hydroxycinnamoyl-CoA Research Articles

Biosynthesis of resveratrol derivatives and evaluation of their anti-inflammatory activity

Resveratrol is a typical plant phenolic compound whose derivatives are synthesized through hydroxylation, O-methylation, prenylation, and oligomerization. Resveratrol and its derivatives exhibit anti-neurodegenerative, anti-rheumatoid, and anti-inflammatory effects. Owing to the diverse biological activities of these compounds and their importance in human health, this study attempted to synthesize five resveratrol derivatives (isorhapontigenin, pterostilbene, 4-methoxyresveratrol, piceatannol, and rhapontigenin) using Escherichia coli. Two-culture system was used to improve the final yield of resveratrol derivatives. Resveratrol was synthesized in the first E. coli cell that harbored genes for resveratrol biosynthesis including TAL (tyrosine ammonia lyase), 4CL (4-coumaroyl CoA ligase), STS (stilbene synthase) and genes for tyrosine biosynthesis such as aroG (deoxyphosphoheptonate aldolase) and tyrA (prephenate dehydrogenase). Thereafter, culture filtrate from the first cell was used for the modification reaction carried out using the second E. coli harboring hydroxylase and/or O-methyltransferase. Approximately, 89.8 mg/L of resveratrol was synthesized and using the same, five derivatives were prepared with a conversion rate of 88.2% to 22.9%. Using these synthesized resveratrol derivatives, we evaluated their anti-inflammatory activity. 4-Methoxyresveratrol, pterostilbene and isorhapontigenin showed the anti-inflammatory effects without any toxicity. In addition, pterostilbene exhibited the enhanced anti-inflammatory effects for macrophages compared to resveratrol.

RNAi Modulation of Chlorogenic Acid and Lignin Deposition in Nicotiana tabacum and Insufficient Compensatory Metabolic Cross-Talk.

Chlorogenic acid (CGA) and guaiacyl/syringyl (G/S) lignin formation involves hydroxycinnamoyl ester intermediacy, the latter formed via hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferase (HCT) and hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase (HQT) activities. HQT and HCT RNAi silencing of a commercial tobacco (Nicotiana tabacum) K326 line was examined herein. NtHQT gene silencing gave relatively normal plant phenotypes, with CGA levels reduced (down to 1% of wild type) with no effects on lignin. RNAi NtHCT silencing had markedly adverse phenotypes (e.g., stunted, multiple stems, delayed flowering, with senescence delayed by several months). Lignin contents were partially lowered, with a small increase in cleavable p-hydroxyphenyl (H) monomers; those plants had no detectable CGA level differences relative to wild type. In vitro NtHCT kinetic parameters revealed preferential p-coumaroyl CoA and shikimate esterification, as compared to other structurally related potential acyl group donors and acceptors. In the presence of coenzyme A, NtHCT catalyzed the reverse reaction. Site-directed mutagenesis of NtHCT (His153Ala) abolished enzymatic activity. NtHQT, by comparison, catalyzed preferential conversion of p-coumaroyl CoA and quinic acid to form p-coumaroyl quinate, the presumed CGA precursor. In sum, metabolic pathways to CGA and lignins appear to be fully independent, and previous conflicting reports of substrate versatilities and metabolic cross-talk are resolved.

Targeting hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase for lignin modification in Brachypodium distachyon

BackgroundHydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) is a central enzyme of the so-called “esters” pathway to monolignols. As originally envisioned, HCT functions twice in this pathway, to form coumaroyl shikimate and then, in the “reverse” direction, to convert caffeoyl shikimate to caffeoyl CoA. The discovery of a caffeoyl shikimate esterase (CSE) that forms caffeic acid directly from caffeoyl shikimate calls into question the need for the reverse HCT reaction in lignin biosynthesis. Loss of function of HCT gives severe growth phenotypes in several dicot plants, but less so in some monocots, questioning whether this enzyme, and therefore the shikimate shunt, plays the same role in both monocots and dicots. The model grass Brachypodium distachyon has two HCT genes, but lacks a classical CSE gene. This study was therefore conducted to evaluate the utility of HCT as a target for lignin modification in a species with an “incomplete” shikimate shunt.ResultsThe kinetic properties of recombinant B. distachyon HCTs were compared with those from Arabidopsis thaliana, Medicago truncatula, and Panicum virgatum (switchgrass) for both the forward and reverse reactions. Along with two M. truncatula HCTs, B. distachyon HCT2 had the least kinetically unfavorable reverse HCT reaction, and this enzyme is induced when HCT1 is down-regulated. Down regulation of B. distachyon HCT1, or co-down-regulation of HCT1 and HCT2, by RNA interference led to reduced lignin levels, with only modest changes in lignin composition and molecular weight.ConclusionsDown-regulation of HCT1, or co-down-regulation of both HCT genes, in B. distachyon results in less extensive changes in lignin content/composition and cell wall structure than observed following HCT down-regulation in dicots, with little negative impact on biomass yield. Nevertheless, HCT down-regulation leads to significant improvements in biomass saccharification efficiency, making this gene a preferred target for biotechnological improvement of grasses for bioprocessing.

Biosynthesis of resveratrol using metabolically engineered Escherichia coli

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a phenolic compound widely used in pharmaceutics and nutraceutics. Although resveratrol is produced by some plant species, including grapes, peanuts, and berries, the content of resveratrol and its derivatives are very low. Therefore, an alternative biosynthetic method using microorganisms, such as Escherichia coli, has been developed over the past two decades. In the present study, a resveratrol-over-producing E. coli strain was developed using three strategies. First, we increased the synthesis of p-coumaric acid, a precursor of resveratrol, by manipulating genes in the shikimate pathway of E. coli. Second, three genes involved in resveratrol biosynthesis, such as tyrosine ammonia lyase (TAL), 4-coumaroyl CoA ligase (4CL), and stilbene synthase (STS), were cloned from diverse sources, such as plants and microorganisms, and the best combination was selected to maximize resveratrol production in E. coli. Finally, culture conditions, such as cell concentration, culture temperature, and carbon sources, were established for optimal resveratrol production. Through these strategies, approximately 80.4 mg/L of resveratrol was biosynthesized after 48 h of culture using glycerol as a carbon source.

Potential application of CHS and 4CL genes from grape endophytic fungus in production of naringenin and resveratrol and the improvement of polyphenol profiles and flavour of wine

4-Coumaroyl-CoA ligase (Al4CL) and chalcone synthase (AlCHS) genes were found in grape endophyte Alternaria sp. MG1, but were not functional verified. A cross-validation method was used in Saccharomyces cerevisiae to identify their functions. AlCHS was identified to synthesize both naringenin and resveratrol, while Al4CL synthesized p-coumaroyl CoA. Co-culture of S. cerevisiae strains separately containing AlCHS and Al4CL resulted in the simultaneous production of naringenin (18.5 mg/L) and resveratrol (113.2 μg/L). Strain S. cerevisiae containing Al4CL was used in winemaking and the chemical and aroma compounds in wine were detected by HPLC and SPME-GC–MS. Results showed that the total contents of polyphenols, anthocyanins, flavonol, ethyl esters and fatty acids significantly increased, while the 4-vinylphenol content decreased, and the fruit and cheese flavour increased but the green aroma declined. This study indicated the potential application of Al4CL and AlCHS genes from Alternaria sp. MG1 for improvement of wine nutrients and flavour.

Two CYP71AJ enzymes function as psoralen synthase and angelicin synthase in the biosynthesis of furanocoumarins in Peucedanum praeruptorum Dunn.

