. Invariant residues that are specificto AtGLRs are shown in green in panel c. All amino acid numbers correspond to the sequence of AtGLR1.1 (accession number AF079998).

fore conserved only in rat iGluRs and ArabidopsisGLRs. By comparing figure 3a and b, we identified otheramino acid residues that are invariant in eukaryotes butare altered in at least one of the two prokaryotic cyano-bacterial iGluRs. These residues include I501, P538,W544, Y619, and L623. Among these five residues,L623 is the only one that is not altered in both cyano-bacterial genes. Whereas L623 is substituted with analanine in Anabaena GluR, it is conserved in Synecho-cystis GluR0. The other four residues that are not con-served between Synechocystis GluR0 and animal iGluRsmay be directly linked to the difference in ligand spec-ificity and ion selectivity between Synechocystis GluR0and animal iGluRs.

In addition to identifying invariant amino acid res-idues for the iGluR gene family, we have identified in-variant residues specifically for the Arabidopsis GLRgene family (fig. 3c). As expected, the conserved resi-dues unique to AtGLR genes span all the important func-tional domains defined for animal iGluRs. Future mu-tagenesis experiments on these residues may be usefulfor examining comparative differences between func-tional properties of animal iGluRs and AtGLRs.

AtGLR Genes from Different Clades Overlap in TheirOrgan Expression Profiles

Our parsimony analysis divides the animal iGluRgene family into three distinct clades (AMPA-KA,NMDA, and delta), with the exception of RNMDAR1(see parsimony analysis results section). These threeclades defined by parsimony analysis agree with the es-tablished classification of these distinct animal iGluR

classes based on biochemical properties, ligand specific-ity, and ion selectivity. The Arabidopsis GLR gene fam-ily is also divided into three clades based on our par-simony analysis. By analogy to animal iGluRs, we hy-pothesize that the three AtGLR clades may represent bio-chemically or functionally distinct glutamate receptorprotein classes. Whereas glutamate-gated calcium fluxeshave been observed in Arabidopsis (Dennison and Spal-ding 2000), no data are currently available for the bio-chemical activity encoded by specific AtGLR genes.Thus, we have begun to address whether the three GLRclades in Arabidopsis represent functionally distinctAtGLRs by first looking at the mRNA expression ofAtGLR genes from each clade.

We first examined the expression of all 20 AtGLRgenes using organ-specific RT-PCR (in situ hybridiza-tion experiments have been attempted for AtGLR genes,but the level of expression is too low to be detected).Because iGluR channels in animals are homotetramersor heterotetramers assembled from proteins encoded bygenes in the same functional class (Rosenmund, Stern-Bach, and Stevens 1998), it is possible that AtGLR genesfrom the same functional class (not defined yet) mayencode proteins that have the ability to form heteromersin vivo. In order for different proteins to form multi-mers, the colocalization of their mRNAs is likely andthat of their proteins is necessary. Therefore, we hy-pothesize that mRNAs of AtGLR genes that are in thesame clade may be present in the same organs. Distinctclade-specific expression patterns may be the first indi-cation that phylogenetically defined AtGLR clades rep-resent functional AtGLR classes. This hypothesis is

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based on the assumption that class-specific expressionis an important mechanism that regulates AtGLR hetero-meric protein coassembly.

Our RT-PCR analysis established that all 20 mem-bers of the AtGLR gene family are expressed genes (fig.4a and b). This finding addresses a previous concernthat several AtGLRs might be pseudogenes becausemany of the AtGLR gene family members are duplicatedgenes, existing in tandem on the chromosomes (notesame BAC clone numbers in table 1). Not only are theyall expressed, potential mRNA splice variants, repre-sented by additional bands on the gel, can be observedfor two AtGLR genes, namely GLR 2.5 and GLR 3.4.Whereas cDNA clones for GLR2.5 are not available atpresent, splice variants for GLR3.4 have already beenisolated (GenBank accession numbers AF167355 andAY072070) and thus confirmed our observation.

