Cis- and Trans-Cinnamic Acids Have Different Effects on the Catalytic Properties of Arabidopsis Phenylalanine Ammonia Lyases PAL1, PAL2, and PAL4

Abstract: Cis-cinnamic acid (CA) is a naturally occurring compound, presumably converted from trans-CA in higher plants. To investigate the effect of cis-CA on the activity of Arabidopsis phenylalanine ammonia lyase (PAL), AtPAL1, AtPAL2, and AtPAL4 genes were isolated using reverse transcription poly-merase chain reaction. These genes were fused to a glutathione S-transferase gene and overexpressed in a heterologous prokaryotic system of Escherichia coli. The purified PAL1, PAL2 and PAL4 enzymes were characterized biochemically to determine the effects of cis-CA on the kinetic parameter Km. The results showed that cis-CA is a competitive inhibitor for PAL1, but not PAL2 and PAL4, whereas trans-CA acts as a competitive inhibitor for all three PAL isomers, suggesting that cis- and trans-CA have different effects on the catalytic activity of PAL.

Activities of lipoxygenase and phenylalanine ammonia lyase in poplar leaves induced by insect herbivory and volatiles

A study was conducted to explore the defense response in woody plants after insect herbivory. The activities of two enzymes, lipoxygenase （LOX）, a key enzyme ofjasmonate （JA） pathway, and phenylalanine ammonia lyase （PAL）, a rate-limiting enzyme of phenyl- propanoid pathway, were measured in the leaves of one-year-old poplar （Populus simonii × P. pyramidalis ＇Opera 8277＇） cuttings after Clostera anachoreta larvae attack. The results show that the increased activities of LOX and PAL were found not only in the leaves wounded by C. anachoreta larvae but also in their tipper systemic leaves, indicating that JA and phenylpropanoid pathways were activated, and the defense response was stimulated systemically. The increase in LOX and PAL activities in neighboring intact poplar cuttings sug- gested that there exists the interplant communication between poplar plants mediated by the herbivore-induced volatiles. Methyl jasmonate （MeJA） was also proved to be an airborne signal to induce defense response in P simonii × P pyramidalis ＇Opera 8277＇ cuttings.

Investigating the mechanisms of isochorismate synthase:An approach to improve salicylic acid synthesis and increase resistance to Fusarium head blight in wheat

Salicylic acid(SA),a vital endogenous hormone,plays a crucial role in plant growth and the response to abiotic and biotic stress.Isochorismate synthase(ICS)and phenylalanine ammonia lyase(PAL)are critical rate-limiting enzymes for SA synthesis.Fusarium head blight(FHB)seriously threatens the safety of wheat production,but increasing the content of SA can enhance FHB resistance.However,the pathway of SA synthesis and regulation in wheat remains unknown.In this study,three wheat ICS(TaICSA,TaICSB,and TaICSD)were identified,and their functions were validated in vitro for isomerizing chorismate to isochorismate.The mutation of one or two homoeoalleles of TaICSA,TaICSB,and TaICSD in the wheat variety‘Cadenza’reduced SA levels under ultraviolet treatment and Fusarium graminearum infection,further enhancing sensitivity to FHB.Overexpression of TaICSA can significantly enhance SA levels and resistance to FHB.To further study SA synthesis pathways in wheat and avoid interference with pathogenicity related genes,the leaves of wild-type Cadenza and different TaICS mutant lines were subjected to ultraviolet treatment for transcriptomic analysis.The results showed that 37 PALs might be involved in endogenous SA synthesis,and 82 WRKY and MYB family transcription factors may regulate the expression of ICS and PAL.These results were further confirmed by RT-PCR.In conclusion,this study expands our knowledge of SA biosynthesis and identifies TaICSA,as well as several additional candidate genes that encode transcription factors for regulating endogenous SA levels,as part of an efficient strategy for enhancing FHB resistance in wheat.

Enhancement of Phenylalanine Ammonia Lyase,Polyphenoloxidase,and Peroxidase in Cucumber Seedlings by Bemisia tabaci (Gennadius) (Hemiptera:Aleyrodidae) Infestation

In this study, the activities of phenylalanine ammonia lyase （PAL）, polyphenoloxidase （PPO）, and peroxidase （POD） were assayed in cucumber seedlings （Cucumis sativus L.） at 0, 6, 12, 24, 48, 72, and 96 h after they were infested by Bemisia tabaci （Gennadius） using spectrophotometric analysis. The results indicated that herbivore infestation increased the activities of PAL, PPO, and POD. The enzymes showed different activity levels at different times after the infestation. The PAL activity reached the first high peak by 23.1% at 6 h and the highest peak by 29.1% at 48 h compared to the control. The PPO activity reached the first high peak by 22.7% at 6 h and the highest peak by 52.6% at 24 h, and the POD activity reached the highest peak by 213.2% at 6 h and another higher peak value by 135.2% at 96 h. The results suggest that the enhanced activities of the enzymes may contribute to bioprotection of cucumber plants against B. tabaci infestation.

