Biosynthesis of resveratrol by an endophytic Priestia megaterium PH3 via the phenylpropane pathway.

Resveratrol (RES) is a secondary metabolite synthesized by plants in response to environmental stress and pathogen infection, which is of great significance for the industrial production of RES by fermentation culture. In this study, we aimed to explore the biosynthesis pathway of RES and its key enzymes in the Priestia megaterium PH3, which was isolated and screened from peanut fruit. Through Liquid Chromatography-Mass Spectrometry (LC-MS) analysis, we quantified the RES content and distribution in the culture medium and determined that Priestia megaterium PH3 mainly secreted RES extracellularly. Furthermore, the highest production of RES was observed in YPD, yielding an impressive 127.46 ± 6.11 µg/L. By optimizing the fermentation conditions, we achieved a remarkable RES yield of 946.82 ± 24.74 µg/L within just 2 days, which represents the highest reported yield for a natural isolate produced in such a short time frame. Our investigation revealed that the phenylpropane pathway is responsible for RES synthesis in this bacterium, with cinnamate 4-hydroxylase (C4H) identified as the main rate-limiting enzyme. Overall, our findings highlight the robust RES production capabilities of Priestia megaterium PH3, offering novel insights and potential applications for bacterial fermentation in RES production. KEY POINTS: • RES synthesized by the bacterium was confirmed through the phenylpropane pathway. • The key rate-limiting enzyme for biosynthesis-RES is C4H. • RES reached 946.82 ± 24.74 µg/L after fermentation for 2 days.

A Cinnamate 4-HYDROXYLASE1 from Safflower Promotes Flavonoids Accumulation and Stimulates Antioxidant Defense System in Arabidopsis.

C4H (cinnamate 4-hydroxylase) is a pivotal gene in the phenylpropanoid pathway, which is involved in the regulation of flavonoids and lignin biosynthesis of plants. However, the molecular mechanism of C4H-induced antioxidant activity in safflower still remains to be elucidated. In this study, a CtC4H1 gene was identified from safflower with combined analysis of transcriptome and functional characterization, regulating flavonoid biosynthesis and antioxidant defense system under drought stress in Arabidopsis. The expression level of CtC4H1 was shown to be differentially regulated in response to abiotic stresses; however, a significant increase was observed under drought exposure. The interaction between CtC4H1 and CtPAL1 was detected using a yeast two-hybrid assay and then verified using a bimolecular fluorescence complementation (BiFC) analysis. Phenotypic and statistical analysis of CtC4H1 overexpressed Arabidopsis demonstrated slightly wider leaves, long and early stem development as well as an increased level of total metabolite and anthocyanin contents. These findings imply that CtC4H1 may regulate plant development and defense systems in transgenic plants via specialized metabolism. Furthermore, transgenic Arabidopsis lines overexpressing CtC4H1 exhibited increased antioxidant activity as confirmed using a visible phenotype and different physiological indicators. In addition, the low accumulation of reactive oxygen species (ROS) in transgenic Arabidopsis exposed to drought conditions has confirmed the reduction of oxidative damage by stimulating the antioxidant defensive system, resulting in osmotic balance. Together, these findings have provided crucial insights into the functional role of CtC4H1 in regulating flavonoid biosynthesis and antioxidant defense system in safflower.

Spatio-temporal control of phenylpropanoid biosynthesis by inducible complementation of a cinnamate 4-hydroxylase mutant.

Cinnamate 4-hydroxylase (C4H) is a cytochrome P450-dependent monooxygenase that catalyzes the second step of the general phenylpropanoid pathway. Arabidopsis reduced epidermal fluorescence 3 (ref3) mutants, which carry hypomorphic mutations in C4H, exhibit global alterations in phenylpropanoid biosynthesis and have developmental abnormalities including dwarfing. Here we report the characterization of a conditional Arabidopsis C4H line (ref3-2pOpC4H), in which wild-type C4H is expressed in the ref3-2 background. Expression of C4H in plants with well-developed primary inflorescence stems resulted in restoration of fertility and the production of substantial amounts of lignin, revealing that the developmental window for lignification is remarkably plastic. Following induction of C4H expression in ref3-2pOpC4H, we observed rapid and significant reductions in the levels of numerous metabolites, including several benzoyl and cinnamoyl esters and amino acid conjugates. These atypical conjugates were quickly replaced with their sinapoylated equivalents, suggesting that phenolic esters are subjected to substantial amounts of turnover in wild-type plants. Furthermore, using localized application of dexamethasone to ref3-2pOpC4H, we show that phenylpropanoids are not transported appreciably from their site of synthesis. Finally, we identified a defective Casparian strip diffusion barrier in the ref3-2 mutant root endodermis, which is restored by induction of C4H expression.

