Lactic acid induced defense responses in tobacco against Phytophthora nicotianae.

Induced resistance is considered an eco-friendly disease control strategy, which can enhance plant disease resistance by inducing the plant's immune system to activate the defense response. In recent years, studies have shown that lactic acid can play a role in plant defense against biological stress; however, whether lactic acid can improve tobacco resistance to Phytophthora nicotianae, and its molecular mechanism remains unclear. In our study, the mycelial growth and sporangium production of P. nicotianae were inhibited by lactic acid in vitro in a dose-dependent manner. Application of lactic acid could reduce the disease index, and the contents of total phenol, salicylic acid (SA), jasmonic acid (JA), lignin and H2O2, catalase (CAT) and phenylalanine ammonia-lyase (PAL) activities were significantly increased. To explore this lactic acid-induced protective mechanism for tobacco disease resistance, RNA-Seq analysis was used. Lactic acid enhances tobacco disease resistance by activating Ca2+, reactive oxygen species (ROS) signal transduction, regulating antioxidant enzymes, SA, JA, abscisic acid (ABA) and indole-3-acetic acid (IAA) signaling pathways, and up-regulating flavonoid biosynthesis-related genes. This study demonstrated that lactic acid might play a role in inducing resistance to tobacco black shank disease; the mechanism by which lactic acid induces disease resistance includes direct antifungal activity and inducing the host to produce direct and primed defenses. In conclusion, this study provided a theoretical basis for lactic acid-induced resistance and a new perspective for preventing and treating tobacco black shank disease.

Genome-Wide Characterization of the Phenylalanine Ammonia-Lyase Gene Family and Their Potential Roles in Response to Aspergillus flavus L. Infection in Cultivated Peanut (Arachis hypogaea L.).

Phenylalanine ammonia-lyase (PAL) is an essential enzyme in the phenylpropanoid pathway, in which numerous aromatic intermediate metabolites play significant roles in plant growth, adaptation, and disease resistance. Cultivated peanuts are highly susceptible to Aspergillus flavus L. infection. Although PAL genes have been characterized in various major crops, no systematic studies have been conducted in cultivated peanuts, especially in response to A. flavus infection. In the present study, a systematic genome-wide analysis was conducted to identify PAL genes in the Arachis hypogaea L. genome. Ten AhPAL genes were distributed unevenly on nine A. hypogaea chromosomes. Based on phylogenetic analysis, the AhPAL proteins were classified into three groups. Structural and conserved motif analysis of PAL genes in A. hypogaea revealed that all peanut PAL genes contained one intron and ten motifs in the conserved domains. Furthermore, synteny analysis indicated that the ten AhPAL genes could be categorized into five pairs and that each AhPAL gene had a homologous gene in the wild-type peanut. Cis-element analysis revealed that the promoter region of the AhPAL gene family was rich in stress- and hormone-related elements. Expression analysis indicated that genes from Group I (AhPAL1 and AhPAL2), which had large number of ABRE, WUN, and ARE elements in the promoter, played a strong role in response to A. flavus stress.

First successful outcomes of pegvaliase (PALYNZIQ) in children.

BACKGROUND: PKU is an autosomal recessive hereditary inborn error of metabolism caused by a lack of phenylalanine hydroxylase enzyme activity. Pegvaliase (PALYNZIQ®) treatment has been approved to reduce blood Phe concentrations in adult phenylketonuria patients with uncontrolled blood Phe concentrations greater than 600 micromol/L on current management. However, data regarding individuals under the age of 16 is still unavailable. CASE REPORT: We report a 12-year-old Saudi girl who underwent pegvaliase therapy and was closely monitored for one year. Remarkably, a positive therapeutic response became apparent six months after commencing pegvaliase treatment. Phenylalanine (Phe) levels showed significant improvement, stabilising within the < 5 to 14 µmol/L range on a regular diet without any restriction. At her current age of 12, the patient maintains an unrestricted dietary regimen, consuming a diverse selection of foods, including poultry, meat, and protein sources, all while consistently maintaining normal Phe levels with no change in mental status after treatment. The parents gave their written, informed consent in allowing the research study to be carried out and clinical data to be published. CONCLUSIONS: This report addresses the potential broader applications of Pegvaliase in children, as well as its safety and tolerability in this age group. However, larger sample sizes and robust methodologies are required to validate such findings.

