Biochemical Pathways of Salicylic Acid Derived from l-Phenylalanine in Plants with Different Basal SA Levels.

As a plant hormone, salicylic acid (SA) has diverse regulatory roles in plant growth and stress resistance. Although SA is widely found in plants, there is substantial variation in basal SA among species. Tea plant is an economically important crop containing high contents of SA whose synthesis pathway remains unidentified. The phenylalanine ammonia-lyase (PAL) pathway is responsible for basal SA synthesis in plants. In this study, isotopic tracing and enzymatic assay experiments were used to verify the SA synthesis pathway in tea plants and evaluate the variation in phenylalanine-derived SA formation among 11 plant species with different levels of SA. The results indicated that SA could be synthesized via PAL in tea plants and conversion efficiency from benzoic acid to SA might account for variation in basal SA among plant species. This research lays the foundation for an improved understanding of the molecular regulatory mechanism for SA biosynthesis.

Comparative analyses reveal a phenylalanine ammonia lyase dependent and salicylic acid mediated host resistance in Zingiber zerumbet against the necrotrophic soft rot pathogen Pythium myriotylum.

Little is known about the molecular basis of host defense in resistant wild species Zingiber zerumbet (L.) Smith against the soil-borne, necrotrophic oomycete pathogen Pythium myriotylum Drechsler, which causes the devastating soft rot disease in the spice crop ginger (Zingiber officinale Roscoe). We investigated the pattern of host defense between Z. zerumbet and ginger in response to P. myriotylum inoculation. Analysis of gene expression microarray data revealed enrichment of phenylpropanoid biosynthetic genes, particularly lignin biosynthesis genes, in pathogen-inoculated Z. zerumbet compared to ginger. RT-qPCR analysis showed the robust activation of phenylpropanoid biosynthesis genes in Z. zerumbet, including the core genes PAL, C4H, 4CL, and the monolignol biosynthesis and polymerization genes such as CCR, CAD, C3H, CCoAOMT, F5H, COMT, and LAC. Additionally, Z. zerumbet exhibited the accumulation of the phenolic acids including p-coumaric acid, sinapic acid, and ferulic acid that are characteristic of the cell walls of commelinoid monocots like Zingiberaceae and are involved in cell wall strengthening by cross linking with lignin. Z. zerumbet also had higher total lignin and total phenolics content compared to pathogen-inoculated ginger. Phloroglucinol staining revealed the enhanced fortification of cell walls in Z. zerumbet, specifically in xylem vessels and surrounding cells. The trypan blue staining indicated inhibition of pathogen growth in Z. zerumbet at the first leaf whorl, while ginger showed complete colonization of the pith within 36 h post inoculation (hpi). Accumulation of salicylic acid (SA) and induction of SA regulator NPR1 and the signaling marker PR1 were observed in Z. zerumbet. Silencing of PAL in Z. zerumbet through VIGS suppressed downstream genes, leading to reduced phenylpropanoid accumulation and SA level, resulting in the susceptibility of plants to P. myriotylum. These findings highlight the essential role of PAL-dependent mechanisms in resistance against P. myriotylum in Z. zerumbet. Moreover, our results suggest an unconventional role for SA in mediating host resistance against a necrotroph. Targeting the phenylpropanoid pathway could be a promising strategy for the effective management of P. myriotylum in ginger.

Efficient biosynthesis of resveratrol via combining phenylalanine and tyrosine pathways in Saccharomyces cerevisiae.

