Genes Encoding Phenylalanine Ammonia-lyase Research Articles

In silico characterization of GbPAL, GbCHS, GbDFR and GbANS structural genes involved in the biosynthesis of flavonoids in Gynura bicolor DC

Gynura bicolor is a member of the Asteraceae family, possessing important pharmaceutical compounds such as flavonoids, anthocyanins, and terpenoids. Genetic and molecular studies focusing on the biosynthesis of flavonoids are indispensable to determining their regulation. GbPAL, GbCHS, GbDFR, and GbANS are genes encoding Phenylalanine ammonia lyase, Chalcone synthase, Dihydroflavonol reductase, and Anthocyanidin synthase enzymes, resepectively and are involved in the biosynthesis of flavonoids in G. bicolor, resepectively. There are still a few studies concerning the characterization of these genes. In this study, the structural enzyme sequences were selected to be characterized in silico using bioinformatics software. The phylogenetic analysis demonstrated that the genes in question were closely related to several species of the Asteraceae family. Our data revealed that the G. bicolor PAL, CHS, DFR and ANS genes are closely related to the sequence of genes found in Chrysanthemum morifolium. Furthermore, the CHS, DFR and ANS of G. bicolor also tightly correlated with sequence from Callistephus chinensis. Further analysis also demonstrated that all conserved motifs contain active functional and catalytic domain supporting the flavonoid-related enzyme functions. All physicochemical parameters used in this study also meet the prediction criteria. The investigation of secondary structure prediction using two instruments (PSIPRED and SOPMA) revealed that all enzymes tested possess similar α-helix (H), β-sheet (E), and coil (C) composition. GbPAL showed a secondary structure dominated by helix (mainly alpha), GbCHS and GbDFR (Alpha-beta), and GbANS (mainly beta). Each enzyme was modeled, with RMSD values superimposed on the template: 0.125 Å (GbPAL), 0.061 Å (GbCHS), 0.065 Å (GbDFR), and 0.089 Å (GbANS). The 3D model evaluation was checked using four parameters, including PROCHECK, Verify3D, ERRAT, and QMEAN-Z score. All the parameters verified that the model showed a high quality (more than 90 % of the favored region), high ressemblence of the 3D and 1D amino acid sequence, the 3D modeles were acceptable since they possesed high resolution of the protein structure (2.5 – 3 Å), and the enzyme models have Z-Score values in the permissible range. The accuracy and reliability of the generated models met Ramachandran value in comparison to experimentally resolved protein templates. The molecular docking analysis showed that each substrate compound docked at the real binding site in every tested enzyme. Furthermore, they also showed similar results compared to other experimentally observed proteins. The overall findings provide an insight into the molecular characteristics of flavonoid-related genes, which might enrich our understanding for further genomic application of the Gynura bicolor.

Physiological response and molecular regulatory mechanism reveal a positive role of nitric oxide and hydrogen sulfide applications in salt tolerance of Cyclocarya paliurus.

As a multifunctional tree species, Cyclocarya paliurus leaves are rich in bioactive substances with precious healthy values. To meet the huge requirement of C. paliurus leaf production, sites with some environmental stresses would be potential land for developing its plantations due to the limitation of land resources in China. Nitric oxide (NO) and hydrogen sulfide (H2S) are common gas messengers used to alleviate abiotic stress damage, whereas the mechanism of these messengers in regulating salt resistance of C. paliurus still remains unclear. We performed a comprehensive study to reveal the physiological response and molecular regulatory mechanism of C. paliurus seedlings to the application of exogenous NO and H2S under salt stress. The results showed that the application of sodium hydrosulfide (NaHS) and sodium nitroprusside (SNP) not only maintained the photosynthetic capacity and reduced the loss of leaf biomass, but also promoted endogenous NO synthesis and reduced oxidative damage by activating antioxidant enzyme activity and increasing the content of soluble protein and flavonoids. Moreover, transcriptome and metabolome analysis indicated the expression of genes encoding phenylalanine ammonia lyase (PAL), cytochromeP450 (CYP), chalcone synthase (CHS), dihydroflavonol 4-reductase (DFR) and flavonol synthase (FLS) in flavonoid biosynthesis pathway was all up-regulated by the application of NO and H2S. Meanwhile, 15 transcriptional factors (TFs) such as WRKY, ERF, bHLH and HY5 induced by NO were found to regulated the activities of several key enzymes in flavonoid biosynthesis pathway under salt stress, via the constructed co-expression network. Our findings revealed the underlying mechanism of NO and H2S to alleviate salt stress and regulate flavonoid biosynthesis, which provides a theoretical basis for establishing C. paliurus plantations in the salt stress areas.