Cloning, Expression and Analysis of pal, a Positive Gene Involved in Capsaicin Biosynthesis in Capsicum chinense Jacq

The cDNA sequence of Capal gene was cloned from Capsicum chinense Jacq by RT-PCR and sequenced. Bioinformatics analysis showed that Capal en- codes a putative polypeptide of 683 amino acids with a calculated molecular mass of 74.2 kD and a theoretical pl of 6.9. Multiple sequence alignments and phyloge- netic tree analyses showed that Capal protein of C. chinense is similar to that of Capsicum annuum var. conoides, with an overall sequence similarity of 96%. The prokaryotic expression vector pET-32a-pal was constructed and induced to express in E. coil BL21. The SDS-PAGE analysis showed that the relative molecular mass of the induced new protein is about 74 kD, which was basically identical with that predicted by DNAMAN software （74.3 kD）, Real-time PCR analysis showed that ex- ogenous jasmonic acid （JA） promoted pal expression. The accumulation of capsaicin in pepper was analyzed by high performance liquid chromatography （HPLC）, and the results indicated that exogenous jasmonic acid （JA） can promote the synthesis of capsaicin. This study will provide valuable experimental basis for the research of transcription regulation and explaining the gene function of pal.

Morphometric and Biochemical Changes in Agave americana L.Plantlets Induced By Ethyl Methanesulfonate

A.americana L.is a crop with very little genetic variability.In order to evaluate the effect of ethyl methanesulfonate(EMS)to induce variability in in vitro plantlets of A.americana,different explants(meristems,leaves and roots)were evaluated for the production of callus.MS medium supplemented with ANA(2.68μM)and BAP(2.68μM)was used.Callus obtained from apical meristem were treated with 15 mM EMS for two hours after which shoot formation was induced using 2,4-D(0.11μM)and BAP(44μM).The EMS induced variations in the morphometric and morphological parameters of the plantlets obtained,with 60%of the plantlets presenting differences such as dwarfism and different leaf forms,without the presence of spines,as well as an increase in fructan content of 30%with respect to the control plantlets.PAL was increased and this activity is related with higher anthocyanins concentration in A.americana L.plantlets.

Expression of phenylalanine ammonia lyase as an intracellularly free and extracellularly cell surface-immobilized enzyme on a gut microbe as a live biotherapeutic for phenylketonuria

Phenylketonuria(PKU),a disease resulting in the disability to degrade phenylalanine(Phe)is an inborn error with a 1 in 10,000 morbidity rate on average around the world which leads to neurotoxicity.As an potential alternative to a protein-restricted diet,oral intake of engineered probiotics degrading Phe inside the body is a promising treatment,currently at clinical stage II(Isabella,et al.,2018).However,limited transmembrane transport of Phe is a bottleneck to further improvement of the probiotic’s activity.Here,we achieved simultaneous degradation of Phe both intracellularly and extracellularly by expressing genes encoding the Phe-metabolizing enzyme phenylalanine ammonia lyase(PAL)as an intracellularly free and a cell surface-immobilized enzyme in Escherichia coli Nissle 1917(EcN)which overcomes the transportation problem.The metabolic engineering strategy was also combined with strengthening of Phe transportation,transportation of PAL-catalyzed trans-cinnamic acid and fixation of released ammonia.Administration of our final synthetic strain TYS8500 with PAL both displayed on the cell surface and expressed inside the cell to the Pah^(F263S)PKU mouse model reduced blood Phe concentration by 44.4%compared to the control Ec N,independent of dietary protein intake.TYS8500 shows great potential in future applications for PKU therapy.

Alteration in Biochemical Response in Sesamum indicum upon Different Plant-Pathogen Interactions

Sesamum, an important oil yielding crop suffers a huge loss in its yield due to attack of large number of fungal pathogens. In semi-arid regions Sesamum is mainly affected by two major plant-pathogenic fungus viz. Macrophomina phaseolina and Fusarium oxysporum. The aim of the study was to analyze the metabolic alterations in Sesamum after infection with both pathogens. This accomplished by individually by （the word estimating is not quantitative） the levels of total phenolic compounds and the activities of phenylalanine ammonia lyase （PAL） of one week old plants. The PAL showed high activity in infected plants, revealing the active phase in the synthesis of secondary metabolites in the Sesamum plant after infection. As a consequence, in infected plants the contents of polyphenols along with salicylic acid （SA） considerably exceeded when compared to control plants. This in vivo study of M. phaseolina and F. oxysporum infection reveals the differences of resistance levels in sesame against these two pathogens. These results provide important information regarding the plant-pathogen interactions and also forfor Sesamum improvement programs seeking the adaptation to diverse range of fungal attack along with adverse environmental factors.

