*Department of Biology, New York University;
Department of Entomology, American Museum of Natural History, New York
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Abstract |
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Introduction |
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Animal iGluR genes encode subunits for ligand-gated ion channels, which account for a major fraction of fast excitatory neurotransmission. They form a large gene family that can be divided into multiple classes (N-methyl-d-aspartate [NMDA], -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid [AMPA]-kainate [KA], and Delta) based on ligand selectivity and ion conductance properties (Sprengel and Seeburg 1995
). NMDA iGluRs have been implicated in neurodegenerative diseases and are believed to be involved in neuronal cell death for pathological conditions such as ischemia (Rameau et al. 2000
). AMPA and KA receptors are often grouped together as non-NMDA iGluRs because of ligand cross-reactivity and their similarities in sequence as well as ion-conducting properties (Sutcliffe, Wo, and Oswald 1996
). We will refer to AMPA and KA iGluRs as a single class in this paper for the reasons mentioned above (AMPA-KA). Lastly, the delta iGluRs do 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
2 subunit results in a constitutively open ion channel with properties similar to AMPA-KA receptor channels and leads to neurodegeneration in mice (Wollmuth et al. 2000
).
2 receptors are found to express predominantly in cerebellar Purkinje cells (Kohda, Wang, and Yuzaki 2000
).
Because animal iGluRs have been studied extensively 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 phylogenetic analysis of animal iGluRs and four Arabidopsis GLR genes, Chiu et al. (1999)
determined that the divergence of animal iGluRs and Arabidopsis GLRs preceded the divergence of animal iGluR classes (AMPA-KA, NMDA, Delta). This preliminary comparative phylogenetic analysis of plant GLRs and animal iGluRs suggested that signaling by amino acids might be a primitive mechanism that existed before the divergence of plants and animals. This hypothesis was subsequently verified with the identification of the first prokaryotic functional ionotropic glutamate receptor, GluR0, in Synechocystis (cyanobacteria) (Chen et al. 1999
).
It is currently unknown whether Arabidopsis GLRs, like animal iGluRs and Synechocystis GluR0, are capable of forming functional ion channels. Glutamate-gated calcium fluxes have been observed in Arabidopsis roots (Dennison and Spalding 2000
), but links of this activity to specific AtGLR genes have not yet been established. Despite the fact that the biochemical properties of GLR proteins remain an open question, the AtGLR genes have already been implicated in processes, such as light signal transduction (Lam et al. 1998
; Brenner et al. 2000
) and calcium homeostasis (Kim et al. 2001
) in plants. We therefore expect that AtGLRs will be involved in many different signaling and physiological processes in plants, especially because a large family of 20 AtGLR genes has been uncovered with the completion of the Arabidopsis genome sequencing project (AGI 2000
). A nomenclature has recently been established for the AtGLR gene family (Lacombe et al. 2001
). The nomenclature is based on a preliminary parsimony analysis that divides the AtGLR gene family into three clades.
In this paper, we confirm the identification of 20 GLR genes in Arabidopsis and report the characterization of five new full-length AtGLR cDNA sequences. We examine the genealogical relationships of the iGluR family and identify residues that are invariant ("invariant" carries the meaning of "unvaried" rather than "invariable" in this paper) in both prokaryotic and eukaryotic iGluRs by performing a parsimony analysis with extensive annotations using the amino acid sequences of a complete family of animal iGluRs, all 20 Arabidopsis GLRs, and two prokaryotic cyanobacterial iGluRs, Synechocystis GluR0 and Anabaena GluR. Synechocystis GluR0 encodes the first identified functional prokaryotic iGluR channel subunit (Chen et al. 1999
). On the other hand, functional studies on the Anabaena GluR have not been published yet. This study demonstrates that phylogenetic analysis can be a powerful tool to guide functional studies of large gene families as well as to understand evolutionary relationships. We also examine mRNA expression patterns of all 20 GLR genes in different organs and determine cell-typespecific expression patterns for a gene from each of the three clades. This analysis has allowed us to investigate whether the phylogenetic division of the AtGLR gene family has functional implications by testing the hypothesis of clade-specific gene expression patterns.