Transcriptomic Analysis on the Peel of UV-B-Exposed Peach Fruit Reveals an Upregulation of Phenolic- and UVR8-Related Pathways.

UV-B treatment deeply influences plant physiology and biochemistry, especially by activating the expression of responsive genes involved in UV-B acclimation through a UV-B-specific perception mechanism. Although the UV-B-related molecular responses have been widely studied in Arabidopsis, relatively few research reports deepen the knowledge on the influence of post-harvest UV-B treatment on fruit. In this work, a transcriptomic approach is adopted to investigate the transcriptional modifications occurring in the peel of UV-B-treated peach (Prunus persica L., cv Fairtime) fruit after harvest. Our analysis reveals a higher gene regulation after 1 h from the irradiation (88% of the differentially expressed genes-DEGs), compared to 3 h recovery. The overexpression of genes encoding phenylalanine ammonia-lyase (PAL), chalcone syntase (CHS), chalcone isomerase (CHI), and flavonol synthase (FLS) revealed a strong activation of the phenylpropanoid pathway, resulting in the later increase in the concentration of specific flavonoid classes, e.g., anthocyanins, flavones, dihydroflavonols, and flavanones, 36 h after the treatment. Upregulation of UVR8-related genes (HY5, COP1, and RUP) suggests that UV-B-triggered activation of the UVR8 pathway occurs also in post-harvest peach fruit. In addition, a regulation of genes involved in the cell-wall dismantling process (PME) is observed. In conclusion, post-harvest UV-B exposure deeply affects the transcriptome of the peach peel, promoting the activation of genes implicated in the biosynthesis of phenolics, likely via UVR8. Thus, our results might pave the way to a possible use of post-harvest UV-B treatments to enhance the content of health-promoting compounds in peach fruits and extending the knowledge of the UVR8 gene network.

Exogenous Glutathione enhances tolerance of the potato (Solanum tuberosum L.) to cadmium stress by regulating the biosynthesis of phenylpropanoid and the signal transduction of plant hormones

BackgroundCadmium (Cd) pollution has brought harm to the growth and development of potato. Glutathione (GSH) is an important antioxidant that may play an active role in the response of a potato to Cd stress. However, how GSH influences the effect of Cd on potatoes is unknown. In this study, we investigated the effects of exogenous GSH on the phenylpropanoid biosynthesis pathway and plant hormone signal transduction pathway in potatoes under Cd stress to explore new ideas for how potatoes respond to Cd stress. We cultured 21-day-old 'Atlantic’ plantlets in Murashige and Skoog (MS) medium supplemented with 500 μmol/L CdCl2 or 500 μmol/L CdCl2 + 400 μmol/L GSH. We then investigated the activities of key enzymes in the phenylpropanoid biosynthesis pathway, hormone levels, and the expression levels of related genes at different time points.ResultsAnalysis showed that 96 h of treatment with glutathione led to an increase in the expression levels of genes encoding phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POD); an increase in the enzymic activities of PAL, CAD and POD; and an increase in the content of lignin. The content of lignin was positively correlated with the expression levels of several genes (PAL: PG0031457, CAD: PG0005359, POD: PG0011640 and PG0015106). In addition, the levels of Salicylic acid (SA) and Jasmonic acid (JA) increased significantly, the expression levels of the genes encoding transcription factor TGA (PG2023696), pathogenesis-related protein 1 (PR1) (PG0005111), and the transcription inhibitor Aux/IAA (PG0006093) all increased while the expression levels of jasmonate ZIM domain-containing protein (JAZ) (PG0004367), auxin influx carrier (AUX) (PG0006550) and auxin response factor (ARF) (PG0005794) all decreased. We also observed a reduction in the content of IAA.ConclusionExogenous GSH improved the tolerance of potato, Atlantic cv. to Cd stress by regulating the phenylpropanoid biosynthesis pathway and the plant hormone signal transduction pathway.Graphical

Proanthocyanidin biosynthesis in the developing wheat seed coat investigated by chemical and RNA-Seq analysis.

