Tyrosine Ammonia-lyase Activity Research Articles

6-BA Reduced Yield Loss under Waterlogging Stress by Regulating the Phenylpropanoid Pathway in Wheat.

Waterlogging stress causes substantial destruction to plant growth and production under climatic fluctuations globally. Plants hormones have been widely explored in numerous crops, displaying an imperative role in crop defense and growth mechanism. However, there is a paucity of research on the subject of plant hormones regulating waterlogging stress responses in wheat crop. In this study, we clarified the role of 6-BA in waterlogging stress through inducing phenylpropanoid biosynthesis in wheat. The application of 6-BA (6-benzyladenine) enhanced the growth and development of wheat plants under waterlogging stress, which was accompanied by reduced electrolyte leakage, high chlorophyll, and soluble sugar content. ROS scavenging was also enhanced by 6-BA, resulting in reduced MDA and H2O2 accumulation and amplified antioxidant enzyme activities. Additionally, under the effect of 6-BA, the acceleration of lignin content and accumulation in the cell walls of wheat tissues, along with the activation of PAL (phenylalanine ammonia lyase), TAL (tyrosine ammonia lyase), and 4CL (4-hydroxycinnamate CoA ligase) activities and the increase in the level of transcription of the TaPAL and Ta4CL genes, were observed under waterlogging stress. Also, 6-BA improved the root growth system under waterlogging stress conditions. Further qPCR analysis revealed increased auxin signaling (TaPR1) in 6-BA-treated plants under waterlogging stress that was consistent with the induction of endogenous IAA hormone content under waterlogging stress conditions. Here, 6-BA also reduced yield loss, as compared to control plants. Thus, the obtained data suggested that, under the application of 6-BA, phenylpropanoid metabolism (i.e., lignin) was stimulated, playing a significant role in reducing the negative effects of waterlogging stress on yield, as evinced by the improved plant growth parameters.

Biocatalysis with In-Situ Product Removal Improves p-Coumaric Acid Production.

Natural and pure p-coumaric acid has valuable applications, and it can be produced via bioprocessing. However, fermentation processes have so far been unable to provide sufficient production metrics, while a biocatalytic process decoupling growth and production historically showed much promise. This biocatalytic process is revisited in order to tackle product inhibition of the key enzyme tyrosine ammonia lyase. In situ product removal is proposed as a possible solution, and a polymer/salt aqueous two-phase system is identified as a suitable system for extraction of p-coumaric acid from an alkaline solution, with a partition coefficient of up to 13. However, a 10 % salt solution was found to reduce tyrosine ammonia lyase activity by 19 %, leading to the need for a more dilute system. The cloud points of two aqueous two-phase systems at 40 °C and pH 10 were found to be 3.8 % salt and 9.5 % polymer, and a 5 % potassium phosphate and 12.5 % poly(ethylene glycol-ran-propylene glycol) mW~2500 system was selected for in situ product removal. An immobilized tyrosine ammonia lyase biocatalyst in this aqueous two-phase system produced up to 33 g/L p-coumaric acid within 24 hours, a 1.9-fold improvement compared to biocatalysis without in situ product removal.

Alginate Oligosaccharides Alleviate Salt Stress in Rice Seedlings by Regulating Cell Wall Metabolism to Maintain Cell Wall Structure and Improve Lodging Resistance.