More importantly, our RT-PCR analysis of all 20AtGLR genes show that although AtGLR genes from thesame clade show similar organ expression profiles, thereis an overlap in expression patterns between genes thatare in different clades. Therefore, we conclude thatAtGLR genes from different clades do not show distinctorgan expression profiles. AtGLR clade I genes are de-tected in all organs examined (leaves, roots, flowers, andsiliques), with highest expression in roots (fig. 4a andb). For AtGLR clade-II genes, the majority is uniquelyexpressed in roots (GLR2.1, GLR2.2, GLR2.3, GLR2.6,and GLR2.9). Exceptions include GLR2.4, which is de-tected in siliques in addition to roots; GLR 2.5, whichis detected in all organs tested; and GLR2.7 and GLR2.8whose transcripts are detectable in all organs, exceptflowers. AtGLR clade-III genes, like clade-I genes, aredetected in all organs tested, with roots and siliquesshowing highest expression for the majority (exceptGLR3.2 and GLR3.7).

The RT-PCR mRNA expression data of AtGLRgenes was mapped onto the AtGLR portion of the iGluRparsimony tree to illustrate the relationship betweenAtGLR gene evolution and organ-specific expression(fig. 5). As shown in figure 5b, root expression is de-tected in all AtGLR genes and is inferred to be an ex-pression character state ancestral to the entire AtGLRgene family. Similarly, mRNA expression in leaves ap-pears to be the ancestral character state of the AtGLRgene family (fig. 5a). However, multiple independentchanges occurred only in AtGLR clade II, resulting inthe loss of leaf expression in six of the nine clade-IIgenes (GLR2.1, GLR2.2, GLR2.3, GLR2.4, GLR2.6, andGLR2.9). Lastly, mRNA expression in reproductive or-gans (flowers and siliques in Arabidopsis) also appearsto be ancestral to the AtGLR gene family (fig. 5c andd). However, once again, multiple independent changesoccurred specifically in AtGLR clade II resulting in (1)a loss of flower expression in all but one of the clade-II genes (GLR2.5), and (2) a loss of silique expressionin five clade-II genes (GLR2.1, GLR2.2, GLR2.3,GLR2.6, and GLR2.9).

Although our RT-PCR results show that AtGLRgenes from different clades overlap in their organ ex-pression profiles, we cannot negate the possibility that

AtGLR genes that are expressed in the same organs maybe present in different cell types. To determine the spe-cific cell types in the organs in which the differentAtGLR genes are expressed, we next examined trans-genic plants that have been transformed with constructsin which the putative promoter of a representative foreach of the three AtGLR clades was fused with the re-porter gene GUS (Jefferson 1989). By this method, weexamined the cell-type–specific expression pattern ofone gene from each AtGLR clade (GLR1.1, GLR2.1,GLR3.1). GUS expression in GLR1.1 promoter::GUStransgenic plants first appears in 7-day-old plants in stip-ules and the collette region (root-shoot junction; fig. 6aand d). Expression is later detected (3- to 4-week-oldplants) in all cell types of lateral roots and at the marginof mature leaves, in addition to all the organs mentionedabove (fig. 7a, d, and g). GLR1.1 expression in repro-ductive organs, flowers, and siliques, is not readily de-tectable (fig. 8a and e). According to promoter::GUSexpression, GLR2.1 (a clade-II gene) has a shoot ex-pression pattern in young seedlings (starting from 5 daysold) that is similar to GLR1.1 in that they are both ex-pressed in stipules (fig. 6e and h). This overlap in ex-pression pattern supports the sister relationship of cladesI and II established in our parsimony tree (fig. 1). Inaddition, GLR2.1 expression is detected in the radicalimmediately after emergence and is clearly observablein the root starting when the seedlings are 3 days old(fig. 6b). GLR2.1 is expressed in all cell types of theroot (including the root hairs), except at the root apex.Root expression of GLR2.1 is also present in more ma-ture seedlings (fig. 7e, h, and k). Although GLR2.1 ex-pression is not readily detectable in flowers and siliques(fig. 8b and f), slight and transient expression has beenobserved in anthers and young ovules (data not shown).Whereas the shoot expression of GLR1.1 and GLR2.1overlap significantly, the shoot expression pattern of-GLR3.1, a clade-III gene, is very different (figs. 6f, 6i,and 7c). GLR3.1 expression is first visible in the vas-culature of the cotyledons in 5-day-old young seedlings(fig. 6f), and its expression is maintained in the leaf andcotyledon vascular tissues as the seedlings mature (fig.7c). GLR3.1 expression is later found in all organs, in-cluding flowers (fig. 8c and d), siliques (fig. 8g and h),and roots (fig. 7f, i, and l), specifically in the vasculatureand in cell types associated with the vasculature and inparticular at contact sites or sites of vascular transferinto plant organs, such as the funiculus and the devel-oping seed. The expression pattern of a second clade-III gene, GLR3.2, is identical to that of GLR3.1 basedon promoter::GUS assays (data not shown).

