Isoforms Of Polyphenol Oxidase Research Articles

Expression and characterization of a novel PPO gene from Mucuna pruriens (L.) DC. var. pruriens involved in catecholamine pathway mediated synthesis of L-DOPA

Mucuna pruriens (L.) DC var. pruriens is a well-known legume of Fabaceae family. The plant is the natural source of l-DOPA, precursor molecule of dopamine neurotransmitter used in the treatment of Parkinson´s disease. In M. pruriens, the Catecholamine (CA) biosynthesis is mediated by Polyphenol oxidase (PPO) as validated in our previous reports. In the present investigation, we identified three PPO isoforms and were quantitatively analyzed by quantitative real-time PCR to understand the expression of PPO´s constitutively in the different tissues of M. pruriens. Consequently, the highly expressed PPO isoform was cloned, and the recombinant protein purified followed by functional characterization. The Kinetic analysis using Lineweaver-Burk curves showed greater binding affinity of enzyme towards its specific substrate. This study provides new insight into plant derived recombinant PPO enzyme, which is involved in CA biosynthetic pathway in this taxon. The Metabolic pathway engineering of PPO step would result in modulation of l-DOPA synthesis in this plant, in future.

Pseudomonas fluorescens VSMKU3054 mediated induced systemic resistance in tomato against Ralstonia solanacearum

Plant growth-promoting rhizobacteria (PGPR) are potential bio agents for the suppression of various soil-borne diseases in crop plants and considered as viable alternative to chemical pesticides. The selected Pseudomonas fluorescens isolate VSMKU3054 is an effective biological control inoculum for the suppression of wilt disease of tomato incited by Ralstonia solanacearum by inducing plant defense enzymes viz., peroxidase (POX), polyphenol oxidase (PPO), lipoxygenase (LOX), phenylalanine ammonia-lyase (PAL) and total phenol content. The highest level of defense enzymes viz., POX, PAL, PPO, LOX and total phenol contents were observed in tomato seedlings treated with P. fluorescens at 24h, 36h, 36h, 48h and 24h after challenge inoculation of R. solanacearum, respectively. Whereas application of P. fluorescens alone in tomato plants also brought about induction of higher levels of POX, PAL, LOX and total phenol contents, and a moderate level of PPO activity compared to pathogen inoculated plants and control plants. The maximum induction of four prominent PPO isoforms was observed in tomato plants upon the treatment of P. fluorescens VSMKU3054 and challenged with R. solanacearum. Thus, this study showed that induction of defense-related genes by P. fluorescens plays an important role against R. solanacearum.

Induction of induced systemic resistance in capsicum chilli against Colletotrichum truncatum through fungal biocontrol agents

To induce the systemic resistance in capsicum against Colletotrichum truncatum an experiment was conducted with two biocontrol agents viz., Trichoderma harzianum and Trichoderma asperellum in pot culture. The induced resistance was monitored by increase in activities of five defense related enzymes viz., peroxidase (PO), polyphenol oxidase (PPO) phenylalanine ammonia lyase (PAL), catalase (CAT), and superoxide dismutase (SOD) and the accumulation of phenols and β -1,3-glucanase were also noticed in capsicum upon challenge inoculation with C. truncatum, the causal agent for anthracnose or fruit rot in capsicum. The activities of defense enzymes reached a peak at eight days after inoculation (DAI) with the pathogen. Native PAGE analysis revealed the expression of an additional isoforms of PO and PPO were observed in biocontrol agents treated seedlings due to induced systemic resistance (ISR) introduction. Prior treatment of capsicum seedlings with biocontrol agents triggered the plant defense mechanism in response to infection by C. capsici. Hence, it is speculated that among the various direct antagonistic tools, ISR is also the one indirect tool by which the tested biocontrol agents afforded resistance to capsicum against the pathogen.

Detection of genetic divergence among some wheat (Triticum aestivum L.) genotypes using molecular and biochemical indicators under salinity stress.