The composition of proanthocyanidins in the testa (seed coat) of bread wheat was analyzed by thiolysis of PA oligomers from developing grain and found to consist of (+)‐catechin monomers, with a small amount of (+)‐gallocatechin. The average chain length of soluble PA stayed relatively constant between 10 and 20 days post‐anthesis, whereas that of unextractable PA increased over the same period, suggesting that increases in chain length might account for the insolubility of PAs from mature wheat grain. We carried out RNA‐Seq followed by differential expression analysis from dissected tissues of developing grain from red‐ and white‐grained near‐isogenic lines differing in the presence of an active R gene that encodes a MYB transcription factor involved in control of PA biosynthesis. In addition to genes already identified encoding chalcone synthase, chalcone isomerase, flavanone 3‐hydroxylase, and dihydroxyflavonoid 4‐reductase, we showed that wheat genes encoding phenylalanine ammonia lyase, flavonoid 3′,5′‐hydroxylase, leucoanthocyanidin reductase, and a glutathione S‐transferase (the orthologue of maize Bronze‐2) were more highly expressed in the red NIL. We also identified candidate orthologues of other catalytic and regulatory components of flavonoid biosynthesis in wheat.

Anthocyanin Accumulation and Differential Expression of the Biosynthetic Genes Result in a Discrepancy in the Red Color of Herbaceous Peony (Paeonia lactiflora Pall.) Flowers

Herbaceous peony (Paeonia lactiflora Pall.) is an ornamental plant with huge potential in the international flower market. Similar to the flowers of most other ornamental plants, the top sellers of P. lactiflora are those with red or pink flowers. However, most studies on flower colors have focused on the novel colors and have neglected the most common red flowers. In this study, a red cultivar of P. lactiflora (‘Dafugui’) and a pink cultivar (‘Qingwen’) were selected in order to study the discrepancy in the red color of the flowers. The results demonstrate that these two cultivars have the same compositions as anthocyanins, flavones, and flavonols but different contents. ‘Dafugui’ was found to have a high accumulation of upstream substances due to the higher expression of the early genes encoding phenylalanine ammonialyase (PlPAL) and flavonoid 3′-hydroxylase (PlF3′H). Moreover, the anthocyanidin synthase gene (PlANS) and UDP-glucose flavonoid 3-O-glucosyltransferase gene (PlUF3GT) encoding enzymes catalyze these upstream substances into anthocyanins, resulting in more redness in ‘Dafugui’ than in ‘Qingwen’. Our study thus provides a better understanding of the anthocyanin accumulation and coloring mechanism of P. lactiflora and can serve as a theoretical basis for breeding more red flowers using genetic engineering techniques to cater to consumers’ preferences.

Integrative analysis of metabolome and transcriptome reveals molecular regulatory mechanism of flavonoid biosynthesis in Cyclocarya paliurus under salt stress

Cyclocarya paliurus is a highly valued and multiple function tree species, and especially its leaves are enriched in diverse secondary metabolites with healthy function. Flavonoids as a kind of secondary metabolites are not only an important part of plant defense system against environmental stresses but also commercial pharmaceutical substances. To meet the leaf production for value-added product development, coastal saline would be a potential land resources for developing C. paliurus plantations, whereas there is limited knowledge on the regulatory mechanisms of flavonoid biosynthesis under salt stress in C. paliurus. Here, we conducted an integrated transcriptomics and metabolomics analysis of C. paliurus under different salt treatments in the short (T1, treatment lasted for 15 days) and long (T2, treatment lasted for 30 days) term. Generally, salt treatments led to increased total flavonoid content in C. paliurus leaves, which increased gradually along with salt stress concentration. For instance, after seedlings were salt-treated with LS (0.15 %), MS (0.30 %) and HS (0.45 %), the total flavonoid content was increased by 26.23 %, 57.54 %, and 74.87 %, respectively, compared with the control (0.00 %, m/v) at T1. Correspondingly, significant enrichment of differentially expressed genes and metabolites was observed in the flavonoid biosynthesis pathways. Weighted gene co-expression network analysis (WGCNA) identified several key genes regulating the responses to salt stress, such as genes encoding phenylalanine ammonia lyase (PAL), chalcone synthase (CHS) and flavonol synthase (FLS). In addition, fifteen transcription factors (TFs) were found to regulate flavonoid biosynthesis by activating or repressing the expression of multiple structural genes in C. paliurus leaves during salt stress. These findings provide insight into the salt stress associated transcriptional regulation, and would drive progress in genetic improvement and plantation development of C. paliurus.