Engineered Zea mays phenylalanine ammonia-lyase for improve the catalytic efficiency of biosynthesis trans-cinnamic acid and p-coumaric acid.

Phenylalanine ammonia-lyase (PAL) plays a pivotal role in the biosynthesis of phenylalanine. PAL from Zea mays (ZmPAL2) exhibits a bi-function of direct deamination of L-phenylalanine (L-Phe) or L-tyrosine(-L-Tyr) to form trans-cinnamic acid or p-coumaric acid. trans-Cinnamic acid and p-coumaric acid are mainly used in flavors and fragrances, food additives, pharmaceutical and other fields. Here, the Activity of ZmPAL2 toward L-Phe or L-Tyr was improved by using semi-rational and rational designs. The catalytic efficiency (kcat/Km) of mutant PT10 (V258I/I459V/Q484N) against L-Phe was 30.8 µM-1 s-1, a 4.5-fold increase compared to the parent, and the catalytic efficiency of mutant PA1 (F135H/I459L) to L-tyrosine exhibited 8.6 µM-1 s-1, which was 1.6-fold of the parent. The yield of trans-cinnamic acid in PT10 reached 30.75 g/L with a conversion rate of 98%. Meanwhile, PA1 converted L-Tyr to yield 3.12 g/L of p-coumaric acid with a conversion rate of 95%. Suggesting these two engineered ZmPAL2 to be valuable biocatalysts for the synthesis of trans-cinnamic acid and p-coumaric acid. In addition, MD simulations revealed that the underlying mechanisms of the increased catalytic efficiency of both mutant PT10 and PA1 are attributed to the substrate remaining stable within the pocket and closer to the catalytically active site. This also provides a new perspective on engineered PAL.

Roles of the N-terminal motif in improving the activity and soluble expression of phenylalanine ammonia lyases in Escherichia coli.

Phenylalanine ammonia-lyase (PAL) has various applications in fine chemical manufacturing and the pharmaceutical industry. In particular, PAL derived from Anabaena variabilis (AvPAL) is used as a therapeutic agent to the treat phenylketonuria in clinical settings. In this study, we aligned the amino acid sequences of AvPAL and PAL derived from Nostoc punctiforme (NpPAL) to obtain several mutants with enhanced activity, expression yield, and thermal stability via amino acid substitution and saturation mutagenesis at the N-terminal position. Enzyme kinetic experiments revealed that the kcat values of NpPAL-N2K, NpPAL-I3T, and NpPAL-T4L mutants were increased to 3.2-, 2.8-, and 3.3-fold that of the wild-type, respectively. Saturation mutagenesis of the fourth amino acid in AvPAL revealed that the kcat values of AvPAL-L4N, AvPAL-L4P, AvPAL-L4Q and AvPAL-L4S increased to 4.0-, 3.7-, 3.6-, and 3.2-fold, respectively. Additionally, the soluble protein yield of AvPAL-L4K increased to approximately 14 mg/L, which is approximately 3.5-fold that of AvPAL. Molecular dynamics studies further revealed that maintaining the attacking state of the reaction and N-terminal structure increased the rate of catalytic reaction and improved the solubility of proteins. These findings provide new insights for the rational design of PAL in the future.

Outcomes in 14 live births resulting from Pegvaliase-treated pregnancies in PKU-affected females.

