Cinnamyl Alcohol Dehydrogenase Activity Research Articles

CAD1 and CCR2 protein complex formation in monolignol biosynthesis in Populus trichocarpa.

Lignin is the major phenolic polymer in plant secondary cell walls and is polymerized from monomeric subunits, the monolignols. Eleven enzyme families are implicated in monolignol biosynthesis. Here, we studied the functions of members of the cinnamyl alcohol dehydrogenase (CAD) and cinnamoyl-CoA reductase (CCR) families in wood formation in Populus trichocarpa, including the regulatory effects of their transcripts and protein activities on monolignol biosynthesis. Enzyme activity assays from stem-differentiating xylem (SDX) proteins showed that RNAi suppression of PtrCAD1 in P.trichocarpa transgenics caused a reduction in SDX CCR activity. RNAi suppression of PtrCCR2, the only CCR member highly expressed in SDX, caused a reciprocal reduction in SDX protein CAD activities. The enzyme assays of mixed and coexpressed recombinant proteins supported physical interactions between PtrCAD1 and PtrCCR2. Biomolecular fluorescence complementation and pull-down/co-immunoprecipitation experiments supported a hypothesis of PtrCAD1/PtrCCR2 heterodimer formation. These results provide evidence for the formation of PtrCAD1/PtrCCR2 protein complexes in monolignol biosynthesis inplanta.

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.

Effect of Burkholderia contaminans on Postharvest Diseases and Induced Resistance of Strawberry Fruits.

This study takes strawberry-fruits as the test material and discusses the effect of Burkholderia contaminans B-1 on preventing postharvest diseases and inducing resistance-related substances in strawberry-fruits. Soaking and wound inoculating is performed to analyze the inhibitory effects of different treatment solutions on the gray mold of postharvest strawberry-fruits. The count of antagonistic bacteria colonies in the wound is found, and the dynamic growth of antagonistic bacteria and the pathogenic fungus is observed by electron microscopy. The results indicated that, either by soaking/wound-inoculating, the fermentation and suspension of antagonistic bacteria significantly reduced the incidence of postharvest diseases of strawberry-fruits. With wound inoculation, the inhibition rate of antagonist fermentation and suspension (1 × 1010 cfu/ml) respectively reached 77.4% and 66.7%. It also led to a significant increase in the activity of resistance-related enzymes, i.e., phenylalanine ammonia lyase (PAL), 4-coumarate coenzyme A ligase (4CL), cinnamate-4-hydroxylase (C4H) and chalcone isomerase (CHI). On 1 d and 2 d post-treatment, the activity of 4CL was respectively 3.78 and 6.1 times of the control, and on 5 d, the activity of PAL was increased by 4.47 times the control. The treatment of antagonistic bacteria delayed the peaking of cinnamyl-alcohol dehydrogenase (CAD) activity and promoted the accumulation of lignin and total phenols. The antagonistic bacteria could be well colonized in the wounds. On 4–5 d post-inoculation, the count of colonies was 108 times of that upon inoculation. Electronmicroscopy indicated that the antagonistic bacteria delayed the germination of pathogenic spores in the wounds, and inhibited further elongations of the mycelia.

Overexpression of Sedum alfredii cinnamyl alcohol dehydrogenase increases the tolerance and accumulation of cadmium in Arabidopsis

