caffeoyl-CoA O-methyltransferase Research Articles

Distinct cinnamoyl CoA reductases involved in parallel routes to lignin in Medicago truncatula

Cinnamoyl CoA reductases (CCR) convert hydroxycinnamoyl CoA esters to their corresponding cinnamyl aldehydes in monolignol biosynthesis. We identified two CCR genes in the model legume Medicago truncatula. CCR1 exhibits preference for feruloyl CoA, but CCR2 prefers caffeoyl and 4-coumaroyl CoAs, exhibits sigmoidal kinetics with these substrates, and is substrate-inhibited by feruloyl and sinapoyl CoAs. M. truncatula lines harboring transposon insertions in CCR1 exhibit drastically reduced growth and lignin content, whereas CCR2 knockouts grow normally with moderate reduction in lignin levels. CCR1 fully and CCR2 partially complement the irregular xylem gene 4 CCR mutation of Arabidopsis. The expression of caffeoyl CoA 3-O-methyltransferase (CCoAOMT) is up-regulated in CCR2 knockout lines; conversely, knockout of CCoAOMT up-regulates CCR2. These observations suggest that CCR2 is involved in a route to monolignols in Medicago whereby coniferaldehyde is formed via caffeyl aldehyde which then is 3-O-methylated by caffeic acid O-methyltransferase.

Genome-wide characterization of the biggest grass, bamboo, based on 10,608 putative full-length cDNA sequences

BackgroundWith the availability of rice and sorghum genome sequences and ongoing efforts to sequence genomes of other cereal and energy crops, the grass family (Poaceae) has become a model system for comparative genomics and for better understanding gene and genome evolution that underlies phenotypic and ecological divergence of plants. While the genomic resources have accumulated rapidly for almost all major lineages of grasses, bamboo remains the only large subfamily of Poaceae with little genomic information available in databases, which seriously hampers our ability to take a full advantage of the wealth of grass genomic data for effective comparative studies.ResultsHere we report the cloning and sequencing of 10,608 putative full length cDNAs (FL-cDNAs) primarily from Moso bamboo, Phyllostachys heterocycla cv. pubescens, a large woody bamboo with the highest ecological and economic values of all bamboos. This represents the third largest FL-cDNA collection to date of all plant species, and provides the first insight into the gene and genome structures of bamboos. We developed a Moso bamboo genomic resource database that so far contained the sequences of 10,608 putative FL-cDNAs and nearly 38,000 expressed sequence tags (ESTs) generated in this study.ConclusionAnalysis of FL-cDNA sequences show that bamboo diverged from its close relatives such as rice, wheat, and barley through an adaptive radiation. A comparative analysis of the lignin biosynthesis pathway between bamboo and rice suggested that genes encoding caffeoyl-CoA O-methyltransferase may serve as targets for genetic manipulation of lignin content to reduce pollutants generated from bamboo pulping.

Assessing functional role of three water deficit stress-induced genes in nonhost disease resistance using virus-induced gene silencing in Nicotiana benthamiana

Nonhost disease resistance is the most common form of disease resistance exhibited by all plants and to date this phenomenon is not yet completely understood. Understanding the mechanisms behind nonhost resistance may facilitate engineering crop plants with durable resistance. Our previous studies identified putative roles for three genes flavonol-3-O-glucosyl transferase (F3OGT), an alcohol dehydrogenase (ADH) and trans caffeoyl coA-3-O methyl transferase (CcoAOMT) in water deficit stress tolerance. Preliminary information from our earlier study also suggested that Arabidopsis null mutants for these genes exhibited altered levels of tolerance to bacterial pathogens. In this manuscript we document more evidences to show the relevance of these genes in nonhost resistance using Nicotiana benthamiana. By using virus-induced gene silencing (VIGS), we independently down regulated these three genes and analyzed the response of gene silenced plants to bacterial pathogens. Our results showed that F3OGT, a gene implicated in anthocyanin biosynthesis, silenced plants compromised resistance against a nonhost pathogen. Based on this and previous results, we propose that anthocyanin might play a role in regulating plant defense against bacterial pathogens. Response of ADH or CcoAOMT gene silenced plants to bacterial nonhost pathogens was similar to wild-type. However, CcoAOMT gene down regulated plants were slightly more susceptibility to a host pathogen.