A Rehmannia glutinosa cinnamate 4-hydroxylase promotes phenolic accumulation and enhances tolerance to oxidative stress.

KEY MESSAGE: RgC4H promotes phenolic accumulation in R. glutinosa, activating the molecular networks of its antioxidant systems, and enhancing the tolerance to oxidative stresses exposed to drought, salinity and H2O2 conditions. Rehmannia glutinosa is of great economic importance in China and increasing R. glutinosa productivity relies, in part, on understanding its tolerance to oxidative stress. Oxidative stress is a key influencing factor for crop productivity in plants exposed to harsh conditions. In the defense mechanisms of plants against stress, phenolics serve an important antioxidant function. Cinnamate 4-hydroxylase (C4H) is the first hydroxylase in the plant phenolics biosynthesis pathway, and elucidating the molecular characteristics of this gene in R. glutinosa is essential for understanding the effect of tolerance to oxidative stress tolerance on improving yield. Using in vitro and in silico methods, a C4H gene, RgC4H, from R. glutinosa was isolated and characterized. RgC4H has 86.34-93.89% amino acid sequence identity with the equivalent protein in other plants and localized to the endoplasmic reticulum. An association between the RgC4H expression and total phenolics content observed in non-transgenic and transgenic R. glutinosa plants suggests that this gene is involved in the process of phenolics biosynthesis. Furthermore, the tolerance of R. glutinosa to drought, salinity and H2O2 stresses was positively or negatively altered in plants with the overexpression or knockdown of RgC4H, respectively, as indicated by the analysis in some antioxidant physiological and molecular indices. Our study highlights the important role of RgC4H in the phenolics/phenylpropanoid pathway and reveals the involvement of phenolic-mediated regulation in oxidative stress tolerance in R. glutinosa.

Developmental role of phenylalanine-ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) genes during adventitious rooting of Juglans regia L. microshoots.

Phenylalanine-ammonia-lyase and cinnamate-4-hydroxylase play important role in the phenylpropanoid pathway, which produces many biologically important secondary metabolites participating in normal plant development. Flavonol quercetin is the main representant of these compounds that has been identified in numerous Juglans spp. In this survey, the developmental expression patterns of PAL and C4H genes during in vitro rooting of two walnut cultivars 'Sunland' and 'Howard' was examined by RT-PCR. To understand the potential role in rooting, the changing pattern of endogenous content of quercetin was also analyzed by HPLC. The 'Sunland' with better capacity to root had more quercetin content during the "inductive phase" of rooting than 'Howard'. In each cultivar, the level of PAL transcripts showed the same behavior with the changing patterns of quercetin during root formation of microshoots. The positive correlation between the changes of quercetin and PAL-mRNA indicated that PAL gene may have an immediate effect on flavonoid pathway metabolites including quercetin. Although the behavioral change of C4H expression was similar in both cultivars during root formation (with significantly more level for 'Howard'), it was not coincide with the changes of quercerin concentrations. Our results showed that C4H function is important for the normal development, but its transcriptional regulation does not correlate with quercetin as an efficient phenolic compound for walnut rhizogenesis.

BrMYB4, a suppressor of genes for phenylpropanoid and anthocyanin biosynthesis, is down-regulated by UV-B but not by pigment-inducing sunlight in turnip cv. Tsuda.

