Extraction, Purification, and Characterization of Olive (Olea europaea L., cv. Chemlal) Polyphenol Oxidase.

Among fruits susceptible to enzymatic browning, olive polyphenol oxidase (OePPO) stood out as being unisolated from a natural source until this study, wherein we successfully purified and characterized the enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of heated and nonheated OePPO revealed distinct molecular weights of 35 and 54 kDa, respectively, indicative of its oligomeric nature comprising active and C-terminal subunits. OePPO displayed latency, fully activating with 5 mM SDS under optimal conditions of pH 7.5 and 15 °C. The enzyme demonstrated monophenolase activity and showcased the highest efficiency toward hydroxytyrosol. Despite its low optimal temperature, OePPO exhibited high thermal resistance, maintaining stability up to 90 °C. However, beyond this threshold, the oligomeric enzyme disassociated, yielding a denatured main subunit and C-terminal fragments. Six OePPO genes were found in the fruits. Tryptic digestion identified the enzyme as mature OePPO1 (INSDC OY733096), while mass spectrometry detected the active form mass alongside several C-terminal fragments, revealing potential cleavage sites (Gly407, Tyr408).

Serine protease homolog pairs CLIPA4-A6, A4-A7Δ, and A4-A12 act as cofactors for proteolytic activation of prophenoloxidase-2 and -7 in Anopheles gambiae.

Phenoloxidase (PO) catalyzed melanization and other insect immune responses are mediated by serine proteases (SPs) and their noncatalytic homologs (SPHs). Many of these SP-like proteins have a regulatory clip domain and are called CLIPs. In most insects studied so far, PO precursors are activated by a PAP (i.e., PPO activating protease) and its cofactor of clip-domain SPHs. Although melanotic encapsulation is a well-known refractory mechanism of mosquitoes against malaria parasites, it is unclear if a cofactor is required for PPO activation. In Anopheles gambiae, CLIPA4 is 1:1 orthologous to Manduca sexta SPH2; CLIPs A5-7, A12-14, A26, A31, A32, E6, and E7 are 11:4 orthologous to M. sexta SPH1a, 1b, 4, and 101, SPH2 partners in the cofactors. Here we produced proCLIPs A4, A6, A7Δ, A12, and activated them with CLIPB9 or M. sexta PAP3. A. gambiae PPO2 and PPO7 were expressed in Escherichia coli for use as PAP substrates. CLIPB9 was mutated to CLIPB9Xa by including a Factor Xa cleavage site. CLIPA7Δ was a deletion mutant with a low complexity region removed. After PAP3 or CLIPB9Xa processing, CLIPA4 formed a high Mr complex with CLIPA6, A7Δ or A12, which assisted PPO2 and PPO7 activation. High levels of specific PO activity (55-85 U/µg for PO2 and 1131-1630 U/µg for PO7) were detected in vitro, indicating that cofactor-assisted PPO activation also occurs in this species. The cleavage sites and mechanisms for complex formation and cofactor function are like those reported in M. sexta and Drosophila melanogaster. In conclusion, these data suggest that the three (and perhaps more) SPHI-II pairs may form cofactors for CLIPB9-mediated activation of PPOs for melanotic encapsulation in A. gambiae.

Identification of an essential role against shrimp pathogens of prophenoloxidase activating enzyme 1 (PPAE1) from Fenneropenaeus merguiensis hemocytes.