Discussion

Arabidopsis expresses a large family of GLR genesthat may be involved in multiple functions, such as lightsignal transduction (Lam et al. 1998; Brenner et al.2000) and calcium homeostasis (Kim et al. 2001). An-imal iGluR genes are traditionally divided into func-tional classes (AMPA-KA, NMDA, and Delta) based onligand specificity as well as electrophysiological prop-


FIG. 4.—RT-PCR mRNA expression analysis reveals overlap in organ expression of AtGLR genes from different clades. a, RT-PCR analysisof the AtGLR gene family using mRNA generated from four different Arabidopsis organs: leaf (L), root (R), flower (F), and silique (S). Theresults shown here represent PCR reactions with 30 amplification cycles. b, Graphic representation of the RT-PCR data of the AtGLR genefamily. Each graph shows the relative intensity of the four AtGLR bands in the different organ lanes on the agarose gel shown in figure 4anormalized against TUB5 (accession number M84702).

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FIG. 4.—Continued.


FIG. 5.—Character-mapping of RT-PCR expression data reveals ancestral and derived states of three AtGLR clades. AtGLR mRNA expressionin (a) leaves, (b) roots, (c) flowers, and (d) siliques as detected by RT-PCR (fig. 4) are converted into binary format (0 5 undetected and 1 5detected) and mapped onto the phylogenetic tree generated from our parsimony analysis (only the AtGLR part of the tree is shown in thisfigure). Light gray: expression is detected by RT-PCR; Black: expression is not detected; Dark gray: equivocal; Asterisks: root-specific AtGLRgenes as detected by RT-PCR performed on 8-week-old Arabidopsis plants (see Materials and Methods for growth conditions).

erties. Genes from different animal iGluR classes en-code proteins that perform different functions in the an-imal nervous system (Sprengel and Seeburg 1995). Inour phylogenetic analyses in this and our previous paper(Chiu et al. 1999), the animal iGluR clade division weobtained using parsimony and Neighbor-Joining analy-ses of animal iGluR amino acid sequences agreed withthe widely accepted class division based on biochem-istry (except with the placement of RNMDAR1 in thecurrent analysis). The well-studied animal iGluR genesthus represent a test case for linking phylogenetic anal-ysis and physiological function. Our results showed thatphylogenetic analysis using sequence data is a usefultool for dividing a large gene family into potential func-tionally distinct classes, when anatomical, biochemical,and other functional data are not yet available. By ap-plying this approach to the GLR gene family in Arabi-dopsis, the 20 AtGLR genes can be divided into threeclades (also shown in Lacombe et al. 2001). The func-tional significance of this classification sets the stage forclade-specific testing of function using expression, bio-chemical, electrophysiological, and in planta physiolog-ical data.

In this paper, we have begun to address whetherthe three AtGLR clades represent functionally distinctprotein classes by examining the mRNA expression pat-

terns of all 20 AtGLR genes and promoter activities ofrepresentatives of each clade. Because iGluRs exist astetramers in animals (Rosenmund, Stern-Bach, and Ste-vens 1998), determining the organ and cell-type–specificexpression patterns of each AtGLR gene will also enableus to predict which gene products could potentially in-teract in vivo. Heteromultimeric coassembly can pro-foundly influence protein function (Sprengel and See-burg 1995; Nitabach et al. 2001). By using RT-PCR, weobtained an mRNA expression profile for the entireAtGLR gene family in four different organ types, namelyleaves, roots, flowers, and siliques. As a cautionary notebecause the phylogenetic analysis was performed usingthe coding sequences of the genes although the mRNAexpression patterns are controlled by regulatory noncod-ing sequences, they might not have direct correlation.Our RT-PCR results show that there is overlap in organexpression profiles between genes from different AtGLRclades. Whereas genes from AtGLR clades I and III areexpressed more ubiquitously, clade-II genes are largelyroot specific (fig. 4a and b). We conclude that the threeAtGLR clades are not distinct based on organ-level ex-pression patterns. Thus, the three AtGLR clades maycontain genes with overlapping functions in vivo. Inter-estingly, five of the nine clade-II genes are root-specificin 8-week-old Arabidopsis plants, and may potentially

