Cinnamyl Alcohol Dehydrogenase Activity Research Articles

Relationship between podophyllotoxin accumulation and soil nutrients and the influence of Fe2+ and Mn2+ on podophyllotoxin biosynthesis in Podophyllum hexandrum tissue culture

Podophyllotoxin (PDT) and its derivatives, which are isolated from the Podophyllum species, are widely used in the clinical setting. The present study was designed to analyze the correlation between PDT levels in the rhizomes of Podophyllum hexandrum (P. hexandrum) and Dysosma versipellis (D. versipellis) and the nutrients in soil. We also aimed to investigate the influence of Fe(2+) and Mn(2+) on the enzyme activity of phenylalanine ammonia lyase (PAL), cinnamyl alcohol-dehydrogenase (CAD), and deoxypodophyllotoxin 6-hydroylase (DOP6H) and PDT accumulation via P. hexandrum tissue culture. The results showed that PDT accumulation was positively correlated with the NO3(-), PO4(3-), Na(+), Fe, and Mn levels and was negatively correlated with the SO4(2-) and K(+) levels, while the correlation with the Mg(2+), Ca(2+), Cu and Zn levels was not significant. The Fe(2+) and Mn(2+) levels were associated with the increased activity of PAL and CAD at 3-18 days; Fe(2+) enhanced the activity levels by 2.66- and 1.76-fold, respectively, and Mn(2+) was associated with a 1.68- and 1.10-fold increase in activity levels, respectively, compared with the control (CK) at 18 days. DOP6H activity was enhanced by Mn(2+), but it was not significantly affected by Fe(2+). Finally, PDT production was enhanced approximately 60% and 34% by Fe(2+) and Mn(2+), respectively, compared with CK at 16 days. These observations may be useful for the generation of PDT and related lignans via commercial cultivation as well as cell and tissue culture of P. hexandrum and other related plant resources.

Ectomycorrhizal colonization and diversity in relation to tree biomass and nutrition in a plantation of transgenic poplars with modified lignin biosynthesis.

Wood from biomass plantations with fast growing tree species such as poplars can be used as an alternative feedstock for production of biofuels. To facilitate utilization of lignocellulose for saccharification, transgenic poplars with modified or reduced lignin contents may be useful. However, the potential impact of poplars modified in the lignification pathway on ectomycorrhizal (EM) fungi, which play important roles for plant nutrition, is not known. The goal of this study was to investigate EM colonization and community composition in relation to biomass and nutrient status in wildtype (WT, Populus tremula × Populus alba) and transgenic poplar lines with suppressed activities of cinnamyl alcohol dehydrogenase, caffeate/5-hydroxyferulate O-methyltransferase, and cinnamoyl-CoA reductase in a biomass plantation. In different one-year-old poplar lines EM colonization varied from 58% to 86%, but the EM community composition of WT and transgenic poplars were indistinguishable. After two years, the colonization rate of all lines was increased to about 100%, but separation of EM communities between distinct transgenic poplar genotypes was observed. The differentiation of the EM assemblages was similar to that found between different genotypes of commercial clones of Populus × euramericana. The transgenic poplars exhibited significant growth and nutrient element differences in wood, with generally higher nutrient accumulation in stems of genotypes with lower than in those with higher biomass. A general linear mixed model simulated biomass of one-year-old poplar stems with high accuracy (adjusted R2 = 97%) by two factors: EM colonization and inverse wood N concentration. These results imply a link between N allocation and EM colonization, which may be crucial for wood production in the establishment phase of poplar biomass plantations. Our data further support that multiple poplar genotypes regardless whether generated by transgenic approaches or conventional breeding increase the variation in EM community composition in biomass plantations.

Elicitor-induced defense responses in Solanum lycopersicum against Ralstonia solanacearum.