Comparative temporal analyses of the Pinus sylvestris L. var. mongolica litv. apical bud proteome from dormancy to growth

Bud dormancy in perennial plants adapts to environmental and seasonal changes. Bud dormancy is of ecological interest because it affects forest population growth characteristics and is of economical interest because it impacts wood production levels. To understand Pinus sylvestris L. var. mongolica litv. bud-dormancy and bud-burst mechanisms, we characterized the proteomes of their apical buds at the four critical stages that occur during the dormancy-to-growth transition. Ninety-six proteins with altered expression patterns were identified using NanoLC-ESI-MS/MS. The majority of these proteins (57%) are involved in metabolic and other cellular processes. For 28% of the proteins, a function could not be assigned. However, because their expression levels changed, they may be potential candidate bud development- or dormancy-related proteins. Of the 75 non-redundant bud proteins identified, ascorbate peroxidase, pathogenesis-related protein PR-10, and heat shock proteins dramatically increased during August and November, suggesting that they may involved in the initiation of bud dormancy. Conversely, S-adenosylmethionine synthetase, abscisic acid/stress-induced proteins, superoxide dismutase (SOD), caffeoyl-CoA O-methyltransferase, actin, and type IIIa membrane protein cp-wap13 had greater expression levels during April, suggesting that they may be involved in the initiation of bud dormancy-release. Cell division cycle protein 48 and eukaryotic initiation factors 4A-15 and 4A had greater expression levels during May, suggesting that they may regulate cell proliferate and differentiation in the shoot apical meristem. These observations provide insights into the molecular mechanisms that induce or break bud dormancy.

Stone formation in peach fruit exhibits spatial coordination of the lignin and flavonoid pathways and similarity to Arabidopsis dehiscence.

BackgroundLignification of the fruit endocarp layer occurs in many angiosperms and plays a critical role in seed protection and dispersal. This process has been extensively studied with relationship to pod shatter or dehiscence in Arabidopsis. Dehiscence is controlled by a set of transcription factors that define the fruit tissue layers and whether or not they lignify. In contrast, relatively little is known about similar processes in other plants such as stone fruits which contain an extremely hard lignified endocarp or stone surrounding a single seed.ResultsHere we show that lignin deposition in peach initiates near the blossom end within the endocarp layer and proceeds in a distinct spatial-temporal pattern. Microarray studies using a developmental series from young fruits identified a sharp and transient induction of phenylpropanoid, lignin and flavonoid pathway genes concurrent with lignification and subsequent stone hardening. Quantitative polymerase chain reaction studies revealed that specific phenylpropanoid (phenylalanine ammonia-lyase and cinnamate 4-hydroxylase) and lignin (caffeoyl-CoA O-methyltransferase, peroxidase and laccase) pathway genes were induced in the endocarp layer over a 10 day time period, while two lignin genes (p-coumarate 3-hydroxylase and cinnamoyl CoA reductase) were co-regulated with flavonoid pathway genes (chalcone synthase, dihydroflavanol 4-reductase, leucoanthocyanidin dioxygen-ase and flavanone-3-hydrosylase) which were mesocarp and exocarp specific. Analysis of other fruit development expression studies revealed that flavonoid pathway induction is conserved in the related Rosaceae species apple while lignin pathway induction is not. The transcription factor expression of peach genes homologous to known endocarp determinant genes in Arabidopsis including SHATTERPROOF, SEEDSTCK and NAC SECONDARY WALL THICENING PROMOTING FACTOR 1 were found to be specifically expressed in the endocarp while the negative regulator FRUITFUL predominated in exocarp and mesocarp.ConclusionsCollectively, the data suggests, first, that the process of endocarp determination and differentiation in peach and Arabidopsis share common regulators and, secondly, reveals a previously unknown coordination of competing lignin and flavonoid biosynthetic pathways during early fruit development.