Transcriptome and metabolite profiling reveals the effects of Funneliformis mosseae on the roots of continuously cropped soybeans

BackgroundArbuscular mycorrhizal fungi are the most widely distributed mycorrhizal fungi, which can form mycorrhizal symbionts with plant roots and enhance plant stress resistance by regulating host metabolic activities. In this paper, the RNA sequencing and ultra-performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS/MS) technologies were used to study the transcriptome and metabolite profiles of the roots of continuously cropped soybeans that were infected with F. mosseae and F. oxysporum. The objective was to explore the effects of F. mosseae treatment on soybean root rot infected with F. oxysporum.ResultsAccording to the transcriptome profiles, 24,285 differentially expressed genes (DEGs) were identified, and the expression of genes encoding phenylalanine ammonia lyase (PAL), trans-cinnamate monooxygenase (CYP73A), cinnamyl-CoA reductase (CCR), chalcone isomerase (CHI) and coffee-coenzyme o-methyltransferase were upregulated after being infected with F. oxysporum; these changes were key to the induction of the soybean’s defence response. The metabolite results showed that daidzein and 7,4-dihydroxy, 6-methoxy isoflavone (glycine), which are involved in the isoflavone metabolic pathway, were upregulated after the roots were inoculated with F. mosseae. In addition, a substantial alteration in the abundance of amino acids, phenolic and terpene metabolites all led to the synthesis of defence compounds. An integrated analysis of the metabolic and transcriptomic data revealed that substantial alterations in the abundance of most of the intermediate metabolites and enzymes changed substantially under pathogen infection. These changes included the isoflavonoid biosynthesis pathway, which suggests that isoflavonoid biosynthesis plays an important role in the soybean root response.ConclusionThe results showed that F. mosseae could alleviate the root rot caused by continuous cropping. The increased activity of some disease-resistant genes and disease-resistant metabolites may partly account for the ability of the plants to resist diseases. This study provides new insights into the molecular mechanism by which AMF alleviates soybean root rot, which is important in agriculture.

Involvement of salicylic acid in cold priming-induced freezing tolerance in wheat plants

Freezing is one of the most damaging abiotic stress factors. Cold priming has been shown to enhance freezing tolerance in wheat. However, the underlying mechanisms are unclear. Salicylic acid (SA) is an important signal molecule involved in plant responses to abiotic and biotic stresses. This study aims to investigate the role of SA in cold priming-induced freezing tolerance in wheat plants. The results showed that the expression of gene encoding phenylalanine ammonia-lyase (PAL, a key enzyme involved in SA biosynthesis) and the level of endogenous SA (both free and conjugated) were significantly up-regulated following cold priming treatment. The role of SA in cold priming-induced freezing tolerance was further explored using the donor and inhibitor of endogenous SA. l-α-aminooxy-β-phenylpropionic acid (AOPP, a specific inhibitor of PAL) pretreatment reversed the increment of SA level induced by cold priming treatment, attenuated the positive effects of cold priming on antioxidant capacity (as indicated by the contents of MDA, ASA and GSH, and the activities of APX, GR, DHAR and MDHAR) and cold-responsive genes expression (such as AOX1a, IRI2 and HSP70) under the later freezing stress as well as the freezing tolerance (as indicated by the photochemical activity, electrolyte leakage and biomass). The application of exogenous SA could obviously counteract the negative effects of AOPP. Findings of the present study suggested that the roles of SA in cold priming-induced freezing tolerance in wheat might involve regulation of antioxidant capacity and cold-responsive gene expression.