We investigated on important parameters of induced resistance in hydroponic tomato (Solanum lycopersicum) against Ralstonia solanacearum using the elicitors chitosan (CHT), salicylic acid (SA), and jasmonic acid (JA). The increase in total phenolic content of roots by the elicitors was significantly higher than control. Most pronounced increase in lignin synthesis was triggered by SA followed by CHT. At 24 h post-elicitation (hpe), the activity of phenylalanine ammonia lyase was 4.5 times higher than control elicited by CHT. The peroxidase activity was about 86 nkat/mg protein at 24 hpe in case of SA and 78 nkat/mg protein in case of CHT. The activity of polyphenol oxidase increased several folds by the elicitors. Cinnamyl alcohol dehydrogenase activity increased to the maximum at 48 hpe under the influence of CHT. The results indicate that the elicitors SA and CHT induced effective defense responses in tomato plants against R. solanacearum. This was evident from reduced vascular browning and wilting symptoms of tomato plants treated with SA and CHT and challenged subsequently with R. solanacearum. This reduced disease incidence in tomato by SA and CHT may be a result of cell wall strengthening through deposition of lignin and the coincident induction of defense enzymes.

Disrupting the cinnamyl alcohol dehydrogenase 1 gene (BdCAD1) leads to altered lignification and improved saccharification in Brachypodium distachyon

Brachypodium distachyon (Brachypodium) has been proposed as a model for grasses, but there is limited knowledge regarding its lignins and no data on lignin-related mutants. The cinnamyl alcohol dehydrogenase (CAD) genes involved in lignification are promising targets to improve the cellulose-to-ethanol conversion process. Down-regulation of CAD often induces a reddish coloration of lignified tissues. Based on this observation, we screened a chemically induced population of Brachypodium mutants (Bd21-3 background) for red culm coloration. We identified two mutants (Bd4179 and Bd7591), with mutations in the BdCAD1 gene. The mature stems of these mutants displayed reduced CAD activity and lower lignin content. Their lignins were enriched in 8-O-4- and 4-O-5-coupled sinapaldehyde units, as well as resistant inter-unit bonds and free phenolic groups. By contrast, there was no increase in coniferaldehyde end groups. Moreover, the amount of sinapic acid ester-linked to cell walls was measured for the first time in a lignin-related CAD grass mutant. Functional complementation of the Bd4179 mutant with the wild-type BdCAD1 allele restored the wild-type phenotype and lignification. Saccharification assays revealed that Bd4179 and Bd7591 lines were more susceptible to enzymatic hydrolysis than wild-type plants. Here, we have demonstrated that BdCAD1 is involved in lignification of Brachypodium. We have shown that a single nucleotide change in BdCAD1 reduces the lignin level and increases the degree of branching of lignins through incorporation of sinapaldehyde. These changes make saccharification of cells walls pre-treated with alkaline easier without compromising plant growth.

Cell wall compositional modifications of Miscanthus ecotypes in response to cold acclimation

Miscanthus, a potential energy crop grass, can be damaged by late frost when shoots emerge too early in the spring and during the first winter after planting. The effects of cold acclimation on cell wall composition were investigated in a frost-sensitive clone of Miscanthus x giganteus compared to frost-tolerant clone, Miscanthus sinensis August Feder, and an intermediate frost-tolerant clone, M. sinensis Goliath. Cellulose and lignin contents were higher in M. x giganteus than in the M. sinensis genotypes. In ambient temperature controls, each clone displayed different glucuronoarabinoxylan (GAX) contents and degree of arabinose substitution on the xylan backbone. During cold acclimation, an increase in (1→3),(1→4)-β-d-glucan content was observed in all genotypes. Uronic acid level increased in the frost sensitive genotype but decreased in the frost tolerant genotypes in response to cold. In all clones, major changes in cell wall composition were observed with modifications in phenylalanine ammonia-lyase (PAL) and cinnamyl alcohol dehydrogenase (CAD) activities in both non- and cold-acclimated experiments. A large increase in CAD activity under cold stress was displayed in each clone, but it was largest in the frost-tolerant clone, M. sinensis August Feder. The marked increase in PAL activity observed in the frost-tolerant clones under cold acclimation, suggests a reorientation of the products towards the phenylpropanoid pathway or aromatic synthesis. How changes in cell wall physical properties can impact frost tolerance is discussed.

