Conservation and diversity of gene families explored using the CODEHOP strategy in higher plants
© Morant et al; licensee BioMed Central Ltd. 2002
Received: 24 April 2002
Accepted: 1 August 2002
Published: 1 August 2002
Availability of genomewide information on an increasing but still limited number of plants offers the possibility of identifying orthologues, or related genes, in species with major economical impact and complex genomes. In this paper we exploit the recently described CODEHOP primer design and PCR strategy for targeted isolation of homologues in large gene families.
The method was tested with two different objectives. The first was to analyze the evolution of the CYP98 family of cytochrome P450 genes involved in 3-hydroxylation of phenolic compounds and lignification in a broad range of plant species. The second was to isolate an orthologue of the sorghum glucosyl transferase UGT85B1 and to determine the complexity of the UGT85 family in wheat. P450s of the CYP98 family or closely related sequences were found in all vascular plants. No related sequence was found in moss. Neither extensive duplication of the CYP98 genes nor an orthologue of UGT85B1 were found in wheat. The UGT85A subfamily was however found to be highly variable in wheat.
Our data are in agreement with the implication of CYP98s in lignification and the evolution of 3-hydroxylation of lignin precursors with vascular plants. High conservation of the CYP98 family strongly argues in favour of an essential function in plant development. Conversely, high duplication and diversification of the UGT85A gene family in wheat suggests its involvement in adaptative response and provides a valuable pool of genes for biotechnological applications. This work demonstrates the high potential of the CODEHOP strategy for the exploration of large gene families in plants.
Plants have evolved extremely diversified gene families as tools to cope with a harsh environment. Some of these families such as cytochromes P450 and UDP-glycosyltransferases (UGT) reflect the extraordinary biochemical versatility of plants and across plant species, and represent a very valuable source of genes for biotechnologies. Both gene families offer a huge potential for bioremediation and control of crop and weed pesticide tolerance [1–3], but obviously also for industrial applications. P450s, considered as the most versatile catalysts known , usually activate dioxygen and transfer one of its atoms into various substrates, but also catalyze a great diversity of reactions ranging from C-C and C=N bond cleavage, phenolic coupling, dehydration, dehydrogenation, isomerizations to reduction . Many of these reactions are important for the biosynthesis of hormones, drugs, pigments, aromas, biopolymer building blocks and defense molecules [6, 7]. Glycosyltransferases are also essential for the production of natural compounds since they control their solubility, stability, transport, storage and sometimes also their bioactivity [8, 9]. Should some of this potential become directly accessible through genomewide sequencing, extensive information is restricted to model plants, usually with a small genome, or to plants with a major economical interest. Exploitation of this knowledge to target genes of other plants that need to be studied or engineered, or to explore gene families in plants with specific biosynthetic capacities is an objective for the next several years.
With the growing availability of gene sequences plus information regarding their diversity and phylogeny, increasingly sophisticated PCR techniques have been developed to target gene families. Plant P450s are low abundant membrane-bound and unstable proteins, usually difficult to purify. For this reason, early on, several groups attempted isolation of P450 genes on the basis of the most conserved consensus regions, after generating probes by conventional PCR at low stringency [10–13]. This approach was later refined and used by several other groups for isolation of P450 genes in various plant species [e.g. [14–18]]. It proved successful in many cases, although only leading to a small number of highly expressed and related P450 families. A significant step forward resulted from coupling degenerate PCR with a heme binding primer and differential display of the amplified fragments, an approach that allowed effective identification of nine P450 genes responsive to elicitor treatment of soybean cell cultures  and 21 unique P450 genes in Taxus cells induced for taxol production . A carefully controlled and strongly differential system is however needed for such an approach. Another interesting improvement was recently reported that involves use of nested primers to increase PCR selectivity . However, the major limitation of all the strategies reported so far is that they did not take into account the huge diversity and low conservation of P450s recently revealed by genome sequencing in higher plants, and allowed neither focused gene selection nor isolation of the most divergent P450 clades, i.e. no systematic exploration of the P450 superfamily in highly divergent species.
