MBE Advance Access originally published online on January 20, 2006
Molecular Biology and Evolution 2006 23(5):941-948; doi:10.1093/molbev/msj097
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Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005 |
Relaxation of Functional Constraint on Light-Independent Protochlorophyllide Oxidoreductase in Thuja
* Department of Biology, Faculty of Sciences, Kyushu University, Ropponmatsu, Chuou-ku, Fukuoka, Japan; and Research and Development Center for Higher Education, Kyushu University, Fukuoka, Japan
E-mail: johtascb{at}mbox.nc.kyushu-u.ac.jp.
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Abstract |
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The light-independent protochlorophyllide oxidoreductase (DPOR) plays a key role in the ability of nonflowering plants and algae to synthesize chlorophyll in darkness. This enzyme consists of three subunits encoded by the chlB, chlL, and chlN genes in the plastid genome. Previously, we found a high nonsynonymous substitution rate (dN) of the chlL gene in the lineage of Thuja standishii, a conifer belonging to the Cupressaceae. Here we revealed that the acceleration of dN in the chlL occurred as well in other species of Thuja, Thuja occidentalis and Thuja plicata. In addition, dark-grown seedlings of T. occidentalis were found to exhibit a pale yellowish color, and their chlorophyll concentration was much lower than that of other species of Cupressaceae. The results suggested that the species of Thuja have lost the ability to synthesize chlorophyll in darkness, and the functional constraint on the DPOR would thus be expected to be relaxed in this genus. Therefore, we expected to find that the evolutionary rates of all subunits of DPOR would in this case be accelerated. Sequence analyses of the chlN and chlB (encoding the other subunits of DPOR) in 18 species of Cupressaceae revealed that the dN of the chlN gene was accelerated in Thuja as was the dN of the chlL gene, but the dN of the chlB gene did not appear to differ significantly among the species of Cupressaceae. Sequencing of reverse transcription–polymerase chain reaction (RT-PCR) products of these genes showed that RNA editing was rare and unlikely to have contributed to the acceleration. Moreover, the RT-PCR analysis indicated that all chl genes were still transcriptionally active in T. occidentalis. Based on these results, it appears that species of Thuja still bear the DPOR protein, although the enzyme has lost its activity because of nonsynonymous mutations of some of the chl genes. The lack of acceleration of the dN of the chlB gene might be accounted for by various unknown functions of its gene product.
Key Words: light-independent protochlorophyllide oxidoreductase • functional constraint • nonsynonymous substitution rate • conifer
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionSupplementary MaterialAcknowledgementsReferences |
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Protochlorophyllide oxidoreductase catalyzes the stereo-specific double-bond reduction of ring-D of protochlorophyllide to form chlorophyllide a. This reduction is a key step in the biosynthesis of chlorophyll and bacteriochlorophyll (Fujita 1996
; Armstrong 1998; Schoefs 2001; Schoefs and Franck 2003). Many photosynthetic organisms have two biochemically and genetically distinct protochlorophyllide oxidoreductases (Suzuki, Bollivar, and Bauer 1997; Armstrong 1998; Fujita and Bauer 2000). One of these two oxidoreductases is a light-dependent protochlorophyllide oxidoreductase (LPOR) encoded by a por gene in the nuclear genome. LPOR utilizes reduced nicotinamide adenine dinucleotide phosphate as a cofactor and requires light absorption by the substrate for its enzymatic activity. The other oxidoreductase is a light-independent protochlorophyllide oxidoreductase (DPOR). Genetic and sequence analyses have indicated that the DPOR consists of three subunits (Suzuki, Bollivar, and Bauer 1997; Armstrong 1998), which are encoded by bch genes (bchL, bchN, and bchB) in photosynthetic bacteria genomes or chl genes (chlL, chlN, and chlB) in the plastid genomes of photosynthetic eukaryotes. In addition, the biochemical analysis of purified DPOR proteins of a purple nonsulfur photosynthetic bacterium, Rhodobacter capsulatus, demonstrated that the DPOR consists of two separable components, the BchL protein (analog of ChlL) and the BchN-BchB (analogues of ChlN and ChlB, respectively) protein complex (Fujita and Bauer 2000). Because the DPOR can catalyze the protochlorophyllide reduction in a light-independent manner, it enables photosynthetic organisms to synthesize bacteriochlorophyll or chlorophyll in the absence of light.