Different response on drought tolerance and post-drought recovery between the small-leafed and the large-leafed white clover (Trifolium repens L.) associated with antioxidative enzyme protection and lignin metabolism

The objective of this present investigation was to study the different response on drought tolerance and post-drought recovery for two different genotypes of white clover (a drought-tolerant small-leafed white clover and a drought-sensitive large-leafed white clover) associating with antioxidative enzyme and lignin metabolism. Two materials were exposed to 12 days of drought stress and 6 days of rewatering in a greenhouse. Significantly higher activities of superoxide dismutase, ascorbate peroxidase, catalase, guaiacol peroxidase and root activity as well as lower electrolyte leakage and malondialdehyde levels of the small-leafed in leaves and roots were observed as compared to the large-leafed under drought stress and rewatering. Lignification-related enzymes (polyphenol oxidase, cinnamyl alcohol dehydrogenase) activities of two materials in leaves gradually increased from 0 to 9 days of drought stress; then declined at 12 days of drought stress and after rewatering. After 12 days of drought stress, lignin content of two materials in roots was significantly higher relative to the well-watered. Lignin content of leaves in the small-leafed also increased significantly after 6 days of rewatering, but not in the large-leafed. The small-leafed exhibited more sensitive lignin metabolism and faster lignification in leaves and roots than the large-leafed regarding the response of drought stress. These results indicated that antioxidant efficiency and lignin metabolism played important roles in response to drought stress and rewatering for different white clovers. Comparatively higher reactive oxygen species scavenging system and more sensitive lignin metabolism could be associated with better drought tolerance and post-drought recovery in white clover.

Transposon insertion in a cinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize

Maize brown midrib1 (bm1) mutant plants have reduced lignin content and offer significant advantages when used in silage and biofuel applications. Cinnamyl alcohol dehydrogenase (CAD) catalyzes the conversion of hydroxycinnamyl aldehydes to monolignols, a key step in lignin biosynthesis. Maize CAD2 has been implicated as the underlying gene for bm1 phenotypes since bm1 plants have reduced CAD activity and lower CAD2 transcript level. Here, we describe a Dow AgroSciences maize bm1 mutant (bm1-das1) that contains a 3,444-bp transposon insertion in the first intron of CAD2 gene. As a result of chimeric RNA splicing, cad2 mRNA from bm1-das1 contains a 409-bp insert between its 1st and 2nd exons. This insertion creates a premature stop codon and is predicted to result in a truncated protein of 48 amino acids (AA), compared to 367 AA for the wild type (WT) CAD2. We have also sequenced cad2 from the reference allele bm1-ref in 515D bm1 stock and showed that it contains a two-nucleotide (AC) insertion in the 3rd exon, which is predicted to result in a truncated protein of 147 AA. The levels of cad2 mRNA in the midribs of bm1-das1 and bm1-ref are reduced by 91 and 86 % respectively, leading to reductions in total lignin contents by 24 and 30 %. Taken together, our data show that mutations in maize CAD2 are responsible for maize bm1 phenotypes. Based on specific changes in bm1-das1 and bm1-ref, high throughput TaqMan and KBioscience's allele specific PCR assays capable of differentiating mutant and WT alleles have been developed to accelerate bm1 molecular breeding.

Effect of heat treatment on lignification of postharvest bamboo shoots (Phyllostachys praecox f. prevernalis.)

In order to evaluate the effects of heat treatment on the quality of postharvest bamboo shoots, the firmness, disease incidence, respiration and ethylene production, ACC synthase (ACS) and ACC oxidase (ACO) activities, lignin and cellulose contents, and the activities of phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POD) were examined during storage at 20°C after heat treatment at 45°C for 5h. Heat treatment inhibited disease incidence and respiration, retarded ethylene production, and decreased ACS and ACO activities in bamboo shoots. Furthermore, heat treatment significantly delayed the rise in the activities of PAL, CAD and POD, which were associated with the inhibition of the synthesis of lignin and the delayed tissue lignification. These findings suggest that heat treatment could be a potential tool to delay lignification and decrease disease incidence in bamboo shoots during storage at 20°C.