Transcriptome Analysis of Early Defenses in Rice against Fusarium fujikuroi

BackgroundBakanae is a seedborne disease caused by Fusarium fujikuroi. Rice seedlings emerging from infected seeds can show diverse symptoms such as elongated and slender stem and leaves, pale coloring, a large leaf angle, stunted growth and even death. Little is known about rice defense mechanisms at early stages of disease development.ResultsThis study focused on investigating early defenses against F. fujikuroi in a susceptible cultivar, Zerawchanica karatals (ZK), and a resistant cultivar, Tainung 67 (TNG67). Quantitative PCR revealed that F. fujikuroi colonizes the root and stem but not leaf tissues. Illumina sequencing was conducted to analyze the stem transcriptomes of F. fujikuroi-inoculated and mock-inoculated ZK and TNG67 plants collected at 7 days post inoculation (dpi). More differentially expressed genes (DEGs) were identified in ZK (n = 169) than TNG67 (n = 118), and gene ontology terms related to transcription factor activity and phosphorylation were specifically enriched in ZK DEGs. Among the complex phytohormone biosynthesis and signaling pathways, only DEGs involved in the jasmonic acid (JA) signaling pathway were identified. Fourteen DEGs encoding pattern-recognition receptors, transcription factors, and JA signaling pathway components were validated by performing quantitative reverse transcription PCR analysis of individual plants. Significant repression of jasmonate ZIM-domain (JAZ) genes (OsJAZ9, OsJAZ10, and OsJAZ13) at 3 dpi and 7 dpi in both cultivars, indicated the activation of JA signaling during early interactions between rice and F. fujikuroi. Differential expression was not detected for salicylic acid marker genes encoding phenylalanine ammonia-lyase 1 and non-expressor of pathogenesis-related genes 1. Moreover, while MeJA did not affect the viability of F. fujikuroi, MeJA treatment of rice seeds (prior to or after inoculation) alleviated and delayed bakanae disease development in susceptible ZK.ConclusionsDifferent from previous transcriptome studies, which analyzed the leaves of infected plants, this study provides insights into defense-related gene expression patterns in F. fujikuroi–colonized rice stem tissues. Twelve out of the 14 selected DEGs were for the first time shown to be associated with disease resistance, and JA-mediated resistance was identified as a crucial component of rice defense against F. fujikuroi. Detailed mechanisms underlying the JA-mediated bakanae resistance and the novel defense-related DEGs are worthy of further investigation.

The effect of cutting style on the biosynthesis of phenolics and cellular antioxidant capacity in wounded broccoli

To explore the effect of cutting style on the biosynthesis of phenolic compounds and cellular antioxidant capacity in wounded broccoli subjected to different cutting styles (heads, florets, 1/2 florets and shredded florets), the mechanism of the accumulation of phenolic compounds was investigated at the transcriptional level, and cellular antioxidant capacity was measured using a breast cancer cell MCF-7 culture model. The results indicated that the relative expression of the genes encoding phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase and 4-coumarin coenzyme A ligase, three enzymes involved in phenylpropanoid metabolism, was upregulated and that contributed to the synthesis of individual phenolic compounds, including catechin, hydroxybenzoic acid, chlorogenic acid, caffeic acid, sinapic acid, catechin gallate, rutin, cinnamic acid and quercetin. This research constructes the phenol synthesis pathway in wounded broccoli. Moreover, the relative expression of critical genes including superoxide dismutase, peroxidase, catalase, glutathione peroxidase and glutathione reductase involved in the metabolism of reactive oxygen species (ROS) increased, resulting in enhanced antioxidant capacity in wounded broccoli. Cell antioxidant capacity (CAA) of heads, florets, 1/2 florets and shredded florets increased by 52.7%, 59.2%, 64.8% and 86.5%, respectively, compared to whole broccoli. The enhancement of CAA was greater as the intensity of wounding increased, indicating that enhancement of antioxidant activity occurred at the cellular level. This research helps provide a reliable and persuasive theoretical basis for nutritional value assessment at the cellular level in wounded broccoli.