BACKGROUND: Adults with PKU have difficulty maintaining plasma phenylalanine (Phe) in the range that is safe for neurologic function. Elevated plasma Phe is a risk factor for congenital anomalies and developmental delay in offspring resulting from pregnancies with poor Phe control in women with PKU. Enzyme supplementation with pegvaliase allows adults with PKU to eat an unrestricted diet and have plasma Phe levels in a safe range for pregnancy but pegvaliase has not been approved for use in pregnant females with PKU. We report the results of chart review of 14 living offspring of females affected with PKU who were responsive to pegvaliase and chose to remain on pegvaliase throughout their pregnancy. METHODS: Fourteen pregnancies (one triplet pregnancy) and their offspring were identified at eight PKU treatment centers and medical records from pregnancy and birth were submitted for this study. Institutional Review Board approval was obtained. Responses to a dataset were provided to a single center and analyzed. RESULTS: Six females and eight males were born without congenital anomalies and all offspring had normal growth parameters. While mothers had preexisting comorbidities, no additional comorbidities were reported in the offspring. Four of eleven infants (excluding triplet pregnancies) were delivered preterm (36%), a higher rate than the general population (12%). A single first trimester (eight weeks) miscarriage in a 40y was not counted in this cohort of 14 live born infants. CONCLUSION: This retrospective study suggests that pegvaliase is effective at maintaining safe maternal blood Phe levels during pregnancy without deleterious effects on mother or child. A tendency toward premature birth (4/11; 36%) is higher than expected.

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.

The phenylalanine ammonia-lyase inhibitor AIP induces rice defence against the root-knot nematode Meloidogyne graminicola.

The phenylalanine ammonia-lyase (PAL) enzyme catalyses the conversion of l-phenylalanine to trans-cinnamic acid. This conversion is the first step in phenylpropanoid biosynthesis in plants. The phenylpropanoid pathway produces diverse plant metabolites that play essential roles in various processes, including structural support and defence. Previous studies have shown that mutation of the PAL genes enhances disease susceptibility. Here, we investigated the functions of the rice PAL genes using 2-aminoindan-2-phosphonic acid (AIP), a strong competitive inhibitor of PAL enzymes. We show that the application of AIP can significantly reduce the PAL activity of rice crude protein extracts in vitro. However, when AIP was applied to intact rice plants, it reduced infection of the root-knot nematode Meloidogyne graminicola. RNA-seq showed that AIP treatment resulted in a rapid but transient upregulation of defence-related genes in roots. Moreover, targeted metabolomics demonstrated higher levels of jasmonates and antimicrobial flavonoids and diterpenoids accumulating after AIP treatment. Furthermore, chemical inhibition of the jasmonate pathway abolished the effect of AIP on nematode infection. Our results show that disturbance of the phenylpropanoid pathway by the PAL inhibitor AIP induces defence in rice against M. graminicola by activating jasmonate-mediated defence.

Evaluating change in diet with pegvaliase treatment in adults with phenylketonuria: Analysis of phase 3 clinical trial data.

Phenylketonuria (PKU), a genetic disorder characterized by phenylalanine hydroxylase (PAH) deficiency and phenylalanine (Phe) accumulation, is primarily managed with a protein-restricted diet and PKU-specific medical foods. Pegvaliase is an enzyme substitution therapy approved for individuals with PKU and uncontrolled blood Phe concentrations (>600 µmol/L) despite prior management. This analysis assessed the effect of pegvaliase on dietary intake using data from the Phase 3 PRISM-1 (NCT01819727), PRISM-2 (NCT01889862), and 165-304 (NCT03694353) clinical trials. Participants (N = 250) had a baseline diet assessment, blood Phe ≥600 µmol/L, and had discontinued sapropterin; they were not required to follow a Phe-restricted diet. Outcomes were analyzed by baseline dietary group, categorized as >75%, some (>0% but ≤75%), or no protein intake from medical food. At baseline, mean age was 29.1 years, 49.2% were female, mean body mass index was 28.4 kg/m2, and mean blood Phe was 1237.0 µmol/L. Total protein intake was stable up to 48 months for all 3 baseline dietary groups. Over this time, intact protein intake increased in all groups, and medical protein intake decreased in those who consumed any medical protein at baseline. Of participants consuming some or >75% medical protein at baseline, 49.1% and 34.1% were consuming no medical protein at last assessment, respectively. Following a first hypophenylalaninemia (HypoPhe; 2 consecutive blood Phe measurements <30 µmol/L) event, consumption of medical protein decreased and consumption of intact protein increased. Substantial and sustained Phe reductions were achieved in all 3 baseline dietary groups. The probability of achieving sustained Phe response (SPR) at ≤600 µmol/L was significantly greater for participants consuming medical protein versus no medical protein in an unadjusted analysis, but no statistically significant difference between groups was observed for probability of achieving SPR ≤360 or SPR ≤120 µmol/L. Participants with alopecia (n = 49) had longer pegvaliase treatment durations, reached HypoPhe sooner, and spent longer in HypoPhe than those who did not have alopecia. Most (87.8%) had an identifiable blood Phe drop before their first alopecia episode, and 51.0% (n = 21/41) of first alopecia episodes with known duration resolved before the end of the HypoPhe episode. In conclusion, pegvaliase treatment allowed adults with PKU to lower their blood Phe, reduce their reliance on medical protein, and increase their intact and total protein intake. Results also suggest that HypoPhe does not increase the risk of protein malnutrition in adults with PKU receiving pegvaliase.