Wheat has remarkable importance among cereals in Egypt. Salt stress affects plant growth, development, and crop productivity. Therefore, salinity tolerance is an essential trait that must be incorporated in crops. This research aimed to investigate molecular and biochemical indicators and defence responses in seedlings of 14 Egyptian wheat genotypes to distinguish the most contrasting salt-responsive genotypes. Analysis of ISSR and SCoT markers revealed high polymorphism and reproducible fingerprinting profiles for evaluating genetic variability within the studied genotypes. The HB-10 and SCoT 1 primers had the highest values for all the studied parameters. All the tested primers generated a set of 66 polymorphic bands among tolerant and sensitive genotypes. The transcript profiles of eight TaWRKY genes showed significant induction under the salinity treatments. Moreover, the expression of TaWRKY6 for genotypes Sids 14 and Sakha 93 sharply increased and recorded the highest expression, while the expression of TaWRKY20 for Misr 1 recorded the lowest expression. Under salt stress, the total sugar, proline, and phenolic contents increased significantly, while the chlorophyll content decreased significantly. Additionally, five peroxidase and polyphenol oxidase isoforms were observed in treated leaves and clustered into five different patterns. Some isoforms increased significantly as salinity levels increased. This increase was clearer in salt-tolerant than in salt-sensitive genotypes. Eighteen protein bands appeared, most of which were not affected by salinity compared with the control, and specific bands were rare. Generally, the Sids 14, Sakha 93, Sohag 4, and Gemmeiza 12 genotypes are considered salt tolerant in comparison to the other genotypes.

Purification, kinetic parameters, and isoforms of polyphenol oxidase from “Xushu 22” sweet potato skin

We purified and compared the polyphenol oxidase (PPO) isoenzymes present in "Xushu 22," a sweet potato. A membrane-bound form (mPPO) and two soluble forms (sPPO1 and sPPO2) were identified and purified using ammonium sulphate precipitation, ion exchange chromatography, gel filtration chromatography, and mass spectrometer. The three PPO isoforms were characterized enzymatically. The specific activity of mPPO was significantly higher than that of the two sPPO isoforms, being 24.55- and 13.89-fold higher than sPPO1 and sPPO2. The preferred substrates for mPPO and the two sPPOs were catechol and chlorogenic acid, respectively. They can be efficiently and safely applied to phenolic wastewater treatment after being immobilized. Both mPPO and the two sPPOs were rapidly inactivated under acid or base conditions and were unstable at 65°C. The most effective inhibitors of mPPO, sPPO1, and sPPO2 were glutathione, ascorbic acid, and L-cysteine, respectively. PRACTICAL APPLICATIONS: According to incomplete statistics, about 5% of sweet potatoes in china are wasted due to enzymatic browning every year. PPO was regarded as a key molecule contributing to enzymatic browning in fruits and vegetables during ripening, processing, and storage and responsible for economic and industrial loss. It's great importance to characterize the PPO from "Xushu 22" sweet potato and provide data on its inactivation. The three PPO isoforms were purified and identified by chromatography and mass spectrometer. This study will provide useful informations to have a better command of PPO from "Xushu 22" sweet potato and give ideals to solve the browning of sweet potato.

Induction of Systemic Resistant Molecules in Phylloplane of Rice Plants against Magnaporthe oryzae by Pseudomonas fluorescens

Rice blast caused by ascomycetes fungus Magnaporthe oryzae, is mostly considered the utmost significant disease of rice worldwide since its widespread dissemination and destructiveness under conducive conditions. Experiments were conducted in phylloplane of rice plants to study the induction of various defense enzymes and accumulation of phenol by three biocontrol agents viz., P. fluorescens talc (Pf1), liquid formulation with TNAU - Pf1 and Biocure - B under pot culture. The application of bioformulatons of P. flourescens triggered the activity of three defense related enzymes viz., peroxidase (PO), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) and SOD were induced and the accumulation of phenol was also noticed in phylloplane rice plant upon challenge inoculation with Magnaporthe oryzae the causal agent for leaf blast disease in rice. The activities of defense enzymes reached a peak at three days after inoculation (DAI) with the pathogen. Native PAGE analysis revealed the expression of an additional isoforms of PO, PPO, SOD and catalase were observed in biocontrol agents treated seedlings due to induced systemic resistance (ISR) induction.