Salt stress is one of the major abiotic stresses that damage the structure and composition of cell walls. Alginate oligosaccharides (AOS) have been advocated to significantly improve plant stress tolerance. The metabolic mechanism by which AOS induces salt tolerance in rice cell walls remains unclear. Here, we report the impact of AOS foliar application on the cell wall composition of rice seedlings using the salt-tolerant rice variety FL478 and the salt-sensitive variety IR29. Data revealed that salt stress decreased biomass, stem basal width, stem breaking strength, and lodging resistance; however, it increased cell wall thickness. In leaves, exogenous AOS up-regulated the expression level of OSCESA8, increased abscisic acid (ABA) and brassinosteroids (BR) content, and increased β-galacturonic activity, polygalacturonase activity, xylanase activity, laccase activity, biomass, and cellulose content. Moreover, AOS down-regulated the expression levels of OSMYB46 and OSIRX10 and decreased cell wall hemicellulose, pectin, and lignin content to maintain cell wall stability under salt stress. In stems, AOS increased phenylalamine ammonia-lyase and tyrosine ammonia-lyase activities, while decreasing cellulase, laccase, and β-glucanase activities. Furthermore, AOS improved the biomass and stem basal width and also enhanced the cellulose, pectin, and lignin content of the stem, As a result, increased resistance to stem breakage strength and alleviated salt stress-induced damage, thus enhancing the lodging resistance. Under salt stress, AOS regulates phytohormones and modifies cellulose, hemicellulose, lignin, and pectin metabolism to maintain cell wall structure and improve stem resistance to lodging. This study aims to alleviate salt stress damage to rice cell walls, enhance resistance to lodging, and improve salt tolerance in rice by exogenous application of AOS.

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.

Extraction and Isolation of Tyrosine Ammonia Lyase (TAL) and Diosgenin from <i>Persea americana</i> as Potent Inhibitors of Melanin Synthesis Involving <i>In Vivo</i> Study on Zebrafish Embryos

Tyrosine ammonia lyase is an enzyme in the natural phenol biosynthesis pathway. L-tyrosine is deaminated to p-Coumaric acid with the help of the enzyme tyrosine ammonia lyase. Tyrosine Ammonia Lyase (TAL) is extracted from the leaves of Persea americana. Diosgenin involved in the inhibition of intracellular tyrosinase is extracted along with the enzyme Tyrosine Ammonia Lyase from leaves of Persea americana, significantly increasing the activity of Tyrosine Ammonia Lyase. Diosgenin in the current study on analysis is highly increasing the activity of enzymes. The ability of the enzyme along with diosgenin to increase the activity of the enzyme is analysed in the current study. The enzyme along with diosgenin is used for depigmentation of skin. The methodology of protein separation on treatment with acetone was utilized for the extraction of the enzyme. p-Coumaric acid formation detected using a UV-Visible spectrophotometer at 380nm confirmed the presence of the enzyme. Defatting followed by microwave-assisted extraction and ultrasound-assisted extraction methodology were used for the extraction of diosgenin from leaves of Persea americana. The role of diosgenin in increasing the activity of the enzyme was carried out by analysing the enzyme activity at 410nm. The enzyme-activating ability of diosgenin was compared with that of magnesium sulphate-purified Tyrosine Ammonia Lyase enzyme and sodium sulphate-purified Tyrosine Ammonia Lyase enzyme. Zebrafish embryos were used as model organisms for the conduction of in vivo studies. kojic acid was used as a control in the in vivo studies to compare the effect of the enzyme extracted along with the diosgenin with that of kojic acid.

Harnessing the metabolic modulatory and antioxidant power of 1-(3-Phenyl-Propyl) cyclopropane and melatonin in maintaining mango fruit quality and prolongation storage life

BackgroundThe aim of this study was to compare and investigate the effects of 1-(3-phenyl-propyl) cyclopropene (PPCP) and melatonin (MT) as anti-ethylene agents on postharvest senescence, quality, chilling tolerance, and antioxidant metabolism in the mango fruit cv. “Keitt”. The study involved exposing the fruit to 20 μL L− 1 PPCP or 200 μM MT, in addition to a control group of untreated fruit, before storing them at 5 ± 1 °C for 28 d. The findings revealed that the treatments with PPCP and MT were effective in reducing chilling injury and preserving fruit quality when compared to the control group.ResultsThe use of 20 μL L− 1 PPCP was an effective treatment in terms of mitigating chilling injury and preserving fruit quality for 28 d. This was attributed to the decrease in metabolic activity, specifically the respiration rate and the production of ethylene, which led to the maintenance of fruit firmness and bioactive compounds, energy metabolism, and antioxidant activity, such as ascorbic acid, total flavonoids, trolox equivalent antioxidant capacity, dehydroascorbate reductase, glutathione reductase activity, ATP, and ATPase activity. The study also found that the MT treatment at 200 μM was effective in reducing chilling injury and weight loss and improving membrane stability. Additionally, it led to a decrease in malondialdehyde content and electrolyte leakage, and the maintenance of fruit quality in terms of firmness, peel and pulp colour values for mango peel and pulp total carotenoid content, as well as phenylalanine ammonia lyase and tyrosine ammonia lyase activity. These findings indicate that PPCP and MT have the potential to be efficient treatments in maintaining mango quality and minimizing post-harvest losses.ConclusionThe utilisation of treatments with 20 μL L− 1 of PPCP or 200 μM MT was found to effectively preserve the postharvest quality parameters, in terms of bioactive compounds, energy metabolism, and antioxidant activity, of mangoes cv. “Keitt” that were stored at 5 ± 1 °C for 28 d.