Prophenoloxidase (proPO) activating enzymes, known as PPAEs, are pivotal in activating the proPO system within invertebrate immunity. A cDNA encoding a PPAE derived from the hemocytes of banana shrimp, Fenneropenaeus merguiensis have cloned and analyzed, referred to as FmPPAE1. The open reading frame of FmPPAE1 encompasses 1392 base pairs, encoding a 464-amino acid peptide featuring a presumed 19-amino acid signal peptide. The projected molecular mass and isoelectric point of this protein stand at 50.5 kDa and 7.82, respectively. Structure of FmPPAE1 consists of an N-terminal clip domain and a C-terminal serine proteinase domain, housing a catalytic triad (His272, Asp321, Ser414) and a substrate binding site (Asp408, Ser435, Gly437). Expression of the FmPPAE1 transcript is specific to hemocytes and is heightened upon encountering pathogens like Vibrio parahaemolyticus, Vibrio harveyi, and white spot syndrome virus (WSSV). Using RNA interference to silence the FmPPAE1 gene resulted in reduced hemolymph phenoloxidase (PO) activity and decreased survival rates in shrimp co-injected with pathogenic agents. These findings strongly indicate that FmPPAE1 plays a vital role in regulating the proPO system in shrimp. Furthermore, upon successful production of recombinant FmPPAE1 protein (rFmPPAE1), it became evident that this protein exhibited remarkable abilities in both agglutinating and binding to a wide range of bacterial strains. These interactions were primarily facilitated through the recognition of bacterial lipopolysaccharides (LPS) or peptidoglycans (PGN) found in the cell wall. This agglutination process subsequently triggered melanization, a critical immune response. Furthermore, rFmPPAE1 exhibited the ability to actively impede the growth of pathogenic bacteria harmful to shrimp, including V. harveyi and V. parahaemolyticus. These findings strongly suggest that FmPPAE1 not only plays a pivotal role in activating the proPO system but also possesses inherent antibacterial properties, actively contributing to the suppression of bacterial proliferation. In summary, these results underscore the substantial involvement of FmPPAE1 in activating the proPO system in F. merguiensis and emphasize its crucial role in the shrimp's immune defense against invading pathogens.

Two clip-domain serine protease homologs, cSPH35 and cSPH242, act as a cofactor for prophenoloxidase-1 activation in Drosophila melanogaster.

Insect phenoloxidases (POs) catalyze phenol oxygenation and o-diphenol oxidation to form reactive intermediates that kill invading pathogens and form melanin polymers. To reduce their toxicity to host cells, POs are produced as prophenoloxidases (PPOs) and activated by a serine protease cascade as required. In most insects studied so far, PPO activating proteases (PAPs) generate active POs in the presence of a high Mr cofactor, comprising two serine protease homologs (SPHs) each with a Gly residue replacing the catalytic Ser of an S1A serine protease (SP). These SPHs have a regulatory clip domain at the N-terminus, like most of the SP cascade members including PAPs. In Drosophila, PPO activation and PO-catalyzed melanization have been examined in genetic analyses but it is unclear if a cofactor is required for PPO activation. In this study, we produced the recombinant cSPH35 and cSPH242 precursors, activated them with Manduca sexta PAP3, and confirmed their predicted role as a cofactor for Drosophila PPO1 activation by MP2 (i.e., Sp7). The cleavage sites and mechanisms for complex formation and cofactor function are highly similar to those reported in M. sexta. In the presence of high Mr complexes of the cSPHs, PO at a high specific activity of 260 U/µg was generated in vitro. To complement the in vitro analysis, we measured hemolymph PO activity levels in wild-type flies, cSPH35, and cSPH242 RNAi lines. Compared with the wild-type flies, only 4.4% and 18% of the control PO level (26 U/µl) was detected in the cSPH35 and cSPH242 knockdowns, respectively. Consistently, percentages of adults with a melanin spot at the site of septic pricking were 82% in wild-type, 30% in cSPH35 RNAi, and 53% in cSPH242 RNAi lines; the survival rate of the control (45%) was significantly higher than those (30% and 15%) of the two RNAi lines. These data suggest that Drosophila cSPH35 and cSPH242 are components of a cofactor for MP2-mediated PPO1 activation, which are indispensable for early melanization in adults.

The Drosophila melanogaster prophenoloxidase system participates in immunity against Zika virus infection.

