Cinnamyl Alcohol Dehydrogenase Research Articles

Comparative transcriptomic analysis provides insights into the genetic networks regulating oil differential production in oil crops

BackgroundPlants differ more than threefold in seed oil contents (SOCs). Soybean (Glycine max), cotton (Gossypium hirsutum), rapeseed (Brassica napus), and sesame (Sesamum indicum) are four important oil crops with markedly different SOCs and fatty acid compositions.ResultsCompared to grain crops like maize and rice, expanded acyl-lipid metabolism genes and relatively higher expression levels of genes involved in seed oil synthesis (SOS) in the oil crops contributed to the oil accumulation in seeds. Here, we conducted comparative transcriptomics on oil crops with two different SOC materials. In common, DIHYDROLIPOAMIDE DEHYDROGENASE, STEAROYL-ACYL CARRIER PROTEIN DESATURASE, PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE, and oil-body protein genes were both differentially expressed between the high- and low-oil materials of each crop. By comparing functional components of SOS networks, we found that the strong correlations between genes in “glycolysis/gluconeogenesis” and “fatty acid synthesis” were conserved in both grain and oil crops, with PYRUVATE KINASE being the common factor affecting starch and lipid accumulation. Network alignment also found a conserved clique among oil crops affecting seed oil accumulation, which has been validated in Arabidopsis. Differently, secondary and protein metabolism affected oil synthesis to different degrees in different crops, and high SOC was due to less competition of the same precursors. The comparison of Arabidopsis mutants and wild type showed that CINNAMYL ALCOHOL DEHYDROGENASE 9, the conserved regulator we identified, was a factor resulting in different relative contents of lignins to oil in seeds. The interconnection of lipids and proteins was common but in different ways among crops, which partly led to differential oil production.ConclusionsThis study goes beyond the observations made in studies of individual species to provide new insights into which genes and networks may be fundamental to seed oil accumulation from a multispecies perspective.

The Integration of Transcriptome and Metabolome Analyses Provides Insights into the Determinants of the Wood Properties in Toona ciliata.

Toona ciliata, also known as Chinese mahogany, is a high-quality and fast-growing wood species with a high economic value. The wood properties of T. ciliata of different provenances vary significantly. In this study, we conducted comprehensive transcriptome and metabolome analyses of red and non-red T. ciliata wood cores of different provenances to compare their wood properties and explore the differential metabolites and genes that govern the variation in their wood properties. Through combined analyses, three differential genes and two metabolites were identified that are possibly related to lignin synthesis. The lignin content in wood cores from T. ciliata of different provenances shows significant variation following systematic measurement and comparisons. The gene Tci09G002190, one of the three differential genes, was identified as a member of the CAD (Cinnamyl alcohol dehydrogenase) gene family of T. ciliata, which is associated with lignin synthesis. Our data provide insights into the determinants of the wood properties in T. ciliata, providing a solid foundation for research into the subsequent mechanisms of the formation of T. ciliata wood.

Physiological and transcriptomic comparisons shed light on the cold stress response mechanisms of Dendrobium spp.

Dendrobium spp. comprise a group of tropical orchids with ornamental and medicinal value. Dendrobium spp. are sensitive to low temperature, and the underlying cold response regulatory mechanisms in this group are unclear. To understand how these plants respond to cold stress, we compared the transcriptomic responses of the cold-tolerant cultivar 'Hongxing' (HX) and the cold-sensitive cultivar 'Sonia Hiasakul' (SH) to cold stress. Chemometric results showed that the physiological response of SH in the later stages of cold stress is similar to that of HX throughout the cold treatment. Orthogonal partial least squares discriminant analysis (OPLS-DA) revealed that soluble protein content and peroxidase activity are key physiological parameters for assessing the cold tolerance of these two Dendrobium spp. cultivars. Additionally, weighted gene co-expression network analysis (WGCNA) results showed that many cold response genes and metabolic pathways significantly associated with the physiological indices were enriched in the 12 detected modules. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) enrichment analyses of the 105 hub genes showed that Dendrobium spp. adapt to cold stress by regulating signal transduction, phytohormones, transcription factors, protein translation and modification, functional proteins, biosynthesis and metabolism, cell structure, light, and the circadian clock. Hub genes of the cold stress response network included the remorin gene pp34, the abscisic acid signaling pathway-related genes PROTEIN PHOSPATASE 2C (PP2C), SNF1-RELATED PROTEIN KINASE 2 (SnRK2), ABRE-BINDING FACTOR 1 (ABF1) and SKI-INTERACTING PROTEIN 17 (SKIP17), the Ca2+ signaling-related GTP diphosphokinase gene CRSH1, the carbohydrate-related gene STARCH SYNTHASE 2 (SS2), the cell wall biosynthesis gene CINNAMYL ALCOHOL DEHYDROGENASE (CAD7), and the endocytosis-related gene VACUOLAR PROTEIN SORTING-ASSOCIATED PROTEIN 52A (VPS52A). The cold-responsive genes and metabolic pathways of Dendrobium spp. revealed in this study provide important insight to enable the genetic enhancement of cold tolerance in Dendrobium spp., and to facilitate cold tolerance breeding in related plants.

