Caffeic Acid O-methyltransferase Research Articles

Genome-Wide Identification of COMT Gene Family in Maize and its Function in Response to Light.

Maize is a major crop, feed, and industrial material. Caffeic acid-O-methyltransferase (COMT) is a methylase closely associated with lignin biosynthesis and plant growth and resistance. In this study, we identified the COMT gene (ZmCOMT) family in maize and further analyzed its phylogenetic evolution, subcellular localization, and its function in response to light. Thirty-one ZmCOMT genes were identified in the maize genome, which were distributed across eight chromosomes and mainly clustered on chromosome 4. Most ZmCOMT proteins were predicted to localize in the cytoplasm. Ten different conserved motifs were present in most ZmCOMT proteins, and motif1, motif6, and motif7 were highly conserved and present in all ZmCOMT proteins. The photoresponsivity elements were conserved among all members, and ZmCOMT22 and ZmCOMT10 genes responsive to light. This result suggests a potential function for these two genes in lignin biosynthesis which a previous study had linked to light regulation. Jasmonic acid responsive and abscisic acid cis-acting elements were present in the promoter regions of family members, thus the family may be regulated by hormone signaling pathways of maize. In summary, ZmCOMT genes are ancient, and the highly conserved motifs may be significant in survival and evolution of maize. Furthermore, light may influence lignin biosynthesis and photosynthesis through ZmCOMT genes. This research provided theoretical basis for lignin biosynthesis of maize and the potential value of ZmCOMT22 and ZmCOMT10 genes to enhance plant photosynthesis for facing global warming.

Genome-Wide Identification of the COMT Gene Family in Juglans regia L. and Response to Drought Stress.

Caffeic acid O-methyltransferase (COMT), as a multifunctional enzyme involved in various physiological and biochemical processes in lignin metabolism, plays an important role in a plant's response to stress. In this study, we isolated COMT family members from the walnut genome by bioinformatics and analyzed their physicochemical properties and their expression under drought stress to provide gene resources for drought resistance in walnut. The results showed that 33 COMT genes were identified from walnuts and distributed on different chromosomes. The molecular weight of proteins varies greatly. According to the phylogenetic tree, the family can be divided into seven subgroups, which are relatively conservative in evolution and closely related to Arabidopsis thaliana. Promoter analysis showed that the promoter of the walnut COMT gene contains rich cis-elements of plant hormone response and stress response, and the real-time fluorescence scale name can be significantly induced by drought stress. Compared with wild-type Arabidopsis, overexpression JrCOMT19 significantly increased the enzyme activity (SOD, POD, and CAT) and proline content. Meanwhile, overexpression of JrCOMT19 significantly increased the lignin content and expression of related genes. Therefore, JrCOMT plays an important role in responding to drought in walnuts, and overexpression JrCOMT19 can improve the resistance to drought stress by increasing lignin content, antioxidant enzyme activity, and osmotic substance content.

Genome-wide investigation of caffeic acid O-methyltransferases and expression analysis under different abiotic stresses and anthracnose infection in pepper (Capsicum annuum L.)

ABSTRACT The caffeic acid O-methyl transferases (COMTs) facilitate the biosynthesis of lignin and play important roles in many processes, including growth, development and response to stresses. Pepper (Capsicum annuum L.) is a major horticultural crop with significant nutritional and medicinal values. Nevertheless, a comprehensive assessment of the COMT genes in pepper and their involvement in drought, salt tolerance and anthracnose resistance remains unestablished. In the present study, 80 pepper COMT genes were identified and classified into four major groups based on domain structure and phylogenetics. Additionally, the sequence analysis, gene structures, and duplication events were analysed. The genes were unevenly distributed across 10 chromosomes, and 10 pairs of tandem and 13 pairs of segmental duplicated genes were detected in the COMT gene family. Examination of cis-acting regulatory elements detected the presence of phytohormone-specific, stress-receptive and light responsive elements in the pepper COMT promoters. Moreover, qRT-PCR analysis revealed significant upregulated expression of CanCOMT48 to drought stress, CanCOMT14 to salinity stress and CanCOMT18 to anthracnose infection by Colletotrichum truncatum, suggesting their important functions in different stresses. Our results provide significant insights into the expression and probable function of selective COMTs which could be used as potential candidate genes for pepper tolerance breeding.

Caffeic acid O-methyltransferase from Ligusticum chuanxiong alleviates drought stress, and improves lignin and melatonin biosynthesis.

