cinnamoyl-CoA Reductase Gene Research Articles

The effect of light on gene expression and podophyllotoxin biosynthesis in Linum album cell culture

Podophyllotoxin (PTOX) is a naturally occurring phenolic compound isolated as an active anti-tumor agent. The stimulatory influence of light on the formation of phenolic compounds has been reported, but the molecular mechanism underlying the effect of light on the expression of genes involved in phenolic biosynthesis, especially of lignans, is still not fully understood. A series of experiments was carried out using ordinary fluorescent lamps to study the influence of light irradiation on growth and PTOX accumulation in Linum album cell cultures by varying the type of light and periods of exposure. The biosynthesis of PTOX was variably affected according to the quality of light. The enhancing effects of red light on PTOX production was correlated with increased activities of the enzyme phenylalanine ammonia-lyase (PAL), and the expression of some key genes involved in the biosynthesis of this compound, including the PAL gene itself and the cinnamoyl-CoA reductase (CCR) gene. Blue light was found to have similar effects but mainly on the expression level of CCR and pinoresinol lariciresinol reductase (PLR) genes.

Characterization of a cinnamoyl-CoA reductase gene in Ginkgo biloba: Effects on lignification and environmental stresses

Cinnamoyl-CoA reductase (CCR, EC 1.2.1.44) catalyzes key steps in the biosynthesis of monolignols, which serve as building blocks in the formation of plant lignin. The full-length cDNA of GbCCR is 1178 bp and contains a 972 bp open reading frame (ORF) encoding a 323 amino acid protein. The deduced GbCCR protein showed high identities with other plant CCRs, and had closer relationship with Picea abies , sharing 56.3% homology. They both contain a common signature which is thought to be involved in the catalytic site of CCR. Phylogenetic tree analysis revealed that GbCCR shared the same ancestor with other CCRs, but the divergence time is early. Southern blot analysis indicated that GbCCR belonged to a multi-gene family. The expression analysis by quantitative real-time polymerase chain reaction (QRT-PCR showed that GbCCR was seen in a tissue specific manner in Ginkgo biloba ; it had the highest expression in injured stems, and a high expression in four years old stems, while it had the lowest in endosperm. GbCCR was also found to be significantly up-regulated by gibberellin (GA), but the expression was weakly induced by Agrobacterium treatment. QRT-PCR analysis showed that GbCCR activity correlated with changes in transcription level of the GbCCR gene, and GbCCR activity was also positively correlated with total lignin accumulation in developments of Ginkgo stem. In light of these properties and expression pattern, we suggested that the corresponding enzyme is probably involved in constitutive lignification and defense. Key words : Ginkgo biloba L., GbCCR, gene expression, lignification, defense.

Gene overexpression and gene silencing in Birch using an Agrobacterium-mediated transient expression system

As transient expression systems are effective methods for the functional characterization of genes, a transient gene expression and silencing system was developed for Betula platyphylla Suk (Chinese Birch). Firstly, the cinnamoyl-CoA reductase (CCR) gene and its promoter were isolated from Chinese Birch. The vectors for overexpression of CCR and RNAi-based silence of CCR were constructed and transformed into Agrobacterium, respectively. Overexpression and silence of the CCR gene were respectively, performed on Birch seedlings using an Agrobacterium-mediated transient expression system. The expression levels of CCR were determined using real-time PCR. The results showed that the transcripts of CCR notably increased in the Birch plants transformed with the CCR overexpression construct, and notably decreased in plants transformed with the silencing construct when compared with nontransgenic plants. These studies confirmed that this transient genetic transformation system works well on Birch plants, and can be used for the functional characterization of genes and protein production in Birch.

Comparative and phylogenomic analyses of cinnamoyl-CoA reductase and cinnamoyl-CoA-reductase-like gene family in land plants