Exogenous Melatonin Application Accelerated the Healing Process of Oriental Melon Grafted onto Squash by Promoting Lignin Accumulation.

Melatonin (MT) is a vital hormone factor in plant growth and development, yet its potential to influence the graft union healing process has not been reported. In this study, we examined the effects of MT on the healing of oriental melon scion grafted onto squash rootstock. The studies indicate that the exogenous MT treatment promotes the lignin content of oriental melon and squash stems by increasing the enzyme activities of hydroxycinnamoyl CoA ligase (HCT), hydroxy cinnamaldehyde dehydrogenase (HCALDH), caffeic acid/5-hydroxy-conifer aldehyde O-methyltransferase (COMT), caffeoyl-CoA O-methyltransferase (CCoAOMT), phenylalanine ammonia-lyase (PAL), 4-hydroxycinnamate CoA ligase (4CL), and cinnamyl alcohol dehydrogenase (CAD). Using the oriental melon and squash treated with the exogenous MT to graft, the connection of oriental melon scion and squash rootstock was more efficient and faster due to higher expression of wound-induced dedifferentiation 1 (WIND1), cyclin-dependent kinase (CDKB1;2), target of monopteros 6 (TMO6), and vascular-related NAC-domain 7 (VND7). Further research found that the exogenous MT increased the lignin content of the oriental melon scion stem by regulating CmCAD1 expression, and then accelerated the graft healing process. In addition, the root growth of grafted seedlings treated with the exogenous MT was more vigorous.

Genome-wide identification and expression patterns in response to signals from cadmium of StCADs gene family in potato (Solanum tuberosum L.)

BackgroundWith the rapid development of the economy and society, soil pollution is becoming more and more serious. Heavy metal cadmium (Cd) pollution is one of the typical problems, which poses a potentially serious threat to crop production and human health. Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin synthesis and plays an important role in plant resistance to external stress. In this study, combined with bioinformatics analysis and expression pattern analysis, the members of the potato CAD family were identified, and their physical and chemical properties, evolutionary characteristics and chromosome location were clarified, as well as their regulatory effects on Cd tolerance.ResultsA total of 50 StCAD genes belonging to 6 subfamilies were obtained, and all of them were located in the cytoplasm. Members of the same family had similar gene structures and functional domains. The promoter region of each StCAD family member contains at least 5 or more abiotic stress response elements, indicating that the family had potential functions in regulating stress. According to the expression pattern analysis, most genes in this family were upregulated after Cd stress, further enhanced CAD activity and significantly promoted lignin accumulation in potato roots.ConclusionIn summary, the StCAD family plays an important role in potato response to Cd stress. This study lays a foundation for further studies on the functions of the StCAD family and provides candidate genes for Cd resistance molecular breeding in potato.Graphical

Integrated metabolomic and transcriptomic dynamic profiles of endopleura coloration during fruit maturation in three walnut cultivars