Effects of Temperature on Seed Germination and Metabolism of Scutellaria baicalensis Georgi

[Objectives]To explore the effects of temperature on the primary and secondary metabolism of Scutellaria baicalensis Georgi during the seed germination.[Methods]The superoxide dismutase(SOD)activity was determined using riboflavin-NBT;peroxidase(POD)activity was determined using guaiacol colorimetric method,catalase(CAT),ascorbate peroxidase(APX),phenylalanine ammonia lyase(PAL)and cinnamic acid-4-hydroxylase(C4H)activity were detected by ultraviolet spectrophotometry,and chalcone synthase(CHS)activity and the content of secondary metabolites were measured by high performance liquid chromatography(HPLC).[Results]The germination rate,germination potential and germination index of S.baicalensis seeds were significantly affected by temperature.The most suitable temperature for the germination of S.baicalensis seeds was 25℃.The activities of SOD,POD and CAT in S.baicalensis seeds treated at low and high temperature were higher than that treated at suitable temperature;the activities of PAL,C4H and CHS of S.baicalensis seeds treated at low and high temperature were lower than that treated at suitable temperature.There was a good positive correlation between flavonoids and soluble sugar,PAL activity and C4H activity,and the correlation coefficients were R=0.894*,R=0.956*and R=0.951*,respectively.[Conclusions]In adverse environment,S.baicalensis seeds have good defense capabilities.During the germination of seeds,the formation of secondary metabolites is significantly correlated to the activity of key enzymes.Therefore,high-quality medicinal materials can be obtained by taking measures to improve the activity of key enzymes.

Effect of selenium application on phenylalanine ammonia-lyase(PAL)activity,phenol leakage and total phenolic content in garlic(Allium sativum L.)under NaCl stress

It is well known that salinity has badly effect on plant growth all over the world and greatly reduces crop production in the affected regions.Selenium can function as an antioxidant in plants and also in low concentration can promotes plant growth and produce tolerance against stress.This study was conducted in order to determine the effects of selenium(Se)application(0,4,8 and 16 mg L^-1)on phenylalanine ammonia-lyase(PAL)activity,phenol leakage and total phenolic content of garlic under salt stress(0,30,60 and 90 mM NaCl).The highest PAL activity was recorded at 60 and 90 mM NaCl salinity with application of 8mg Se L^-1.Also,when Se was added to the salt-stress garlic,the level of phenol leakage was decreased significantly at two levels of NaCl concentration(by 52%and 40%at 30 mM NaCl with application of 4 and 16 mg Se L^-1,and by 50%at 90 mM NaCl with application of 4mg Se L^-1,respectively)in comparison to the salt-stressed garlic without Se.The results showed that Se can increase the salt tolerance of garlic by protecting the cell membrane against lipid peroxidation.The highest concentration of phenols was recorded at 90 mM NaCl salinity level with application of 4 and 8 mg Se L^-1,that respectively produced 59%and 51%higher phenols than control treatment without Se.So,application of optimal Se level can increase the potential of garlic in a medium with relatively high level of NaCl.

Engineering of triterpene metabolism and overexpression of the lignin biosynthesis gene PAL promotes ginsenoside Rg3 accumulation in ginseng plant chassis

The ginsenoside Rgfound in Panax species has extensive pharmacological properties,in particular anti-cancer effects.However,its natural yield in Panax plants is limited.Here,we report a multimodular strategy to improve yields of Rgin a Panax ginseng chassis,combining engineering of triterpene metabolism and overexpression of a lignin biosynthesis gene,phenylalanine ammonia lyase(PAL).We first performed semi-rational design and site mutagenesis to improve the enzymatic efficiency of Pq3-O-UGT2,a glycosyltransferase that directly catalyzes the biosynthesis of Rgfrom Rh.Next,we used clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)gene editing to knock down the branch pathway of protopanaxatriol-type ginsenoside biosynthesis to enhance the metabolic flux of the protopanaxadiol-type ginsenoside Rg.Overexpression of PAL accelerated the formation of the xylem structure,significantly improving ginsenoside Rgaccumulation(to 6.19-fold higher than in thecontrol).Wecombinedoverexpression of the ginsenoside aglycon synthetic genes squalene epoxidase,Pq3-O-UGT2,and PAL with CRISPR/Cas9-based knockdown of CYP716A53v2 to improve ginsenoside Rgaccumulation.Finally,we produced ginsenoside Rgat a yield of 83.6 mg/L in a shake flask(7.0 mg/g dry weight,21.12-fold higher than with wild-type cultures).The highproduction system established in this study could be a potential platform to produce the ginsenoside Rgcommercially for pharmaceutical use.