Characterization of three polyphenol oxidase isoforms in royal dates and inhibition of its enzymatic browning reaction by indole-3-acetic acid

In this study, Polyphenol oxidases (PPO) from royal dates fruit was purified and characterized. These procedures led to 8-fold purification with 14.72% recovery. Three isoenzymes of royal dates PPO exhibited a molecular weight of 20.,45 and 64 kDa. The royal date PPOs had maximum activity at pH 4.6 and 8, 3.6–5.6 and 5.6 with pyrogallol, 4-methylcatechol and pyrocatechol substrates, respectively. The enzyme showed high stability in the temperature range of 30–60 °C.4-Methylcatechol was the most suitable substrate, due to the lowest Km and the biggest Vmax/Km values. The kinetic of thermal inactivation were performed in a temperature range of 60–75 °C. A biphasic model provided a good description of dates PPO thermal inactivation. Indole-3acetic acid markedly inhibited Royal dates PPO activity. Its Anti-browning effect on dates was investigated. IAA-treatment reduced weight loss, pericarp decay, browning index and membrane electrolyte leakage. Thus, a higher total anthocyanins contents and total phenolic contents was correlated with higher DPPH scavenging activity and lower MDH contents Such effect was accredited to maintain of membrane integrity and inhibition of oxidative enzymes by IAA. In conclusion, IAA could be used as a potent inhibitor of fruits enzymatic browning reaction.

Role of lipopolysaccaride extracted from Alcaligenes faecalis as elicitor for the induction of plant defense against fusarium wilt

In the present investigation, emphasis was placed on the efficacy of lipopolysaccharide (LPS) extracted from the plant beneficial rhizobacterium Alcaligenes faecalis MSS8 for the induction of disease resistance by enhancing immune response in plants against phytopathogen Fusarium oxysporum f. sp. lycopersici (FOL). Characterization and proficiency of the rhizobacterial LPS for the induced defense response in plant against FOL was revealed by the gel activity staining of Peroxidase (PO) and Polyphenol oxidase (PPO) isoforms as compared to the untreated and only FOL treated plants. It has been observed that after 48 h of FOL challenge inoculation, in the LPS pretreated plant there was significant induction of PO and PPO isoforms. Furthermore, RT-PCR analysis for the study of relative gene expression of pathogenesis related (PR) proteins confirms the result of gel activity staining. It has been observed that expression level of LeChi9, LeGluB, LePR1a was significantly upregulated in LPS treated plants 24 h after FOL inoculation and maximum level was observed at 48 h as compared to the control and FOL treated plants. This clearly indicates the role of LPS extracted from A. faecalis as a potential biocontrol agent against fusarium wilt.

Biocontrol of bacterial speck of tomato by aqueous extract of Tagetes erecta

Abstract Biocontrol of plant diseases has emerged as an eco-friendly measure of plant protection and has experienced a lot of devotion in the last two decades. Biocontrol agents include application of microbial agents, their secretion products and natural extracts from different parts of several plants. The present study, therefore, aimed at evaluating the potency of aqueous extracts of Tagetes erecta L. (marigold) in controlling bacterial speck disease in tomato plants. The experimental design consisted of two groups of 50 plants each: group 1 – sprayed with sterile water (control); and group 2 – sprayed with marigold extract. Spraying was performed under aseptic conditions at the third node from the base of each plant. Challenge inoculation with the bacterial speck pathogen Pseudomonas syringae pv. tomato was performed to analyze the disease severity on the test plants. The parameters of study were analysis of alteration in the activity and gene expression of peroxidase (POX), phenyl ammonia lyase (PAL), and polyphenol oxidase (PPO), as well as isoform expression of POX and PPO. The results demonstrated strong inductive effects of the extract on the activity and genes of POX, PAL and PPO. De novo expression of POX and PPO isoforms following marigold extract treatment was also observed. The observations indicate that marigold extract could be a promising biopesticide.

