* Division of Biological Sciences, University of Missouri
Department of Ecology and Evolutionary Biology, Yale University
Correspondence: E-mail: mathewss{at}missouri.edu.
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Abstract |
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Key Words: phytochrome A positive selection adaptive evolution early angiosperms
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Introduction |
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Recent phylogenetic analyses have identified the extant remnants of the earliest-diverging angiosperm lineages, which include shrubs and woody vines that occur in shady habitats (Mathews and Donoghue 1999, 2000; Parkinson, Adams, and Palmer 1999; Qiu et al. 1999; Soltis, Soltis, and Chase 1999; Barkman et al. 2000; Doyle and Endress 2000; Graham et al. 2000; Savolainen et al. 2000; Soltis et al. 2000; Zanis et al. 2002); these are Amborella and a clade including the families Austrobaileyaceae, Illiciaceae, Schisandraceae, and Trimeniaceae. The water lilies also diverged early, but probably not before the lineage containing the woody understory species Amborella trichopoda of New Caledonia (Zanis et al. 2002). Implications for character evolution of the recently discovered fossil aquatic plant Archaefructus liaoningensis remain unclear in light of the low support for its placement in phylogenetic trees (Sun et al. 2002); however, similarities to Cabomba suggest that it may be a member of the water lily lineage (James Doyle, personal communication). Thus, the preponderance of phylogenetic evidence is consistent with the hypothesis of Feild et al. (2003) that the earliest angiosperms occupied shady, disturbed, and/or streamside habitats. In such an environment, innovations that enhanced their ability to establish in dimly lit environments may have been important to their early survival.
Photomorphogenesis, the control of plant form by ambient light conditions, is mediated by phytochrome photoreceptors, which absorb red light (R) and far-red light (FR), and by distinct blue- and ultraviolet-absorbing receptors (reviewed in Fankhauser and Chory 1997). Phytochrome evolution in land plants is marked by a series of gene duplications that have led to independently evolving and functionally distinct lines (Mathews and Sharrock 1997). A duplication preceding the origin of seed plants resulted in two distinct lines that persist in all extant seed plants. Phylogenetic analyses suggest that subsequent duplications occurred in each of these lines, leading to the four major forms found in angiosperms, phytochromes A, B, C, and E (phyAC, E), encoded by PHYAC, and E (Mathews, Lavin, and Sharrock 1995; Mathews and Sharrock 1997). PHYA and PHYC form one duplicate pair; PHYB and PHYE form a second duplicate pair. In cycads, Ginkgo, and conifers, a duplication in the PHYA/C-related line led to PHYN and PHYO, but the PHYB/E-related line, PHYP, did not diversify in other seed plants except for Pinaceae (Schneider-Poetsch et al. 1998; Clapham et al. 1999; Schmidt and Schneider-Poetsch 2002; Mathews and Donoghue, unpublished data). These phylogenetic analyses also suggest that the duplication leading to PHYA and PHYC occurred prior to the origin of angiosperms (Mathews, Lavin, and Sharrock 1995; Mathews and Sharrock 1997; Mathews and Donoghue 1999). PHYA and PHYC are about 50% identical (Sharrock and Quail 1989) and are functionally distinct (Casal, Sanchez, and Yanovsky 1997; Halliday, Thomas, and Whitelam 1997; Qin et al. 1997). The functional divergence of this gene pair is of interest in the context of early angiosperm history because phytochrome A (phyA) confers a competitive advantage for Arabidopsis seedlings establishing in dense shade (Yanovsky, Casal, and Whitelam 1995).