Interestingly, these two enzymes, DPOR and LPOR, coexist in most cyanobacteria, algae, and land plants but not in those species that have lost the chl genes during the process of evolution processes (Armstrong 1998). Angiosperms are typical organisms lacking all the chl genes. The absence of the DPOR necessitates light for chlorophyll synthesis, thus dark-grown angiosperm seedlings fail to accumulate chlorophyll and become yellowish (etiolation). Such a lack of the chl genes has also been observed in some algae and nonflowering land plants (e.g., the brown alga Odontella sinensis, the euglenoid Euglena gracilis, the whisk fern Psilotum nudum, and the gnetophytes Welwitschia mirabilis). In addition, dark-grown ginkgo and larch seedlings fail to accumulate chlorophyll or have low levels of chlorophyll, in spite of the fact that these species have intact chl genes (Mariani et al. 1990; Chinn and Silverthorne 1993; Richard, Tremblay, and Bellemare 1994; Karpinska, Karpinski, and Hallgren 1997; Armstrong 1998). When taken together, the findings of these previous studies suggest that light-independent chlorophyll synthesis may be less important than light-dependent chlorophyll synthesis and that the DPOR is not essential in some photosynthetic organisms. On the other hand, the amino acid sequences of the chl genes are highly conserved among other photosynthetic organisms ranging from cyanobacteria to gymnosperms (i.e., the sequence similarities range from 60% to 90%) (Armstrong 1998). High sequence similarity between the chl genes of various species suggests that the DPOR is under strong functional constraints and is necessary at least in cyanobacteria, algae, and many nonflowering plants.
Previously, we sequenced four plastid genes, including the chlL gene, in order to reconstruct a molecular phylogeny of Cupressaceae, which is the most widely distributed conifer family; in that study, we found an acceleration of the nonsynonymous substitution rate (dN) of chlL in the lineage of Thuja standishii (Kusumi et al. 2000). There are five Thuja species, three of which (T. standishii, Thuja koraiensis, and Thuja sutchuenensis) are endemic to Eastern Asia and two of which are found in eastern and western North America (Thuja occicidentalis and Thuja pilicata, respectively). All species are found in cool and moist mixed conifer forests and can be found growing at sea level and up into mountain regions (Farjon 1990; Page 1990). Although we did not detect any nonsense mutations or frameshift mutations in the chlL gene of T. standishii, we expect that a functional constraint in the DPOR might have been altered in the case of T. standishii, and if so, the chlL gene might be on its way to becoming a pseudogene. If this were indeed the case, then we would expect to observe an impairment of DPOR function, which would in turn lead to a reduced level of chlorophyll in species grown in the dark, as well as an acceleration of the dN of all DPOR genes, that is, the chlL, chlN, and chlB genes and/or a change in transcriptional regulation of these genes. Alternatively, RNA editing may have caused inaccurate estimation of dN in the Thuja lineage or might have caused some defective change to the chlL transcripts, such as generating a premature termination codon. Premature termination would affect not only the DPOR function but also the dN of the chlL gene. Here, in order to assess these possibilities, we carried out the following analyses: (1) sequencing of all chl genes from 18 species of Cupressaceae, including novel sequencing studies of two additional Thuja species, and tests of the heterogeneity of the dNs in the Thuja lineage; (2) observation of dark-grown Thuja seedlings; (3) reverse transcription–polymerase chain reaction (RT-PCR) analysis of the chl genes in the Thuja species; and (4) sequencing of the RT-PCR products to examine the extents of RNA editing in these genes.
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Materials and Methods |
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DNA Sequencing
In addition to considering previously sequenced genes, we determined in the course of the present study the three loci of the chl genes in 18 species of Cupressaceae (table 1) and one species of Pinaceae, that is, Larix kaempferi. Most of the DNA materials were the same as those used in our previous study (Kusumi et al. 2000
), but some of these materials were newly extracted from leaves using a modified cetyldimethylammonium bromide method (Murray and Thompson 1980). PCR amplifications were performed with the Taq polymerase (Promega, Madison, Wisc.) or ExTaq polymerase (Takara, Shiga, Japan) using the following primer pairs (see also Supplementary Figure 1, Supplementary Material online): ChlL-F and ChlN-R for the chlL and chlN genes, ChlB-F and ChlB-R for the chlB gene, ChlL5'-F and ChlL-IR for the 5'-upstream region of the chlL gene, CHLB5'-F and ChlB-IR for the 5'-upstream region of the chlB gene, and ChlB-IF and ChlB-R3 for the 3'-downstream region of the chlB gene. The PCR products were purified using the MinElute PCR Purification kit (Qiagen, Hilden, Germany) and then the products were directly sequenced for both strands on an ABI 3100 automated sequencer using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Carlsbad, Calif.). All novel sequences were submitted to the DNA Data Bank of Japan. The accession numbers for the sequences are listed in table 1.