The regulation of light-dependent anthocyanin biosynthesis in Brassica rapa subsp. rapa cv. Tsuda turnip was investigated using an ethyl methanesulfonate (EMS)-induced mutant R30 with light-independent pigmentation. TILLING (targeting induced local lesions in genomes) and subsequent analysis showed that a stop codon was inserted in the R2R3-MYB transcription factor gene BrMYB4 and that the encoded protein (BrMYB4mu) had lost its C-terminal region. In R30, anthocyanin accumulated in the below-ground portion of the storage root of 2-month-old plants. In 4-day-old seedlings and 2-month-old plants, expression of BrMYB4 was similar between R30 and the wild type (WT), but the expression of the cinnamate 4-hydroxylase gene (BrC4H) was markedly enhanced in R30 in the dark. In turnip seedlings, BrMYB4 expression was suppressed by UV-B irradiation in the WT, but this negative regulation was absent in R30. Concomitantly, BrC4H was repressed by UV-B irradiation in the WT, but stayed at high levels in R30. A gel-shift assay revealed that BrMYB4 could directly bind to the promoter region of BrC4H, but BrMYB4mu could not. The BrMYB4-enhanced green fluorescent protein (eGFP) protein could enter the nucleus in the presence of BrSAD2 (an importin ß-like protein) nuclear transporter, but BrMYB4mu-eGFP could not. These results showed that BrMYB4 functions as a negative transcriptional regulator of BrC4H and mediates UV-B-dependent phenylpropanoid biosynthesis, while BrMYB4mu has lost this function. In the storage roots, the expression of anthocyanin biosynthesis genes was enhanced in R30 in the dark and in sunlight in both the WT and R30. However, in the WT, anthocyanin-inducing sunlight did not suppress BrMYB4 expression. Therefore, sunlight-induced anthocyanin biosynthesis does not seem to be regulated by BrMYB4.

Isolation and Characterization of Phenylalanine Ammonia Lyase (PAL) Genes in Ferula pseudalliacea: Insights into the Phenylpropanoid Pathway.

Phenylalanine ammonia lyase (PAL) is a key enzyme regulating the biosynthesis of the compounds of the phenylpropanoid pathway. This study aimed to isolate and characterize PAL genes from Ferula pseudalliacea Rech.f. (Apiales: Apiaceae) to better understand the regulation of metabolite production. Three PAL gene isoforms (FpPAL1-3) were identified and cloned using the 3'-RACE technique and confirmed by sequencing. Bioinformatics analysis revealed important structural features, such as phosphorylation sites, physicochemical properties, and evolutionary relationships. Expression analysis by qPCR demonstrated the differential transcription profiles of each FpPAL isoform across roots, stems, leaves, flowers, and seeds. FpPAL1 showed the highest expression in stems, FpPAL2 in roots and flowers, and FpPAL3 in flowers. The presence of three isoforms of PAL in F. pseudalliacea, along with the diversity of PAL genes and their tissue-specific expression profiles, suggests that complex modes of regulation exist for phenylpropanoid biosynthesis in this important medicinal plant. The predicted interaction network revealed associations with key metabolic pathways, emphasizing the multifaceted roles of these PAL genes. In silico biochemical analyses revealed the hydrophilicity of the FpPAL isozyme; however, further analysis of substrate specificity and enzyme kinetics can clarify the specific role of each FpPAL isozyme. These comprehensive results increase the understanding of PAL genes in F. pseudalliacea, helping to characterize their contributions to secondary metabolite biosynthesis.

Cloning and functional characterization of a cinnamate 4-hydroxylase gene from the hornwort Anthoceros angustus.

Hornworts, as the sister group to liverworts and mosses, comprise bryophytes, which are critical in understanding the evolution of key land plant traits. Cinnamate 4-hydroxylase (C4H) catalyzes the second step of the phenylpropanoid pathway to synthesize the precursor of numerous phenolic compounds, such as lignin and flavonoids. However, C4H in the hornwort Anthoceros angustus has not yet been cloned and functionally characterized. In this work, we screened the transcriptome database of A. angustus and identified one C4H gene, AnanC4H. AnanC4H maintained conserved cytochrome P450 domains with other typical plant C4Hs. Ultraviolet B irradiation and exogenous application of methyl jasmonate (MeJA) induced the expression of AnanC4H to varying degrees. The coding sequence of AnanC4H was expressed in yeast, and the recombinant proteins were isolated. The recombinant proteins of AnanC4H catalyzed the conversion of trans-cinnamic acid to p-coumaric acid and catalyzed the conversion of 3-hydroxycinnamic acid to caffeic acid. AnanC4H showed higher affinity for trans-cinnamic acid than for 3-hydroxycinnamic acid, but there was no significant difference in the catalytic efficiency of AnanC4H for the two substrates in vitro. Moreover, the expression of AnanC4H in Arabidopsis thaliana led to an increase in both the lignin content and the number of lignified cells in stems. However, there was no significant change in flavonoid content in transgenic Arabidopsis plants.