Putrescin modulates metabolic and physiological characteristics of Thymus daenensis under drought stress

This experiment was conducted to understand the protective impact of exogenous putrescine (Put) in Thymus daenensis under drought stress. Plants of T. daenensis were subjected to drought stress (15% polyethylene glycol) and Put (0.2 mM). Drought stress lowered growth attributes and photosynthetic pigments in T. daenensis plants. The increase in the content of malondialdehyde (MDA) and hydrogen peroxide (H2O2) as oxidative stress indicators showed that drought stress caused oxidative stress in T. daenensis plants. Drought stress augmented proline content, phenylalanine ammonia-lyase activity, tyrosine ammonia-lyase activity, indole-3-acetic acid content and methyl jasmonate content in T. daenensis plants. Put treatment improved growth and regulated adaptative mechanisms in T. daenensis plants. Decrease in H2O2 and lipid peroxidation (MDA content) by Put displayed that Put helped to alleviate drought-trigged oxidative stress in T. daenensis plants. Put application strengthened the antioxidant system as a resistance response in the drought-treated T. daenensis plants thereby induction in enzymes activities (superoxide dismutase, catalase and peroxidase) and non-enzymatic antioxidants (total phenol and flavonoid). Put treatment increased coumaric acid and resveratrol content in stressed plants. These results propose a multifaceted contribution of Put on adaptative mechanisms in T. daenensis plants under drought stress.

Components of the Phenylpropanoid Pathway in the Implementation of the Protective Effect of Sodium Nitroprusside on Wheat under Salinity.

Nitric oxide (NO) is a multifunctional, gaseous signaling molecule implicated in both physiological and protective responses to biotic and abiotic stresses, including salinity. In this work, we studied the effects of 200 µM exogenous sodium nitroprusside (SNP, a donor of NO) on the components of the phenylpropanoid pathway, such as lignin and salicylic acid (SA), and its relationship with wheat seedling growth under normal and salinity (2% NaCl) conditions. It was established that exogenous SNP contributed to the accumulation of endogenous SA and increased the level of transcription of the pathogenesis-related protein 1 (PR1) gene. It was found that endogenous SA played an important role in the growth-stimulating effect of SNP, as evidenced by the growth parameters. In addition, under the influence of SNP, the activation of phenylalanine ammonia lyase (PAL), tyrosine ammonia lyase (TAL), and peroxidase (POD), an increase in the level of transcription of the TaPAL and TaPRX genes, and the acceleration of lignin accumulation in the cell walls of roots were revealed. Such an increase in the barrier properties of the cell walls during the period of preadaptation played an important role in protection against salinity stress. Salinity led to significant SA accumulation and lignin deposition in the roots, strong activation of TAL, PAL, and POD, and suppression of seedling growth. Pretreatment with SNP under salinity conditions resulted in additional lignification of the root cell walls, decreased stress-induced endogenous SA generation, and lower PAL, TAL, and POD activities in comparison to untreated stressed plants. Thus, the obtained data suggested that during pretreatment with SNP, phenylpropanoid metabolism was activated (i.e., lignin and SA), which contributed to reducing the negative effects of salinity stress, as evidenced by the improved plant growth parameters.