In this paper we report on the high potential of the recently described COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOP) strategy  of primer design, ensuring optimal match and PCR amplification focused on very short conserved sequences, for the isolation of orthologues in evolutionarily distant species and for the focused or systematic exploration of gene families in plants with a very large genome. The method was tested to analyze both the duplication and conservation of the CYP98 family of P450 genes in many plant species. This family was recently suggested to play an essential role in lignification and plant development [23–25]. The same approach was also used for the analysis of the UGT85 family in wheat.
Chasing the CYP98 genes in wheat
The CYP98 family of cytochrome P450 genes encodes the 3'-hydroxylases of coumaroyl esters, which catalyze an essential step in the synthesis of lignin monomers and chlorogenic acid [23–25]. CYP98 activity is also needed for the biosynthesis of many phenolic flavouring compounds such as eugenol, safrole or vanilin. Engineering the expression of this family of P450s has important agro-industrial applications, including enhancement of plant defense and modification of lignin composition to improve forage digestibility and wood pulping [26, 27]. Access to CYP98 genes from major crop and forage plants and most common woody species is a necessary step for modifying their expression. If some partial sequences are made available by EST sequencing of a limited number of species, they do not reflect the whole range of gene isoforms expressed in a plant or plant tissue, especially for large genome plants.
CODEHOP primers used for CYP98s amplification. For each primer the consensus clamp (XXX.XXX) is given in upper case and degenerate core (xxx.xxx) in lower case. y = [C, T], n = [A, G, C, T], s = [G, C], r = [A, G]. BLASTp analysis was performed with the consensus protein sequence corresponding to each primer. Hits: best scoring P450, with expectation values lower than 0.1, are listed in the order of scores.
BLASTp hits on P450 database (1)
Expected size of PCR product (bp)
The CODEHOP strategy thus appears well suited for the very focused to broader exploration of gene families in plants with a large genome, depending on primer selectivity. Selectivity of each set of primers can be predicted by a BLAST analysis. In young wheat seedlings, focused CODEHOP screening allowed the detection of three clearly different CYP98 genes. All three genes are apparently related to A. thaliana CYP98A3.
Isolation of CYP98 ortho/homologues in other plant species
The second step of this investigation was aimed at testing the possibility to isolate CYP98A3 orthologues in a broad range of distantly related species. The most selective primer pair P98c/P98cr, without modification for codon usage, was first assayed with the same amplification programme or after shifting the initial annealing temperature from 70 to 65°C using various cDNA libraries, prepared from Capsicum annuum fruit (Solanaceae), Ceratopteris richardii (fern), Coleus blumei cell culture (Lamiaceae), Eucalyptus globulus xylem (Myrtaceae), Helianthus annuus stem and leaf (Asteraceae), Lycopersicon esculentum shoots (Solanaceae), Picea abies cell culture (Coniferales), Pinus pinaster stem and root (Coniferales), Populus trichocarpa x deltoides stem, root and leaf (Salicaceae), Physcomitrella patens protonemal tissue (moss). CYP98-related sequences were amplified from the libraries of C. blumei, E. globulus, H. annuus, P. abies, and Populus. After optimization of primer codon usage, a CYP98-like fragment was also amplified from the C. richardii cDNA library. No amplicon was obtained however for P. patens, P. pinaster or the Solanaceae, neither after optimizing codon usage, nor after further decreasing the initial temperature of the touch-down PCR.
Limits of the method
To find an explanation, EST and cDNA sequences now available for pine and tomato, another Solanaceae, were examined and aligned with our primers and consensus sequences. This comparison to the authentic sequences revealed significant divergences from most frequent codon usage in addition to the selected consensus sequence. The assumption that the clamp segment of the primers has a very minor impact on the amplification  thus probably should be reconsidered. The dissimilarity present in this example is however likely to be local and thus successful amplification should be obtained by using multiple primers distributed along the full sequence.
Exploration of another gene family in wheat: UGT85
CODEHOP primers used for UGTs amplification. y = [C, T], n = [A, G, C, T], r = [A, G], w = [A, T], k = [G, T]. BLAST analysis of protein consensus sequences corresponding to each primer was performed as indicated in the legend of Table 1.