INDUCTION OF ANTIOXIDANT ENZYMES AND ISOZYMES PROFILE OF CHICKPEA GENOTYPES UNDER COMPATIBLE AND INCOMPATIBLE INTERACTIONS WITH FUSARIUM WILT

Chickpea genotypes (ten desi and eight kabuli), differing in their reaction to Fusarium wilt, were grown in Fusarium infested soil and screened for wilt resistance and susceptibility. The root and leaf tissues of highly resistant (desi WR 315 and kabul KAK 2) and highly susceptible (desi JG 62 and kabul L 550) chickpea genotypes were collected during disease development stages under pathogen infestation and non-infestation conditions. They were analyzed for antioxidant enzyme activities and isozymes profiling. The specific activities of ROS scavenging enzymes superoxide dismutase (SOD), glutathione peroxidase (GPX), ascorbate peroxidase (APX), polyphenol oxidase (PPO) and catalase (CAT) were found higher in the root and leaf tissues under compatible interactions (infection) compared with incompatible (non-infested or non-infected?) during disease development stages. The susceptible compatible interactions of desi and kabuli chickpea genotypes induced systemic antioxidant bursts (SOD, GPX, APX, PPO, CAT) efficiently after Fusarium infection. The isozymes profiling of SOD, APX, PPO, CAT agreed with the enzyme activities and also showed differences in GPX zymogram pattern distinctive in the root of kabuli resistant KAK 2 during infection stage (S2). However, nativePAGE electrophoregram revealed distinctive bands for infected root tissues of desi resistant WR 315 (novel) and kabuli resistant KAK 2 (correlated with GPX isoforms) at S2 stage. The SOD, GPX, and PPO isoforms are more diverse and associated with Fusarium interactions during disease development stages. The results explained production of an oxidative burst and related responses during Fusarium wilt infection of desi and kabuli chickpea genotypes.

Induced systemic resistance (ISR) in hot pepper against Phytophthora capsici infection triggered by cell wall oligosaccharide elicitors from Trichoderma species

Induced systemic resistance, one of the mechanisms of biological control is elicited either by oligosaccharides or glycoproteins released from cell wall of fungal bioagents like Trichoderma species. Oligosaccharide elicitors from 10 Trichoderma isolates having biocontrol potential were tested for their ability to elicit ISR in hot pepper against Phytophthora capsici infection. Treatment with elicitors from isolates Th10, Th9, Th33 and Th28 reduced P. capsici infection in hot pepper by 70–80% compared to 100% infection in pathogen inoculated control. Assays of peroxiase (PO), polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL) and glucanase revealed that elicitor treatment significantly induced higher enzyme activity in elicitor treated plants compared to control. Induction of more number of isoforms of PO and PPO was also observed. Elicitors from Th10 and Th33 induced two fold increase in PPO and PAL and three-fold increase in PO, while Th28 and Th9 increased PPO and glucanase by two folds. Different enzymes (involved in phenyl propanoid metabolism or hydryolytic enzymes) contributed to ISR in different isolates.