Drought stress is a major constraint on plant growth and agricultural productivity. Caffeic acid O-methyltransferase (COMT), an enzyme involved in the methylation of various substrates, plays a pivotal role in plant responses to abiotic stress. The involvement of COMTs in drought response, particularly through the enhancement of lignin and melatonin biosynthesis, remains poorly understood. In this study, LcCOMT was firstly proposed to be associated with the biosynthesis of both lignin and melatonin, as demonstrated through sequence comparison, phylogenetic analysis, and conserved motif identification. In vitro enzymatic assays revealed that LcCOMT effectively methylates N-acetylserotonin to melatonin, albeit with a higher Km value compared to caffeic acid. Site-directed mutagenesis of residues Phe171 and Asp269 resulted in a significant reduction in catalytic activity for caffeic acid, with minimal impact on N-acetylserotonin, underscoring the specificity of these residues in substrate binding and catalysis. Under drought conditions, LcCOMT expression was significantly upregulated. Overexpression of LcCOMT gene in Arabidopsis plants conferred enhanced drought tolerance, characterized by elevated lignin and melatonin levels, increased chlorophyll and carotenoid content, heightened activities of antioxidant enzymes peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD), and reduced malondialdehyde (MDA) and hydrogen peroxide (H2O2) accumulation. This study is among the few to demonstrate that COMT-mediated drought tolerance is achieved through the simultaneous promotion of lignin and melatonin biosynthesis. LcCOMT represents the first functionally characterized COMT in Apiaceae family, and it holds potential as a target for genetic enhancement of drought tolerance in future crop improvement strategies.

Target of rapamycin coordinates auxin are involved in exogenous melatonin regulated low temperature tolerance in cucumber seedlings

Low temperature (LT) is an important environmental factor affecting the growth and yield of plants. Melatonin (MT) can effectively enhance the LT tolerance of cucumber. This study found that LT stress induced the expression of Comt1 (caffeic acid O-methyltransferase 1), with the highest expression being about 2-times that of the control. Meanwhile, the content of MT was found to be roughly 63.16% of that in the control samples. Compared with LT treatment alone, exogenous MT pretreatment upregulated the expression levels of TOR (Target of rapamycin), PIN1 (Pin-formed 1), and YUC4 (YUCCA 4), with maximum upregulations reaching approximately 66.67%, 79.32%, and 42.86%, respectively. These results suggest that MT may modulate the tolerance of cucumber seedlings to LT stress by regulating the expression of TOR, PIN1, and YUC4. In addition, co-treatment with AZD-8055 (a TOR inhibitor) or NPA (N-1-naphthylphthalamic acid, an auxin polar transport inhibitor) and MT attenuated MT-induced resistance to LT stress, leading to higher levels of reactive oxygen species (ROS), reduced antioxidant defense capacity, and increased damage to the membrane system in cucumber seedlings. Concurrently, the content of osmoregulatory substances and the photosynthesis decreased. These results demonstrate that both TOR and auxin were required for MT to alleviate LT-induced damage in cucumber. In summary, the present study demonstrates that TOR and auxin signaling synergistically contribute to alleviating LT damage in cucumber seedlings by exogenous MT. These findings help us understand the function of MT and provide insights into the regulatory network of MT that regulates the LT tolerance of plants.

Genome-Wide Identification of Caffeic Acid O-Methyltransferase Gene Family in Medicago truncatula: MtCOMT13-Mediated Salt and Drought Tolerance Enhancement

Legumes are important grains and forages, providing high-quality proteins, vitamins, and micronutrients to humans and animals. Medicago truncatula is a close relative of alfalfa (Medicago sativa). Caffeic acid O-methyltransferase (COMT), a key gene that is identified to be essential for melatonin synthesis, plays a significant role in plant growth, development, and abiotic stress responses. However, a systematic study on the COMT gene family in M. truncatula has still not been reported. In this study, 63 MtCOMT genes were identified and categorized into three groups. Gene structure and conserved motif analyses revealed the relative conservation of closely clustered MtCOMTs within each group. Duplicated events in MtCOMT members were identified, and segmental duplication was the main mean. Cis-acting element prediction revealed the involvement of MtCOMTs in growth and development and response to light, stress, and plant hormones. RNA-seq data analysis showed that 57 MtCOMTs varied under salt and drought stresses. The RT-qPCR expression patterns showed that MtCOMT9, MtCOMT13, MtCOMT22, MtCOMT24, MtCOMT43, and MtCOMT46 were related to salt and drought responses in M. truncatula. Additionally, Arabidopsis thaliana overexpressing MtCOMT13 displayed superior plant growth phenotypes and enhanced tolerance to salt and drought stresses through higher photosynthetic parameters and activities of antioxidant enzymes, which indicated that MtCOMT13 played an important role in positively regulating plant salt and drought tolerance. These findings contribute to an improved understanding of MtCOMTs’ roles in abiotic stress responses in M. truncatula, providing an important theoretical basis and genetic resource for legume species resistance breeding in the future.