Plant cell walls protect plants from heavy metal stress by resistance, transport and the adsorption process. Cell walls are impregnated with lignin, which enables structural integrity, long-distance water transport and protects plants from many types of stress. Cinnamyl alcohol dehydrogenase (CAD) is an essential enzyme that participates in the final step of the phenylpropanoid lignin biosynthetic pathway and plays an important role in the stress resistance process. In this study, we demonstrated that a CAD-encoding gene in the hyper-accumulating Sedum alfredii Hance, SaCAD, is constitutively expressed in all tissues. While the abundance of the SaCAD transcript is affected by cadmium (Cd) stress, it is upregulated in roots, stems and leaves during Cd treatment. Overexpression of SaCAD in transgenic Arabidopsis thaliana significantly increased CAD activities. Compared with the wild type (WT) plants, the Cd concentrations of SaCAD-overexpressing plants increased in the leaves and roots under Cd stress. The increased fixation of Cd to the thickened cell wall in the SaCAD-overexpressing A. thaliana plants resulted in better growth when the plants were grown in Cd stress conditions. In agreement with these data, SaCAD-overexpressing plants exhibited higher Cd tolerance compared to the wild type (WT) with higher chlorophyll and proline (Pro) contents and antioxidant enzyme activity, as well as a lower methane dicarboxylic aldehyde (MDA) content, electric conductivity and reactive oxygen species when exposed to Cd stress due to a lower amount of Cd distributed in the cytoplasm which is the most site of cytosolic metabolism. SaCAD was found to localize to the cytoplasm of tobacco cells. NMT analysis of the root tips from transgenic A. thaliana lines during Cd stress confirmed that SaCAD-overexpressing plants were capable of retaining more Cd in the cell wall in the Cd-supplied growth medium, which provided additional evidence for the potential role of SaCAD in heavy metal ion compartmentation and detoxification. In summary, we concluded that SaCAD performs critical functions in plants: Cd absorption and fixation to lignified cell wall during stress conditions.

Comparative effects of L-DOPA and velvet bean seed extract on soybean lignification

ABSTRACTVelvet bean (Mucuna pruriens) is an efficient cover forage that controls weeds, pathogens and nematodes, and the non-protein amino acid L-3,4-dihydroxyphenylalanine (L-DOPA) is its main allelochemical. The effects of 3 g L−1 of an aqueous extract of velvet bean seeds, along with 0.5 mM L-DOPA for comparison, were evaluated in roots, stems and leaves of soybean (Glycine max). The activities of phenylalanine ammonia lyase (PAL) and cinnamyl alcohol dehydrogenase (CAD) were determined, along with the lignin content and its monomeric composition. The results revealed similar effects caused by L-DOPA and the aqueous extract. Both treatments reduced PAL and CAD activities, lignin, and lignin monomer contents in roots; PAL and CAD activities in stems, and CAD activity in leaves. These findings provide further evidence that the effects of velvet bean cover forage on root lignification were due to the L-DOPA, its major allelochemical.

Characteristics of chilling injury-induced lignification in kiwifruit with different sensitivities to low temperatures

The extent of chilling injury (CI) of kiwifruit varies greatly among cultivars. The characteristics of CI-induced lignification and the effects of 1-methylcyclopropene (1-MCP) on CI of kiwifruit has been studied on Actinidia chinensis var. chinensis ‘Hongyang’ and A. chinensis var. deliciosa ‘Xuxiang’. 1-MCP delayed and reduced the CI proportion of the subcutaneous flesh. CI-induced lignification was mainly concentrated in the flesh of ‘Hongyang’ fruit, but in the core of ‘Xuxiang’. The lignified cells were formed by the deposition of lignin particles on the inside of the cell wall. The accumulation of lignin in the flesh of both cultivars was decreased after treating with 1-MCP, but the more serious lignification was observed in core tissues, especially those of ‘Xuxiang’. Activities of phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), and peroxidase (POD) involved in lignin synthesis were analyzed. 1-MCP increased PAL and POD activities in both flesh and core tissues of the two cultivars, whereas CAD activity was enhanced in the core and decreased in the flesh. The results indicated that 1-MCP is effective in controlling the occurrence of CI, but the associated induced fruit core lignification in particular cultivars can be serious.