Benzylaminopurine and Abscisic Acid Mitigates Cadmium and Copper Toxicity by Boosting Plant Growth, Antioxidant Capacity, Reducing Metal Accumulation and Translocation in Bamboo [Pleioblastus pygmaeus (Miq.)] Plants.

An in vitro experiment was conducted to determine the influence of phytohormones on the enhancement of bamboo resistance to heavy metal exposure (Cd and Cu). To this end, one-year-old bamboo plants (Pleioblastus pygmaeus (Miq.) Nakai.) contaminated by 100 µM Cd and 100 µM Cu both individually and in combination were treated with 10 µM, 6-benzylaminopurine and 10 µM abscisic acid. The results revealed that while 100 µM Cd and 100 µM Cu accelerated plant cell death and decreased plant growth and development, 10 µM 6-benzylaminopurine and 10 µM abscisic acid, both individually and in combination, increased plant growth by boosting antioxidant activities, non-antioxidants indices, tyrosine ammonia-lyase activity (TAL), as well as phenylalanine ammonia-lyase activity (PAL). Moreover, this combination enhanced protein thiol, total thiol, non-protein, glycine betaine (GB), the content of proline (Pro), glutathione (GSH), photosynthetic pigments (Chlorophyll and Carotenoids), fluorescence parameters, dry weight in shoot and root, as well as length of the shoot. It was then concluded that 6-benzyl amino purine and abscisic acid, both individually and in combination, enhanced plant tolerance under Cd and Cu through several key mechanisms, including increased antioxidant activity, improved photosynthesis properties, and decreased metals accumulation and metal translocation from root to shoot.

Molecular characterization of the Bambusa oldhamii BoPAL3–encoded phenylalanine ammonia-lyase

• BoPAL3 gene was cloned from Bambusa oldhamii and heterologously expressed in Escherichia coli . • Recombinant BoPAL3 protein is an active enzyme, exhibiting obvious PAL activity and minuscule TAL activity. • BoPAL3 had similar biochemical properties with BoPAL2. Phenylalanine ammonia-lyase (PAL), which is ubiquitous in plants, catalyzes the formation of trans -cinnamic acid via the nonoxidative deamination of l -phenylalanine. Bambusa oldhamii contains four different forms of PAL proteins that differ in substrate specificity. Full-length BoPAL3 cDNA was cloned by a combination bamboo cDNA library screening and PCR-based cloning methods. Sequence alignment showed high homology between the deduced amino acid sequences of the BoPAL2 and BoPAL3 proteins (90%). Obvious PAL and tyrosine ammonia-lyase (TAL) activities were detected in Escherichia coli Top10 expressing recombinant BoPAL3 protein. Size-exclusion chromatography and denatured SDS–PAGE showed that the estimated molecular mass of recombinant BoPAL3 and the subunit form were approximately 330 kDa and 80 kDa, indicating that BoPAL3 presents as a tetrameric protein. The optimum temperature and pH for BoPAL3 activity were 50 °C and 8.5, respectively. The K m value of BoPAL3 for l -phenylalanine was 200 μM.

Zinc Oxide Nanoparticles Improve Pleioblastus pygmaeus Plant Tolerance to Arsenic and Mercury by Stimulating Antioxidant Defense and Reducing the Metal Accumulation and Translocation.

