Cinnamyl Alcohol Dehydrogenase Gene Research Articles

Biochemical Characterization of the Rice Cinnamyl Alcohol Dehydrogenase Gene Family.

Cinnamyl alcohol dehydrogenase (CAD) is involved in the final step of the phenylpropanod pathway, catalyzing the NADPH-dependent reduction of hydroxy-cinnamaldehydes into the corresponding alcohols. The rice genome contains twelve CAD and CAD-like genes, collectively called OsCADs. To elucidate the biochemical function of the OsCADs, OsCAD1, 2, 6, and 7, which are highly expressed in rice, were cloned from rice tissues. The cloned OsCADs were heterologously expressed in Escherichia coli as His-tag fusion proteins. The activity assay of the recombinant OsCADs showed that OsCAD2, 6, and 7 have CAD activity toward hydroxycinnamaldehydes, but OsCAD1 has no detectable catalytic activity. The kinetic parameters of the enzyme reactions demonstrated that OsCAD2 has the highest catalytic activity among the examined enzymes. This result agrees well with the finding that the Zn binding and NADPH binding motifs and the residues constituting the substrate binding pocket in bona fide plant CADs were fully conserved in OsCAD2. Although they have large variations in the residue for the substrate binding pocket, OsCAD6 and 7 catalyzed the reduction of hydroxycinnamaldehydes with a similar efficiency. Alignment of amino acid sequences showed that OsCAD1 lacks the GxxxxP motif for NADPH binding and has mismatches in residues important in the reduction process, which could be responsible for the loss of catalytic activity. OsCAD2 belongs to CAD Class I with bona fide CADs from other plant species and is constitutively expressed throughout the developmental stages of rice, with preferential expression in actively lignifying tissues such as the root, stem, and panicle, suggesting that it is mainly involved in developmental lignification in rice. The expression of OsCAD2 was also induced by biotic and abiotic stresses such as Xanthomonas oryzae pv. oryzae (Xoo) infection and UV-irradiation, suggesting that it plays a role in the defense response of rice, in addition to a bona fide role in developmental lignification. OsCAD6 and 7 belong in CAD Class II. Their expression is relatively lower than that of OsCAD2 and is confined to certain tissues, such as the leaf sheath, stem, and panicle. The expression of OsCAD6 was stimulated by Xoo infection and UV-irradiation. Thus OsCAD6 appears to be an inducible OsCAD that is likely involved in the defense response of rice against biotic and abiotic stresses.

Vascular preferential activity of the Pennisetum purpureum cinnamyl alcohol dehydrogenase promoter in transgenic tobacco plants

Little is known about the cross talk between the lignin biosynthesis gene promoters and the regulatory proteins that modulate molecular signaling and respond to various stresses. In this study, we characterized the promoter region of the lignin biosynthesis pathway cinnamyl alcohol dehydrogenase (CAD) gene in elephant grass, Pennisetum purpureum. Quantification of the transcript levels of the PpCAD promoter revealed it is preferentially expressed in vascular tissue, especially xylem. Histochemical and fluorometric assays confirmed the vascular-preferential expression of the PpCAD promoter, as the highest β-glucuronidase (GUS) activity was found in the basal stem in transgenic tobacco plants expressing a 1154-bp PpCAD promoter-GUS fusion construct. Moreover, 5′-deleted PpCAD promoter analyses showed that the 1154-bp PpCAD promoter fragment had the highest transcriptional activity, whereas the 2054-bp fragment had multifarious inducible activity responding to gibberellin (GA), methyl jasmonate (MeJA), abscisic acid (ABA), and wounding. The regions from −248 to −243 bp and −1416 to −1411 bp contained W-box cis-elements, which were detected by electrophoretic mobility shift assay (EMSA). The binding effects of the GA-responsive elements (from −561 to −555 bp and −1077 to −1071 bp), MeJA-responsive element (from −1146 to −1142 bp), and the ABA-responsive cis-element (from −1879 to −1874 bp) were also validated by EMSA. Based on our results, we suggest that lignin deposition associated with PpCAD promoter activity adapts to the environment through molecular signaling involving GA, MeJA, and ABA.

