Plant P450s as versatile drivers for evolution of species-specific chemical diversity

The irreversible nature of reactions catalysed by P450s makes these enzymes landmarks in the evolution of plant metabolic pathways. Founding members of P450 families are often associated with general (i.e. primary) metabolic pathways, restricted to single copy or very few representatives, indicative of purifying selection. Recruitment of those and subsequent blooms into multi-member gene families generates genetic raw material for functional diversification, which is an inherent characteristic of specialized (i.e. secondary) metabolism. However, a growing number of highly specialized P450s from not only the CYP71 clan indicate substantial contribution of convergent and divergent evolution to the observed general and specialized metabolite diversity. We will discuss examples of how the genetic and functional diversification of plant P450s drives chemical diversity in light of plant evolution. Even though it is difficult to predict the function or substrate of a P450 based on sequence similarity, grouping with a family or subfamily in phylogenetic trees can indicate association with metabolism of particular classes of compounds. Examples will be given that focus on multi-member gene families of P450s involved in the metabolic routes of four classes of specialized metabolites: cyanogenic glucosides, glucosinolates, mono- to triterpenoids and phenylpropanoids.     

Molecular evolution and functional divergence of the cytochrome P450 3 (CYP3) Family in Actinopterygii (ray-finned fish)

Background

The cytochrome P450 (CYP) superfamily is a multifunctional hemethiolate enzyme that is widely distributed from Bacteria to Eukarya. The CYP3 family contains mainly the four subfamilies CYP3A, CYP3B, CYP3C and CYP3D in vertebrates; however, only the Actinopterygii (ray-finned fish) have all four subfamilies and detailed understanding of the evolutionary relationship of Actinopterygii CYP3 family members would be valuable.

Methods and Findings

Phylogenetic relationships were constructed to trace the evolutionary history of the Actinopterygii CYP3 family genes. Selection analysis, relative rate tests and functional divergence analysis were combined to interpret the relationship of the site-specific evolution and functional divergence in the Actinopterygii CYP3 family. The results showed that the four CYP3 subfamilies in Actinopterygii might be formed by gene duplication. The first gene duplication event was responsible for divergence of the CYP3B/C clusters from ancient CYP3 before the origin of the Actinopterygii, which corresponded to the fish-specific whole genome duplication (WGD). Tandem repeat duplication in each of the homologue clusters produced stable CYP3B, CYP3C, CYP3A and CYP3D subfamilies. Acceleration of asymmetric evolutionary rates and purifying selection together were the main force for the production of new subfamilies and functional divergence in the new subset after gene duplication, whereas positive selection was detected only in the retained CYP3A subfamily. Furthermore, nearly half of the functional divergence sites appear to be related to substrate recognition, which suggests that site-specific evolution is closely related with functional divergence in the Actinopterygii CYP3 family.

Conclusions

The split of fish-specific CYP3 subfamilies was related to the fish-specific WGD, and site-specific acceleration of asymmetric evolutionary rates and purifying selection was the main force for the origin of the new subfamilies and functional divergence in the new subset after gene duplication. Site-specific evolution in substrate recognition was related to functional divergence in the Actinopterygii CYP3 family.     

Brassinosteroids Regulate Plant Growth through Distinct Signaling Pathways in Selaginella and Arabidopsis