Psoralen synthase and angelicin synthase responsible for the formation of psoralen and angelicin in Peucedanum praeruptorum Dunn were identified and functionally characterized, respectively. Furanocoumarins were reported to possess several activities such as anticancer, anti-inflammatory and neuroprotective, and function as phytotoxin and allelochemical in plants. Furanocoumarins are the main bioactive ingredient in P. praeruptorum which is a commonly used traditional Chinese medicine. Phenylalanine ammonia lyase (PAL), 4-coumarate: CoA ligase (4CL), p-coumaroyl CoA 2'-hyfroxylase (C2'H) were cloned previously to elucidate the biosynthetic mechanism of coumarin lactone ring. However, the genes involved in complex coumarins in P. praeruptorum have not been explored. Herein, putative psoralen synthase CYP71AJ49 and angelicin synthase CYP71AJ51 were cloned from P. praeruptorum. In vivo and in vitro yeast assays were conducted to confirm their activities. Furthermore, the results of High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry (HPLC-ESI-MS) verified that CYP71AJ49 catalyzed the conversion of marmesin to psoralen, and CYP71AJ51 catalyzed columbianetin to angelicin. Subsequently, the expression profile showed that CYP71AJ49 and CYP71AJ51 were easily affected by environmental conditions, especially UV and temperature. The genes tissue-specific expression and compounds tissue-specific distribution pattern indicated the existence of substance transport in P. praeruptorum. Phylogenetic analysis was conducted with 27 CYP71AJs, CYP71AJ49 and CYP71AJ51 were classified in I-4 and I-2, respectively. These results provide further insight to understand the biosynthetic mechanism of complex coumarins.

Structure and Monomer Ratio of Lignin in C3H and HCT RNAi Transgenic Poplar Saplings

AbstractWild type poplar hybrid (Populus alba × P. glandulosa cv‘84k′), p‐coumaroyl shikimate 3′‐hydroxylase (C3H) downregulated transgenic poplar and hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) downregulated transgenic poplar were used as experimental materials. The techniques of two‐dimensional heteronuclear single‐quantum coherence nuclear magnetic resonance (2D‐HSQC‐NMR) spectroscopy and pyrolysis coupled to gas chromatography‐mass spectrometry (Py‐GC/MS) were used for the analysis and comparison of the lignin structure and monomer ratio between the wild type poplar hybrid and transgenic plants. The results of these analyses showed that the lignin of all poplar samples contained β‐O‐4′ aryl ether linkages and their derivative substructures, resinol substructures, spirodienone substructures, phenylcoumarane substructures, p‐hydroxycinnamyl alcohol end groups, syringyl units, p‐hydroxyphenyl units, guaiacyl units, p‐hydroxybenzoate substructures, and ferulate. Downregulation of C3H and HCT in double transgenic poplar reduced the β‐O‐4′ relative content of lignin in poplar. The S unit, H unit, and S/G ratio in the C3H‐downregulated transgenic poplar were increased, while its G unit was decreased. The S unit and S/G ratio in HCT‐downregulated transgenic poplar were decreased while its G unit was increased. The C3H‐downregulated transgenic poplar was a superior material for biomass energy, pulping, and papermaking than the HCT‐downregulated transgenic poplar.

Identification and tissue-specific expression of rutin biosynthetic pathway genes in Capparis spinosa elicited with salicylic acid and methyl jasmonate

