Comparative and phylogenomic analyses of cinnamoyl-CoA reductase and cinnamoyl-CoA-reductase-like gene family in land plants
Highlights
► Characterization of motifs associated with CCR activity and annotation of hundreds CCR and ► CCR-like genes. ► The evolution of CCR, CCR-like, and DFR classes is the result of a duplication that happened prior to the evolution of lycophytes. ► Duplications as well as recombination contributed to the evolution of this gene family. ► Nucleotide variation at the catalytic site of CCR proteins contributed to the functional divergence of CCR and CCR-like genes. ► CCR and CCR-like genes evolved specialized expression profiles and different functions.
Introduction
Lignin is one of the most abundant biopolymers on earth after cellulose; it represents about 20–30% of the weight of wood [1]. Lignin plays major biological roles by maintaining the structure of plants and defending them against biotic and abiotic stresses. The biosynthesis of monolignols (p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol), which are the basic components of lignin, requires the action of several enzymes. One of these enzymes is cinnamoyl-CoA reductase (CCR; EC 1.2.1.44); it converts cinnamoyl-CoAs into their corresponding cinnamaldehydes. CCR catalyze the first specific step in lignin biosynthesis and as such it has been considered the main target for controlling lignin biosynthesis. Down-regulation of CCR and CCR-like genes resulted in several phenotypic and developmental abnormalities such as dwarfism, reduced number of seeds, small stem diameter, shorter floral stems, and limited growth [2], [3], [4], [5].
The CCR and CCR-like gene family has been partially studied in the plant models (Arabidopsis, Oryza, and Populus). These studies reported 11 Arabidopsis [6], eight Populus [7], and 26 Oryza genes [8]. Genes encoding CCR proteins have also been partially identified from several economic species such as Eucalyptus gunnii [9], Triticum aestivum [10], and Zea mays [4]. The highest number of CCR and CCR-like genes identified was from Oryza [8] and E. gunnii [11] where 26 genes were identified from each of these species. Since studies during the past decades have focused mainly on reducing the lignin amount in plants, most of the CCR and CCR-like genes identified so far are involved in monolignol biosynthesis [3], [12], [13], [14], [15], [16]. Previous studies [7], [10], [17], [18] showed that the CCR enzyme uses four cinnamoyl CoA esters (p-coumaryl-CoA, caffeoyl-CoA, feruloyl-CoA, 5-hydroxyferuloyl-CoA, and sinapoyl-CoA) as substrates. Some CCR genes have preference for certain substrates such as feruloyl-CoA. For instance, biochemical function analyses of Arabidopsis genes (AtCCR1 and AtCCR2), which were reported to be involved in lignin biosynthesis [18], showed that they preferentially use feruloyl-CoA and sinapoyl-CoA than p-coumaryl-CoA. Similarly, CCR from Eucalyptus [17] and Populus [7] showed similar affinities for all substrates but have a higher conversion rate for feruloyl-CoA [17] suggesting a preferential role in the biosynthesis of G-type lignin. However, the substrate specificity seems to be different between CCR enzymes from different species and also between iso-enzymes from the same species. For example, while wheat Ta-CCR1 showed a preference for feruloyl-CoA, Ta-CCR2 used feruloyl CoA, 5-OH- feruloyl CoA, sinapoyl CoA, and caffeoyl CoA with equal efficiency [10], [14]. Similarly, the Arabidopsis CCR enzymes (AtCCR1 and AtCCR2) showed a similar efficiency towards caffeoyl-CoA. However, AtCCR2 affinity and conversion efficiency of feruloyl-CoA and sinapoyl-CoA were approximately five times lower compared to AtCCR1 [18].
The large number of CCR and CCR-like genes in plants and the diversity of isoforms seem to be associated with the functional divergence of CCR and CCR-like genes [19]. CCR genes function through monolignol biosynthesis in plant development as well as in defense against pests and pathogens. It was suggested that only one to two genes (real CCR genes) were involved in lignin biosynthesis during development. For instance, functional analyses showed that AtCCR1 is the only real CCR gene in Arabidopsis [5]. The other genes may still function in monolignol biosynthesis and compensate for the loss of function of the real CCR gene. Indeed, AtCCR2, which was more expressed during plant defense and compensated for the AtCCR1 mutation during development [5]. The biological role of the few CCR and CCR-like genes in lignin biosynthesis was well studied using mutagenesis in several plant species. These studies showed that the down-regulation of real CCR and CCR-like genes resulted in reduced amounts of lignin and various phenotypic abnormalities [1], [5], [20], [21]. For example, the Arabidopsis CCR mutant Irx4 (irregular xylem 4) exhibited a defective secondary cell wall, reduced plant growth, dark green leaves, growth delay, and collapsed xylem vessels [21].
