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Brown planthopper (BPH) is one of the most destructive insects affecting rice (Oryza sativa L.) production. Phenylalanine ammonialyase (PAL) is a key enzyme involved in plant defense against pathogens, but the role of PAL in insect resistance is still poorly understood. Here we show that expression of the majority of PALs in rice is significantly induced by BPH feeding. Knockdown of OsPALs significantly reduces BPH resistance, whereas overexpression of OsPAL8 in a susceptible rice cultivar significantly enhances its BPH resistance. We found that OsPALs mediate resistance to BPH by regulating the biosynthesis and accumulation of salicylic acid and lignin. Furthermore, we show that expression of OsPAL6 and OsPAL8 in response to BPH attack is directly up-regulated by OsMYB30, an R2R3 MYB transcription factor. Taken together, our results demonstrate that the phenylpropanoid pathway plays an important role in BPH resistance response, and provide valuable targets for genetic improvement of BPH resistance in rice.

Phenylalanine ammonia-lyase (PAL) is the first enzyme of the general phenylpropanoid pathway catalyzing the nonoxidative elimination of ammonia from L-phenylalanine to give trans-cinnamate. In monocots, PAL also displays tyrosine ammonia lyase (TAL) activity, leading to the formation of p-coumaric acid. The catalytic mechanism and substrate specificity of a major PAL from sorghum (Sorghum bicolor; SbPAL1), a strategic plant for bioenergy production, were deduced from crystal structures, molecular docking, site-directed mutagenesis, and kinetic and thermodynamic analyses. This first crystal structure of a monocotyledonous PAL displayed a unique conformation in its flexible inner loop of the 4-methylidene-imidazole-5-one (MIO) domain compared with that of dicotyledonous plants. The side chain of histidine-123 in the MIO domain dictated the distance between the catalytic MIO prosthetic group created from ¹⁸⁹Ala-Ser-Gly¹⁹¹ residues and the bound L-phenylalanine and L-tyrosine, conferring the deamination reaction through either the Friedel-Crafts or E₂ reaction mechanism. Several recombinant mutant SbPAL1 enzymes were generated via structure-guided mutagenesis, one of which, H123F-SbPAL1, has 6.2 times greater PAL activity without significant TAL activity. Additional PAL isozymes of sorghum were characterized and categorized into three groups. Taken together, this approach identified critical residues and explained substrate preferences among PAL isozymes in sorghum and other monocots, which can serve as the basis for the engineering of plants with enhanced biomass conversion properties, disease resistance, or nutritional quality.

Salicylic acid (SA), an essential regulator of plant defense, is derived from chorismate via either the phenylalanine ammonia lyase (PAL) or the isochorismate synthase (ICS) catalyzed steps. The ICS pathway is thought to be the primary contributor of defense-related SA, at least in Arabidopsis. We investigated the relative contributions of PAL and ICS to defenserelated SA accumulation in soybean (Glycine max). Soybean plants silenced for five PAL isoforms or two ICS isoforms were analyzed for SA concentrations and SA-derived defense responses to the hemibiotrophic pathogens Pseudomonas syringae and Phytophthora sojae. We show that, unlike in Arabidopsis, PAL and ICS pathways are equally important for pathogen-induced SA biosynthesis in soybean. Knock-down of either pathway shuts down SA biosynthesis and abrogates pathogen resistance. Moreover, unlike in Arabidopsis, pathogen infection is associated with the suppression of ICS gene expression. Pathogen-induced biosynthesis of SA via the PAL pathway correlates inversely with phenylalanine concentrations. Although infections with either virulent or avirulent strains of the pathogens increase SA concentrations, resistance protein-mediated response to avirulent P. sojae strains may function in an SA-independent manner. These results show that PAL- and ICS-catalyzed reactions function cooperatively in soybean defense and highlight the importance of PAL in pathogen-induced SA biosynthesis.