BackgroundThe color of endopleura is a vital factor in determining the economic value and aesthetics appeal of nut. Walnuts (Juglans) are a key source of edible nuts, high in proteins, amino acids, lipids, carbohydrates. Walnut had a variety endopleura color as yellow, red, and purple. However, the regulation of walnut endopleura color remains little known.ResultsTo understand the process of coloration in endopleura, we performed the integrative analysis of transcriptomes and metabolomes at two developmental stages of walnut endopleura. We obtained total of 4,950 differentially expressed genes (DEGs) and 794 metabolites from walnut endopleura, which are involved in flavonoid and phenolic biosynthesis pathways. The enrichment analysis revealed that the cinnamic acid, coniferyl alcohol, naringenin, and naringenin-7-O-glucoside were important metabolites in the development process of walnut endopleura. Transcriptome and metabolome analyses revealed that the DEGs and differentially regulated metabolites (DRMs) were significantly enriched in flavonoid biosynthesis and phenolic metabolic pathways. Through co-expression analysis, CHS (chalcone synthase), CHI (chalcone isomerase), CCR (cinnamoyl CoA reductase), CAD (cinnamyl alcohol dehydrogenase), COMT (catechol-Omethyl transferase), and 4CL (4-coumaroyl: CoA-ligase) may be the key genes that potentially regulate walnut endopleura color in flavonoid biosynthesis and phenolic metabolic pathways.ConclusionsThis study illuminates the metabolic pathways and candidate genes that underlie the endopleura coloration in walnuts, lay the foundation for further study and provides insights into controlling nut’s colour.

Enhancing productivity, modifying biochemical parameters, and regulating the phenylpropanoid pathway in 'Le-Conte' pears through optimal protocatechuic acid treatments

BackgroundThis study aimed to investigate the impact of protocatechuic acid (PRC) treatments on the productivity and fruit quality of 'Le-Conte' pears, with a specific focus on productivity, stone cells content, and antioxidant activity. The research spanned over three consecutive cultivating seasons, with the first season serving as a preliminary study to determine the optimal PRC concentrations and the most effective number of spray applications. During the initial season, response surface methodology (RSM) was employed to optimize PRC concentration and application frequency. PRC was evaluated at concentrations ranging from 50 to 400 ppm, with treatment frequencies of either once or twice. Considering the optimal conditions obtained from RSM results, PRC treatments at 200 ppm and 300 ppm were applied twice, and their respective effects were studied in comparison to the control in the following seasons.ResultsRSM results indicated that PRC at 200 and 300 ppm, applied twice, once during full bloom and again three weeks later, yielded the most significant effects. Subsequent studies revealed that PRC treatments had a substantial impact on various aspects of fruit production and quality. Applying 300 ppm PRC once during full bloom and again three weeks later resulted in higher fruit set percentages, lower fruit abscission, and enhanced fruit yield compared to untreated trees. Additionally, the 200 ppm PRC treatment maintained physicochemical characteristics such as fruit color, increased total soluble solids (TSS), and total sugar, and maintained higher ascorbic acid content and antioxidant capacity in the fruits while reducing stone cells content and lignin. Notably, enzyme activities related to phenylpropanoid metabolism and stone cells, including phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-Coumarate-CoA Ligase (4CL), cinnamyl alcohol dehydrogenase (CAD), and cinnamoyl-CoA reductase (CCR), as well as peroxidase, polyphenol oxidase, and laccase, were significantly regulated by PRC treatments.ConclusionOverall, this study suggests that PRC treatments are suitable for enhancing pear yield and quality, with PRC at 200 ppm being the more recommended option over 300 ppm. This approach serves as an effective strategy for achieving a balance between enhancing the productivity and fruit quality of 'Le-Conte' pears.

Influence of myoinositol on post-ripening and softening of Prunus salicina ‘Wushan plum’

To investigate the impact of myoinositol (MI) immersion treatment on the storage characteristics of Prunus salicina ‘Wushan Plum’, we assessed myoinositol content, ascorbic acid content, weight loss, decay rate, alteration in color, titratable acidity (TA), soluble solids (TSS), firmness, cell wall metabolism-related compounds, enzyme activities, and levels of gene expression throughout a 15-day storage period at 25 °C. The results revealed that MI treatment could increase myoinositol and ascorbic acid contents of fruit during the pre-storage period, effectively delay the color transformation and decay progression of P. salicina ‘Wushan Plum’ while maintaining elevated levels of TA and TSS. Furthermore, it suppressed the increase in weight loss and the decline in firmness. Notably, the delay in softening was more pronounced in the group treated with 100 mg L−1 MI compared to other groups. MI treatment regulated the expression levels of various genes associated with cell wall metabolism, which resulted in reduced activities of polygalacturonase (PG), pectate lyase (PL), pectin methylesterase (PME), cellulase (Cx), and β-glucosidase (β-Glu), and increased activities of phenylalanine ammonia-lyase (PAL) and cinnamyl alcohol dehydrogenase (CAD). Furthermore, MI treatment maintained higher levels of covalently bound pectin (CSP), cellulose, and lignin, while decreasing the levels of water-soluble pectin (WSP) in the fruit. As a result, MI treatment could delay the depolymerization of cell wall polysaccharides and further delay the quality deterioration of P. salicina ‘Wushan Plum’ fruits under ambient storage environment and prolong the storage period.