Reference gene identification for reliable normalisation of quantitative RT-PCR data in Setaria viridis

BackgroundQuantitative real-time polymerase chain reaction (RT-qPCR) is the key platform for the quantitative analysis of gene expression in a wide range of experimental systems and conditions. However, the accuracy and reproducibility of gene expression quantification via RT-qPCR is entirely dependent on the identification of reliable reference genes for data normalisation. Green foxtail (Setaria viridis) has recently been proposed as a potential experimental model for the study of C4 photosynthesis and is closely related to many economically important crop species of the Panicoideae subfamily of grasses, including Zea mays (maize), Sorghum bicolor (sorghum) and Sacchurum officinarum (sugarcane). Setaria viridis (Accession 10) possesses a number of key traits as an experimental model, namely; (i) a small sized, sequenced and well annotated genome; (ii) short stature and generation time; (iii) prolific seed production, and; (iv) is amendable to Agrobacterium tumefaciens-mediated transformation. There is currently however, a lack of reference gene expression information for Setaria viridis (S. viridis). We therefore aimed to identify a cohort of suitable S. viridis reference genes for accurate and reliable normalisation of S. viridis RT-qPCR expression data.ResultsEleven putative candidate reference genes were identified and examined across thirteen different S. viridis tissues. Of these, the geNorm and NormFinder analysis software identified SERINE/THERONINE-PROTEIN PHOSPHATASE 2A (PP2A), 5′-ADENYLYLSULFATE REDUCTASE 6 (ASPR6) and DUAL SPECIFICITY PHOSPHATASE (DUSP) as the most suitable combination of reference genes for the accurate and reliable normalisation of S. viridis RT-qPCR expression data. To demonstrate the suitability of the three selected reference genes, PP2A, ASPR6 and DUSP, were used to normalise the expression of CINNAMYL ALCOHOL DEHYDROGENASE (CAD) genes across the same tissues.ConclusionsThis approach readily demonstrated the suitably of the three selected reference genes for the accurate and reliable normalisation of S. viridis RT-qPCR expression data. Further, the work reported here forms a highly useful platform for future gene expression quantification in S. viridis and can also be potentially directly translatable to other closely related and agronomically important C4 crop species.

Characterization of Cinnamyl Alcohol Dehydrogenase gene family in lignifying tissues of Tectona grandis L.f.

Abstract The cinnamyl alcohol dehydrogenase (CAD) enzyme catalyzes the last step of monolignols synthesis in the lignin pathway. Tectona grandis (teak) is a tropical tree with high valuable tim­ber. As there is few genetic information about lignin formation in teak, the purpose of this study is to characterize members of CAD family in this species. As methodology, PCR amplification using cDNA samples, vector cloning, sequencing, bioinforma­tics analyses and gene expression studies using real time RT-qPCR were performed. As results, four members (TgCAD1- TgCAD4) were obtained. Comparative analyses showed that all of them have conserved residues for catalytic zinc action, structural zinc ligation, NADPH binding and substrate specifici­ty, consistent with the mechanism of alcohol dehydrogenases. Phylogenetic analysis showed that TgCADs are present in three main classes and seven groups. Expression analyses revealed that TgCAD1 was highly expressed in leaves and could be rela­ted with pathogen defense. TgCAD2 was more expressed in branches and roots. Differently, TgCAD3 and TgCAD4 were highly expressed in juvenile and mature sapwood, suggesting a crucial role in wood development and lignin biosynthesis, with tissue-specialized expression profiles. Furthermore, TgCAD4 could be related with teak maturation for being more expressed in sapwood of mature teak trees. As conclusion, this work is the first to characterize genes of CAD family in Tectona grandis. These genes could be interesting to develop transge­nic plants for basic research and field applications.