Capparis spinosa is an edible medicinal plant which is considered as an excellent source of rutin. Rutin is a glycoside of the flavonoid quercetin that has been reported to have a beneficial role in controlling various diseases such as hypertension, arteriosclerosis, diabetes, and obesity. In this study, the partial cDNA of four genes involved in the rutin biosynthetic pathway including 4-coumaroyl CoA ligase (4CL), flavonoid 3′-hydroxylase (F3′H), flavonol synthase (FLS) and flavonol-3-O-glucoside L-rhamnosyltransferase (RT) were identified in C.spinosa plants for the first time. The protein sequences of these genes shared high similarity with the same proteins in other plant species. Subsequently, the expression patterns of these genes as well as rutin accumulation in C.spinosa leaves treated with different concentrations of salicylic acid (SA) and methyl jasmonate (MeJA) and also in different tissues of Caper plants treated with 100 mgL−1 SA and 150 μM MeJA were evaluated. The expression of all four genes was clearly up-regulated and rutin contents increased in response to MeJA and SA treatments after 24 h. The highest rutin contents (5.30 mgg−1 DW and 13.27 mgg−1 DW), as well as the highest expression levels of all four genes, were obtained using 100 mgL−1 SA and 150 μM MeJA, respectively. Among the different tissues, the highest rutin content was observed in young leaves treated with 150 μM MeJA, which corresponded to the expression of related genes, especially RT, as a key gene in the rutin biosynthetic pathway. These results suggest that rutin content in various tissues of C. spinosa can be enhanced to a significant extent by MeJA and SA treatments and the gene expression patterns of rutin-biosynthesis-related genes are regulated by these elicitors.

Metabolic Flux Analysis of Catechin Biosynthesis Pathways Using Nanosensor.

(+)-Catechin is an important antioxidant of green tea (Camelia sinensis (L.) O. Kuntze). Catechin is known for its positive role in anticancerous activity, extracellular matrix degradation, cell death regulation, diabetes, and other related disorders. As a result of enormous interest in and great demand for catechin, its biosynthesis using metabolic engineering has become the subject of concentrated research with the aim of enhancing (+)-catechin production. Metabolic flux is an essential concept in the practice of metabolic engineering as it helps in the identification of the regulatory element of a biosynthetic pathway. In the present study, an attempt was made to analyze the metabolic flux of the (+)-catechin biosynthesis pathway in order to decipher the regulatory element of this pathway. Firstly, a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor (FLIP-Cat, fluorescence indicator protein for (+)-catechin) was developed for real-time monitoring of (+)-catechin flux. In vitro characterization of the purified protein of the nanosensor showed that the nanosensor was pH stable and (+)-catechin specific. Its calculated Kd was 139 µM. The nanosensor also performed real-time monitoring of (+)-catechin in bacterial cells. In the second step of this study, an entire (+)-catechin biosynthesis pathway was constructed and expressed in E. coli in two sets of plasmid constructs: pET26b-PT7-rbs-PAL-PT7-rbs-4CL-PT7-rbs-CHS-PT7-rbs-CHI and pET26b-T7-rbs-F3H-PT7-rbs- DFR-PT7-rbs-LCR. The E. coli harboring the FLIP-Cat was transformed with these plasmid constructs. The metabolic flux analysis of (+)-catechin was carried out using the FLIP-Cat. The FLIP-Cat successfully monitored the flux of catechin after adding tyrosine, 4-coumaric acid, 4-coumaroyl CoA, naringenin chalcone, naringenin, dihydroquercetin, and leucocyanidin, individually, with the bacterial cells expressing the nanosensor as well as the genes of the (+)-catechin biosynthesis pathway. Dihydroflavonol reductase (DFR) was identified as the main regulatory element of the (+)-catechin biosynthesis pathway. Information about this regulatory element of the (+)-catechin biosynthesis pathway can be used for manipulating the (+)-catechin biosynthesis pathway using a metabolic engineering approach to enhance production of (+)-catechin.

ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE 1 (ADPG1) releases latent defense signals in stems with reduced lignin content

There is considerable interest in engineering plant cell wall components, particularly lignin, to improve forage quality and biomass properties for processing to fuels and bioproducts. However, modifying lignin content and/or composition in transgenic plants through down-regulation of lignin biosynthetic enzymes can induce expression of defense response genes in the absence of biotic or abiotic stress. Arabidopsis thaliana lines with altered lignin through down-regulation of hydroxycinnamoyl CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) or loss of function of cinnamoyl CoA reductase 1 (CCR1) express a suite of pathogenesis-related (PR) protein genes. The plants also exhibit extensive cell wall remodeling associated with induction of multiple cell wall-degrading enzymes, a process which renders the corresponding biomass a substrate for growth of the cellulolytic thermophile Caldicellulosiruptor bescii lacking a functional pectinase gene cluster. The cell wall remodeling also results in the release of size- and charge-heterogeneous pectic oligosaccharide elicitors of PR gene expression. Genetic analysis shows that both in planta PR gene expression and release of elicitors are the result of ectopic expression in xylem of the gene ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE 1 (ADPG1), which is normally expressed during anther and silique dehiscence. These data highlight the importance of pectin in cell wall integrity and the value of lignin modification as a tool to interrogate the informational content of plant cell walls.