Comparative Genome Analysis of Scutellaria baicalensis and Scutellaria barbata Reveals the Evolution of Active Flavonoid Biosynthesis

Scutellaria baicalensis (S. baicalensis) and Scutellaria barbata (S. barbata) are common medicinal plants of the Lamiaceae family. Both produce specific flavonoid compounds, including baicalein, scutellarein, norwogonin, and wogonin, as well as their glycosides, which exhibit antioxidant and antitumor activities. Here, we report chromosome-level genome assemblies of S. baicalensis and S. barbata with quantitative chromosomal variation (2n = 18 and 2n = 26, respectively). The divergence of S. baicalensis and S. barbata occurred far earlier than previously reported, and a whole-genome duplication (WGD) event was identified. The insertion of long terminal repeat elements after speciation might be responsible for the observed chromosomal expansion and rearrangement. Comparative genome analysis of the congeneric species revealed the species-specific evolution of chrysin and apigenin biosynthetic genes, such as the S. baicalensis-specific tandem duplication of genes encoding phenylalanine ammonia lyase and chalcone synthase, and the S. barbata-specific duplication of genes encoding 4-CoA ligase. In addition, the paralogous duplication, colinearity, and expression diversity of CYP82D subfamily members revealed the functional divergence of genes encoding flavone hydroxylase between S. baicalensis and S. barbata. Analyzing these Scutellaria genomes reveals the common and species-specific evolution of flavone biosynthetic genes. Thus, these findings would facilitate the development of molecular breeding and studies of biosynthesis and regulation of bioactive compounds.

Analysis of the Transcriptome of Polygonatum odoratum (Mill.) Druce Uncovers Putative Genes Involved in Isoflavonoid Biosynthesis

Polygonatum odoratum (Mill.) Druce (P. odoratum) is a traditional Chinese herb. The isoflavonoids of P. odoratum are important medicinal bioactive compounds for the treatment of hypoimmunity, rheumatic heart disease, cardiovascular diseases, and diabetes. This study used RNA sequencing to identify potential genes regulating isoflavonoid biosynthesis in P. odoratum. To do this, we generated an overview of metabolic and secondary metabolic pathways, using MAPMAN, and identified 96 genes encoding seven key enzymes involved in isoflavonoid biosynthesis through their KEGG annotation. Alignment of phenylalanine ammonia-lyase or chalcone synthase amino acid sequences revealed well-conserved sequences, spatial structures, and active sites. We also verified the expression of key genes encoding phenylalanine ammonia-lyase and chalcone synthase by quantitative real-time PCR (qRT-PCR). This analysis of the isoflavonoid biosynthesis pathway and its crucial enzymes in P. odoratum lays the foundation for uncovering the regulatory mechanism of isoflavonoid biosynthesis.

Application of sodium salicylate up-regulates defense responseagainst Fusarium graminearum in wheat spikes

Fusarium head blight caused by the hemibiotrophic fungus Fusarium graminearum is one of the most devastating diseases of wheat which reduces both grain yield and quality. To better understand mechanism underlying wheat resistance to this pathogen, the expressions of five candidate genes encoding phenylalanine ammonia-lyase (PAL), glucanase-2 (Gl 2), class IV chitinase (Cht-4), cytochrome P450 (CYP), and pleiotropic drug resistance (PDR) following spike inoculation with F. graminearum was compared in susceptible cv. Falat and resistant cv. Sumai3 at three time points (48, 96, 144 h after inoculation). Real-time quantitative PCR analysis indicated earlier and greater inductions of PAL, Glu-2, and Cht-4 in spikes of 'Sumai3' as compared to 'Falat' in response to F. graminearum inoculation. The expression of CYP in the resistant 'Sumai3' was about three times higher than in 'Falat' at 144 h after pathogen inoculation. Moreover, soil drench application of sodium salicylate (SA) one day before pathogen inoculation drastically curtailed pathogen infection in both the cultivars. Furthermore, SA treatment caused an induction of these genes in spikes of the susceptible cultivar to show a similar pattern as in the resistant one when inoculated with F. graminearum. Proteomics analysis of F. graminearum treated spikes 96 h after inoculation confirmed an increase of Glu and Cht spot volume in 'Sumai3' whereas a decrease in 'Falat'. The SA treatment also caused significant increases in Glu and Cht spot volumes in both the cultivars. Our findings show an association between SA improvement of wheat defense against F. graminearum infection and induction of genes encoding proteins involved in pathogen response (Glu-2, Cht-4), secondary metabolite biosyntheses (PAL), and xenobiotic detoxification (CYP and PDR).