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Materials and Methods |
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In order to strengthen and confirm our analysis, we also performed a second parsimony analysis where we processed the data matrix using elision (Wheeler, Gatesy, and DeSalle 1995
) instead of a combination of culling and gap-coding. As in the first parsimony analysis, we used CLUSTAL X to align the sequences using different sets of alignment parameters (gap-to-change = 10, 15, and 20; gap extension cost = 1; amino acid substitution matrix = Blosum 30). We then combined the three matrices to generate a larger matrix, which we used for parsimony analysis.
We used PAUP* (phylogenetic analysis using parsimony; Swofford 1998
) to infer phylogeny. Ten random-addition heuristic searches with TBR branch swapping were implemented, and all characters were equally weighted in our analyses. We did not place a limit on the number of trees saved in each search. When more than one equally parsimonious tree was obtained, we generated strict consensus trees to represent our phylogenetic hypothesis. To measure the robustness of all nodes in our parsimony analysis, bootstrap analysis (100 replicates) was performed using PAUP*. Ten random-addition heuristic searches with TBR branch swapping were used for each replicate of the bootstrap analysis.
Computation of Percent Identity Values
We used CLUSTAL X to generate the percent identity values between different iGluRs when comparing the entire amino acid sequences. The default parameters (gap-to-change cost = 10; gap extension cost = 1; amino acid substitution matrix = Blosum 30) were used for the alignment, and percent identity values were generated along with the alignment. We then compiled these values to generate(1) the percent identity values between genes within the same rat iGluR classes or AtGLR clades (AMPA and KA iGluRs are grouped together as a single class), (2) the percent identity values between genes that are in different rat iGluR classes or AtGLR clades, and (3) the percent identity values between rat and Arabidopsis iGluR genes. The percent identity values for the glutamate-binding domains as well as the pore (P) and surrounding transmembrane regions (M1 and M2) were generated using the same method. The sequence boundaries for the functional domains in rat iGluR genes used in this study follow the boundaries that were previously established by structural and functional studies (Hollmann and Heinemann 1994
; Stern-Bach et al. 1994
). The sequence boundaries of transmembrane domains in AtGLR genes were predicted by the software PHD version 1996.1 (Rost 1996
). The computer predictions generally coincided with the AtGLR transmembrane domain boundaries predicted when using the alignment of the AtGLR genes and rat iGluR genes as guidelines. In cases where the two predictions did not match exactly, they only differed by one or two amino acids. The sequence boundaries for the glutamate-binding domains were predicted using the alignment of AtGLRs and rat iGluRs as guidelines because programs specializing in predicting glutamate-binding domains are not available. Amino acid residues numbers 406527 and 628737 were used for the alignment for the glutamate-binding domains, and amino acid residues numbers 543626 were used for the alignment for the P and surrounding transmembrane regions. The amino acid numbering follows the sequence of AtGLR1.1 (AF079998). The compiled percent identity values were then mapped onto a schematized version of a tree generated from our parsimony analysis.
Character Mapping
We imported the processed data matrix used for parsimony analysis and the consensus tree obtained from parsimony analysis into MacClade version 4.0 (Maddison WP and Maddison DR 1992
) for character-mapping analysis. We identified conserved residues at different nodes by using the trace character function in MacClade.
For mapping RT-PCR expression data on the parsimony tree, we converted AtGLR mRNA expression in leaves, roots, flowers, and siliques as detected by RT-PCR into binary format (0 = undetected and 1 = detected) and appended the four additional characters to the matrix used to generate the parsimony tree shown in figure 1 . The corresponding characters for the rat and cyanobacteria genes are coded as missing. We used this new matrix, with four additional characters, to generate a second parsimony tree. The resulting phylogenetic tree is identical to the one shown in figure 1 . We then used the trace-character function in MacClade to map each of the four expression characters onto the phylogenetic tree.