The utilization of nanoparticles to potentially reduce toxicity from metals/metalloids in plants has increased in recent years, which can help them to achieve tolerance under the stressful conditions. An in vitro experiment was conducted to investigate five different levels of zinc oxide nanoparticles (ZnO-NPs; 0, 50, 100, 150, and 200 μM) both alone and in combination with 150 μM arsenic (As) and 150 μM mercury (Hg) in one-year-old Pleioblastus pygmaeus (Miq.) Nakai plants through four replications. The results demonstrated that As and Hg alone had damaging effects on the plant growth and development. However, the addition of various concentrations of ZnO-NPs led to increased antioxidant activity, proline (79%) content, glycine betaine (71%) content, tyrosine ammonia-lyase (43%) activity, phenylalanine ammonia-lyase (69%) activity, chlorophyll indices, and eventually plant biomass, while the lipoxygenase activity, electrolyte leakage, soluble protein, hydrogen peroxide content, and thiobarbituric acid reactive substances were reduced. We concluded that ZnO-NPs detoxified As and Hg toxicity in the plants through increasing antioxidant activity, reducing As and Hg accumulation, As and Hg translocation from roots to shoots, and adjusting stomatal closure. This detoxification was further confirmed by the reduction of the translocation factor of As and Hg and the enhancement of the tolerance index in combination with ZnO-NPs. However, there is a need for further investigation with different metals/metalloids.

An Exogenous Pre-Storage Melatonin Alleviates Chilling Injury in Some Mango Fruit Cultivars, by Acting on the Enzymatic and Non-Enzymatic Antioxidant System.

Melatonin (MT) treatment (100 µM, 2 h) was applied to four mango fruit cultivars (‘Langra’, ‘Chaunsa’, ‘Dashehari’, and ‘Gulab Jamun’), before being stored at 5 ± 1 °C for 28 d, in order to alleviate chilling injury (CI). Maximum CI reduction was observed in ‘Langra’ mangoes, and minimum in ‘Gulab Jamun’ mangoes. This positive effect on quality preservation was associated with an increased concentration of endogenous MT, which prevented the accumulation of reactive oxygen species (H2O2 and O2·−) and stimulated non-enzymatic antioxidants (total phenolic compounds and total flavonoids), possibly due to higher activity of phenylalanine ammonia lyase and tyrosine ammonia lyase. Increased antioxidant activity was also documented in MT-treated ‘Langra’ mangoes, according to four different assays (DPPH, TEAC, FRAP, and CUPRAC) and higher activity of six antioxidant enzymes (superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, glutathione reductase, and dehydroascorbate reductase). In contrast, ‘Gulab Jamun’ mangoes showed minimal or no positive effects on the aforementioned variables in response to the exogenous MT application. ‘Chaunsa’ and ‘Dashehari’ mangoes had some intermediate effects on their antioxidant system (enzymatic and non-enzymatic) and alleviation of CI, when treated with exogenous MT. We conclude that exogenous MT exerts a cultivar-dependent stimulating effect on the antioxidant system of mangoes, which results in an increase in the fruits’ resistance to low temperature.

Seasonal changes of physiological and biochemical characteristics of Garcinia brasiliensis leaves

The present study aimed to determine seasonal influences on the physiological characteristics of G. brasiliensis that are associated with the production of secondary metabolites, as well as the yield and composition of leaf essential oil (EO). We evaluated leaves of six-year-old plants at four cardinal points in the middle third of a canopy with four replicates during May, August, November, and February. There were assessed photosynthesis rate, total phenolic and total flavonoid contents, phenylalanine (PAL) and tyrosine (TAL) ammonia-lyase activities and the leaves were histochemically stained. We quantified isolated phenolic compounds from leaf methanolic and hexanic extracts. The EO yield and composition, and the numbers and diameters of oil secretory channels in leaves were also evaluated. The photosynthetic rate peaked during November, whereas the total phenolic content and PAL and TAL activities peaked in February. Phenolic compounds were dispersed in the central rib and limbus in idioblasts. Only morelloflavone (fukugetin) was detected in methanolic extracts, and the concentration in leaves peaked during November. The yield and composition of the EO varied with the seasons, and the EO yield was maximal during November. Sesquiterpenes were the main components in the leaf EO. Our findings indicate that spring is the most suitable season for obtaining morelloflavone and a higher yield of EO from G. brasiliensis leaves in tropical regions.

