JXB Advance Access originally published online on April 23, 2007
Journal of Experimental Botany 2007 58(8):2011-2021; doi:10.1093/jxb/erm064
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESEARCH PAPER |
Characterization of a cinnamoyl-CoA reductase that is associated with stem development in wheat
Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, 20 Nanxin Cun, Xiangshan, Beijing 100093, China
* To whom correspondence should be addressed. E-mail: mqh{at}ibcas.ac.cn
Received 21 November 2006; Revised 28 January 2007 Accepted 6 March 2007
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Cinnamoyl-CoA reductase (CCR) is responsible for the CoA ester to aldehyde conversion in monolignol biosynthesis, which diverts phenylpropanoid-derived metabolites into the biosynthesis of lignin. To gain a better understanding of lignin biosynthesis and its biological function, a cDNA encoding CCR was identified from wheat (Triticum aestivum L.), and designated as Ta-CCR1. Phylogenetic analysis indicated that Ta-CCR1 grouped together with other monocot CCR sequences while it diverged from Ta-CCR2. DNA gel-blot and mapping analyses demonstrated that Ta-CCR1 is present as a single copy gene in the wheat genome. Recombinant Ta-CCR1 protein converted feruloyl CoA, 5-OH-feruloyl CoA, sinapoyl CoA, and caffeoyl CoA, but feruloyl-CoA was the best substrate, suggesting the preferential biosynthesis of G-type lignin. RNA gel-blot analysis indicated that Ta-CCR1 was highly expressed in stem, with lower expression in leaves, and undetectable expression in roots. CCR enzyme activity was increased progressively along with the lignin biosynthesis and stem maturity. During stem development, Ta-CCR1 mRNA levels remained high at elongation, heading, and milky stages in the wheat H4564 cultivar, while they declined dramatically at the heading and milky stages in stems of the C6001 cultivar. Ta-CCR1 mRNA expression paralleled extractable CCR enzyme activity in these two cultivars. Furthermore, high Ta-CCR1 mRNA levels and high CCR enzyme activity in wheat stem were correlated with a higher Klason lignin content and greater stem mechanical strength in the H4564 cultivar. This suggests that Ta-CCR1 and its related CCR enzyme may be involved in the regulation of lignin biosynthesis during stem maturity and then contributes to stem strength support in wheat.
Key words: Cinnamoyl-CoA reductase, lignin biosynthesis, stem strength support, Triticum aestivum L
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Lignin is a phenolic cell wall polymer closely linked to cellulose and hemicelluloses, and is, second to cellulose, the most abundant biopolymer on earth. The presence of lignin is exclusively confined to vascular plants including pteridophytes, gymnosperms, and angiosperms. Polyphenolic material, which is not lignin but has similar functions to lignin, has been found in bryophytes. This suggests that the evolution of lignin was an important factor in the successful colonization of land by plants. In present day plants, lignin is mainly deposited in the walls of certain specialized cells such as tracheary elements, sclerenchyma, phloem fibres, and periderm. This leads to a dramatic change in the cell wall properties, which imparts rigidity and structural support to the wall and assists in the transport of water and nutrients within xylem tissue by decreasing the permeability of the cell wall. In addition, lignification of plant tissue also occurs in response to various environmental cues such as mechanical stress or pathogen attacks (Dixon, 2001).
In the past decade, cloning and characterization of genes involved in lignin biosynthesis and modification of lignin content and composition in plants have provided new insight into the lignin biosynthesis pathway. The majority of this work has been done in dicotyledonous angiosperms such as trees and forage crops (see review by Baucher et al., 2003, and references therein). Lignin is derived from the dehydrogenation and polymerization of three different hydroxycinnamyl alcohols (monolignols), namely 
-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol. They give rise to the -hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units of the lignin polymer, respectively. The three monolignols differ from each other only by their degree of methoxylation. Lignin composition is different among the different classes of plants. In general, the lignin of gymnosperms and pteridophytes consists mainly of guaiacyl units, whereas dicotyledonous angiosperm lignin is composed of both guaiacyl and syringyl units. Monocot lignin also contains very high -hydroxyphenyl subunits. Moreover, the lignin of monocots exhibits additional complexity since it contains ester- and ester-linked hydroxycinnamic acid, which can account for up 1% of dry matter (Humphreys and Chapple, 2002). To understand the biosynthesis and functions of lignin in monocots, detailed analyses need to be done to uncover any unique features of lignin in this special class of plants.
The biochemical pathways leading to the formation of lignin subunits (monolignols) consists of successive hydroxylation and O-methylation of the aromatic ring and conversion of the side-chain carboxyl to an alcohol function. The current model of the monolignol biosynthetic pathway is a metabolic grid leading to guaiacyl and syringyl subunits, in which the hydroxylation and O-methylation reactions may occur at different levels of side-chain oxidation. However, recent studies from biochemical and transgenic approaches suggest a new model for monolignol biosynthesis, in which different pathways may operate independently to form guaiacyl and syringyl subunits (Guo et al., 2001; Dixon et al., 2001). Further investigations into the monolignol biosynthetic pathway in different plants will help to resolve these conflicting models. The monocot plant is a suitable system to investigate monolignol biosynthesis because of the unique biosynthetic features of monocot lignin. Analysis of lignin biosynthetic genes and the biochemical characterization of the enzymes will help to decode the ambiguous points in the lignin biosynthetic pathway.
There have only been a few reports of cloning lignin genes from monocots. Cinnamoyl-CoA reductase (CCR) cDNAs have been isolated from maize (Pichon et al., 1998). Caffeic acid O-methyltransferase (COMT) (McAlister et al., 1998), cinnamyl alcohol dehydrogenase (CAD) (Lynch et al., 2002), and CCR (McInnes et al., 2002; Larsen, 2004) genes have also been isolated from ryegrass. Some brown-midrib mutants (bm) of maize have been characterized, in which bm1 was shown to affect expression of the CAD directly (Halpin et al., 1998), while the bm3 mutation was found to occur in the gene encoding COMT (Vignols et al., 1995). By using a map-based cloning approach, the gh2 mutant, which is GOLD HULL AND INTERNODE, has been shown to encode a CAD in rice (Zhang et al., 2006). The enzyme kinetics of proteins encoded by these genes, however, has not been studied and the biochemical data of lignin biosynthetic enzymes in monocot plants are still limited. Furthermore, the evidence for physiological functions of lignin biosynthesis in gene levels is very scarce.