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.

Engineering phenylalanine ammonia lyase to limit feedback inhibition by cinnamate and enhance biotransformation.

Phenylalanine ammonia-lyase (PAL) is a crucial enzyme for various biotechnology applications, such as producing phenols, antioxidants, and nutraceuticals. However, feedback inhibition from its product, cinnamic acid, limits its forward reaction rate. Therefore, this study aims to address the feedback inhibition in PAL using enzyme engineering strategies. Random and site-directed mutagenesis approaches were utilized to screen mutant enzymes with ameliorated tolerance against cinnamic acid. A thermotolerant and cinnamate-tolerant mutant was rationally identified using a high throughput screening method and subsequent biochemical characterization. We evaluated cinnamate affinity among the seven rationally selected mutations, and the T102E mutation was identified as the most promising mutant. This mutant showed a six-fold reduction in the affinity of PAL for cinnamic acid and a two-fold increase in operational stability compared with native PAL. Furthermore, the enzyme was immobilized on carbon nanotubes to increase its robustness and reusability. The immobilized mutant PAL showed greater efficiency in the deamination of phenylalanine present in protein hydrolysate than its free form. The rationale behind the enhancement of cinnamate tolerance was validated using molecular dynamic simulations. Overall, the knowledge of the sequence-function relationship of PAL was applied to drive enzyme engineering to develop highly tolerant PAL.

Best practice recommendations for the management of anxiety during the pegvaliase journey.

BACKGROUND: Pegvaliase, an enzyme substitution therapy, is a treatment option for phenylketonuria (PKU). Due to the neuropathophysiology and disease burden of PKU, individuals can experience baseline anxiety unrelated to pegvaliase therapy. In addition, there are aspects of pegvaliase therapy that may be anxiety-inducing for those considering or receiving treatment. The aim of this manuscript is to present best practice recommendations for the identification and management of anxiety symptoms that can occur along the pegvaliase journey. METHODS: A modified Delphi approach was used to seek consensus among a multidisciplinary panel of experts. To this end, an in-person meeting was held that was preceded by a medical specialist- and patient-specific survey to develop preliminary recommendations on ways to address anxiety along the pegvaliase journey. After the meeting, an additional survey was conducted to rank the proposed solutions and mitigation strategies from which a set of recommendations was developed. All recommendations were voted on with the aim of consensus generation, defined as achieving ≥75% agreement among experts. RESULTS: The panel reached consensus on a total of 28 best practice recommendations for the management of anxiety during the pre-treatment, induction and titration, early maintenance (pre-efficacy), and late maintenance (post-efficacy) stages. The recommendations offer strategies to identify and address the most common causes of pegvaliase-related anxiety, including self-injection, side effects, the titration schedule, prescribed dietary changes, and variable time to efficacy. Overall, managing anxiety in those considering or receiving pegvaliase involves patient-centered communication, shared decision-making, and personalized treatment plans. CONCLUSIONS: The best practice recommendations described herein can guide healthcare providers in proactively addressing anxiety during the different stages of pegvaliase treatment, and support providers with initiating and managing pegvaliase in individuals who may experience baseline and treatment-related anxiety.

Insights to Phenylalanine Ammonia Lyase (PAL) and Secondary Metabolism in Orchids: An in silico Approach.