Lactic acid bacteria mediated induction of defense enzymes to enhance the resistance in tomato against Ralstonia solanacearum causing bacterial wilt

The biocontrol agent Lactic acid bacterium (LAB) was used against the bacterial wilt caused by Ralstonia solanacearum. The present investigation focuses on the role of defense related enzymes in imparting resistance to tomato plants against R. solanacearum. The LAB isolate was tested for its ability to induce the production of defense-related enzymes in treated tomato seedlings. Tomato seedlings were raised from LAB pretreated seeds, were challenge inoculated with R. solanacearum, harvested at different time intervals (0–72h) and assayed for defense enzyme activity. The LAB treated seeds showed increase in germination percentage (6%) and seedling vigour index (259) compared with control. Treatment of tomato seedlings with LAB isolate induced a significant amount of Peroxidase (POX), Polyphenol oxidase (PPO), Phenylalanine ammonialyase (PAL), total phenolics and β-1,3-glucanase activities. The activities of PAL, POX, PPO and β-1,3-glucanase reached maximum at 24h, 24h, 32h and 24h respectively after challenge inoculation. Increased accumulation of phenolics was noticed in plants pre-treated with LAB. Native PAGE analyses of POX and PPO were carried out for the time course of enzyme activities and the isoforms of POX and PPO were examined. In field study, ten isolates of R. solanacearum treated plots yielded an average of 32.4–50kg/m2 and LAB treated plots an average of 153.5kg/m2. As compared to the control, LAB increased the yield by 15.3% (8.2kg/m2) and the pathogen infected plants and pre-treated with LAB gave an average of 55% (28.3kg/m2 compared to the infected plots). Field experiment results indicated that LAB exhibited 61.1% of disease reduction of bacterial wilt in tomato.

Polyphenol Oxidase Containing Sidestreams as Emulsifiers of Rumen Bypass Linseed Oil Emulsions: Interfacial Characterization and Efficacy of Protection against in Vitro Ruminal Biohydrogenation.

The low transfer in ruminants of dietary polyunsaturated fatty acids to the milk or peripheral tissues is largely due to ruminal biohydrogenation. Lipids emulsified by a polyphenol oxidase (PPO) rich protein extract of red clover were shown before to be protected against this breakdown after cross-linking with 4-methylcatechol. Protein extracts of 13 other vegetal resources were tested. Surprisingly, the effectiveness to protect emulsified lipids against in vitro ruminal biohydrogenation largely depended on the origin of the extract and its protein concentration but was not related to PPO activity. Moreover, PPO isoforms in vegetal sources, effectively protecting emulsified lipids, were diverse and their presence at the emulsion interface did not seem essential. Potato tuber peels were identified as an interesting biological source of emulsifying proteins and PPO, particularly since protein extracts of industrial potato sidestreams proved to be suitable for the current application.

The Use of Polyphenol Oxidase Activity to Identify a Potential Raisin Variety

ABSTRACTThe biochemical characterization of polyphenol oxidase (PPO) in the white grape Bianchetta Trevigiana is addressed in this research to fulfil the requirement for raisin varieties with high concentrations of phenolic compounds and low PPO activity not only to avoid enzymatic browning but also for maintaining the nutritional value of raisins. Spectrophotometric assays and electrophoresis were used to study this ancient variety exhibiting low PPO activity (only cathecolase activity) which reached at the low pH value of grape berry and was about 15% of the maximum enzymatic activity measured. In addition, the visualization of the active isoforms of grape PPO after 2-DE separation of proteins was a key step for further studies using MALDI-TOF-MS and bioinformatic tools. Based on the thermal inactivation profiles, heating treatment at 70°C may be considered a way to inhibit Bianchetta Trevigiana PPO activity by preserving also its high phenolic content. Therefore PPO characterization may be proposed as a marker for identifying potential high value raisin varieties.

