Ocimum sanctum, OscWRKY1, regulates phenylpropanoid pathway genes and promotes resistance to pathogen infection in Arabidopsis

WRKY transcription factor (TF) family regulates various developmental and physiological functions in plants. PAL genes encode enzymes which are involved in plant defense responses, but the direct regulation of PAL genes and phenylpropanoid pathway through WRKY TF is not well characterized. In the present study, we have characterized an OscWRKY1 gene from O. sanctum which shows induced expression after methyl jasmonate (MeJA), salicylic acid (SA), and wounding. Recombinant OscWRKY1 protein binds to the W-box cis-element TTGAC[C/T] and activates the reporter gene in yeast. Overexpression of OscWRKY1 enhances Arabidopsis resistance towards Pseudomonas syringae pv. tomato Pst DC3000. Upstream activator sequences of PAL and C4H have identified the conserved W-box cis-element (TTGACC) in both O. sanctum and Arabidopsis. OscWRKY1 was found to interact with W-box cis-element present in the PAL and C4H promoters. Silencing of OscWRKY1 using VIGS resulted in reduced expression of PAL, C4H, COMT, F5H and 4CL transcripts. OscWRKY1 silenced plants exhibit reduced PAL activity, whereas, the overexpression lines of OscWRKY1 in Arabidopsis exhibit increased PAL activity. These results revealed that OscWRKY1 positively regulates the phenylpropanoid pathway genes and enhances the resistance against bacterial pathogen in Arabidopsis.

Characteristics of healthy German children and adolescents across tertiles of calcaneal stiffness index

Aim Identifying risk factors for low bone stiffness index (SI) might be one crucial strategy for osteoporosis prevention. Purpose was to characterize healthy schoolchildren across tertiles of SI. Subject and methods In 248 girls (13.4 ± 1.9 years, BMI: 20.2 ± 4.8 kg/m2) and 231 boys (13.6 ± 1.7 years, BMI: 19.3 ± 3.3 kg/m2), the following parameters were assessed: calcaneal SI (quantitative ultrasound), body composition (bioelectrical impedance analysis), Bone Healthy Eating Index (BoneHEI; food frequency questionnaire), and physical activity level (PAL; activity questionnaire). Participants were classified according to age- and sex-specific SI tertiles (low, medium, and high). Between-group comparisons were achieved by Kruskal–Wallis-H-tests (α = 0.05). Results Girls with low SI had significantly lower body mass (49.2 ± 16.7 vs 54.8 ± 12.2 kg; p < 0.01), BMI (19.6 ± 5.4 vs 21.3 ± 3.9 kg/m2; p < 0.0001), fat-free mass (36.3 ± 8.3 vs 39.5 ± 6.0 kg; p < 0.01), and fat mass (23.7 ± 9.1 vs 26.8 ± 7.2%; p < 0.05) compared to those with high SI. In boys, significant differences between low and high SI were obtained for PAL (1.49 ± 0.12 vs 1.56 ± 0.14; p < 0.01). BoneHEI was not significantly different between tertiles in both sexes. Conclusion Girls with low body mass and boys with low PAL have a higher risk for low SI. Schoolchildren should strive for normal body mass and perform regular physical activity.

The Effect of Simulated Microgravity On The Antioxidant Capacity And Alkaloid Formation In The Hyoscyamus Niger

Microgravity is one of the most important abiotic stresses in space. In the case of plant exposure to short term microgravity, plants establish strategies to response to these stresses and promote growth and survival. We hypothesized that the simulated microgravity can promote the antioxidant capacity and the formation of secondary metabolites such as tropane alkaloids in the Hyoscyamus niger. Callus induction was conducted by putting hypocotyl segments of H. niger seedlings in solid MS medium supplemented with 1 mg L−1 2,4-D and 1 mg L−1 BAP growth regulators. Then, the sub-cultured calli were placed on a clinostat for 3, 7 and 10 days. We performed Atropine and Scopolamine determination through HPLC. PAL (Phenyle alanine amonalyase) and antioxidant activity were also determined. Gene expression analysis of jasmonic acid (JA), Hyoscyamine 6-beta Hydroxylase (H6H), Putrescine N-methyltransferase (PMT), mitogen-activated protein kinase (MAPK) and ethylene responsive element binding (EREB) was performed using quantitative real time PCR. Findings showed that microgravity had a positive effect on the antioxidant capacity, Atropine and Scopolamine production in the H. niger calli. However, microgravity had a negative effect on the PAL activity. Furthermore, gene expression analysis indicated that microgravity significantly induced gene expression of the H6H, PMT and JA. It was also revealed that callus growth, carbohydrate and protein content increased in response to microgravity treatment. We conclude that microgravity can be considered as a potent factor to induce plant antioxidant activity and tropane alkaloids formation to be applicable in the pharmaceutical and medicinal industries.

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.