Colonization with arbuscular mycorrhizal fungus Funneliformis mosseae enhanced the responses of tomato plants to mechanical wounding

Insect herbivore feeding causes mechanical damage to plants, which can activate plant defense responses. Whether symbiosis with beneficial microorganisms can enhance the responses of plants to mechanical damage is of importance for plant anti-herbivore resistance. In this study, defense responses of tomato (Lycopersicon esculentum) plants to mechanical wounding was investigated after the tomato roots being infected by arbuscular mycorrhizal fungus (AMF) Funneliformis mosseae. The results showed that in response to leaf mechanical wounding, the activities of phenylalanine ammonia-lyase (PAL), superoxide dismutase (SOD), peroxidase (POD), polyphenol oxidase (PPO) and catalase (CAT) in the leaves of tomato pre-inoculated with AMF (FD), as well as transcript levels of genes encoding phenylalanine ammonia lyase (PAL) and β-1,3-glucanase (PR2) in the leaves and roots were significantly higher in relative to sole mechanical wounding (D), sole mycorrhizal inoculation (F), and control without mechanical wounding and mycorrhizal inoculation (CK). Although the activities of protective enzyme and transcript levels of the two defense-related genes were induced in the plants of sole mechanical wounding (D) and sole mycorrhizal inoculation (F), the induction was faster and stronger in the plants with leaf mechanical wounding and mycorrhizal pre-inoculation (FD). Our findings indicated that arbuscular mycorrhizal colonization could prime quicker and stronger defense responses of tomato plants to mechanical damage.

Morphological and transcript changes in the biosynthesis of lignin in oil palm (Elaeis guineensis) during Ganoderma boninense infections in vitro.

Lignification of the plant cell wall could serve as the first line of defense against pathogen attack, but the molecular mechanisms of virulence and disease between oil palm and Ganoderma boninense are poorly understood. This study presents the biochemical, histochemical, enzymology and gene expression evidences of enhanced lignin biosynthesis in young oil palm as a response to G. boninense (GBLS strain). Comparative studies with control (T1), wounded (T2) and infected (T3) oil palm plantlets showed significant accumulation of total lignin content and monolignol derivatives (syringaldehyde and vanillin). These derivatives were deposited on the epidermal cell wall of infected plants. Moreover, substantial differences were detected in the activities of enzyme and relative expressions of genes encoding phenylalanine ammonia lyase (EC 4.3.1.24), cinnamate 4-hydroxylase (EC 1.14.13.11), caffeic acid O-methyltransferase (EC 2.1.1.68) and cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195). These enzymes are key intermediates dedicated to the biosynthesis of lignin monomers, the guaicyl (G), syringyl (S) and ρ-hydroxyphenyl (H) subunits. Results confirmed an early, biphasic and transient positive induction of all gene intermediates, except for CAD enzyme activities. These differences were visualized by anatomical and metabolic changes in the profile of lignin in the oil palm plantlets such as low G lignin, indicating a potential mechanism for enhanced susceptibility toward G. boninense infection.

De novo biosynthesis of trans-cinnamic acid derivatives in Saccharomyces cerevisiae.

The production of natural aroma compounds is an expanding field within the branch of white biotechnology. Three aromatic compounds of interest are cinnamaldehyde, the typical cinnamon aroma that has applications in agriculture and medical sciences, as well as cinnamyl alcohol and hydrocinnamyl alcohol, which have applications in the cosmetic industry. Current production methods, which rely on extraction from plant materials or chemical synthesis, are associated with drawbacks regarding scalability, production time, and environmental impact. These considerations make the development of a sustainable microbial-based production highly desirable. Through steps of rational metabolic engineering, we engineered the yeast Saccharomyces cerevisiae as a microbial host to produce trans-cinnamic acid derivatives cinnamaldehyde, cinnamyl alcohol, and hydrocinnamyl alcohol, from externally added trans-cinnamic acid or de novo from glucose as a carbon source. We show that the desired products can be de novo synthesized in S. cerevisiae via the heterologous overexpression of the genes encoding phenylalanine ammonia lyase 2 from Arabidopsis thaliana (AtPAL2), aryl carboxylic acid reductase (acar) from Nocardia sp., and phosphopantetheinyl transferase (entD) from Escherichia coli, together with endogenous alcohol dehydrogenases. This study provides a proof of concept and a strain that can be further optimized for production of high-value aromatic compounds.