Regulation of growth response to water stress in the soybean primary root. I. Proteomic analysis reveals region‐specific regulation of phenylpropanoid metabolism and control of free iron in the elongation zone

In water-stressed soybean primary roots, elongation was maintained at well-watered rates in the apical 4 mm (region 1), but was progressively inhibited in the 4-8 mm region (region 2), which exhibits maximum elongation in well-watered roots. These responses are similar to previous results for the maize primary root. To understand these responses in soybean, spatial profiles of soluble protein composition were analysed. Among the changes, the results indicate that region-specific regulation of phenylpropanoid metabolism may contribute to the distinct growth responses in the different regions. Several enzymes related to isoflavonoid biosynthesis increased in abundance in region 1, correlating with a substantial increase of isoflavonoid content in this region which could contribute to growth maintenance via various potential mechanisms. In contrast, caffeoyl-CoA O-methyltransferase, which is involved in lignin synthesis, was highly up-regulated in region 2. This response was associated with enhanced accumulation of lignin, which may be related to the inhibition of growth in this region. Several proteins that increased in abundance in both regions of water-stressed roots were related to protection from oxidative damage. In particular, an increase in the abundance of ferritin proteins effectively sequestered more iron and prevented excess free iron in the elongation zone under water stress.

Towards a Systems Approach for Lignin Biosynthesis in Populus trichocarpa: Transcript Abundance and Specificity of the Monolignol Biosynthetic Genes

As a step toward a comprehensive description of lignin biosynthesis in Populus trichocarpa, we identified from the genome sequence 95 phenylpropanoid gene models in 10 protein families encoding enzymes for monolignol biosynthesis. Transcript abundance was determined for all 95 genes in xylem, leaf, shoot and phloem using quantitative real-time PCR (qRT-PCR). We identified 23 genes that most probably encode monolignol biosynthesis enzymes during wood formation. Transcripts for 18 of the 23 are abundant and specific to differentiating xylem. We found evidence suggesting functional redundancy at the transcript level for phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL), p-hydroxycinnamoyl-CoA:quinate shikimate p-hydroxycinnamoyltransferase (HCT), caffeoyl-CoA O-methyltransferase (CCoAOMT) and coniferyl aldehyde 5-hydroxylase (CAld5H). We carried out an enumeration-based motif identification and discriminant analysis on the promoters of all 95 genes. Five core motifs correctly discriminate the 18 xylem-specific genes from the 77 non-xylem genes. These motifs are similar to promoter elements known to regulate phenylpropanoid gene expression. This work suggests that genes in monolignol biosynthesis are regulated by multiple motifs, often related in sequence.

Functional characterization of three water deficit stress-induced genes in tobacco and Arabidopsis: An approach based on gene down regulation

Functional characterization of water deficit stress responsive genes is important to understand their role in stress tolerance. RNAi-based silencing of gene of interest and studying the stress response of knockdown plants under stress can be one of the potential options for assessing functional significance of these genes. Several genes showing higher transcript expression under water deficit stress were cloned earlier from a stress adapted crop species, groundnut. In this study, a few selected gene homologs have been characterized in Nicotiana tabacum and Arabidopsis. Using post transcriptional gene silencing (PTGS) based RNAi approach we developed N. tabacum knockdown lines for three of the genes namely alcohol dehydrogenase (ADH), trans caffeoyl coA-3-O-methyl transferase (CcoAOMT) and flavonol-3-O-glucosyl transferase (F3OGT). By quantitative RT-PCR we demonstrated that the RNAi lines showed significant reduction in target gene transcripts. We followed a stress imposition protocol that allows the plants to experience initial gradual acclimation stress and subsequently severe stress for a definite period. The RNAi knockdown lines generated against ADH and F3OGT, when subjected to water deficit stress showed susceptible symptoms signifying the relevance of these genes under stress. Knockdown of CcoAOMT showed higher chlorophyll degradation and less cell viability upon stress compared to control plants. Further, the Arabidopsis mutant lines clearly showed susceptibility to salinity and water deficit stresses validating relevance of these three genes under abiotic stresses.

