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PubMed2002

The 5' leader of plant PgiC has an intron: the leader shows both the loss and maintenance of constraints compared with introns and exons in the coding region.

PgiC, a complex gene with 23 coding exons and 22 intervening introns, encodes the cytosolic isozyme of phosphoglucose isomerase (EC 5.3.1.9) in higher plants. Here, we report RNA ligase-mediated rapid amplification of cDNA ends experiments that showed that PgiC in Clarkia (Onagraceae) and Arabidopsis thaliana has an intron in the 5' leader. Comparison of the EMBL accessions of the cDNA and genomic sequences showed that this is also the case in rice (Oryza sativa), suggesting that a leader intron is generally present in higher plant PgiC. The intron is bounded by consensus 5'-GT and AG-3' splice sites but showed alternative splicing in Clarkia, resulting in mature transcripts that differ by 8-19 nt in length. The intron is located 18 or 10 nt upstream of the start codon in Clarkia, 2 nt upstream in Arabidopsis, and 9 nt in rice. PgiC in Clarkia was duplicated before the divergence of the extant species, many of which have two expressed genes PgiC1 and PgiC2. Full-length transcripts of both genes identified the transcription start and made it possible to identify the leader intron and leader exon (between the transcription start and leader intron) from previously obtained genomic sequences of both genes in other Clarkia species. These data permit the comparison of evolution in the leader exon and intron with the exons and introns of the coding region, a topic that has not been studied previously. Both the leader exon and the leader intron resemble introns of the coding region in base substitution rate and accumulation of gaps. But the leader intron splice junctions are not strictly conserved in position as are those of the coding region introns. Also, in base composition, the leader intron resembles the other introns, whereas the leader exon more nearly resembles the coding exons. A difference in base composition between coding exons and flanking introns is known to be important for the recognition of splice sites. Thus, the marked difference in base composition between the leader exon and leader intron is probably maintained by selection despite a high rate of sequence divergence.

5' Untranslated Regions↗
PubMed1992

An rbcL sequence from a Miocene Taxodium (bald cypress).

During the past decade, ancient DNAs from both animals and plants have been successfully extracted and analyzed. Recently, the age of DNA that can be recovered and sequenced was increased manyfold by the amplification and sequencing of a DNA fragment from a Magnolia fossil obtained from the Miocene Clarkia deposit (17-20 million yr old). However, the validity of this report has been questioned based on models predicting that DNA should be completely degraded after 4 million yr. We report here the successful amplification, sequencing, and analysis of a 1320-base-pair portion of the chloroplast gene rbcL from a Miocene Taxodium specimen, also from the Clarkia site. These data not only validate the earlier report of sequence data for a Magnolia species from the same site but also suggest that it may be possible to isolate and sequence DNAs routinely from the Clarkia deposit. The ability to recover and sequence DNAs of such age offers enormous research possibilities in the areas of molecular evolution, biogeography, and systematics.

Base Sequence↗
PubMed2006

Geographic structure of pollinator communities, reproductive assurance, and the evolution of self-pollination.

Reproductive assurance is often invoked as an explanation for the evolution of self-fertilization in plants. However, key aspects of this hypothesis have received little empirical support. In this study, I use geographic surveys of pollinator communities along with functional studies of floral trait variation to examine the role of pollination ecology in mating system differentiation among populations and subspecies of the annual plant Clarkia xantiana. A greenhouse experiment involving 30 populations from throughout the species' range indicated that variation in two floral traits, herkogamy and protandry, was closely related to levels of autofertility and that trait variation was partitioned mainly among populations. Emasculation experiments in the field showed that autonomous selfing confers reproductive assurance by elevating fruit and seed production. Surveys of pollinator communities across the geographic range of the species revealed that bee pollinator abundance and community composition differed dramatically between populations of the outcrossing subspecies xantiana and the selfing subspecies parviflora despite their close proximity. Specialist bee pollinators of Clarkia were absent from selfing populations, but they were the most frequent visitors to outcrossing populations. Moreover, within the outcrossing subspecies xantiana, there was a close correspondence between specialist abundance and population differentiation in herkogamy, a key mating system trait. This spatial covariation arose, in part, because geographically peripheral populations had reduced herkogamy, higher autofertility, and lower pollinator abundance compared to central populations of xantiana. Finally, I detected strong spatial structure to bee communities both across the range of the species and within the outcrossing subspecies. In both cases, spatial structure was stronger for specialist bees compared to generalist bees, and pollinator communities varied in parallel with population variation in herkogamy. These results provide evidence that mating system differentiation parallels spatial variation in pollinator abundance and community composition at both broad and more restricted spatial scales, consistent with the hypothesis that pollinator abundance and reproductive assurance are important drivers of plant mating system evolution.