Stimulatory involvement of abscisic acid in wound suberization of postharvest kiwifruit

Wound-induced suberization is an essentially protective healing process for fruit to reduce water loss and avoid infecting. However, cognate mechanisms that regulate this process are little known. To expand our knowledge of suberization induced by wounding, a wound-healing investigation together with metabolite profiling study was conducted in postharvest kiwifruit (Actinidia deliciosa). The development of suberization in wounded fruit was demonstrated by autofluorescence observation and toluidine blue staining at 1–4day (d) after wounding. Activities of phenylalanine ammonia-lyase (PAL), cinnamyl-alcohol dehydrogenase (CAD) and peroxidase (POD) in wound-healing tissue were enhanced by abscisic acid (ABA). The constituent analysis of suberin including polyphenolics (SPP) and polyaliphatics (SPA) proved that exogenous ABA increased the content levels of total phenols, total flavonoids and alkanes, alkenes, alcohols, alkane acids, olefine acids, esters, glycerides and vitamin E in wound-healing tissue. Results suggested that ABA stimulated suberization through the activation of PAL, CAD and POD to accelerate wound-healing of wounded kiwifruit.

The Enzyme Activity and Substrate Specificity of Two Major Cinnamyl Alcohol Dehydrogenases in Sorghum (Sorghum bicolor), SbCAD2 and SbCAD4.

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step in monolignol biosynthesis, reducing sinapaldehyde, coniferaldehyde, and p-coumaraldehyde to their corresponding alcohols in an NADPH-dependent manner. Because of its terminal location in monolignol biosynthesis, the variation in substrate specificity and activity of CAD can result in significant changes in overall composition and amount of lignin. Our in-depth characterization of two major CAD isoforms, SbCAD2 (Brown midrib 6 [bmr6]) and SbCAD4, in lignifying tissues of sorghum (Sorghum bicolor), a strategic plant for generating renewable chemicals and fuels, indicates their similarity in both structure and activity to Arabidopsis (Arabidopsis thaliana) CAD5 and Populus tremuloides sinapyl alcohol dehydrogenase, respectively. This first crystal structure of a monocot CAD combined with enzyme kinetic data and a catalytic model supported by site-directed mutagenesis allows full comparison with dicot CADs and elucidates the potential signature sequence for their substrate specificity and activity. The L119W/G301F-SbCAD4 double mutant displayed its substrate preference in the order coniferaldehyde > p-coumaraldehyde > sinapaldehyde, with higher catalytic efficiency than that of both wild-type SbCAD4 and SbCAD2. As SbCAD4 is the only major CAD isoform in bmr6 mutants, replacing SbCAD4 with L119W/G301F-SbCAD4 in bmr6 plants could produce a phenotype that is more amenable to biomass processing.

The effects of ultraviolet radiation on the brown midrib mutants of Zea mays and Sorghum bicolor

The brown-midrib varieties (bm/bmr) of corn (Zea mays) and sorghum (Sorghum bicolor) have gained attention as both forage material and feedstock for biofuel. These varieties are attractive due to their low-lignin content resulting from alterations in the phenylpropanoid pathway. These bm/bmr varieties have impaired activity of cinnamyl alcohol dehydrogenase (CAD) or caffeate/5 hydroxyferulate O-methyl transferase (COMT) enzymes. We examined the effects of ultraviolet-B radiation (UV-B; 280–320 nm) on bm3 corn and bmr6 sorghum growing in a UV-transparent greenhouse in southern Minnesota. Plants grew under filters that either transmitted 2.8% (mylar) or 90% (aclar) of UV-B for 71 days. We monitored UV-screening effectiveness and chlorophyll fluorescence of leaves with pulse-amplitude modulated fluorometers. In order to explain differences in UV-screening effectiveness, we estimated concentrations of bulk-soluble UV-absorbing compounds (λ = 300 and 370 nm). We also measured final cellulose, hemicellulose and lignin concentrations of plants using a fiber analyzer. Epidermal transmittance of UV was reduced under aclar filters in both species and decreased from 41.6 to 1.9% in corn and 66.4 to 0.98% in sorghum. Increased epidermal screening may be related to the 35–39 and 37–54% higher concentrations of UV-absorbing compounds in plants growing under aclar. Concentrations of lignin were 1.7% lower in corn and 1.1% higher in sorghum in plants under aclar treatments. These contrasting results may be the result of alterations in the phenylpropanoid pool associated with each bm/bmr variety.