The biosynthesis of monolignols, the main components of lignin, involves many intermediates and enzymes. The cinnamoyl-CoA reductase (CCR) enzyme catalyzes the conversion of cinnamoyl-CoAs to cinnamaldehydes, i.e. the first specific step in lignin synthesis. The CCR and CCR-like gene family was studied partially in several plant species. This is a comprehensive study of the CCR and CCR-like gene family including genome organization, gene structure, phylogeny across land plant species, and, expression profiling in Populus. Analysis of amino acid motifs enabled the identification of sequence variations in the CCR catalytic site and annotates CCR and CCR-like genes. CCR and CCR-like genes were distributed in three major phylogenetic classes of which one includes the bona fide CCR genes. The other two classes include CCR and CCR-like, of which several genes present a high similarity to cinnamyl alcohol dehydrogenase, or dihydroflavonol reductase ( DFR) genes. All CCR, CCR-like, and DFR classes were deeply rooted in the phylogeny of land plants suggesting that their evolution preceded the evolution of lycophytes. Over two thirds of CCR and CCR-like Populus genes were physically distributed on duplicated regions. This suggests that these duplication/retention processes contributed significantly to the size of the CCR and CCR-like gene family. The Populus CCR and CCR-like genes showed six expression patterns in the tissues studied with a preferential expression of PoptrCCR12 in xylem. The other genes present divergent expression profiles with some preferentially expressed in leaves, bark, or both. Several CCR and CCR-like genes were induced or repressed under various abiotic stresses suggesting that their duplication was followed by the evolution of divergent expression profiles and divergence of functions.

TaMYB4 cloned from wheat regulates lignin biosynthesis through negatively controlling the transcripts of both cinnamyl alcohol dehydrogenase and cinnamoyl-CoA reductase genes

The subgroup 4 of R2R3-MYB transcription factors has been proposed as repressors regulating the phenylpropanoid pathway. Here, we report a cDNA encoding a subgroup 4 R2R3-MYB factor from wheat, designated as TaMYB4. A phylogenetic analysis showed that TaMYB4 is in a subclade that is specific to monocot plants. The TaMYB4 gene was highly expressed in stem and root tissues. In vitro binding analysis in yeast cells showed TaMYB4 interacted with OsCAD2 promoter characterized by an AC-II element that has been considered as the MYB-binding site in lignin biosynthetic genes. The overexpression of TaMYB4 in transgenic tobacco led to transcriptional reduction of both cinnamyl alcohol dehydrogenase (CAD) and cinnamoyl-CoA reductase (CCR) genes involved in the lignin biosynthesis. Enzymatic assay showed reduction of CAD and CCR activities in the transgenic tobacco plants that substantially decreased the levels of total lignin but increased it’s ratio of S/ G. In addition, the total flavonoid content was increased in transgenic tobacco leaves, suggesting that the overexpression of TaMYB4 likely led to a redirection of the metabolic flux from the lignin pathway to the flavonoid pathway. These data suggest that TaMYB4 negatively regulates the lignin biosynthesis in wheat.

Molecular Diversity Among Members of the Saccharum Complex Assessed Using TRAP Markers Based on Lignin-Related Genes

In addition to the cultivation of sugarcane for sugar, the crop is considered seriously as an important bioenergy grass crop for its high biomass production ability. But, lignin is a serious bottleneck in the bioconversion of lignocellulosic biomass to ethanol. Hence, genetic relationships among 64 genotypes within the Saccharum complex were studied with respect to lignin-related genes using target region amplified polymorphic (TRAP) primers derived from caffeic acid O-methyltransferase (COMT), cinnamoyl alcohol dehydrogenase (CAD), cinnamoyl coA reductase (CCR), and ferrulate 5-hydroxylase (F5H) genes. While the average polymorphism detected by the TRAP markers was 43%, the markers derived from F5H gene (34%) were less polymorphic in comparison to those derived from COMT (46%), CCR (44%), and CAD (46%) genes. The lignin gene-based TRAP markers differentiated members of the Saccharum complex broadly according to previously established genetic relationships in the order of Miscanthus > Erianthus > Saccharum spontaneum > Saccharum robustum/Saccharum barberi/Saccharum sinense > Saccharum officinarum/cultivars. Principal coordinate analysis showed that 29% of the total variation was explained by the genotypes with respect to the lignin-related genes. The association of genetic variation revealed in this study with the biomass composition-related genes of the genotypes within a species will be helpful to design breeding strategies to develop superior energy cane cultivars with improved biomass quality of the sugarcane.