Isolation and in silico characterization of full-length cinnamyl alcohol dehydrogenase gene involved in lignin biosynthesis in Neolamarckia cadamba

Cinnamyl alcohol dehydrogenase (CAD) is one of the regulating enzymes that catalyzes the reduction of cinnamaldehydes to ρ-coumaryl, coniferyl and sinapyl alcohols during the final stage of lignin biosynthesis pathway. The expression of CAD genes is believed to be important towards the phenotype characteristics of plants. A full-length CAD cDNA sequence was successfully inferred from the Neolamarckia cadamba wood formation genomic resource database (10,368 ESTs) through the contig mapping approach. Further validation by RT-PCR had confirmed the identity of the gene discovered. The full-length CAD cDNA, designated as NcCAD was 1,240 bp long with a 1,086 bp open reading frame encoding a protein of 361 amino acids, a 68 bp 5’-UTR and a 86 bp 3’-UTR. Phylogenetic analysis showed that NcCAD was grouped in the cluster containing both CAD and SAD genes, in which both genes were involved in lignin biosynthesis. This also showed that the NcCAD cDNA discovered may poses intermediate characteristics of both CAD and SAD genes. The full-length NcCAD cDNA discovered can serve as a good candidate gene for further insight into the wood properties of N. cadamba through association genetics study approach.

Biosynthesis of biodegradable polymer by a potent soil bacterium from a stress-prone environment

Isolation and in silico characterization of full-length cinnamyl alcohol dehydrogenase gene involved in lignin biosynthesis in Neolamarckia cadambaBoon-Ling Tchin, Wei-Seng Ho, Shek-Ling Pang

Heterogenous expression of Pyrus pyrifolia PpCAD2 and PpEXP2 in tobacco impacts lignin accumulation in transgenic plants

Lignin, a natural macromolecular compound, plays an important role in the texture and taste of fruit. Hard end is a physiological disorder of pear fruit, in which the level of lignification in fruit tissues is dramatically elevated. Cinnamyl alcohol dehydrogenase and expansin genes (PpCAD2 and PpEXP2, respectively) exhibit higher levels of expression in ‘Whangkeumbae’ (Pyrus pyrifolia) pear fruit exhibiting this physiological disorder, relative to control fruit without symptoms. These genes were isolated from pear fruit and subsequently expressed in tobacco (Nicotiana tabacum) to investigate their function. Histochemical staining for lignin revealed that the degree of lignification in leaf veins and stem tissues increased in plants transformed with sense constructs and decreased in plants transformed with antisense constructs of PpCAD2. The expression of native NtCADs was also inhibited in the antisense PpCAD2 transgenic tobacco. Sense and antisense PpCAD2 transgenic tobacco exhibited an 86.7% increase and a 60% decrease in CAD activity, respectively, accompanied by a complementary response in lignin content in root tissues. The basal portion of the stem in PpEXP2 transgenic tobacco was bent and highly lignified. Additionally, the level of cellulose also increased in the stem of PpEXP2 transgenic tobacco. Collectively, these results suggested that PpCAD2 and PpEXP2 genes play a significant role in lignin accumulation in transgenic tobacco plants, and it is inferred that these two genes may also participate in the increased lignification observed in hard end pear fruit.

The Phenylpropanoid Pathway and Lignin in Defense against Ganoderma boninense Colonized Root Tissues in Oil Palm (Elaeis guineensis Jacq.).

Basal stem rot, caused by the basidiomycete fungus, Ganoderma boninense, is an economically devastating disease in Malaysia. Our study investigated the changes in lignin content and composition along with activity and expression of the phenylpropanoid pathway enzymes and genes in oil palm root tissues during G. boninense infection. We sampled control (non-inoculated) and infected (inoculated) seedlings at seven time points [1, 2, 3, 4, 8, and 12 weeks post-inoculation (wpi)] in a randomized design. The expression profiles of phenylalanine ammonia lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), and peroxidase (POD) genes were monitored at 1, 2, and 3 wpi using real-time quantitative polymerase chain reaction. Seedlings at 4, 8, and 12 wpi were screened for lignin content, lignin composition, enzyme activities (PAL, CAD, and POD), growth (weight and height), and disease severity (DS). Gene expression analysis demonstrated up-regulation of PAL, CAD, and POD genes in the infected seedlings, relative to the control seedlings at 1, 2, and 3 wpi. At 2 and 3 wpi, CAD showed highest transcript levels compared to PAL and POD. DS increased progressively throughout sampling, with 5, 34, and 69% at 4, 8, and 12 wpi, respectively. Fresh weight and height of the infected seedlings were significantly lower compared to the control seedlings at 8 and 12 wpi. Lignin content of the infected seedlings at 4 wpi was significantly higher than the control seedlings, remained elicited with no change at 8 wpi, and then collapsed with a significant reduction at 12 wpi. The nitrobenzene oxidation products of oil palm root lignin yielded both syringyl and guaiacyl monomers. Accumulation of lignin in the infected seedlings was in parallel to increased syringyl monomers, at 4 and 8 wpi. The activities of PAL and CAD enzymes in the infected seedlings at DS = 5–34% were significantly higher than the control seedlings and thereafter collapsed at DS = 69%.