Fungal soil communities in a young transgenic poplar plantation form a rich reservoir for fungal root communities

Fungal communities play a key role in ecosystem functioning. However, only little is known about their composition in plant roots and the soil of biomass plantations. The goal of this study was to analyze fungal biodiversity in their belowground habitats and to gain information on the strategies by which ectomycorrhizal (ECM) fungi form colonies. In a 2-year-old plantation, fungal communities in the soil and roots of three different poplar genotypes (Populus × canescens, wildtype and two transgenic lines with suppressed cinnamyl alcohol dehydrogenase activity) were analyzed by 454 pyrosequencing targeting the rDNA internal transcribed spacer 1 (ITS) region. The results were compared with the dynamics of the root-associated ECM community studied by morphotyping/Sanger sequencing in two subsequent years. Fungal species and family richness in the soil were surprisingly high in this simple plantation ecosystem, with 5944 operational taxonomic units (OTUs) and 186 described fungal families. These findings indicate the importance that fungal species are already available for colonization of plant roots (2399 OTUs and 115 families). The transgenic modification of poplar plants had no influence on fungal root or soil communities. Fungal families and OTUs were more evenly distributed in the soil than in roots, probably as a result of soil plowing before the establishment of the plantation. Saprophytic, pathogenic, and endophytic fungi were the dominating groups in soil, whereas ECMs were dominant in roots (87%). Arbuscular mycorrhizal diversity was higher in soil than in roots. Species richness of the root-associated ECM community, which was low compared with ECM fungi detected by 454 analyses, increased after 1 year. This increase was mainly caused by ECM fungal species already traced in the preceding year in roots. This result supports the priority concept that ECMs present on roots have a competitive advantage over soil-localized ECM fungi.

Altered Lignin Biosynthesis Improves Cellulosic Bioethanol Production in Transgenic Maize Plants Down-Regulated for Cinnamyl Alcohol Dehydrogenase

Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme involved in the last step of monolignol biosynthesis. The effect of CAD down-regulation on lignin production was investigated through a transgenic approach in maize. Transgenic CAD-RNAi plants show a different degree of enzymatic reduction depending on the analyzed tissue and show alterations in cell wall composition. Cell walls of CAD-RNAi stems contain a lignin polymer with a slight reduction in the S-to-G ratio without affecting the total lignin content. In addition, these cell walls accumulate higher levels of cellulose and arabinoxylans. In contrast, cell walls of CAD-RNAi midribs present a reduction in the total lignin content and of cell wall polysaccharides. In vitro degradability assays showed that, although to a different extent, the changes induced by the repression of CAD activity produced midribs and stems more degradable than wild-type plants. CAD-RNAi plants grown in the field presented a wild-type phenotype and produced higher amounts of dry biomass. Cellulosic bioethanol assays revealed that CAD-RNAi biomass produced higher levels of ethanol compared to wild-type, making CAD a good target to improve both the nutritional and energetic values of maize lignocellulosic biomass.

EFFECT OF GIBBERELLIC ACID AND 6-BENZYLAMINOPURINE ON LIGNIFICATION OF FRESH-CUT ZIZANIA LATIFOLIA DURING REFRIGERATED (1C) STORAGE

ABSTRACT The effect of gibberellic acid (GA3) and 6-benzylaminopurine (6-BA) on lignification of fresh-cut Zizania latifolia were investigated for 15 days at 1C. Results showed that treatment of Z. latifolia slices with GA3 and 6-BA slowed down the increase in cellulose and lignin contents, maintained relatively high cellulase (CEL) activities and total phenol contents. Both treatments also inhibited phenylalanine ammonia-lyase, 4-coumarate CoA ligase, cinnamyl alcohol dehydrogenase and peroxidase activities. GA3 treatment promoted the accumulation of superoxide anion radical (O2·-) and hydrogen peroxide (H2O2) over the first 9 days of storage but reduced their accumulation after 9 days of storage. 6-BA treatment significantly inhibited O2- and H2O2 accumulation throughout storage period. These results indicated that GA3 and 6-BA could retard lignification thereby maintaining a better texture of fresh-cut Z. latifolia during storage at 1C, which provides a theoretical basis for commercial application. PRACTICAL APPLICATIONS The marketing of packaged and refrigerated fresh-cut Zizania latifolia is a choice of increasing interest for vegetables processors. However, fresh-cut Z. latifolia have a faster rate of lignification during retail because of wounding. Therefore, this study was conducted to develop treatments to reduce tissue lignification of fresh-cut Z. latifolia. Results showed that GA3 and 6-BA treatments can effectively inhibit lignification in fresh-cut Z. latifolia, which provides a theoretical basis for commercial application.