Phytochrome null mutants of Arabidopsis, tomato, and rice demonstrate that phyA induces seedling de-etiolation in response to continuous far-red light (FRc) via a far-red high irradiance response (FR-HIR; Nagatani, Reed, and Chory 1993; Parks and Quail 1993; Whitelam et al. 1993; van Tuinen et al. 1995; Takano et al. 2001). Etiolation is viewed as a specialized developmental pathway that is used to delay development in the dark or under low light conditions (e.g., McNellis and Deng 1995). Etiolated angiosperm seedlings require a light signal to de-etiolate, or to develop into a compact, green seedling with fully expanded seed leaves. Canopy shade is FR-enriched because chlorophyll strongly absorbs R; thus, in dense shade phyA induces de-etiolation. Conversely, open sunlight is R-enriched, and in these habitats, phyB induces de-etiolation. Additionally, phyA and phyB have contrasting antagonistic roles such that FRc suppresses phyB-mediated de-etiolation and Rc suppresses phyA-mediated de-etiolation (McCormac et al. 1992). The antagonistic role of phyA may best be understood in the context of the shade avoidance syndrome, controlled primarily by phyB, which induces stem elongation and accelerated development in response to neighbor detection (reviewed in Smith 2000). These responses may be too costly for seedlings, which have only limited resources (Smith, Xu, and Quail 1997). Subsequent to de-etiolation, shade avoidance responses come into play because phyA is lost rapidly in the light (Somers et al. 1991). It has been argued that seedlings emerging in the shade require a mechanism through which early shade avoidance reactions are suppressed, and furthermore, that the FR-HIR mediated by phyA provides such a mechanism (Smith, Xu, and Quail 1997). The importance of phyA in low light conditions is supported by the observation that phyA mutants of Arabidopsis die prematurely when grown in deep shade (Yanovsky, Casal, and Whitelam 1995). And while one Arabidopsis ecotype has a reduced sensitivity to FR (Maloof et al. 2001), the ability of this ecotype to survive in deep shade awaits further investigation.
Ginkgo and some conifers have elements of the FR-HIR de-etiolation response (Burgin et al. 1999; Christensen et al. 2002), but the response may be less important in conifer seedlings, many of which are compact and green (not etiolated) in the dark (Burgerstein 1900; Bogorad 1950; Mukai et al. 1992). Moreover, none of the seed plant phytochromes are unambiguous homologs of PHYA (e.g., Schmidt and Schneider-Poetsch 2002; Mathews and Donoghue, unpublished data), suggesting that the FR-HIRs of angiosperms and other seed plants may have independent origins. Phytochrome A also allows dark-imbibed seeds to germinate in response to millisecond pulses of broad spectrum light via the very low fluence response (VLFR; Botto et al. 1996), a response that is unknown outside of angiosperms and that operates via a downstream pathway different from that of the FR-HIR (Yanovsky, Casal, and Luppi 1997; Cerdán et al. 1999; Casal 2000; Yanovsky et al. 2002). The VLF germination response may be important for germination in open environments, allowing germination in response to brief soil disturbances (Casal, Sanchez, and Yanovsky 1997), and/or it may be important in shady, disturbed sites where light intensities fluctuate. Evidence from analyses of spectral characteristics indicates that phyC is likely to differ from phyA in its photoresponse to shade (Eichenberg et al. 2000). PHYC transcripts occur in low abundance in Arabidopsis (Sharrock and Quail 1989) and rice (Basu et al. 2000), and they may be constitutively expressed (Basu et al. 2000). Although little is known about the function of phyC, overexpression of Arabidopsis PHYC in tobacco suggests that it is involved in expansion of cotyledons in response to red light (Halliday, Thomas, and Whitelam 1997; Qin et al. 1997) and thus may be more important in open habitats. Thus, if the first angiosperms were understory colonizers, and if innovations in phytochrome function were important to their success, it is the evolution of phyA function that might have been important.
Gene duplications are considered to be a significant force in genome evolution (e.g., Wagner 2001) and may also play a significant role in speciation (Lynch and Conery 2000). When a gene duplicates, one copy may be silenced or evolve a novel function, or the two copies may subdivide functions of the ancestral gene (e.g., Ohno 1970; Walsh 1995; Force et al. 1999; Hughes 1999; Lynch and Force 2000). In the case of PHYA and PHYC, both copies have been maintained and they encode different functions. Moreover, as noted above, at least some of the functions of phyA are unique to angiosperms, suggesting that functional diversification has involved processes other than, or in addition to, subdivision of ancestral functions. Thus, two alternative models of functional diversification following gene duplication might explain the divergence of phyA from phyC. First, the function of one copy might be selectively maintained while the other copy evolves without selective constraints, obtaining a new function (Ohno 1970; Dykhuizen and Hartl 1980). This model is supported by evidence that nucleotide substitution rates in phyA differ significantly from those in phyC (Mathews and Sharrock 1997; Alba et al. 2000). Second, one copy might evolve a new function via positive selection. This model would be supported by evidence that rates of nonsynonymous substitution exceed rates of synonymous substitution (Hill and Hastie 1987). Yang and Nielsen (2002) report evidence of positive selection along a branch separating phyA and phyC sequences from species that are nested well within the angiosperms. Thus, the divergence of phyA from phyC may have involved both the relaxation of selective constraints and positive selection.