Summary Sugarcane mosaic virus (SCMV) is a pathogen of worldwide importance that causes dwarf mosaic disease on maize (Zea mays). Until now, few maize genes/proteins have been shown to be involved in resistance to SCMV. In this study, we characterized the role of maize phenylalanine ammonia‐lyases (ZmPALs) in accumulation of the defence signal salicylic acid (SA) and in resistance to virus infection. SCMV infection significantly increased SA accumulation and expression of SA‐responsive pathogenesis‐related protein genes (PRs). Interestingly, exogenous SA treatment decreased SCMV accumulation and enhanced resistance. Both reverse transcription‐coupled quantitative PCR and RNA‐Seq data confirmed that expression levels of at least four ZmPAL genes were significantly up‐regulated upon SCMV infection. Knockdown of ZmPAL expression led to enhanced SCMV infection symptom severity and virus multiplication, and simultaneously resulted in decreased SA accumulation and PR gene expression. Intriguingly, application of exogenous SA to SCMV‐infected ZmPAL‐silenced maize plants decreased SCMV accumulation, showing that ZmPALs are required for SA‐mediated resistance to SCMV infection. In addition, lignin measurements and metabolomic analysis showed that ZmPALs are also involved in SCMV‐induced lignin accumulation and synthesis of other secondary metabolites via the phenylpropanoid pathway. In summary, our results indicate that ZmPALs are required for SA accumulation in maize and are involved in resistance to virus infection by limiting virus accumulation and moderating symptom severity.

To gain a better insight into the selenium nanoparticle (nSe) benefits/toxicity, this experiment was carried out to address the behavior of bitter melon seedlings to nSe (0, 1, 4, 10, 30, and 50 mgL-1) or bulk form (selenate). Low doses of nSe increased biomass accumulation, while concentrations of 10 mgL-1 and above were associated with stem bending, impaired root meristem, and severe toxicity. Responses to nSe were distinct from that of bulk in that the nano-type exhibited a higher efficiency to stimulate growth and organogenesis than the bulk. The bulk form displayed higher phytotoxicity than the nano-type counterpart. According to the MSAP-based analysis, nSe mediated substantial variation in DNA cytosine methylation, reflecting the epigenetic modification. By increasing the concentration of nSe, the expression of the WRKY1 transcription factor linearly up-regulated (mean = 7.9-fold). Transcriptions of phenylalanine ammonia-lyase (PAL) and 4-Coumarate: CoA-ligase (4CL) genes were also induced. The nSe treatments at low concentrations enhanced the activity of leaf nitrate reductase (mean = 52%) in contrast with the treatment at toxic concentrations. The toxic concentration of nSe increased leaf proline concentration by 80%. The nSe supplement also stimulated the activities of peroxidase (mean = 35%) and catalase (mean = 10%) enzymes. The nSe-treated seedlings exhibited higher PAL activity (mean = 39%) and soluble phenols (mean = 50%). The nSe toxicity was associated with a disrupted differentiation of xylem conducting tissue. The callus formation and performance of the explants originated from the nSe-treated seedlings had a different trend than that of the control. This experiment provides new insights into the nSe-associated advantage/ cytotoxicity and further highlights the necessity of designing convincing studies to introduce novel methods for plant cell/tissue cultures and agriculture.

Main conclusionScreening 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), β-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.

• Plants produce several hundreds of thousands of secondary metabolites that are important for adaptation to various environmental conditions. Although different groups of secondary metabolites are synthesized through unique biosynthetic pathways, plants must orchestrate their production simultaneously. Phenylpropanoids and glucosinolates are two classes of secondary metabolites that are synthesized through apparently independent biosynthetic pathways. Genetic evidence has revealed that the accumulation of glucosinolate intermediates limits phenylpropanoid production in a Mediator Subunit 5 (MED5)-dependent manner. • To elucidate the molecular mechanism underlying this process, we analyzed the transcriptomes of a suite of Arabidopsis thaliana glucosinolate-deficient mutants using RNAseq and identified misregulated genes that are rescued by the disruption of MED5. • The expression of a group of Kelch Domain F-Box genes (KFBs) that function in PAL degradation is affected in glucosinolate biosynthesis mutants and the disruption of these KFBs restores phenylpropanoid deficiency in the mutants. • Our study suggests that glucosinolate/phenylpropanoid metabolic crosstalk involves the transcriptional regulation of KFB genes that initiate the degradation of the enzyme phenylalanine ammonia-lyase, which catalyzes the first step of the phenylpropanoid biosynthesis pathway. Nevertheless, KFB mutant plants remain partially sensitive to glucosinolate pathway mutations, suggesting that other mechanisms that link the two pathways also exist.