The expression of lignin biosynthesis genes during plant development and effects of downregulated cinnamyl alcohol dehydrogenase (CAD) gene in bahiagrass (Paspalum notatum Flügge)

AbstractBahiagrass (Paspalum notatum Flügge) is a warm‐season grass where high lignin content limits its forage quality, negatively affecting animal performance. To create a new breeding method by genome editing, isolation and characterization of lignin biosynthesis genes and identification of their molecular relationships are essential. The caffeic acid O‐methyltransferase (COMT) and cinnamyl alcohol dehydrogenase (CAD) cDNA clones were isolated from bahiagrass, and their protein sequences showed high similarity to other C4 monocot species through phylogenetic analysis. Gene expression analysis of phenylalanine ammonia‐lyase (PAL), COMT and CAD, involved in crucial stages of lignin biosynthesis and lignin content, was conducted at different plant developmental stages. The highest gene expression levels of these genes were observed in the vegetative (V) and early reproductive stages, while the lignin content increased until the middle reproductive stage and remained constant thereafter. Additionally, to further understand the molecular relationships in lignin biosynthesis, the effect of CAD downregulation was analyzed in transgenic bahiagrass lines introduced with sorghum CAD antisense and RNAi vector obtained from a previous report. This led to a reduced lignin content and affected the expression of PAL and COMT working upstream of CAD. In the V stage, PAL expression was lower in transgenic lines compared to wild type (WT), while COMT expression showed no significant differences. However, PAL and COMT expression of transgenic lines in the middle reproductive stage (R2) was significantly higher than in the WT. These findings suggest that the downregulation of CAD gene expression affects PAL and COMT expression and induces a feedback system in the R2 stage. Lignin content influenced the phenotype of the transgenic plants with significantly reduced lignin, exhibiting a dwarf phenotype with shorter plant heights. The findings of this study can be applied to genome editing for the development of practical new breeding materials with improved digestibility in bahiagrass.

Postharvest caffeic acid dipping enhances disease resistance and storage capacity of ‘Zaosu’ pear fruit via regulating phenylpropane metabolism

Caffeic acid, a naturally occurring and safe phenolic compound, acts as a precursor in lignin synthesis within the phenylpropane metabolism pathway. The aims of this study was to evaluate the impacts of postharvest caffeic acid immersion on Alternaria rot and postharvest quality of ‘Zaosu’ pear fruit, as well as elucidate the underlying mechanisms involved. Results showed that postharvest caffeic acid dipping effectively inhibited spore germination and colony diameter of Alternaria alternata, as well as lesion diameter in A. alternata-inoculated pears. Caffeic acid treatment also delayed the increase of weight loss and decline in flesh firmness, increased total soluble solids content, and reduced surface yellowing of pears. Additionally, caffeic acid treatment enhanced the enzymatic activities and gene transcription levels of phenylalanine ammonialyase, 4-coumarate/coenzyme A ligase, cinnamate 4-hydroxylase, peroxidase, cinnamyl alcohol dehydrogenase, and polyphenol oxidase in pears. Meanwhile, the transcription levels of p-coumarate-3-hydroxylase, caffeic acid O-methyltransferase, cinnamoyl CoA reductase, caffeoyl shikimate esterase, hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase, caffeoyl CoA O-methyltransferase, ferulic acid 5-hydroxylase, and laccase genes were also up-regulated by caffeic acid treatment in pears. Furthermore, caffeic acid dipping enhanced phenylalanine, flavonoids, caffeic acid, total phenolic compounds, and lignin levels in the fruit. Therefore, caffeic acid exhibits promising potential for enhancing resistance against A. alternata and maintaining the storage capacity of pears via inducing phenylpropane metabolism.