BACKGROUND: Resveratrol is a commercially available stilbenoid widely used as dietary supplements, functional food ingredients, and cosmetic ingredients due to its diverse physiological activities. The production of resveratrol in microorganisms provides an ideal source that reduces the cost of resveratrol, but the titer in Saccharomyces cerevisiae was still much lower than that in other hosts. RESULTS: To achieve enhanced production of resveratrol in S. cerevisiae, we constructed a biosynthetic pathway via combining phenylalanine and tyrosine pathways by introducing a bi-functional phenylalanine/tyrosine ammonia lyase from Rhodotorula toruloides. The combination of phenylalanine pathway with tyrosine pathway led to a 462% improvement of resveratrol production in yeast extract peptone dextrose (YPD) medium with 4% glucose, suggesting an alternative strategy for producing p-coumaric acid-derived compounds. Then the strains were further modified by integrating multi-copy biosynthetic pathway genes, improving metabolic flux to aromatic amino acids and malonyl-CoA, and deleting by-pathway genes, which resulted in 1155.0 mg/L resveratrol in shake flasks when cultured in YPD medium. Finally, a non-auxotrophic strain was tailored for resveratrol production in minimal medium without exogenous amino acid addition, and the highest resveratrol titer (4.1 g/L) ever reported was achieved in S. cerevisiae to our knowledge. CONCLUSIONS: This study demonstrates the advantage of employing a bi-functional phenylalanine/tyrosine ammonia lyase in the biosynthetic pathway of resveratrol, suggesting an effective alternative in the production of p-coumaric acid-derived compounds. Moreover, the enhanced production of resveratrol in S. cerevisiae lays a foundation for constructing cell factories for various stilbenoids.

Effect of co-treatment of microwave and exogenous l-phenylalanine on the enrichment of flavonoids in Tartary buckwheat sprouts.

BACKGROUND: Tartary buckwheat is rich in flavonoids. The application of physical processing technology and exogenous materials treatment can effectively promote grain germination and the accumulation of bioactive secondary metabolites. The content of four flavonoids, the activities of key enzymes (phenylalanine ammonia-lyase (PAL), chalcone isomerase (CHI), flavonol synthase (FLS)) and the expression of key enzyme genes (FtPAL, FtCHI, FtFLS1, FtFLS2) in Tartary buckwheat sprouts treated with microwave and l-phenylalanine (l-Phe) were investigated, and the relationship between them was analyzed to explore the mechanism of promoting flavonoid accumulation, and to provide a theoretical basis for the development of functional Tartary buckwheat sprout food. RESULTS: Germination can promote the synthesis of flavonoids. The contents of chlorogenic acid and rutin in 7-day sprouts increased by 13 420.63% and 225.12% compared with seeds, respectively. Under the best treatment condition T3 (microwave 250 W, 90 s, 2.9 mmol L-1 L-Phe), the specific activities of PAL, CHI and FLS in 5-day-old sprouts increased by 47.84%, 53.04% and 28.02% compared with control check (CK), respectively; and the expression of FtPAL, FtCHI and FtFlS1 increased by 39.84%, 24.78% and 33.72% compared with CK, respectively. Correlation analysis showed that the content of flavonoids in Tartary buckwheat sprouts was significantly positively correlated with the specific activities of key enzymes (P < 0.01) and dynamically correlated with genes related to the synthesis of three enzymes. CONCLUSION: It suggested that microwave and l-Phe treatment may promote the synthesis of flavonoids by promoting the expression of key enzymes genes in phenylpropane metabolism and controlling the activity of key enzymes in phenylpropane metabolism. © 2022 Society of Chemical Industry.

Biochemical defense mechanism associated with host-specific disease resistance pathways against Rhizoctonia solani AG3-PT potatoes canker disease.