Brassinosteroids (BRs) are growth-promoting steroid hormones that regulate diverse physiological processes in plants. Most BR biosynthetic enzymes belong to the cytochrome P450 (CYP) family. The gene encoding the ultimate step of BR biosynthesis in Arabidopsis likely evolved by gene duplication followed by functional specialization in a dicotyledonous plant-specific manner. To gain insight into the evolution of BRs, we performed a genomic reconstitution of Arabidopsis BR biosynthetic genes in an ancestral vascular plant, the lycophyte Selaginella moellendorffii. Selaginella contains four members of the CYP90 family that cluster together in the CYP85 clan. Similar to known BR biosynthetic genes, the Selaginella CYP90s exhibit eight or ten exons and Selaginella produces a putative BR biosynthetic intermediate. Therefore, we hypothesized that Selaginella CYP90 genes encode BR biosynthetic enzymes. In contrast to typical CYPs in Arabidopsis, Selaginella CYP90E2 and CYP90F1 do not possess amino-terminal signal peptides, suggesting that they do not localize to the endoplasmic reticulum. In addition, one of the three putative CYP reductases (CPRs) that is required for CYP enzyme function co-localized with CYP90E2 and CYP90F1. Treatments with a BR biosynthetic inhibitor, propiconazole, and epi-brassinolide resulted in greatly retarded and increased growth, respectively. This suggests that BRs promote growth in Selaginella, as they do in Arabidopsis. However, BR signaling occurs through different pathways than in Arabidopsis. A sequence homologous to the Arabidopsis BR receptor BRI1 was absent in Selaginella, but downstream components, including BIN2, BSU1, and BZR1, were present. Thus, the mechanism that initiates BR signaling in Selaginella seems to differ from that in Arabidopsis. Our findings suggest that the basic physiological roles of BRs as growth-promoting hormones are conserved in both lycophytes and Arabidopsis; however, different BR molecules and BRI1-based membrane receptor complexes evolved in these plants.     

Genome-wide identification and evolutionary analysis of Arabidopsis sm genes family

Sm proteins are members of a family of small proteins that are widespread in biosphere and found associated with RNA metabolism. To date, to our knowledge, only Arabidopsis SAD1 gene has been studied functionally in plant. In this study, 42 Sm genes are identified through comprehensive analysis in Arabidopsis. And a complete overview of this gene family is presented, including the gene structures, phylogeny, chromosome locations, selection pressure and expression. The results reveal that gene duplication contributes to the expansion of the Sm gene family in Arabidopsis genome, diverse expression patterns suggest their functional differentiation and divergence analysis indicates purifying selection as a key role in evolution. Our comparative genomics analysis of Sm genes will provide the first step towards the future experimental research on determining the functions of these genes.     

Structural divergence and adaptive evolution in mammalian cytochromes P450 2C

Cytochromes P450 (CYPs) comprise a superfamily of enzymes involved in various physiological functions, including the metabolism of drugs and carcinogenic compounds present in food, making them of great importance for human health. The possibility that CYPs could be broadening or changing substrate specificity in accordance to the high diversity of xenobiotics compounds environmentally available suggests that their metabolic function could be under adaptive evolution. We evaluated the existence of functional divergence and signatures of selection on mammalian genes from the drug-metabolizing CYP2 family. Thirteen of the sites found to be functionally divergent and the eight found to be under strong positive selection occurred in important functional domains, namely on the substrate entrance channel and within the active site. Our results provide insight into CYPs evolution and the role of molecular adaptation in enzyme substrate-specificity diversification.     

CYP6B1 and CYP6B3 of the black swallowtail (Papilio polyxenes): adaptive evolution through subfunctionalization

Gene duplication provides essential material for functional divergence of proteins and hence allows organisms to adapt to changing environments. Following duplication events, redundant paralogs may undergo different evolutionary paths via processes known as nonfunctionalization, neofunctionalization, or subfunctionalization. Studies of adaptive evolution at the molecular level have progressed rapidly by computationally analyzing nucleotide substitution patterns but such studies are limited by the absence of information relating to alterations of function of the encoded enzymes. In this respect, evolution of the Papilio polyxenes cytochrome P450 monooxygenases (P450s) responsible for the adaptation of this insect to furanocoumarin-containing host plants provides an excellent model for elucidating the evolutionary fate of duplicated genes. Evidence from sequence and functional analysis in combination with molecular modeling indicates that the paralogous CYP6B1 and CYP6B3 genes in P. polyxenes have probably evolved via subfunctionalization after the duplication event by which they arose. Both enzymes have been under independent purifying selection as evidenced by the low dN/dS ratio in both the coding region and substrate recognition sites. Both enzymes have maintained their ability to metabolize linear and angular furanocoumarins albeit at different efficiencies. Comparisons of molecular models developed for the CYP6B3 and CYP6B1 proteins highlight differences in their binding modes that account for their different activities toward linear and angular furanocoumarins. That P. polyxenes maintains these 2 furanocoumarin-metabolizing loci with somewhat different activities and expression patterns provides this species with the potential to acquire P450s with novel functions while maintaining those most critical to its exclusive feeding on its current range of host plants.     