The particular interest in lignin biosynthesis in wheat is because of its agricultural importance and biological significance. Some important agricultural traits, such as lodging-resistance, are likely to link with lignin biosynthesis (Berry et al., 2004). Initially, efforts were focused on characterizing cinnamoyl-CoA reductase (EC 1.2.1.44 [EC] ) in wheat, since it diverts phenylpropanoid-derived metabolites into the lignin-specific branch pathway, and CCR may be a potential control point in regulating carbon flux towards lignin. The biochemical characterization of one CCR (Ta-CCR2) from wheat has already been reported (Ma and Tian, 2005). Here, the characterization of another CCR gene in wheat is reported, which is associated with stem development and strength support.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant materials and nucleic acid isolation
Wheat (Triticum aestivum L.) plants were grown in a naturally lit glasshouse with normal irrigation and fertilization. Two cultivars, C6001 and H4564, were chosen for the analysis. C6001 and H4564 have the same growth period (240 d) and vernalization type (winter), similar developmental and agricultural phenotypes, and are planted in the same region (Hebei province of China), but differ in lodging phenotype, C6001 is lodging-sensitive while H4564 is lodging-resistant. Stem, leaf, and root tissues were collected from the wheat plants with 2–3 internodes, and then collected at 3-week intervals until 1 week before anthesis. In the analyses, the tissues collected on 20 April, 11 May, and 1 June corresponded to the elongation, heading, and milky stages of development, respectively. Tissues were immediately frozen and stored in liquid nitrogen until used for RNA, DNA, and enzyme isolation. Total RNA was isolated from wheat tissues by TRI reagent (Molecular Research Center, Inc, Cincinnati, USA) according to the manufacturer's instructions. Poly(A)+ RNA was isolated using PolyAT tractR mRNA Isolation Kit (Promega). Genomic DNA was purified from young wheat leaf tissues according to the protocol described by Dellaporta et al. (1983).
Isolation of CCR sequence for use as a probe
cDNA synthesis was based on the rapid amplification of cDNA ends method (Frohman et al., 1988) using oligonucleotide primer 5'-GACTCGAGTCGACATCGA(T)17-3'. RT-PCR reactions were carried out using following primers: 5'-primers: C15: 5'-GA(A/G)AA(A/G)GG(T/C/A/G)TA(T/C)AC(T/C/A/G)GT-3', C20: 5'-GT(T/C/A/G)AC(T/C/A/G)GA(T/C)GA(T/C)CC(T/C/A/G)GA(A/G)CA-3', and 3'-primers: C2: 5'-GACTCGAGTCGACATCG-3', C16: 5'-AG(A/G)TG(T/C/A/G)CC(T/C)TT(T/C)TC(T/C)TG(T/C/A/G)AG-3'. The degenerate primers (C15, C16, and C20) were designed based on the consensus regions of all known plant CCR amino acid sequences (shown in Fig. 1). Two-round PCR reactions were conducted to amplify the specific CCR sequence. The first-round PCR reaction used the primers C15 and C2, and second-round PCR (nested PCR) reaction used primers C20 and C16. The PCR products were resolved on a 1.0% agarose gel and purified by using a GlassMAX® DNA Isolation Kit (Gibco). The purified fragments were cloned into pGEM-T Easy vector (Promega). After sequencing, positive clones that showed high similarity to other CCR genes were used as a probe for screening the wheat stem cDNA library.
|
Construction and screening of the wheat stem cDNA library
A wheat stem cDNA library was constructed following the manufacturer's instructions (Stratagene). cDNA was prepared using the ZAP-cDNA synthesis Kit in conjunction with the Uni-ZAP unidirectional vector. This phage vector was multiplied in E. coli XL1-Blue cells. About 2x105 recombinant phages were screened by lifting onto Hybond-N+ membrane (Amersham) and hybridizing with a 32P-labelled, PCR-generated CCR probe using standard procedure at 42 °C (Sambrook et al., 1989). Positive isolates were purified by three rounds of plating and hybridization. cDNA inserts were sequenced using an ABI 377 automated DNA sequencing machine following established protocols. Sequence similarities were analysed using the SIM-Alignment Tool (Altschul et al., 1997) and data from the GenBank database. Evolutionary relationships were determined using the Clustal W method with a PAM 250 residue weight table (Thompson et al., 1994). Hydrophobicity was analysed by the DNASIS program as described by Kyte and Doolittle (1982).
DNA and RNA gel blot analysis
Genomic DNA from wheat leaf tissues was digested with appropriate restriction enzymes and resolved on a 1.0% agarose gel. Ten µg of total RNA was electrophoresed on 1.4% (w/v) formaldehyde agarose gels. DNA and RNA were blotted onto Hybond-N+ membrane (Amersham) using established protocols (Sambrook et al., 1989). The blots were hybridized at 42 °C in 6x SSC, 5x Denhardt, 0.5% SDS, 100 µg ml–1 salmon sperm DNA with 50% formamide, and washed with 0.1x SSC plus 0.1% SDS at 65 °C. Probes were 32P-labelled using a Ready-to-Go DNA Labelling Kit (Amersham). RNA hybridization signals were normalized by a soybean 18S ribosomal RNA.
Mapping analysis
The mapping population consists of 114 RILs and has been the subject of an extensive genome mapping effort by investigators of the International Triticeae Mapping Initiative (ITMI) (Marino et al., 1996). The first 60 RILs of the ITMI population and 56 F2 plants were used for genetic mapping. Linkage relationships were evaluated with MAPMAKER (Lander et al., 1987) using a minimum LOD of 2.0 and the Kosambi mapping function (Kosambi, 1944).