Phytomelatonin: From Intracellular Signaling to Global Horticulture Market.

Melatonin (N-acetyl-5-methoxytryptamine), a well-known mammalian hormone, has been having a great relevance in the Plant World in recent years. Many of its physiological actions in plants are leading to possible features of agronomic interest, especially those related to improvements in tolerance to stressors and in the postharvest life of fruits and vegetables. Thus, through the exogenous application of melatonin or by modifying the endogenous biosynthesis of phytomelatonin, some change can be made in the functional levels of melatonin in tissues and their responses. Also, acting in the respective phytomelatonin biosynthesis enzymes, regulating the expression of tryptophan decarboxylase (TDC), tryptamine 5-hydroxylase (T5H), serotonin N-acetyltransferase (SNAT), N-acetylserotonin O-methyltransferase (ASMT), and caffeic acid O-methyltransferase (COMT), and recently the possible action of deacetylases on some intermediates offers promising opportunities for improving fruits and vegetables in postharvest and its marketability. Other regulators/effectors such as different transcription factors, protein kinases, phosphatases, miRNAs, protein-protein interactions, and some gasotransmitters such as nitric oxide or hydrogen sulfide were also considered in an exhaustive vision. Other interesting aspects such as the role of phytomelatonin in autophagic responses, the posttranslational reprogramming by protein-phosphorylation, ubiquitylation, SUMOylation, PARylation, persulfidation, and nitrosylation described in the phytomelatonin-mediated responses were also discussed, including the relationship of phytomelatonin and several plant hormones, for chilling injury and fungal decay alleviating. The current data about the phytomelatonin receptor in plants (CAND2/PMTR1), the effect of UV-B light and cold storage on the postharvest damage are presented and discussed. All this on the focus of a possible new action in the preservation of the quality of fruits and vegetables.

Spatial accumulation of lignin monomers and cellulose underlying stalk strength in maize

Lodging largely affects yield, quality and mechanical harvesting of maize. Stalk strength is one of the major factors that affect maize lodging. Although plant cell wall components including lignin and cellulose were known to be associated with stalk strength and lodging resistance, spatial accumulation of specific lignin monomers and cellulose in different tissues and their association with stalk strength in maize was not clearly understood. In this study, we found that both G and S lignin monomers accumulate highest in root, stem rind and leaf vein. Consistently, most lignin biosynthetic genes were expressed higher in root and stem than in other tissues. However, cellulose appears to be lowest in root. There are only mild changes of G lignin and cellulose in different internodes. Instead, we noticed a dramatic decrease of S-lignin accumulation and lignin biosynthetic gene expression in 2nd to 4th internodes wherein stem breakage usually occurs, thereby revealing a few candidate lignin biosynthetic genes associated with stalk strength. Moreover, stalk strength is positively correlated with G, S lignin, and cellulose, but negatively correlated with S/G ratio based on data of maize lines with high or low stalk strength. Loss-of-function of a caffeic acid o-methyltransferase (COMT), which is involved in S lignin biosynthesis, in the maize bm3 mutant, leads to lower stalk strength. Our data collectively suggest that stalk strength is determined by tissue-specific accumulation of lignin monomers and cellulose, and manipulation of the cell wall components by genetic engineering is vital to improve maize stalk strength and lodging resistance.

De Novo Biosynthesis of Curcumin in Saccharomyces cerevisiae.