Research on energy metabolism, lignification and veil opening in postharvest white mushroom ( Agaricus bisporus ) under high O 2 /CO 2 controlled atmospheres (HOC-CA)

White mushroom is an abundant nutritional but perishable product; veil opening and stipe lignification are two of the obvious characte-ristics of senescence. In order to study the relationship between the energy charge, veil opening and lignification of the white mushroom, high O2/CO2 controlled atmospheres (HOC-CA) were utilized at 2±1 °C. The treatments comprised the following 100% O2, 80% O2 + 20% CO2, 60% O2 + 40% CO2, 40% O2 + 60% CO2 respectively, with air as the control. In this study, sensorial and physiological qualities, adenosine triphosphate (ATP) content, energy charge level, lignin content, phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POD) activity were studied. The optimal condition 80% O2 + 20% CO2 treatment inhibited both respiration rate and veil opening of the mushroom; suppressed PAL, POD and CAD activity. It also retarded the reduction of ATP level, energy charge level, and growth of lignin content. Exogenous ATP had an effect on inhibiting veil opening and lignification. These results indicated that there was a negative correlation between the ATP content, cap opening and stipe lignification. Appropriate HOC-CA maintained high energy, preserved the white mushroom well and extended the storage time to 24 days.

The effect of 1-methylcyclopropene, methyl jasmonate and methyl salicylate on lignin accumulation and gene expression in postharvest ‘Xuxiang’ kiwifruit during cold storage

Kiwifruit is susceptible to chilling injury during storage at 0°C, which causes lignin accumulation, a substantial impact to fruit quality. Lignin has been observed in the pulp and core of kiwifruit (Actinidia deliciosa cv. Xuxiang), developing gradually from the stem end to the calyx end. Fruit firmness can be maintained at a higher level during cold storage through application of 1-methylcyclopropene (1-MCP), although with dramatically enhanced activities of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), cinnamyl-alcohol dehydrogenase (CAD, EC 1.1.1.195), and peroxidase (POD, EC 1.11.1.7) as well as lignification, which occurred during the later storage period. Methyl jasmonate (MeJA) and methyl salicylate (MeSA) significantly inhibited lignification at later stages. Expression profiles of the four lignin-related genes (i.e., AcPAL, AcCAD, AcPOD1, and AcPOD2) were assessed, 1-MCP markedly increased the expression levels of AcPOD1 in fruit core tissue. Together, these results illustrate that MeJA and MeSA can inhibit lignin accumulation to some degree; however, 1-MCP may aggravate lignin deposition, especially in core tissue, mainly via up-regulating AcPOD1, which is involved in lignin synthesis.

Characterization of novel Brown midrib 6 mutations affecting lignin biosynthesis in sorghum.

The presence of lignin reduces the quality of lignocellulosic biomass for forage materials and feedstock for biofuels. In C4 grasses, the brown midrib phenotype has been linked to mutations to genes in the monolignol biosynthesis pathway. For example, the Bmr6 gene in sorghum (Sorghum bicolor) has been previously shown to encode cinnamyl alcohol dehydrogenase (CAD), which catalyzes the final step of the monolignol biosynthesis pathway. Mutations in this gene have been shown to reduce the abundance of lignin, enhance digestibility, and improve saccharification efficiencies and ethanol yields. Nine sorghum lines harboring five different bmr6 alleles were identified in an EMS-mutagenized TILLING population. DNA sequencing of Bmr6 revealed that the majority of the mutations impacted evolutionarily conserved amino acids while three-dimensional structural modeling predicted that all of these alleles interfered with the enzyme's ability to bind with its NADPH cofactor. All of the new alleles reduced in vitro CAD activity levels and enhanced glucose yields following saccharification. Further, many of these lines were associated with higher reductions in acid detergent lignin compared to lines harboring the previously characterized bmr6-ref allele. These bmr6 lines represent new breeding tools for manipulating biomass composition to enhance forage and feedstock quality.