Water Deficit-Induced Changes in Phenolic Acid Content in Maize Leaves Is Associated with Altered Expression of Cinnamate 4-Hydroxylase and p-Coumaric Acid 3-Hydroxylase.

The amino acid phenylalanine is a precursor to phenolic acids that constitute the lignin biosynthetic pathway. Although there is evidence of a role of some phenolic acids in plant responses to pathogens and salinity, characterization of the involvement of phenolic acids in plant responses to drought is limited. Drought reduces water content in plant tissue and can lead to decreased cell viability and increased cell death. We thus subjected maize seedlings to water deficit and evaluated relative water content and cell viability together with p-coumaric acid, caffeic acid and ferulic acid contents in the leaves. Furthermore, we measured the enzymatic activity of cinnamate 4-hydroxylase (EC 1.14.13.11) and p-coumarate 3-hydroxylase (EC 1.14.17.2) and associated these with the expression of genes encoding cinnamate 4-hydroxylase and p-coumarate-3 hydroxylase in response to water deficit. Water deficit reduced relative water content and cell viability in maize leaves. This corresponded with decreased p-coumaric acid but increased caffeic and ferulic acid content in the leaves. Changes in the phenolic acid content of the maize leaves were associated with increased enzymatic activities of cinnamate 4-hydroxylase and p-coumarate hydroxylase. The increased enzymatic activity of p-coumarate 3-hydroxylase was associated with increased expression of a gene encoding p-coumarate 3-hydroxylase. We thus conclude that metabolic pathways involving phenolic acids may contribute to the regulation of drought responses in maize, and we propose that further work to elucidate this regulation may contribute to the development of new maize varieties with improved drought tolerance. This can be achieved by marker-assisted selection to select maize lines with high levels of expression of genes encoding cinnamate 4-hydroxylase and/or p-coumarate 3-hydroxylase for use in breeding programs aimed and improving drought tolerance, or by overexpression of these genes via genetic engineering to confer drought tolerance.

Combination of calcium lactate impregnation with UV-C irradiation maintains quality and improves antioxidant capacity of fresh-cut kiwifruit slices.

This study investigated the combined effects of calcium lactate (CA-L, 3 g L-1) and shortwave ultraviolet (UV-C, 4.0 kJ m-2) irradiation on quality attributes and antioxidant defense capacity of fresh-cut kiwifruits at refrigerated storage for 7 d. The results indicated that CA-L and UV-C joint treatment, compared to either treatment alone, alleviated microbial load, showed higher quality on ascorbic acid (AsA), green color, total chlorophyll, flesh hardness, total sugar, total acid and malonaldehyde (MDA) content. Besides, it inhibited O 2 · - and •OH generation, induced H2O2 production, improved the activity of antioxidant enzymes (SOD, CAT and APX), activated critical enzymes (PAL, C4H and 4CL) in phenylpropanoid metabolism pathway and further enhanced total phenolic and proanthocyanidin content. Above results demonstrated that UV-C together with CA-L treatment could synergistically maintain overall quality and improve antioxidant capacity of kiwifruit slices. Therefore, the combination of CA-L and UV-C treatment showed a potential practical application in fresh-cut kiwifruits.

Behind the Scenes: The Impact of Bioactive Phenylpropanoids on the Growth Phenotypes of Arabidopsis Lignin Mutants.

The phenylpropanoid pathway converts the aromatic amino acid phenylalanine into a wide range of secondary metabolites. Most of the carbon entering the pathway incorporates into the building blocks of lignin, an aromatic polymer providing mechanical strength to plants. Several intermediates in the phenylpropanoid pathway serve as precursors for distinct classes of metabolites that branch out from the core pathway. Untangling this metabolic network in Arabidopsis was largely done using phenylpropanoid pathway mutants, all with different degrees of lignin depletion and associated growth defects. The phenotypic defects of some phenylpropanoid pathway mutants have been attributed to differentially accumulating phenylpropanoids or phenylpropanoid-derived compounds. In this perspectives article, we summarize and discuss the reports describing an altered accumulation of these bioactive molecules as the causal factor for the phenotypes of lignin mutants in Arabidopsis.

Cloning and functional characterization of two cinnamate 4-hydroxylase genes from Pyrus bretschneideri.