Curcumin, a natural polyphenol derived from turmeric, has attracted immense interest due to its diverse pharmacological properties. Traditional extraction methods from Curcuma longa plants present limitations in meeting the growing demand for this bioactive compound, giving significance to its production by genetically modified microorganisms. Herein, we have developed an engineered Saccharomyces cerevisiae to produce curcumin from glucose. A pathway composed of the 4-hydroxyphenylacetate 3-monooxygenase oxygenase complex from Pseudomonas aeruginosa and Salmonella enterica, caffeic acid O-methyltransferase from Arabidopsis thaliana, feruloyl-CoA synthetase from Pseudomonas paucimobilis, and diketide-CoA synthase and curcumin synthase from C. longa was introduced in a p-coumaric acid overproducing S. cerevisiae strain. This strain produced 240.1 ± 15.1 μg/L of curcumin. Following optimization of phenylpropanoids conversion, a strain capable of producing 4.2 ± 0.6 mg/L was obtained. This study reports for the first time the successful de novo production of curcumin in S. cerevisiae.

An integrated multi-omics approach reveals polymethoxylated flavonoid biosynthesis in Citrus reticulata cv. Chachiensis

Citrus reticulata cv. Chachiensis (CRC) is an important medicinal plant, its dried mature peels named “Guangchenpi”, has been used as a traditional Chinese medicine to treat cough, indigestion, and lung diseases for several hundred years. However, the biosynthesis of the crucial natural products polymethoxylated flavonoids (PMFs) in CRC remains unclear. Here, we report a chromosome-scale genome assembly of CRC with the size of 314.96 Mb and a contig N50 of 16.22 Mb. Using multi-omics resources, we discover a putative caffeic acid O-methyltransferase (CcOMT1) that can transfer a methyl group to the 3-hydroxyl of natsudaidain to form 3,5,6,7,8,3’,4’-heptamethoxyflavone (HPMF). Based on transient overexpression and virus-induced gene silencing experiments, we propose that CcOMT1 is a candidate enzyme in HPMF biosynthesis. In addition, a potential gene regulatory network associated with PMF biosynthesis is identified. This study provides insights into PMF biosynthesis and may assist future research on mining genes for the biosynthesis of plant-based medicines.

Integrative Dissection of Lignin Composition in Tartary Buckwheat Seed Hulls for Enhanced Dehulling Efficiency.

The rigid hull encasing Tartary buckwheat seeds necessitates a laborious dehulling process before flour milling, resulting in considerable nutrient loss. Investigation of lignin composition is pivotal in understanding the structural properties of tartary buckwheat seeds hulls, as lignin is key determinant of rigidity in plant cell walls, thus directly impacting the dehulling process. Here, the lignin composition of seed hulls from 274 Tartary buckwheat accessions is analyzed, unveiling a unique lignin chemotype primarily consisting of G lignin, a common feature in gymnosperms. Furthermore, the hardness of the seed hull showed a strong negative correlation with the S lignin content. Genome-wide detection of selective sweeps uncovered that genes governing the biosynthesis of S lignin, specifically two caffeic acid O-methyltransferases (COMTs) and one ferulate 5-hydroxylases, are selected during domestication. This likely contributed to the increased S lignin content and decreased hardness of seed hulls from more domesticated varieties. Genome-wide association studies identified robust associations between FtCOMT1 and the accumulation of S lignin in seed hull. Transgenic Arabidopsis comt1 plants expressing FtCOMT1 successfully reinstated S lignin content, confirming its conserved function across plant species. These findings provide valuable metabolic and genetic insights for the potential redesign of Tartary buckwheat seed hulls.

Overexpression of caffeic acid O-methyltransferase from Lycoris aurea (LaCOMT) confers tolerance to mercury in Arabidopsis thaliana by modulating ROS homeostasis

Overexpression of caffeic acid O-methyltransferase from Lycoris aurea (LaCOMT) confers tolerance to mercury in Arabidopsis thaliana by modulating ROS homeostasis

Caffeic Acid O-Methyltransferase Gene Family in Mango (Mangifera indica L.) with Transcriptional Analysis under Biotic and Abiotic Stresses and the Role of MiCOMT1 in Salt Tolerance.