Drosophila melanogaster relies on an evolutionarily conserved innate immune system to protect itself from a wide range of pathogens, making it a convenient genetic model to study various human pathogenic viruses and host antiviral immunity. Here we explore for the first time the contribution of the Drosophila phenoloxidase (PO) system to host survival and defenses against Zika virus (ZIKV) infection by analyzing the role of mutations in the three prophenoloxidase (PPO) genes in female and male flies. We show that only PPO1 and PPO2 genes contribute to host survival and appear to be upregulated following ZIKV infection in Drosophila. Also, we present data suggesting that a complex regulatory system exists between Drosophila PPOs, potentially allowing for a sex-dependent compensation of PPOs by one another or other immune responses such as the Toll, Imd, and JAK/STAT pathways. Furthermore, we show that PPO1 and PPO2 are essential for melanization in the hemolymph and the wound site in flies upon ZIKV infection. Our results reveal an important role played by the melanization pathway in response to ZIKV infection, hence highlighting the importance of this pathway in insect host defense against viral pathogens and potential vector control strategies to alleviate ZIKV outbreaks.

Structural and expression analysis of polyphenol oxidases potentially involved in globe artichoke (C. cardunculus var. scolymus L.) tissue browning.

Globe artichoke capitula are susceptible to browning due to oxidation of phenols caused by the activity of polyphenol oxidases (PPOs), this reduces their suitability for fresh or processed uses. A genome-wide analysis of the globe artichoke PPO gene family was performed. Bioinformatics analyses identified eleven PPOs and their genomic and amino acidic features were annotated. Cis-acting element analysis identified a gene regulatory and functional profile associated to plant growth and development as well as stress response. For some PPOs, phylogenetic analyses revealed a structural and functional conservation with different Asteraceae PPOs, while the allelic variants of the eleven PPOs investigated across four globe artichoke varietal types identified several SNP/Indel variants, some of which having impact on gene translation. By RTqPCR were assessed the expression patterns of PPOs in plant tissues and in vitro calli characterized by different morphologies. Heterogeneous PPO expression profiles were observed and three of them (PPO6, 7 and 11) showed a significant increase of transcripts in capitula tissues after cutting. Analogously, the same three PPOs were significantly up-regulated in calli showing a brown phenotype due to oxidation of phenols. Our results lay the foundations for a future application of gene editing aimed at disabling the three PPOs putatively involved in capitula browning.

A Polyphenol Oxidase Catalyzes Aurone Synthesis in Marchantia polymorpha.

Aurones constitute one of the major classes of flavonoids, with a characteristic furanone structure that acts as the C-ring of flavonoids. Members of various enzyme families are involved in aurone biosynthesis in different higher plants, suggesting that during evolution plants acquired the ability to biosynthesize aurones independently and convergently. Bryophytes also produce aurones, but the biosynthetic pathways and enzymes involved have not been determined. The present study describes the identification and characterization of a polyphenol oxidase (PPO) that acts as an aureusidin synthase (MpAS1) in the model liverwort, Marchantia polymorpha. Crude enzyme assays using an M. polymorpha line overexpressing MpMYB14 with high accumulation of aureusidin showed that aureusidin was biosynthesized from naringenin chalcone and converted to riccionidin A. This activity was inhibited by N-phenylthiourea, an inhibitor specific to enzymes of the PPO family. Of the six PPOs highly induced in the line overexpressing MpMyb14, one, MpAS1, was found to biosynthesize aureusidin from naringenin chalcone when expressed in Saccharomyces cerevisiae. MpAS1 also recognized eriodictyol chalcone, isoliquiritigenin and butein, showing the highest activity for eriodictyol chalcone. Members of the PPO family in M. polymorpha evolved independently from PPOs in higher plants, indicating that aureusidin synthases evolved in parallel in land plants.

Analysis of oxidase activity and transcriptomic changes related to cutting propagation of hybrid larch.

Hybrid larch is the main timber and afforestation tree species in Northeast China. To solve the problem of rooting difficulties in larch cutting propagation, enzyme activity determination and transcriptome sequencing were carried out on the rooting tissues at five timepoints after cutting. peroxidase (POD), indole acetic acid oxidase (IAAO) and polyphenol oxidase (PPO) play important roles in the larch rooting process after cutting. A total of 101.20 Gb of clean data was obtained by transcriptome sequencing, and 43,246 unigenes were obtained after further screening and assembly. According to GO analysis and KEGG enrichment analysis, we think that plant hormones play an important role in the rooting process of larch stem cuttings. in the plant hormone signal transduction pathway, a larch gene c141104.graph_c0 that is homologous to the Arabidopsis AUX1 was found to be significantly up-regulated. We suggest that AUX1 may promote IAA transport in larch, thus affecting adventitious root development. According to the results of POD, PPO IAAO indexes and GO analysis, we think s1 and s2 periods may be important periods in the rooting process of larch stem cuttings, so we built a gene regulatory network, a total of 14genes, including LBD, NAC, AP2/ERF, bHLH and etc., may be important in different stages of cutting propagation. As the rooting rate after cutting inhibits the development of larch clone propagation, identifying the genes that regulate rooting could help us to preliminarily understand the molecular mechanism of adventitious root formation and select a better treatment method for cutting propagation.