Selection in the evolution of gene duplications

Background  

Gene duplications have a major role in the evolution of new biological functions. Theoretical studies often assume that a duplication per se is selectively neutral and that, following a duplication, one of the gene copies is freed from purifying (stabilizing) selection, which creates the potential for evolution of a new function.     

Why do paralogs persist? Molecular evolution of CYCLOIDEA and related floral symmetry genes in Antirrhineae (Veronicaceae)

CYCLOIDEA (CYC) and DICHOTOMA (DICH) are paralogous genes that determine adaxial (dorsal) flower identity in the bilaterally symmetric flowers of Antirrhinum majus (snapdragon). We show here that the duplication leading to the existence of both CYC and DICH in Antirrhinum occurred before the radiation of the Antirrhineae (the tribe to which snapdragon belongs). We find no additional gene duplications within Antirrhineae. Using explicit codon-based models of evolution in a likelihood framework, we show that patterns of molecular evolution after the duplication that gave rise to CYC and DICH are consistent with purifying selection acting at both loci, despite their known functional redundancy in snapdragon. However, for specific gene regions, purifying selection is significantly relaxed across DICH lineages, relative to CYC lineages. In addition, we find evidence for relaxed purifying selection along the lineage leading to snapdragon in one of two putative functional domains of DICH. A model of selection accounting for the persistence of paralogous genes in the absence of diversifying selection is presented. This model takes into account differences in the degree of purifying selection acting at the two loci and is consistent with subfunctionalization models of paralogous gene evolution.     

Rapid birth-death evolution specific to xenobiotic cytochrome P450 genes in vertebrates

Genes vary greatly in their long-term phylogenetic stability and there exists no general explanation for these differences. The cytochrome P450 (CYP450) gene superfamily is well suited to investigating this problem because it is large and well studied, and it includes both stable and unstable genes. CYP450 genes encode oxidase enzymes that function in metabolism of endogenous small molecules and in detoxification of xenobiotic compounds. Both types of enzymes have been intensively studied. My analysis of ten nearly complete vertebrate genomes indicates that each genome contains 50-80 CYP450 genes, which are about evenly divided between phylogenetically stable and unstable genes. The stable genes are characterized by few or no gene duplications or losses in species ranging from bony fish to mammals, whereas unstable genes are characterized by frequent gene duplications and losses (birth-death evolution) even among closely related species. All of the CYP450 genes that encode enzymes with known endogenous substrates are phylogenetically stable. In contrast, most of the unstable genes encode enzymes that function as xenobiotic detoxifiers. Nearly all unstable CYP450 genes in the mouse and human genomes reside in a few dense gene clusters, forming unstable gene islands that arose by recurrent local gene duplication. Evidence for positive selection in amino acid sequence is restricted to these unstable CYP450 genes, and sites of selection are associated with substrate-binding regions in the protein structure. These results can be explained by a general model in which phylogenetically stable genes have core functions in development and physiology, whereas unstable genes have accessory functions associated with unstable environmental interactions such as toxin and pathogen exposure. Unstable gene islands in vertebrates share some functional properties with bacterial genomic islands, though they arise by local gene duplication rather than horizontal gene transfer.     