Cinnamate 4-hydroxylase (C4H) is a key enzyme in the phenylpropanoid pathway in plants and is involved in the biosynthesis of secondary metabolites such as lignin and flavonoids. However, the function of C4H in pear plants (Pyrus bretschneideri) has not yet been fully elucidated. By searching pear genome databases, we identified three C4H genes (PbC4H1, PbC4H2 and PbC4H3) encoding proteins that share higher identity with bonafide C4Hs from several species with typical cytochrome P450 domains, suggesting that all three PbC4Hs are also bonafide C4Hs that have close evolutionary relationships with C4Hs from other land plants. Quantitative real-time PCR (qRT-PCR) results indicated that the three PbC4Hs were specifically expressed in one or more tissues. The expression levels of PbC4H1 and PbC4H3 first increased and then decreased during pear fruit development. Treatment with exogenous hormones (ABA, MeJA, and SA) altered the expression of the three PbC4Hs to varying degrees. The expression levels of the PbC4Hs were first induced and then decreased under ABA treatment, while MeJA treatment significantly increased the expression levels of the PbC4Hs. Following treatment with SA, expression levels of PbC4H1 and PbC4H2 increased, while expression levels of PbC4H3 decreased. Enzymatic analysis of the recombinant proteins expressed in yeast indicated that PbC4H1 and PbC4H3 catalysed the conversion of trans-cinnamic acid to p-coumaric acid. Moreover, the expression of PbC4H1 and PbC4H3 in Arabidopsis resulted in an increase in both the lignin content and the thickness of cell walls for intervascular fibres and xylem cells. Taken together, the results of our study not only revealed the potential role of PbC4H1 and PbC4H3 in lignin biosynthesis but also established a foundation for future investigations of the regulation of lignin synthesis and stone cell development in pear fruit by molecular biological techniques.

De novo biosynthesis of liquiritin in Saccharomyces cerevisiae.

Liquiritigenin (LG), isoliquiritigenin (Iso-LG), together with their respective glycoside derivatives liquiritin (LN) and isoliquiritin (Iso-LN), are the main active flavonoids of Glycyrrhiza uralensis, which is arguably the most widely used medicinal plant with enormous demand on the market, including Chinese medicine prescriptions, preparations, health care products and even food. Pharmacological studies have shown that these ingredients have broad medicinal value, including anti-cancer and anti-inflammatory effects. Although the biosynthetic pathway of glycyrrhizin, a triterpenoid component from G. uralensis, has been fully analyzed, little attention has been paid to the biosynthesis of the flavonoids of this plant. To obtain the enzyme-coding genes responsible for the biosynthesis of LN, analysis and screening were carried out by combining genome and comparative transcriptome database searches of G. uralensis and homologous genes of known flavonoid biosynthesis pathways. The catalytic functions of candidate genes were determined by in vitro or in vivo characterization. This work characterized the complete biosynthetic pathway of LN and achieved the de novo biosynthesis of liquiritin in Saccharomyces cerevisiae using endogenous yeast metabolites as precursors and cofactors for the first time, which provides a possibility for the economical and sustainable production and application of G. uralensis flavonoids through synthetic biology.

Induced biosynthesis of chlorogenic acid in sweetpotato leaves confers the resistance against sweetpotato weevil attack.

Sweetpotato weevil is among the most harmful pests in some major sweetpotato growing areas with warm climates. To enable the future establishment of safe weevil-resistance strategies, anti-weevil metabolites from sweetpotato should be investigated. In the present study, we pretreated sweetpotato leaves with exogenous chlorogenic acid and then exposed them to sweetpotato weevils to evaluate this compound's anti-insect activity. We found that chlorogenic acid applied to sweetpotato conferred significant resistance against sweetpotato-weevil feeding. We also observed enhanced levels of chlorogenic acid in response to weevil attack in sweetpotato leaves. To clarify how sweetpotato weevils regulate the generation of chlorogenic acid, we examined key elements of plant-herbivore interaction: continuous wounding and phytohormones participating in chlorogenic acid formation. According to our results, sweetpotato weevil-derived continuous wounding induces increases in phytohormones, including jasmonic acid, salicylic acid, and abscisic acid. These phytohormones can upregulate expression levels of genes involved in chlorogenic acid formation, such as IbPAL, IbC4H and IbHQT, thereby leading to enhanced chlorogenic acid generation. This information should contribute to understanding of the occurrence and formation of natural anti-weevil metabolites in sweetpotato in response to insect attack and provides critical targets for the future breeding of anti-weevil sweetpotato cultivars.