A Transcriptional Network Makes Normal Tomato Fruit Not Purple

A Transcriptional Network Makes Normal Tomato Fruit Not Purple

Identification and Functional Characterization of Genes Involved in the Biosynthesis of Caffeoylquinic Acids in Sunflower (Helianthus annuus L.).

Sunflower (Helianthus annuus L.) sprouts accumulate high amounts of caffeoylquinic acids (CQAs) including chlorogenic acid (5-CQA) and 1,5-diCQA. These compounds, which can be found in many plants, including tomato, globe artichoke, and chicory, have many health benefits, including antioxidant, antihepatotoxic, and antiglycative activities. However, CQA profiles and biosynthesis have not previously been studied in sunflower sprouts. In the present study, we found that 5-CQA and 1,5-diCQA were the major CQAs found in sunflower sprouts. We also identified minor accumulation of other CQAs, namely 3-CQA, 4-CQA, 3,4-diCQA, and 4,5-diCQA. According to genome-wide identification and phylogenetic analysis of genes involved in CQA biosynthesis in sunflower, three genes (HaHQT1, HaHQT2, and HaHQT3) encoding hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase (HQT) and two genes (HaHCT1 and HaHCT2) encoding hydroxycinnamoyl CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) were identified. Expression analysis of these five genes in hypocotyls and cotyledons strongly suggested that HaHQT2 could be the main enzyme responsible for CQA biosynthesis, as HaHQT2 had the highest expression levels. In addition, when transiently expressed in the leaves of Nicotiana benthamiana, all three HaHQTs, which were soluble and not membrane-bound enzymes, could increase the content of 5-CQA by up to 94% compared to that in a control. Overall, our results increase understanding of CQA biosynthesis in sunflower sprouts and could be exploited by plant breeders to enhance accumulation of health-promoting CQAs in these plants.

Elucidation of the biosynthesis pathway and heterologous construction of a sustainable route for producing umbelliferone

BackgroundCoumarins play roles in many biological processes. Angelica decursiva is one of the major sources of coumarins in China. Due to increasing demand for coumarins in the marketplace, traditional extraction from plants is now considered economically insufficient and unsustainable. Microbial synthesis is a promising strategy for scalable production of coumarins. However, the biosynthetic pathway of coumarin remains poorly understood, and even more, the genes associated with this process have not been characterized in A. decursiva.ResultsRNA-seq was employed to elucidate the umbelliferone biosynthetic pathway. The results indicated that three enzymes, phenylalanine ammonia-lyase (PAL), 4-Coumarate: Coenzyme A Ligase (4CL), and p-coumaroyl CoA 2'-hydroxylase (C2’H) were involved in umbelliferone biosynthesis. Using the cloned genes, we generated a synthetic biology based microbial cell factory that produces coumarins from tyrosine utilizing Rhodotorula glutinis tyrosine ammonia lyase (RgTAL) to bypass cinnamic acid 4-hydroxylase (C4H). With metabolic engineering strategies, we deleted prephenate dehydratase (pheA), anthranilate synthase (trpE) and transcriptional regulatory protein (tyrR) and overexpressed six related genes involved in tyrosine biosynthesis, to drive the carbon flux from tyrosine. To overcome the limitation of 4CL, a virtual screening and site-specific mutagenesis-based protein engineering approach was applied. In addition, induction/culture conditions and different ions were employed to further improve the yield of umbelliferone. Finally, a yield of 356.59 mg/L umbelliferone was obtained.ConclusionsThe current study elucidated the umbelliferone biosynthesis pathway in A. decursiva. The results also demonstrated the feasibility of integrating gene mining with synthetic biology techniques to produce natural compounds.

