Inhibition of downy blight and enhancement of resistance in litchi fruit by postharvest application of melatonin.

Litchis are tasty fruit with economic importance. However, the extreme susceptibility of harvested litchis to litchi downy blight caused by Peronophythora litchii leads to compromised quality. This study aimed to study the effects of melatonin on postharvest resistance to P. litchii in 'Feizixiao' litchis. Results showed that melatonin restricted lesion expansion in litchis after P. litchi inoculation. Melatonin enhanced the activities of phenylalanine ammonia-lyase, cinnamate-4-hydroxylase and 4-hydroxycinnamate CoA ligase while promoting the accumulations of phenolics and flavonoids. Nicotinamide adenine dinucleotide phosphate content and glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase activities were higher in treated fruit than control fruit. Higher energy status along with elevated H+-ATPase, Ca2+-ATPase, succinate dehydrogenase and cytochrome C oxidase activities were observed in treated fruit. Ultrastructural observation showed reduced damage in mitochondria in treated fruit. The results suggest that melatonin induced resistance in litchis by modulating the phenylpropanoid and pentose phosphate pathways as well as energy metabolism. .

Chemical composition and deterioration mechanism of Pleurotus tuoliensis during postharvest storage.

Pleurotus tuoliensis is a popular edible and medical mushroom, but it is highly perishable during postharvest storage. The quality parameters, chemical composition, malondialdehyde (MDA) concentration, and activity of metabolic enzymes were studied during 12 days of storage at 4 °C and 6 days of storage at 25 °C. Degradation was well described by changes in quality parameters, losses in nutritional value, increased metabolic enzyme activity, the accumulation of MDA concentrations, and the increase of total phenolic (TP) content. The phenylalanine ammonia lyase (PAL) significantly positively correlated with TP, which suggested an underlying mechanism of browning that the increased PAL activity stimulates the biosynthesis of phenols through the phenylalanine pathway. These results suggest that increased activity of laccase, lipoxygenase, PAL, TP and MDA accumulation, together with polysaccharide degradation, are the main factors involved in the deterioration of P. tuoliensis during storage.

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.

Induction of Natural Defenses in Tomato Seedlings by Using Alginate and Oligoalginates Derivatives Extracted from Moroccan Brown Algae.

Polysaccharides extracted from marine algae have attracted much attention due to their biotechnological applications, including therapeutics, cosmetics, and mainly in agriculture and horticulture as biostimulants, biofertilizers, and stimulators of the natural defenses of plants. This study aimed to evaluate the ability of alginate isolated from Bifurcaria bifurcata from the Moroccan coast and oligoalginates derivatives to stimulate the natural defenses of tomato seedlings. Elicitation was carried out by the internodal injection of bioelicitor solutions. The elicitor capacities were evaluated by monitoring the activity of phenylalanine ammonia-lyase (PAL) as well as polyphenols content in the leaves located above the elicitation site for 5 days. Alginate and oligoalginates treatments triggered plant defense responses, which showed their capacity to significantly induce the PAL activity and phenolic compounds accumulation in the leaves of tomato seedlings. Elicitation by alginates and oligoalginates showed an intensive induction of PAL activity, increasing from 12 h of treatment and remaining at high levels throughout the period of treatment. The amount of polyphenols in the leaves was increased rapidly and strongly from 12 h of elicitation by both saccharide solutions, representing peaks value after 24 h of application. Oligoalginates exhibited an effective elicitor capacity in polyphenols accumulation compared to alginate polymers. The alginate and oligosaccharides derivatives revealed a similar elicitor capacity in PAL activity whereas the accumulation of phenolic compounds showed a differential effect. Polysaccharides extracted from the brown seaweed Bifurcaria bifurcate and oligosaccharides derivatives induced significantly the phenylpropanoid metabolism in tomato seedlings. These results contribute to the valorization of marine biomass as a potential bioresource for plant protection against phytopathogens in the context of eco-sustainable green technology.

Postharvest application of pectic-oligosaccharides on quality attributes, activities of defense-related enzymes, and anthocyanin accumulation in strawberry.

BACKGROUND: The postharvest application of pectic-oligosaccharides (POS) as an elicitor to improve the postharvest shelf-life and nutritional quality by stimulating natural defense mechanisms in strawberries was studied. Strawberries (cv. Festival) were treated with POS (at 0, 2, 5, and 9 g L-1 ) and evaluated for firmness, weight loss, color, soluble solids, titratable acidity (TA), total phenolic and anthocyanin content, antioxidant capacity, decay, and some defense-related enzyme activity during storage at 2 ± 0.5 °C for 14 days. RESULTS: Treatment with POS significantly delayed (P < 0.05) strawberry decay, and reduced the water loss and softening of fruit during storage. Strawberries treated with POS showed a significant increase in total phenolic and anthocyanin content, and antioxidant capacity when compared with controls. Interestingly, POS induced higher activity of phenylalanine ammonia-lyase (PAL), chitinase, and ß-1,3-glucanase in strawberries. Compared to the control, the activity of enzymes was markedly increased in fruit treated with all tested POS concentrations, particularly chitinase, and ß-1,3-glucanase activities, but 5 and 9 g L-1 POS were the most effective treatments for maintaining the quality attributes and improving anthocyanin accumulation and antioxidant capacity of strawberries. CONCLUSION: These findings suggest that POS treatment could potentially be applied to maintain quality attributes, reduce decay, and further enrich the anthocyanin content and antioxidant capacity of strawberries during postharvest storage. The results also suggest that the positive effects of POS on strawberries could be associated with the rapid accumulation of chitinase and ß-1,3-glucanase activities, and the increase of PAL enzyme activity leading to the synthesis and accumulation of anthocyanins. © 2019 Society of Chemical Industry.