Distribution of Silicified Microstructures, Regulation of Cinnamyl Alcohol Dehydrogenase and Lodging Resistance in Silicon and Paclobutrazol Mediated Oryza sativa.

Lodging is a phenomenon that affects most of the cereal crops including rice, Oryza sativa. This is due to the fragile nature of herbaceous plants whose stems are non-woody, thus affecting its ability to grow upright. Silicon (Si), a beneficial nutrient is often used to toughen and protect plants from biotic and abiotic stresses. Deposition of Si in plant tissues enhances the rigidity and stiffness of the plant as a whole. Silicified cells provide the much needed strength to the culm to resist breaking. Lignin plays important roles in cell wall structural integrity, stem strength, transport, mechanical support, and plant pathogen defense. The aim of this study is to resolve effects of Si on formation of microstructure and regulation of cinnamyl alcohol dehydrogenase (CAD), a key gene responsible for lignin biosynthesis. Besides evaluating silicon, paclobutrazol (PBZ) a plant growth retartdant that reduces internode elongation is also incorporated in this study. Hardness, brittleness and stiffness were improved in presence of silicon thus reducing lodging. Scanning electron micrographs with the aid of energy dispersive x-ray (EDX) was used to map silicon distribution. Presence of trichomes, silica cells, and silica bodies were detected in silicon treated plants. Transcripts of CAD gene was also upregulated in these plants. Besides, phloroglucinol staining showed presence of lignified vascular bundles and sclerenchyma band. In conclusion, silicon treated rice plants showed an increase in lignin content, silicon content, and formation of silicified microstructures.

Genome-wide identification and characterization of the cinnamyl alcohol dehydrogenase gene family in Zoysia japonica

Genome-wide identification and characterization of the cinnamyl alcohol dehydrogenase gene family in Zoysia japonica

Identification of the CAD gene from Eucalyptus urophylla GLU4 and its functional analysis in transgenic tobacco.

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step in lignin biosynthesis. The genus Eucalyptus belongs to the family Myrtaceae, which is the main cultivated species in China. Eucalyptus urophylla GLU4 (GLU4) is widely grown in Guangxi. It is preferred for pulping because of its excellent cellulose content and fiber length. Based on GLU4 and CAD gene expression, a Eucalyptus variety low in lignin content should be obtained using transgenic technology, which could reduce the cost of pulp and improve the pulping rate, and have favorable prospects for application. However, the role and function of CAD in GLU4 is still unclear. In the present study, EuCAD was cloned from GLU4 and identified using bioinformatic tools. Subsequently, in order to evaluate its impact on lignin synthesis, a full-length EuCAD RNAi vector was constructed, and transgenic tobacco was obtained via Agrobacterium-mediated transformation. A significant decrease in CAD expression and lignin content in transgenic tobacco demonstrated a key role for EuCAD in lignin biosynthesis and established a regulatory role for RNAi. In our study, the direct molecular basis of EuCAD expression was determined, and the potential regulatory effects of this RNAi vector on lignin biosynthesis in E. urophylla GLU4 were demonstrated. Our results provide a theoretical basis for the study of lignin biosynthesis in Eucalyptus.