MAIN CONCLUSION: Screening for resistance in 40 potato genotypes to Rhizoctonia solani AG-3PT-stem-canker, antioxidant enzymes activity as well as total phenol compounds were documented. Rhizoctonia solani AG-3PT-stem-canker is one of the most devastating diseases that leads to severe economic losses in potatoes, Solanum tuberosum globally. Crop management and eugenic practices, especially the use of resistance can be effective in reducing the disease incidence. However, the information about potato-R. Solani interaction is still limited. This study explored screening for resistance in forty potato genotypes to R. solani, analyzing biomass growth parameters (BGPs), as well as antioxidant enzymes activity of which peroxidase/peroxide-reductases (POXs), superoxide dismutase (SOD), polyphenol oxidase (PPO), catalase (CAT), phenylalanine ammonia-lyase (PAL), ß-1,3-glucanase (GLU) and total phenol compounds (TPCs) were taken into account. In addition, we analyzed up-regulation of two gene markers (PR-1 and Osmotin), using reverse transcription quantitative PCR (RT-qPCR). For which, the resistant 'Savalan', partially resistant 'Agria', partially susceptible 'Sagita' and susceptible 'Pashandi' were selected to explore the trails in their roots and leaves over the time courses of 1, 2 and 3-weeks post inoculation (wpi) following inoculation. Cluster analysis divided potatoes into four distinct groups, based on disease severity scales (0-100%) significance. The BGPs, shoot and root length, fresh and dry weight, and root volume were also significantly higher in infected potatoes compared to non-inoculated controls. Antioxidant enzymes activity also indicated the highest increased levels for POX (fourfold at 3wpi), CAT (1.5-fold at 3wpi), SOD (6.8-fold at 1wpi), and PAL (2.7-fold at 3wpi) in the resistant genotype, 'Savalan', whereas the highest activity was recorded in TPC (twofold at 1 wpi), PPO (threefold at 3wpi), and GLU (2.3-fold at 1wpi) in partially resistant genotypes. Although the defense-related enzymatic activities were sharply elevated in the resistant and partially resistant genotypes following inoculation, no significant correlations were between the activity trends of the related enzymes. The two related gene markers also showed comprehensive transcriptional responses up to 3.4-fold, predominantly in resistant genotypes. Surprisingly, the PR-1 gene marker, basically resistant to Wilting agent Verticillium dahlia was overexpressed in resistant 'Savalan' and 'Agria' against R. solani AG3-PT. Similar results were obtained on Osmotin gene marker resistant to late-blight P. infestans, and early-blight Alternaria solani that similarly modulates immunity against R. solani. Furthermore, there was a significant correlation between resistance, enzyme activity, and gene expression in the aforesaid cultivars. Studying the physiological metabolic pathways of antioxidant enzymes activity appears to be an important direction in research to elucidate resistance to R. solani in potatoes.

An insight into the mechanisms underpinning the anti-browning effect of Codium tomentosum on fresh-cut apples.

This work focuses on understanding the action of a novel seaweed extract with anti-browning functionality in fresh-cut apples. Organic fresh-cut apples were coated by immersion in an aqueous Codium tomentosum seaweed extract (0.5 % w/v), packaged under ambient atmospheric conditions in plastic bags, and stored at 4 ˚C for 15 days. Browning-related enzymatic activities, as well as targeted gene expression related to superficial browning, were monitored immediately after coating and followed at five-day intervals, until a final storage period of 15 days. Gene expression was particularly affected one hour after coating application (day 0), with no expression registered for peroxidase (mdPOD) and phenylalanine ammonia-lyase (mdPAL) genes in the coated samples. A reduction in polyphenol oxidase expression levels was also observed. After 15 days of storage, the coated samples developed lower browning levels and presented distinctly lower activities of polyphenol oxidase and peroxidase - the oxidative enzymes predominantly involved in enzymatic browning. The observed post-coating suppression of mdPAL and mdPOD expression, and reduction in mdPPO expression, suggest that the seaweed C. tomentosum extract delays the activation of these genes, and decreases enzymatic activity, which in turn accounts for the coating's anti-browning effect.

Integrated Metabolite and Transcriptome Profiling-Mediated Gene Mining of Sida cordifolia Reveals Medicinally Important Genes.

