Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts
Correspondence: E-mail: ekramer{at}oeb.harvard.edu.
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Abstract |
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Key Words: APETALA3 PISTILLATA MADS-box gene gene duplication
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Introduction |
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One of the best-understood lineages of MIKC-type genes in terms of evolution is the clade of B class genes, including the closely related paralogous lineages represented in Arabidopsis by APETALA3 (AP3) and PISTILLATA (PI ) (Bowman, Smyth, and Meyerowitz 1989). The products of AP3 and PI function as obligate heterodimers (Riechmann, Krizek, and Meyerowitz 1996) to establish the identity of petals and stamens in the developing floral meristem (Bowman, Smyth, and Meyerowitz 1989). Both the developmental and the biochemical aspects of AP3 and PI function appear to be conserved across orthologs analyzed in many core eudicots (reviewed in Irish and Kramer [1998] and Theissen et al. [2000]). Outside the core eudicots, however, greater variability has been observed (Kramer and Irish 2000), and at least one PI homolog has been shown to have the capacity to bind DNA as a homodimer (Winter et al. 2002). Along with this flexibility, many ancient and recent gene duplications have been characterized in the B gene lineage (Kramer, Dorit, and Irish 1998; Kramer, Di Stilio, and Schluter 2003). These comparative approaches have facilitated the identification of highly conserved C-terminal sequences known as the PI, paleoAP3, and euAP3 motifs (Kramer, Dorit, and Irish 1998). The paleoAP3 motif represents the ancestral C-terminal sequence within the angiosperm AP3 lineage but was replaced by the euAP3 motif in one paralogous AP3 lineage found only in the core eudicots (Kramer, Dorit, and Irish 1998). Our understanding of the evolution of the B gene lineages has been further elucidated by gymnosperm studies that have identified putative homologs of AP3/PI (Mouradov et al. 1999; Sundstrom et al. 1999). These genes appear to represent an ancestral lineage that predates the AP3/PI duplication (Winter, Saedler, and Theissen 2002) but retains C-terminal sequences similar to the PI and paleoAP3 motifs and is exclusively expressed in male reproductive organs (Mouradov et al. 1999; Sundstrom et al. 1999). An additional lineage, known as the Bsister (Bs) genes, has recently been identified as a closely related paralogous lineage to the angiosperm and gymnosperm B class genes (Becker et al. 2002). Although the members of this lineage possess PI and paleoAP3 motifs, they appear to be involved in aspects of carpel or ovule development (Becker et al. 2002; Nesi et al. 2002).
The emerging picture of B lineage evolution has been somewhat obscured by our lack of data from basal angiosperm lineages, particularly the primitive ANITA grade families (Qiu et al. 1999). Obtaining a clearer understanding of the early patterns of gene duplication and sequence evolution during the initial angiosperm radiations is particularly dependent on sampling from these groups. Because of the critical roles that the A, B, and C class genes play in flower development, the evolution of the corresponding lineages has been suggested to be connected to the evolution of the flower itself (Theissen et al. 2000; Theissen et al. 2002), underscoring the importance of obtaining information from basal angiosperms. To these ends, we have identified B and Bs lineage representatives from 10 angiosperm taxa primarily drawn from the ANITA grade and magnoliid dicots. Phylogenetic analyses of these genes in combination with earlier characterized homologs indicate that the evolution of the AP3 and PI lineages has been complex and dynamic from the earliest stages of angiosperm diversification.