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FIG. 6.—Three AtGLR clades exhibit cell-specific expression patterns in young seedlings. Anatomical and developmental expression of GLR1.1, GLR2.1, and GLR3.1 as seen in transgenic Arabidopsis seedlings harboring promoter::GUS fusions. Early after germination (3–5 days),GLR2.1 shows strong expression in all cell types of the root except the apex (b). Similarly, weak expression of GLR1.1 can be seen in the root(a). GLR3.1 expression cannot be detected at this early stage (c). In more mature seedlings (5–14 days), GLR1.1 and GLR2.1 are expressed instipules (d, e, g, and h), whereas GLR3.1 expression is found in the vasculature and in cells neighboring the vasculature (f and i).

represent a functional class. Additional data from phys-iological studies are necessary to verify this propositionbecause genes that have the same expression patternmay not share the same function.

By mapping the RT-PCR mRNA expression dataonto the iGluR parsimony tree (fig. 5), we are able tomake phylogenetic inferences relating organ-specific ex-pression and AtGLR gene evolution. Ubiquitous expres-sion in leaves, roots, and reproductive organs appears tobe the ancestral character states of the AtGLR gene fam-ily, but multiple independent character state changes oc-curred specifically in clade-II lineages, resulting in theloss of expression in leaves, flowers, and siliques insome of the clade-II genes. It is interesting to note thatall changes in expression character state occurred spe-cifically in clade-II genes. Perhaps the gene duplicationevent leading to the divergence of GLR clades I and IIprovided the opportunity for one of these two AtGLRclades (clade II) to diverge in terms of function, as in-dicated by the change in expression patterns.

To test the possibility that mRNA of differentAtGLR genes that are detected in the same organs maybe present in different cell types, we examined trans-genic plants harboring promoter::reporter gene con-structs to determine the cell types in the organs in whichdifferent AtGLR genes are expressed. For this, we ana-lyzed one representative gene from each of the threeAtGLR clades. Although genes from both clades I andIII are expressed in all organs tested as shown by RT-PCR, promoter::GUS analyses of representative clade-I(GLR1.1) and clade-III (GLR3.1) genes indicate thatgenes from clades I and III may be expressed in differ-ent cell types. The expression of GLR3.1 (a clade-IIIgene) is limited to the vasculature in all organs tested(as is a second clade-III gene, GLR3.2 [data notshown]). In contrast, GLR1.1 (a clade-I gene) is nothighly expressed in the vasculature but is expressed in-stead in distinct cell types (figs. 6–8). Future experi-ments (protein fusions and antibody staining) are nec-essary to increase the resolution of this analysis. Cur-


FIG. 7.—Cell-type–specific expression patterns of three AtGLR clades in 3- to 4-week-old plants. Ultrastructural expression of AtGLR genes invegetative organs using GUS as a reporter gene. Compound micrographs of the leaf show GUS expression in the leaf margins of GLR1.1 (a).GLR2.1 is not detected in the leaf (b). GLR3.1 expression is seen in the vasculature and in cell types associated with the vasculature in the leaf(c), the root (f), and emerging lateral roots (i). Expression of GLR1.1 is detected in most layers of the root and the root hairs (d and g). GLR2.1is strongly expressed throughout the root (e) and in root hairs (h and k) and emerging lateral roots (h). Expression of all three genes is missingin the apex of the root (j, k, and l).

rently, we can only conclude that GLR1.1 and GLR3.1are expressed in distinct cell types, although they areexpressed in the same organs. More experiments areneeded to examine if this holds true for all other genesfrom clades I and III. If AtGLR genes from clades I andIII are indeed expressed in distinct cell types, it will bemore likely for clades I and III to represent distinct func-tional classes.