Adaptation, Biological↗
PubMed2002

Molecular cloning and characterization of a new linalool synthase.

Mentha citrata Ehrh. (bergamot mint; Lamiaceae) produces an essential oil containing only the acyclic monoterpenol (-)-3R-linalool and its acetate ester. A cloning strategy based upon the assumption that the responsible monoterpene synthase would resemble, in sequence, monoterpene cyclases from this plant family yielded a cDNA encoding the (--)-3R-linalool synthase. The nucleotide sequence of this monoterpene synthase is similar to those of several monoterpene cyclases from the mint (Lamiaceae) family (62-72% identity), but differs substantially from that of 3S-linalool synthase from Clarkia (41% identity; this composite gene appears to be of recent origin) and from that of 3R-linalool synthase from Artemisia (52% identity; the functional role of this gene is uncertain). Heterologous expression in Escherichia coli of a truncated version of the cDNA (in which the plastidial transit peptide was deleted) allowed purification and characterization of the enzyme, which was shown to possess most properties similar to other known monoterpene cyclases, but with a K(m) value for the natural substrate, geranyl diphosphate, of 56 microM with k(cat) of 0.83 s(-1). These kinetic constants for this 3R-linalool synthase are higher than those of any defined monoterpene cyclase, but the kinetic efficiency does not approach that reported for the 3S-linalool synthase from Clarkia. Although linalyl diphosphate is an enzyme-bound intermediate of monoterpene cyclase reactions, this tertiary allylic isomer of the geranyl substrate is not an efficient precursor of linalool with the M. citrata synthase. Modeling of the active site of this linalool synthase from Mentha and comparison to the modeled active sites of phylogenetically related monoterpene cyclases revealed structural differences in the binding of the diphosphate moiety which initiates the ionization step of the electrophilic reaction sequence and in the access of water to the active site to permit stereoselective quenching of the initially formed carbocationic intermediate to produce 3R-linalool.

Binding Sites↗
PubMed2003

Evidence for positive selection on the floral scent gene isoeugenol-O-methyltransferase.

Isoeugenol-O-methyltransferase (IEMT) is an enzyme involved in the production of the floral volatile compounds methyl eugenol and methyl isoeugenol in Clarkia breweri (Onagraceae). IEMT likely evolved by gene duplication from caffeic acid-O-methyltransferase followed by amino acid divergence, leading to the acquisition of its novel function. To investigate the selective context under which IEMT evolved, maximum likelihood methods that estimate variable d(N)/d(S) ratios among lineages, among sites, and among a combination of both lineages and sites were utilized. Statistically significant support was obtained for a hypothesis of positive selection driving the evolution of IEMT since its origin. Subsequent Bayesian analyses identified several sites in IEMT that have experienced positive selection. Most of these positions are in the active site of IEMT and have been shown by site-directed mutagenesis to have large effects on substrate specificity. Although the selective agent is unknown, the adaptive evolution of this gene may have resulted in increased effectiveness of pollinator attraction or herbivore repellence.

Amino Acid Sequence↗
PubMed2003

Structural basis for substrate recognition in the salicylic acid carboxyl methyltransferase family.