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Materials and Methods |
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Cloning and Characterization of AP3 and PI Homologs
Isolation of AP3 and PI homologs was performed using RT-PCR in a manner similar to that described in Kramer, Dorit, and Irish (1998). Initial amplification of first-strand cDNA used one of two degenerate forward primers: primer 1, 5'-GGIMGIGGIAARATIGARATIAARMGIAT-3', or primer 2, 5'-ATGGSIMGIGGIAARATISARAT-3', with a poly-T reverse primer, 5'-CCGGATCTCTAGACGGCCGC(T)17. The products of the primary PCR reaction were cleaned with the QIAquick PCR purification kit (Qiagen, Valencia, Calif.), diluted 1:10, and used as template in a second PCR reaction using one of two degenerate primers: primer 3, 5'-AAYMGMCAMGTIACITWYTCIAARMGRMG-3', or primer 4, 5'-WCIAAYMGRCARGTIACITWWTC-3', with the same anchored poly-T reverse primer. All PCR amplifications were performed in 100 ml of PCR buffer (200 mM Tris-HCl, pH 8.4; 500 mM KCl; 50 mM MgCl2) containing 50 pmol and 10 pmol of 5' and 3' primer, respectively, 200 mmol of each dTNP, and 2 units of PlatinumTaq Polymerase (Invitrogen, Carlsbad, Calif.). The amplification program began with a 12 min activation step at 95°C, followed by a 1 min incubation step at 95°C, a 30 s annealing step at temperatures ranging from 50°C to 65°C, and a 1 min extension at 72°C. The program was repeated for 37 cycles and was terminated by a 10 min incubation step at 72°C. The amplified PCR products were cloned using the TOPO TA Cloningâ Kit (Invitrogen, Carlsbad, Calif.) as per manufacturer's instructions. For each taxon, 100 to 400 clones of more than 650 bp were characterized by sequencing (BigDye Terminator version 3.0, ABI prism 3100, Applied Bioscience, Foster City, Calif.) and/or restriction analysis. At least five independent clones were sequenced for every putative locus. Criteria used to distinguish putative loci include the degree of nucleotide identity and presence of unique indels. Sequence variants that contained no indels and differed by less than 5% identity were treated as alleles. All cDNA sequences have been deposited in GenBank (accession numbers AY436707 to AY436746). The Aquilegia alpina gene AqaBS was identified in the context of a separate screen (Kramer, Di Stilio, and Schluter 2003) but is being reported here for the first time.
Phylogenetic Analysis
Alignments were initially compiled using ClustalW and then refined by hand, taking into consideration both nucleotide and amino acid sequences (see Supplementary Material online [www.mbe.oupjournals.org] for NEXUS files and accession numbers). Five different alignments were created from the amino acid and nucleotide data sets. The first amino acid alignment contained all new AP3, PI, and BS homologs, previously identified Magnoliid dicot B and Bs lineage representatives, gymnosperm B and Bs homologs, and sequences from the AGL15 and AGL17/ANR1 MADS-box gene lineages, which have been identified as closely related to the B genes (Hasebe and Banks 1997; Shindo et al. 1999). This amino acid alignment excluded the C-terminal region of the predicted protein sequences because of difficulty aligning this region between angiosperm and gymnosperm B class genes and between the outgroup sequences. We delimited the K domain as originally defined in Ma, Yanofsky, and Meyerowitz (1991), resulting in an MIK data set of 171 characters. Separate full-length nucleotide and predicted protein sequence alignments were created for the angiosperm AP3 and PI data sets. Based on the position of NymPI in the MIK analysis (see below), this PI homolog was chosen as the outgroup for both the AP3 and the PI data sets.
All amino acid alignments were analyzed using PAUP* version 4.03b (Swofford 2001). Maximum-parsimony trees were generated through heuristic searches with 1,000 random stepwise additions, with tree bisection-reconnection (TBR) branch swapping and saving multiple parsimonious trees (MULTREES on). Gaps were encoded as missing data, and all characters were weighted equally. Bootstrap support for the full-length AP3 and PI data sets was estimated by performing 1,000 heuristic searches with 10 addition sequence replicates per bootstrap, using the same criteria as in the original search. Bootstrap support for the MIK data set was estimated by performing 1,000 nonparametric bootstrap replicates with random taxon addition, TBR branch-swapping, and MULTREES turned off. Wilcoxon sign-rank (known as the Templeton test [Templeton 1983]) and Kishino-Hasegawa (Kishino and Hasegawa 1989) tests were conducted on the MP trees to determine whether the data could reject topologies that were found in the Bayesian trees. Tests were also performed to explore topologies that would suggest alternative patterns of gene duplication.
Bayesian phylogenetic analyses were conducted on the nucleotide alignments using the program MrBayes version 3.0 (Huelsenbeck and Ronquist 2001). The best model of evolution was determined using Modeltest version 3.06 (Posada and Crandall 1998). The model of DNA substitution selected for both AP3 and PI was GTR+ I + , which assumes general time reversibility (GTR), certain proportion of invariable sites (I), and a gamma approximation of the rate-variation among sites (
). The option "codon" was used for the nucleotide substitution model, following the probabilistic model of codon evolution by Muse and Gaut (1994). We ran four chains of the Markov chain Monte Carlo method, sampling one tree every 100 generations for 1,000,000 generations starting with a random tree. Both searches reached stationarity after about 63,000 generations. The first 63,000 generations were considered the "burn-in" period and were not included in generating the consensus phylogenies.