O -Methyltransferase from Soybean Uses Both Caffeoyl-CoA and Flavonoids as Substrates

A gene encoding O-methyltransferase (SOMT)-10 from soybean, SOMT-10, was cloned by reverse transcription polymerase chain reaction. Phylogenetic analysis revealed that SOMT-10 belonged to caffeoyl-CoA O-methyltransferase (CCoAOMT) and contained conserved catalytic residues found in CCoAOMT. SOMT-10 was expressed in Escherichia coli as a glutathione S-transferase fusion protein and purified to determine its substrate. Several compounds including caffeoyl-CoA, naringenin, quercetin, caffeic acid, kaempferol and luteonin were tested as substrates for the purified recombinant SOMT-10. Analysis of reaction products using high performance liquid chromatography revealed that SOMT-10 used caffeoyl-CoA, quercetin and luteolin as substrates. This result indicated that SOMT-10 used flavones having vicinal hydroxyl groups. The methylation position was determined to be the 3′ hydroxyl group. It is likely that SOMT-10 is a new class of OMT that uses not only caffeoyl-CoA, but also flavonoids. Molecular docking of tricetin with the modeled structure SOMT-10 disclosed that SOMT-10 showed all combinations of the O-methylated products.

Gene expression profiling and silencing reveal that monolignol biosynthesis plays a critical role in penetration defence in wheat against powdery mildew invasion.

Cell wall apposition (CWA) formation is one of the first lines of defence used by plants to halt invading fungi such as powdery mildew. Lignin is a complex polymer of hydroxylated and methoxylated phenylpropane units (monolignols) and lignification renders the cell wall more resistant to pathogen attack. The role of monolignol biosynthesis in CWA-mediated defence against powdery mildew penetration into cereals is demonstrated here using RNA interference (RNAi)-mediated gene silencing and enzyme-specific inhibitors. Thirteen cDNAs representing eight genes involved in monolignol biosynthesis were cloned from an expression sequence tag (EST) library derived from the epidermis of diploid wheat (Triticum monococcum) infected with Blumeria graminis f. sp. tritici (Bgt). Differential expression patterns were found for these genes in susceptible and resistant plants after infection. Transcripts of phenylalanine ammonia lyase (PAL), caffeic acid O-methyltransferase (CAOMT), ferulic acid hydroxylase (FAH), caffeoyl-CoA O-methyltransferase (CCoAMT), and cinnamyl alcohol dehydrogenase (CAD) were accumulated, particularly in the epidermis. RNAi-mediated transient gene silencing in the epidermis led to a higher penetration efficiency of Bgt than in the controls. Gene silencing also compromised penetration resistance to varying degrees with different genes against an inappropriate pathogen, B. graminis f. sp. hordei (Bgh). Co-silencing led to greater penetration of Bgt or Bgh than when the genes were silenced separately. Fluorescence emission spectra analyses revealed that gene silencing hampered host autofluorescence response at fungal contact sites. These results illustrate that monolignol biosynthesis is critically important for host defence against both appropriate and inappropriate pathogen invasion in wheat.