An insight into the mechanisms underpinning the anti-browning effect of Codium tomentosum on fresh-cut apples.

This work focuses on understanding the action of a novel seaweed extract with anti-browning functionality in fresh-cut apples. Organic fresh-cut apples were coated by immersion in an aqueous Codium tomentosum seaweed extract (0.5 % w/v), packaged under ambient atmospheric conditions in plastic bags, and stored at 4 ˚C for 15 days. Browning-related enzymatic activities, as well as targeted gene expression related to superficial browning, were monitored immediately after coating and followed at five-day intervals, until a final storage period of 15 days. Gene expression was particularly affected one hour after coating application (day 0), with no expression registered for peroxidase (mdPOD) and phenylalanine ammonia-lyase (mdPAL) genes in the coated samples. A reduction in polyphenol oxidase expression levels was also observed. After 15 days of storage, the coated samples developed lower browning levels and presented distinctly lower activities of polyphenol oxidase and peroxidase - the oxidative enzymes predominantly involved in enzymatic browning. The observed post-coating suppression of mdPAL and mdPOD expression, and reduction in mdPPO expression, suggest that the seaweed C. tomentosum extract delays the activation of these genes, and decreases enzymatic activity, which in turn accounts for the coating's anti-browning effect.

Chromosomal-level genome of velvet bean (Mucuna pruriens) provides resources for L-DOPA synthetic research and development.

Mucuna pruriens, commonly called velvet bean, is the main natural source of levodopa (L-DOPA), which has been marketed as a psychoactive drug for the clinical management of Parkinson's disease and dopamine-responsive dystonia. Although velvet bean is a very important plant species for food and pharmaceutical manufacturing, the lack of genetic and genomic information about this species severely hinders further molecular research thereon and biotechnological development. Here, we reported the first velvet bean genome, with a size of 500.49 Mb and 11 chromosomes encoding 28,010 proteins. Genomic comparison among legume species indicated that velvet bean speciated ∼29 Ma from soybean clade, without specific genome duplication. Importantly, we identified 21 polyphenol oxidase coding genes that catalyse l-tyrosine to L-DOPA in velvet bean, and two subfamilies showing tandem expansion on Chr3 and Chr7 after speciation. Interestingly, disease-resistant and anti-pathogen gene families were found contracted in velvet bean, which might be related to the expansion of polyphenol oxidase. Our study generated a high-quality genomic reference for velvet bean, an economically important agricultural and medicinal plant, and the newly reported L-DOPA biosynthetic genes could provide indispensable information for the biotechnological and sustainable development of an environment-friendly L-DOPA biosynthesis processing method.

Molecular characterization of polyphenol oxidase between small and large leaf tea cultivars.

Tea is a widely consumed beverage prepared using the fresh leaves of Camellia sinensis L. Tea plants are classified into small- and large-leaf varieties. Polyphenol oxidase (PPO), a crucial enzyme in tea manufacturing, catalyzes essential phenolic metabolites into different derivatives. To compare the molecular characteristics of CsPPO between cultivars, we cloned the full-length sequence of CsPPO cDNA from four representative tea cultivars in Taiwan. Amino acid sequence alignment analyses indicated that CsPPO is highly conserved. PPO exhibited similar enzymatic activity in different tea cultivars. Quantitative real-time polymerase chain reaction revealed no significant differences in the CsPPO transcript level between the small- and large-leaf varieties. However, tea harvested in summer and from low-altitude areas had a higher CsPPO transcript level than that harvested in winter and from high-altitude areas. Regulation of CsPPO by temperature was more significant in the small-leaf variety than in the large-leaf variety. The content of flavonoids and the expression of chalcone synthase, anthocyanidin synthase, and anthocyanidin reductase genes involved in the tea flavonoid biosynthesis pathway were higher in the large-leaf than in the small-leaf varieties, suggesting that the large-leaf variety had a higher antioxidative capacity than did the small-leaf variety. Our study compared the molecular properties of CsPPO between two tea varieties and clarified the physiological role of PPO in tea.