The cinnamyl alcohol dehydrogenase (CAD) gene family in flax (Linum usitatissimum L.): Insight from expression profiling of cads induced by elicitors in cultured flax cells

Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in the biosynthesis of lignin and lignans as it catalyzes the final step of monolignol biosynthesis, using NADPH as a cofactor. In higher plants, CAD is encoded by a multigene family consisting of three major classes. Based on the recently released flax (Linum usitatissimum L.) whole-genome sequences, in this study we identified six CAD family genes that contain an ADH_N domain and an ADH_zinc_N domain, which suggests that the putative flax CADs (LuCADs) are zinc-dependent alcohol dehydrogenases and members of the plant CAD family. In addition, expression analysis using quantitative real-time PCR revealed spatial variations in the expression of LuCADs in different organs. Comparative analysis between LuCAD enzymatic activity and LuCAD transcripts indicates that the variation of LuCAD enzymatic activities by elicitors is reflected by transcription of LuCADs in flax suspension-cultured cells. Taken together, our genome-wide analysis of CAD genes and the expression profiling of these genes provide valuable information for understanding the function of CADs, and will assist future studies on the physiological role of monolignols associated with plant defense.

Expression of three phenylpropanoid pathway genes in Scots pine (Pinus sylvestris L.) in open-pollinated families with differing relative wood densities during early and late wood formation

Abstract Wood volume and quality are the most important aspects of commercial forestry production, and studies of wood formation are important in order to increase the value and efficiency of forestry production. The phenylpropanoid pathway produces various compounds with diverse functions both for plant defence against biotic and abiotic stress as well as structural development. One of the main roles is monolignol production for lignin biosynthesis, which is a crucial aspect of wood formation. For this study three candidate genes involved in lignin biosynthesis were selected: phenylalanine ammonialyase (PAL1), cinnamyl alcohol dehydrogenase (CAD) and cinnamoyl-CoA reductase (CCR). Candidate gene expression was analysed in selected individuals with high and low wood density from open-pollinated Scots pine families during early wood (EW) and late wood (LW) formation and correlation between expression of these genes, total lignin content, and wood density was determined. Wood density values for analysed trees were similar within tree families but differed significantly between families with high and low wood density (p=1,06E-20). Wood density was slightly negatively correlated with lignin content (r=-0.36, p=0.038), but only in individuals in the high density wood group. In trees with low wood density, expression of the CAD gene was significantly lower in late wood formation compared to early wood (p=0.00179). In trees with high wood density, expression of the PAL1 gene was five times higher during early wood formation compared to late wood formation. A positive correlation was detected between PAL1 and CCR gene expression during early wood formation (r=0.804) and late wood formation (r=0.466).

Phenylpropanoid and antioxidant changes in chickpea plants during cold stress

In creating cold tolerance, plant cells induce cascades of alterations in metabolic pathways including, phenylpropanoid pathway, the activity of antioxidant enzymes and the regulation of gene expression. In this study, some such responses were comparatively studied in two chickpea (Cicer arietinum L.) genotypes, Kabuli (Sel96Th11439) and Desi (4322) under control (23°C) and days 2 and 4 after exposing the seedlings to cold stress (4°C) in acclimated and non-acclimated plants. Under cold, the contents of hydrogen peroxide (H2O2) in Kabuli plants were lower than that of Desi plants particularly in acclimated plants. In acclimated plants, antioxidants and antioxidant enzymes such as ascorbate peroxidase (APX) and guaiacol peroxidase (GPX) and total phenolic compounds had an important role in creating greater cold tolerance. Under these conditions, a high transcription level of phenylalanine ammonia-lyase (CaPAL), cinnamyl alcohol dehydrogenase (CaCAD) and CaLaccase genes was detected in acclimated plants which was accompanied with an increase in lignin contents. The above results indicate the crucial role of phenylpropanoid pathway in creating cold tolerance particularly in Kabuli chickpea plants, which leads to decreased levels of injuries and increased capacity in defense systems.

Root Heterogeneity and Developmental Stage Determine the Pattern of Cellulose Synthase and Cinnamyl Alcohol Dehydrogenase Gene Expression Profiles during Xylogenesis in Populus trichocarpa (Torr. et Gray)

Premise of research. Woody plants show clear divergence among first-order roots. The functions of pioneer roots are mostly for structural support and transport, whereas fibrous roots are responsibl...