Purifying selection and birth-and-death evolution in the histone H4 gene family

Histones are small basic proteins encoded by a multigene family and are responsible for the nucleosomal organization of chromatin in eukaryotes. Because of the high degree of protein sequence conservation, it is generally believed that histone genes are subject to concerted evolution. However, purifying selection can also generate a high degree of sequence homogeneity. In this study, we examined the long-term evolution of histone H4 genes to determine whether concerted evolution or purifying selection was the major factor for maintaining sequence homogeneity. We analyzed the proportion (p(S)) of synonymous nucleotide differences between the H4 genes from 59 species of fungi, plants, animals, and protists and found that p(S) is generally very high and often close to the saturation level (p(S) ranging from 0.3 to 0.6) even though protein sequences are virtually identical for all H4 genes. A small proportion of genes showed a low level of p(S) values, but this appeared to be caused by recent gene duplication. Our findings suggest that the members of this gene family evolve according to the birth-and-death model of evolution under strong purifying selection. Using histone-like genes in archaebacteria as outgroups, we also showed that H1, H2A, H2B, H3, and H4 histone genes in eukaryotes form separate clusters and that these classes of genes diverged nearly at the same time, before the eukaryotic kingdoms diverged.     

Molecular evolution and functional divergence of HAK potassium transporter gene family in rice （Oryza sativa L.）

The high-affinity K＋ （HAK） transporter gene family is the largest family in plant that functions as potassium transporter and is important for various aspects of plant life. In the present study, we identified 27 members of this family in rice genome. The phylogenetic tree divided the land plant HAK transporter proteins into 6 distinct groups. Although the main characteristic of this family was established before the origin of seed plants, they also showed some differences between the members of non-seed and seed plants. The HAK genes in rice were found to have expanded in lineage-specific manner after the split of monocots and dicots, and both segmental duplication events and tandem duplication events contributed to the expansion of this family. Functional divergence analysis for this family provided statistical evidence for shifted evolutionary rate after gene duplication. Further analysis indicated that both point mutant with positive selection and gene conversion events contributed to the evolution of this family in rice.     

Enhancement of metabolizing herbicides in young tubers of transgenic potato plants with the rat CYP1A1 gene

A rat P450 monooxygenase gene (CYP1A1) was introduced into potato plants to enhance the metabolism of the environmental contaminants in subterranean organs. The CYP1A1 gene was kept under the control of the potato patatin promoter to enhance tuber-specific expression. A total of 106 transgenic plants (PAT1A1 plants) were obtained following selection by a resistance test to kanamycin and PCR analysis. PAT1A1 plants treated with 10% exogenous sucrose showed a higher activity of monooxgenase in the leaves than the non-transgenic plants. This indicated that the activity enhanced by 10% sucrose was due to the patatin promoter containing the sucrose-inducted elements. One representative transgenic plant, Ag2197, was selected on the basis of monooxgenase activity in the leaves and Western blot analysis. Ag2197 was found to accumulate a large amount of CYP1A1 mRNA and protein in the developing tuber but not in the mature tuber. The residual herbicides, atrazine and chlortoluron, were analyzed in the micro-tubers of Ag2197 and non-transgenic plants. The amount of residual herbicides in Ag2197 was much lower than that in the non-transgenic plant, indicating that the transgenic plant metabolized the herbicides to a detoxified form. The transgenic plants produced in this study might be useful for the phytoremediation of chemical pollution in the soil.     

Degree of Functional Divergence in Duplicates Is Associated with Distinct Roles in Plant Evolution

Gene duplication is a major mechanism to create new genes. After gene duplication, some duplicated genes undergo functionalization, whereas others largely maintain redundant functions. Duplicated genes comprise various degrees of functional diversification in plants. However, the evolutionary fate of high and low diversified duplicates is unclear at genomic scale. To infer high and low diversified duplicates in Arabidopsis thaliana genome, we generated a prediction method for predicting whether a pair of duplicate genes was subjected to high or low diversification based on the phenotypes of knock-out mutants. Among 4,017 pairs of recently duplicated A. thaliana genes, 1,052 and 600 are high and low diversified duplicate pairs, respectively. The predictions were validated based on the phenotypes of generated knock-down transgenic plants. We determined that the high diversified duplicates resulting from tandem duplications tend to have lineage-specific functions, whereas the low diversified duplicates produced by whole-genome duplications are related to essential signaling pathways. To assess the evolutionary impact of high and low diversified duplicates in closely related species, we compared the retention rates and selection pressures on the orthologs of A. thaliana duplicates in two closely related species. Interestingly, high diversified duplicates resulting from tandem duplications tend to be retained in multiple lineages under positive selection. Low diversified duplicates by whole-genome duplications tend to be retained in multiple lineages under purifying selection. Taken together, the functional diversities determined by different duplication mechanisms had distinct effects on plant evolution.     