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We analyzed the PCR products using gel electrophoresis followed by NIH image version 1.62. The graphic representation of the RT-PCR data is generated by normalizing AtGLR expression against TUB5 expression. To construct each AtGLR graph, we measured the integrated density values of the TUB5 band and the AtGLR band for each of the four organ lanes on the digitalized picture of the agarose gel (samples are from 30 PCR cycles) using NIH image. The integrated density values of the four AtGLR bands were then normalized using the integrated density values of the four TUB5 bands as control. The normalized integrated density values of the four AtGLR bands were then used to calculate the relative intensity values shown on the graph. The highest of the four intensity values was regarded as 1, and the others were calculated and represented as a fraction of the highest value.
The above RT-PCR protocol was also used to obtain full-length cDNA clones for GLR1.2, GLR1.4, GLR2.2, and GLR2.7. However, instead of using the gene-specific primers used to examine expression patterns, primers that were predicted from genomic sequence analysis to represent the beginning and end of each full-length cDNA sequence were used in the PCR amplification reactions.
Examining AtGLR Expression in Transgenic Plants
Representative AtGLR genes from each of the three GLR clades were examined for cell-type expression patterns using a reporter gene system. Transcriptional fusions were made between the promoters of GLR1.1, GLR2.1 or GLR3.1, and the reporter gene ß-glucuronidase (GUS) (Jefferson 1989
). AtGLR promoter regions were amplified by PCR from Columbia (Col-0) genomic DNA using Ex-Taq polymerase (Panvera). A 1,713-bp region upstream of the start codon of GLR1.1 was amplified at the 5' end with the primer JC99 (5'-CGTCGAAGCTTATAAGAAACG-3') and at the 3' end with the primer JC101 (5'-CATATCTACTTGTGCCATGG-3'). The amplified fragment has a HindIII site at the 5' end and an NcoI site at the 3' end so that GUS is translated at the presumptive ATG start site for GLR1.1. A 1,553-bp region upstream of the start codon of GLR2.1 was amplified at the 5' end with the primer EBP027 (5'-GTCGACGATCAAAGGTTATGTCGCTAAAGGAG-3') and at the 3' end with the primer EBP017 (5'-GTGGATCCTACTTAGCCGAAAAAGAATGAAACTTG-3'). This introduced a SalI restriction site at the 5' end and an NcoI site at the 3' end. The NcoI site facilitated the translation of GUS at the presumptive start ATG site for GLR2.1. A 1,780-bp upstream region from GLR3.1 was amplified at the 5' end with the primer EBP4 (5'-TGAAGCTTCGTTCACTAATTGGAGTGCAT-3') and at the 3' end with EBP1 (5'-ATGACCATGGAGCTTAACATTGAACAACAAAAAGAG-3'). This introduced a HindIII restriction site at the 5' end and an NcoI site at the 3' end so that GUS is translated at the presumptive start ATG site for GLR3.1. All fragments were cut at the introduced restriction sites and cloned into pTZGUS (Ngai, Tsai, and Coruzzi 1997
). The promoter::GUS fusions were excised with EcoRI and HindIII for GLR1.1 and GLR3.1 and with EcoRI and SalI for GLR2.1 and independently subcloned into the binary Agrobacterium vector pBI101 (Jefferson, Kavanaugh, and Bevan 1987
) at those respective sites. Binary constructs were transformed into Agrobacterium strain GV3101, and Arabidopsis plants (Columbia Col-0 ecotype) were transformed (Clough and Bent 1998
). At least 20 independent transgenic lines for each construct were selected for analysis. Transgenic Arabidopsis seedlings were germinated on MS media containing 1% sucrose and grown in 16 h light-8 h dark day-night cycles. GUS staining was performed according to the method of Jefferson, Kavanaugh, and Bevan (1987)
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Results |
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Parsimony Analysis of iGluRs from Prokaryotes and Eukaryotes Defines Three AtGLR Clades
Previously, a phylogenetic analysis of the iGluR gene family was performed using animal iGluRs and the four AtGLR genes (GLR1.1, GLR2.1, GLR3.1, and GLR3.4) that were available at the time (Chiu et al. 1999
). This initial analysis showed that the divergence of animal iGluRs and AtGLRs preceded the divergence of animal iGluR classes (AMPA-KA, NMDA, and Delta). The completion of the Arabidopsis genome sequencing project led to the identification of 16 additional AtGLR genes. We therefore performed a new, more comprehensive phylogenetic analysis using all the 20 AtGLR gene family members of the model plant Arabidopsis (table 1
) and all known iGluR genes of a single animal, rat (except NR3; table 2
). In addition, two prokaryotic iGluRs, Synechocystis GluR0 (Chen et al. 1999
) and Anabaena GluR (table 2
), were also included in this phylogenetic analysis to examine where they fit into the evolutionary history, relative to the complete plant and animal glutamate receptor gene families.