A kinetic model for flavonoid production in tea cell culture.

As one of the strategies for efficient production of a metabolite from cell cultures, a kinetic model is very useful tool to predict productivity under various culture conditions. In this study, we propose a kinetic model for flavonoid production in tea cell culture based on the cell life cycle and expression of PAL, the gene encoding phenylalanine ammonia-lyase (PAL)-the key enzyme in flavonoid biosynthesis. The flavonoid production rate was considered to be related to the amount of active PAL. Synthesis of PAL was modelled based on a general gene expression/translation mechanism, including the transcription of DNA encoding PAL into mRNA and the translation of PAL mRNA into the PAL protein. The transcription of DNA was assumed to be promoted at high light intensity and suppressed by a feedback regulatory mechanism at high flavonoid concentrations. In the model, mRNA and PAL were considered to self-decompose and to be lost by cell rupture. The model constants were estimated by fitting the experimental results obtained from tea cell cultures under various light intensities. The model accurately described the kinetic behaviors of dry and fresh cell concentrations, glucose concentration, cell viability, PAL specific activity, and flavonoid content under a wide range of light intensities. The model simulated flavonoid productivity per medium under various culture conditions. Therefore, this model will be useful to predict optimum culture conditions for maximum flavonoid productivity in cultured tea cells.

The mvp2 mutation affects the generative transition through the modification of transcriptome pattern, salicylic acid and cytokinin metabolism in Triticum monococcum

Wild type and mvp2 (maintained vegetative phase) deletion mutant T. monococcum plants incapable of flowering were compared in order to determine the effect of the deleted region of chromosome 5A on transcript profile and hormone metabolism. This region contains the vernalization1 (VRN1) gene, a major regulator of the vegetative/generative transition. Transcript profiling in the crowns of T. monococcum during the transition and the subsequent formation of flower primordia showed that 306 genes were affected by the mutation, 198 by the developmental phase and 14 by the interaction of these parameters. In addition, 546 genes were affected by two or three factors. The genes controlled by the deleted region encode transcription factors, antioxidants and enzymes of hormone, carbohydrate and amino acid metabolism. The observed changes in the expression of the gene encoding phenylalanine ammonia lyase (PAL) might indicate the effect of mvp2 mutation on the metabolism of salicylic acid, which was corroborated by the differences in 2-hydroxycinnamic acid and cinnamic acid contents in both of the leaves and crowns, and in the concentrations of salicylic acid and benzoic acid in crowns during the vegetative/generative transition. The amount and ratio of active cytokinins and their derivatives (ribosides, glucosides and phosphates) were affected by developmental changes as well as by mvp2 mutation, too.

Kaolin exogenous application boosts antioxidant capacity and phenolic content in berries and leaves of grapevine under summer stress

Heat waves, high light intensities and water deficit are becoming important threats in many important viticultural areas worldwide, so the implementation of efficient and cost-effective mitigation strategies is crucial for the production of premium wines while maintaining productivity. In this context, the foliar application of kaolin, a chemically inert mineral with excellent reflective properties, is being developed and experimented as a strategy to reduce the impact of heat and drought in Douro vineyards (Northern Portugal), already revealing promising results. In the present study we investigated if an improved antioxidant capacity is part of the beneficial effects of kaolin, by studying changes in the enzymatic and nonenzymatic antioxidant system in leaves and berries (cv Touriga Nacional). Results showed that mature grape berries contained higher amounts of total phenols (40%), flavonoids (24%), anthocyanins (32%) and vitamin C (12%) than fruits from control vines, and important changes were also measured in leaves. In parallel, kaolin application improved the antioxidant capacity in berries, which was correlated with the observed increased content in secondary metabolites. Kaolin application also regulated secondary metabolism at the transcriptional level through the increase in the transcript abundance of genes encoding phenylalanine ammonia lyase and chalcone synthase.