Functional expression of Ta-CCR1 in E. coli
PCR was used to introduce a HindIII site at the 5' end and a NotI site at the 3' end of the coding region of Ta-CCR1. The primers used were: 5'-CACAAGCTTATGACCGTCGTCGCCGCCGC-3' (5'-primer) and 5'-ATAAGAATGCGGCCGCTCACGCTGTTGCACCGTCCAG-3' (3'-primer). The fidelity of the PCR amplification was confined by DNA sequencing. The amplified product was digested with HindIII plus NotI and cloned into the same sites of the pET-29b vector (Novagen), and plasmids were transformed into BL21 (DE3) and pLysS (Novagen) or RIP and RP (Stratagene) bacterial cells. The BL21(DE3) cell strain transformed with the pET-29b vector without insert was used as a control. The growth and induction of bacterial cells were optimized according to the manufacturer's instructions. Proteins were subjected to denaturing SDS–PAGE on precast 12% TRIS–glycine gels (Novex, San Diego, CA) and stained by Coomassie blue R250. A Kaleidoscope prestained standard (Bio-Rad) was used as a molecular weight marker.
Purification of recombinant CCR protein
All purification steps were carried out at 4 °C. The bacterial culture was grown at 37 °C until OD600=0.7, then moved to 16 °C for 1 h. IPTG was then added to 0.5 mM. Cells were harvested after 24 h at 16 °C. Induced E. coli cells were pelleted at 2000 g for 10 min and resuspended in extraction buffer (50 mM TRIS–HCl, pH 8.5, 500 mM NaCl, 10% glycerol, 1% Triton X-100, with 20 mM mercaptoethanol, and 1 mM PMSF freshly added). Lysozyme was then added to 100 µg ml–1, and the cells were incubated at 30 °C for 15 min. The extracts were sonicated three times for 2 min (at 20 cycles per minute) and the extract was then spun at 15 000 g for 15 min. The supernatant was used for further purification using the S-Tag Thrombin Purification Kit (Novagen, USA) according to the manufacturer's instructions. Protein concentrations were determined by the Bradford assay (Bradford, 1976) with BSA as standard.
CCR activity and enzyme kinetic assay
CCR enzyme activity was measured according to the method of Goffner et al. (1994). For each reaction, 12 min of declination at OD336 was monitored automatically with 1 min intervals. A non-induced E. coli extract was used as a control. Km and Vmax values were determined by extrapolation from Lineweaver–Burke plots.
Enzyme extraction and assay
Wheat stem tissues were collected and ground under liquid nitrogen. The powdered tissues were extracted for 1 h at 4 °C in extraction buffer (100 mM TRIS–HCl pH 7.5, 0.2 mM MgCl2, 2 mM DTT, 10% glycerol). The samples were spun at 12 000 g for 10 min at 4 °C and the resulting supernatant was desalted on PD-10 columns (Pharmacia, Piscataway, NJ, USA). The soluble protein fraction was used for determination of CCR enzyme activity.
Lignin content analysis
Lignin content was quantitatively measured by using the Klason method (Kirk and Obst, 1988). Briefly, air-dried stem tissues were ground into powder and exhaustively extracted in a Soxhlet apparatus with toluene-ethanol (2:1,v/v), followed by 95% ethanol and water. Samples were then vacuum dried and 200 mg was hydrolysed in 3 ml of 72% H2SO4 at 25 °C for 3 h with occasional stirring. The hydrolysate was diluted with the addition of 190 ml H2O and then autoclaved for 1 h. The sample was filtered through a fritted glass crucible, and then washed with hot water. The crucible was dried at 105 °C and weighed. The filtered solution was diluted to 500 ml and A205 was determined spectrophotometrically using a 1 cm long cuvette. The Klason lignin was expressed as a percentage of the cell wall residue (CWR). The acid-soluble lignin was calculated by following formula: Acid lignin (g l–1)=A205/110.
Stem strength determination
The wheat stem samples were collected from the field. The stems from the same position were cut into segments. Freshly collected stem segments were placed horizontally and the force exerted to break the stem was recorded with a universal force testing device (model DC-KZ300, Sichuan, China) to determine the stem rigidity, which was normalized with the stem length and diameter. The experimental results were statistically analysed.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Isolation and sequence analysis of wheat CCR cDNA clone
RT-PCR using degenerate primers was used to amplify CCR sequences from wheat mRNA (see the Materials and methods). After RT-PCR with primers C15 plus C2, and nested-PCR with primers C20 and C16, a single fragment of about 700 bp in size was detected (data not shown). This fragment was ligated to the pGEM-T Easy vector. Sequencing analysis indicated that this fragment showed high similarity (more than 60%) with the corresponding region of CCR cDNAs from other plants. Using this cDNA fragment as a probe, six positively hybridizing plaques were detected after high-stringency hybridization of a wheat stem cDNA library. Restriction analysis showed that all of the isolates had the same restriction fragment pattern. Complete DNA sequence analysis of three positive clones showed that they belonged to the same cDNA, designated as Ta-CCR1 (GenBank Accession no. DQ449508).