Sida cordifolia is a medicinal shrub that is conventionally used in the Indian system of medicine;however, the genes contributing to its medicinal properties have been minimally explored, thus limiting its application. High-throughputsequencing and Liquid Chromatography with tandem mass spectrometry(LC-MS/MS) technologies were applied to unravel the medicinally important bioactive compounds. As a result, transcriptomic sequencing generated more than 12 GB of clean data, and 187,215 transcripts were obtained by de novoassembly. These transcripts were broadly classified into 20 classes, based on the gene ontology classification, and 6551 unigenes were annotated using Kyoto Encyclopedia of Genes and Genomes (KEGG) database with more than 142 unigenes involved in the biosynthesis of secondary metabolites. LC-MS/MS analysis of three tissues of Sida cordifolia revealed that acacetin and procyanidin are some important metabolites identified thatcontribute to its medicinal value. Several key enzymes witha crucial role in phenylpropanoid and flavonoid biosynthetic pathways were identified, especially phenylalanine ammonia lyase, which might be an important rate-limiting enzyme. Real-Time Quantitative Reverse Transcription Polymerase chain reaction (qRT-PCR) analysis revealed enzymes, such as Phenylalanine ammonia lyase (PAL), Cinnamyl alcohol dehydrogenase 1 (CAD), Cinnamoyl-CoA reductase 1 (CF1) and Trans cinnamate 4-monooxygenase(TCM), which were predominantly expressed in root compared to leaf and stem tissue. The study provides a speculative insight for the screening of active metabolites and metabolic engineering in Sida cordifolia.

Characterization of phenylalanine ammonia-lyase genes facilitating flavonoid biosynthesis from two species of medicinal plant Anoectochilus.

Background: Anoectochilus roxburghii and Anoectochilus formosanus, belong to the Anoectochilus genus, have been used for Chinese herbal drugs as well as health food. Phenylalanine ammonia-lyase (PAL), the key enzyme in primary metabolism and phenylpropanoid metabolism, produces secondary metabolites (flavonoids) in plants, which are beneficial for the biosynthesis of phenylpropanoid metabolites. Methods: The PAL genes were cloned from A. formosanus and A. roxburghii according to our previous transcriptomic analysis. The PALs were introduced into pCAMBIA2300-35S-PAL-eGFP to generate 35S-PAL-eGFP. The constructs were further used for subcellular localization and transgenic Arabidopsis. The expression of AfPAL and ArPAL under precursor substance (L-Phe), NaCl, UV, and red-light were analyzed by real-time quantitative PCR (RT-qPCR). Results: AfPAL and ArPAL , encoding 2,148 base pairs, were cloned from A. formosanus and A. roxburghii. The subcellular localization showed that the ArPAL and AfPAL were both localized in the nucleus with GPF. Quantitative RT-PCR analysis indicated that the ArPAL and AfPAL genes function in the phenylalanine pathway as well as response to induced conditions. Overexpression of the AfPAL and ArPAL could increase flavonoids and anthocyanin content in the transgenic Arabidopsis. Discussion: The results suggest that AfPAL and ArPAL play a crucial role in the flavonoid biosynthesis in Anoectochilus. Also, our study provides new insights into the enrichment of secondary metabolites of traditional Chinese medicines A. formosanus and A. roxburghii, which can improve their medicinal active ingredients and be used for drug discovery in plants.

Genome-Wide Analysis and Expression Profiling of the Phenylalanine Ammonia-Lyase Gene Family in Solanum tuberosum.

Phenylalanine ammonia-lyase is one of the most widely studied enzymes in the plant kingdom. It is a crucial pathway from primary metabolism to significant secondary phenylpropanoid metabolism in plants, and plays an essential role in plant growth, development, and stress defense. Although PAL has been studied in many actual plants, only one report has been reported on potato, one of the five primary staple foods in the world. In this study, 14 StPAL genes were identified in potato for the first time using a genome-wide bioinformatics analysis, and the expression patterns of these genes were further investigated using qRT-PCR. The results showed that the expressions of StPAL1, StPAL6, StPAL8, StPAL12, and StPAL13 were significantly up-regulated under drought and high temperature stress, indicating that they may be involved in the stress defense of potato against high temperature and drought. The expressions of StPAL1, StPAL2, and StPAL6 were significantly up-regulated after MeJa hormone treatment, indicating that these genes are involved in potato chemical defense mechanisms. These three stresses significantly inhibited the expression of StPAL7, StPAL10, and StPAL11, again proving that PAL is a multifunctional gene family, which may give plants resistance to multiple and different stresses. In the future, people may improve critical agronomic traits of crops by introducing other PAL genes. This study aims to deepen the understanding of the versatility of the PAL gene family and provide a valuable reference for further genetic improvement of the potato.