Caffeic acid O-methyltransferase (COMT) participates in various physiological activities in plants, such as positive responses to abiotic stresses and the signal transduction of phytohormones. In this study, 18 COMT genes were identified in the chromosome-level reference genome of mango, named MiCOMTs. A phylogenetic tree containing nine groups (I-IX) was constructed based on the amino acid sequences of the 71 COMT proteins from seven species. The phylogenetic tree indicated that the members of the MiCOMTs could be divided into four groups. Quantitative real-time PCR showed that all MiCOMT genes have particularly high expression levels during flowering. The expression levels of MiCOMTs were different under abiotic and biotic stresses, including salt and stimulated drought stresses, ABA and SA treatment, as well as Xanthomonas campestris pv. mangiferaeindicae and Colletotrichum gloeosporioides infection, respectively. Among them, the expression level of MiCOMT1 was significantly up-regulated at 6-72 h after salt and stimulated drought stresses. The results of gene function analysis via the transient overexpression of the MiCOMT1 gene in Nicotiana benthamiana showed that the MiCOMT1 gene can promote the accumulation of ABA and MeJA, and improve the salt tolerance of mango. These results are beneficial to future researchers aiming to understand the biological functions and molecular mechanisms of MiCOMT genes.

Salicylic acid promotes phenolic acid biosynthesis for the production of phenol acid-rich barley sprouts.

Phenolic acid exhibits a variety of well-known physiological functions. In this study, optimal germination conditions to ensure total phenolic acid enrichment in barley sprouts induced by salicylic acid treatment and its effects on sprout physiology and activity, as well as the gene expression of key enzymes for phenolic acid biosynthesis, were investigated. When sprouts were treated with 1 mmol L-1 salicylic acid during germination and germinated at 25 °C for 4 days, the phenolic acid content was 1.82 times that of the control, reaching 1221.54 μg g-1 fresh weight. Salicylic acid significantly increased the activity of phenylalanine aminolase and cinnamic acid-4-hydroxylase and the gene expression of phenylalanine aminolase, cinnamic acid-3-hydroxylase, cinnamic acid-4-hydroxylase, 4-coumaric acid-coenzyme A, caffeic acid O-methyltransferase, and ferulate-5-hydroxylase in barley sprouts. However, salicylic acid treatment significantly increased malondialdehyde and H2O2 content, H2O2 and O2 - fluorescence intensity, as well as significantly decreasing sprout length and fresh weight. Salicylic acid treatment markedly increased the activity of peroxidase and catalase and the gene expression of peroxidase, catalase, and ascorbate peroxidase in barley sprouts. Salicylic acid treatment during barley germination significantly promoted the enrichment of total phenolic acid by increasing the activities and gene expression levels of enzymes involved in the phenolic acid biosynthesis pathway. Salicylic acid induced the accumulation of reactive oxygen species, inhibited sprout growth, and activated the antioxidant system. This study provides a basis for the future development of functional foods using phenol acid-rich plants as raw materials. © 2024 Society of Chemical Industry.

Silicon nanoparticles (SiNPs) stimulated secondary metabolism and mitigated toxicity of salinity stress in basil (Ocimum basilicum) by modulating gene expression: a sustainable approach for crop protection.

The underlying mechanisms through which silicon oxide nanoparticles (SiNPs) can confer salinity resistance to plants are poorly understood. This study explored the efficacy of supplementing nutrient solution with SiNPs (20-30nm; 10 mg kg-1 soil) to stimulate metabolism and alleviate the risks associated with salinity (0.73 g kg-1 soil) in basil seedlings. For this purpose, variations in photosynthetic indices, proline osmoprotectant, antioxidant markers, phenylpropanoid metabolism, and transcriptional behaviors of genes were investigated. SiNPs increased shoot fresh weight (38%) and mitigated the risk associated with the salinity stress by 14%. SiNPs alleviated the inhibitory effects of salinity on the total chlorophyll concentration by 15%. The highest increase (twofold) in proline content was recorded in the SiNP-treated seedlings grown under salinity. The nano-supplement enhanced the activity of enzymatic antioxidants, including peroxidase (2.5-fold) and catalase (4.7-fold). SiNPs induced the expression of gamma-cadinene synthase (CDS) and caffeic acid O-methyltransferase (COMT) genes by 6.5- and 18.3-fold, respectively. SiNPs upregulated the eugenol synthase (EGS1) and fenchol synthase (FES) genes by six- and nine-fold, respectively. Salinity transcriptionally downregulated the geraniol synthase (GES) gene, while this gene displayed an upward trend in response to SiNPs by eight-fold. The nano-supplement transcriptionally stimulated the R-linalool synthase (LIS) gene by 3.3-fold. The terpinolene synthase (TES) gene displayed a similar trend to that of the GES gene. The highest expression (25-fold) of the phenylalanine ammonia-lyase (PAL) gene was recorded in seedlings supplemented with SiNPs. The physiological and molecular assessments demonstrated that employing SiNPs is a sustainable strategy for improving plant primary/secondary metabolism and crop protection.