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Growth of Seedlings in the Dark
Growth Conditions of Seedlings
We analyzed dark-grown seedlings of T. occidentalis, Chamaecyparis obtusa, Cunninghamia lanceolata because seeds of these species were readily available, and their germination rates under dark conditions were higher than those of other conifer species reared in this study. These seeds were provided either by H. Yoshimaru of the Forestry and Forest Products Research Institute or by the Forest Tree Breeding Center of Japan. The seeds were sown on soil and grown under continuous exposure to light or dark (23°C) for 3 weeks.
Pigment Analysis
On the harvesting of 3-week-old T. occidentalis, C. obtusa, or C. lanceolata seedlings, we measured the fresh weight of each sample and immediately ground the sample in 250 µl of 80 % (v/v) acetone. The extract was centrifuged at 93,000xg for 10 min, and the pellet was extracted once again. A total of 500 µl of supernatant was used for the measurement of absorbance. The concentrations of chlorophyll a and chlorophyll b were calculated based on their absorbance values at 663 and 645 nm according to the method of Mackinney (1941).
RT-PCR
Total RNA was isolated from 3-week-old T. occidentalis and C. obtusa seedlings using CONCERT reagent (Invitrogen). Genomic DNA was removed by incubation of the RNA preparation with RQ1 DNase (Promega), followed by heat inactivation of DNase for 10 min at 70°C. The absence of genomic DNA in the RNA preparations was confirmed by PCR amplification with primers specific to the rbcL gene (RbcL-F and RbcL-R, see Supplementary Figure 1, Supplementary Material online) using 100 ng of total RNA as the template and genomic DNA as a positive control. RT-PCR was performed by using 2.5 µg total RNA with MMLV-Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions. Subsequent PCR amplifications were performed with the primers specific to the chl genes (chlL, ChlL-F and CHL-R; chlN, ChlN-IF and ChlN-R; and chlB, CHLB-F and CHLB-R; see Supplementary Figure 1, Supplementary Material online). The primers specific to the rbcL gene were also used as positive control primers. To determine the editing sites, purified RT-PCR products of the chl genes were directly sequenced.
Sequence Analysis
Sequence alignments of the obtained sequences were first performed by ClustalX (Thompson et al. 1997) and then the alignments were refined manually. Phylogenetic trees were reconstructed by the neighbor-joining (NJ) method (Nei and Gojobori 1986) implemented in MEGA3 (Kumar, Tamura, and Nei 2004). In the NJ trees, we used the Kumar model for the nucleotide sequences or the p-distance for the amino acid sequences to infer the topology. To test the rate constancy of the chl genes between genera, we used Relativerate.bf as implemented in the HYPHY program, which carries out a maximum likelihood (ML)-based analysis for the molecular evolution (Pond, Frost, and Muse 2005). We applied Relativerate.bf to the sequence data from the following representatives of 13 genera: C. lanceolata, Metasequoia glyptostroboides, Sequoia sempervirens, Sequoiadendron giganteum, Cryptomeria japonica, Glyptostrobus pensilis, Taxodium distichum, T. standishii, Thujopsis dolobrata, Platycladus orientalis, Juniperus rigida, Cupressus sempervirens, C. obtusa. We used C. lanceolata as an outgroup, which is a basal lineage of Cupressaceae (Kusumi et al. 2000). We used the codon substitution model "MGW9" (Muse and Gaut 1994) with a model option, "Local," [this model assumes that each branch has two parameters, synonymous substitution rate (dS) and dN]. Tests for the constancy of the 
= dS/dN ratio were conducted by the codeml program implemented in the PAML package, which also uses ML estimations (Yang 1997). Using the sequence data from the above 13 representatives, likelihood values were calculated with respect to two models, namely, a null model, in which all branches have a constant ratio 0, and an alternative model, in which the branch leading to Thuja has the ratio 1, whereas all of the other branches have a background ratio of 0. To test the constancy of among the branches, we compared the two likelihood values in a likelihood ratio test (LRT) (see Yang 1997, for details).