The Ta-CCR1 cDNA is 1317 nucleotides in length with a single open reading frame (ORF) of 1047 nucleotides flanked by 5' and 3' untranslated regions of 72 and 198 nucleotides, respectively. The ORF encodes a predicted protein of 349 amino acids with a molecular mass of 37.4 kDa and a calculated isoelectric point of 6.3. Based on the Kozak consensus sequence for eukaryotes, the ATG at position 72 is the real initiation codon with an A at position –3, and a C at position +5, as frequently observed in plants (Joshi et al., 1997). The 3’ end terminates in a 24 bp poly(A) tail, preceded by a 174 bp untranslated A+T rich region (59%). One potential polyadenylation signal (AAAGTGA) is located 13 bp upstream of the polyadenylation site. This 3’ end structure of Ta-CCR1 is generally in accordance with other plant genes (Joshi, 1987). The coding region of Ta-CCR1 is very G+C-rich (68%), characteristic of many monocot genes (Campbell and Gowri, 1990). Ta-CCR1 showed a high degree of similarity with published CCR sequences both at the nucleotide and amino acid levels. In particular, a highly conserved motif, NWYCY, was present in the Ta-CCR1 amino acid sequence, which is proposed to be involved in the catalytic activity of the CCR enzyme (Lacombe et al., 1997). Hydropathy analysis revealed a hydrophobic domain in the first 20 amino acids in N-terminus of Ta-CCR1 protein (Fig. 1), which may be functioned as a signal domain.
Phylogenetic reconstruction with CCR amino acid sequences from the GenBank database revealed three groups, and these were identified as Dicot CCR, Monocot CCR, and a group contained CCR, aldehyde reductase (AR), and dihydroflavonol reductase (DFR) (Fig. 2). CCRs from the monocot plants (including maize, sugarcane, and ryegrass) clustered to the Monocot CCR group, while most of the dicot CCRs clustered to the Dicot CCR group. However, Ta-CCR2 (Ma and Tian, 2005) together with Zm-CCR2 and Os-CCR separated far from the other monocot CCRs. Furthermore, some interspecies sequence similarities exceeded intraspecies sequence similarities between the various forms of CCR; for example, Ta-CCR1 is more similar to Zm-CCR1 than to Ta-CCR2. This indicates that an early gene duplication event may have taken place during monocot evolution. The analysis also showed that CCR from Malusxdomestica (Md-CCR) was closely related to aldehyde reductase (AR) and dihydroflavonol reductase (DFR). This indicates that CCR, AR, and DFR may be a part of a larger superfamily of enzymes that share a common mechanism of catalysis, consistent with former report that CCR and DFR are clustered together (Lacombe et al., 1997).
|
Genomic complexity and chromosome location of Ta-CCR1
To determine the copy number of the Ta-CCR1 gene, DNA gel-blot analysis was performed using wheat genomic DNA digested individually with the restriction enzymes EcoRI, HindIII, XbaI, and NcoI, which are predicted not to cut (HindIII and XbaI) or cut once (EcoRI and NcoI) within the Ta-CCR1 cDNA. The blot was probed with part of the coding region and 3’ untranslated region of Ta-CCR1 cDNA under high-stringency conditions. As shown in Fig. 3, two bands were detected in the EcoRI and NcoI digests, while only one band was detected in the HindIII digest. These results suggest that Ta-CCR1 is present as a single-copy gene in the wheat genome. The six positive CCR clones isolated from the wheat cDNA library gave the same restriction fragment pattern, which further substantiates this conclusion. The three bands with similar molecular weight in the XbaI digest suggests that there may be allelic polymorphism at this site, which may stem from the complexity of the wheat genome and its hexaploidy characteristics.
|
Chromosome location of Ta-CCR1 was further investigated by genetic mapping. Nulli-tetrasomic analyses localized Ta-CCR1 to group-1 chromosmes (1A, 1B, and 1D). Linkage mapping using ITMI mapping population indicated that Ta-CCR1 locus was mapped to the proximal region of 1AL arm (Fig. 4). These results are consistent with the data from Southern hybridization.
|
Heterologous expression of Ta-CCR1 cDNA in E. coli
In order to characterize the activity of Ta-CCR1 further, Ta-CCR1 cDNA was cloned into the pET-29b vector for expression in E. coli. Expression of Ta-CCR1 was tested in four E. coli strains: BL21(DE3), BL21(DE3)pLysS, RIL, and RP. RIL and RP strains are BL21 derivatives that carry extra copies of tRNA genes for rarely used codons such as AGG, AUA, and CUA to alleviate poor expression of genes (in particular G+C rich genes) due to codon bias. Ta-CCR1 cDNA is G+C rich (68%) and contains five AGG codons. After induction of protein expression with 1 mM IPTG, a new protein of
43 kDa appeared in all of the strains tested (Fig. 5). This protein was not present in uninduced cells or in cells transformed with the empty vector. Contrary to what was expected, the BL21(DE3) and BL21(DE3)pLysS strains produced a much higher amount of Ta-CCR1 protein compared with the RIL and RP strains.
|
Kinetic analysis of Ta-CCR1 protein
Ta-CCR1 protein was purified to homogeneity (as determined by SDS–PAGE) from the soluble fraction of induced E. coli extracts (Fig. 6) and its kinetic parameters were determined (Table 1). The calculated catalytic efficiency (Kcat/Km) of Ta-CCR1 indicated that feruloyl-CoA was the best substrate, in consideration of both its low Km and high Vmax values relative to the other substrates tested. The catalytic efficiency towards the other three substrates (5-OH-feruloyl-CoA, sinapoyl-CoA, and caffeoyl-CoA) was lower than feruloyl-CoA. Compared with feruloyl-CoA, 5-OH-feruloyl-CoA had a similar Vmax value, but higher Km value, while sinapoyl-CoA and caffeoyl-CoA had lower Km values with correspondingly lower Vmax values.
|
|
Expression patterns of the Ta-CCR1 gene
In order to examine the expression pattern of Ta-CCR1 in wheat, total RNA were isolated from various wheat organs and hybridized with the same 32P-labelled Ta-CCR1 probe used in the DNA gel-blot analysis. Results showed that the Ta-CCR1 transcript was most abundant in stem tissue with a lower, but still detectable abundance in leaf tissue, while no signal was detected in root tissue (Fig. 7).
|
Expression of Ta-CCR1 gene in relation to wheat stem development
In order to determine if Ta-CCR1 was related to stem development in wheat, the expression of Ta-CCR1 in different developmental stages of wheat was analysed. Furthermore, any links between Ta-CCR1 gene action, lignin biosynthesis, and stem strength support were analysed in two wheat cultivars with different characters.