Lignan enhancement in hairy root cultures of Linum album using coniferaldehyde and methylenedioxycinnamic acid

ABSTRACTFeeding experiments with hairy root cultures of Linum album have established that the extracellular coniferaldehyde is a good precursor for production of two lignans: lariciresinol (LARI) and pinoresinol (PINO). The accumulation of the LARI, PINO, and podophyllotoxin (PTOX) in hairy roots were enhanced about 14.8-, 8.7-, and 1.5-fold (107.61, 8.7 and 6.42 µg g−1 Fresh Wight), respectively, by the addition of coniferaldehyde (2 mM) to the culture media (after 24 hr). This result was correlated with an increase pinoresinol/lariciresinol reductase (PLR) expression gene and cinnamyl alcohol dehydrogenase (CAD) activity in the fed hairy roots. Adding 3,4-(methylendioxy)cinnamic acid (MDCA) precursor did not influence on the lignans accumulation, but the lignin content of the hairy roots was increased. Moreover, the expression genes of phenylalanine ammonialyase (PAL), CAD, and cinnamoyl-CoA reductase (CCR) were influenced after feeding hairy roots with MDCA.

Effects of Combined Aqueous Chlorine Dioxide and Chitosan Coatings on Microbial Growth and Quality Maintenance of Fresh-Cut Bamboo Shoots (Phyllostachys praecox f. prevernalis.) During Storage

This study was conducted to investigate the effects of aqueous chlorine dioxide (ClO2) combined with chitosan coating on microbial growth and quality maintenance of fresh-cut bamboo shoots during cold storage. Fresh-cut shoots respiration rate, weight loss, firmness, lignin, colour, microbial and activities of phenylalanine ammonia lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), peroxidase (POD) and polyphenol oxidase (PPO) were measured. The results indicated that treatment with 28 mg L−1 ClO2 plus chitosan coating inhibited increase in respiration rate and firmness, delayed browning and lignification and reduced microorganism counts of total aerobic bacteria and yeasts and moulds compared to control treatment. The efficiency was better than that of ClO2 or chitosan treatment alone. Furthermore, combination of ClO2 and chitosan inhibited the activities of PAL, CAD, POD and PPO throughout the storage period. Our study suggests that ClO2 plus chitosan coating treatment may be a promising method of maintaining fresh-cut bamboo shoot quality and extending their postharvest life.

Role of phenolic acids and enzymes of phenylpropanoid pathway in resistance of chayote fruit (Sechium edule) against infestation by melon fly, Bactrocera cucurbitae