In vitro evaluation of hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase expression and regulation in Taraxacum antungense in relation to 5-caffeoylquinic acid production

Chlorogenic acids (CGA; including 5-caffeoylquinic acid and its regio-isomers) in Taraxacum antungense Kitag. have antioxidant and anti-inflammatory properties and exert other pharmacological effects. T. antungense hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (TaHQT)1 and TaHQT2, which belong to the BAHD acyltransferase family, are candidates for synthesizing 5-caffeoylquinic acid and that have not been extensively characterized. In this study, we cloned the TaHQT1 and TaHQT2 genes and analysed the properties of the expressed enzymes both in vitro and in vivo. Quantitative reverse transcription PCR analysis revealed that TaHQT1 was highly expressed in the root, whereas the strongest TaHQT2 expression was observed in T. antungense leaves. In Nicotiana benthamiana leaf cells, TaHQT1 and TaHQT2 were localized at the cell periphery as well as in the cytoplasm and nucleus. The 5-caffeoylquinic acid concentrations in T. antungense calli were reduced by TaHQT1 and TaHQT2 knockdown relative to the control. Conversely, inoculation of T. antungense plants tissues with recombinant TaHQT1 and TaHQT2 increased 5-caffeoylquinic acid levels in situ. These in vitro and in vivo findings demonstrate that both HQTs are involved in regulating 5-caffeoylquinic acid biosynthesis in T. antungense, which can be exploited to increase 5-caffeoylquinic acid production in plants for medicinal or other beneficial purposes.

Functional characterization and correlation analysis of phenylalanine ammonia-lyase (PAL) in coumarin biosynthesis from Peucedanum praeruptorum Dunn

Coumarins exhibit many biological activities and are the main specialised metabolites of Peucedanum praeruptorum Dunn, an important plant used in traditional Chinese medicine. In preliminary studies, we cloned several genes involved in coumarin biosynthesis in P. praeruptorum, such as 4-coumarate: CoA ligase (4CL), p-coumaroyl CoA 2′-hydroxylase (C2′H), feruloyl CoA 6′-hydroxylase (F6′H) and bergaptol O-methyltransferase (BMT). However, phenylalanine ammonia-lyase (PAL) in P. praeruptorum (PpPAL) has not yet been studied. In the present study, we cloned one novel PpPAL gene. Subsequently, the relationship between gene and compounds was studied using quantitative real-time PCR (qRT-PCR) and High Performance Liquid Chromatography (HPLC) analysis. Then, enzyme function was analyzed with L-phenylalanine (L-Phe) as substrate. These experiments showed that the coumarin content could be upregulated by methyl jasmonate (MeJA), UV irradiation and cold, which was consistent with increased expression levels of PpPAL. In addition, correlation analysis indicated that coumarins were partially related to PpPAL. And the recombinant protein could catalyze the conversion of L-Phe to trans-cinnamic acid (t-CA) with a Km of 120 ± 33 μM and a Kcat of 117 ± 32 min−1. Besides, Tyr110, Phe116, Gly117, Ser206, Leu209, Leu259, Tyr354, Arg357, Asn387 and Phe403 were essential for enzymatic activity based on three-dimensional modeling and site-directed mutagenesis experiments. Altogether these results highlight the importance of PpPAL in abiotically induced coumarin biosynthesis and provide further insights regarding the structure-function relationships of this protein.