Visual inspection of the alignment of all Arabidopsis, rat, and two prokaryotic glutamate receptor genes reveals a high degree of similarity in most of the iGluR functional domains. These include the two glutamate-binding domains (GlnH1 and 2), transmembrane domain M1, the P, and transmembrane domain M2. On the other hand, most of the sequences before GlnH1 and the regions after GlnH2, which includes the last transmembrane domain M3 (absent in Synechocystis GluR0 and Anabaena GluR), show limited similarity. These regions of low conservation and ambiguous alignment were culled (Gatesy, DeSalle, and Wheeler 1993
) accordingly during matrix processing before the first parsimony analysis. Although these highly variable regions may not be absolutely required for protein activity, they may contain domains with modulatory functions, e.g., regulatory kinase-binding sites (Nitabach et al. 2001
).
Parsimony analysis of the iGluR gene family using bacterial sequences as outgroups gave two equally parsimonious trees of 4,235 steps, with a consistency index of 0.616 and a retention index of 0.730. Figure 1
shows the consensus parsimony tree with bootstrap values. As shown in our previous analysis (Chiu et al. 1999
), the divergence of animal iGluR and AtGLR precedes the divergence of animal iGluR classes (AMPA-KA, NMDA, and Delta). This phylogenetic inference is supported by in vivo data showing that glutamate-gated calcium fluxes in Arabidopsis roots, which may be linked to AtGLR gene products, cannot be induced when l-glutamate is replaced by animal iGluR class-specific ligands, such as AMPA or NMDA (Dennison and Spalding 2000
). The two prokaryotic iGluRs, Synechocystis GluR0 and Anabaena GluR, are sister taxa, and they fall outside the multicellular clade. The well-characterized rat iGluR genes belonging to different animal iGluR classes all reside within their appropriate clades, with the exception of RNMDAR1. Instead of forming a monophyletic clade with the other four NMDA iGluRs (RNR2A to 2D), it groups with the rest of the animal iGluRs with a weak bootstrap value of 54% (fig. 1
). It is important to note that although RNMDAR1 has indeed diverged greatly from the other four NMDA iGluRs, its placement outside the clade consisting of the other four NMDA genes is only weakly supported. In fact, in a similar analysis, where all but the Anabaena GluR sequence is included, RNMDAR1 forms a monophyletic clade with the other four NMDA genes (bootstrap value = 62%, data not shown). In general, the animal iGluR clades resulting from this parsimony analysis coincide with the classes established in functional studies. Like animal iGluRs, the AtGLR gene family also separates into clades (Clades I, II, and III) with strong node support (Bootstrap values = 95%100%).
The division of the AtGLR gene family into three clades is also demonstrated by the comparison of amino acid percent identity values between genes that are in the same AtGLR clade (40%57%) and genes that are from different AtGLR clades (21%33%; fig. 2a ). As shown by the amino acid percent identity values generated by comparing entire proteins, there is a clear boundary between AtGLR clades based on sequence identity. As a test case, amino acid percent identity values between different rat iGluRs also support the tree topology established in our parsimony analysis, which agrees with the established animal iGluR classes. There is a clear separation between animal iGluR classes based on amino acid percent identity values, with the exception of the NMDA class. The amino acid percent identity is relatively low (19%38%) within the NMDA class because NMDAR1 has diverged greatly from the other four NMDA genes (NR2A-D). When the percent identity values generated for the genes within the NMDA class are excluded, the percent identity values between genes that are within the same animal iGluR class are relatively high (29%56%), whereas that between genes from different animal iGluR classes are relatively low (10%26%).