The phenylalanine ammonia lyase (PAL) catalyses the first step of phenylpropanoid metabolic pathway which leads to the biosynthesis of a diverse group of secondary metabolites. Orchids serve as a rich source of metabolites and the availability of genome or transcriptome for selected orchid species provides an opportunity to analyse the PAL genes in orchids. In the present study, 21 PAL genes were characterized using bioinformatics tools in nine orchid species (Apostasia shenzhenica, Cypripedium formosanum, Dendrobium catenatum, Phalaenopsis aphrodite, Phalaenopsis bellina, Phalaenopsis equestris, Phalaenopsis lueddemanniana, Phalaenopsis modesta and Phalaenopsis schilleriana). Multiple sequence alignment confirmed the presence of PAL-specific conserved domains (N-terminal, MIO, core, shielding and C-terminal domain). All these proteins were predicted to be hydrophobic in nature and to have cytoplasmic localisation. Structural modelling depicted the presence of alpha helices, extended strands, beta turns and random coils in their structure. Ala-Ser-Gly triad known for substrate binding and catalysis of MIO-domain was found to be completely conserved in all the proteins. Phylogenetic study showed that the PALs of pteridophytes, gymnosperms and angiosperms clustered together in separate clades. Expression profiling showed tissue-specific expression for all the 21 PAL genes in the various reproductive and vegetative tissues which suggested their diverse role in growth and development. This study provides insights to the molecular characterization of PAL genes which may help in developing biotechnological strategies to enhance the synthesis of phenylpropanoids in orchids and other heterologous systems for pharmaceutical applications.

Long-term comparative effectiveness of pegvaliase versus medical nutrition therapy with and without sapropterin in adults with phenylketonuria.

Phenylketonuria is characterized by intellectual disability and behavioral, psychiatric, and movement disorders resulting from phenylalanine (Phe) accumulation. Standard-of-care treatment involves a Phe-restricted diet plus medical nutrition therapy (MNT), with or without sapropterin dihydrochloride, to reduce blood Phe levels. Pegvaliase is an injectable enzyme substitution treatment approved for adult patients with blood Phe >600 µmol/L despite ongoing management. A previous comparative effectiveness analysis using data from the Phase 3 PRISM trials of pegvaliase (NCT01819727 and NCT01889862) and the Phenylketonuria Demographics, Outcomes and Safety Registry (PKUDOS; NCT00778206) suggested that pegvaliase was more effective at lowering mean blood Phe levels than sapropterin + MNT or MNT alone at 1 and 2 years of treatment. The current work augments and complements the previous analysis by including additional follow-up from the completed studies, robust methods reflecting careful consideration of issues with the distribution of Phe, and alternative methods for adjustment that are important for control of potential confounding in comparative effectiveness. Median blood Phe levels were lower, and median intact protein intakes were higher, in the pegvaliase group (n = 183) than in the sapropterin + MNT (n = 82) and MNT (n = 67) groups at Years 1, 2, and 3. In the pegvaliase group, median blood Phe levels decreased from baseline (1244 µmol/L) to Year 1 (535 µmol/L), Year 2 (142 µmol/L), and Year 3 (167 µmol/L). In the sapropterin + MNT group, median blood Phe levels decreased from baseline (900 µmol/L) to Year 1 (588 µmol/L) and Year 2 (592 µmol/L), and increased at Year 3 (660 µmol/L). In the MNT group, median blood Phe levels decreased slightly from baseline (984 µmol/L) to Year 1 (939 µmol/L) and Year 2 (941 µmol/L), and exceeded baseline levels at Year 3 (1157 µmol/L). The model-estimated proportions of participants achieving blood Phe ≤600 µmol/L were 41%, 100%, and 100% in the pegvaliase group at Years 1, 2, and 3, respectively, compared with 55%, 58%, and 38% in the sapropterin + MNT group and 5%, 16%, and 0% in the MNT group. The estimated proportions of participants achieving more stringent blood Phe targets of ≤360 µmol/L and ≤120 µmol/L were also higher in the pegvaliase group than in the other groups at Years 2 and 3. Overall, our results indicate that, compared with standard therapy, pegvaliase induces a substantial, progressive, and sustained decrease in blood Phe levels - to a much greater extent than sapropterin + MNT or MNT alone - which is expected to improve long-term outcomes in patients with phenylketonuria.