Previous studies suggested that lignin plays a major role in plant defense [22], [23], [24], [25]. The increase in lignin content reinforces the cell walls by surrounding a wound or infected area and stops the progression of the pathogens. It was also suggested that lignin increase in leaves makes the leaves difficult to chew [26]. Previous studies showed that CCR and CCR-like genes play a role in plant defense by increasing the biosynthesis of monolignols which are the basic component of the cell wall. For instance, Arabidopsis AtCCR2 was induced following the infection of plants with Xanthomonas campestris pv. campestris and may function in defense against plant pathogens [18], [19]. Studies on Oryza CCR and CCR-like genes showed that OsCCR1, which is induced by sphingolipid elicitor, is involved in defense against Xanthomonas oryza and blast Magnaporthe grisea infection [8], [27]. Other studies showed that CCR and CCR-like genes are involved in defense against UV light exposure [5].
Few phylogenetic analyses were reported on CCR and CCR-like genes [3], [10], [12], [28]. However, most of these analyses were done on a small subset of genes mainly from monocots and eudicots and did not provide a clear idea about the evolution of CCR and CCR-like genes. Another study [10] showed that CCR and CCR-like genes were divided into three phylogenetic groups represented by eudicots, monocots, and a third group including CCR and CCR-like, aldehyde reductase (AR), and dihydroflavonol reductase (DFR, EC 1.1.1.219) sequences. These gene families share common catalytic mechanisms and appear to belong to one super-family [10]. Similar phylogenetic distribution of CCR and CCR-like genes was published by other groups [3], [12]. On the contrary, Raes et al. showed that CCR and CCR-like genes grouped in several classes with several genes distributed separately from the main classes [28]. Similarly, Hamberger et al. [29] reported four subfamilies of CCR and CCR-like genes.
In this study, CCR and CCR-like sequences from a wide variety of plants were retrieved and annotated, making full use of the available plant genome sequences including non-seed plants like mosses and lycopods. This dataset was used to analyze the phylogeny and to get insight into the evolution of the CCR and CCR-like gene family. The organization, structure, and expression of CCR and CCR-like genes in Populus were also analyzed.
Section snippets
CCR sequences used in phylogenetic analysis
This study used sequences from model species representing various land plant lineages (Supplementary Table 1). Sequences were identified by querying genome sequences using the Arabidopsis CCR and CCR-like proteins. CCR sequences from Arabidopsis, and Oryza, were retrieved from TAIR [29], and TIGR [30]. Sequences from Populus (version 2), Selaginella moellendorffii, Sorghum bicolor, Chlamydomonas reinhardtii, and Physcomitrella patens were imported from the Joint Genome Institute [31]. CCR
Annotation of CCR and CCR-like genes
A query of various genome sequences using the Arabidopsis CCR proteins identified 364 sequences. After removing sequences that were short or not having the NAD(P) binding domain, 146 sequences covering at least 70% of the Arabidopsis CCR protein were left (Supplementary Table 1). Since DFR, CCR, and CCR-like genes present high sequence similarity, the first set of sequences identified using similarity included sequences from all these classes of genes. Sequence analysis using aa motifs involved
Discussion
Mining the proteomes of several model species with fully sequenced genomes resulted in the identification of 146 sequences. However, the annotation of these sequences was limited to bioinformatics prediction. This study analyzed the distribution of the CCR catalytic domain signature motif in CCR and CCR-like sequences retrieved by sequence similarity to Arabidopsis CCR proteins. This analysis enabled the identification of a large group of sequences corresponding to DFR genes suggesting that the
Acknowledgements
The authors thank The Joint Genome Institute for providing access to the genome sequence of several model species. The authors thank also K. Mohammed and P. Breen for their help with the intro-exon structure analysis. Many thanks to Dr. Dawn Luthe for access to the real time RT-PCR machine and to Wen-Po Chuang for her help in analyzing the RT-PCR results. This work was supported by The Schatz Center for Tree Molecular Genetics at Penn State.
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Present address: The Department of Bioenergy Science & Technology. Chonnam National University, Buk-Gu, Gwangju, 500-757, Republic of Korea.