Preliminary study of Cell Wall Structure and its Mechanical Properties of C3H and HCT RNAi Transgenic Poplar Sapling

This research focused on the cell wall structure and its mechanical properties of down-regulated Coumaroyl shikimate 3-hydroxylase (C3H) transgenic poplar and down-regulated hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) transgenic poplar (Populus alba × P. glandulosa cv ‘84 k’). The wood samples with respect to microstructure, the longitudinal elastic modulus (MOE) and hardness of wood fiber secondary cell wall were investigated. The results show that the lignin contents in the two transgenic poplar woods were lower than non-modified wood. The C3H transgenic poplar and HCT transgenic poplar have more than 18.5% and 16.1% cellulose crystalline regions than non-modified poplar respectively. The diameter of the fiber cell and the vessel element of transgenic poplars are smaller. Double radial vessel cell wall thicknesses of both transgenic poplars were smaller than non-modified poplar. Cell wall ratios for the transgenic poplar were higher than non-modified poplar and cell wall density was significantly lower in both C3H and HCT transgenic poplar. The cell wall MOEs of C3H and HCT transgenic poplar was 5.8% and 7.0% higher than non-modified poplar. HCT can be more effective than C3H to modify the trees by considerably increasing mechanical properties of the cell wall.

Agronomic Performance and Lignin Content of HCT Down-Regulated Alfalfa (Medicago sativa L.)

Lignin is a cell wall polymer that reduces the enzymatic digestibility and conversion efficiency of lignocellulosic biomass to ethanol. Down-regulation of the hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) gene reduces lignin content in alfalfa (Medicago sativa L.). The objective of this study was to evaluate HCT down-regulated alfalfa plants for their forage composition and agronomic performance in the greenhouse and under field conditions. Individual plants with two separate T0HCT down-regulation events 3a and 30a were used to make crosses with a common male sterile line to generate T1 progenies (3A and 30A, respectively). The experimental design was a randomized complete block with four replications per line and ten individuals per replication. Two-row plots with full-sib progeny rows with (+) and without (−) the transgene were planted along with commercial cultivars used as checks. Most differential responses in forage quality and agronomic characteristics between full-sib down-regulated and non-down-regulated progenies were event-specific and found mainly between the HCT30A+ and HCT30A− progenies. Variation between HCT− and HCT+ alfalfa plants was observed in the HCT gene transcript levels, acid detergent lignin (ADL), relative feed value (RFV), and saccharification efficiency. Although differential responses in agronomic performance of field-grown HCT down-regulated alfalfa plants were identified for biomass yield and plant height, HCT+ and HCT− progenies had similar spring growth and fall dormancy. The reduction in lignin content of alfalfa plants via modification of HCT transcript levels increased forage quality and efficiency of sugar release of plants grown under greenhouse and field conditions.

Development and use of a switchgrass (Panicum virgatum L.) transformation pipeline by the BioEnergy Science Center to evaluate plants for reduced cell wall recalcitrance

BackgroundThe mission of the BioEnergy Science Center (BESC) was to enable efficient lignocellulosic-based biofuel production. One BESC goal was to decrease poplar and switchgrass biomass recalcitrance to biofuel conversion while not affecting plant growth. A transformation pipeline (TP), to express transgenes or transgene fragments (constructs) in these feedstocks with the goal of understanding and decreasing recalcitrance, was considered essential for this goal. Centralized data storage for access by BESC members and later the public also was essential.ResultsA BESC committee was established to codify procedures to evaluate and accept genes into the TP. A laboratory information management system (LIMS) was organized to catalog constructs, plant lines and results from their analyses. One hundred twenty-eight constructs were accepted into the TP for expression in switchgrass in the first 5 years of BESC. Here we provide information on 53 of these constructs and the BESC TP process. Eleven of the constructs could not be cloned into an expression vector for transformation. Of the remaining constructs, 22 modified expression of the gene target. Transgenic lines representing some constructs displayed decreased recalcitrance in the field and publications describing these results are tabulated here. Transcript levels of target genes and detailed wall analyses from transgenic lines expressing six additional tabulated constructs aimed toward modifying expression of genes associated with wall structure (xyloglucan and lignin components) are provided. Altered expression of xyloglucan endotransglucosylase/hydrolases did not modify lignin content in transgenic plants. Simultaneous silencing of two hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferases was necessary to decrease G and S lignin monomer and total lignin contents, but this reduced plant growth.ConclusionsA TP to produce plants with decreased recalcitrance and a LIMS for data compilation from these plants were created. While many genes accepted into the TP resulted in transgenic switchgrass without modified lignin or biomass content, a group of genes with potential to improve lignocellulosic biofuel yields was identified. Results from transgenic lines targeting xyloglucan and lignin structure provide examples of the types of information available on switchgrass lines produced within BESC. This report supplies useful information when developing coordinated, large-scale, multi-institutional reverse genetic pipelines to improve crop traits.