Phylogenetic analysis, homology modelling, molecular dynamics and docking studies of caffeoyl–CoA-O- methyl transferase (CCoAOMT 1 and 2) isoforms isolated from subabul (Leucaena leucocephala)

Caffeoyl coenzyme A O-methyltransferase (CCoAOMT) is an important enzyme that participates in lignin biosynthesis especially in the formation of cell wall ferulic esters of plants. It plays a pivotal role in the methylation of the 3-hydroxyl group of caffeoyl CoA. Two cDNA clones that code CCoAOMT were isolated earlier from subabul and in the present study; 3D models of CCoAOMT1 and CCoAOMT2 enzymes were built using the MODELLER7v7 software to find out the substrate binding sites. These two proteins differed only in two amino acids and may have little or no functional redundancy. Refined models of the proteins were obtained after energy minimization and molecular dynamics in a solvated water layer. The models were further assessed by PROCHECK, WHATCHECK, Verify_3D and ERRAT programs and the results indicated that these models are reliable for further active site and docking analysis. The refined models showed that the two proteins have 9 and 10 alpha-helices, 6 and 7 beta-sheets respectively. The models were used for docking the substrates CoA, SAM, SAH, caffeoyl CoA, feruloyl CoA, 5-hydroxy feruloyl CoA and sinapyl CoA which showed that CoA and caffeoyl CoA are binding with high affinity with the enzymes in the presence and absence of SAM. It appears therefore that caffeoyl CoA is the substrate for both the isoenzymes. The results also indicated that CoA and caffeoyl CoA are binding with higher affinity to CCoAOMT2 than CCoAOMT1. Therefore, CCoAOMT2 conformation is thought to be the active form that exists in subabul. Docking studies indicated that conserved active site residues Met58, Thr60, Val63, Glu82, Gly84, Ser90, Asp160, Asp162, Thr169, Asn191 and Arg203 in CCoAOMT1 and CCoAOMT2 enzymes create the positive charge to balance the negatively charged caffeoyl CoA and play an important role in maintaining a functional conformation and are directly involved in donor-substrate binding.

Pulping performance of transgenic poplar with depressed Caffeoyl-CoA O-methyltransferase

This paper evaluated pulping performance of 3-year-old field-grown transgenic poplar (Populus tremula × Populus alba). The transgenic poplar with anti-sense CCoAOMT had an about 13% decreased lignin content, in which a slight increment was found in S/G ratio. Chemical analysis showed that the transgenic poplar had significantly less benezene-ethanol extractive than that of control wood, but no significant differences were found in contents of ash, cold water extractive, hot water extractive, 1% NaOH extractive, holocellulose, pentosans and cellulose. Fiber assay demonstrated that down-regulation of CCoAOMT expression improved the fiber quality in transgenic poplar. Kraft pulping showed that lower lignin in transgenic poplar led to remarkable improved pulp quality and increased pulp yield.

Scopoletin is biosynthesized viaortho‐hydroxylation of feruloyl CoA by a 2‐oxoglutarate‐dependent dioxygenase inArabidopsis thaliana

Coumarins are derived via the phenylpropanoid pathway in plants. The 2H-1-benzopyran-2-one core structure of coumarins is formed via the ortho-hydroxylation of cinnamates, trans/cis isomerization of the side chain, and lactonization. Ortho-hydroxylation is a key step in coumarin biosynthesis as a branch point from lignin biosynthesis; however, ortho-hydroxylation of cinnamates is not yet fully understood. In this study, scopoletin biosynthesis was explored using Arabidopsis thaliana, which accumulates scopoletin and its beta-glucopyranoside scopolin in its roots. T-DNA insertion mutants of caffeoyl CoA O-methyltransferase 1 (CCoAOMT1) showed significant reduction in scopoletin and scopolin levels in the roots, and recombinant CCoAOMT1 exhibited 3'-O-methyltransferase activity on caffeoyl CoA to feruloyl CoA. These results suggest that feruloyl CoA is a key precursor in scopoletin biosynthesis. Ortho-hydroxylases of cinnamates were explored in the oxygenase families in A. thaliana, and one of the candidate genes in the Fe(II)- and 2-oxoglutarate-dependent dioxygenase (2OGD) family was designated as F6'H1. T-DNA insertion mutants of F6'H1 showed severe reductions in scopoletin and scopolin levels in the roots. The pattern of F6'H1 expression is consistent with the patterns of scopoletin and scopolin accumulation. The recombinant F6'H1 protein exhibited ortho-hydroxylase activity for feruloyl CoA (K(m) = 36.0 +/- 4.27 microM; k(cat) = 11.0 +/- 0.45 sec(-1)) to form 6'-hydroxyferuloyl CoA, but did not hydroxylate ferulic acid. These results indicate that Fe(II)- and 2-oxoglutarate-dependent dioxygenase is the pivotal enzyme in the ortho-hydroxylation of feruloyl CoA in scopoletin biosynthesis.