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Results and Discussion |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionSupplementary MaterialAcknowledgementsReferences |
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dN of chlL Genes in Thuja
In a previous study, we observed an acceleration of the dN in the chlL gene of T. standishii, but it remained unclear whether or not this phenomenon is common among Thuja species. Thus, we determined the chlL sequences of T. occidentalis and T. plicata, and we reconstructed a NJ tree based on the nonsynonymous substitutions of the complete coding sequences of the chlL gene (
876 bp) (fig. 1, left). The NJ tree, including the three species of Thuja, revealed that T. occidentalis and T. plicata form a clade with T. standishii, and this clade was found to have a long branch. Therefore, the increase in dN appears to have occurred prior to the divergence of the Thuja species.
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Phenotypes of Dark-Grown Seedlings
Next, we examined whether or not the Thuja and other Cupressaceae species have retained the ability to synthesize chlorophyll in the dark. We used T. occidentalis, C. obtusa, and C. lanceolata as representative species for this analysis. Cunninghamia lanceolata is one of the basal lineages of Cupressaceae, and C. obtusa and T. occidentalis belong to the same subfamily, Cuprressoidiae (Gadek et al. 2000
). The cotyledons of dark-grown C. lanceolata and C. obtusa seedlings were green, which indicated the accumulation of chlorophyll (fig. 2A). However, the cotyledons of dark-grown T. occidentalis seedlings were yellowish. Apparently, the T. occidentalis seedlings were unable to accumulate chlorophyll in the dark, as is also the case with angiosperms. We also measured the chlorophyll concentrations in these species (fig. 2B). When the samples were grown in light, the average chlorophyll concentrations of C. lanceolata, T. occidentalis, and C. obtusa were 1.45, 1.66, and 1.41 µg/mg, respectively. Therefore, light-grown T. occidentalis seedlings exhibited normal chlorophyll concentrations. However, when grown in the dark, the chlorophyll concentration of T. occidentalis was 0.019 µg/mg, which was less than one-tenth of that of C. lanceolata and C. obtusa (0.295 and 0.390 µg/mg). This finding demonstrated that T. occidentalis has certain defects in terms of its ability to synthesize chlorophyll in the dark. Although it remains unclear whether or not the nonsynonymous changes at chlL of Thuja are a cause or a consequence of the defect in the ability to synthesize chlorophyll in the dark, the DPOR appears to have become nonessential in Thuja. Moreover, the results of the phenotype analysis provided further support for our expectation that Thuja species exhibit only low-level chlorophyll accumulation when grown in the dark.
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Comparison of the dNs of chl Genes Among Cupressaceae Species
According to the neutral model, the dN is expected to be higher in functionally less constrained genes than in strongly constrained genes; this expectation is based on purifying selection (Kimura 1983
). According to the results of the phenotype analysis, the functional constraint in the DPOR appears to be relaxed in species of Thuja. If this is indeed the case, the genes for all three components of the DPOR, that is, the chl genes, are expected to have an accelerated dN in the Thuja lineage. To examine this expectation, we compared the dNs of all chl genes among Cupressaceae species. For the chlB gene, we were able to obtain complete coding sequences (1,553 bp) in most species analyzed here, but in C. lanceolata, G. pensilis, J. chinensis var procumbens, and T. plicata, we were only able to obtain partial sequences (17 amino acids shorter than that of the expected length in the C-terminal region). For the chlN gene, we sequenced a partial coding region (1,166 bp), which was 70 amino acids shorter than a complete coding region of the chlN gene in Pinus thunbergii in the C-terminal region. We did not find any nonsense or frameshift mutations among any of the sequences analyzed here. Initially, we reconstructed a NJ tree based on the nonsynonymous substitutions for each chl gene (fig. 1). As in the case of the chlL gene tree (fig. 1, left), the chlN gene tree also has a long branch in the Thuja lineage (fig. 1, middle). To examine the rate constancy, we performed pairwise relative rate tests between the genera by using the dNs of 13 representative species. For the chlL gene, 11 tests revealed significant differences between the dNs of various genera (P < 0.05, after Bonferroni correction). Among the significant comparisons, 10 pairs included Thuja, that is, 10 out of 11 tests between Thuja and the other genera showed significant differences. For the chlN gene, four tests were significant, and all significant results were from comparisons between Thuja and the other genera (P < 0.05, after Bonferroni correction). These results are consistent with our expectation that the relaxed functional constraint on the DPOR accelerated the dNs of the chl genes. In contrast, the branch lengths of the chlB tree were found to be similar among the species analyzed (fig. 2, right), and the relative rate tests for the dN of the chlB gene did not reveal any significant differences between genera. In the case of the chlB gene analysis, the results were inconsistent with our expectations. The apparent phenotypic difference between Thuja and the other genera was not always associated to the dNs of the chl genes, and interactive proteins do not appear to have consistently evolved in concert, even when they formed a protein complex.