Engineering of triterpene metabolism and overexpression of the lignin biosynthesis gene PAL promotes ginsenoside Rg3 accumulation in ginseng plant chassis.

The ginsenoside Rg3 found in Panax species has extensive pharmacological properties, in particular anti-cancer effects. However, its natural yield in Panax plants is limited. Here, we report a multi-modular strategy to improve yields of Rg3 in a Panax ginseng chassis, combining engineering of triterpene metabolism and overexpression of a lignin biosynthesis gene, phenylalanine ammonia lyase (PAL). We first performed semi-rational design and site mutagenesis to improve the enzymatic efficiency of Pq3-O-UGT2, a glycosyltransferase that directly catalyzes the biosynthesis of Rg3 from Rh2 . Next, we used clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing to knock down the branch pathway of protopanaxatriol-type ginsenoside biosynthesis to enhance the metabolic flux of the protopanaxadiol-type ginsenoside Rg3 . Overexpression of PAL accelerated the formation of the xylem structure, significantly improving ginsenoside Rg3 accumulation (to 6.19-fold higher than in the control). We combined overexpression of the ginsenoside aglycon synthetic genes squalene epoxidase, Pq3-O-UGT2, and PAL with CRISPR/Cas9-based knockdown of CYP716A53v2 to improve ginsenoside Rg3 accumulation. Finally, we produced ginsenoside Rg3 at a yield of 83.6 mg/L in a shake flask (7.0 mg/g dry weight, 21.12-fold higher than with wild-type cultures). The high-production system established in this study could be a potential platform to produce the ginsenoside Rg3 commercially for pharmaceutical use.

Comparative transcriptome and tissue-specific expression analysis of genes reveal tissue-cultured plants as an alternative source for phenylethanoids and phenylpropanoids in Rhodiola imbricata (Edgew.).

Rhodiola imbricata (Crassulaceae) is a traditional trans-Himalayan endangered medicinal herb with immense therapeutic applications. Over the years, over-exploitation, un-managed harvesting, and lack of captive cultivation procedures persuaded threat to its wild habitat. Plant tissue culture and RNA-Seq-based molecular bioprospection of key regulatory genes aid the understanding of molecular dynamics involved in specialized metabolites (phenylethanoids and phenylpropanoids) biosynthesis and its sustainable production. Hence, comparative transcriptomic analysis was performed using leaf and root tissues from the wild and tissue-cultured plants, revealing tissue-specific production of salidroside and rosavin. The transcriptome profiling resulted in 345 million high-quality reads yielding 92,380 unique transcripts with an N50 of 1260 bp. Tissue-specific gene expression analysis revealed that both phenylethanoids and phenylpropanoids biosynthesis are predominantly associated with the shikimate pathway. In addition to RNA-Seq data, the downstream biosynthesis pathways genes viz., phospho-2-dehydro-3-deoxyheptonate aldolase (DAHPS), 3-dehydroquinate synthase (DHQS), shikimate kinase (SK), chorismate mutase (CM), arogenate dehydrogenase (TYRAAT), aromatic-L-amino-acid decarboxylase (TDC), phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4-CL), cinnamoyl-CoA reductase (CCR), and cinnamyl alcohol dehydrogenase (CAD) showed higher expression pattern in wild plant tissues compared to tissue-cultured plants. The transcript fold expression determined by RT-qPCR results followed similar patterns as those observed in RNA-seq and targeted metabolite profiling data. Salidroside and rosavin content in wild plants exhibited 2.40 fold and 1.77 fold increase accumulation compared to the tissue-cultured plant. The present investigation explained the tissue and condition-specific significant differences between the expression of proposed biosynthetic pathway genes and salidroside and rosavin content. Additionally, NAC, bHLH, and ARF were the most abundant transcription factor families found in the transcriptomic analysis of R. imbricata. The generated transcriptome dataset provides a valuable gene(s)/transcription factors hub that can be used for the sustainable production of salidroside and rosavin in R. imbricata under tissue culture conditions.