Production of Trans-Cinnamic Acid by Immobilization of the Bambusa oldhamii BoPAL1 and BoPAL2 Phenylalanine Ammonia-Lyases on Electrospun Nanofibers

Phenylalanine ammonia-lyase (PAL) catalyzes the nonoxidative deamination of phenylalanine to yield trans-cinnamic acid and ammonia. Recombinant Bambusa oldhamii BoPAL1/2 proteins were immobilized onto electrospun nanofibers by dextran polyaldehyde as a cross-linking agent. A central composite design (CCD)-response surface methodology (RSM) was utilized to optimize the electrospinning parameters. Escherichia coli expressed eBoPAL2 exhibited the highest catalytic efficiency among four enzymes. The optimum conditions for fabricating nanofibers were determined as follows: flow rate of 0.10 mL/h, voltage of 13.8 kV, and distance of 13 cm. The response surface models were used to obtain the smaller the fiber diameters as well as the highest PAL activity in the enzyme immobilization. Compared with free BoPALs, immobilized BoPALs can be reused for at least 6 consecutive cycles. The remained activity of the immobilized BoPAL proteins after storage at 4 °C for 30 days were between 75 and 83%. In addition, the tolerance against denaturants of the immobilized BoPAL proteins were significantly enhanced. As a result, the dextran polyaldehyde natural cross-linking agent can effectively replace traditional chemical cross-linking agents for the immobilization of the BoPAL enzymes. The PAL/nylon 6/polyvinyl alcohol (PVA)/chitosan (CS) nanofibers made are extremely stable and are practical for industrial applications in the future.

Enzymatic Browning in Banana Blossoms and Techniques for Its Reduction

Banana blossoms are rich in fiber and nutrients and are a popular plant-based, vegan alternative to fish. However undesirable browning, usually visible at the peduncle cut-end, negatively impacts consumer acceptability of banana blossoms. The aim of this work was to develop safe alternatives to prevent browning in banana blossoms. First, the activities of primary enzymes associated with tissue browning, i.e., polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonia lyase (PAL), were assayed. Our data showed that PPO and POD were the key enzymes responsible for blossom browning as they increased in activity, reaching a maximum at pH 7, as browning developed. In contrast, PAL activity decreased, and total phenolic content did not change as browning progressed, indicating PAL was not induced by cutting and may not be involved in blossom browning. Second, to find antibrowning agents for banana blossoms that can substitute for the use of sodium metabisulfite (SMS), different organic acids of varying concentrations were tested. Among organic acids studied, treatment with 3% (w/v) oxalic acid was the most effective method and thus could be a safe substitution for SMS to prevent browning in banana blossoms.

Assessment of the Role of PAL in Lignin Accumulation in Wheat (Tríticum aestívum L.) at the Early Stage of Ontogenesis

The current study evaluates the role of phenylalanine ammonia-lyase (PAL) and the associated metabolic complex in the accumulation of lignin in common wheat plants (Tríticum aestívum L.) at the early stages of ontogenesis. The data analysis was performed using plant samples that had reached Phases 4 and 5 on the Feekes scale—these phases are characterized by a transition to the formation of axial (stem) structures in cereal plants. We have shown that the substrate stimulation of PAL with key substrates, such as L-phenylalanine and L-tyrosine, leads to a significant increase in lignin by an average of 20% in experimental plants compared to control plants. In addition, the presence of these compounds in the nutrient medium led to an increase in the number of gene transcripts associated with lignin synthesis (PAL6, C4H1, 4CL1, C3H1). Inhibition was the main tool of the study. Potential competitive inhibitors of PAL were used: the optical isomer of L-phenylalanine—D-phenylalanine—and the hydroxylamine equivalent of phenylalanine—O-Benzylhydroxylamine. As a result, plants incubated on a medium supplemented with O-Benzylhydroxylamine were characterized by reduced PAL activity (almost one third). The lignin content of the cell wall in plants treated with O-Benzylhydroxylamine was almost halved. In contrast, D-phenylalanine did not lead to significant changes in the lignin-associated metabolic complex, and its effect was similar to that of specific substrates.

Improving the Berry Quality and Antioxidant Potential of Flame Seedless Grapes by Foliar Application of Chitosan–Phenylalanine Nanocomposites (CS–Phe NCs)