Allelic Variation in Cinnamyl Alcohol Dehydrogenase (LoCAD) Associated with Wood Properties of Larix olgensis

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the key step in the lignin monomer biosynthesis pathway, but little is known about CADs in larch (Larix olgensis). Larch is one of the most important conifer plantation species and is used worldwide for reforestation and paper making. However, the presence of lignin is a significant barrier in the conversion of plant biomass to bioethanol. In the current study, 240 individuals from the Northeast Forest University provenance progeny trial population were evaluated, and 47 single-nucleotide polymorphisms (SNPs) were identified in the CAD gene. We used a candidate gene-based association mapping approach to identify CAD gene allelic variants that were associated with growth and wood property traits in L. olgensis. We found that LoCAD harbors high single nucleotide polymorphism (SNP) diversity (πT = 0.00622 and θW = 0.00646). The results of an association analysis indicated that nine SNPs and six haplotypes were significantly associated with wood property and growth traits, explaining between 1.35% and 18.4% of the phenotypic variance. There were strong associations between SNP (g.590G > T) and SNP (g.1184A > T) in LoCAD. These SNPs might represent two quantitative trait nucleotides that are important for the analysis of lignin content.

Regulation of gibberellin on gene expressions related with the lignin biosynthesis in ‘Wangkumbae’ pear (Pyrus pyrifolia Nakai) fruit

‘Wangkumbae’ pear (Pyrus pyrifolia Nakai) was used as material and genes encoding enzymes involved in lignin biosynthesis were cloned: two of 4-coumarate:coenzyme A ligase (4CL) genes Pp4CL1 and Pp4CL2, another two cinnamyl alcohol dehydrogenase (CAD) genes PpCAD1 and PpCAD2, and four peroxidase (POD) genes PpPOD1, PpPOD2, PpPOD3 and PpPOD4. The result of real-time PCR analysis showed a similar expression pattern of PpPAL1 and PpPAL2 with higher accumulation of transcripts at fruit developing early stage and low accumulation at late stage. The expression of PpPAL1 and PpPAL2 revealed down-regulation in gibberellin (GA)-treated fruit. The accumulations of Pp4CL1 and Pp4CL2 transcripts were higher in fruit developing early stage compared with those in late stage, and Pp4CL2 expression level was higher than Pp4CL1. Pp4CL1 and Pp4CL2 expression levels in GA-treated fruit were lower compared with in the control fruit. The relative expression level of PpCAD2 showed higher than PpCAD1 and reached a peak at 53 day after blooming. The expressions of PpCAD2 in GA-treated fruit was significantly lower compared with those in the control fruit in early developmental stage, which was consistent with developmental dynamics of sclereid and dynamic changes in lignin contents during the development. The expression of PpPOD4 gene was significantly higher than PpPOD1, PpPOD2 and PpPOD3 during developmental stages. The expressions of PpPOD1, PpPOD2, and PpPOD4 increased firstly and then decreased during the development of the control fruit. PpPOD1 expression in GA-treated fruit was lower than that in the control fruit, which was consistent with the changes in lignin contents.

A mechanistic study on the toxic effect of copper oxide nanoparticles in soybean (Glycine max L.) root development and lignification of root cells.

Copper oxide nanoparticles (CuONPs) are widely used in several products and their release into the environment can cause toxicity to major food crops. In this study, toxic responses as a result of CuONPs exposure were studied in soybean (Glycine max L.) seedlings. The plants were grown in 1/2 strength Murashige and Skoog medium containing 0, 50, 100, 200, 400, and 500 mg/L of CuONPs in a growth chamber at 26 ± 2 °C with 16/8 h light/dark photoperiod for 14 days. The toxic effects of CuONPs were tested on the shoot and root development, total chlorophyll content, hydrogen peroxide generation, peroxidase (POD) enzyme activity, and lignification of root cells. The mRNA expression of different genes involved in lignin biosynthesis viz. phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), cinnamyl alcohol dehydrogenase (CAD), peroxidase 2 (POD2), peroxidase 4 (POD4), and peroxidase 7 (POD7) was studied using real-time polymerase chain reaction. Exposure to 500 mg/L of CuONPs significantly reduced the shoot growth, weight, and total chlorophyll content. However, the root length and fresh weights were significantly reduced at all concentrations of CuONPs exposure. Exposure to 100, 200, 400, and 500 mg/L of CuONPs significantly increased the hydrogen peroxide level, POD activity, and lignin contents of roots. Treatment with 2,7-dichlorofluorescein diacetate indicated a concentration-dependent increase in reactive oxygen species generation in roots. Staining with phloroglucinol-HCl revealed a concentration dependant increase in lignification of root cells. The expression levels of PAL, C4H, and CAD genes were significantly up-regulated upon exposure to 100, 200, and 400 mg/L of CuONPs. Significant up-regulation in the expression levels of POD2 and POD4 genes was observed upon exposure to 100, 200, 400, and 500 mg/L of CuONPs. Exposure to 200, 400, and 500 mg/L of CuONPs resulted in significant up-regulation of POD7 gene. These results for the first time show that exposure to CuONPs causes enhanced lignification of root cells and thereby affect root development in soybean seedlings.