The loss of polyphenol oxidase function is associated with hilum pigmentation and has been selected during pea domestication.

Seed coats serve as protective tissue to the enclosed embryo. As well as mechanical there are also chemical defence functions. During domestication, the property of the seed coat was altered including the removal of the seed dormancy. We used a range of genetic, transcriptomic, proteomic and metabolomic approaches to determine the function of the pea seed polyphenol oxidase (PPO) gene. Sequencing analysis revealed one nucleotide insertion or deletion in the PPO gene, with the functional PPO allele found in all wild pea samples, while most cultivated peas have one of the three nonfunctional ppo alleles. PPO functionality cosegregates with hilum pigmentation. PPO gene and protein expression, as well as enzymatic activity, was downregulated in the seed coats of cultivated peas. The functionality of the PPO gene relates to the oxidation and polymerisation of gallocatechin in the seed coat. Additionally, imaging mass spectrometry supports the hypothesis that hilum pigmentation is conditioned by the presence of both phenolic precursors and sufficient PPO activity. Taken together these results indicate that the nonfunctional polyphenol oxidase gene has been selected during pea domestication, possibly due to better seed palatability or seed coat visual appearance.

Natural history-guided omics reveals plant defensive chemistry against leafhopper pests.

Although much is known about plant traits that function in nonhost resistance against pathogens, little is known about nonhost resistance against herbivores, despite its agricultural importance. Empoasca leafhoppers, serious agricultural pests, identify host plants by eavesdropping on unknown outputs of jasmonate (JA)-mediated signaling. Forward- and reverse-genetics lines of a native tobacco plant were screened in native habitats with native herbivores using high-throughput genomic, transcriptomic, and metabolomic tools to reveal an Empoasca-elicited JA-JAZi module. This module induces an uncharacterized caffeoylputrescine-green leaf volatile compound, catalyzed by a polyphenol oxidase in a Michael addition reaction, which we reconstitute in vitro; engineer in crop plants, where it requires a berberine bridge enzyme-like 2 (BBL2) for its synthesis; and show that it confers resistance to leafhoppers. Natural history-guided forward genetics reveals a conserved nonhost resistance mechanism useful for crop protection.

The unexpected flavone synthase-like activity of polyphenol oxidase in tomato.

The biosynthesis of flavones has drawn considerable attention. However, the presence of flavones and their biosynthesis in tomato (Solanum lycopersicum) remain unclear. Here, we confirmed that flavones are present in MicroTom tomato and unexpectedly found that a tomato polyphenol oxidase (SlPPO F) possesses a flavone synthase-like activity and catalyzes the conversion of eriodictyol to luteolin without the need for any cofactor. SlPPO F showed a similar Km value to that of other polyphenol oxidases, and could be inhibited by ascorbic acid. The flavone synthase-like activity of SlPPO F exhibited strict substrate specificity and only accepted flavanones with two hydroxyl groups (3' and 4') on the B ring as substrates. SlPPO F showed higher catalytic efficiency and better thermostability than type I flavone synthase from Apium graveolens, suggesting its possible application in enzyme engineering. In summary, we identified flavones in tomato and unraveled a polyphenol oxidase exhibiting flavone synthase-like activity.

Prophenoloxidase of Odontotermes formosanus (Shiraki) (Blattodea: Termitidae) Is a Key Gene in Melanization and Has a Defensive Role during Bacterial Infection.