If photoreceptor evolution played a role in the initial establishment of angiosperms, we would expect to find evidence of altered selective constraints in sequences from the earliest diverging angiosperms. Previously published analyses could not address this question because no early-diverging species were sampled (Yang and Nielsen 2002). In this analysis we have explored a published data set that sampled the photosensory domain sequences of phyA and phyC from species representing all of the early-diverging lines of angiosperms (Mathews and Donoghue 2000) to determine if evolutionary constraints on phyA and phyC were altered early in the history of angiosperms, or if they were under positive selection. A further test of the hypothesis that the evolution of phyA function was important early in the history of angiosperms is not addressed in this study. It will require characterization of phyA responses in early-diverging angiosperms to determine whether they have the same responses as those described from derived eudicots (e.g., Arabidopsis and tomato) and a derived monocot (rice).
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Materials and Methods |
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The 3-dimensional structure of phytochromes remains undetermined, but functional domains have been identified through biochemical, genetic, and bioinformatics approaches (reviewed in Montgomery and Lagarias 2002). At a broad level, the molecule can be divided in half, with photosensory activity attributed to the n-terminal half and the regulatory activity attributed to the c-terminal half. Within each half, domains that confer specific functions have been identified. The photosensory domain comprises four subdomains (P1P4) necessary for photosensory specificity. Three of these, P2P4, are conserved among eukaryotic and prokaryotic phytochromes. The regulatory domain comprises two subdomains necessary for transducing light signals, a PAS-related subdomain and a histidine kinaserelated subdomain (Montgomery and Lagarias 2002). Our sequences correspond to photosensory subdomains P3 and P4. Based on sequence homology, the 3-dimensional structure of these individual subdomains is inferred to be similar to that of the of the small yeast protein YLK9 (Montgomery and Lagarias 2002). The phytochrome chromophore is covalently bound to a conserved cysteine in the pocket formed by the P3 subdomain.
Rate Tests
We estimated the coefficient of functional divergence () in order to test the hypothesis that functional constraints on PHYA and PHYC did not change after gene duplication (Gu 1999, 2001) by using DIVERGE version 1.04 (Gu 1999). DIVERGE uses the amino acid substitution rate at a site as an indication of positive selection on that site, and it examines whether rapidly evolving sites in one paralog are rapidly evolving in the other paralog. A shift in the distribution of rates among sites between the two paralogs may indicate functional diversification. In this test, theta (
) measures the coefficient of rate correlation over sites between paralogs. Assuming a Poisson model of amino acid substitution, the Gu test (1999, 2001) estimates subtree likelihoods for a given tree. In this case, the tree was similar to the tree in figure 1, pruned as described above (see Taxonomic Sampling). After obtaining maximum likelihood (ML) estimates of branch lengths, the gamma shape parameter (
) of rate variation across sites, and the coefficient of functional divergence (
AC), a likelihood ratio test (LRT) can be constructed to test the null hypothesis that
AC = 0 (with the alternative hypothesis being that
AC > 0). Rejection of the null hypothesis provides statistical evidence that functional constraints shifted after gene duplication (Gu 1999, 2001).
Tests for Selection
We compared the rates of synonymous (silent; dS) and nonsynonymous (amino-acid replacement; dN) substitutions in PHYA and PHYC to distinguish whether or not positive selection had a role in functional divergence. The dN/dS ratio measures selective pressure on amino acids. If functional divergence occurs by neutral mutations that are fixed by random genetic drift (Ohno 1970), or by neutral mutations that preadapt it for a new function (Dykhuizen and Hartl 1980), dN will not exceed dS. If functional divergence occurs by positive selection, dN may significantly exceed dS. Thus, the ratio of dN/dS () under the three conditions will vary. Under the first two conditions,
< 1; relaxing selective constraints should elevate the nonsynonymous substitution rate but the dN/dS ratio should never exceed the neutral expectation that
= 1. However,
may exceed one if an amino acid change is selectively advantageous and it is fixed at a higher rate than a synonymous mutation. Thus, finding that
is significantly greater than one provides convincing evidence of positive selection (Hill and Hastie 1987).