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In addition to establishing the three clades of AtGLR genes, our phylogenetic analysis also showed that clades I and II are sisters to each other (bootstrap value = 58%). To confirm the results of our analysis, we performed a second parsimony analysis using elision (Wheeler, Gatesy, and DeSalle 1995
) as an alternative method to process the matrix, instead of a combination of culling and gap-coding. The topology of the resulting parsimony tree is essentially identical to the one discussed above (data not shown).
Defining Invariant Amino Acid Residues for the iGluR Gene Family
With the inclusion of all 20 AtGLR gene family members and the two prokaryotic iGluRs (Synechocystis GluR0 and Anabaena GluR) in our analysis, we can now identify potential functionally important iGluR amino acid residues that are absolutely conserved before the divergence of plants and animals and those that are conserved even before the divergence of prokaryotes and eukaryotes. These iGluR invariant residues are presented in figure 3a.
The present analysis is more comprehensive than a previous one (Chiu et al. 1999
) because the sequences for the entire AtGLR gene family (16 more genes) and two prokaryotic iGluRs are now included. Another difference between this and our previous analysis is that animal iGluR sequences included in this analysis are limited to the sequences of a single animal, rat. In our previous analysis, animal iGluR sequences from other species, such as Drosophila melanogaster, Caenorhabditis elegans, pigeon, and fish were included. The exclusion of non-rat animal iGluR sequences has allowed us to directly compare the entire iGluR gene family of a single plant (Arabidopsis) and a single animal (rat). Such an analysis could potentially increase the number of conserved residues, but this does not appear to be the case. The only invariant residue identified 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 of AtGLR1.1), which is found in all animal iGluRs, except Drosophila GluR2 (M73271, data not shown). G701 is also replaced by proline and asparagine in Synechocystis GluR0 and Anabaena GluR, respectively, and is therefore conserved only in rat iGluRs and Arabidopsis GLRs. By comparing figure 3a and b,
we identified other amino acid residues that are invariant in eukaryotes but are altered in at least one of the two prokaryotic cyanobacterial 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 cyanobacterial genes. Whereas L623 is substituted with an alanine in Anabaena GluR, it is conserved in Synechocystis GluR0. The other four residues that are not conserved between Synechocystis GluR0 and animal iGluRs may be directly linked to the difference in ligand specificity and ion selectivity between Synechocystis GluR0 and animal iGluRs.
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AtGLR Genes from Different Clades Overlap in Their Organ Expression Profiles
Our parsimony analysis divides the animal iGluR gene family into three distinct clades (AMPA-KA, NMDA, and delta), with the exception of RNMDAR1 (see parsimony analysis results section). These three clades defined by parsimony analysis agree with the established classification of these distinct animal iGluR classes based on biochemical properties, ligand specificity, and ion selectivity. The Arabidopsis GLR gene family is also divided into three clades based on our parsimony analysis. By analogy to animal iGluRs, we hypothesize that the three AtGLR clades may represent biochemically or functionally distinct glutamate receptor protein classes. Whereas glutamate-gated calcium fluxes have been observed in Arabidopsis (Dennison and Spalding 2000
), no data are currently available for the biochemical activity encoded by specific AtGLR genes. Thus, we have begun to address whether the three GLR clades in Arabidopsis represent functionally distinct AtGLRs by first looking at the mRNA expression of AtGLR genes from each clade.