Calcium chloride treatment maintains quality and enhances antioxidant capacity of fresh-cut carrots through modulating DcCMLs-DcCAMTA3-mediated phenylpropanoid pathway

Whitening and microbial infection are two major reasons to induce quality deterioration in fresh-cut carrots. Calcium chloride (CaCl2) could enhance the quality attributes in fresh-cut carrots, but the molecular mechanism is still not illuminated. In this study, CaCl2 treatment suppressed the increases of whiteness index and total bacterial count as well as enhanced the antioxidant capacity of fresh-cut carrots. In addition, CaCl2 treatment promoted phenolics accumulation by upregulating the transcripts and enzyme activity of phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H) and 4-coumarate coenzyme A ligase (4CL) in phenylpropanoid pathway. However, this treatment inhibited the increase of enzyme activity and gene expression of cinnamyl alcohol dehydrogenase (CAD), thus suppressing lignin synthesis and whitening process in fresh-cut carrots. Moreover, a calmodulin-binding transcription activator (CAMTA) transcription factor DcCAMTA3, was induced in response to CaCl2 treatment. DcCAMTA3 activated the transcription of DcPAL1, DcC4H and Dc4CL1 genes and repressed the expression of DcCAD1 gene by directly targeting the CG-box. DcCML3/11, a calmodulin-like protein, interacted with DcCAMTA3, and this interaction enhanced DcCAMTA3-mediated transcriptional activation of DcPAL1, DcC4H and Dc4CL1 and repression of DcCAD1. Therefore, CaCl2 treatment is effective in maintaining quality and enhancing antioxidant capacity of fresh-cut carrots by the cooperative action of DcCMLs and DcCAMTA3 in positively regulating phenylpropanoid pathway.

Comparative characterization and expression analysis revealed cinnamyl alcohol dehydrogenase genes differentially responding to Colletotrichum fructicola in woodland and cultivated strawberries

BACKGROUND: Strawberry (Fragaria×ananassa) production is hampered by anthracnose caused by Colletotrichum spp. Cinnamyl alcohol dehydrogenase (CAD) catalyzes lignin biosynthesis to fortify plant cell wall upon various stresses, but their involvement in strawberry resistance is elusive. OBJECTIVE: Current work aims to uncover the involvement of CAD genes in both woodland and cultivated strawberry responses to Colletotrichum spp. METHODS: Genome-wide identification of CAD genes was updated in cultivated strawberry. Comparative molecular characterization integrated with pathogenesis phenotyping after C. fructicola inoculation, were followed by RT-qPCR analysis and RNAseq data retrieval. RESULTS: Cultivated strawberry comprises 32 CAD genes of three classes. Thirteen syntenic relationships were observed between cultivated and woodland strawberry, with high homology in protein coding regions while promoters being greatly diversified. F. vesca ‘Hawaii4’ was least susceptible, followed by octoploid cultivar ‘ShenQi’, and ‘Benihoppe’ the most susceptible to C. fructicola. The class I CAD4 and CAD5, preferentially expressed in leaves and crowns, respectively, were up-regulated in susceptible varieties at 72 h post inoculation (hpi). Of class II, CAD8 was up-regulated in three genotypes at distinct timepoints, while CAD12 was quickly down-regulated in ‘ShenQi’ upon inoculation. CAD1 and CAD2 of class III were down-regulated in ‘ShenQi’ and ‘Hawaii4’ at distinct stages. The strong involvement of CAD genes in strawberry response to Colletotrichum spp. sets a basis for future functional study. CONCLUSION: F. vesca is less susceptible to Colletotrichum spp. than cultivated strawberries but the expression of CAD family in F. vesca and F.×ananassa were differentially provoked in timing and magnitude hinting no correlation with strawberry resistance to this pathogen.

Combined metabolome and transcriptome analysis reveals a critical role of lignin biosynthesis and lignification in stem-like pneumatophore development of the mangrove Avicennia marina.

Transcriptional and metabolic regulation of lignin biosynthesis and lignification plays crucial roles in Avicennia marina pneumatophore development, facilitating its adaptation to coastal habitats. Avicennia marina is a pioneer mangrove species in coastal wetland. To cope with the periodic intertidal flooding and hypoxia environment, this species has developed a complex and extensive root system, with its most unique feature being a pneumatophore with a distinct above- and below-ground morphology and vascular structure. However, the characteristics of pneumatophore lignification remain unknown. Studies comparing the anatomy among above-ground pneumatophore, below-ground pneumatophore, and feeding root have suggested that vascular structure development in the pneumatophore is more like the development of a stem than of a root. Metabolome and transcriptome analysis illustrated that the accumulation of syringyl (S) and guaiacyl (G) units in the pneumatophore plays a critical role in lignification of the stem-like structure. Fourteen differentially accumulated metabolites (DAMs) and 10 differentially expressed genes involved in the lignin biosynthesis pathway were targeted. To identify genes significantly associated with lignification, we analyzed the correlation between 14 genes and 8 metabolites and further built a co-expression network between 10 transcription factors (TFs), including 5 for each of MYB and NAC, and 23 enzyme-coding genes involved in lignin biosynthesis. 4-Coumarate-CoA ligase, shikimate/quinate hydroxycinnamoyl transferase, cinnamyl alcohol dehydrogenase, caffeic acid 3-O-methyltransferase, phenylalanine ammonia-lyase, and peroxidase were identified to be strongly correlated with these TFs. Finally, we examined 9 key candidate genes through quantitative real-time PCR to validate the reliability of transcriptome data. Together, our metabolome and transcriptome findings reveal that lignin biosynthesis and lignification regulate pneumatophore development in the mangrove species A. marina and facilitate its adaptation to coastal habitats.