Recently, a novel family of methyltransferases was identified in plants. Some members of this newly discovered and recently characterized methyltransferase family catalyze the formation of small-molecule methyl esters using S-adenosyl-L-Met (SAM) as a methyl donor and carboxylic acid-bearing substrates as methyl acceptors. These enzymes include SAMT (SAM:salicylic acid carboxyl methyltransferase), BAMT (SAM:benzoic acid carboxyl methyltransferase), and JMT (SAM:jasmonic acid carboxyl methyltransferase). Moreover, other members of this family of plant methyltransferases have been found to catalyze the N-methylation of caffeine precursors. The 3.0-A crystal structure of Clarkia breweri SAMT in complex with the substrate salicylic acid and the demethylated product S-adenosyl-L-homocysteine reveals a protein structure that possesses a helical active site capping domain and a unique dimerization interface. In addition, the chemical determinants responsible for the selection of salicylic acid demonstrate the structural basis for facile variations of substrate selectivity among functionally characterized plant carboxyl-directed and nitrogen-directed methyltransferases and a growing set of related proteins that have yet to be examined biochemically. Using the three-dimensional structure of SAMT as a guide, we examined the substrate specificity of SAMT by site-directed mutagenesis and activity assays against 12 carboxyl-containing small molecules. Moreover, the utility of structural information for the functional characterization of this large family of plant methyltransferases was demonstrated by the discovery of an Arabidopsis methyltransferase that is specific for the carboxyl-bearing phytohormone indole-3-acetic acid.

Amino Acid Sequence↗
PubMed1999

S-Adenosyl-L-methionine:salicylic acid carboxyl methyltransferase, an enzyme involved in floral scent production and plant defense, represents a new class of plant methyltransferases.

S-Adenosyl-L-methionine:salicylic acid carboxyl methyltransferase (SAMT) was partially purified from petals of the annual California plant Clarkia breweri. SAMT catalyzes the formation of methylsalicylate, an important floral scent compound in C. breweri, from salicylic acid and S-adenosyl-L-methionine (SAM). The native enzyme is a dimer with a subunit molecular weight of 40.3 kDa, and it has a Km for salicylic acid of 24 microM and a Km for SAM of 9 microM. A cDNA encoding SAMT was isolated from a C. breweri cDNA library prepared from floral mRNA. The sequence of the protein encoded by SAMT cDNA shows no significant sequence similarity to any protein in the data bank whose biochemical function is known. It does show significant sequence similarity (20-40% identity) to proteins encoded by at least seven Arabidopsis thaliana genes whose sequences have recently been determined in large-scale sequencing projects. The C. breweri SAMT cDNA was expressed in E. coli and the bacterial cells synthesized a functional SAMT protein with properties nearly identical to those of the plant-purified enzyme.

Amino Acid Sequence↗
PubMed1999

Identification of specific residues involved in substrate discrimination in two plant O-methyltransferases.

Among the large number of plant O-methyltransferases that are involved in secondary metabolism, only a few have been enzymatically characterized, and little information is available on the structure of their substrate binding site and the mechanism which determines their substrate specificity and methylation regiospecificity. We have previously reported the isolation of two O-methyltransferases, S-adenosyl-l-methionine:(iso)eugenol O-methyltransferase (IEMT) and S-adenosyl-l-methionine:caffeic acid O-methyltransferase (COMT) from Clarkia breweri, an annual plant from California. While IEMT and COMT (which methylate eugenol/isoeugenol and caffeic acid/5-hydroxyferulic acid, respectively) share 83% identity at the amino acid level, they have distinct substrate specificity and methylation regiospecificity. We report here that seven amino acids play a critical role in discriminating between eugenol/isoeugenol and caffeic acid/5-hydroxyferulic acid. When these amino acids in IEMT were replaced by the corresponding residues of COMT, the hybrid protein showed activity only with caffeic acid/5-hydroxyferulic acid. Conversely, when these amino acids in COMT were replaced by corresponding IEMT residues, the hybrid protein had activity only with eugenol/isoeugenol. These results provide strong evidence that O-methyltransferase substrate preference could be determined by a few amino acid residues and that new OMTs with different substrate specificity could begin to evolve from an existing OMT by mutation of a few amino acids. Phylogenetic analysis confirms that C. breweri IEMT evolved recently from COMT.

Amino Acid Sequence↗
PubMed1992

Molecular analysis of the plant gene encoding cytosolic phosphoglucose isomerase.