Cloning and Characterization of the NymAP3 and NymPI Genomic Loci
Nymphaea sp. genomic DNA was prepared from leaf tissue using the DNeasy Plant Mini Kit (Qiagen, Valencia, Calif.). To obtain fragments of the NymAP3 genomic locus, the DNA was amplified using a specific forward primer, NymAP3F 5'-CATTCTGAGCTGTGCGGTCTTGAGCAA-3', in combination with one of two specific reverse primers: NymAP3R1, 5'-CTTTGTTTCTAGGGTCATCGGCTAACCT-3' or NymAP3R2, 5'-GGATTCATAATTATCTTCACTTCCATCGAA-3'. The primers were designed to regions of the NymAP3 cDNA predicted to fall within exon 3 for NymAP3F and within exons 5 and 6 for NymAP3R1 and NymAP3R2, respectively. PCR amplification was performed using BD Advantage Genomic PCR Kit (BD Biosciences Clonetech, Palo Alto, Calif.) as per manufacturer's instructions. The amplification program began with a 1 min activation step at 94°C, followed by a 30 s denaturing step at 94°C, a 30 s annealing step at 50°C to 60°C, and a 3 min extension step at 68°C, repeated for 30 cycles. The resulting genomic fragments were cloned using the TOPO TA Cloning® Kit (Invitrogen, Carlsbad, Calif.). Approximately 60 clones were screened for size, and 24 clones of either 350 bp (generated with NymAP3R1) or 470 bp (generated with NymAP3R2) were sequenced as described above. The resulting consensus genomic sequence (GenBank accession number AY436747) was aligned to the NymAP3 cDNAs (GenBank accession numbers AY436740 to AY436743) using ClustalW and then refined by hand (see figure S2 in Supplementary Material online [www.mbe.oupjournals.org]).
A region of the NymPI genomic locus was similarly obtained using the following primers: NymPIF, 5'-GACCTGAGCTCGTTGTCTGTTGTCGAACTTCGAA-3' and NymPIR, 5'-CCAATGTCGATGTCTCCCAGCTCGCGCATT-3'. These primers were predicted to fall within exons 4 and 7, respectively. PCR was performed on Nymphaea genomic DNA as described above. Intron position was assessed by aligning the sequence of the genomic fragment (GenBank accession number AY4366748) to the NymPI cDNA sequence (GenBank accession number AY436744).
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Results |
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Close examination of the Nymphaea PI homolog revealed a notable difference in the structure of this gene. All Bs homologs, gymnosperm B lineage representatives, and AP3 homologs sequenced to date have 42 nucleotides in exon 5, whereas all previously examined PI representatives have only 30 nucleotides in exon 5 (Johansen et al. 2002). This difference is caused by a 12-nucleotide deletion that appears to have occurred in the center of the exon (Purugganan et al. 1995). As can be seen in figure 5, NymPI lacks this 12-nucleotide deletion, which has been confirmed by sequencing this region from genomic DNA (see figure S1 in Supplementary Material online). In contrast, both PI homologs from Illicium have the deletion, as do all other PI homologs recovered in this analysis. The absence of this deletion indicates that NymPI is indeed ancestral to the other PI orthologs included in this analysis.
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The multiple AP3 homologs found in Drimys and Lindera appear to be derived from relatively recent duplications, but the Illicium AP3 homologs have a more complex evolutionary history. IhAP3-1 does not form a clade with IhAP3-2 and IhAP3-3, being positioned as a separate branch in both analyses. The MP topology would suggest that a duplication occurred in the AP3 lineage before the diversification of all the angiosperms sampled in this study, with one paralogous lineage represented by NymAP3, IhAP3-2, and IhAP3-3 and the other represented by IhAP3-1 and the balance of the AP3 homologs. The topology from the Bayesian analysis indicates that AP3 was duplicated somewhat later, along the branch between Nymphaea and Illicium, but similarly suggests that orthologs of IhAP3-2/-3 are absent in angiosperm lineages above Illicium. Maximum-parsimony analyses in which all Illicium paralogs were constrained together yielded two trees, each 10 steps longer than the maximum-parsimony tree. These trees were found to be significantly less parsimonious than the original MP tree by both the KH and Templeton tests (table 2).