Similar, but different: structurally related azelaic acid and hexanoic acid trigger differential metabolomic and transcriptomic responses in tobacco cells

BackgroundPlants respond to various stress stimuli by activating an enhanced broad-spectrum defensive ability. The development of novel resistance inducers represents an attractive, alternative crop protection strategy. In this regard, hexanoic acid (Hxa, a chemical elicitor) and azelaic acid (Aza, a natural signaling compound) have been proposed as inducers of plant defense, by means of a priming mechanism. Here, we investigated both the mode of action and the complementarity of Aza and Hxa as priming agents in Nicotiana tabacum cells in support of enhanced defense.ResultsMetabolomic analyses identified signatory biomarkers involved in the establishment of a pre-conditioned state following Aza and Hxa treatment. Both inducers affected the metabolomes in a similar manner and generated common biomarkers: caffeoylputrescine glycoside, cis-5-caffeoylquinic acid, feruloylglycoside, feruloyl-3-methoxytyramine glycoside and feruloyl-3-methoxytyramine conjugate. Subsequently, quantitative real time-PCR was used to investigate the expression of inducible defense response genes: phenylalanine ammonia lyase, hydroxycinnamoyl CoA quinate transferase and hydroxycinnamoyl transferase to monitor activation of the early phenylpropanoid pathway and chlorogenic acids metabolism, while ethylene response element-binding protein, small sar1 GTPase, heat shock protein 90, RAR1, SGT1, non-expressor of PR genes 1 and thioredoxin were analyzed to report on signal transduction events. Pathogenesis-related protein 1a and defensin were quantified to investigate the activation of defenses regulated by salicylic acid and jasmonic acid respectively. The qPCR results revealed differential expression kinetics and, in general (except for NPR1, Thionin and PR1a), the relative gene expression ratios observed in the Hxa-treated cells were significantly greater than the expression observed in the cells treated with Aza.ConclusionsThe results indicate that Aza and Hxa have a similar priming effect through activation of genes involved in the establishment of systemic acquired resistance, associated with enhanced synthesis of hydroxycinnamic acids and related conjugates.

Engineering of Escherichia coli for the synthesis of N-hydroxycinnamoyl tryptamine and serotonin.

Plants synthesize various phenol amides. Among them, hydroxycinnamoyl (HC) tryptamines and serotonins exhibit antioxidant, anti-inflammatory, and anti-atherogenic activities. We synthesized HC-tryptamines and HC-serotonin from several HCs and either tryptamine or serotonin using Escherichia coli harboring the 4CL (4-coumaroyl CoA ligase) and CaHCTT [hydroxycinnamoyl-coenzyme A:serotonin N-(hydroxycinnamoyl)transferase] genes. E. coli was engineered to synthesize N-cinnamoyl tryptamine from glucose. TDC (tryptophan decarboxylase) and PAL (phenylalanine ammonia lyase) along with 4CL and CaHCTT were introduced into E. coli and the phenylalanine biosynthetic pathway of E. coli was engineered. Using this strategy, approximately 110.6mg/L of N-cinnamoyl tryptamine was synthesized. By feeding 100μM serotonin into the E. coli culture, which could induce the synthesis of cinnamic acid or p-coumaric acid, more than 99μM of N-cinnamoyl serotonin and N-(p-coumaroyl) serotonin were synthesized.