The gene encoding a cytosolic isozyme of phosphoglucose isomerase (PGI, EC 5.3.1.9) was isolated from Clarkia lewisii, a wild flower native to California, and the structure and sequence of the entire coding region determined. PGI catalyzes an essential step in glycolysis and carbohydrate biosynthesis in plants. Spanning about 6 kb, the gene has 23 exons and 22 introns, the highest number yet reported in plants. The exons range in size from 43 to 156 nt and encode a protein of 569 amino acids. The protein is about 44-46% identical to the inferred protein sequences of pig, Escherichia coli and Saccharomyces cerevisiae. All of the introns are bordered with the consensus 5'-GT...AG-3' dinucleotides. The longest intron includes a large stem-loop structure bounded by a perfect 9 nt direct repeat. We cloned the PGI gene from a genomic library prepared from a single plant of known PGI genotype. The locus and allele of the clone were identified by matching restriction fragments to fragments from genetically defined genomic DNAs by Southern hybridization.

Amino Acid Sequence↗
PubMed1979

Distinguishing allozymes and isozymes of phosphoglucoisomerases by electrophoretic comparisons of pollen and somatic tissues.

The different electrophoretic patterns of dimeric phosphoglucoisomerases extracted from haploid pollen and diploid somatic tissues of plants may be used to distinguish allozymes and isozymes. The analysis depends on the presence of two alleles at each locus in somatic tissues but only one or the other allele in pollen grains. Consequently, in heterozygotes, heterodimeric allozymes can be identified because they are formed in stems and leaves but not in pollen. The procedure is described in enzymes extracted from the diploid annual plant Clarkia dudleyana, which possesses three gene loci for PGI subunits. Comparison of the electrophoretic patterns of stem and pollen extracts makes it possible in many cases to identify allelic state without breeding tests. The technique also is likely to be useful in the interpretation of zymograms of other multimeric enzymes coded by more than one gene locus.

Alleles↗
PubMed1984

Subunit hybridization and immunological studies of duplicated phosphoglucose isomerase isozymes.

We examined structural and functional properties of the recently duplicated cytosolic isozymes of phosphoglucose isomerase (PGI) (EC 5.3.1.9) of the wild plant Clarkia xantiana and related species. A new purification protocol yielded PGIs with high specific activities. The duplicated PGI isozymes showed similar substrate affinities (apparent Km) in both catalytic directions. A newly devised competitive enzyme-linked immunosorbent assay (ELISA), using polyclonal antibodies, distinguished sodium dodecyl sulfate (SDS)-denatured PGIs coded by the duplicated loci but did not discriminate between allelic products of one of the loci. In vitro dissociation and reassociation experiments revealed that the duplicated subunits differed in their efficiency of reassociation. The difference was closely correlated with differences between the isozymes in their in vivo accumulation. In contrast, allelic subunits in species with or without the duplication were able to reassociate with similar efficiency. The duplicated PGI isozymes have diverged more in structural properties than kinetic ones. The total evidence suggests that mechanisms have evolved which reduce the potential consequences of the duplication.

Cytosol↗
PubMed2005

An Arabidopsis thaliana methyltransferase capable of methylating farnesoic acid.

We previously reported the identification of a new family of plant methyltransferases (MTs), named the SABATH family, that use S-adenosyl-L-methionine (SAM) to methylate a carboxyl moiety or a nitrogen-containing functional group on a diverse array of plant compounds. The Arabidopsis genome alone contains 24 distinct SABATH genes. To identify the catalytic specificities of members of this protein family in Arabidopsis, we screened recombinantly expressed and purified enzymes with a large number of potential substrates. Here,we report that the Arabidopsis thaliana gene At3g44860 encodes a protein with high catalytic specificity towards farnesoic acid (FA). Under steady-state conditions, this farnesoic acid carboxyl methyltransferase (FAMT) exhibits KM values of 41 and 71 microM for FA and SAM, respectively. A three-dimensional model of FAMT constructed based upon similarity to the experimentally determined structure of Clarkia breweri salicylic acid methyltransferase (SAMT) suggests a reasonable model for FA recognition in the FAMT active site. In planta, the mRNA levels of At3g44860 increase in response to the exogenous addition of several compounds previously shown to induce plant defense responses at the transcriptional level. Although methyl farnesoate (MeFA) has not yet been detected in Arabidopsis, the presence of a FA-specific carboxyl methyltransferase in Arabidopsis capable of producing MeFA, an insect juvenile hormone made by some plants as a presumed defense against insect herbivory, suggests that MeFA or chemically similar compounds are likely to serve as new specialized metabolites in Arabidopsis.