Effect of Cinnamic acid on the activity of phenylalanine ammonialyase (PAL) and tyrosine ammonialyase (TAL) enzymes and some physiological characteristics of tobacco plant (Nicotiana rustica L.) under salinity stress in vitro calture

هدف: هدف از این مطالعه بررسی اثر سینامیک‌اسید (CA) بر برخی خصوصیات فیزیولوژیکی و میزان تحمل‌به‌شوری گیاه‌تنباکو (Nicotiana rustica L.) می‌باشد.مواد و روش‌ها: در این مطالعه قطعات جداکشت گیاه تنباکو رشد‌یافته در محیط MS به محیط‌های کشت MS حاوی سینامیک‌اسید (غلظت‌های 0، 5، 10 و 20 میلی‌گرم‌در‌لیتر) و نمک NaCl (غلظت‌های 0، 100 و 200 میلی‌مولار) منتقل‌گردید. پس از چهار هفته میزان فعالیت آنزیم فنیل‌آلانین‌آمونیالیاز (PAL) و تیروزین‌آمونیالیاز (TAL) ، میزان ترکیبات‌فنلی‌کل و پروتئین‌کل، میزان تجمع فلاونوئید، آنتوسیانین، ظرفیت‌آنتی‌اکسیدانی‌کل، هیدروژن‌پراکسید (H2O2)، میزان پتاسیم، سدیم و پرولین اندازه گیری شد..نتایج: نتایج نشان‌داد در اثر تیمار گیاهان با CA میزان فعالیت آنزیم PAL (غلظت‌های 5و10 میلی‌گرم‌در‌لیتر CA به ترتیب 25 و 27 درصد افزایش در میزان فعالیت این آنزیم نسبت به گیاهان شوری 100 میلی‌مولار بدون CA نشان دادند) و TAL (در شوری 200 میلی‌مولار، غلظت 5 میلی‌گرم‌در‌لیتر CA باعث افزایش 20درصدی میزان فعالیت آنزیم‌TAL نسبت به گیاهان شوری 200 میلی‌مولار بدون CA شده‌اند)، میزان ترکیبات‌فنلی‌کل و پروتئین‌کل نیز نسبت به گیاهان شاهد افزایش‌یافت. تیمار CA باعث افزایش میزان تجمع فلاونوئید، آنتوسیانین و پرولین و نیز میزان پتاسیم و ظرفیت‌آنتی‌اکسیدانی‌کل نسبت به گیاهان شاهد گردید. اما میزان سدیم و H2O2 در نتیجه تیمار CA در مقایسه با گیاهان شاهد کاهش‌یافت.نتیجه‌گیری: می‌توان احتمال داد که CA با افزایش میزان ترکیبات‌فنلی‌کل و نیز پرولین در شرایط تنش‌شوری موجب افزایش تحمل به شوری گیاه تنباکو می شود.

Enzymatic and biochemical alterations in relation to lignin deposition at different growth stages of tall fescue

AbstractLignin is a phenolic biopolymer that limits the intake potential and energy availability of forage crops. In order to understand the lignification process, enzymatic and biochemical studies were conducted at four different growth stages of tall fescue (Festuca arundinacea Schreb.). An increase in phenylalanine ammonia lyase and tyrosine ammonia lyase activity was observed with advancement of growth from first node palpable stage to third node palpable stage with a decrease at spikelet emergence stage. Cinnamoyl‐CoA reductase and peroxidase exhibited a continuous significant (p < .05) increase and positive correlation with lignin concentration. Cinnamyl alcohol dehydrogenase exhibited a significant (p < .05) positive correlation with lignin concentration, suggesting its probable exclusive role in tall fescue lignification. Cellulose and total phenol exhibited a 13.0 and 71.8% increase, whereas pectin and crude protein exhibited a significant (p < .05, p = .0001) decrease of 65.1 and 15.4%, respectively, with the growth advancement from first node palpable to spikelet emergence stage. Klason lignin also exhibited a dramatic 9.4‐fold increase from vegetative to reproductive stage. The enzymatic and biochemical alterations were closely associated with the lignification process. This study provides an integrated picture of the enzymatic and biochemical alterations that accompany changes in lignin deposition in tall fescue.