We used a tree-based likelihood ratio approach (e.g., Yang 1998), implemented in PAML (Yang 1997), to test for variable selective pressures among branches in the phylogeny of PHYA and PHYC. We compared the likelihoods of the one-ratio model, which assumes the same dN/dS ratio for each branch in the tree (table 1, model 1A), with two-ratio models, which assume two dN/dS ratios. One of the two-ratio models assumes that all PHYA branches have one ratio and that the PHYC and outgroup branches have another (table 1, model 2A), while the other assumes that the branch to all PHYA has a different ratio than all the other branches in the phylogeny (table 1, model 3A). We also compared the two-ratio models with a three-ratio model (table 1, model 4A), which assumes one dN/dS ratio for the branch to all PHYA, one for the internal PHYA branches, and one for all the rest. Likelihood estimation assumed the codon substitution model of Goldman and Yang (1994), which estimates separate nucleotide frequencies for each codon position and includes parameters for the transition/tranversion ratio and the dN/dS ratio.
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Character Mapping
Amino acid substitutions that result in a new function may occur once, early in the history of a protein lineage, to character states that are conserved through subsequent speciation events. Tests for selection using branch-specific or site-specific models may not identify these sites, because they are most suitable for detecting recurrent diversifying selection (e.g., Anisimova, Bielawski, and Yang 2002). Similarly, the prediction of sites of functional specification using DIVERGE (Gu 1999) identifies just those sites that have changed often through time (Gu 2001), although sites that change infrequently are included in the estimation of (Gu 1999, 2001). To identify the full set of the amino acid substitutions that occurred on the branches to all PHYA and all PHYC, we translated the nucleotide alignment of Mathews and Donoghue (2000) and mapped all unambiguous changes onto the branches of their phylogeny by using MacClade (Maddison and Maddison 2000). The alignment is available at TreeBase (www.treebase.org) under accession number SN543. This data set includes singletons that we did not include in our pairwise comparisons and comprises a total of 100 PHYA and PHYC sequences to which we added the sequence from Picea. The species phylogeny inferred by Mathews and Donoghue (2000) in their duplicate gene rooting analysis unambiguously identifies the earliest diverging branches of the angiosperm phylogeny to be the same as those converged upon in many other analyses (Mathews and Donoghue 1999; Parkinson, Adams, and Palmer 1999; Qiu et al. 1999; Soltis, Soltis, and Chase 1999; Barkman et al. 2000; Doyle and Endress 2000; Graham et al. 2000; Savolainen et al. 2000; Soltis et al. 2000; Zanis et al. 2002). We used parsimony to infer ancestral states at the root node, because our focus was on sites that change just once or very rarely on the treethat is, those that are conserved in the outgroups and in one gene copy but change to a conserved state on the branch to the other copy. When the focus is on rare changes, parsimony and maximum likelihood often yield the same results (e.g., Schluter et al. 1997; Cunningham, Omland, and Oakley 1998). To determine if this was the case in our study, we compared the ancestral states inferred using parsimony with those inferred using the maximum likelihood approach of Yang, Kumar, and Nei (1995) that is implemented in PAML (Yang 1997). We further tested the results from the mapping exercise by adding some unpublished data (Mathews and Donoghue) from additional seed plant phytochromes to the alignment described above. In addition to the PHYO from Picea, a conifer, we included PHYN from Pinus, also a conifer, and we included both PHYO and PHYN from Cycas, a cycad, and from Ginkgo. As noted above, PHYO and PHYN of other seed plants are related to PHYA and PHYC of angiosperms, whereas PHYP from other seed plants is related to PHYB and PHYE of angiosperms.
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Results |
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Tests for Selection
Tests for selection using branch-specific models indicate that the dN/dS ratio varies significantly between the branch to all PHYA and all other branches in the phylogeny of PHYA and PHYC. Just one of the two-ratio models fits the data significantly better than the one-ratio model. This is the model that assumes one ratio for the branch to all PHYA and one ratio for all other branches in the phylogeny (table 1, model 3A). The test statistic is 2l = 5.63; P = 0.02. The two-ratio model that assumes one ratio for all PHYA branches (table 1, model 2A) does not fit the data better than the one-ratio model. Moreover, the three-ratio model (table 1, model 4A) does not fit the data significantly better than two-ratio model 3A (2
l = 0.02; P = 0.99). Together these results indicate that all the variation in the dN/dS ratio can be explained by separating the branch to all PHYA from all other branches. They also suggest that the dN/dS ratio on the internal PHYA branches is not significantly different from the ratio on the PHYC branches. Thus, it does not appear that there was a general relaxation of constraints on PHYA.