This paper focuses on class-specific mRNA ex-pression as being an important mechanism for the reg-ulation of heteromeric coassembly between proteinswithin the same functional class. It is important to notethat other regulatory mechanisms might be involved aswell. Moreover, it is also important to keep in mind thatgenes that have the same expression pattern may not

have the same function. In addition, it is possible thatunlike the case of animal iGluRs, in which phylogenet-ically defined clades coincide with true functional clas-ses, phylogenetically defined AtGLR clades may not rep-resent functional AtGLR classes. Future biochemical andphysiological experiments will help to confirm the va-lidity of the root-specific class of five clade-II genes, aswell as to define other functional classes.

Both RT-PCR and promoter::GUS analyses haveshown that all AtGLR genes are strongly expressed inroots as a general rule. If AtGLR genes indeed functionas ion channels, their high expression in roots may po-tentially be important for regulating ion uptake fromsoil. It is interesting to note that glutamate-gated calciumfluxes across the plasma membrane have been observed

1080 Chiu et al.

FIG. 8.—AtGLR genes show distinct expression patterns in flowers and reproductive organs. Expression of AtGLR genes in reproductive organsusing GUS as a reporter gene. Expression of GLR1.1 (a and e) and GLR2.1 (b and f) are not readily detectable in reproductive organs (slightand transient GUS expression has been observed in anthers and ovules for GLR2.1). GLR3.1 GUS expression is found in the vasculature andneighboring organs, including the petal, the ovary, and the filament (c, d, g, and h). In the vasculature, GLR3.1 expression is particularly strongat contact sites between the anther and the filament, the receptacle and the flower, and the funiculus and developing seed (c, d, g, and h).

in Arabidopsis roots (Dennison and Spalding 2000). Fu-ture experiments using mutagenesis and reverse geneticsapproaches will be necessary to investigate the possiblelink between this phenomenon and specific AtGLRgenes. In addition to the strong root expression for themajority of AtGLR genes, which suggest a role in ionuptake from soil, the strong vascular expression ofclade-III genes (GLR3.1 and GLR3.2) suggests thatAtGLR genes may potentially play a role in regulatingamino acid transport in the phloem. It has been shownthat glutamate is transported in the phloem (Lam et al.1995) as well as to siliques (H. M. Lam, personal com-munication) in Arabidopsis, as in other higher plants.

In addition to testing the hypothesis that the divi-sion of the AtGLR gene family into three clades hasfunctional significance, we have identified potentialfunctionally important amino acid residues that are in-variant in both eukaryotic and prokaryotic iGluR genesin conserved functional domains (fig. 3a). Mutagenesisexperiments have been conducted for most of these res-idues in animal iGluRs or bacterial periplasmic-bindingproteins, and they have indeed been shown to be func-tionally important. R505 has been shown to interact withall known animal iGluR agonists and is therefore crucialfor the ligand-binding capability of iGluR proteins(Armstrong et al. 1998). F511, G703, and W736 havebeen shown to be important to the structural integrity ofiGluR and bacterial periplasmic-binding proteins (Chenet al. 1999). A621 is part of the YTANLAAL motif thatis believed to shape iGluR ligand-gated channel kinetics(Kohda, Wang, and Yuzaki 2000). This analysis shows

that invariant residues identified in this manner are al-most always functionally important residues.

We have also identified amino acid residues infunctional domains that are conserved in eukaryoticiGluRs (rat and Arabidopsis) but not in the functionalprokaryotic Synechocystis GluR0 (fig. 3b). AnabaenaGluR has not been shown to be a functional ion channelto date and is therefore excluded in this discussion. Thesequence divergence at these particular amino acid res-idues may play a part in creating the observed differ-ences in properties between eukaryotic iGluR and Sy-nechocystis GluR0. Synechocystis GluR0 has a differentligand selectivity profile when compared with animaliGluRs (Chen et al. 1999). It does not bind subtype-specific eukaryotic iGluR agonists, such as kainate,NMDA, and AMPA, but does bind a wider variety ofamino acids in addition to L-glutamate, such as L-glu-tamine and L-serine, as compared with animal iGluRs.In addition, Synechocystis GluR0 encodes a protein thatforms a homomeric channel permeable only to potassi-um ions as tested in heterologous systems (Chen et al.1999), whereas animal iGluR channels are permeable tosodium, potassium, as well as calcium depending ontheir subunit composition (Sprengel and Seeburg 1995).It is possible that the amino acid residues identified heremight be involved in the difference in ligand selectivityand channel kinetics between eukaryotic iGluRs and Sy-nechocystis GluR0. In fact, one of the residues, Y619,has been shown to be involved in influencing animaliGluR channel kinetics (Kohda, Wang, and Yuzaki2000).