4种模式植物LRR VIII-2亚家族基因的鉴定和进化历史分析

全基因组重复与串联重复是发生基因重复的重要机制, 也是基因组和遗传系统多样化的重要动力。LRR-RLK编码富含亮氨酸重复的类受体蛋白激酶, 是被子植物进化史上发生大规模扩张而形成的多基因家族。拟南芥(Arabidopsis thaliana) AtLRR-RLK包含15个亚家族, AtLRR VIII-2是其中发生串联重复比例最高的亚家族。通过分析拟南芥、杨树(Populus trichocarpa)、葡萄(Vitis vinifera)和番木瓜(Carica papaya) 4种模式植物中LRR VIII-2亚家族基因的扩张及差异保留情况, 结果显示, LRR VIII-2在杨树中的扩张程度最高, 在拟南芥和葡萄中的扩张程度居中, 但在番木瓜中发生丢失。拟南芥、杨树和葡萄LRR VIII-2亚家族具有旁系同源基因对, 但在番木瓜中未发现旁系同源基因。除杨树中的1对旁系同源基因外, 4种模式植物中LRR VIII-2亚家族的旁系和直系同源基因都受到较强的纯化选择作用。对LRR VIII-2亚家族进化历史的深入分析有助于理解基因重复在植物进化中的作用和意义, 可为预测同源基因功能及解析其它基因家族进化历史提供参考。     

Duplicated genes evolve slower than singletons despite the initial rate increase

Background

Gene duplication is an important mechanism that can lead to the emergence of new functions during evolution. The impact of duplication on the mode of gene evolution has been the subject of several theoretical and empirical comparative-genomic studies. It has been shown that, shortly after the duplication, genes seem to experience a considerable relaxation of purifying selection.

Results

Here we demonstrate two opposite effects of gene duplication on evolutionary rates. Sequence comparisons between paralogs show that, in accord with previous observations, a substantial acceleration in the evolution of paralogs occurs after duplication, presumably due to relaxation of purifying selection. The effect of gene duplication on evolutionary rate was also assessed by sequence comparison between orthologs that have paralogs (duplicates) and those that do not (singletons). It is shown that, in eukaryotes, duplicates, on average, evolve significantly slower than singletons. Eukaryotic ortholog evolutionary rates for duplicates are also negatively correlated with the number of paralogs per gene and the strength of selection between paralogs. A tally of annotated gene functions shows that duplicates tend to be enriched for proteins with known functions, particularly those involved in signaling and related cellular processes; by contrast, singletons include an over-abundance of poorly characterized proteins.

Conclusions

These results suggest that whether or not a gene duplicate is retained by selection depends critically on the pre-existing functional utility of the protein encoded by the ancestral singleton. Duplicates of genes of a higher biological import, which are subject to strong functional constraints on the sequence, are retained relatively more often. Thus, the evolutionary trajectory of duplicated genes appears to be determined by two opposing trends, namely, the post-duplication rate acceleration and the generally slow evolutionary rate owing to the high level of functional constraints.