We first examined the expression of all 20 AtGLR genes using organ-specific RT-PCR (in situ hybridization experiments have been attempted for AtGLR genes, but the level of expression is too low to be detected). Because iGluR channels in animals are homotetramers or heterotetramers assembled from proteins encoded by genes in the same functional class (Rosenmund, Stern-Bach, and Stevens 1998
), it is possible that AtGLR genes from the same functional class (not defined yet) may encode proteins that have the ability to form heteromers in vivo. In order for different proteins to form multimers, the colocalization of their mRNAs is likely and that of their proteins is necessary. Therefore, we hypothesize that mRNAs of AtGLR genes that are in the same clade may be present in the same organs. Distinct clade-specific expression patterns may be the first indication that phylogenetically defined AtGLR clades represent functional AtGLR classes. This hypothesis is based on the assumption that class-specific expression is an important mechanism that regulates AtGLR heteromeric protein coassembly.
Our RT-PCR analysis established that all 20 members of the AtGLR gene family are expressed genes (fig. 4a and b ). This finding addresses a previous concern that several AtGLRs might be pseudogenes because many of the AtGLR gene family members are duplicated genes, existing in tandem on the chromosomes (note same BAC clone numbers in table 1 ). Not only are they all expressed, potential mRNA splice variants, represented by additional bands on the gel, can be observed for two AtGLR genes, namely GLR 2.5 and GLR 3.4. Whereas cDNA clones for GLR2.5 are not available at present, splice variants for GLR3.4 have already been isolated (GenBank accession numbers AF167355 and AY072070) and thus confirmed our observation.
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The RT-PCR mRNA expression data of AtGLR genes was mapped onto the AtGLR portion of the iGluR parsimony tree to illustrate the relationship between AtGLR gene evolution and organ-specific expression (fig. 5 ). As shown in figure 5b, root expression is detected in all AtGLR genes and is inferred to be an expression character state ancestral to the entire AtGLR gene family. Similarly, mRNA expression in leaves appears to be the ancestral character state of the AtGLR gene family (fig. 5a ). However, multiple independent changes occurred only in AtGLR clade II, resulting in the loss of leaf expression in six of the nine clade-II genes (GLR2.1, GLR2.2, GLR2.3, GLR2.4, GLR2.6, and GLR2.9). Lastly, mRNA expression in reproductive organs (flowers and siliques in Arabidopsis) also appears to be ancestral to the AtGLR gene family (fig. 5c and d ). However, once again, multiple independent changes occurred 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 expression in five clade-II genes (GLR2.1, GLR2.2, GLR2.3, GLR2.6, and GLR2.9).
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Discussion |
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In this paper, we have begun to address whether the three AtGLR clades represent functionally distinct protein classes by examining the mRNA expression patterns of all 20 AtGLR genes and promoter activities of representatives of each clade. Because iGluRs exist as tetramers in animals (Rosenmund, Stern-Bach, and Stevens 1998
), determining the organ and cell-typespecific expression patterns of each AtGLR gene will also enable us to predict which gene products could potentially interact in vivo. Heteromultimeric coassembly can profoundly influence protein function (Sprengel and Seeburg 1995
; Nitabach et al. 2001
). By using RT-PCR, we obtained an mRNA expression profile for the entire AtGLR gene family in four different organ types, namely leaves, roots, flowers, and siliques. As a cautionary note because the phylogenetic analysis was performed using the coding sequences of the genes although the mRNA expression patterns are controlled by regulatory noncoding sequences, they might not have direct correlation. Our RT-PCR results show that there is overlap in organ expression profiles between genes from different AtGLR clades. Whereas genes from AtGLR clades I and III are expressed more ubiquitously, clade-II genes are largely root specific (fig. 4a and b
). We conclude that the three AtGLR clades are not distinct based on organ-level expression patterns. Thus, the three AtGLR clades may contain genes with overlapping functions in vivo. Interestingly, five of the nine clade-II genes are root-specific in 8-week-old Arabidopsis plants, and may potentially represent a functional class. Additional data from physiological studies are necessary to verify this proposition because genes that have the same expression pattern may not share the same function.