Finally, we have identified amino acid residues inimportant functional domains that are invariant in allmembers of the AtGLR gene family. These include ami-no acid residues that are: (1) conserved in both eukary-otes (rat and Arabidopsis) and prokaryotes (fig. 3a), (2)those that are conserved only in eukaryotes (rat and Ar-abidopsis, fig. 3b), and (3) those that are only conservedin the AtGLR gene family (fig. 3c). In addition, AtGLRinvariant residues will naturally include residues that areconserved in prokaryotic iGluRs but not in all ratiGluRs (not illustrated in fig. 3). Results of mutagenesisexperiments for residues that are conserved in both eu-karyotes and prokaryotes were discussed earlier, andthey represent obvious targets in future mutagenesis ex-periments that can help to elucidate the function of GLRgenes in plants. Mutagenesis experiments for AtGLR in-variant residues that are conserved in some rat iGluRgenes as well as in prokaryotic iGluRs have been per-formed in animal iGluR genes and bacterial periplasmicamino acid–binding proteins, and results showed thatthey are functionally important. For example, P514 hasbeen found to be critical to the structural integrity of thebacterial amino acid–binding protein (Chen et al. 1999).The F583A mutation in animal AMPA subunit GluR3(F605 in GluR3) produces a killer subunit that has adominant negative effect when expressed with wild-typesubunits (Wo and Oswald 1995). Mutagenesis studies onthe AtGLR invariant residues identified in this study willhelp us to understand the electrophysiological propertiesof these AtGLR channels if these are indeed channels,as well as their function in planta.

Conclusions

We have conducted the first comprehensive mRNAexpression analysis of the Arabidopsis GLR gene family.This analysis is the first step to examining whether thethree AtGLR clades, defined by parsimony analysis, rep-resent functionally distinct classes. On the basis of ourRT-PCR analysis, we conclude that genes from the threeAtGLR clades do not show distinct organ expressionprofiles. However, five of the nine clade-II genes areroot-specific in 8-week-old Arabidopsis plants and mayrepresent a functional class. Future physiological andbiochemical experiments are necessary to verify thissuggestion. In examining cell-type–specific expression,we point out that genes that are expressed in the sameorgans may be expressed in different cell types withinthe organs. As a result, the question of whether cladesI and III indeed have distinct expression patterns stillremains open.

It is important to understand that genes that showthe same expression pattern may not necessarily havethe same in vivo function. This is why future expression,biochemical, electrophysiological, and in planta physi-ological experiments are necessary to increase the res-olution of the expression analyses presented in this pa-per and continue to classify AtGLR genes into distinctfunctional classes as well as elucidating their functionin vivo. It is important to note that organ or cell-type–specific expression may only be one of the mechanisms

to restrict protein coassembly within each GLR func-tional class, assuming GLR proteins are capable offorming heteromultimers. The phylogeny and expressionstudies presented here will help to drive future biochem-ical and electrophysiological analyses. AtGLR genes thatare coexpressed in specific cell or organ types will betargeted for tests of coassembly and cofunction in vivo.

Supplementary Material

The processed data matrix (after culling and gap-coding) used for our parsimony analysis is available on-line at the MBE website.

Acknowledgments

This work was supported by NIH grant GM32877to Gloria M. Coruzzi. R.D. is supported in part by theLewis B. and Dorothy Cullman Program for molecularsystematics and the Ambrose Monell collection for mo-lecular and microbial research. E.D.B. is supported byan NIH postdoctoral fellowship. M.N.N. is supported byan NIH NRSA individual fellowship.

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ELIZABETH KELLOGG, reviewing editor

Accepted February 11, 2002

Phylogenetic and Expression Analysis of the Glutamate...

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