Improvement of a synthetic live bacterial therapeutic for phenylketonuria with biosensor-enabled enzyme engineering.

In phenylketonuria (PKU) patients, a genetic defect in the enzyme phenylalanine hydroxylase (PAH) leads to elevated systemic phenylalanine (Phe), which can result in severe neurological impairment. As a treatment for PKU, Escherichia coli Nissle (EcN) strain SYNB1618 was developed under Synlogic's Synthetic Biotic™ platform to degrade Phe from within the gastrointestinal (GI) tract. This clinical-stage engineered strain expresses the Phe-metabolizing enzyme phenylalanine ammonia lyase (PAL), catalyzing the deamination of Phe to the non-toxic product trans-cinnamate (TCA). In the present work, we generate a more potent EcN-based PKU strain through optimization of whole cell PAL activity, using biosensor-based high-throughput screening of mutant PAL libraries. A lead enzyme candidate from this screen is used in the construction of SYNB1934, a chromosomally integrated strain containing the additional Phe-metabolizing and biosafety features found in SYNB1618. Head-to-head, SYNB1934 demonstrates an approximate two-fold increase in in vivo PAL activity compared to SYNB1618.

Safety and pharmacodynamics of an engineered E. coli Nissle for the treatment of phenylketonuria: a first-in-human phase 1/2a study.

Phenylketonuria (PKU) is a rare disease caused by biallelic mutations in the PAH gene that result in an inability to convert phenylalanine (Phe) to tyrosine, elevated blood Phe levels and severe neurological complications if untreated. Most patients are unable to adhere to the protein-restricted diet, and thus do not achieve target blood Phe levels. We engineered a strain of E. coli Nissle 1917, designated SYNB1618, through insertion of the genes encoding phenylalanine ammonia lyase and L-amino acid deaminase into the genome, which allow for bacterial consumption of Phe within the gastrointestinal tract. SYNB1618 was studied in a phase 1/2a randomized, placebo-controlled, double-blind, multi-centre, in-patient study ( NCT03516487 ) in adult healthy volunteers (n = 56) and patients with PKU and blood Phe level ≥600 mmol l-1 (n = 14). Participants were randomized to receive a single dose of SYNB1618 or placebo (part 1) or up to three times per day for up to 7 days (part 2). The primary outcome of this study was safety and tolerability, and the secondary outcome was microbial kinetics. A D5-Phe tracer (15 mg kg-1) was used to study exploratory pharmacodynamic effects. SYNB1618 was safe and well tolerated with a maximum tolerated dose of 2 × 1011 colony-forming units. Adverse events were mostly gastrointestinal and of mild to moderate severity. All participants cleared the bacteria within 4 days of the last dose. Dose-responsive increases in strain-specific Phe metabolites in plasma (trans-cinnamic acid) and urine (hippuric acid) were observed, providing a proof of mechanism for the potential to use engineered bacteria in the treatment of rare metabolic disorders.

Production of trans-cinnamic acid by whole-cell bioconversion from L-phenylalanine in engineered Corynebacterium glutamicum.

BACKGROUND: trans-cinnamic acid (t-CA) is a phenylpropanoid with a broad spectrum of biological activities including antioxidant and antibacterial activities, and it also has high potential in food and cosmetic applications. Although significant progress has been made in the production of t-CA using microorganisms, its relatively low product titers still need to be improved. In this study, we engineered Corynebacterium glutamicum as a whole-cell catalyst for the bioconversion of L-phenylalanine (L-Phe) into t-CA and developed a repeated bioconversion process. RESULTS: An expression module based on a phenylalanine ammonia lyase-encoding gene from Streptomyces maritimus (SmPAL), which mediates the conversion of L-Phe into t-CA, was constructed in C. glutamicum. Using the strong promoter PH36 and ribosome binding site (RBS) (in front of gene 10 of the T7 phage), and a high-copy number plasmid, SmPAL could be expressed to levels as high as 39.1% of the total proteins in C. glutamicum. Next, to improve t-CA production at an industrial scale, reaction conditions including temperature and pH were optimized; t-CA production reached up to 6.7 mM/h in a bioreactor under optimal conditions (50 °C and pH 8.5, using NaOH as base solution). Finally, a recycling system was developed by coupling membrane filtration with the bioreactor, and the engineered C. glutamicum successfully produced 13.7 mM of t-CA (24.3 g) from 18.2 mM of L-Phe (36 g) and thus with a yield of 75% (0.75 mol/mol) through repetitive supplementation. CONCLUSIONS: We developed a highly efficient bioconversion process using C. glutamicum as a biocatalyst and a micromembrane-based cell recycling system. To the best of our knowledge, this is the first report on t-CA production in C. glutamicum, and this robust platform will contribute to the development of an industrially relevant platform for the production of t-CA using microorganisms.