Similar to what was seen in the PI nucleotide trees, the Piperaceae and Saururaceae AP3 homologs are placed together with high support but have long branch lengths. Although the Piperales AP3 homologs do not form a clade in the MP phylogeny, they do in the Bayesian analysis. Constraining all Piperales AP3 homologs into a monophyletic clade was tested with MP analysis, yielding four trees each only two steps longer than the maximum-parsimony tree. These trees were not found to be significantly different by the KH and Templeton tests (table 2).
As shown in figure 2, AP3 homologs from the Magnoliales and CfAP3-1 from Calycanthus appear to have similar deletions in the PI motif-derived region. However, neither phylogenetic analysis groups CfAP3-1 with the AP3 representatives from the Magnoliales containing the similar deletion. MP analysis where the Magnoliales AP3s and CfAP3-1 were constrained to form a single clade yielded 12 trees, each three steps longer than the unconstrained tree. When analyzed using the KH and Templeton tests, it was found that these trees are not significantly different from unconstrained MP trees (table 2). If the deletion in the PI motif region does represent a synapomorphy uniting CfAP3-1 and the Magnoliales AP3 homologs, it would suggest that a duplication occurred in the AP3 lineage before the split of the Magnoliales and the Laurales, followed by the deletion of the PI motif region in one of the paralogous lineages. Such an event may have been followed by different patterns of paralog loss in the separate Magnoliales and Laurales lineages. Alternatively, the PI motif deletion could have occurred independently in the Magnoliales and the CfAP3-1 lineages, with the CfAP3-1/-2 duplication occurring more recently in the Laurales. The MP and Bayesian phylogenies both suggest that the CfAP3-1/-2 duplication predated the last common ancestor of Lindera and Calycanthus, although a CfAP3-1 ortholog was not detected in Lindera. If the two Calycanthus AP3 homologs are constrained together, however, MP analysis produces two trees only four steps longer then the original MP tree, a difference that cannot be rejected with the current data set (table 2). Therefore, the timing of the CfAP3-1/-2 duplication and the homology of the PI motif deletion remains unclear.
In the course of characterizing the putative AP3 homolog from Nymphaea, four different classes of polyadenylated NymAP3 cDNAs were obtained. These classes were identical in sequence, except for four distinct patterns of indels observed in the K domain (fig. 7B). There is also slight variation in the 3' UTR polyadenylation position, but this does not correspond with the four indel classes (data not shown). Given the high frequency with which three of the classes were recovered (fig. 7B), it seemed possible that the different transcript types were produced through an alternative splicing mechanism. To investigate this possibility, a genomic fragment of NymAP3 corresponding to the region showing the indels was amplified and sequenced. All genomic clones (a total of 24 generated using two different primer pairs) were identical (data not shown), indicating that the different transcripts are all derived from one locus. Alignment of the genomic DNA and cDNA sequences (see figure S2 in Supplementary Material online) revealed a complex pattern of alternative splicing. Comparison of the class I cDNAs, which appear to contain the complete reading frame, with the characterized genomic DNA indicates that three introns designated I3, I4, and I5 (fig. 7A) are present in this region. These introns correspond to the same positions as introns 3, 4, and 5 in the Arabidopsis AP3 genomic sequence (Jack, Brockman, and Meyerowitz 1992). Based on the NymAP3 genomic and cDNA alignment (figure S2 in Supplementary Material online) and the consensus donor/acceptor site sequences from Arabidopsis (Hebsgaard et al. 1996; Brown and Simpson 1998; Lorkovic et al. 2000), the putative acceptor and donor sites for each NymAP3 intron/exon boundary were determined (fig. 7C). All of these sites appear to be correctly utilized in the class I clones, yielding a complete reading frame that encodes a predicted protein of 207 aa, including a C-terminal region with a paleoAP3 motif (fig. 2). In the class II clones, the I4 region has not been removed before polyadenylation of the transcript. Class III clones also retain I4 but appear to have utilized an alternative donor splice site located 5 bp within the E5 exon to splice to the E6 acceptor site. Because of the inclusion of I4 in the mature transcript, the translation products of both class II and class III cDNAs would be truncated by an in-frame stop codon that occurs in the center of I4. The resulting proteins would have 15 novel amino acids after those encoded by E4 and would lack almost all of the C-terminal domain. The class IV cDNA exhibits direct splicing of the E4 donor site to the E6 acceptor site, causing the omission of the entire E5 exon (fig. 7B). The protein product of the class IV transcript would lack the amino acids encoded by E5 but would remain in frame in E6. The resulting protein would lack most of the N-terminal end of the C domain.