Potato skin proteome is enriched with plant defence components

Periderm is a tissue of secondary origin that replaces damaged epidermis. It can be found in underground plant organs, as an above-ground tissue of woody species (cork), and as a wound-healing tissue. Its outer layers are composed of phellem cells with suberized walls that constitute a protective barrier, preventing pathogen invasion and fluid loss. In potato, a model for periderm studies, periderm tissue replaces the epidermis early in tuber development and the suberized phellems constitute the tuber's skin. To identify factors involved in phellem/skin development and that play a role in its defensive characteristics, two-dimensional gel electrophoresis was used to compare the skin and parenchymatic flesh proteomes of young developing tubers. Proteins exhibiting differentially high signal intensity in the skin were sorted by functional categories. As expected, the differential skin proteome was enriched in proteins whose activity is characteristic of actively dividing tissues such as cell proliferation, C1 metabolism, and the oxidative respiratory chain. Interestingly, the major functional category consisted of proteins (63%) involved in plant defence responses to biotic and abiotic stresses. This group included three isozymes of caffeoyl-CoA O-methyltransferase and five isozymes of peroxidase that may play a role in suberization processes. The differential expression of these proteins in the skin was further verified by RT-PCR of their corresponding transcripts in skin and tuber flesh samples. The results presented here shed light on the early events in skin development and further expand the concept of the periderm as a protective tissue containing an array of plant defence components.

Isolation of a Novel Tomato Caffeoyl CoA 3-O-methyltransferase Gene Following Infection with the BacteriumRalstonia solanacearum

We combined cDNA amplified fragment length polymorphism (cDNA-AFLP) with bulked segregant analysis (BSA) to detect genes that control tomato (Lycopersicon esculentum) bacterial wilt infected with Ralstonia solanacearum, resistance in F 2 population derived from a cross between a bacterial wilt-resistant variety, T51A, and a bacterial wilt-susceptible variety, T9230. In cDNA-AFLP analysis among bulked-resistant F 2 (B R ) pool, bulked-susceptible F 2 (Bs) pool, bulked-resistant T51A (B A ) pool and bulked-susceptible T9230 (B B ) pool, 34 differentially expressed transcript-derived fragments (DE-TDFs) that were present in only B R and B A pools were detected. Analysis of differential DE-TDF expression in individual resistant F 2 resulted in the isolation of a caffeoyl CoA 3-O-methyltransferase (CCoAOMT) gene not previously described from tomato and which showed similarity to an CCoAOMT gene from tobacco and potato plants. This CCoAOMT gene may play a role in innate generalized response to pathogen infection as it was downregulated in susceptible tomato following infection with the bacterium. CCoAOMT gene plays an essential role in the synthesis of guaiacyl lignin units and supply substrates for the synthesis of syringyl lignin units.