AbstractMelon fly is a serious pest of cucurbits all over the world causing huge losses to yield. However, the only exception is the chayote fruit (Sechium edule) that shows resistance to melon fly infestation. Studies on culture of melon fly indicated the absence of plant traits resisting oviposition on chayote fruit. However, the melon fly was unable to complete its life cycle successfully on chayote showing that factors inhibiting larval development in melon fly could be attributed to biochemical constituents. Studies were, therefore, carried out to compare the biochemical responses of chayote, a melon fly resistant species and bitter gourd, a susceptible species to melon fly infestation with regard to the levels of phenolic acids and activities of the enzymes of phenylpropanoid pathway (PPP) leading to synthesis of lignin. The resistant chayote exhibited significantly higher accumulation of lignin associated with higher activities of phenylalanine ammonia‐lyase (PAL), tyrosine ammonia‐lyase (TAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POD). On the contrary, the susceptible bitter gourd recorded lower activities of PAL, CAD and POD and a decreasing trend of TAL during infestation associated with a lower lignin content. The monomer composition of lignin in the resistant chayote showed twofold higher level of guaiacyl (G) and syringyl (S) units compared to susceptible bitter gourd and the G/S ratio during infestation increased in chayote while decreasing in bitter gourd. The levels of PPP intermediates, p‐coumaric acid was higher in chayote while p‐hydroxy benzoic acid, a chemo‐attractant, was higher in bitter gourd. Incorporation of p‐coumaric acid in the larval diet strongly inhibited larval growth even as p‐hydroxy benzoic acid promoted growth confirming the direct role of p‐coumaric acid in conferring resistance to chayote. The level of salicylic acid, a signal molecule involved in induction of defence response, was higher in chayote compared to bitter gourd. Chayote also exhibited higher level of activity of POD in the phloem exudates compared to bitter gourd. The higher concentration of sugars in exudates of chayote might act like signalling molecules causing activation of plant genes, especially of the phenylpropanoid biosynthesis pathway and possibly produce an osmotic effect inducing resistance against the melon fly. Thus, the study revealed that the resistance in chayote to melon fly infestation is a complex, multi‐layered process in which the activities of PPP enzymes generating phenolic intermediates leading to lignin biosynthesis and the composition of exudates appear to play significant roles. Besides, the study also indicated that different forms of lignin might play a role in the resistance of chayote against melon fly infestation.

Ozone-elicited secondary metabolites in shoot cultures of Melissa officinalis L.

The effects of ozone treatment (200 ppb, 3 h) on the accumulation of main secondary metabolites have been investigated in Melissa officinalis (lemon balm) aseptic shoot cultures in order to evaluate the biotechnological application of this gas for improving the yield of secondary metabolites of medicinal plants. During the treatment, we found (1) an activation of enzymes involved in phenolic metabolism [as confirmed by the increase of shikimate dehydrogenase, phenylalanine ammonia-lyase and cinnamyl alcohol dehydrogenase activities (about twofold higher than controls)], (2) a development of cellular barriers with a higher degree of polymerization of monolignols [as indicated by the increase of lignin (+23 % compared to controls)], (3) an accumulation of phenolic compounds, in particular rosmarinic acid (about fourfold compared to control plants cultivated in filtered air) and (4) an increase of antioxidant capacity (as documented by the improved 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity). The effect of ozone as elicitor of the production of secondary metabolites in lemon balm shoot cultures was dependent on the specific regime, the time of exposure and the concentration of the stressor. After the end of the treatment, we found cell death and hydrogen peroxide (H2O2) deposition concomitant with a prolonged superoxide anion-generation, suggesting that a transient oxidative burst had occurred.

Relationship Between Lignin Metabolism and Lodging Resistance of Culm in Buckwheat

To disclose the relationship between lignin content and lodging resistance, field experiments were conducted in 2012 and 2013 to investigate the dynamic changes of lignin content, the activities of four enzymes involved in lignin metabolism in culm, as well as the snapping resistance and lodging index of buckwheat. 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 2 nd internode of four cultivars with different lodging resistance. The snapping resistance and culm lodging index were determined at anthesis, grain filling, and maturity stages. The lignin content varied significantly among cultivars. The XD cultivar, which showed high resistance to culm snapping and lodging, had higher lignin content and higher activity of PAL, TAL, 4CL, and CAD than other cultivars. The lignin content was significantly negatively correlated with the actual lodging percent ( r = -0.844, P 0.05), 0.927 ( P < 0.01), and 0.862 ( P < 0.01), respectively. In conclusion, our results indicate that the activities of PAL and 4CL were the key enzymes that influenced the lignin content, and that the lignin content can be used as main indicators to evaluate the lodging resistance of buckwheat.