Phenylalanine ammonia-lyase (PAL) has a crucial role in secondary phenylpropanoid metabolism and is one of the most extensively studied enzymes with respect to plant responses to biotic and abiotic stress. Here, we identified the pepper (Capsicum annuum) PAL (CaPAL1) gene, which was induced in pepper leaves by avirulent Xanthomonas campestris pv. vesicatoria (Xcv) infection. CaPAL1-silenced pepper plants exhibited increased susceptibility to virulent and avirulent Xcv infection. Reactive oxygen species (ROS), hypersensitive cell death, expression of the salicylic acid (SA)-dependent marker gene CaPR1, SA accumulation, and induction of PAL activity were significantly compromised in the CaPAL1-silenced pepper plants during Xcv infection. Overexpression (OX) of CaPAL1 in Arabidopsis conferred increased resistance to Pseudomonas syringae pv. tomato (Pst) and Hyaloperonospora arabidopsidis infection. CaPAL1-OX leaves exhibited restricted Pst growth, increased ROS burst and cell death, and induction of PR1 expression and SA accumulation. The increase in PAL activity in healthy and Pst-infected leaves was higher in CaPAL1-OX plants than in wild-type Arabidopsis. Taken together, these results suggest that CaPAL1 acts as a positive regulator of SA-dependent defence signalling to combat microbial pathogens via its enzymatic activity in the phenylpropanoid pathway.

In the course of their life, plants face a multitude of environmental anomaly that affects their growth and production. In recent decades, lead (Pb) gained an increasing attention as it is among the most significant contaminants in the environment. Therefore, in this study the effects of Pb concentrations (0, 50 and 100 ppm) on Vicia faba plants and attempts to alleviate this stress using chitosan (Chs; 0 and 0.1%) were performed. The results validated that with increasing Pb concentrations, a decline in growth, pigments and protein contents was observed. In the same time, a significant upsurge in the stress markers, both malondialdehyde (MDA) and H 2 O 2 , was observed under Pb stress. Nonetheless, foliar spraying with Chs improves the faba bean growth, pigment fractions, protein, carbohydrates, reduces MDA and H 2 O 2 contents and decreases Pb concentrations under Pb stress. Pb mitigation effects by Chs are probably related with the activity of antioxidant enzymes, phenylalanine ammonia lyase (PAL) and proline. The application of Chs enhanced the activities of peroxidase, catalase and PAL by 25.77, 17.71 and 20.07%, respectively at 100 ppm Pb compared to their control. Plant genomic material exhibits significant molecular polymorphism, with an average polymorphism of 91.66% across all primers. To assess the genetic distance created among treatments, the dendrogram was constructed and the results of the similarity index ranged from 0.75 to 0.95, indicating genetic divergence. Our research offers a thorough comprehension of the role of Chs in lessening the oxidative stress, which will encourage the use of Chs in agricultural plant protection.

In this study, rational design and saturation mutagenesis efforts for engineering phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) provided tailored PALs active towards challenging, highly valuable di-substituted substrates, such as the l-DOPA precursor 3,4-dimethoxy-l-phenylalanine or the 3-bromo-4-methoxy-phenylalanine. The rational design approach and saturation mutagenesis strategy unveiled identical PcPAL variants of improved activity, highlighting the limited mutational variety of the substrate specificity-modulator residues, L134, F137, I460 of PcPAL. Due to the restricted catalytic efficiency of the best performing L134A/I460V and F137V/I460V PcPAL variants, we imprinted these beneficial mutations to PALs of different origins. The variants of PALs from Arabidopsis thaliana (AtPAL) and Anabaena variabilis (AvPAL) showed higher catalytic efficiency than their PcPAL homologues. Further, the engineered PALs were also compared in terms of catalytic efficiency with a novel aromatic ammonia-lyase from Loktanella atrilutea (LaAAL), close relative of the metagenome-derived aromatic ammonia-lyase AL-11, reported recently to possess atypically high activity towards substrates with electron-donor aromatic substituents. Indeed, LaAAL outperformed the engineered Pc/At/AvPALs in the production of 3,4-dimethoxy-l-phenylalanine; however, in case of 3-bromo-4-methoxy derivatives it showed no activity, with computational results supporting the occurrence of steric hindrance. Transferring the unique array of selectivity modulator residues from LaAAL to the well-characterized PALs did not enhance their activity towards the targeted substrates. Moreover, applying the rational design strategy valid for these well-characterized PALs to LaAAL decreased its activity. These results suggest that distinct tailoring rationale is required for LaAAL/AL-11-like aromatic ammonia-lyases, which might represent a distinct PAL subclass, with natural reaction and substrate scope modified through evolutionary processes.Key points• PAL-activity for challenging substrates generated by protein engineering• Rational/semi-rational protein engineering reveals constrained mutational variability• Engineered PALs are outperformed by novel ALs of distinct catalytic site signature