Stem tissues from two cultivars were collected at the elongation, heading, and milky stages. The rigidity of stems from the three stages was determined and the results shown in Table 2 indicated that stem rigidity increased during wheat development. Comparison of the two cultivars indicated that the stem rigidity of H4564 was always higher than that of C6001, in accordance with its lodging-resistant phenotype.
|
The pattern of Ta-CCR1 expression at different stem developmental stages showed differences in the two cultivars (Fig. 8). Ta-CCR1 mRNA was highly expressed in both C6001 and H4564 at the elongation stage, however, Ta-CCR1 mRNA levels declined at the heading and milky stages in C6001, but remained high in H4564 (Table 3). In addition, the relative levels of Ta-CCR1 mRNA at the different developmental stages in these two wheat cultivars paralleled extractable CCR enzyme activity in these tissues (Table 4). While CCR activity remained fairly high at each developmental stage in H4564, CCR activity declined markedly at the heading and milky stages in C6001, relative to the level of activity present at the elongation stage. CCR activity in C6001 stem tissues was only 44.4% and 41.5% of the activity present in H4564 at the heading and milky stages, respectively. These results strongly suggest that Ta-CCR1 gene expression is associated with stem development and also contributes to stronger stem support in the H4564 cultivar.
|
|
|
Lignin synthesis in relation to wheat stem development
To determine whether lignin synthesis was associated with stem development in wheat, the lignin content was analysed at different developmental stages of stem tissues in the two wheat cultivars (Table 5). From the elongation to the milky stage, the content of Klason lignin increased accordingly, reflecting active lignin synthesis during wheat stem maturity. The Klason lignin content of H4564 was significantly higher than that of C6001 at heading and milky stages (60.6% and 37.7% higher, respectively). By contrast with the Klason lignin content, the content of acid-soluble lignin decreased accordingly from the elongation to the milky stage. The acid-soluble lignin content of H4564 was lower than that of C6001 at heading and milky stages (16.4% and 32.2% lower, respectively).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Two CCR genes exist in wheat which have different properties
CCR is believed to play a vital part in lignin biosynthesis. CCR is responsible for the CoA ester to aldehyde conversion in monolignol biosynthesis, which diverts phenylpropanoid-derived metabolites into the biosynthesis of lignin. CCR activity is generally low in plants, indicating that this enzymatic step might be an important control point to regulate metabolite flux into lignin biosynthesis (Lacombe et al., 1997). Transgenic approaches have demonstrated that CCR activity has profound influences on lignin synthesis and plant development. CCR down-regulated tobacco plants have a strong reduction in lignin content which is associated with a profound alteration of development (Piquemal et al., 1998). However, after these lines were crossed with homozygous CAD down-regulated lines, the hybrid lines showed a similar decline in lignin content without affecting plant development (Chabannes et al., 2001). These data highlight the necessity to study CCR in more detail in order to elucidate its actions, especially its link with plant development.
Although CCR has been demonstrated to have an important role in lignin biosynthesis, many questions remain concerning its in vivo function. Only a few CCR genes have been cloned and characterized. Cloning of the CCR gene was first reported from Eucalyptus gunnii, and the enzymatic activity of this CCR gene was demonstrated through expression of a functional recombinant enzyme in E. coli (Lacombe et al., 1997). Two cDNAs have been identified to encode CCR in maize (Pichon et al., 1998) and ryegrass (McInnes et al., 2002; Larsen, 2004), but their biochemical properties have not been analysed. Two cDNAs encoding CCR have also been identified from the Arabidopsis EST database, and the recombinant proteins were expressed in E. coli (Lauvergeat et al., 2001). Several other CCR gene sequences have been deposited in the GenBank database (Fig. 2), but their functions have still not been demonstrated.
In this report, a CCR cDNA from wheat has been isolated, which was designated as Ta-CCR1. Compared with another wheat CCR gene, Ta-CCR2 that was reported before (Ma and Tian, 2005), the two CCR genes have very different characteristics. Ta-CCR2 exists as three copies in the wheat genome, in contrast to the single copy of Ta-CCR1. Ta-CCR1 and Ta-CCR2 share only 60% similarity in nucleotide sequence and 62% identity in amino acid sequence. This is also reflected in the phylogenetic analysis of CCR sequences, which shows that Ta-CCR1 is more similar to Zm-CCR1 than to Ta-CCR2 (Fig. 2). By contrast, two CCR genes identified from Arabidopsis showed a closer relationship to each other than to the CCR genes from other plants. Therefore, at least in monocot plants, there are two classes of CCR that may have the different functions.
Although the primary amino acid sequences indicated many differences between Ta-CCR1 and Ta-CCR2, both Ta-CCR1 and Ta-CCR2 have almost identical motifs for NADPH binding and the reaction active site. It is proposed that all CCR enzymes have a similar catalysing mechanism for converting the CoA ester to aldehyde in monolignol biosynthesis.
Kinetic analysis of bacterially-expressed and purified Ta-CCR1 protein against four possible substrates showed that this enzyme preferred feruloyl CoA over 5-OH- feruloyl CoA, sinapoyl CoA, and caffeoyl CoA. This is quite different with Ta-CCR2, which can convert feruloyl CoA, 5-OH- feruloyl CoA, sinapoyl CoA, and caffeoyl CoA with almost similar efficiency (Ma and Tian, 2005). As proposed by Dixon et al. (2001), feruloyl CoA would be converted into guaiacyl monomers via an independent pathway. Since Ta-CCR1 is mainly expressed in the stem, while Ta-CCR2 is in the root, more guaiacyl units may be incorporated into lignin in wheat stem tissues than in root tissues. How different structures of Ta-CCR1 and Ta-CCR2 affect this different substrate specificity is still not clear at present and warrants further investigation.