Tanshinone and salvianolic acid biosynthesis are regulated by SmMYB98 in Salvia miltiorrhiza hairy roots.

Salvia miltiorrhiza Bunge is an herb rich in bioactive tanshinone and salvianolic acid compounds. It is primarily used as an effective medicine for treating cardiovascular and cerebrovascular diseases. Liposoluble tanshinones and water-soluble phenolic acids are a series of terpenoids and phenolic compounds, respectively. However, the regulation mechanism for the simultaneous promotion of tanshinone and salvianolic acid biosynthesis remains unclear. This study identified a R2R3-MYB subgroup 20 transcription factor (TF), SmMYB98, which was predominantly expressed in S. miltiorrhiza lateral roots. The accumulation of major bioactive metabolites, tanshinones, and salvianolic acids, was improved in SmMYB98 overexpression (OE) hairy root lines, but reduced in SmMYB98 knockout (KO) lines. The qRT-PCR analysis revealed that the transcriptional expression levels of tanshinone and salvianolic acid biosynthesis genes were upregulated by SmMYB98-OE and downregulated by SmMYB98-KO. Dual-Luciferase (Dual-LUC) assays demonstrated that SmMYB98 significantly activated the transcription of SmGGPPS1, SmPAL1, and SmRAS1. These results suggest that SmMYB98-OE can promote tanshinone and salvianolic acid production. The present findings illustrate the exploitation of R2R3-MYB in terpenoid and phenolic biosynthesis, as well as provide a feasible strategy for improving tanshinone and salvianolic acid contents by MYB proteins in S. miltiorrhiza.

trans-Cinnamic acid-induced leaf expansion involves an auxin-independent component.

The phenylpropanoid pathway, the source of a large array of compounds with diverse functions, starts with the synthesis of trans-cinnamic acid (t-CA) that is converted by cinnamate-4-hydroxylase (C4H) into p-coumaric acid. We have recently shown that in Arabidopsis, exogenous t-CA promotes leaf growth by increasing cell expansion and that this response requires auxin signaling. We have also shown that cell expansion is increased in C4H loss-of-function mutants. Here we provide further evidence that leaf growth is enhanced by either t-CA or a t-CA derivative that accumulates upstream of C4H. We also show that this growth response pathway has two components: one that requires auxin signaling and another which employs a currently unknown mechanism.

Functional characterization of phenylalanine ammonia-lyase- and cinnamate 4-hydroxylase-encoding genes from Lycoris radiata, a galanthamine-producing plant.

Galanthamine (GAL), the well-known Amaryllidaceae alkaloid, is a clinically used drug for the treatment of Alzheimer's disease. L-Phenylalanine (Phe) and trans-cinnamic acid (CA) were enzymatically transformed into the catechol portion of GAL. Herein, a Phe ammonia-lyase-encoding gene LrPAL3 and a cinnamate 4-hydroxylase-encoding gene LrC4H were cloned from Lycoris radiata, a GAL-producing plant. LrPAL3 was overexpressed in Escherichia coli and purified to homogeneity. LrPAL3 catalyzes the forward deamination conversion of L-Phe into trans-CA. The 3-chloro- and 4-fluoro-L-Phe were deaminated to generate the corresponding 3-chloro- and 4-fluoro-trans-CA by LrPAL3. LrPAL3-catalyzed reverse hydroamination was confirmed by the conversion of trans-CA into L-Phe with exceptional regio- and stereo-selectivity. LrC4H was overexpressed in E. coli with tCamCPR, a cytochrome P450 reductase-encoding gene. LrC4H catalyzes the regioselective para-hydroxylation on trans-CA to form p-coumaric acid. The transcriptional levels of both LrPAL3 and LrC4H were positively associated with the GAL contents within the leaves and flowers of L. radiata, which suggested that their expression and function are co-regulated and involved in the biosynthesis of GAL. The present investigations on the biosynthetic genes of GAL will promote the development of synthetic biology platforms for this kind of important drug via metabolic engineering.

Stacking of a low-lignin trait with an increased guaiacyl and 5-hydroxyguaiacyl unit trait leads to additive and synergistic effects on saccharification efficiency in Arabidopsis thaliana.