Tests for selection using branch-site models identify sites under positive selection along the branch to all PHYA. Parameter estimates under model A suggest that 16% of amino acid sites are highly conserved across all lineages with 0 = 0, and 79% are nearly neutral, with
1 = 1, while a handful of sites, 5%, are under strong selection along the branch to all PHYA. Model A fits the data significantly better than model M1 (2
l = 119.68; P < 0.001 with df = 2). Parameter estimates under model B (table 1) suggest that about 5% of the sites are under strong positive selection (
2 = 9.29). In multiple analyses under the assumptions of model B, the
2 values ranged from 9.29 to 17.53, indicating the computational difficulty of the analysis (Z. Yang, personal communication). In the table we report the value corresponding to the tree with the highest likelihood. Model B fits the data significantly better than the site-specific model M3 (2
l = 11.22; P = 0.004 with df = 2). Regardless of the
2 value, at P > 0.5, model B identifies eleven sites potentially under selection along the branch to all PHYA (table 1). This is approximately one third of the sites inferred by character mapping to have changed along this branch (see below).
Character Mapping
We found that 32 amino acid substitutions map unambiguously to the branch leading to all PHYA in the tree with just Picea PHYO as the outgroup, while just seven substitutions map unambiguously to the branch leading to all PHYC. The same ancestral states at these sites are inferred by parsimony using MacClade (Maddison and Maddison 2000) and by maximum likelihood using PAML (Yang 1997). Of the substitutions occurring along the branch to all PHYA, 12 involve changes in charge, and three of these are at sites where changes within the PHYA clade do not alter the charge. Of the substitutions occurring along the branch leading to all PHYC, three involve changes in charge, and one of these is at a site where changes within the PHYC clade do not alter the charge. None of these sites is inferred to have been under selection. When PHYN and additional PHYO sequences representing other seed plant groups are added to the base of the tree, the number of substitutions that map to the foreground branch decreases to 30 while the number and identity of the sites that change on the branch to all PHYC remains the same. The sites that no longer map to the foreground branch are not inferred to have been under selection.
Our alignment corresponds to subdomains P3 and P4 of the phytochrome photosensory domain. The structure of these domains is inferred to be similar to that of the yeast protein YLK9 (Montgomery and Lagarias 2002). The chromophore is bound to a conserved cysteine residue in P3. Substitutions that involve charged residues are of particular interest because they might influence interactions between the protein and chromophore and/or between protein domains (Wu and Lagarias 2000). Three of the amino acid substitutions that occur along the branch to all PHYA result in a change in charge that is not altered within the PHYA clade. At position 119, the change is from an uncharged proline to a potentially negatively charged aspartic acid or glutamic acid residue very near the point of chromophore attachment. This site is inferred to have been under positive selection (P = 0.98). The position lies within a predicted loop between the third and fourth beta strands of the P3 domain; this likely is an important region for modulating the entry of the chromophore, its dynamics, its protonation state and/or its spectrum. At position 188, the change is from an uncharged glutamine to a positively charged histidine residue within a predicted alpha helix of the P3 domain that might serve as a connector to other domains. At position 360, the change is from a negatively charged glutamic acid residue shared by PHYC and the outgroup sequences to a potentially uncharged lysine or a neutral uncharged residue in all PHYA. This site is variable in PHYA but is never a negatively charged residue. It lies within a predicted alpha helix of the P4 domain that may serve as a connector to other domains. The single amino acid substitution along the branch to all PHYC that results in a change in charge replaces an uncharged glutamine at position nine with a positively charged arginine or lysine residue that is not altered within the PHYC clade. The position might lie in the terminal alpha helix in the P2 domain that may serve as a connector to other domains. The substitutions at each of these four sites may have functional implications, although just one is inferred to have been at a positively selected site (P119, P = 0.98). Thus, character mapping may provide insight into sites of potential functional significance that is not gained from tests for selection and functional divergence.