Physiological functions of Ta-CCR1 in stem development and strength support
It is generally believed that lignin deposition imparts rigidity and structural support to the cell wall, which may be involved in some physiological functions of lignin such as lodging-resistance. However, the data of lignin biosynthesis in relation to stem development and strength support in gene level are still limited. In this report, the Ta-CCR1 expression has been investigated in wheat stem development since this gene is specifically expressed in stem tissues.
Ta-CCR1 transcripts were most strongly expressed in wheat stem (Fig. 7) and this pattern of expression is quite distinct from the expression of Ta-CCR2 and other CCR genes such as Eg-CCR (Lacombe et al., 1997), Zm-CCR1 and Zm-CCR2 (Pichon et al., 1998), and At-CCR1 and At-CCR2 (Lauvergeat et al., 2001) for which expression data have been reported. The expression of Eg-CCR and Zm-CCR1 is generally associated with tissues undergoing active lignification, including stem and root tissues. The expression of At-CCR1 was detected in stem and leaf tissues, but it was not reported whether or not this gene was expressed in root tissues. The expression of Zm-CCR2 and At-CCR2 was barely detectable in vegetatively growing tissues, which suggests that these genes may be associated with other physiological processes such as the defence response. A recent report showed that Zm-CCR2 was strongly induced by water-deficit treatment in the root elongation zone of maize seedlings, suggesting that it may facilitate root acclimation to drying environments (Fan et al., 2006). The only Lp-CCR gene from ryegrass showed a stem-specific expression pattern like Ta-CCR1 (Larsen, 2004). The almost exclusive expression of Ta-CCR1 in stem tissues suggests it is involved in stem-specific biological processes.
Stem strength support is an important phenotype in crop plants such as wheat, maize, and rice. This issue is especially important in crop plants, where weak stem strength will lead to a lodging phenotype (Berry et al., 2004). To date, however, there is little information at the molecular level as to how the regulation of lignin synthesis affects stem development and strength support. Reduced lignin levels have been observed in maize brown-midrib (bm) mutants and this has been associated with reduced stem strength. Recent work has confirmed that the bm3 mutant harbours a mutation in the gene for COMT (Vignols et al., 1995). A COMT cDNA fragment from wheat also showed differential expression in lodging-sensitive and resistant cultivars (Ma et al., 2002).
Two wheat cultivars (C66001 [GenBank] and H4564) with different stem strengths were chosen for this study. Analysis of stem tissues at different developmental stages showed the significant increase in stem rigidity and Klason lignin levels during stem maturity in two wheat cultivars (Tables 2, 5), suggesting the close relationship among lignin biosynthesis, stem development, and rigidity. This is consistent with the results from other crops such as alfalfa (Inoue et al., 1998). Furthermore, these data also showed the significant differences between the C6001 and H4564 cultivars. H4564 stem tissues contained more Klason lignin than C6001 stem tissues at heading and milky stages (Table 5). This was correlated with its higher stem rigidity at these stages. On the other hand, acid-soluble lignin levels decreased with maturity in wheat tissues. It is proposed that the low-molecule of acid-soluble lignin might convert into Klason lignin in cell wall. The above data suggest that intensive lignin synthesis to impart stem strength and rigidity at the heading and milky stages contributes to the stronger strength support in the H4564 cultivar.
CCR enzyme activity was compared between the C6001 and H4564 cultivars at different stem developmental stages (Table 4). A large increase in CCR activity occurred at the heading and milky stages in H4564. By contrast, CCR activity exhibited a remarkable decrease in C6001 during the same period. As proposed by Lacombe et al. (1997), CCR might be an important control point in regulating metabolite flux to lignin synthesis. Therefore, the higher enzyme activity of CCR at the heading and milky stages in H4564 would increase metabolite flux towards lignin synthesis and lead to higher lignin levels in H4564 stem tissues at these stages.
Although two CCR genes were found in wheat (Ta-CCR1 and Ta-CCR2), the expression of Ta-CCR2 was most abundant in root tissues (Ma and Tian, 2005) while Ta-CCR1 was highly expressed in stem tissues. Therefore, the majority, if not all, of the CCR enzyme activity in wheat stem tissues can be attributed to Ta-CCR1. Analysis of Ta-CCR1 transcripts during stem development showed that Ta-CCR1 mRNA levels remained high at the elongation, heading, and milky stages in H4564, while Ta-CCR1 mRNA levels declined dramatically at the heading and milky stages in C6001, relative to the level present at elongation stage (Fig. 8; Table 3). The regulation of Ta-CCR1 transcripts during stem development paralleled extractable CCR enzyme activity (Table 4), again suggesting that CCR is involved in regulating lignin synthesis and stem rigidity in wheat stem tissues.
It should be noted that the increase of Ta-CCR1 gene expression and enzyme activity are not in proportion to the increase in lignin content and stem rigidity. Several reasons may explain this phenomenon. First, mRNA levels and enzyme activity just reflect a time-point value when plant tissues are measured, while Klason lignin is accumulated progressively. Once lignin is deposited in plant tissues, it will not be converted or degraded, and stem rigidity will follow the same rule. Therefore, it can be found that Klason lignin content and stem rigidity increase continually from elongation to the heading and milky stages in both wheat cultivars. Secondly, as lignin constitutes a large portion of plant dry mass (up to 25%) and its biosynthesis is a metabolically costly process that requires large quantities of carbon skeletons and reducing equivalents (Lewis and Yamamoto, 1990), the increase in lignin biosynthesis not only needs the higher enzyme activity, but also plenty of carbon and other metabolites. For this reason, it is often seen that the higher expression activity of lignin biosynthetic gene just leads to a small increase in lignin content. Finally, other cell wall component, like cellulose (Tanaka et al., 2003), may also contribute to stem strength, which makes the lignin content out of proportion to stem strength. Whether and how higher stem strength links with the high lodging-resistant phenotype has not been established and warrants further investigation. Recent reports showed that water-deficit treatment increases transcript levels of Zm-CCR1 and Zm-CCR2 genes in the maize root elongation zone, in particular it was more than 10-fold up-regulated after 48 h of water-deficit treatment in Zm-CCR2 transcript levels. This led to a detectable increase in lignin staining in vascular regions of the stele in water-deficit-treated roots, although not proportional to the increase in Zm-CCR2 transcript levels. The authors suggest that water deficit increased the expression of CCR genes and lignin biosynthesis in the root elongation zone. This contributes to progressive inhibition of wall extensibility and root growth, which may facilitate root acclimation to drying environments (Fan et al., 2006). Combined with these data, it suggests that the regulation of lignin biosynthesis in gene levels will finally affect physiological processes, such as stem strength support and root adaptation to drought environments.