Functional and kinetics of two efficient phenylalanine ammonia lyase from Pyrus bretschneideri.

BACKGROUND: The enzyme phenylalanine ammonia lyase (PAL) controls the transition from primary to secondary metabolism by converting L-phenylalanine (L-Phe) to cinnamic acid. However, the function of PAL in pear plants (Pyrus bretschneideri) has not yet been fully elucidated. RESULTS: We identified three PAL genes (PbPAL1, PbPAL2 and PbPAL3) from the pear genome by exploring pear genome databases. The evolutionary tree revealed that three PbPALs were classified into one group. We expressed PbPAL1 and PbPAL2 recombinant proteins, and the purified PbPAL1 and PbPAL2 proteins showed strict substrate specificity for L-Phe, no activity toward L-Tyr in vitro, and modest changes in kinetics and enzyme characteristics. Furthermore, overexpression of PbAL1 and PbPAL1-RNAi, respectively, and resulted in significant changes in stone cell and lignin contents in pear fruits. The results of yeast one-hybrid (Y1H) assays that PbWLIM1 could bind to the conserved PAL box in the PbPAL promoter and regulate the transcription level of PbPAL2. CONCLUSIONS: Our findings not only showed PbPAL's potential role in lignin biosynthesis but also laid the foundation for future studies on the regulation of lignin synthesis and stone cell development in pear fruit utilizing molecular biology approaches.

Deciphering morphological and biochemical responses of Salvia leriifolia to seed cold plasma treatment, priming, and foliar spraying with nano-salicylic acid.

The pretreatment of seeds with cold plasma (CP) (0 and 100 w for 240 s), and salicylic acid priming (SA) (0 and 2 mM normal and nano form), and foliar spraying of SA at the six-leaf stage (0 and 2 mM normal and nano form) of Salvia leriifolia plants in field condition was studied. Compared to the control plants of S. leriifolia, the results showed that CP + both forms of SA priming + nano-SA spraying increased plant height, leaf length, plant dry weight, total phenol, and the activities of phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) enzymes. The chlorophyll a and b contents in all treated plants remained either unchanged or decreased when compared to the control. The highest PAL activity was obtained in CP-free + hydro-priming + nano-SA foliar spraying. The highest content of caffeic acid was achieved in CP + SA priming + SA foliar spraying in the leaf. The maximum contents of rosmarinic and salvianolic acid were obtained in the control plants. In conclusion, CP and nano-SA can increase PAL and TAL activity and total phenol accumulation in S. leriifolia plants, but not rosmarinic and salvianolic acid contents. Other phenolic compound enzymes and their production require further study.

Expression profiling of the phenylalanine ammonia-lyase (PAL) gene family in ginkgo biloba L.

The PAL gene family plays an important role in plant growth, development, and response to abiotic stresses and has been identified in a variety of plants. However, a systematic characterization is still lacking in Ginkgo biloba. Using a bioinformatics approach, 11 GbPAL members of the PAL gene family identified in ginkgo were identified in this study. The protein structure and physicochemical properties indicated that the GbPAL genes were highly similar. Based on their exon-intron structures, they can be classified into three groups. A total of 62 cis-elements for hormone, light, and abiotic stress responses were identified in the promoters of GbPAL genes, indicating that PAL is a multifunctional gene family. GbPAL genes were specifically expressed in different tissues and ploidy of ginkgo. These results provide a theoretical basis for further studies on the functional expression of the GbPAL genes.

Ancestral l-amino acid oxidase: From substrate scope exploration to phenylalanine ammonia-lyase assay.