Environmental stresses of field growth allow cinnamyl alcohol dehydrogenase-deficient Nicotiana attenuata plants to compensate for their structural deficiencies.

The organized lignocellulosic assemblies of cell walls provide the structural integrity required for the large statures of terrestrial plants. Silencing two CINNAMYL ALCOHOL DEHYDROGENASE (CAD) genes in Nicotiana attenuata produced plants (ir-CAD) with thin, red-pigmented stems, low CAD and sinapyl alcohol dehydrogenase activity, low lignin contents, and rubbery, structurally unstable stems when grown in the glasshouse (GH). However, when planted into their native desert habitat, ir-CAD plants produced robust stems that survived wind storms as well as the wild-type plants. Despite efficient silencing of NaCAD transcripts and enzymatic activity, field-grown ir-CAD plants had delayed and restricted spread of red stem pigmentation, a color change reflecting blocked lignification by CAD silencing, and attained wild-type-comparable total lignin contents. The rubbery GH phenotype was largely restored when field-grown ir-CAD plants were protected from wind, herbivore attack, and ultraviolet B exposure and grown in restricted rooting volumes; conversely, it was lost when ir-CAD plants were experimentally exposed to wind, ultraviolet B, and grown in large pots in growth chambers. Transcript and liquid chromatography-electrospray ionization-time-of-flight analysis revealed that these environmental stresses enhanced the accumulation of various phenylpropanoids in stems of field-grown plants; gas chromatography-mass spectrometry and nuclear magnetic resonance analysis revealed that the lignin of field-grown ir-CAD plants had GH-grown comparable levels of sinapaldehyde and syringaldehyde cross-linked into their lignins. Additionally, field-grown ir-CAD plants had short, thick stems with normal xylem element traits, which collectively enabled field-grown ir-CAD plants to compensate for the structural deficiencies associated with CAD silencing. Environmental stresses play an essential role in regulating lignin biosynthesis in lignin-deficient plants.

The effects of l-DOPA on root growth, lignification and enzyme activity in soybean seedlings

In the present study, we investigated the effects of l-DOPA (l-3,4-dihydroxyphenylalanine), an allelochemical exuded from the velvetbean (Mucuna pruriens L DC. var. utilis), on the growth and cell viability of soybean (Glycine max L. Merrill) roots. We analyzed the effects of l-DOPA on phenylalanine ammonia lyase (PAL), cinnamyl-alcohol dehydrogenase (CAD) and cell wall-bound peroxidase (POD) activities as well as its effects on phenylalanine, tyrosine and lignin contents in the roots. 3-day-old seedlings were cultivated in half-strength Hoagland nutrient solution (pH 6.0), with or without 0.5 mM l-DOPA, in a growth chamber at 25 °C for 6, 12, 18 or 24 h with a day/night regime of 1:1, and a photon flux density of 280 μmol m−2 s−1. In general, the length, fresh weight and dry weight of the roots decreased followed by a significant loss of cell viability. Phenylalanine, tyrosine and lignin contents as well as PAL, CAD and cell wall-bound POD activities increased after l-DOPA treatment. These results reinforce the susceptibility of soybean to l-DOPA, which increases the enzyme activity in the phenylpropanoid pathway and, therefore, provides precursors for the polymerization of lignin. In brief, these findings suggest that the inhibition of soybean root growth induced by exogenously applied l-DOPA may be due to excessive production of lignin in the cell wall.