In conclusion, stem development and rigidity associated with lignin synthesis and CCR enzyme activity during the different development stages of wheat stem tissues have been analysed. These results showed that CCR enzyme activity increased progressively with lignin biosynthesis and stem maturity. Comparing two cultivars, CCR enzyme activity was much higher in the H4564 cultivar than in the C6001 cultivar, at the heading and milky stages. This is predicted to lead to more lignin synthesis and to provide a greater mechanical strength to the stem of the H4564 cultivar. A cDNA encoding CCR (Ta-CCR1) was isolated from a wheat stem cDNA library and confirmed to encode CCR by functional expression in E. coli. The gene encoding Ta-CCR1 is present as a single copy gene in the wheat genome. Recombinant Ta-CCR1 protein converted feruloyl CoA, 5-OH-feruloyl CoA, sinapoyl CoA, and caffeoyl CoA, but feruloyl-CoA was the best substrate, suggesting the preferential biosynthesis of a G-type lignin. The expression of Ta-CCR1 was exclusively confined to stem and leaf tissues, and was undetectable in root tissues. Furthermore, Ta-CCR1 mRNA levels remained high at elongation, heading, and milky stages in the stem tissues of the H4564 cultivar, while Ta-CCR1 mRNA levels declined dramatically at the heading and milky stages in the stem tissues of the C6001 cultivar. This expression pattern is in good agreement with CCR enzyme activity in these two cultivars. Furthermore, high Ta-CCR1 mRNA levels and high CCR enzyme activity in the wheat stem were correlated with a higher Klason lignin content and greater stem mechanical strength in the H4564 cultivar. It is suggested that Ta-CCR1 may be involved in the regulation of lignin biosynthesis during stem maturity and then contributes to stem strength support in wheat.
|
Acknowledgements |
|---|
This work was supported by grants from the National Natural Science Foundation of China (No.30570133), the Chinese National Special Foundation for Transgenic Plant Research and Commercialization, and the Innovation Project of the Chinese Academy of Sciences. We wish to sincerely thank Dr Bettina Deavours (Plant Biology Division, The Samuel Roberts Noble Foundation, USA) for critical reading of the manuscript.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman JD. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research (1997) 25:3389–3402.
Baucher M, Halpin C, Petit-Conil M, Boerjan W. Lignin: genetic engineering and impact on pulping. Critical Reviews in Biochemistry and Molecular Biology (2003) 38:305–350.[Web of Science][Medline]
Berry PM, Sterling M, Spink JH, Baker CJ, Sylvester-Bradley R, Mooney SJ, Tams AR, Ennos AR. Understanding and reducing lodging in cereals. Advances in Agronomy (2004) 84:217–271.[CrossRef][Web of Science]
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry (1976) 72:248–254.[CrossRef][Web of Science][Medline]
Campbell WH, Gowri G. Codon usage in higher plants, green algae, and cyanobacteria. Plant Physiology (1990) 92:1–11.
Chabannes M, Barakate A, Lapierre C, Marita JM, Ralph J, Pean M, Danoun S, Halpin C, Grima-Pettenati J, Boudet AM. Strong decrease in lignin content without significant alternation of plant development is induced by simultaneous down-regulation of cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) in tobacco plants. The Plant Journal (2001) 28:257–270.[CrossRef][Web of Science][Medline]
Dellaporta SL, Wood J, Hicks JB. A plant DNA minipreparation: versionII. Plant Molecular Biology Reporter (1983) 1:19–21.[CrossRef]
Dixon RA. Natural products and disease resistance. Nature (2001) 411:843–847.[CrossRef][Medline]
Dixon RA, Chen F, Guo D, Parvathi K. The biosynthesis of monolignols: a ‘metabolic grid’, or independent pathways to guaiacyl and syringyl units. Phytochemistry (2001) 57:1069–1084.[CrossRef][Web of Science][Medline]
Fan L, Linker R, Gepstein S, Tanimoto E, Yamamoto R, Neumann PM. Progressive inhibition by water deficit of cell wall extensibility and growth along the elongation zone of maize roots is related to lncreased lignin metabolism and progressive stelar accumulation of wall phenolics. Plant Physiology (2006) 140:603–612.
Frohman MA, Dush MK, Martin GR. Rapid amplification of full-length cDNA ends from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proceedings of the National Academy of Sciences, USA (1988) 85:8998–9002.
Goffner D, Campbell MM, Campargue C, Clastre M, Borderies G, Boudet A, Boudet AM. Purification and characterization of cinnamoyl-CoA:NADP oxidoreductase in Eucalyptus gunnii. Plant Physiology (1994) 106:625–632.[Abstract]
Guo DJ, Chen F, Inoue K, Blount JW, Dixon RA. Downregulation of caffeic acid 3-O-methyltransferase and caffeoyl CoA 3-O-methyltransferase in transgenic alfalfa: impacts on lignin structure and implications for the biosynthesis of G and S lignin. The Plant Cell (2001) 13:73–88.
Halpin C, Holt K, Chojecki J, Oliver D, Chabbert B, Monties B, Edwards K, Barakats A, Foxon GA. Brown-midrib maize (bm1): a mutation affecting the cinnamyl alcohol dehydrogenase gene. The Plant Journal (1998) 14:545–553.[CrossRef][Web of Science][Medline]
Humphreys JM, Chapple C. Rewriting the lignin roadmap. Current Opinion in Plant Biology (2002) 5:224–229.[CrossRef][Web of Science][Medline]
Inoue K, Sewalt VJH, Balance GM, Ni W, Sturzer C, Dixon RA. Developmental expression and substrate specificities of alfalfa caffeic acid 3-O-methyltransferase and caffeoyl CoA 3-O-methyltransferase in relation to lignification. Plant Physiology (1998) 117:761–770.