Metabolic Profiling and Molecular Mechanisms Underlying Melatonin-Induced Secondary Metabolism of Postharvest Goji Berry (Lycium barbarum L.).

Postharvest decay of goji berries, mainly caused by Alternaria alternata, results in significant economic losses. To investigate the effects of melatonin (MLT) on resistance to Alternaria rot in goji berries, the fruits were immersed in the MLT solutions with varying concentrations (0, 25, 50, and 75 μmol L-1) and then inoculated with A. alternata. The results showed that the fruits treated with 50 μmol L-1 MLT exhibited the lowest disease incidence and least lesion diameter. Meanwhile, endogenous MLT in the fruits treated with 50 μmol L-1 MLT showed higher levels than in the control fruits during storage at 4 ± 0.5 °C. Further, the enzymatic activities and expressions of genes encoding peroxidase, phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, 4-coumarate-CoA ligase, chalcone synthase, chalcone isomerase, and cinnamyl alcohol dehydrogenase were induced in the treated fruit during storage. UPLC-ESI-MS/MS revealed that secondary metabolites in the fruits on day 0, in order of highest to lowest levels, were rutin, p-coumaric acid, chlorogenic acid, ferulic acid, caffeic acid, naringenin, quercetin, kaempferol, and protocatechuic acid. MLT-treated fruits exhibited higher levels of secondary metabolites than the control. In conclusion, MLT treatment contributed to controlling the postharvest decay of goji fruit during storage by boosting endogenous MLT levels, thus activating the antioxidant system and secondary metabolism.

Carboxylic acid reductase-dependent biosynthesis of eugenol and related allylphenols

Background(Hydroxy)cinnamyl alcohols and allylphenols, including coniferyl alcohol and eugenol, are naturally occurring aromatic compounds widely utilised in pharmaceuticals, flavours, and fragrances. Traditionally, the heterologous biosynthesis of (hydroxy)cinnamyl alcohols from (hydroxy)cinnamic acids involved CoA-dependent activation of the substrate. However, a recently explored alternative pathway involving carboxylic acid reductase (CAR) has proven efficient in generating the (hydroxy)cinnamyl aldehyde intermediate without the need for CoA activation. In this study, we investigated the application of the CAR pathway for whole-cell bioconversion of a range of (hydroxy)cinnamic acids into their corresponding (hydroxy)cinnamyl alcohols. Furthermore, we sought to extend the pathway to enable the production of a variety of allylphenols and allylbenzene.ResultsBy screening the activity of several heterologously expressed enzymes in crude cell lysates, we identified the combination of Segniliparus rugosus CAR (SrCAR) and Medicago sativa cinnamyl alcohol dehydrogenase (MsCAD2) as the most efficient enzymatic cascade for the two-step reduction of ferulic acid to coniferyl alcohol. To optimise the whole-cell bioconversion in Escherichia coli, we implemented a combinatorial approach to balance the gene expression levels of SrCAR and MsCAD2. This optimisation resulted in a coniferyl alcohol yield of almost 100%. Furthermore, we extended the pathway by incorporating coniferyl alcohol acyltransferase and eugenol synthase, which allowed for the production of eugenol with a titre of up to 1.61 mM (264 mg/L) from 3 mM ferulic acid. This improvement in titre surpasses previous achievements in the field employing a CoA-dependent coniferyl alcohol biosynthesis pathway. Our study not only demonstrated the successful utilisation of the CAR pathway for the biosynthesis of diverse (hydroxy)cinnamyl alcohols, such as p-coumaryl alcohol, caffeyl alcohol, cinnamyl alcohol, and sinapyl alcohol, from their corresponding (hydroxy)cinnamic acid precursors but also extended the pathway to produce allylphenols, including chavicol, hydroxychavicol, and methoxyeugenol. Notably, the microbial production of methoxyeugenol from sinapic acid represents a novel achievement.ConclusionThe combination of SrCAR and MsCAD2 enzymes offers an efficient enzymatic cascade for the production of a wide array of (hydroxy)cinnamyl alcohols and, ultimately, allylphenols from their respective (hydroxy)cinnamic acids. This expands the range of value-added molecules that can be generated using microbial cell factories and creates new possibilities for applications in industries such as pharmaceuticals, flavours, and fragrances. These findings underscore the versatility of the CAR pathway, emphasising its potential in various biotechnological applications.