Changes in cinnamyl alcohol dehydrogenase activities from sugarcane cultivars inoculated with Sporisorium scitamineum sporidia

This study describes a method for determining cinnamyl alcohol dehydrogenase activity in sugarcane stems using reverse phase (RP) high-performance liquid chromatography to elucidate their possible lignin origin. Activity is assayed using the reverse mode, the oxidation of hydroxycinnamyl alcohols into hydroxycinnamyl aldehydes. Appearance of the reaction products, coniferaldehyde and sinapaldehyde is determined by measuring absorbance at 340 and 345 nm, respectively. Disappearance of substrates, coniferyl alcohol and sinapyl alcohol is measured at 263 and 273 nm, respectively. Isocratic elution with acetonitrile:acetic acid through an RP Mediterranea sea C18 column is performed. As case examples, we have examined two different cultivars of sugarcane; My 5514 is resistant to smut, whereas B 42231 is susceptible to the pathogen. Inoculation of sugarcane stems elicits lignification and produces significant increases of coniferyl alcohol dehydrogenase (CAD) and sinapyl alcohol dehydrogenase (SAD). Production of lignin increases about 29% in the resistant cultivar and only 13% in the susceptible cultivar after inoculation compared to uninoculated plants. Our results show that the resistance of My 5514 to smut is likely derived, at least in part, to a marked increase of lignin concentration by the activation of CAD and SAD.

Increased activities of phenyalanine ammonia lyase, peroxidase, and cinnamyl alcohol dehydrogenase in relation to pericarp hardening after physical impact in mangosteen (Garcinia mangostana L.)

SummaryWe previously reported that physical (mechanical) impact results in increased lignin concentrations in the pericarp of mangosteen (Garcinia mangostana L.) fruit. Here, we observed the effects of physical impact on fruit firmness and enzyme activities. Increases in fruit firmness and in lignin levels were detectable within 15 min of impact. Cellulose levels in the pericarp and water contents changed significantly, suggesting that the main increase in pericarp firmness was due cell wall cross-linking, possibly due to lignification. However, the increase in pericarp lignin level was not directly related to a concomitant decrease in the levels of free phenolics, potential substrates for lignin synthesis. The activities of pericarp phenyalanine ammonia lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), and peroxidase (POD), all enzymes involved in lignin biosynthesis, showed a significant increase 15 min after impact, then decreased. It was concluded that the increase in the firmness of mangosteen pericarp after impact was related to increased activities of the enzymes required for lignin biosynthesis and not to the biosynthesis of phenolic compounds.

Cloning, characterization and impact of up- and down-regulating subabul cinnamyl alcohol dehydrogenase (CAD) gene on plant growth and lignin profiles in transgenic tobacco

Both cDNA including 5′UTR and 3′UTR and genomic clones of cinnamyl alcohol dehydrogenase (CAD) were isolated and characterized from a pulp-yielding leguminous tree Leucaena leucocephala (LlCAD1). The deduced amino acid sequence shared high identity with orthologous sequences of Acacia mangium × Acacia auriculiformis (83%), Medicago sativa (83%), Nicotiana tabaccum (83%) and Aralia cordata (81%). Full length cDNA contained 78 bases of 5′UTR and 283 bases of 3′UTR, while the genomic clone contained 5 exons and 4 introns. Western blot analysis revealed elevated expression of LlCAD1 in seedling roots and shoots compared to leaves. Sense and antisense CAD tobacco transgenics showed increased and reduced CAD activity accompanied by a change in monomeric lignin composition. Histochemical staining of lignin in down-regulated plants suggested an increase in aldehyde units and a decrease in S/G ratio. Down-regulation of CAD resulted in accumulation of syringic, ferulic, p-coumaric and sinapic acids compared to untransformed controls. These observations were validated by anatomical studies of down-regulated transgenic stems which showed thin walled, elongated phloem and xylem fibres, accompanied by a reduction in the density of vessel elements and amount of secondary xylem when compared to untransformed plants. Furthermore, Klason lignin analysis of CAD antisense transgenics showed 7–32% reduced lignin and normal phenotype as compared to untransformed plants. Such a reduction was not noticed in up-regulated transgenics. These results demonstrate a unique opportunity to explore the significant role that down-regulation of CAD gene plays in reducing lignin content thereby offering potential benefits to the pulp and paper industry.