Amino Acid Sequence↗
PubMed2005

Floral benzenoid carboxyl methyltransferases: from in vitro to in planta function.

Benzenoid carboxyl methyltransferases synthesize methyl esters (e.g., methyl benzoate and methyl salicylate), which are constituents of aromas and scents of many plant species and play important roles in plant communication with the surrounding environment. Within the past five years, eleven such carboxyl methyltransferases were isolated and most of them were comprehensively investigated at the biochemical, molecular and structural level. Two types of enzymes can be distinguished according to their substrate preferences: the SAMT-type enzymes isolated from Clarkia breweri, Stephanotis floribunda, Antirrhinum majus, Hoya carnosa, and Petunia hybrida, which have a higher catalytic efficiency and preference for salicylic acid, while BAMT-type enzymes from A. majus, Arabidopsis thaliana, Arabidopsis lyrata, and Nicotiana suaveolens prefer benzoic acid. The elucidation of C. breweri SAMT's three-dimensional structure allowed a detailed modelling of the active sites of the carboxyl methyltransferases and revealed that the SAM binding pocket is highly conserved among these enzymes while the methyl acceptor binding site exhibits some variability, allowing a classification into SAMT-type and BAMT-type enzymes. The analysis of expression patterns coupled with biochemical characterization showed that these carboxyl methyltransferases are involved either in floral scent biosynthesis or in plant defense responses. While the latter can be induced by biotic or abiotic stress, the genes responsible for floral scent synthesis exhibit developmental and rhythmic expression pattern. The nature of the product and efficiency of its formation in planta depend on the availability of substrates, the catalytic efficiency of the enzyme toward benzoic acid and/or salicylic acid, and the transcriptional, translational, and post-translational regulation at the enzyme level. The biochemical properties of benzenoid carboxyl methyltransferases suggest that the genes involved in plant defenses might represent the ancestor for the presently existing floral genes which during evolution gained different expression profiles and encoded enzymes with the ability to accept structurally similar substrates.

Amino Acid Sequence↗
PubMed2002

Novel S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase, an enzyme responsible for biosynthesis of methyl salicylate and methyl benzoate, is not involved in floral scent production in snapdragon flowers.

Using a functional genomic approach we have isolated and characterized a cDNA that encodes a salicylic acid carboxyl methyltransferase (SAMT) from Antirrhinum majus. The sequence of the protein encoded by SAMT has higher amino acid identity to Clarkia breweri SAMT than to snapdragon benzoic acid carboxyl methyltransferase (BAMT) (55 and 40% amino acid identity, respectively). Escherichia coli-expressed SAMT protein catalyzes the formation of the volatile ester methyl salicylate from salicylic acid with a K(m) value of 83 microM. It can also methylate benzoic acid to form methyl benzoate, but its K(m) value for benzoic acid is 1.72 mM. Snapdragon flowers do not emit methyl salicylate. The potential involvement of SAMT in production and emission of methyl benzoate in snapdragon flowers was analyzed by RNA gel blot analysis. SAMT mRNA was not detected in floral tissues by RNA blot hybridization, but low levels of SAMT gene expression were detected after real-time RT-PCR in the presence of SAMT-specific primers, indicating that this gene does not contribute significantly, if at all, in methyl benzoate production and emission in snapdragon flowers. Expression of SAMT in petal tissue was found to be induced by salicylic and jasmonic acid treatments.

Amino Acid Sequence↗
PubMed2003

Characterization of the glucose-6-phosphate isomerase gene in Phytophthora infestans reveals the presence of multiple alleles.

Glucose-6-phosphate isomerase (GPI) plays a key role in both glycolysis and gluconeogenesis. Isoforms of GPI are common, and therefore, its isozyme pattern is widely used to characterize isolates of Phytophthora infestans. Despite the importance of GPI in P. infestans studies, the gene encoding this enzyme has not yet been characterized. Furthermore, it has been suggested that P. infestans contains multiple copies of the gene but this hypothesis remains to be demonstrated. We have cloned and characterized GPI in various isolates of P. infestans as well as in several species of the genus Phytophthora. The gene contains 1671bp and encodes a protein with a predicted molecular weight of 60.8kDa. Multiple different alleles were identified and Southern analysis indicated certain P. infestans isolates carry several copies of the gene. Phylogenetic analysis revealed that P. infestans GPI is most closely related to sequences from Toxoplasma gondii, Arabidopsis thaliana, and Clarkia lewisii.