The Simultaneous Repression of CCR and CAD, Two Enzymes of the Lignin Biosynthetic Pathway, Results in Sterility and Dwarfism in Arabidopsis thaliana

Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the last steps of monolignol biosynthesis. In Arabidopsis, one CCR gene (CCR1, At1g15950) and two CAD genes (CAD C At3g19450 and CAD D At4g34230) are involved in this pathway. A triple cad c cad d ccr1 mutant, named ccc, was obtained. This mutant displays a severe dwarf phenotype and male sterility. The lignin content in ccc mature stems is reduced to 50% of the wild-type level. In addition, stem lignin structure is severely affected, as shown by the dramatic enrichment in resistant inter-unit bonds and incorporation into the polymer of monolignol precursors such as coniferaldehyde, sinapaldehyde, and ferulic acid. Male sterility is due to the lack of lignification in the anther endothecium, which causes the failure of anther dehiscence and of pollen release. The ccc hypolignified stems accumulate higher amounts of flavonol glycosides, sinapoyl malate and feruloyl malate, which suggests a redirection of the phenolic pathway. Therefore, the absence of CAD and CCR, key enzymes of the monolignol pathway, has more severe consequences on the phenotype than the individual absence of each of them. Induction of another CCR (CCR2, At1g80820) and another CAD (CAD1, At4g39330) does not compensate the absence of the main CCR and CAD activities. This lack of CCR and CAD activities not only impacts lignification, but also severely affects the development of the plants. These consequences must be carefully considered when trying to reduce the lignin content of plants in order to facilitate the lignocellulose-to-bioethanol conversion process.

Expression analysis of cinnamoyl-CoA reductase (CCR) gene in developing seedlings of Leucaena leucocephala: A pulp yielding tree species

Removal of lignin is a major hurdle for obtaining good quality pulp. Leucaena leucocephala (subabul) is extensively used in paper industry in India; therefore, as a first step to generate transgenic plants with low lignin content, cDNA and genomic clones of CCR gene were isolated and characterized. The cDNA encoding CCR (EC 1.2.1.44) was designated as Ll-CCR; the sequence analysis revealed an Open Reading Frame (ORF) of 1005bp. Phylogenetic analysis showed that Ll-CCR sequence is highly homologous to CCRs from other dicot plants. The 2992bp genomic clone of Leucaena CCR consists of 5 exons and 4 introns. The haploid genome of L. leucocephala contains two copies as revealed by DNA blot hybridization. Ll-CCR gene was over-expressed in Escherichia coli, which showed a molecular mass of approximately 38kDa. Protein blot analysis revealed that Ll-CCR protein is expressed at higher levels in root and in stem, but undetectable in leaf tissues. Expression of CCR gene in Leucaena increased up to 15d in case of roots and stem as revealed by QRT-PCR studies in 0-15d old seedlings. ELISA based studies of extractable CCR protein corroborated with QRT-PCR data. CCR protein was immuno-cytolocalized around xylem tissue. Lignin estimation and expression studies of 5, 10 and 15d old stem and root suggest that CCR expression correlates with quantity of lignin produced, which makes it a good target for antisense down regulation for producing designer species for paper industry.

Isolation and characterization of a gene encoding cinnamoyl-CoA reductase from Isatis indigotica Fort.

A novel cinnamoyl-CoA reductase gene, designated as Iiccr (GenBank Accession No. GQ872418) was cloned from Isatis indigotica Fort. The full-length cDNA of Iiccr was 1368 bp with an ORF of 1026 bp that putatively encoded a polypeptide of 341 amino acids, with a predicted molecular mass of 37.50 kDa. The deduced amino acid sequence of IiCCR shared high homology with other known CCRs. No intron was detected in the genomic sequence of Iiccr. Southern-blot analysis revealed that Iiccr was a high-copy gene and real-time quantitative PCR analysis indicated that Iiccr was constitutively expressed in roots, stems and leaves of I. indigotica, with the highest expression level in roots. The results from treatment experiments using different signaling components for plant defense responses including methyl jasmonate (MeJA), gibberellins (GA(3)), abscisic acid (ABA) and ultraviolet-B revealed that expression of IiCCR had a prominent diversity. The full-length of ORF was sub-cloned into prokaryotic expression vector pET32a(+), which was then transferred into E. coli BL21(DE3). The recombinant protein had high expression level in E. coli BL21(DE3) with IPTG induction. A 2.6 kb long promoter sequence was isolated and its putative regulatory elements and potential specific transcription factor binding sites were analyzed. This study will enable us to further understand the role of IiCCR in the synthesis of phenylpropanoid compounds in I. indigotica Fort. at the molecular level.