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Discussion |
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Along the lineage leading to the angiosperms, the B lineage was duplicated to give rise to what are referred to as the AP3 and PI gene lineages. These two paralogous lineages acquired many clear synapomorphies before the diversification of extant angiosperms, including a truncation in the PI lineage that eliminated the paleoAP3 motif (Kramer, Dorit, and Irish 1998). One of the characters previously considered to be diagnostic for the PI lineage, a 12-nucleotide difference in the length of exon 5 (Johansen et al. 2002; Winter, Saedler, and Theissen 2002), has now been shown to have evolved within the ANITA grade. This finding provides further confirmation of phylogenetic analyses that have placed the Nymphaeales at or close to the base of the angiosperms (Parkinson, Adams, and Palmer 1999; Qiu et al. 2001; Zanis et al. 2002) and may also have implications for the evolution of PI function. The four amino acids, which are present in Nymphaea but absent in Illicium, are located at the beginning of the recently proposed third -helix of the K domain (Yang, Fanning, and Jack 2003). Although several studies have shown that the first two K region helices are critical for the heterodimerization of AP3 and PI (Krizek and Meyerowitz 1996; Riechmann, Krizek, and Meyerowitz 1996; Yang, Fanning, and Jack 2003), evidence now suggests that the putative third
-helix may mediate higher-order protein interactions between AP3/PI heterodimers and the E class SEPALLATA proteins (Yang and Jack, personal communication). The observed 4-aa deletion would shift the pattern of hydrophobic residues, which appear to be organized in heptad repeats in this region (Yang, Fanning, and Jack 2003), possibly changing the capacity of the domain to mediate certain protein interactions.
Further evidence of potential changes in the specificity of protein interactions is found in the first -helix of the K domain. Residues corresponding to positions 113 and 118 in our MIK amino acid alignment (Supplementary Material online at www.mbe.oupjournals.org) have been identified as critical to the specific formation of heterodimers between Arabidopsis AP3 and PI (Yang, Fanning, and Jack 2003). In eudicot AP3 homologs, position 113 is typically uncharged, whereas position 118 is basic. Eudicot PI homologs generally have an acidic residue at 113 but an uncharged amino acid at 118. The potential ionic interaction between amino acids at position 118 in AP3 and 113 in PI has been proposed to be similar to the i +5 ionic interactions that promote the formation of specific dimer pairs in leucine zipper proteins (Yang, Fanning, and Jack 2003). Moreover, the ability of the protein product of the gymnosperm B gene GGM2 to bind DNA as a homodimer (Winter et al. 2002) is correlated with the presence of oppositely charged residues at these positions. It is interesting to note, therefore, that the majority of magnoliid dicot and ANITA grade AP3 homologs encode acidic residues at 113 and basic residues at 118, which under the theory of Yang, Fanning, and Jack (2003) could indicate that they have the capacity to function as homodimers. Similarly, several PI homologs encode basic residues at position 118, along with the highly conserved acidic residue at 113. Several studies have now shown that changes in coding sequence can have a significant impact on gene function (Galant and Carroll 2002; Ronshaugen, McGinnis, and McGinnis 2002), including evidence of functional divergence between the euAP3 and paleoAP3 motifs (Lamb and Irish 2003). It remains to be determined whether the deletions and character state changes that occurred during the course of AP3/PI evolution have had a similar impact on biochemical aspects of gene function.