Next, we tested the constancy of the ratio in the Thuja lineage by using an LRT (Yang 1997). The LRT was conducted by comparison of a null model, in which all branches had the same ratio, to an alternative model, in which the Thuja lineage had a different ratio from the other branches. In the case of the chlL gene, the null model was rejected (df = 1, P < 0.001). Under the alternative ratio model, the ratio in the Thuja lineage (1) was estimated to be 0.303, which was fivefold that of the other lineages (0 = 0.058). In the case of the chlN gene, although the LRT result was not significant (df = 1, P = 0.089), the estimated 1 was approximately 2.5 times higher than the 0 ratio (0.425 and 0.171, respectively) in the alternative ratio model. On the other hand, the estimated 0 and 1 of the chlB genes were similar (0.151 and 0.112) under the alternative ratio model. Therefore, the functional constraint at chlL and chlN appears to have been diminished but not at chlB in Thuja. Alternatively, the higher ratio in the Thuja lineage may be explained by positive selection. However, we consider this explanation unlikely in this case because the Thuja species have lost the ability to synthesize chlorophyll in darkness. Even if the loss was advantageous, the accumulation of the amino acid changes in the chl L and N genes would not be advantageous except for the first one that caused the loss.
Expression of chl genes in Thuja Species
When we investigated the alignments of the 5'-flanking regions of the chlL and chlB genes, we found many indels, some of which were Thuja specific. In fact, a number of defective phenotypes have been shown to appear when the expression of a gene is inhibited due to mutations of regulatory elements (Wray et al. 2003). Therefore, we analyzed the expression of the chl genes under light and dark conditions in T. occidentalis and C. obtusa by using RT-PCR analysis. The expression of all chl genes was always detected in both species under both conditions, even in the etiolated seedlings of T. occidentalis (fig. 3). The expression patterns of the chl genes in these species are similar to those found in Pinus teada L. (Skinner and Timko 1999) and P. thunbergii (data not shown). Thus, the chl genes do appear to be expressed in T. occidentalis.
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RNA Editing
RNA editing has been observed in the plastid transcripts of some algae and land plants, and most of this type of editing involves conversions from a cytidine (C) to uridine (U) residue (and in some bryophytes also from U to C) by base modification (Freyer, Kiefer-Meyer, and Kossel 1997
; Bock 2000). In P. thunbergii, 14 plastid genes with putative editing sites were investigated, and 26 edited sites in transcripts from 12 genes were identified (Wakasugi et al. 1996). Two more editing sites were found in the chlB transcripts of some Pinus species (Karpinska, Karpinski, and Hallgren 1997). Thus, RNA editing may also take place in Cupressaceae species, and this process may affect the dN of the chl genes and/or may generate a termination codon in the chl genes of Thuja. We therefore sequenced the cDNA of the chl genes in T. occidentalis, C. obtusa, and P. thunbergii. Among the three chl genes, we identified only one editing site in the chlN genes of C. obtusa and P. thunbergii. This site restores the conserved site for serine via a CCU (Pro) to UCU (Ser) codon. On the other hand, the corresponding site in T. occidentalis showed a C-to-G substitution. This substitution was found in the three Thuja species analyzed here, and such a substitution would be expected to prevent RNA editing at the identified site in Thuja. Because editing sites were found to be rare in the chl genes of Cupressaceae and were not found among Thuja species, we concluded that RNA editing does not contribute substantially to the heterogeneity of the dN. In addition, the products of the chl genes are not thought to be truncated by RNA editing in species of Thuja. Although we have not yet investigated the expression of these products at the protein level, we think that species of Thuja may have retained the DPOR proteins, and it is expected that certain amino acid substitutions may have reduced the enzymatic activity of this enzyme.