Isolation of a cinnamoyl CoA reductase gene involved in formation of stone cells in pear (Pyrus pyrifolia)

Stone cell which appears in most pear cultivars contributes badly to pear internal quality. Lignin is the important component of stone cells and its first biosynthetic but specific step is catalyzed by cinnamoyl CoA reductase (CCR). In the present study, a cDNA encoding CCR, named as PpCCR (GenBank accession no. GU138672), was isolated from pear (Pyrus pyrifolia) fruit. Its full length is 1,247 bp with a putative maximal open reading frame of 1,017 bp, encoding a protein of 339 amino acids. Sequence alignment revealed that the deduced protein contained a typical CCR catalytic site and NADP(H) binding site. Phylogenetic analysis showed that PpCCR belonged to dicot CCR class II in which most CCRs participated in the regulation of lignin biosynthesis. PpCCR transcript could be detected in leaves, flowers and fruits. In addition, PpCCR expression profiles during fruit development were compared between two cultivars with significant difference in stone cell content in the flesh. Although PpCCR expressed in a similar pattern between the two cultivars and their transcript level consisted strictly with the change of lignin content, its transcript level was always higher in the high-lignin content cultivar than in the low-lignin content cultivar during fruit development. Therefore, it could be suggested that PpCCR is involved in the regulation of lignin biosynthesis and plays an important role in stone cell formation in pear flesh.

Switchgrass (Panicum virgatum) possesses a divergent family of cinnamoyl CoA reductases with distinct biochemical properties

The down-regulation of enzymes of the monolignol pathway results in reduced recalcitrance of biomass for lignocellulosic ethanol production. Cinnamoyl CoA reductase (CCR) catalyzes the first step of the phenylpropanoid pathway specifically dedicated to monolignol biosynthesis. However, plants contain multiple CCR-like genes, complicating the selection of lignin-specific targets. This study was undertaken to understand the complexity of the CCR gene family in tetraploid switchgrass (Panicum virgatum) and to determine the biochemical properties of the encoded proteins. Four switchgrass cDNAs (most with multiple variants) encoding putative CCRs were identified by phylogenetic analysis, heterologously expressed in Escherichia coli, and the corresponding enzymes were characterized biochemically. Two cDNAs, PvCCR1 and PvCCR2, encoded enzymes with CCR activity. They are phylogenetically distinct, differentially expressed, and the corresponding enzymes exhibited different biochemical properties with regard to substrate preference. PvCCR1 has higher specific activity and prefers feruloyl CoA as substrate, whereas PvCCR2 prefers caffeoyl and 4-coumaroyl CoAs. Allelic variants of each cDNA were detected, but the two most diverse variants of PvCCR1 encoded enzymes with similar catalytic activity. Based on its properties and expression pattern, PvCCR1 is probably associated with lignin biosynthesis during plant development (and is therefore a target for the engineering of improved biomass), whereas PvCCR2 may function in defense.

Isolation and Characterization of Cinnamoyl-CoA Reductase Gene from Panax ginseng C. A. Meyer

Cinnamoyl-CoA reductase (CCR, EC 1.2.1.44) catalyses the reduction of cinnamic acid CoA esters into their corresponding aldehydes, the first step of the phenylpropanoid pathway specially dedicated to monolignol biosynthesis. A cDNA clones encoding CCR have been isolated from Panax ginseng C.A. Meyer and its expression was investigated in response to abiotic stresses. The cDNA, designated PgCCR which is 865 nucleotides long and has an open reading frame of 590 bp with a deduced amino acid sequence of 176 residues. The PgCCR encoded protein possesses substantial homology with CCRs isolated and cloned from other sources; the highest identity (51.8%) was observed with CCR from Tomato (Lycopersicon esculentum). Under various stress conditions, expression patterns of the PgCCR were highly induced in adventitious and hairy roots by several abiotic stresses. These results indicated that PgCCR plays protective role against diverse environmental stresses.