Relationship between Lignin Metabolism and Lodging Resistance in Wheat

As a component of cell wall,lignin is believed to be relevant to mechanical intensity of culm.In this study,we investigated the dynamic changes of lignin content and activities of four enzymes involved in lignin metabolism in culm as well as the snapping resistance and lodging index of wheat(Tritcum aestivum L.) to disclose the relationship between lignin content and lodging resistance.The lignin content and activities of phenylalanine ammonia-lyase(PAL),tyrosine ammonia-lyase(TAL),cinnamyl alcohol dehydrogenase(CAD),and 4-coumarate:CoA ligase(4CL) were tested with the second internode of six cultivars with different lodging resistance.The snapping resistance and culm lodging index were determined at anthesis,milking,and maturity stages.The lignin content varied significantly among cultivars.Jimai 22 and Jimai 20,which showed high resistance to culm snapping and loging,had higher lignin content and the activities of PAL,TAL,CAD,and 4CL than other cultivars.The lignin content was negatively correlated with the actual lodging ratio(r = -0.83,P 0.05),and positively correlated with the snapping resistance of culm(r = 0.86,P 0.05).The activities of PAL,TAL,and CAD were positively correlated with lignin content with correlation coefficients of 0.78(P 0.05),0.77(P 0.05),and 0.85(P 0.05),respectively.Based on these results,we conclude that high activities of PAL and CAD are the enzymatic basis of increased lignin content,and the lignin content in culm can be used as an important indicator to evaluate lodging resistance of wheat.

Early detection of wood quality in Hevea brasiliensis (Rubber wood) using cinnamyl alcohol dehydrogenase (CAD) activity and lignification

Lignins are phenolic polymers of the cell wall and cinnamyl alcohol dehydrogenase (CAD) is the key enzyme involved in the synthesis of lignin monomers. A study was conducted in stems of 18 wild Hevea germplasm accessions and five popular clones to localize and correlate CAD activity and lignification at various stages of xylogenesis through tissue printing and in situ localization as well as quantitative image analysis techniques. Simultaneously, quantification of lignin produced was also done by classical histochemistry and gravimetric estimation. CAD activity was maximum during the early stages of stem development and minimum during the mature stage of xylogenesis whereas the pattern of lignification showed a reverse trend indicating that as xylogenesis increased, CAD activity decreased. The quantity of lignin produced was also increased in association with progress of secondary thickening. The percentage of lignin at different developmental stages of the stem was highly significant. The percentage of lignin ranged from 21.04–27.4 % in wild Hevea germplasm and 20.01–23.00 % in popular clones. Of the 18 wild accessions screened, nine showed significantly higher percentage of lignin over five popular clones. The present study revealed that CAD activity is involved in the lignification process in Hevea stem and its activity is restricted in the mature xylem. The CAD activity and lignification pattern in the juvenile growth phase can be used as reliable tools for the early detection of wood quality in Heveabrasiliensis.

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

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

Nucleotide diversity and linkage disequilibrium in Populus nigra cinnamyl alcohol dehydrogenase (CAD4) gene

Cinnamyl alcohol dehydrogenase (CAD) is involved in the biosynthesis of lignin, a component of plant cell wall which negatively impacts paper pulp processing and biomass fermentation to ethanol. Transgenic poplars with depressed CAD activity show structural alterations of lignin. Natural CAD mutants have been identified in several plants; however, no natural CAD mutants have been identified in poplar. We surveyed the natural genetic variation in CAD4, a gene coding for CAD, in 360 poplar trees from Western Europe. We measured linkage disequilibrium (LD) between single-nucleotide polymorphisms (SNPs), performed neutrality tests and estimated diversity indexes, and investigated their dependence from sample size. We identified 45 SNPs, six of which caused an amino acid substitution. Our results suggest a short span of LD in Populus nigra CAD4 gene. We identified carriers of different nonsynonymous SNPs in CAD4; those subjects are candidate to be used in classical breeding programs to obtain carriers of different combinations of functional polymorphisms. We showed that use of small sample size might lead to biased estimates of LD, neutrality tests, and diversity indexes.