Phenotype and Sequence Analyses of Other Coniferous Species
The defective phenotype of T. occidentalis suggests that the DPOR is not necessary in Thuja (fig. 2). However, Thuja was found to have a dN in the chlB that was similar to that of other species of Cupressaceae (fig. 1), and all chl genes were found to be expressed in T. occidentalis, even in darkness (fig. 3). In the present study, we also grew seeds of Ginkgo biloba, L. kaempferi, and P. thunbergii in the dark, and we found that the seedlings of P. thunbergii were able to accumulate chlorophyll, whereas the G. biloba and L. kaempferi seedlings exhibited etiolation (data not shown), as has also been reported previously (Mariani et al. 1990; Chinn and Silverthorne 1993). To determine whether or not the chl genes of those conifers which fail to synthesize chlorophyll in darkness exhibit an elevated dN, we analyzed the corresponding amino acid sequences in these species. Despite the distinct phenotypes of dark-grown seedlings, all chl genes of L. kaempferi (accession number: AB232500, AB232501) and the chlB gene of G. biloba (accession number: GBU01531) were intact, and their evolutionary rates were not significantly higher than those of P. thunbergii (accession number: NC001631). These results were analogous to those obtained in the case of Thuja, in that the phenotype defect was not found to be associated with the changes in the evolutionary rates of the contributing genes.
A Possible Explanation for Why the Evolutionary Rates of All Genes Contributing to the DPOR Did Not Change in Concert with One Another
The failure to find an acceleration of the dN in the chlB gene in Thuja may be accounted for by assuming that there has not been a sufficient amount of time for the accumulation of substitutions which could be detected as a rate heterogeneity between Thuja and the other genera with respect to all chl genes. To test this assumption, we estimated the time lapse since the functional constraint in the DPOR was relaxed in the Thuja lineage by using dN and dS of T. standishii, T. dolobrata (the closest genus), and C. japonica (for the calibration of divergence time). We then estimated the divergence time (Td) of Thujopsis from Thuja by using the average dS of the chl and matK genes (Kusumi et al. 2000). Assuming that the Cupressoideae and Taxodioideae diverged at 100 MYA (Miller 1977, 1988), the value of Td was estimated to be approximately 17 MYA. According to Li, Gojobori, and Nei (1981) and Miyata and Yasunaga (1981), the following equations hold:
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Because the average dSThujopsis is slightly lower than the average dSThuja, we did not use dSThujopsis in order to remain conservative in our estimation. We then obtained t = 9.3 MYA for the chlL gene and t = 6.4 MYA for the chlN gene. We assumed that dNThuja of the chlB gene accelerated 6.4 MYA. Then, using the same equations and based on dSThuja and dNThujopsis of the chlB gene, the expected dNThuja of the chlB gene could be estimated to be 0.008, which was four times greater than the observed value (0.002). If the substitution process is a Poisson process at a rate of 0.008, the probability that less than three nonsynonymous substitutions would occur in 1195 sites is 0.014 (i.e., the number of nonsynonymous sites in the chlB gene is 1195.8). Although our calculations include some degree of uncertainty due to the large variance of the estimate for the expected dNThuja of the chlB gene, we concluded that the dN of the chlB gene did not appear to have changed in the Thuja lineage. An alternative explanation for the failure to find an acceleration of the dN in chlB would be to assume some additional function of the gene product other than that of becoming a component of the DPOR. This might in turn prevent the acceleration of the dN of the chlB gene in the Thuja lineage and prevent chlB from becoming a pseudogene. At present, it remains unknown what this other putative function of the chlB might be. The deduced amino acids suggested that the ChlL protein is associated with membranes and that it may function as an adenosine triphosphate–dependent electron donor; in addition, it has already been demonstrated that ChlN and ChlB proteins are soluble and provide the catalytic site for the protochlorophyllide reduction (Fujita et al. 1989; Liu, Xu, and Huang 1993; Fujita and Bauer 2000). Such differences between the biochemical characteristics of the gene products may have led to differences between chl genes in terms of evolutionary rates.