Lignin biosynthesis in transgenic Norway spruce plants harboring an antisense construct for cinnamoyl CoA reductase (CCR)

An attractive objective in tree breeding is to reduce the content of lignin or alter its composition, in order to facilitate delignification in pulping. This has been achieved in transgenic angiosperm tree species. In this study we show for the first time that changes in lignin content and composition can be achieved in a conifer by taking a transgenic approach. Lignin content and composition have been altered in five-year-old transgenic plants of Norway spruce (Picea abies [L.] Karst) expressing the Norway spruce gene encoding cinnamoyl CoA reductase (CCR) in antisense orientation. The asCCR plants had a normal phenotype but smaller stem widths compared to the transformed control plants. The transcript abundance of the sense CCR gene was reduced up to 35% relative to the transformed control. The corresponding reduction in lignin content was up to 8%, which is at the lower limit of the 90-99% confidence intervals reported for natural variation. The contribution of H-lignin to the non-condensed fraction of lignin, as judged by thioacidolysis, was reduced up to 34%. The H-lignin content was strongly correlated with the total lignin content. Furthermore, the kappa number of small-scale Kraft pulps from one of the most down-regulated lines was reduced 3.5%. The transcript abundances of the various lignin biosynthetic genes were down-regulated indicating co-regulation of the biosynthetic pathway.

Polymorphisms in Cinnamoyl CoA Reductase (CCR) Are Associated With Variation in Microfibril Angle in Eucalyptus spp.

Linkage disequilibrium (LD) mapping using natural populations results in higher resolution of marker-trait associations compared to family-based quantitative trait locus (QTL) studies. Depending on the extent of LD, it is possible to identify alleles within candidate genes associated with a trait. Analysis of a natural mutant in Arabidopsis has shown that mutations in cinnamoyl CoA reductase (CCR), a key lignin gene, affect physical properties of the secondary cell wall such as stiffness and strength. Using this gene, we tested whether LD mapping could identify alleles associated with microfibril angle (MFA), a wood quality trait affecting stiffness and strength of wood. We identified 25 common single-nucleotide polymorphism (SNP) markers in the CCR gene in Eucalyptus nitens. Using single-marker and haplotype analyses in 290 trees from a E. nitens natural population, two haplotypes significantly associated with MFA were found. These results were confirmed in two full-sib families of E. nitens and Eucalyptus globulus. In an effort to understand the functional significance of the SNP markers, we sequenced the cDNA clones and identified an alternatively spliced variant from the significant haplotype region. This study demonstrates that LD mapping can be used to identify alleles associated with wood quality traits in natural populations of trees.

Isolation and characterization of a cinnamoyl-CoA reductase gene from perennial ryegrass ( Lolium perenne)

Cinnamoyl-CoA reductase (CCR, EC 1.2.1.44) catalyses the conversion of hydroxycinnamoyl-CoA thioesters to cinnamaldehydes at the entry point to the monolignol-specific branch of the lignin biosynthetic pathway. A gene encoding CCR in perennial ryegrass (LpCCR1) was isolated and fully sequenced. LpCCR1 contains five exons of similar sizes to other CCRs and four introns in conserved positions. The 5′ untranslated region contains motifs common to lignin biosynthetic genes. LpCCR1 is present as a single copy gene and maps to LG7 in perennial ryegrass. LpCCR1 is constitutively expressed, is stimulated by mechanical wounding, and transcript abundance in stems increases with maturity.

Characterization of cis-elements required for vascular expression of the cinnamoyl CoA reductase gene and for protein-DNA complex formation.

Cinnamoyl-CoA reductase (CCR) catalyses the first specific step in the biosynthesis of monolignols, the monomeric units of lignins. We examined the developmental regulation of the Eucalyptus gunnii EgCCR promoter by analysing the expression of EgCCR-GUS fusions in tobacco. EgCCR promoter activity was strongest in lignified organs (stems and roots) consistent with the EgCCR mRNA level in these organs. Histochemical analysis showed expression in vascular tissues (cambium, young differentiating xylem, ray cells, internal and external phloem) of stems and roots in agreement with in situ hybridization data. Promoter deletion analysis and gain-of-function experiments identified the sequences between positions -119 and -77 as necessary and sufficient for expression in vascular tissues of stems. Electrophoretic mobility-shift assays showed that this region is specifically recognized by nuclear proteins present in tobacco stems, giving rise to two retarded complexes, LMC1 and LMC2. Using overlapping EgCCR fragments and mutated oligonucleotides as competitors in gel-shift assays, two DNA-protein interaction sites were mapped. Finally, the role of protein-protein interactions in the formation of the LMC1 and LMC2 complexes was investigated using the detergent sodium deoxycholate, and protein fractionation onto a heparin Sepharose column.