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Discussion |
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Strong evidence of positive selection has been reported mostly from analyses of recently diverged genes (Yang and Bielawski 2000), from analyses of genes that are expected to be under constant pressure to diversify, such as human immunodeficiency virus (HIV) and human influenza virus (Nielsen and Yang 1998; Crandall et al. 1999; Yang et al. 2000; Yang, Swanson, and Vacquier 2000), and from genes sampled from closely related species (Yang, Swanson, and Vacquier 2000). In plants, such evidence has been detected in disease-resistance genes (Bishop, Dean, and Mitchell-Olds 2000; Bergelson et al. 2001), where frequent diversification likely is advantageous. Evidence of positive selection may be much more difficult to obtain when adaptive change occurs early and is fixed, especially becausee adaptive change may result from evolution at a single site or at just a handful of sites (e.g., Perutz 1983; Hall 1984; Wilks et al. 1988; Chen, Greer, and Dean 1995, 1996; Newcomb et al. 1997). The nature of the challenge was emphasized by Golding and Dean (1998), who noted the exceeding difficulty of finding the few substitutions of large effect among the many other substitutions that have occurred in genes that diverged millions of years ago (Golding and Dean 1998:364). When adaptive evolution involves only a handful of amino acid sites, tests for selection that average rates across all sites of a locus may not detect evidence of positive selection (Endo, Ikeo, and Gojobori 1996; Crandall et al. 1999; Anisimova, Bielawski, and Yang 2001 ). Conversely, tests that allow the dN/dS ratio to vary among codons assume the same ratio on all branches of a phylogeny and thus may not detect episodic selection (Yang, Swanson, and Vacquier 2000).
Yang and Nielsen (2002) have addressed this in their branch-sites models, which show greater sensitivity to episodic selection. In the phytochrome data set that they analyzed they found no evidence of selection using either branch-specific or site-specific models alone, but they did find evidence of selection using branch-site models (Yang and Nielsen 2002), a finding similar to ours. However, their phylogeny of full-length PHYA and PHYC included only monocots (grasses) and eudiocots nested well within angiosperms. No outgroup was included and sites potentially under selection were identified with reference to the PHYA sequence from maize, a derived monocot (in figure 1 it would occur in the clade with the PHYA from other grasses, Oryza and Sorghum). Thus, although their analysis sampled more characters of the phytochrome molecule, they could not determine when in the history of the angiosperms adaptive evolution occurred; nor could they determine if selection occurred on the branch to PHYA or the branch to PHYC.
Episodic selection on PHYA very early in the history of flowering plants suggests that innovation in this photoreceptor may have given the first angiosperms some adaptive advantage. Among angiosperm phytochromes, phyA is unique in its capacity to serve a transient role under conditions where an extremely high sensitivity is required (Furuya and Schäfer 1996). This results from the fact that phyA is abundant in dark-grown seedlings but disappears rapidly in the light, due both to degradation and downregulation (Somers et al. 1991), and from its role as mediator of very low fluence responses (VLFRs), which require exposure to only millisecond pulses of broad spectrum light (Botto et al. 1996). It also is unique in that the far-red high irradiance response (FR-HIR) depends on the conversion of Pfr to Pr induced by the absorption of continuous far-red light (FRc; Shinomura, Uchida, and Furuya 2000). Although angiosperm phyE is known to induce Arabidopsis seeds to germinate in response to FRc, seed germination in many species is inhibited by FRc (Hennig et al. 2002), and the primary activities of phyE and of the remaining angiosperm phytochromes are dependent primarily on the conversion of Pr to Pfr, induced by absorption of either continuous or pulsed red light.
Phylogenetic and ecophysiological evidence suggests that the earliest angiosperms may have occupied dimly lit, disturbed, and moist understory habitats, but that the aquatic habit, which requires the capacity to begin life underwater, also arose early (Mathews and Donoghue 1999, 2000; Parkinson, Adams, and Palmer 1999; Qiu et al. 1999; Soltis, Soltis, and Chase 1999; Barkman et al. 2000; Doyle and Endress 2000; Graham et al. 2000; Savolainen et al. 2000; Soltis et al. 2000; Zanis et al. 2002; Feild, Arens, and Dawson 2003). We speculate that having an extremely sensitive photoreceptor capable of inducing responses with minimal exposure to light might have conferred an adaptive advantage in these habitats, and that this is reflected in the signal of molecular adaptation in phyA along the branch leading to all PHYA.