SmKFB5 protein regulates phenolic acid biosynthesis by controlling the degradation of phenylalanine ammonia-lyase in Salvia miltiorrhiza.

Phenolic acids are the major secondary metabolites and significant bioactive constituents of the medicinal plant Salvia miltiorrhiza. Many enzyme-encoding genes and transcription factors involved in the biosynthesis of phenolic acids have been identified, but the underlying post-translational regulatory mechanisms are poorly understood. Here, we demonstrate that the S. miltiorrhiza Kelch repeat F-box protein SmKFB5 physically interacts with three phenylalanine ammonia-lyase (PAL) isozymes and mediates their proteolytic turnover via the ubiquitin-26S proteasome pathway. Disturbing the expression of SmKFB5 reciprocally affected the abundance of SmPAL protein and the accumulation of phenolic acids, suggesting that SmKFB5 is a post-translational regulator responsible for the turnover of PAL and negatively controlling phenolic acids. Furthermore, we discovered that treatment of the hairy root of S. miltiorrhiza with methyl jasmonate suppressed the expression of SmKFB5 while inducing the transcription of SmPAL1 and SmPAL3. These data suggested that methyl jasmonate consolidated both transcriptional and post-translational regulation mechanisms to enhance phenolic acid biosynthesis. Taken together, our results provide insights into the molecular mechanisms by which SmKFB5 mediates the regulation of phenolic acid biosynthesis by jasmonic acid, and suggest valuable targets for plant breeders in tailoring new cultivars.

Pathway-based analysis of anthocyanin diversity in diploid potato.

Anthocyanin biosynthesis is one of the most studied pathways in plants due to the important ecological role played by these compounds and the potential health benefits of anthocyanin consumption. Given the interest in identifying new genetic factors underlying anthocyanin content we studied a diverse collection of diploid potatoes by combining a genome-wide association study and pathway-based analyses. By using an expanded SNP dataset, we identified candidate genes that had not been associated with anthocyanin variation in potatoes, namely a Myb transcription factor, a Leucoanthocyanidin dioxygenase gene and a vacuolar membrane protein. Importantly, a genomic region in chromosome 10 harbored the SNPs with strongest associations with anthocyanin content in GWAS. Some of these SNPs were associated with multiple anthocyanin compounds and therefore could underline the existence of pleiotropic genes or anthocyanin biosynthetic clusters. We identified multiple anthocyanin homologs in this genomic region, including four transcription factors and five enzymes that could be governing anthocyanin variation. For instance, a SNP linked to the phenylalanine ammonia-lyase gene, encoding the first enzyme in the phenylpropanoid biosynthetic pathway, was associated with all of the five anthocyanins measured. Finally, we combined a pathway analysis and GWAS of other agronomic traits to identify pathways related to anthocyanin biosynthesis in potatoes. We found that methionine metabolism and the production of sugars and hydroxycinnamic acids are genetically correlated to anthocyanin biosynthesis. The results contribute to the understanding of anthocyanins regulation in potatoes and can be used in future breeding programs focused on nutraceutical food.

Physiological and metabolic analysis of winter jujube after postharvest treatment with calcium chloride and a composite film.