     

Expression of a cytochrome P450 gene family in maize

Maize seedlings, like seedlings of many other plants, are rich in cytochrome P450 (P450) enzyme activity. Four P450 genes (CYPzm1–4), isolated from a seedling-specific cDNA library, are characterised by a transient and seedling-specific expression pattern. The maximum steady state mRNA levels are reached at 3 days in root and at 7 days in shoot tissue, respectively. All four genes belong to one gene family and are closely related to the CYP71 family of plant P450 genes, which includes the enzymes of the ripening avocado fruit (CYP71A1) and eggplant hypocotyls (CYP71A2, A3, A4). The expression of these related P450 genes in monocot and dicot plants indicates that these enzymes play a significant role in plants; however, the in vivo enzyme functions are unknown. The divergence of the four members of the maize gene family is sufficiently high to account for different substrate and/or reaction specificity. Although the general expression pattern of the four genes is identical, the maximum steady-state mRNA levels vary in different maize lines. In situ hybridisation reveals the highest mRNA levels in the coleoptile, the first developed leaflets, the ground tissue of the nodular complex, and in the cortex and pith of the region of cell division in the root. The mapping of the maize CYPzm genes shows that, as in animals, P450 genes of the same family can be clustered. The presence of the CYPzm gene cluster in maize argues for generation of distinct plant P450 gene families by gene duplication.     

The evolutionary fate of recently duplicated retrogenes in mice

Inferences about the evolutionary impact of gene duplications often rely on the analysis of their long-term outcome. The fate of the majority of them must, however, be decided shortly after duplication. Here we analysed the evolutionary pattern of 10 mouse genes very recently duplicated by retrotransposition, by sequencing the retroposed copy in five to 10 closely related mouse species. In all cases the retroposed copy experienced accelerated nonsynonymous evolution whereas the divergence pattern of the source copy appeared unaffected by the duplication, consistent with the neofunctionalization model. The analysis further revealed that most retrogenes, including pseudogenes, did not experience a period of relaxed neutral evolution, but have been submitted to purifying selection ever since their retroposition. We propose that these duplicates play a biochemical role but are not indispensable. Purifying selection prevents them from acquiring a negative role until they are lost or silenced. This period of unnecessary redundancy could in rare cases give the time for new functions to evolve.     

Evolutionary patterns of RNA-based duplication in non-mammalian chordates

The role of RNA-based duplication, or retroposition, in the evolution of new gene functions in mammals, plants, and Drosophila has been widely reported. However, little is known about RNA-based duplication in non-mammalian chordates. In this study, we screened ten non-mammalian chordate genomes for retrocopies and investigated their evolutionary patterns. We identified numerous retrocopies in these species. Examination of the age distribution of these retrocopies revealed no burst of young retrocopies in ancient chordate species. Upon comparing these non-mammalian chordate species to the mammalian species, we observed that a larger fraction of the non-mammalian retrocopies was under strong evolutionary constraints than mammalian retrocopies are, as evidenced by signals of purifying selection and expression profiles. For the Western clawed frog, Medaka, and Sea squirt, many retrogenes have evolved gonad and brain expression patterns, similar to what was observed in human. Testing of retrogene movement in the Medaka genome, where the nascent sex chrosomes have been well assembled, did not reveal any significant gene movement. Taken together, our analyses demonstrate that RNA-based duplication generates many functional genes and can make a significant contribution to the evolution of non-mammalian genomes.     

Human cytochromes P450

The cytochrome P450 proteins (CYPs) are a family of haem proteins resulting from expression of a gene super-family that currently contains around 1000 members in species ranging from bacteria through to plants and animals. In humans, about 40 different CYPs are present and these play critical roles by catalyzing reactions in: (a) the metabolism of drugs, environmental pollutants and other xenobiotics; (b) the biosynthesis of steroid hormones; (c) the oxidation of unsaturated fatty acids to intracellular messengers; and (d) the stereo- and regio-specific metabolism of fat-soluble vitamins. This review deals with aspects of cytochrome P450s of relevance to human physiology, biochemistry, pharmacology and medicine. Topics reviewed include: pharmacogenetics of CYPs, induction and inhibition of these haem proteins, their role in metabolism of endogenous compounds such as steroids and eicosanoids, the effect of disease on CYP function, CYPs and cancer, and CYPs as targets of antibodies in immune-mediated diseases.