BACKGROUND: Lignocellulosic biomass, such as wood and straw, is an interesting feedstock for the production of fermentable sugars. However, mainly due to the presence of lignin, this type of biomass is recalcitrant to saccharification. In Arabidopsis, lignocellulosic biomass with a lower lignin content or with lignin with an increased fraction of guaiacyl (G) and 5-hydroxyguaiacyl (5H) units shows an increased saccharification efficiency. Here, we stacked these two traits and studied the effect on the saccharification efficiency and biomass yield, by combining either transaldolase (tra2), cinnamate 4-hydroxylase (c4h-3), or 4-coumarate:CoA ligase (4cl1-1) with caffeic acid O-methyltransferase (comt-1 or comt-4) mutants. RESULTS: The three double mutants (tra2 comt-1, c4h-3 comt-4, and 4cl1-1 comt-4) had a decreased lignin amount and an increase in G and 5H units in the lignin polymer compared to wild-type (WT) plants. The tra2 comt-1 double mutant had a better saccharification efficiency compared to the parental lines when an acid or alkaline pretreatment was used. For the double mutants, c4h-3 comt-4 and 4cl1-1 comt-4, the saccharification efficiency was significantly higher compared to WT and its parental lines, independent of the pretreatment used. When no pretreatment was used, the saccharification efficiency increased even synergistically for these mutants. CONCLUSION: Our results show that saccharification efficiency can be improved by combining two different mutant lignin traits, leading to plants with an even higher saccharification efficiency, without having a yield reduction of the primary inflorescence stem. This approach can help improve saccharification efficiency in bio-energy crops.

Expression profile of phenylpropanoid pathway genes in Decalepis hamiltonii tuberous roots during flavour development.

To explore the transcriptional control of phenylpropanoid pathway (PPP) involved in vanillin flavour metabolites production in tuberous roots of Decalepis hamiltonii, four PPP key genes expressed during the tuber development were identified and their mRNA expression profiles were evaluated using quantitative real-time PCR. Flavour metabolite quantification by HPLC analysis confirmed 10, 170 and 500 µg/g 2-hydroxy-4-methoxy benzaldehyde and 4, 20 and 40 µg/g vanillin in first- (3-month-old plant), second- (18-month-old plant) and third-stage tubers (60-month-old matured plant), respectively. The expression of all four genes phenylalanine ammonia lyase (DhPAL), cinnamate-4-hydroxylase (DhC4H), caffeic acid-O-methyltransferase (DhCOMT) and vanillin synthase (DhVAN) increased with flavour development from first stage to second stage. A decrease in expression from 1.9-folds to 0.1-folds and 19.2-folds to 5.2-folds for DhCOMT and DhVAN was recorded for second stage to third stage, respectively. However, a gradual increase in expression of DhPAL (up to 26.4-folds) and a constant expression pattern for DhC4H (up to 7.1-folds) was evident from second stage to third stage of flavour development. The decrease in the expression levels of DhCOMT and DhVAN in third stage shows that the second-stage tubers are more transcriptionally active towards flavour biosynthesis.

Characterization and Expression Profiling of Camellia sinensis Cinnamate 4-hydroxylase Genes in Phenylpropanoid Pathways.

Cinnamate 4-hydroxylase (C4H), a cytochrome P450-dependent monooxygenase, participates in the synthesis of numerous polyphenoid compounds, such as flavonoids and lignins. However, the C4H gene number and function in tea plants are not clear. We screened all available transcriptome and genome databases of tea plants and three C4H genes were identified and named CsC4Ha, CsC4Hb, and CsC4Hc, respectively. Both CsC4Ha and CsC4Hb have 1518-bp open reading frames that encode 505-amino acid proteins. CsC4Hc has a 1635-bp open reading frame that encodes a 544-amino acid protein. Enzymatic analysis of recombinant proteins expressed in yeast showed that the three enzymes catalyzed the formation of p-coumaric acid (4-hydroxy trans-cinnamic acid) from trans-cinnamic acid. Quantitative real-time PCR (qRT-PCR) analysis showed that CsC4Ha was highly expressed in the 4th leaf, CsC4Hb was highly expressed in tender leaves, while CsC4Hc was highly expressed in the young stems. The three CsC4Hs were induced with varying degrees by abiotic stress treatments. These results suggest they may have different subcellular localization and different physiological functions.