Induction of Defense-Related Enzymes and Pathogenesis-Related Proteins Imparts Resistance to Barley Genotypes Against Spot Blotch Disease

Spot blotch caused by Bipolaris sorokiniana is a major foliar disease of barley affecting its productivity worldwide. The barley cultivar with complete resistance to this pathogen is not yet identified. In the present study, leaves of six barley genotypes namely PL 807, BH 946, DWRUB 52, BL 1338, BL 1369 and PL 426 inoculated with Bipolaris sorokiniana were evaluated for levels of H2O2, malondialdehyde, antioxidative enzymes, non-enzymatic antioxidants, pathogenesis-related (PR) proteins and β-glucan. The disease score was assessed at flag leaf (F) and one leaf lower to the flag leaf (F1) at two growth stages, i.e. GS 55 and GS 87. The genotype BL 1369 showed moderately resistant nature, PL 807, DWRUB 52 and BL 1338 moderately susceptible whereas other genotypes remained susceptible in response to this pathogen. During GS 87 stage, maximum increase of catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), phenylalanine ammonia lyase (PAL), tyrosine ammonia lyase (TAL) and polyphenol oxidase (PPO) activities along with concomitant decrease of H2O2 content in the infected F1 leaves of BL 1369 may contribute towards its moderately resistant character. However, downregulation of APX and GR enzymes in PL 426, BH 946 and DWRUB 52 may be responsible for their sensitivity towards this pathogen. Although on fungal inoculation, chitinase activity increased in all the genotypes except DWRUB 52 and BL 1338 but maximum increase was reported in BL 1369. β-glucanase activity also increased significantly in both the leaf tissues of BL 1369. It showed positive correlation with chitinase in the flag leaf at 1% level of significance. The β-glucan content decreased in DWRUB 52 and PL 426 upon fungal infection while it increased in BL 1338 and BL 1369 which further pointed out towards resistant character of BL 1369 as the cell wall is a physical barrier which constitutes the first line defence against pathogen attack.

Effect of Elicitation on Polyphenol Metabolism and Antioxidant Properties of Broccoli and Knolkhol Sprouts

The present study evaluated the effect of different elicitors (salicylic acid, methyl salicylate and neem leaf extract) along with precursor feeding on phenolic content and antioxidant activity of broccoli and knolkhol sprouts. The results of the study revealed that elicitation increased the PAL and TAL enzyme activity which resulted in the enhancement of antioxidant and phenolic content of broccoli and knolkhol sprouts. Salicylic acid (200μM) plus L-phenylalanine treatment was found to be optimum as it showed the highest increase of phenolics, flavonoids, condensed tannin content and antioxidant activity for both broccoli and knolkhol sprouts. The optimum treatment also enhanced the phenylalanine ammonia-lyase (PAL), tyrosine ammonia-lyase (TAL), and peroxidase (POD) activity in broccoli sprouts by the 1.45-fold, 1.61-fold, and 1.51-fold, respectively and in knolkhol sprouts, the increase was 2.25-fold, 0.68-fold and 1.44-fold, respectively. Based on the findings of the present research, elicitation supported by precursor feeding appeared to be a easy and cheap method for increasing the bioactive components and antioxidant potential of broccoli and knolkhol sprouts.

Directed evolution of tyrosine ammonia-lyase to improve the production of p-coumaric acid in Escherichia coli