Trichoderma harzianumelicits induced resistance in sunflower challenged byRhizoctonia solani

To investigate the efficacy of Trichoderma harzianum NBRI-1055 (denoted as 'T-1055') in suppression of seedling blight of sunflower caused by Rhizoctonia solani Kühn and their impact on host defence responses. T-1055 was applied as seed treatment, soil application and combined application (seed treatment+soil application). Higher protection afforded by combined application of T-1055 was associated with the marked induction of phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO), peroxidase (PO) and cinnamyl alcohol dehydrogenase (CAD) activities. The activities of PAL and PPO reached maximum at 10days after sowing (DAS), while PO and CAD levels reached maximum at 12 DAS. This was further supported by the accumulation of total phenolic content that showed an increase up to threefold at 14 DAS. In addition, HPLC analysis revealed that the contents of ferulic and p-coumaric acids increased by 6·3 and 4·6 times, respectively, at 14 DAS. Amount of gallic acid was also little more than double. Lignin deposition in sunflower root increased by 2·7, 3·4 and 3·7 times through combined application of T-1055 at 16, 18 and 20 DAS, respectively. Combined application also increased the accumulation of PR-2 and PR-3 proteins by 3·3 and 3·9 times, respectively, at 12 DAS in followed by seed treatment alone. The combined application of T-1055 triggered defence responses in an enhanced level in sunflower than the soil and seed alone and provided better protection against Rhizoctonia seedling blight. Rhizospheric fungal bioagent 'T-1055' can enhance protection in sunflower against the R.solani pathogen through augmented elicitation of host defence responses.

Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon.

BackgroundLignin is a significant barrier in the conversion of plant biomass to bioethanol. Cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the pathway of lignin monomer biosynthesis. Brown midrib mutants in Zea mays and Sorghum bicolor with impaired CAD or COMT activity have attracted considerable agronomic interest for their altered lignin composition and improved digestibility. Here, we identified and functionally characterized candidate genes encoding CAD and COMT enzymes in the grass model species Brachypodium distachyon with the aim of improving crops for efficient biofuel production.ResultsWe developed transgenic plants overexpressing artificial microRNA designed to silence BdCAD1 or BdCOMT4. Both transgenes caused altered flowering time and increased stem count and weight. Downregulation of BdCAD1 caused a leaf brown midrib phenotype, the first time this phenotype has been observed in a C3 plant. While acetyl bromide soluble lignin measurements were equivalent in BdCAD1 downregulated and control plants, histochemical staining and thioacidolysis indicated a decrease in lignin syringyl units and reduced syringyl/guaiacyl ratio in the transgenic plants. BdCOMT4 downregulated plants exhibited a reduction in total lignin content and decreased Maule staining of syringyl units in stem. Ethanol yield by microbial fermentation was enhanced in amiR-cad1-8 plants.ConclusionThese results have elucidated two key genes in the lignin biosynthetic pathway in B. distachyon that, when perturbed, may result in greater stem biomass yield and bioconversion efficiency.

English

The present study evaluated the effects of heat-shock treatment (washing in 48&deg;C distilled water for 5 min) on quality of green asparagus spears stored at 25 or 4&deg;C for 7 days. The results showed that the quality characteristics including toughness, lignin and cellulose contents, and phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase and peroxidase activities were increasing in both heat-shocked and control spears stored at 25&deg;C; however, the heat-shocked spears exhibited lower degrees of increases than control spears. Similar increasing trends but with even smaller magnitudes in these characteristics were obtained from the heat-shocked spears stored at 4&deg;C. Heat-shocked spears stored at 25 or 4&deg;C also demonstrated lesser proliferations of total mesophilic aerobes,&nbsp;Escherichia&nbsp;coli&nbsp;and mold than their respective controls. Thus, a combination of 48&deg;C heat-shock treatment and 4&deg;C cold storage can be used to conserve the quality of stored green asparagus spears. &nbsp; Key words:&nbsp;Green asparagus, heat-shock, lignification, microbial load, storage.