Joshi CP. Putative polyadenylation signals in nuclear genes of higher plants: a compilation and analysis. Nucleic Acids Research (1987) 15:9627–9640.
Joshi CP, Zhou H, Huang X, Chiang V. Context sequences of translation initiation codon in plants. Plant Molecular Biology (1997) 35:993–1001.[CrossRef][Web of Science][Medline]
Kirk TK, Obst JR. Lignin determination. Methods in Enzymology (1988) 161:87–101.[CrossRef][Web of Science]
Kosambi DD. The estimation of map distances from recombination values. Annual of Eugene (1944) 12:172–175.
Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology (1982) 157:105–132.[CrossRef][Web of Science][Medline]
Lacombe E, Hawkins S, Doorsselaers JV, Piquemal J, Goffner D, Poeydomege O, Boudet AM, Grima-Pettenati J. Cinnamoyl-CoA reductase, the first committed enzyme of lignin branch biosynthetic pathway: cloning, expression, and phylogenetic relationships. The Plant Journal (1997) 11:429–441.[CrossRef][Web of Science][Medline]
Lander ES, Green P, Abrahamson J, Barlow A, Daly MJ, Lincoln SE, Newburg L. MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics (1987) 1:174–181.[CrossRef][Medline]
Larsen K. Cloning and characterization of a ryegrass (Lolium perenne) gene encoding cinnamoyl-CoA reductase (CCR). Plant Science (2004) 166:569–581.
Lauvergeat V, Lacommer C, Lacmbe E, Lasserre E, Roby D, Grima-Pettenati J. Two cinnamoyl-CoA reductase (CCR) genes from Arabidopsis thaliana are differentially expressed during development and in response to infection with pathogenic bacteria. Phytochemistry (2001) 57:1187–1193.[CrossRef][Web of Science][Medline]
Lewis NG, Yamamoto E. Lignin: occurrence, biogenesis and biodegradation. Annual Review of Plant Physiology and Plant Molecular Biololgy (1990) 41:455–496.[CrossRef]
Lynch D, Lidgett A, Mclnnes R, Huxley H, Jones E, Mahoney N, Spangenberg G. Isolation and characterization of three cinnamyl alcohol dehydrogenase homologue cDNAs from perennial ryegrass (Lolium perenne L.). Journal of Plant Physiology (2002) 159:653–660.[CrossRef][Web of Science]
McAlister FM, Jenkins CLD, Watson JM. Sequence and expression of a stem-abundance caffeic acid O-methyltransferase cDNA from perennial ryegrass (Lolium perenne). Australian Journal of Plant Physiology (1998) 25:225–235.[Web of Science]
McInnes R, Lidgett A, Lynch D, Huxley H, Jones E, Mahoney N, Spanganberg G. Isolation and characterization of cinnamoyl-CoA reductase gene from perennial ryegrass (Lolium perenne L.). Journal of Plant Physiology (2002) 159:415–422.[CrossRef][Web of Science]
Ma QH, Tian B. Biochemical characterization of a cinnamoyl-CoA reductase from wheat. Biological Chemistry (2005) 386:553–560.[CrossRef][Web of Science][Medline]
Ma QH, Xu Y, Lin ZB, He P. Cloning of cDNA encoding COMT from wheat which is differentially expressed in lodging-sensitive and-resistant cultivars. Journal of Experimental Botany (2002) 53:2281–2282.
Marino CL, Nelson JC, Lu YH, Sorrells ME, Leroy P, Tuleen NA, Lopes CR, Hart GE. Molecular genetic maps of the group 6 chromosomes of hexaploid wheat (Triticum aestivum L. em Thell). Genome (1996) 39:359–366.[Medline]
Pichon M, Courbon I, Bechert M, Boudet AM, Grima-Pettenati J. Cloning and characterization of two maize cDNAs encoding cinnamoyl-CoA reducatase (CCR) and differential expression of the corresponding genes. Plant Molecular Biology (1998) 38:671–676.[CrossRef][Web of Science][Medline]
Piquemal J, Lapierre C, Myton K, O'Connell A, Schuch W, Grima-Pettenati J, Boudet AM. Down-regulation of cinnamoyl-CoA reductase induces significant changes of lignin profiles in transgenic tobacco plants. The Plant Journal (1998) 13:71–83.[CrossRef][Web of Science]
Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual (1989) 2nd edn. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Tanaka K, Murata K, Yamazaki M, Onosato K, Miyao A, Hirochika H. Three distinct rice cellulose synthase catalytic subunit genes required for cellulose synthesis in the secondary wall. Plant Physiology (2003) 133:73–83.
Thompson JD, Higgins DG, Gibson TJ, Clustal W. Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choices. Nucleic Acids Research (1994) 22:4673–4680.
Vignols F, Rigau J, Torres MA, Capeliades M, Puigdomenech P. The brown midrib3 (bm3) mutation in maize occurs in the gene encoding caffeic acid O-methyltransferase. The Plant Cell (1995) 7:407–416.[Abstract]
Zhang KW, Qian Q, Huang ZJ, Wang YQ, Li M, Hong LL, Zeng DL, Gu MH, Chu CC, Cheng ZK. GOLD HULL AND INTERNODE2 (GH2) encodes a primarily multifunctional cinnamyl-alcohol dehydrogenase (CAD) in Oryza sativa. Plant Physiology (2006) 140:972–983.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  Q.-H. Ma The expression of caffeic acid 3-O-methyltransferase in two wheat genotypes differing in lodging resistance J. Exp. Bot., July 1, 2009; 60(9): 2763 - 2771. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||