Lignin and lignans in plant defence: Insight from expression profiling of cinnamyl alcohol dehydrogenase genes during development and following fungal infection in Populus

Cinnamyl alcohol dehydrogenase (CAD) catalyses the final step in the biosynthesis of monolignol, the main component of lignin. Lignins, deposited in the secondary cell wall, play a role in plant defence against pathogens. We re-analysed the phylogeny of CAD/CAD-like genes using sequences from recently sequenced genomes, and analysed the temporal and spatial expression profiles of CAD/CAD-like genes in Populus trichocarpa healthy and infected plants. Three fungal pathogens (Rhizoctonia solani, Fusarium oxysporum, and Cytospora sp.), varying in lifestyle and pathogenicity, were used for plant infection. Phylogenetic analyses showed that CAD/CAD-like genes were distributed in classes represented by all members from angiosperm lineages including basal angiosperms and Selaginella. The analysed genes showed different expression profiles during development and demonstrated that three genes were involved in primary xylem maturation while five may function in secondary xylem formation. Expression analysis following inoculation with fungal pathogens, showed that five genes were induced in either stem or leaves. These results add further evidence that CAD/CAD-like genes have evolved specialised functions in plant development and defence against various pest and pathogens. Two genes (PoptrCAD11 and PoptrCAD15), which were induced under various stresses, could be treated as universal markers of plant defence using lignification or lignan biosynthesis.

Molecular cloning and functional analysis of nine cinnamyl alcohol dehydrogenase family members in Populus tomentosa.

Nine CAD/CAD-like genes in P. tomentosa were classified into four classes based on expression patterns, phylogenetic analysis and biochemical properties with modification for the previous claim of SAD. Cinnamyl alcohol dehydrogenase (CAD) functions in monolignol biosynthesis and plays a critical role in wood development and defense. In this study, we isolated and cloned nine CAD/CAD-like genes in the Populus tomentosa genome. We investigated differential expression using microarray chips and found that PtoCAD1 was highly expressed in bud, root and vascular tissues (xylem and phloem) with the greatest expression in the root. Differential expression in tissues was demonstrated for PtoCAD3, PtoCAD6 and PtoCAD9. Biochemical analysis of purified PtoCADs in vitro indicated PtoCAD1, PtoCAD2 and PtoCAD8 had detectable activity against both coniferaldehyde and sinapaldehyde. PtoCAD1 used both substrates with high efficiency. PtoCAD2 showed no specific requirement for sinapaldehyde in spite of its high identity with so-called PtrSAD (sinapyl alcohol dehydrogenase). In addition, the enzymatic activity of PtoCAD1 and PtoCAD2 was affected by temperature. We classified these nine CAD/CAD-like genes into four classes: class I included PtoCAD1, which was a bone fide CAD with the highest activity; class II included PtoCAD2, -5, -7, -8, which might function in monolignol biosynthesis and defense; class III genes included PtoCAD3, -6, -9, which have a distinct expression pattern; class IV included PtoCAD12, which has a distinct structure. These data suggest divergence of the PtoCADs and its homologs, related to their functions. We propose genes in class II are a subset of CAD genes that evolved before angiosperms appeared. These results suggest CAD/CAD-like genes in classes I and II play a role in monolignol biosynthesis and contribute to our knowledge of lignin biosynthesis in P. tomentosa.