Defense response of ‘Cuimili’ plum / ‘Maotao’ to Agrobacterium tumefaciens: A combined physiological and transcriptomic analysis

Crown gall, a tumor disease caused by Agrobacterium tumefaciens, results in considerable economic losses to orchards. Following pathogen invasion, plants trigger a series of defense responses. However, little is known about the physiological and molecular mechanisms of plum in response to A. tumefaciens infection. In this study, we characterized the changes in physiological and transcriptional levels of ‘Cuimili’ plum / ‘Maotao’ after infection with A. tumefaciens. The root vigor was decreased. The malondialdehyde and soluble sugar content, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities were elevated. Additionally, there was an increase in the lignin content and the activities of enzymes related to lignin biosynthesis, including phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate CoA ligase (4CL), cinnamoyl-CoA reductase (CCR), and cinnamyl-alcohol dehydrogenase (CAD). Notably, the expression levels of the corresponding genes of the lignin biosynthesis pathway were also found to be up-regulated. Antioxidant system and lignin play a crucial role in the early defense response of ‘Cuimili’ plum / ‘Maotao’. Transcriptome analysis revealed that 1,348 differentially expressed genes (DEGs) were involved in the response to A. tumefaciens infection. DEGs were enriched in the biosynthesis of secondary metabolites, monoterpenoid biosynthesis, ascorbate and aldarate metabolism, carbohydrate metabolism, and plant hormone signal transduction. Our findings provide new insights into the defense mechanisms and adaptive changes in plants against A. tumefaciens infection.

Differential response of phenylpropanoid pathway as linked to hormonal change in two Brassica napus cultivars contrasting drought tolerance.

Oilseed rape (Brassica napus L.) is a significant agro-economic crop with a wide range of uses. Drought is the most frequent unfavourable environmental stressor restraining its growth and development worldwide. This study was conducted to characterize the drought-responsive phenylpropanoid pathway and its link to hormonal changes in two cultivars, drought-resistant "Saturnin" and drought-susceptible "Mosa." Drought susceptibility in cv. Mosa was confirmed by its lower water use efficiency and higher lipid peroxidation levels with reactive oxygen species (ROS) accumulation. In cv. Saturnin, higher salicylic acid (SA) levels and expression of dehydration-responsive element binding 2 (DREB2) and non-expressor of pathogenesis-related gene 1 (NPR1) led to an upregulation of production of anthocyanin pigment 1 (PAP1) and phenylpropanoid pathway-related gene (CHS, F5H and COMT1) expression, increasing hydroxycinnamic acid and flavonoid compound concentrations. However, in cv. Mosa, higher increases in the activity of lignifying enzymes (polyphenol oxidase, coniferyl alcohol peroxidase, syringaldazine peroxidase, guaiacol peroxidase) and expression of the lignin synthesis-related gene cinnamyl alcohol dehydrogenase 2 (CAD2) were found along with greater increases in abscisic acid (ABA) levels and upregulation of ABA-responsive element binding 2 (AREB2) and basic helix-loop-helix transcription factor MYC2. These results indicate that drought-induced SA-mediated activation of the hydroxycinnamic acid and flavonoid pathways contributes to drought resistance, whereas ABA-mediated lignification contributes to drought susceptibility.