By mapping the RT-PCR mRNA expression data onto the iGluR parsimony tree (fig. 5 ), we are able to make phylogenetic inferences relating organ-specific expression and AtGLR gene evolution. Ubiquitous expression in leaves, roots, and reproductive organs appears to be the ancestral character states of the AtGLR gene family, but multiple independent character state changes occurred specifically in clade-II lineages, resulting in the loss of expression in leaves, flowers, and siliques in some of the clade-II genes. It is interesting to note that all changes in expression character state occurred specifically in clade-II genes. Perhaps the gene duplication event leading to the divergence of GLR clades I and II provided the opportunity for one of these two AtGLR clades (clade II) to diverge in terms of function, as indicated by the change in expression patterns.
To test the possibility that mRNA of different AtGLR genes that are detected in the same organs may be present in different cell types, we examined transgenic plants harboring promoter::reporter gene constructs to determine the cell types in the organs in which different AtGLR genes are expressed. For this, we analyzed one representative gene from each of the three AtGLR clades. Although genes from both clades I and III 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 that genes from clades I and III may be expressed in different cell types. The expression of GLR3.1 (a clade-III gene) is limited to the vasculature in all organs tested (as is a second clade-III gene, GLR3.2 [data not shown]). In contrast, GLR1.1 (a clade-I gene) is not highly expressed in the vasculature but is expressed instead in distinct cell types (figs. 68 ). Future experiments (protein fusions and antibody staining) are necessary to increase the resolution of this analysis. Currently, we can only conclude that GLR1.1 and GLR3.1 are expressed in distinct cell types, although they are expressed in the same organs. More experiments are needed to examine if this holds true for all other genes from clades I and III. If AtGLR genes from clades I and III are indeed expressed in distinct cell types, it will be more likely for clades I and III to represent distinct functional classes.
This paper focuses on class-specific mRNA expression as being an important mechanism for the regulation of heteromeric coassembly between proteins within the same functional class. It is important to note that other regulatory mechanisms might be involved as well. Moreover, it is also important to keep in mind that genes that have the same expression pattern may not have the same function. In addition, it is possible that unlike the case of animal iGluRs, in which phylogenetically defined clades coincide with true functional classes, phylogenetically defined AtGLR clades may not represent functional AtGLR classes. Future biochemical and physiological experiments will help to confirm the validity of the root-specific class of five clade-II genes, as well as to define other functional classes.
Both RT-PCR and promoter::GUS analyses have shown that all AtGLR genes are strongly expressed in roots as a general rule. If AtGLR genes indeed function as ion channels, their high expression in roots may potentially be important for regulating ion uptake from soil. It is interesting to note that glutamate-gated calcium fluxes across the plasma membrane have been observed in Arabidopsis roots (Dennison and Spalding 2000
). Future experiments using mutagenesis and reverse genetics approaches will be necessary to investigate the possible link between this phenomenon and specific AtGLR genes. In addition to the strong root expression for the majority of AtGLR genes, which suggest a role in ion uptake from soil, the strong vascular expression of clade-III genes (GLR3.1 and GLR3.2) suggests that AtGLR genes may potentially play a role in regulating amino acid transport in the phloem. It has been shown that glutamate is transported in the phloem (Lam et al. 1995
) as well as to siliques (H. M. Lam, personal communication) in Arabidopsis, as in other higher plants.
In addition to testing the hypothesis that the division of the AtGLR gene family into three clades has functional significance, we have identified potential functionally important amino acid residues that are invariant in both eukaryotic and prokaryotic iGluR genes in conserved functional domains (fig. 3a
). Mutagenesis experiments have been conducted for most of these residues in animal iGluRs or bacterial periplasmic-binding proteins, and they have indeed been shown to be functionally important. R505 has been shown to interact with all known animal iGluR agonists and is therefore crucial for the ligand-binding capability of iGluR proteins (Armstrong et al. 1998
). F511, G703, and W736 have been shown to be important to the structural integrity of iGluR and bacterial periplasmic-binding proteins (Chen et al. 1999
). A621 is part of the YTANLAAL motif that is believed to shape iGluR ligand-gated channel kinetics (Kohda, Wang, and Yuzaki 2000
). This analysis shows that invariant residues identified in this manner are almost always functionally important residues.