It is important to use two approaches to test this hypothesis. First, phytochrome-mediated responses in early-diverging flowering plants and additional outgroups need to be characterized. If our hypothesis were correct, we would expect extant remnants of the earliest-diverging lineages to show the full range of responses that are attributed to phyA in model species. Similarly, we would expect a full expression of these responses to be lacking in other seed plants. Both of these expectations need to be tested. We know that phyA null mutants of Arabidopsis fail to de-etiolate and then die prematurely when grown in deep shade (Yanovsky, Casal, and Whitelam 1995), possibly as a consequence of the inappropriate expression of shade avoidance responses (Smith, Xu, and Quail 1997). But phytochrome-mediated responses of early-diverging angiosperms are uncharacterized, and there are only limited data from other seed plants. Some members of the conifer family Pinaceae have a rudimentary FR-HIR (Burgin et al. 1999). However, the degree of etiolation, and thus the need to de-etiolate, varies among conifers; many are green in the dark (Burgerstein 1900; Bogorad 1950; Mukai et al. 1992). Gingko etiolates (Burgerstein 1900; Chinn and Silverthorne 1993) and also shows rudimentary elements of the FR-HIR de-etiolation response (Christensen et al. 2002). Some cycads (Cycas and Zamia) and some Gnetales (Ephedra and Gnetum) also etiolate (Bower 1882; Burgerstein 1900), but their ability to respond to FRc needs to be investigated. The VLFR also contributes to de-etiolation under low light conditions, although perhaps not enough to confer an advantage (Smith, Xu, and Quail 1997). No evidence has been presented that plants other than angiosperms have VLFRs, and we do not know if they occur in early-diverging angiosperms. It has been suggested that the very low fluence germination response is most important in open environments because seeds of many angiosperms germinate well in darkness, especially large seeds with adequate resources that are produced by many shade-tolerant species (Smith 1995). Small seeds, such as those that characterize early-diverging flowering plants (Feild, Arens, and Dawson 2003), however, are known to be viable in shady but disturbed habitats (Metcalfe and Grubb 1997; Grubb 1998), where the ability to respond to brief pulses of light may be important.
Second, in addition to characterizing phytochrome-mediated responses in early-diverging flowering plants and in other seed plants, mutagenesis experiments should be used to compare phytochrome-mediated responses of wild-type Arabidopsis with those of Arabidopsis phyA null mutants bearing sequence constructs with the ancestral states at the sites identified in our analyses as being of potential functional significance. If there is a connection between these sites and the known functions of phyA, altering them should also alter one or more of these functions. The fitness of phyA null mutants carrying these constructs can be determined to test our hypothesis that one or more of these functions could have conferred an adaptive advantage. The fitness of shade-avoidance phenotypes has been similarly tested using phyB mutants (Schmitt, McCormac, and Smith 1995; Pigliucci and Schmitt 1999). An important complementary test remains to be devised. Although it is possible to place a modified PHYA sequence in Arabidopsis, its ecology differs greatly from species like Amborella trichopoda. One is a weedy herb of open habitats; the other, a woody shade-tolerant shrub. It would be desirable to test the constructs in Amborella, which is not currently possible, or to find a way to test the fitness of phyA-mediated responses of woody shade-tolerant plants by somehow altering phyA expression. For example, it might be possible to test the far-red high irradiance de-etiolation response by using a brief light exposure to reduce the phyA pool before subjecting the emerging seedlings to continuous FR.
Many of the phytochrome mutants so far isolated have identified amino acid residues that are conserved, and possibly important, in all phytochromes (e.g., Quail et al. 1995; Maloof et al. 2001; Yanovsky et al. 2002). For example, one natural accession of Arabidopsis thaliana shows decreased sensitivity to FR, and its PHYA differs by a single amino acid from that of the lab strain Col-O. When this site was altered in PHYB, sensitivity to low-fluence R was also reduced, suggesting that the site is generally important for modulating light response in different phytochrome family members (Maloof et al. 2001). Similarly, an amino acid residue that is necessary for the phyA-mediated FR-HIR (Yanovsky et al. 2002) is also necessary for phyB-mediated response to continuous red light (Wagner and Quail 1995), suggesting that this site is important in different phytochromes. Our approach has identified amino acid residues that are uniquely altered in phyA. Manipulation of these sites has the potential to provide novel insight into the function of phyA.
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Acknowledgements |
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Footnotes |
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Keith Crandall, Associate Editor
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Literature Cited |
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