BACKGROUND: Ziziphus jujuba Miller cv. Dongzao is extremely susceptible to reddening, browning, nutritional loss, and perishability after harvest. In this study, we evaluated the mechanisms of calcium chloride and chitosan/nano-silica composite film treatments on the quality, especially in reddening, by physiological and metabolomic assays. RESULTS: The treatment delayed the decline of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), and chalcone isomerase (CHI) activities. Meanwhile, the treated groups retarded the increases in anthocyanin and quercetin contents by inhibiting the gene expressions of flavonol synthase (ZjFLS), dihydroflavonol 4-reductase (ZjDFR), and anthocyanidin synthase (ZjANS), while promoting leucoanthocyanidin reductase (ZjLAR) expression, which leads to retardation of fruit reddening. Anthocyanins were found to be responsible for post-harvest winter jujube reddening through principal component analysis. Results from the technique for order preference by similarity to an ideal solution indicated that the treated group delayed the decline of the quality of 'Dongzao' and extended its shelf life. CONCLUSION: The treatment induced the heightening of flavonoids metabolism. They enhanced the nutritional value and the ability to resist stress by delaying the decline of PAL, CHS, and CHI activities. Meanwhile, the treated groups retarded the increase in anthocyanin and quercetin contents by inhibiting the gene expressions of ZjFLS, ZjDFR, and ZjANS and promoting ZjLAR expression, which leads to retardation of fruit reddening. Anthocyanins are responsible for post-harvest winter jujube reddening. Coating treatment effectively delayed the decline of winter jujube quality. © 2020 Society of Chemical Industry.

Effect of benzothiadiazole treatment on quality and anthocyanin biosynthesis in plum fruit during storage at ambient temperature.

BACKGROUND: Plums tend to experience a reduction in fruit quality due to ripening and they deteriorate quickly during storage at room temperature. Benzothiadiazole (BTH) is a plant elicitor capable of inducing disease resistance in many crops. In this study, the effect of BTH treatment on fruit ripening, fruit quality, and anthocyanin biosynthesis in 'Taoxingli' plum was investigated. RESULTS: The results showed that BTH treatment could accelerate fruit ripening without affecting the incidence of fruit decay or the shelf life. Benzothiadiazole treatment improved the quality and consumer acceptability of 'Taoxingli' plums during storage by increasing the sweetness, red color formation, and the concentration of healthy antioxidant compounds. The BTH treatment could also effectively promote the biosynthesis of anthocyanin by enhancing the enzyme activities of phenylalanine ammonia-lyase (PAL), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and uridine diphosphate flavonoid 3-O-glucosyltransferase (UFGT) and up-regulating the gene expressions of PsPAL, PsCHI, PsDFR, PsANS, and PsUFGT during storage. CONCLUSION: Benzothiadiazole treatment could be a potential postharvest technology for improving fruit quality and consumer acceptability in harvested plum fruit. © 2020 Society of Chemical Industry.

Biomedical applications of microbial phenylalanine ammonia lyase: Current status and future prospects.

Phenylalanine ammonia lyase (PAL) has recently emerged as an important therapeutic enzyme with several biomedical applications. The enzyme catabolizes l-phenylalanine to trans-cinnamate and ammonia. PAL is widely distributed in higher plants, some algae, ferns, and microorganisms, but absent in animals. Although microbial PAL has been extensively exploited in the past for producing industrially important metabolites, its high substrate specificity and catalytic efficacy lately spurred interest in its biomedical applications. PEG-PAL drug named Palynziq™, isolated from Anabaena variabilis has been recently approved for the treatment of adult phenylketonuria (PKU) patients. Further, it has exhibited high potency in regressing tumors and treating tyrosine related metabolic abnormalities like tyrosinemia. Several therapeutically valuable metabolites have been biosynthesized via its catalytic action including dietary supplements, antimicrobial peptides, aspartame, amino-acids, and their derivatives. This review focuses on all the prospective biomedical applications of PAL. It also provides an overview of the structure, production parameters, and various strategies to improve the therapeutic potential of this enzyme. Engineered PAL with improved pharmacodynamic and pharmacokinetic properties will further establish this enzyme as a highly efficient biological drug.

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

KEY MESSAGE: 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.