Alleles↗
PubMed2000

NTR1 encodes a floral nectary-specific gene in Brassica campestris L. ssp. pekinensis.

We have characterized a gene specifically expressed in the floral nectaries of Brassica campestris L. ssp. pekinensis. Differential screening led to the isolation of a floral nectary-specific cDNA clone. Northern hybridization indicated that its mRNA transcript is 1450 nucleotides long and specific to the flower base. In situ hybridization and immunolocalization showed that its mRNA and protein are localized specifically to both the lateral and median nectaries of flowers. The cDNA codes for a 43.8 kDa polypeptide 392 amino acids long. The protein was named nectarin1 (NTR1) after floral nectary protein. NTR1 was located in the cytoplasm of nectariferous cells in the nectaries and was also observed in nuclei at a much lower level. The level of the transcript increases with flower development, especially during nectary development, but decreases abruptly with the opening of the flower. Genomic Southern blot analysis indicated that at least three copies of homologous genes were present in the genome of B. campestris, but that only a single copy was present in both Arabidopsis thaliana and Lycopersicon esculentum. The deduced amino acid sequence of NTR1 shows similarity to S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase of Clarkia breweri which is expressed mostly in petals. The function of the gene is speculated to be involved in the methylation of a plant secondary metabolite in the floral nectaries.

Amino Acid Sequence↗
PubMed1990

Chloroplast DNA sequence from a miocene Magnolia species.

DNA has been successfully extracted from several samples of preserved tissue, the oldest so far reported originating from a 13,000-year-old ground sloth. Both severe damage to the preserved DNA, primarily due to oxidation of the pyrimidines, has prevented the acquisition of sequence data from ancient samples except in a few cases. We report here the extraction of DNA from fossil leaf samples from the Miocene Clarkia deposit (17-20 Myr old), the amplification of an 820-base pair (bp) DNA fragment from the chloroplast gene rbcL from a fossil of the genus Magnolia, and its subsequent sequencing. The sequence was verified by comparison with published and unpublished rbcL sequences. These results extend our ability to analyse ancient DNA and may open new avenues into problems in palaeobotany, biogeography, and in the calibration of mutation rates.

Base Sequence↗
PubMed2004

Biochemical and structural characterization of benzenoid carboxyl methyltransferases involved in floral scent production in Stephanotis floribunda and Nicotiana suaveolens.

Flower-specific benzenoid carboxyl methyltransferases from Stephanotis floribunda and Nicotiana suaveolens were biochemically and structurally characterized. The floral scents of both these species contain higher levels of methyl benzoate and lower levels of methyl salicylate. The S. floribunda enzyme has a 12-fold lower K(m) value for salicylic acid (SA) than for benzoic acid (BA), and results of in silico modeling of the active site of the S. floribunda enzyme, based on the crystal structure of Clarkia breweri salicylic acid methyltransferase (SAMT), are consistent with this functional observation. The enzyme was therefore designated SAMT. The internal concentration of BA in S. floribunda flowers is three orders of magnitude higher than the SA concentration, providing a rationale for the observation that these flowers synthesize and emit more methyl benzoate than methyl salicylate. The N. suaveolens enzyme has similar K(m) values for BA and SA, and the in silico modeling results are again consistent with this in vitro observation. This enzyme was therefore designated BSMT. However, the internal concentration of BA in N. suaveolens petals was also three orders of magnitude higher than the concentration of SA. Both S. floribunda SAMT and N. suaveolens BSMT are able to methylate a range of other benzenoid-related compounds and, in the case of S. floribunda SAMT, also several cinnamic acid derivatives, an observation that is consistent with the larger active site cavity of each of these two enzymes compared to the SAMT from C. breweri, as shown by the models. Broad substrate specificity may indicate recent evolution or an adaptation to changing substrate availability.

Amino Acid Sequence↗