BLASTp hits on arabidopsis UGT database
In this paper we investigated the potential of the CODEHOP strategy for targeted isolation of genes from organisms not yet submitted to extensive sequencing and for exploration of the complexity of selected gene families in large plant genomes. Special emphasis was given to wheat as representative of plants with a complex genome. The CODEHOP method proved extremely useful for the characterization of gene orthologues or homologues in a broad range of plants species. Focus of the gene search can be controlled by changing the degree of specificity of the primers (easily checked by a BLAST analysis) and by the choice of touch-down PCR temperature. In our hands, the method was successful where cDNA library screening with heterologous probes (e.g. cDNAs from maize for screening wheat libraries) had failed. The main advantage of this method is to rapidly provide a representative sample of either the allelic variants and recently evolved paralogues, or of the homologues of a given gene in complex genomes. Compared to other methods, a very high proportion of useful sequences (more than 90% with some primer couples) is obtained. Besides targeted search for specific expressed sequences, it allows complete exploration of the different P450 clades in a single organism, which was not possible using previously described methods. The main source of failure seems to be local sequence or codon divergence from the consensus or most frequent usage. This problem should be easily circumvented by using several primer couples chosen from different regions of the gene. Using the CODEHOP strategy on subgroups of phylogenetically related genes, families or clades, within large superfamilies such as UGTs or P450s is a powerful approach for exploring their complexity in various genomes. It is a very effective tool for the construction of expression libraries for agrochemical and other industrial applications. It can also be used for identifying genes from a given subgroup expressed at a specific stage of development.
Some plant P450 families result from extensive duplications, some of them forming clusters of up to 13 genes in Arabidopsis. Some P450 families or subfamilies are found in only subsets of plant species. Extensive search for CYP98 genes expressed in wheat seedlings did not reveal more than 3 different sequences, all related to A. thaliana CYP98A3. The bread wheat genome is hexaploid and results of successive hybridizations and rearrangements . This suggests that the three CYP98A10, A11 and A12 genes may correspond each to one of the three wheat genomes, with CYP98A12 which is the most divergent possibly resulting from the most recent genome introduction. This hypothesis is currently investigated. In agreement with the low number of CYP98s found expressed in wheat, no extensive duplication of CYP98 was detected in other plant species. This observation, together with the high conservation of the CYP98As gene across evolution argue for essential functions of the CYP98 genes in higher plant development. Accordingly, strongly impaired growth and fertility are observed in cyp98A3 mutants [, S. Goepfert, personal communication]. Similar conservation is observed for other P450 genes participating in the early phenylpropanoid pathway and hormone homeostasis.
No obvious homologue of the sbHMNGT gene was found in wheat. This may be connected to the fact that large amounts of dhurrin are not reported to accumulate in wheat, since sbHMNGT is described as showing a strong preference for mandelonitrile substrates . A large number of related genes, all belonging to the UGT85A subfamily were however detected. This is not surprising considering that 6 UGT85A genes have been reported in the small Arabidopsis genome . Such a duplication of genes in this family probably reflects an adaptative evolution and their implication in some type of stress/defense response rather than a developmentally essential function. Significantly, a large number of UGT85A-related sequences are found among wheat ESTs isolated from wheat challenged with pathogens. Recombinant expression of such genes should provide a valuable library for pharmacological and toxicological investigations, and for studying evolutionary ecology of plant pathogen interactions.
The CODEHOP strategy appears as a powerful method for exploring the complexity of gene families in plants with a large genome, and conservation of genes across evolution. CYP98s are genes evolved early and are highly conserved during evolution, as expected for genes with an essential role in homeostasis and development of vascular plants. Conversely, the UGT85s are more variable. No orthologue of the sorghum UGT85B1 gene was detected in wheat, while UGT85As, found in many plants species, are also present in wheat. The great variability of this subfamily in wheat strongly suggests a role in environmental adaptation and plant defense.