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.

Characterization of sugarcane mutants developed through gamma irradiations for their lignin content and caffeic acid-O-methyl transferase (COMT) gene mutations

Purpose Bagasse, the residue left after extracting juice from sugarcane stalks, is rich in lignocellulosic biomass. The lignin present in this plant biomass is the key factor that hinders the efficient extraction of ethanol from the bagasse. In the current study, γ-irradiated sugarcane mutants were evaluated for variation in lignin content and its corresponding caffeic acid-O-methyl transferase (COMT) gene. Materials and methods The acetyl bromide method was used to estimate lignin content in sugarcane mutants. PCR-based cloning of the COMT gene was performed in low lignin mutants as well as control plants in E. coli (strain DH5α) to understand the mechanism of variation at the molecular level. The Sanger sequencing for cloned gene was performed to check variation in gene sequence. Results In comparison to the control (21.5%), the mutant plants’ lignin content ranged from 13 to 28%. The Sanger sequencing revealed approximately the same length of the gene from mutants as well as a control plant. In comparison to the reference gene, the mutated gene showed SNPs and indels in different regions, which may have an impact on lignin content. Conclusions Therefore, γ-irradiated mutagenesis is an acceptable approach to develop novel mutants of sugarcane with low lignin content to enhance bioethanol production from waste material using bioprocess technology.

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.

Understanding the mechanism of red light-induced melatonin biosynthesis facilitates the engineering of melatonin-enriched tomatoes

Melatonin is a functionally conserved broad-spectrum physiological regulator found in most biological organisms in nature. Enrichment of tomato fruit with melatonin not only enhances its agronomic traits but also provides extra health benefits. In this study, we elucidate the full melatonin biosynthesis pathway in tomato fruit by identifying biosynthesis-related genes that encode caffeic acid O-methyltransferase 2 (SlCOMT2) and N-acetyl-5-hydroxytryptamine-methyltransferases 5/7 (SlASMT5/7). We further reveal that red light supplementation significantly enhances the melatonin content in tomato fruit. This induction relies on the “serotonin—N-acetylserotonin—melatonin” biosynthesis route via the SlphyB2-SlPIF4-SlCOMT2 module. Based on the regulatory mechanism, we design a gene-editing strategy to target the binding motif of SlPIF4 in the promoter of SlCOMT2, which significantly enhances the production of melatonin in tomato fruit. Our study provides a good example of how the understanding of plant metabolic pathways responding to environmental factors can guide the engineering of health-promoting foods.

A Rehmannia glutinosa caffeic acid O-methyltransferase functional identification: Reconstitution of the ferulic acid biosynthetic pathway in Saccharomyces cerevisiae using Rehmannia glutinosa enzymes.

Rehmannia glutinosa produces many pharmacological natural components, including ferulic acid (FA) which is also an important precursor of some medicinal ingredients, so it is very significant to explore FA biosynthesis for enhancing the production of FA and its derivations. This study aimed to determine and reconstitute the R. glutinosa FA biosynthetic pathway from phenylalanine (Phe) metabolism in Saccharomyces cerevisiae as a safe host for the biosynthesis of plant-derived products. Although plant caffeic acid O-methyltransferases (COMTs) are thought to be a vital catalytic enzyme in FA biosynthesis pathways, to date, none of the RgCOMTs in R. glutinosa has been characterized. This study identified an RgCOMT and revealed its protein enzymatic activity for FA production in vitro. The RgCOMT overexpression in R. glutinosa significantly increased FA yield, suggesting that its molecular function is involved in FA biosynthesis. Heterologous expression of the RgCOMT and reported R. glutinosa genes, RgPAL2 (encoding phenylalanine ammonia-lyase [PAL] protein), RgC4H (cinnamate 4-hydroxylase [C4H]), and RgC3H (p-coumarate-3-hydroxylase [C3H]), in S. cerevisiae confirmed their catalytic abilities in the reaction steps for the FA biosynthesis. Importantly, in this study, these genes were introduced into S. cerevisiae and coexpressed to reconstitute the R. glutinosa FA biosynthetic pathway from Phe metabolism, thus obtaining an engineered strain that produced an FA titer of 148.34mgL-1 . This study identified the functional activity of RgCOMT and clarified the R. glutinosa FA biosynthesis pathway in S. cerevisiae, paving the way for the efficient production of FA and its derivatives.