Relative Timing of Mutations and Changes in Phenotype
Although chlorophyll syntheses under both light and dark conditions share enzymes at most of the reaction steps, the reduction of the protochlorophyllide to form the chlorophyllide is catalyzed by two different enzymes under these two conditions. Because the gene is expressed even in species which do not accumulate much chlorophyll in the absence of light, a mutation in the regulatory region did not precede the accumulation of substitutions in the gene to render it less functional, and we think that some mutations at the chlL or chlN genes may have directly induced the etiolated phenotype in Thuja. To identify possible amino acid substitutions that inactivated the DPOR in Thuja, we aligned the available amino acid sequence of the chl genes among a broad range of plant species (alga to gymnosperms). However, we did not find any substitutions that had occurred only in the Thuja lineage, which might have exerted substantial influence in this regard. The measurement of the protochlorophyllide concentration in dark-grown Thuja seedlings could be expected to clarify whether or not chlorophyll synthesis is actually interrupted at the step of the protochlorophyllide reduction. Moreover, an assay system for the DPOR enzymatic activity recently established in the purple bacterium R. capsulatus (Fujita and Bauer 2000) may enable the elucidation of the biochemical properties of each subunit of the DPOR in Thuja.
A Possible Explanation for Why Thuja Exhibits Reduced Accumulation of Chlorophyll in the Absence of Light
Because angiosperms that lack the DPOR flourish worldwide, having only the light-dependent pathway to reduce protochlorophyllide may confer a selective advantage in certain environments. In fact, chlorophyll synthesis in the absence of photosynthesis appears to be costly (Armstrong 1998). In addition, the accumulation of tetrapyrroles must be accompanied by efficient protection against photodestruction (Schoefs and Franck 2003). However, chlorophyll synthesis in the dark may lead to a temporary accumulation of free precursors of chlorophylls and/or to the delayed binding of chlorophyll to their protein partners. Subsequent irradiation by sunlight (at daybreak or at germination) of such molecules produces singlet oxygen and oxygen radicals, which are known to damage the photosynthetic apparatus (Schoefs and Franck 2003). On the other hand, most photosynthetic organisms from cyanobacteria to gymnosperms synthesize chlorophyll in the light as well as in the dark using both the LPOR and DPOR (Suzuki, Bollivar, and Bauer 1997; Armstrong 1998), thus suggesting that the presence of parallel pathways to reduce protochlorophyllide would provide a selective advantage in some environments other than those of angiosperms. However, the reason for which these parallel pathways exist in these species remains unknown (Armstrong 1998). Low temperature may be among the key factors related to altering the selective advantage of the DPOR in Thuja species. Because exposure to low temperatures blocks the Calvin cycle in photosynthesis, an excess of light energy is generated, which causes photooxidative damage. Yamazaki et al. (2003) recently reported that the winter conversion from chlorophyll to protochlorophyllide observed in Abies veitchii, which can live at the tree line in Central Japan, may provide effective protection from photooxidative damage of the photosynthetic apparatus. As species of Thuja grow primarily in regions where the climate is cool and humid, that is, where the winters are snowy and cool to cold (Farjon 1990; Page 1990), the loss of DPOR activity may be related to an acclimation to a cold climate. Further molecular evolutionary analyses and comparisons of the phenotypes of a broad range of species of conifers may clarify whether changes in light and temperature are related to defects in light-independent chlorophyll synthesis.
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Supplementary Material |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionSupplementary MaterialAcknowledgementsReferences |
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Supplementary Figure 1 is available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
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Acknowledgements |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionSupplementary MaterialAcknowledgementsReferences |
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The authors would like to thank Y. Tsumura and H. Yoshimaru (The Forestry and Forest Products Research Institute, Japan) for providing the DNA and plant materials. We are also grateful to the organizers and participants of the Society for Molecular Biology and Evolution Young Investigator Workshop for their helpful comments and L. Katz and two anonymous reviewers for many helpful suggestions on earlier drafts of the manuscript. This work was partially supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no. 16370101).
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Footnotes |
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Laura Katz, Associate Editor
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References |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionSupplementary MaterialAcknowledgementsReferences |
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Armstrong, G. A. 1998. Greening in the dark:light-independent chlorophyll biosynthesis from anoxygenic photosynthetic bacteria to gymnosperms. J. Photochem. Photobiol. B Biol. 43:87–100.[CrossRef]
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