In this study we assessed the applicability of the recently reported ancestral l-amino acid oxidase (AncLAAO), for the development of an enzyme-coupled phenylalanine ammonia-lyase (PAL) activity assay. Firstly, the expression and isolation of the AncLAAO-N1 was optimized, followed by activity tests of the obtained octameric N-terminal His-tagged enzyme towards various phenylalanine analogues to assess the compatibility of its substrate scope with that of the well-characterized PALs. AncLAAO-N1 showed high catalytic efficiency towards phenylalanines mono-, di-, or multiple-substituted in the meta- or para-positions, with ortho- substituted substrates being poorly transformed, these results highlighting the significant overlap between its substrate scope and those of PALs. After successful set-up of the AncLAAO-PAL coupled solid phase assay, in a 'proof of concept' approach we demonstrated its applicability for the high-throughput activity screens of PAL-libraries, by screening the saturation mutagenesis-derived I460NNK variant library of PAL from Petroselinum crispum, using p-MeO-phenylalanine as model substrate. Notably, the hits revealed by the coupled assay comprised all the active PAL variants: I460V, I460T, I460S, I460L, previously identified from the tested PAL-library by other assays. Our results validate the applicability of AncLAAO for coupled enzyme systems with phenylalanine ammonia-lyases, including cell-based assays suitable for the high-throughput screening of directed evolution-derived PAL-libraries.

Drought stress-responsive abscisic acid and salicylic acid crosstalk with the phenylpropanoid pathway in soybean seeds.

Crosstalk between hormones and secondary metabolites regulates the interactions between plants and stress. However, little is known about the effects of hormone crosstalk on the concentration of flavonoids in seeds. In this study, we identified abscisic acid (ABA) as a negative regulator of flavonoid accumulation in soybean seeds under drought-stress conditions. Alterations in flavonoid accumulation at several intensities of water stress, followed by a recovery period, were measured during the soybean seed-filling stage. Low soil moisture (SM 10%) significantly decreased the total flavonoid content in seeds. The decline in flavonoid content was proportional to the severity of drought stress and was dependent on the activities of phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS), two key phenylpropanoid pathway enzymes. The expression of phenylalanine ammonia-lyase 1 (GmPAL1), chalcone isomerase 1A (GmCHI1A), and chalcone synthase 8 (GmCHS8) was associated with phenolic and flavonoid accumulation in soybean seeds of plants subjected to drought stress. Interestingly, the expression levels of GmCHS8 were highly correlated with flavonoid levels under drought stress and water recovery conditions. Cinnamic acid, which is a biosynthesis precursor shared by both phenylpropanoid metabolism and salicylic acid (SA) biosynthesis, decreased under drought stress conditions. Notably, exogenous ABA suppressed the expression of GmPAL1, which encodes the first rate-limiting enzyme in the phenylpropanoid biosynthesis pathway and affects downstream products such as SA and flavonoids. In conclusion, drought stress altered the phenylpropanoid-derived compounds, at least with regard to flavonoid and SA accumulation in seeds, which was regulated by antagonistic interactions with ABA.

Genome-wide identification and expression analysis of phenylalanine ammonia-lyase (PAL) family in rapeseed (Brassica napus L.).

BACKGROUND: Phenylalanine ammonia-lyase (PAL), as a key enzyme in the phenylalanine metabolism pathway in plants, plays an important role in the response to environmental stress. However, the PAL family responding to abiotic stress has not been fully characterized in rapeseed. RESULTS: In this study, we conducted a genome-wide study of PAL family, and analyzed their gene structure, gene duplication, conserved motifs, cis-acting elements and response to stress treatment. A total of 17 PALs were identified in the rapeseed genome. Based on phylogenetic analysis, the BnPALs were divided into four clades (I, II, IV, and V). The prediction of protein structure domain presented that all BnPAL members contained a conservative PAL domain. Promoter sequence analysis showed that the BnPALs contain many cis-acting elements related to hormone and stress responses, indicating that BnPALs are widely involved in various biological regulatory processes. The expression profile showed that the BnPALs were significantly induced under different stress treatments (NaCl, Na2CO3, AlCl3, and PEG), suggesting that BnPAL family played an important role in response to abiotic stress. CONCLUSIONS: Taken together, our research results comprehensively characterized the BnPAL family, and provided a valuable reference for revealing the role of BnPALs in the regulation of abiotic stress responses in rapeseed.