Bacterial antagonists and hexanal-induced systemic resistance of mango fruits against Lasiodiplodia theobromae causing stem-end rot

ABSTRACTInduction of defense-related enzymes, such as phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO), superoxide dismutase (SOD) and catalase (CAT) due to bacterial antagonists viz., Pseudomonas fluorescens (Pf1) and Bacillus subtilis (EPCO16) and plant-derived lipoxygenase volatile compound hexanal, were studied in mango fruits against Lasiodiplodia theobromae causing stem-end rot disease. The results showed increased induction of all the defense-related enzymes in mango fruits 3–5 days after dipping treatment with combination of bacterial antagonists and hexanal when compared to untreated control treatment and treatment with fungicide carbendazim in storage condition. The increased activity was observed up to 3 days after treatment and thereafter declined. Further, increased expression of specific isoforms of PO, PPO, SOD and CAT were also observed in the treatment effect of P. fluorescens (0.5%) + hexanal (0.02%) treated fruits against L. theobromae. From the results obtained, it is inferred that due to the enhancement of defense-related enzymes via the phenylpropanoid pathway and due to secretion of secondary metabolites that would play significant role in hindering the pathogen quiescence and further invasion in mango fruits and thereby prevent the fruit rot.

ISOLATION AND PROPERTIES OF POLYPHENOL OXIDASE FROM BASIDIOCARPS OF Lactarius pergamenus Fr. (Fr.) FUNGI.

Fresh juice of basidiocarps of Lactarius pergamenus Fr. (Fr.) fungi was subjected to ion exchange chromatography with used DEAE-toyopearl and CM-cellulose columns, as well as preparative electrophoresis in 7.5% polyacrylamide gels (pH 8.6). Three isoforms of polyphenol oxidase (PPO) were discovered and two isoforms (1-l and 1-2) were purified with a release of protein 0.42 mg/kg and 0.15 mg/kg of basidiocarps, respectively. These isoforms differ in the mobility at disc-electrophoresis in 7.5% PAGE in alkaline buffer system (pH 8.6). Specfic activity of isoform 1-2 is 4.8 times higher than that of the isoforms 1-1. The molecular weight determination by gel chromatography on the Toyopearl HW-55 demonstrated that both isoforms 1-1 and 1-2 have the same 64 ± 2 kDa molecular mass. Electrophoresis in 15% PAGE in the presence of sodium dodecylsulphate and β-mercaptoethanol revealed one band with molecular mass of 64 ± 1 kDa which suggests the presence of one polypeptide chain in the molecule ofthe enzyme. The enzyme has demonstrated the highest activity at pH 6.0 and temperature +10 °C, and at +70 °C the enzyme was inactivated. The PPO activity was the highest in young mushrooms and it decreased with their age and positively correlated with the content ofthe milky juice. Ortho-aminophenol was most effective among all the tested substrates to determine the activity of PPO (o-, m- and p-aminophenol, catechol, tyrosine, resorcinol, phloroglucinol) and its relative activity was 129% of the activity of catechol. Ascorbic acid was the most effective inhibitor of the polyphenol oxidase activity which was completely blocked at 1 mM concentration, whereas the same concentration of thiourea and sodium sulphite decreased the enzymatic activity by 40-45%. The PPO in L. pergamenus fungi basidiocarps was mainly localized in the mushroom milky juice where its high activity may be associated with protection of basidiocarps against various pathogens.

Polyphenol oxidase as a biochemical seed defense mechanism.

Seed dormancy and resistance to decay are fundamental survival strategies, which allow a population of seeds to germinate over long periods of time. Seeds have physical, chemical, and biological defense mechanisms that protect their food reserves from decay-inducing organisms and herbivores. Here, we hypothesize that seeds also possess enzyme-based biochemical defenses, based on induction of the plant defense enzyme, polyphenol oxidase (PPO), when wild oat (Avena fatua L.) caryopses and seeds were challenged with seed-decaying Fusarium fungi. These studies suggest that dormant seeds are capable of mounting a defense response to pathogens. The pathogen-induced PPO activity from wild oat was attributed to a soluble isoform of the enzyme that appeared to result, at least in part, from proteolytic activation of a latent PPO isoform. PPO activity was also induced in wild oat hulls (lemma and palea), non-living tissues that cover and protect the caryopsis. These results are consistent with the hypothesis that seeds possess inducible enzyme-based biochemical defenses arrayed on the exterior of seeds and these defenses represent a fundamental mechanism of seed survival and longevity in the soil. Enzyme-based biochemical defenses may have broader implications since they may apply to other defense enzymes as well as to a diversity of plant species and ecosystems.