In addition to these trends of sequence evolution, we see dynamic patterns of gene lineage evolution in both the AP3 and PI lineages throughout the evolution of the basal angiosperms. For example, in the PI lineage, there is clear evidence for a duplication predating the last common ancestor of Lindera of the Lauraceae, a derived family within the Laurales, and Calycanthus of the Calycanthaceae, which is basal in the order and has a fossil record going back 100 Myr (Renner 1999). Similarly, paralogs derived from a duplication predating the last common ancestor of Piperaceae and Saururaceae have been retained in Piper. The IhAP3-1 and IhAP3-2/-3 lineages from Illicium may represent the most ancient example of this phenomenon. There are also cases of multiple recent duplications, most notably the two PI and four AP3 homologs in Drimys, which most likely reflect its polyploid genome (Sun, Stuessy, and Crawford 1990). These findings highlight the complicated nature of gene lineage evolution and exemplify the important caution raised by Theissen (2002) that simple genetic orthology is unlikely to exist between distantly related taxa. It is currently unclear as to what the functional implications of the long-term retention of AP3 or PI paralogs might be. None of the duplications detected in this study appear to be followed by dramatic patterns of sequence change, such as what has been observed in the case of the euAP3 and TM6 gene lineages (Kramer, Dorit, and Irish 1998). However, the fact that, for instance, two PI paralogs have been retained in the Laurales for approximately 100 Myr does suggest that they are being selectively maintained (Otto and Yong 2002). This does not necessarily mean that novel functions have evolved, because some type of subfunctionalization (Force et al. 1999; Lynch and Conery 2000) may have occurred. It is also unknown as to whether patterns of neofunctionalization or subfunctionalization are highly conserved within orthologous lineages. One possible indication of functional plasticity is the long branch lengths observed for both AP3 and PI homologs of the Piperaceae and Saururaceae. Although this trend could be a genome-wide phenomenon, it may be specific to these genes and related to the highly derived floral morphology found in these families (Tucker, Douglas, and Liang 1993).
The evidence for alternative splicing of the NymAP3 transcript is unique among loci identified in this study or any other survey of AP3 and PI homologs to date (Irish and Kramer 1998; Kramer, Dorit, and Irish 1998; Kramer and Irish 2000; Kramer, Di Stilio, and Schluter 2003). Instances of alternative RNA processing are not especially uncommon in plants (Reddy 2001), however, and examples have been identified in other MADS-box-containing genes (Krogen and Ashton 2000; Cheng et al. 2003), including a minor splicing difference in transcripts of ABS (Nesi et al. 2002), the Arabidopsis Bs lineage representative. The critical question is whether the phenomenon observed for NymAP3 is regulatory in nature. The high frequency with which alternatively spliced NymAP3 transcripts were recovered, especially compared with the perfect splicing of all isolated NymPI cDNAs, suggests that unspecific sloppiness is not a suitable explanation. There are at least two potential mechanisms by which the production of alternative transcripts could regulate NymAP3. If the class II and III mRNAs are not translated, their production could serve to reduce the concentration of functional NymAP3 transcript, thereby attenuating gene function. Alternatively, if the transcripts are translated, the truncated products may be capable of functioning as dominant negative factors, similar to what has been found for truncated AP3 in Arabidopsis (Krizek, Riechmann, and Meyerowitz 1999). Whether this would be in the context of homodimer or heterodimer formation is currently not known. Class IV transcripts would also produce an altered protein, but it is unclear what effect deleting the exon 5 region would have on protein function, although the whole C-terminal domain has been implicated in the formation of higher-order protein complexes (Egea-Cortines, Saedler, and Sommer 1999). It has previously been found that the autoregulatory and cross-regulatory interactions that characterize AP3 and PI homologs in the core eudicots (reviewed in Irish and Kramer [1998]) are not universally conserved (Kramer and Irish 2000). The NymAP3 findings provide further evidence that diverse gene regulatory mechanisms are acting on AP3 or PI homologs across divergent taxa.
Thus, the emerging picture of AP3 and PI lineage evolution is one of dynamic and stochastic evolution. Even if the developmental function of these genes in determining the identity of petaloid organs and stamens is broadly conserved, the exact parsing of ancestral functions among paralogs, along with possible instances of neofunctionalization, is likely to vary. Furthermore, this study demonstrates that these processes have been acting on the AP3 and PI lineages at every phylogenetic level. Our findings underscore the potential role of gene duplication in the process of developmental system drift (True and Haag 2001) and suggest that different degrees of conservation may be observed across biochemical, regulatory, and developmental aspects of gene function.
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Acknowledgements |
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Footnotes |
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Claudia Kappen, Associate Editor
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