We have also identified amino acid residues in functional domains that are conserved in eukaryotic iGluRs (rat and Arabidopsis) but not in the functional prokaryotic Synechocystis GluR0 (fig. 3b
). Anabaena GluR has not been shown to be a functional ion channel to date and is therefore excluded in this discussion. The sequence divergence at these particular amino acid residues may play a part in creating the observed differences in properties between eukaryotic iGluR and Synechocystis GluR0. Synechocystis GluR0 has a different ligand selectivity profile when compared with animal iGluRs (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 of amino acids in addition to l-glutamate, such as l-glutamine and l-serine, as compared with animal iGluRs. In addition, Synechocystis GluR0 encodes a protein that forms a homomeric channel permeable only to potassium ions as tested in heterologous systems (Chen et al. 1999
), whereas animal iGluR channels are permeable to sodium, potassium, as well as calcium depending on their subunit composition (Sprengel and Seeburg 1995
). It is possible that the amino acid residues identified here might be involved in the difference in ligand selectivity and channel kinetics between eukaryotic iGluRs and Synechocystis GluR0. In fact, one of the residues, Y619, has been shown to be involved in influencing animal iGluR channel kinetics (Kohda, Wang, and Yuzaki 2000
).
Finally, we have identified amino acid residues in important functional domains that are invariant in all members of the AtGLR gene family. These include amino acid residues that are: (1) conserved in both eukaryotes (rat and Arabidopsis) and prokaryotes (fig. 3a
), (2) those that are conserved only in eukaryotes (rat and Arabidopsis, fig. 3b
), and (3) those that are only conserved in the AtGLR gene family (fig. 3c
). In addition, AtGLR invariant residues will naturally include residues that are conserved in prokaryotic iGluRs but not in all rat iGluRs (not illustrated in fig. 3
). Results of mutagenesis experiments for residues that are conserved in both eukaryotes and prokaryotes were discussed earlier, and they represent obvious targets in future mutagenesis experiments that can help to elucidate the function of GLR genes in plants. Mutagenesis experiments for AtGLR invariant residues that are conserved in some rat iGluR genes as well as in prokaryotic iGluRs have been performed in animal iGluR genes and bacterial periplasmic amino acidbinding proteins, and results showed that they are functionally important. For example, P514 has been found to be critical to the structural integrity of the bacterial amino acidbinding protein (Chen et al. 1999
). The F583A mutation in animal AMPA subunit GluR3 (F605 in GluR3) produces a killer subunit that has a dominant negative effect when expressed with wild-type subunits (Wo and Oswald 1995
). Mutagenesis studies on the AtGLR invariant residues identified in this study will help us to understand the electrophysiological properties of these AtGLR channels if these are indeed channels, as well as their function in planta.
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Conclusions |
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It is important to understand that genes that show the same expression pattern may not necessarily have the same in vivo function. This is why future expression, biochemical, electrophysiological, and in planta physiological experiments are necessary to increase the resolution of the expression analyses presented in this paper and continue to classify AtGLR genes into distinct functional classes as well as elucidating their function in vivo. It is important to note that organ or cell-typespecific expression may only be one of the mechanisms to restrict protein coassembly within each GLR functional class, assuming GLR proteins are capable of forming heteromultimers. The phylogeny and expression studies presented here will help to drive future biochemical and electrophysiological analyses. AtGLR genes that are coexpressed in specific cell or organ types will be targeted for tests of coassembly and cofunction in vivo.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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Abbreviations: AMPA, -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; GlnH, glutamate-binding domain; iGluR, ionotropic glutamate receptor; KA, kainate; M, transmembrane domain; NMDA, N-methyl-d-aspartate; P, pore.
Keywords: glutamate receptor
plant
Arabidopsis thaliana
mRNA expression
character mapping
Address for correspondence and reprints: Gloria Coruzzi, Department of Biology, New York University, 100 Washington Sq. East, New York 10003. gloria.coruzzi{at}nyu.edu
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