The production and sustainability of grape berries with high quality and health-promoting properties is a major goal. In this regard, nano-engineered materials are being used for improving the quality and marketability of berries. In this study, we investigated the potential role of chitosan–phenylalanine nanocomposites (CS–Phe NCs) in improving the quality of Flame Seedless (Vitis vinifera L.) grape berries, such as titratable acidity (TA), pH, total soluble solids (TSS), ascorbic acid, total phenolics, total flavonoids, anthocyanin, 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging activity, and phenylalanine ammonia-lyase (PAL) activity. In this context, grape berries collected in two growing seasons (2018–2019) were screened. Regarding the experimental design, the treatments included chitosan at a 0.5% concentration (CS 0.5%), phenylalanine at 5 mM and 10 mM concentrations (Phe 5 mM and Phe 10 mM), and chitosan–phenylalanine nanocomposites (CS–Phe NCs) at 5 mM and 10 mM concentrations. The lowest TA was recorded in grape berries treated with CS–Phe NCs with a 10 mM concentration. However, treatments enhanced with TSS, which reached the highest value with 10 mM of CS–Phe NCs, were reflected as the highest ratio of TSS/TA with 10 mM of CS–Phe NC treatment. Nanocomposites (NCs) also increased pH values in both study years compared to the control. Similarly, the ascorbic acid and total phenolic content increased in response to NP treatment, reaching the highest value with 5 mM and 10 mM of CS–Phe NCs in 2018 and 2019, respectively. The highest flavonoid content was observed with 5 mM of CS–Phe NCs in both study years. In addition, the anthocyanin content increased with 5 and 10 mM of CS–Phe NCs. PAL activity was found to be the highest with 5 mM of CS–Phe NCs in both study years. In addition, in accordance with the increase in PAL activity, increased total phenolics and anthocyanin, and higher DPPH radical scavenging activity of the grapes were recorded with the treatments compared to the control. As deduced from the findings, the coating substantially influenced the metabolic pathway, and the subsequent alterations induced by the treatments were notably appreciated due to there being no adverse impacts perceived.

Induced Resistance by Ascorbate Oxidation Involves Potentiating of the Phenylpropanoid Pathway and Improved Rice Tolerance to Parasitic Nematodes

Anticipating an increased ecological awareness, scientists have been exploring new strategies to reduce the use of chemical pesticides to control pests and diseases. Triggering the intrinsic plant defense system is one of the promising strategies to reduce yield loss by pathogenic organisms, such as nematodes. Ascorbate oxidase (AO) enzyme plays an important role in plant defense by regulating the apoplastic ascorbate/dehydroascorbate (DHA) ratio via the ascorbate oxidation process. Ascorbate oxidation is known to induce systemic resistance in rice against parasitic root-knot nematodes (RKN). Here, we sought to evaluate if AO- or DHA-induced resistance (IR) against RKN M. graminicola involves activation of the phenylpropanoid pathway and whether this IR phenotype has potential effects on growth of rice seedlings under stressed and unstressed conditions. Our results show that AO/DHA-IR against these parasitic nematodes is dependent on activation of phenylalanine ammonia lyase (PAL). However, application of reduced ascorbic acid (AA) did not induce this response. Gene expression analysis via qRT-PCR showed that OsPAL2 and OsPAL4 are highly expressed in AO/DHA-sprayed nematode-infected roots and PAL-activity measurements confirmed that AO/DHA spraying triggers the plants for primed activation of this enzyme upon nematode infection. AO/DHA-IR is not effective in plants sprayed with a chemical PAL inhibitor confirming that AO/DHA-induced resistance is dependent on PAL activity. Improved plant growth and low nematode infection in AO/DHA-sprayed plants was found to be correlated with an increase in shoot chlorophyll fluorescence (Fv/Fm), chlorophyll index (ChlIdx), and modified anthocyanin reflection index which were proven to be good above-ground parameters for nematode infestation. A detailed growth analysis confirmed the improved growth of AO/DHA-treated plants under nematode-infected conditions. Taken together, our results indicate that ascorbate oxidation enhances the phenylpropanoid-based response to nematode infection and leads to a tolerance phenotype in treated rice plants.

In-depth sequence-function characterization reveals multiple paths to enhance phenylalanine ammonia-lyase (PAL) activity.

Phenylalanine ammonia-lyases (PALs) deaminate L-phenylalanine to trans-cinnamic acid and ammonium and have widespread application in chemo-enzymatic synthesis, agriculture, and medicine. In particular, the PAL from Anabaena variabilis has garnered significant attention as the active ingredient in Pegvaliase®, the only FDA-approved drug treating classical phenylketonuria (PKU). Although an extensive body of literature exists on structure, substrate-specificity, and catalytic mechanism, protein-wide sequence determinants of function remain unknown, which limits the ability to rationally engineer these enzymes. Previously, we developed a high-throughput screen (HTS) for PAL, and here, we leverage it to create a detailed sequence-function landscape of PAL by performing deep mutational scanning (DMS). Our method revealed 79 hotspots that affected a positive change in enzyme fitness, many of which have not been reported previously. Using fitness values and structure-function analysis, we picked a subset of residues for comprehensive single- and multi-site saturation mutagenesis to improve the catalytic activity of PAL and identified combinations of mutations that led to improvement in reaction kinetics in cell-free and cellular contexts. To understand the mechanistic role of the most beneficial mutations, we performed QM/MM and MD and observed that different mutants confer improved catalytic activity via different mechanisms, including stabilizing first transition and intermediate states and improving substrate diffusion into the active site, and decreased product inhibition. Thus, this work provides a comprehensive sequence-function relationship for PAL, identifies positions that improve PAL activity when mutated and assess their mechanisms of action.