p-coumaric acid is an important natural phenolic compound with a variety of pharmacological activities, and also a precursor for the biosynthesis of many natural compounds. It is widely used in foods, cosmetics and medicines. Compared with the chemical synthesis and plant extraction, microbial production of p-coumaric acid has many advantages, such as energy saving and emission reduction. However, the yield of p-coumaric acid by microbial synthesis is too low to meet the requirements of large-scale industrial production. Here, to further improve p-coumaric acid production, the directed evolution of tyrosine ammonia lyase (TAL) encoded by Rhodotorula glutinis tal gene was conducted, and a high-throughput screening method was established to screen the mutant library for improve the property of TAL. A mutant with a doubled TAL catalytic activity was screened from about 10,000 colonies of the mutant library. There were three mutational amino acid sites in this TAL, namely S9Y, A11N, and E518A. It was further verified by a single point saturation mutation. When S9 was mutated to Y, I or N, or A11 was mutated to N, T or Y, the catalytic activity of TAL increased by more than 1-fold. Through combinatorial mutation of three types of mutations at the S9 and A11, the TAL catalytic activity of S9Y/A11N or S9N/A11Y mutants were significantly higher than that of other mutants. Then, the plasmid containing S9N/A11Y mutant was transformed into CP032, a tyrosine-producing E. coli strain. The engineered strain produced 394.2 mg/L p-coumaric acid, which is 2.2-fold higher than that of the control strain, via shake flask fermentation at 48 h. This work provides a new insight for the biosynthesis study of p-coumaric acid.

Fluorescent enzyme-coupled activity assay for phenylalanine ammonia-lyases

Phenylalanine ammonia-lyases (PALs) catalyse the non-oxidative deamination of l-phenylalanine to trans-cinnamic acid, while in the presence of high ammonia concentration the reverse reaction occurs. PALs have been intensively studied, however, their industrial applications for amino acids synthesis remained limited, mainly due to their decreased operational stability or limited substrate specificity. The application of extensive directed evolution procedures to improve their stability, activity or selectivity, is hindered by the lack of reliable activity assays allowing facile screening of PAL-activity within large-sized mutant libraries. Herein, we describe the development of an enzyme-coupled fluorescent assay applicable for PAL-activity screens at whole cell level, involving decarboxylation of trans-cinnamic acid (the product of the PAL reaction) by ferulic acid decarboxylase (FDC1) and a photochemical reaction of the produced styrene with a diaryltetrazole, that generates a detectable, fluorescent pyrazoline product. The general applicability of the fluorescent assay for PALs of different origin, as well as its versatility for the detection of tyrosine ammonia-lyase (TAL) activity have been also demonstrated. Accordingly, the developed procedure provides a facile tool for the efficient activity screens of large mutant libraries of PALs in presence of non-natural substrates of interest, being essential for the substrate-specificity modifications/tailoring of PALs through directed evolution-based protein engineering.

Effects of cobalt oxide nanoparticles (Co3O4 NPs) on ion leakage, total phenol, antioxidant enzymes activities and cobalt accumulation in Brassica napus L.

Interaction of nanoparticles (NPs) as a significant threat to ecosystems with biological processes of plants is very important. Here, the effects of cobalt oxide (Co3O4) NPs on some physio-biochemical characteristics of Brassica napus L. were investigated. The two-weeks seedlings were sprayed with different concentrations of Co3O4 NPs (0, 50, 100, 250, 500, 1000, 2000, and 4000 mg L-1). The results showed that this treatment significantly affected the fresh and dry weights, area, relative water content (RWC) and relative chlorophyll value (SPAD) of leaves. The highest reduction of growth and biomass indexes occurred at 4000 mg L-1 NPs. The content of H2O2 and electrolyte leakage (EL) increased respectively, after 100 and 250 mg L-1 of Co3O4 NPs and showed a maximum level at 4000 mg L-1. The activities of phenylalanine ammonia lyase (PAL), ascorbate peroxidase (APX) and superoxide dismutase (SOD) increased after 100 mg L-1 of Co3O4 NPs. However, tyrosine ammonia lyase (TAL) activity enhanced after 500 mg L-1. The catalase (CAT) activity and protein content decreased after 1000 mg L-1 of Co3O4 NPs. Application of concentrations higher than 500 mg L-1 of Co3O4 NPs induced polyphenol oxidase (PPO) activity but reduced glutathione reductase (GR). The activities of guaiacol peroxidase (GPX) and glutathione S-transferase (GST) increased at 250-1000 mg L-1 of Co3O4 NPs and then decreased. These results suggested that low concentrations of Co3O4 NPs induced a positive effect on growth parameters but high levels caused extensive oxidative damage and mediated defense responses by organization of phenolic compounds and antioxidative system.