Identification of dehydration responsive genes from two non-nodulated alfalfa cultivars using Medicago truncatula microarrays

To have a comprehensive understanding of how legume plants respond to drought at the gene expression level and examine whether legume plants that are not fixing nitrogen would behave similar to non-legume plants in drought response, transcriptomes were studied in two non-nodulated alfalfa (Medicago sativa L.) cultivars, Ladak and 53V08, when plants were subjected to dehydration stress. Two heat shock-related protein genes were up-regulated in the 3-h stressed shoots in both cultivars. One of them was also up-regulated in the 8-h stressed shoots, along with dehydrin and LEA. A xyloglucan endotransglycosylase and a gene with unknown function were down-regulated in both 3- and 8-h stressed shoots. In roots, nearly half of the 55 genes commonly up-regulated at 3 h are involved in pathogen resistance, insect defense and flavonoid synthesis, which differs from other dehydration-responsive transcriptomes in the literature. Many known drought-responsive genes, such as LEA and dehydrin, were up-regulated after 8 h of treatment. The genes encoding caffeoyl-CoA O-methyl transferase and dirigent were up-regulated in the 3-h stressed roots, while two aquaporin genes were down-regulated, suggesting that lignification and prevention of water loss in roots in initial dehydration stress is a common strategy for both cultivars. The results also indicate the involvement of some specific signal transduction pathways, osmotic adjustment and ion homeostasis regulation during dehydration response. Besides those known dehydration-responsive genes in the literature, some dehydration responses and genes in alfalfa appear to be unique. Our results provide valuable insight into a comprehensive understanding of dehydration response in alfalfa at the molecular level.

Proteome-level changes in the roots of Brassica napus as a result of Plasmodiophora brassicae infection

Clubroot, caused by the obligate parasite Plasmodiophora brassicae, has emerged as an important new disease of canola ( Brassica napus) in Alberta, Canada. Changes in the root protein profile were examined by two-dimensional gel electrophoresis at 12, 24, 48 and 72 h after inoculation of a susceptible canola genotype with the pathogen. A total of 20 protein spots were identified as either increasing (13 spots) or decreasing (7 spots) in intensity relative to water-treated controls. The identities of these proteins were established by MS/MS, and included proteins involved in lignin biosynthesis, cytokinin metabolism, glycolysis, intracellular calcium homeostasis, and the detoxification of ROS. Decreased abundance of adenosine kinase, which is involved in cytokinin homeostasis, supported previous reports indicating a key role for cytokinin in the early phases of clubroot infection. An approximately sixfold reduction in caffeoyl-CoA O-methyltransferase abundance suggested a reduction in host lignin biosynthesis after pathogen challenge, and is consistent with the compatible nature of the interaction examined. Interestingly, levels of enzymes involved in ROS metabolism declined sharply at 12 h after inoculation, but increased at 24–72 h. Collectively, these data suggest major changes in canola metabolism shortly after challenge by the pathogen, which may result in the susceptibility of the host.

Both caffeoyl Coenzyme A 3-O-methyltransferase 1 and caffeic acid O-methyltransferase 1 are involved in redundant functions for lignin, flavonoids and sinapoyl malate biosynthesis in Arabidopsis

Two methylation steps are necessary for the biosynthesis of monolignols, the lignin precursors. Caffeic acid O-methyltransferase (COMT) O-methylates at the C5 position of the phenolic ring. COMT is responsible for the biosynthesis of sinapyl alcohol, the precursor of syringyl lignin units. The O-methylation at the C3 position of the phenolic ring involves the Caffeoyl CoA 3-O-methyltransferase (CCoAOMT). The CCoAOMT 1 gene (At4g34050) is believed to encode the enzyme responsible for the first O-methylation in Arabidopsis thaliana. A CCoAOMT1 promoter-GUS fusion and immunolocalization experiments revealed that this gene is strongly and exclusively expressed in the vascular tissues of stems and roots. An Arabidopsis T-DNA null mutant named ccomt 1 was identified and characterised. The mutant stems are slightly smaller than wild-type stems in short-day growth conditions and has collapsed xylem elements. The lignin content of the stem is low and the S/G ratio is high mainly due to fewer G units. These results suggest that this O-methyltransferase is involved in G-unit biosynthesis but does not act alone to perform this step in monolignol biosynthesis. To determine which O-methyltransferase assists CCoAOMT 1, a comt 1 ccomt1 double mutant was generated and studied. The development of comt 1 ccomt1 is arrested at the plantlet stage in our growth conditions. Lignins of these plantlets are mainly composed of p-hydroxyphenyl units. Moreover, the double mutant does not synthesize sinapoyl malate, a soluble phenolic. These results suggest that CCoAOMT 1 and COMT 1 act together to methylate the C3 position of the phenolic ring of monolignols in Arabidopsis. In addition, they are both involved in the formation of sinapoyl malate and isorhamnetin.