Wheat cDNA libraries were constructed from poly(A)+ mRNA from 3–5 mm Triticum aestivum (L. cv. Darius) seedlings, both control or pre-coated with cloquintocet-mexyl (0.1% seed dry weight) and phenobarbital, as described previously , in λ ZipLox (GIBCOBRL) by T. Thomas (College Station, Texas). Other cDNA libraries were kindly provided by B. Camara, IBMP Strasbourg (Capsicum annuum), W. Faigl, MPI Cologne (Ceratopteris richardii), M. Petersen, Philipps Universität Marburg (Coleus blumei), J. Grimma-Pettenati, CNRS Toulouse (Eucalyptus globulus), J. L. Evrard, IBMP Strasbourg (Helianthus annuus), A. Schaller, ETH Zurich (Lycopersicon esculentum), D. Ernst, GSF Munich (Picea abies), J. M. Frigerio, INRA Gazinet (Pinus pinaster), C. Douglas, University of British Columbia Vancouver (Populus trichocarpa x deltoides), and Leeds University (Physcomitrella patens).
CODEHOP primers are designed as to ensure a very high probability of annealing on the gene of interest with 11–12 completely degenerate core nucleotides at their 3'-end and efficient amplification with a consensus 18–25 nucleotide clamp sequence at the 5'-end. Design of the clamp is based on an alignment of available related sequences and codon usage of the target organism. Primers completely degenerate at their 3'-end and ensuring high probability of annealing to CYP98s or UGTs specific sequences were designed using the CODEHOP strategy  based on the multiply-aligned sequences: AF029856, AF022458, AA86449, C74921, D47937, AI881302, AI734373, AAG52369, AAG52373 for the P450 family CYP98, and AAF17077, AAF18537, BAA34687 for UGTs. The multiple alignment was first generated using ClustalW , then cut into blocks using the BlockMaker server . Primers were designed using the default parameters of the CODEHOP server . It was assumed that barley codon usage proposed by the server was close enough to that of wheat to obtain effective primer design.
From the primer solutions proposed by the server, five P450 primers were selected, which both provided the largest PCR fragments and avoided the most conserved consensus regions common to a large number of P450s. In the case of UGTs, a broader choice was offered by the server, thus 5 sense and 5 reverse primers could be selected on the same basis as for P450s.
Probe amplification and analysis
In the present work, the CODEHOP approach was not used with genomic DNA but with cDNA libraries, our main objective being to identify genes expressed in specific plant tissues. Screening can be directly performed on cDNA libraries but better results were obtained after preliminary extraction of cDNA from phages. For extraction, an aliquot of the cDNA library was heated 10 min at 70°C, then extracted with one volume of phenol-chloroform. PCR screening was then performed on 50 ng of this template using 2.6 U of HiFi (Expand High Fidelity, Roche), 1.5 mM Mg2+, 0.3 mM dNTP and 0.5 μM primers in the polymerase manufacturer's buffer. The PCR program was designed according to the CODEHOP server's tips, including successively a touch down (A) and a classical (B) PCR as follows: first 3 min initial denaturation at 94°C, then (A) 20 cycles of 1 min at 94°C, 2 min at 70°C (-1°C/cycle), and 2 min at 72°C, then (B) 20 cycles of 1 min at 94°C, 2 min at 58°C, and 2 min at 72°C, and finally a 5 min extension. Elongation time was adapted to the largest expected fragment in each screening. When no amplified fragment was obtained with a primer pair, the touch-down starting temperature of annealing was decreased by 5°C until a successful amplification was achieved.
Analysis and cloning of PCR products
PCR products were analyzed on 1% agarose gels, and fragments of expected size were eluted by centrifugation on Ultrafree-DA columns (Millipore), precipitated and cloned into the pGEM-T vector (Promega). E. Coli XL1blue (Stratagene) was electroporated with 1/10 of the ligation volume. Inserts from five white colonies were sequenced.
Sequences were analyzed by BLASTx against a local database generated from the data found [33, 38]. Non-P450 sequences were xblasted on the NCBI site. The CYP98A10, CYP98A11 and CYP98A12 names were assigned on the basis of the full-length coding sequences subsequently isolated from the cDNA library.
The support of Aventis Crops Science and of the Association Nationale de la Recherche Technique to M. M. is gratefully acknowledged. We thank D. Little for critical reading of the manuscript and B. Camara, W. Faigl, M. Petersen, J. Grimma-Pettenati, J. L. Evrard, A. Schaller, D. Ernst, J. M. Frigerio, C. Douglas and Leeds University for providing cDNA libraries.
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