Melanization mediated by the prophenoloxidase (PPO)-activating system is an important innate immunity to fight pathogens in insects. In this study, the in vitro time-dependent increase in the intensity of melanization and phenoloxidase (PO) activity from the hemolymph of Odontotermes formosanus (Shiraki) challenged by pathogenic bacteria was detected. PPO is one of the key genes in melanization pathway, whereas the molecular characteristics and functions of O. formosanus PPO are unclear. The OfPPO gene was cloned and characterized. The open reading frame of OfPPO is 2085 bp in length and encodes a 79.497 kDa protein with 694 amino acids. A BLASTx search and phylogenetic analyses revealed that OfPPO shares a high degree of homology to the Blattodea PPOs. Moreover, real-time fluorescent quantitative PCR analysis showed that OfPPO is ubiquitously expressed in all castes and tissues examined, with the highest expression in workers and variable expression patterns in tissues of different termite castes. Furthermore, the expression of OfPPO was significantly induced in O. formosanus infected by pathogenic bacteria. Intriguingly, in combination with silencing of OfPPO expression, pathogenic bacteria challenge caused greatly increased mortality of O. formosanus. These results suggest that OfPPO plays a role in defense against bacteria and highlight the novel termite control strategy combining pathogenic bacteria application with termite PPO silencing.

Improving the Tea Withering Process Using Ethylene or UV-C.

Using a combination of biochemical, transcriptomic, and physiological analyses, we elucidated the mechanisms of physical and chemical withering of tea shoots subjected to UV-C and ethylene treatments. UV-C irradiation (15 kJ m-2) initiated oxidation of catechins into theaflavins, increasing theaflavin-3-monogallate and theaflavin digallate by 5- and 13.2-4.4-fold, respectively, at the end of withering. Concomitantly, a rapid change to brown/red, an increase in electrolyte leakage, and the upregulation of peroxidases (viz. Px2, Px4, and Px6) and polyphenol oxidases (PPO-1) occurred. Exogenous ethylene significantly increased the metabolic rate (40%) and moisture loss (30%) compared to control during simulated withering (12 h at 25 °C) and upregulated transcripts associated with responses to dehydration and abiotic stress, such as those in the ethylene signaling pathway (viz. EIN4-like, EIN3-FBox1, and ERFs). Incorporating ethylene during withering could shorten the tea manufacturing process, while UV-C could enhance the accumulation of flavor-related compounds.

Genome-wide investigation and expression profiling of polyphenol oxidase (PPO) family genes uncover likely functions in organ development and stress responses in Populus trichocarpa.

BACKGROUND: Trees such as Populus are planted extensively for reforestation and afforestation. However, their successful establishment greatly depends upon ambient environmental conditions and their relative resistance to abiotic and biotic stresses. Polyphenol oxidase (PPO) is a ubiquitous metalloproteinase in plants, which plays crucial roles in mediating plant resistance against biotic and abiotic stresses. Although the whole genome sequence of Populus trichocarpa has long been published, little is known about the PPO genes in Populus, especially those related to drought stress, mechanical damage, and insect feeding. Additionally, there is a paucity of information regarding hormonal responses at the whole genome level. RESULTS: A genome-wide analysis of the poplar PPO family was performed in the present study, and 18 PtrPPO genes were identified. Bioinformatics and qRT-PCR were then used to analyze the gene structure, phylogeny, chromosomal localization, gene replication, cis-elements, and expression patterns of PtrPPOs. Sequence analysis revealed that two-thirds of the PtrPPO genes lacked intronic sequences. Phylogenetic analysis showed that all PPO genes were categorized into 11 groups, and woody plants harbored many PPO genes. Eighteen PtrPPO genes were disproportionally localized on 19 chromosomes, and 3 pairs of segmented replication genes and 4 tandem repeat genomes were detected in poplars. Cis-acting element analysis identified numerous growth and developmental elements, secondary metabolism processes, and stress-related elements in the promoters of different PPO members. Furthermore, PtrPPO genes were expressed preferentially in the tissues and fruits of young plants. In addition, the expression of some PtrPPOs could be significantly induced by polyethylene glycol, abscisic acid, and methyl jasmonate, thereby revealing their potential role in regulating the stress response. Currently, we identified potential upstream TFs of PtrPPOs using bioinformatics. CONCLUSIONS: Comprehensive analysis is helpful for selecting candidate PPO genes for follow-up studies on biological function, and progress in understanding the molecular genetic basis of stress resistance in forest trees might lead to the development of genetic resources.