Partial purification and characterization of polyphenol oxidase from persimmon

Activity of polyphenol oxidase (PPO) from “Rojo Brillante” persimmon (Diospyros kaki L.) fruits was characterized. Crude extracts were used for characterization of enzyme activity and stability at different temperatures (60, 70 and 80°C), pHs (from 3.5 to 7.5) and substrate concentrations (catechol from 0 to 0.5M). Maximum enzyme activity was reached at pH 5.5 and 55°C. Enzyme stability was higher than PPO activities found in other natural sources, since above pH 5.5 the minimum time needed to achieve an enzyme inactivation of 90% was 70min at 80°C. However, at pH 4.0 the enzyme stability decreased, reaching inactivation levels above 90% after 10min even at 60°C. Thus it was concluded that acidification can circumvent browning problems caused by PPO activity. Moreover, polyacrylamide gel electrophoresis of the enriched extract revealed the presence of at least four bands with strong oxidase activity, suggesting the existence of different PPO isoforms.

Development and Validation of MRM Methods to Quantify Protein Isoforms of Polyphenol Oxidase in Loquat Fruits

Multiple reaction monitoring (MRM) is emerging as a promising technique for the detection and quantification of protein biomarkers in complex biological samples. Compared to Western blotting or enzyme assays, its high sensitivity, specificity, accuracy, assay speed, and sample throughput represent a clear advantage for being the approach of choice for the analysis of proteins. MRM assays are capable of detecting and quantifying proteolytic peptides differing in mass unique to particular proteins, that is, proteotypic peptides, through which different protein isoforms can be distinguished. We have focused on polyphenol oxidase (PPO), a plant conspicuous enzyme encoded by a multigenic family in loquat (Eriobotrya japonica Lindl.) and other related species. PPO is responsible for both the protection of plants from biotic stress as a feeding deterrent for herbivore insects and the enzymatic browning of fruits and vegetables. The latter makes fruit more attractive to seed dispersal agents but is also a major cause of important economic losses in agriculture and food industry. An adequate management of PPO at plant breeding level would maximize the benefits and minimize the disadvantages of this enzyme, but it would require a precise knowledge of the biological role played by each isoform in the plant. Thus, for the functional study of the PPOs, we have cloned and overexpressed fragments of three PPO isoforms from loquat to develop MRM-based methods for the quantification of each isoform. The method was developed using an ion trap instrument and validated in a QQQ instrument. It resulted in the selection of at least two peptides for each isoform that can be monitored by at least three transitions. A combination of SDS-PAGE and MRM lead to detect two out of three monitored isoforms in different gel bands corresponding to different processing stages of PPO. The method was applied to determine the amount of the PPO2 isoform in protein extracts from fruit samples using external calibrants.

Stage-specific induction of systemic acquired resistance by fruit extracts of Azadirachta indica

In the present study, the efficacy of aqueous fruit extract of Azadirachta indica A. Juss. (Neem) on induction of systemic acquired resistance (SAR) in tomato against Pseudomonas syringae pv. tomato through enhancement in the activity of polyphenol oxidase (PPO) at different ages was studied. Plants at 6, 8, 10 and 12 weeks of age were selected. A single leaf at the third node from the base of each plant was treated either singly or with different combinations of the pathogen and Neem fruit extract. Samples were collected from the non-treated leaves at an interval of 24 h up to five days for enzyme assay and after two weeks for disease development from both treated and untreated plants. The results demonstrate that the neem fruit extract could induce additional PPO isoforms both locally as well as systemically. The PPO activity was observed to be elevated in both the treated and non-treated leaves leading to induction of SAR. The induction of SAR enhances with the increase in the age of the plant.