MePOD12 participates the regulation to postharvest physiological deterioration by ROS scavenging and lignin accumulation in cassava tuberous roots

Cassava tuberous root, as a staple food crop and industrial raw material worldwide, is highly sensitive to postharvest physiological deterioration (PPD) caused by wound. However, the molecular mechanism underlying PPD tolerance remains elusive in cassava. The reactive oxygen species (ROS) scavenging system is activated at the preliminary stage of PPD and peroxidase (POD) can effectively eliminate ROS. The expression level of MePOD12 and POD enzymatic activity in cassava tuberous roots was considerably up-regulated with PPD occurrence. Yeast two-hybrid and luciferase reporter assay showed that MePOD12 physically interacted with lignin-related cinnamyl alcohol dehydrogenase 15 (MeCAD15) in vitro and in vivo. The MePOD12-silenced tuberous roots decayed more than control, especially stored at room temperature for 3 and 5 days after harvesting. The total antioxidant capacity, activities of POD, SOD, CAT, CAD and lignin content were lower than those of control. Particularly, after harvest for 3 days, the accumulation of hydrogen peroxide and superoxide anion substantially increased compared to control. In addition, a proposed model was generated to elaborate the response mechanism of MePOD12 participating in PPD regulation via ROS elimination and lignin accumulation. It provides a new clue for breeding cassava PPD-tolerant varieties.

Effects of Planting Density—Potassium Interaction on the Coordination among the Lignin Synthesis, Stem Lodging Resistance, and Grain Yield in Oil Flax

To clarify the effects of planting density (PD) and potassium (K) application on the lignin synthesis of oil flax stalks and their relationship with lodging resistance, a two-year field experiment was conducted in the 2020 and 2021 growing seasons. The planting densities were 600 grains·m−2 (D1), 750 grains·m−2 (D2), and 900 grains·m−2 (D3); the potassium application levels were 0 kg K·hm−2 (K0), 60 kg K·hm−2 (K1,) and 90 kg K·hm−2 (K2). Then, the effects of PD and K on the stalk agronomic traits, lignin synthesis, lodging resistance, and grain yield (GY) of oil flax were studied. The results show that D3 increased the oil flax plant height and stem fresh weight, and K1 raised the height of the center of gravity. The enzyme activities of phenylalanine aminolyase (PAL), cinnamyl alcohol dehydrogenase (CAD), and peroxidase (POD) increased with an increase in planting density, whereby K1 promoted and K2 inhibited the lignin metabolic enzymes, respectively. The lignin and cellulose were significantly positively correlated with PAL. The combination of K with the D2 treatment increased the lignin and cellulose content in stems of oil flax at the kernel and maturity stages, and the effect of K1 on lignin metabolism and lodging resistance was better than that of K2. The increased GY was significantly correlated with the number of effective oil flax capsules per plant and the lodging resistance index, which were directly affected by K application. In conclusion, under the same ecological conditions as the experiment, the field management strategy of medium PD (750 grains·m−2) and low K (60 kg K·hm−2) was the best for improving the lodging resistance and GY formation of oil flax.

Hydroxycinnamaldehyde-derived benzofuran components in lignins.

Lignin is an abundant polymer in plant secondary cell walls. Prototypical lignins derive from the polymerization of monolignols (hydroxycinnamyl alcohols), mainly coniferyl and sinapyl alcohol, via combinatorial radical coupling reactions and primarily via the endwise coupling of a monomer with the phenolic end of the growing polymer. Hydroxycinnamaldehyde units have long been recognized as minor components of lignins. In plants deficient in cinnamyl alcohol dehydrogenase, the last enzyme in the monolignol biosynthesis pathway that reduces hydroxycinnamaldehydes to monolignols, chain-incorporated aldehyde unit levels are elevated. The nature and relative levels of aldehyde components in lignins can be determined from their distinct and dispersed correlations in 2D 1H-13C-correlated nuclear magnetic resonance (NMR) spectra. We recently became aware of aldehyde NMR peaks, well resolved from others, that had been overlooked. NMR of isolated low-molecular-weight oligomers from biomimetic radical coupling reactions involving coniferaldehyde revealed that the correlation peaks belonged to hydroxycinnamaldehyde-derived benzofuran moieties. Coniferaldehyde 8-5-coupling initially produces the expected phenylcoumaran structures, but the derived phenolic radicals undergo preferential disproportionation rather than radical coupling to extend the growing polymer. As a result, the hydroxycinnamaldehyde-derived phenylcoumaran units are difficult to detect in lignins, but the benzofurans are now readily observed by their distinct and dispersed correlations in the aldehyde region of NMR spectra from any lignin or monolignol dehydrogenation polymer. Hydroxycinnamaldehydes that are coupled to coniferaldehyde can be distinguished from those coupled with a generic guaiacyl end-unit. These benzofuran peaks may now be annotated and reported and their structural ramifications further studied.