Polyphenol oxidase (PPO) arm of catecholamine pathway catalyzes the conversion of L-tyrosine to L-DOPA in Mucuna pruriens (L.) DC var. pruriens: An integrated pathway analysis using field grown and in vitro callus cultures.

Mucuna pruriens (L.) DC var. pruriens is the natural source for L-DOPA, precursor of the neurotransmitter dopamine, used widely in the treatment of Parkinson's disease. However, L-DOPA synthesis in plants is mediated either by Catecholamine (CA) pathway or alternate pathway catalyzed by Cytochrome P450 (CYP450) class of enzymes. Interestingly, the CA pathway itself can be initiated either by tyrosine hydroxylase (TH) or polyphenol oxidase (PPO). The CA pathway mediated synthesis of L-DOPA has not yet been proved in M. pruriens albeit strong indications. Therefore, the present investigation is focused on metabolite analysis of major intermediates of CA pathway up to the formation of dopamine and expression analysis of the selected genes, in different tissues and callus cultures. The four major intermediates, L-tyrosine, tyramine, L-DOPA and dopamine, were detected using NMR spectroscopy and quantified by HPLC in the callus cultures and in different tissues of the field plant, respectively. The various stages of leaf tissue were also analyzed for metabolite profiling. The relative amount of intermediates detected during the ontogeny of leaf indicates that PPO mediated conversion of L-tyrosine to dopamine through L-DOPA is relatively higher compared to dopamine production from tyramine. Among the two possible enzymes, activity of PPO was 6.5-fold more than TH in metabolically active young leaves compared to intermediate leaves. The gene expression profiles comprising upstream genes of L-tyrosine synthesis and downstream up to dopamine synthesis shows strong correlation with L-DOPA synthesis. The study validates CA pathway mediated synthesis of L-DOPA with PPO as candidate enzyme, in M. pruriens.

The basicity of an active-site water molecule discriminates between tyrosinase and catechol oxidase activity.

Tyrosinase (Ty) and catechol oxidase (CO) are members of type-3 copper enzymes. While Ty catalyzes both phenolase and catecholase reactions, CO catalyzes only the latter reaction. In the present study, Ty was found to catalyze the catecholase reaction, but hardly the phenolase reaction in the presence of the metallochaperon called "caddie protein (Cad)". The ability of the substrates to dissociate the motif shielding the active-site pocket seems to contribute critically to the substrate specificity of Ty. In addition, a mutation at the N191 residue, which forms a hydrogen bond with a water molecule near the active center, decreased the inherent ratio of phenolase versus catecholase activity. Unlike the wild-type complex, reaction intermediates were not observed when the catalytic reaction toward the Y98 residue of Cad was progressed in the crystalline state. The increased basicity of the water molecule may be necessary to inhibit the proton transfer from the conjugate acid to a hydroxide ion bridging the two copper ions. The deprotonation of the substrate hydroxyl by the bridging hydroxide seems to be significant for the efficient catalytic cycle of the phenolase reaction.

Polyphenol oxidase genes as integral part of the evolutionary history of domesticated tetraploid wheat.

Studying and understanding the genetic basis of polyphenol oxidases (PPO)-related traits plays a crucial role in genetic improvement of crops. A tetraploid wheat collection (T. turgidum ssp., TWC) was analyzed using the 90K wheat SNP iSelect assay and phenotyped for PPO activity. A total of 21,347 polymorphic SNPs were used to perform genome-wide association analysis (GWA) in TWC and durum wheat sub-groups, detecting 23 and 85 marker-trait associations (MTA). In addition, candidate genes responsible for PPO activity were predicted. Based on the 23 MTAs detected in TWC, two haplotypes associated with low and high PPO activity were identified. Four SNPs were developed and validated providing one reliable marker (IWB75732) for marker assisted selection. The 23 MTAs were used to evaluate the genetic divergence (FST > 0.25) between the T. turgidum subspecies, providing new information important for understanding the domestication process of Triticum turgidum ssp. and in particular of ssp. carthlicum.