Isolation and Identification of Peanut Leaf Proteins Regulated by Water Stress

Water deficits trigger signaling cascades leading to modulation of protein expression in plant tissues. Identification of peanut leaf proteins regulated by water stress provides some insights of cellular and molecular response of peanut plants to drought stress. Peanut variety Khon Kaen 4, a water-stress sensitive variety, was grown in a growth chamber under controlled environment. Water stress was imposed on day 30 after seedling emergence by withholding watering peanut plants for 6 days as compared to plants adequately supplied with water. Total protein were prepared from a leaflet of fully expanded leaf on the main stem. Proteins were separated in duplicated gels using two-dimensional gel electrophoresis and visualized by silver nitrate staining. Image analysis was performed using ImageMaster 2D Platinum 5.0 to determine proteins regulated by water stress. Molecular mass and isoelectric point of each regulated protein were used in database queries for protein identification. One protein was induced under water stress and the homologous protein was identified as Serine/threonine-protein phosphatase PP 1. Five proteins were down-regulated by water deficit. The homologous proteins were chaperone protein DNAJ, auxin-responsive protein IAA29, peroxidase 43, caffeoyl-CoA O-methyltransferase and SNF1-related protein kinase regulatory subunit beta-2. Down-regulated proteins may be associated with sensitivity of the peanut variety to water stress.

Biosynthesis of curcuminoids and gingerols in turmeric ( Curcuma longa) and ginger ( Zingiber officinale): Identification of curcuminoid synthase and hydroxycinnamoyl-CoA thioesterases

Members of the Zingiberaceae such as turmeric ( Curcuma longa L.) and ginger ( Zingiber officinale Rosc.) accumulate at high levels in their rhizomes important pharmacologically active metabolites that appear to be derived from the phenylpropanoid pathway. In ginger, these compounds are the gingerols; in turmeric these are the curcuminoids. Despite their importance, little is known about the biosynthesis of these compounds. This investigation describes the identification of enzymes in the biosynthetic pathway leading to the production of these bioactive natural products. Assays for enzymes in the phenylpropanoid pathway identified the corresponding enzyme activities in protein crude extracts from leaf, shoot and rhizome tissues from ginger and turmeric. These enzymes included phenylalanine ammonia lyase, polyketide synthases, p-coumaroyl shikimate transferase, p-coumaroyl quinate transferase, caffeic acid O-methyltransferase, and caffeoyl-CoA O-methyltransferase, which were evaluated because of their potential roles in controlling production of certain classes of gingerols and curcuminoids. All crude extracts possessed activity for all of these enzymes, with the exception of polyketide synthases. The results of polyketide synthase assays showed detectable curcuminoid synthase activity in the extracts from turmeric with the highest activity found in extracts from leaves. However, no gingerol synthase activity could be identified. This result was explained by the identification of thioesterase activities that cleaved phenylpropanoid pathway CoA esters, and which were found to be present at high levels in all tissues, especially in ginger tissues. These activities may shunt phenylpropanoid pathway intermediates away from the production of curcuminoids and gingerols, thereby potentially playing a regulatory role in the biosynthesis of these compounds.