Cysteine enhances the content of betalains and polyphenols in fresh-cut red beet.

This study investigated the effectiveness of cysteine in conservation of bioactive compounds and the antioxidant capacity of minimally processed red beet. After red beet minimal processing increasing cysteine concentrations were applied, corresponding to control, 2 mM, 4 mM, 8 mM and 16 mM. Assay was performed over 15 d to evaluate the polyphenols, betalains, antioxidant capacity and enzymatic activity of polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL). Cysteine enhanced the gallic acid, caffeic acid, chlorogenic acid, kaempferol and betalain contents until 6 d of storage. Subsequently, dosages of cysteine above 4 mM maintained gallic acid, kaempferol and betalains contents. Cysteine appears to influence the phenylpropanoid pathway, favoring the accumulation of polyphenols and betalains. In red beet, cysteine did not inhibit PPO activity but enhanced PAL activity. Betalains contribute more than phenolics to the antioxidant capacity, and their relationship with cysteine has not been thoroughly elucidated to date.

Phenylalanine ammonia lyase from Arabidopsis thaliana (AtPAL2): A potent MIO-enzyme for the synthesis of non-canonical aromatic alpha-amino acids.: Part II: Application in different reactor concepts for the production of (S)-2-chloro-phenylalanine.

Phenylalanine ammonia lyase (PAL) from Arabidopsis thaliana (AtPAL2) is in general a very good catalyst for the amination of fluoro- and chloro-cinnamic acid derivatives yielding halogenated (S)-phenylalanine derivatives with ≥85% conversion and excellent ee values >99%. We have studied the application of this enzyme as whole cell biocatalyst and immobilized on the cellulose carrier Avicel® for the production of the hypertension drug precursor (S)-2-chloro-phenylalanine using batch, fed-batch, as well as continuous membrane reactor and plug-flow reactor. For immobilization, a C-terminal fusion of the enzyme with a carbohydrate binding module (CBM) was produced, which selectively binds to Avicel® directly from crude cell extracts, thus enabling a fast and cheap immobilization, stabilization and recycling of the enzyme. 1g Avicel was loaded with 10mg enzyme. Best results were obtained with whole cells using the continuous membrane reactor (47gproduct/gDryCellWeight) and using the immobilized enzyme in a repetitive fed-batch (274gproduct/gimmobilized enzyme) or in a continuous plug-flow reactor (288gproduct/gimmobilize enzyme). Therewith the productivity of AtPAL2 outperforms the established fed-batch process at DSM using PAL from Rhodotorula glutinis in E. coli as whole cell biocatalyst with a productivity of 0.14gproduct/gWetCellWeight (ca. 0.7gproduct/gDryCellWeight) (de Lange et al., 2011; doi:10.1002/cctc.201000435).

Phenylalanine ammonia lyase from Arabidopsis thaliana (AtPAL2): A potent MIO-enzyme for the synthesis of non-canonical aromatic alpha-amino acids: Part I: Comparative characterization to the enzymes from Petroselinum crispum (PcPAL1) and Rhodosporidium toruloides (RtPAL).

Phenylalanine ammonia lyase (PAL) from Arabidopsis thaliana (AtPAL2) was comparatively characterized to the well-studied enzyme from parsley (PcPAL1) and Rhodosporidium toruloides (RtPAL) with respect to kinetic parameters for the deamination and the amination reaction, pH- and temperature optima and the substrate range of the amination reaction. Whereas both plant enzymes are specific for phenylalanine, the bifunctional enzyme from Rhodosporidium toruloides shows KM-values for L-Phe and L-Tyr in the same order of magnitude and, compared to both plant enzymes, a 10-15-fold higher activity. At 30°C all enzymes were sufficiently stable with half-lives of 3.4days (PcPAL1), 4.6days (AtPAL2) and 9.7days (RtPAL/TAL). Very good results for the amination of various trans-cinnamic acid derivatives were obtained using E. coli cells as whole cell biocatalysts in ammonium carbonate buffer. Investigation of the substrate ranges gave interesting results for the newly tested enzymes from A. thaliana and R. toruloides. Only the latter accepts besides 4-hydroxy-CA also 3-methoxy-4-hydroxy-CA as a substrate, which is an interesting intermediate for the formation of pharmaceutically relevant L-Dopa. AtPAL2 is a very good catalyst for the formation of (S)-3-F-Phe, (S)-4-F-Phe and (S)-2-Cl-Phe. Such non-canonical amino acids are valuable building blocks for the formation of various drug molecules.

Phenylalanine ammonia-lyase, flavanone 3ß-hydroxylase and flavonol synthase enzyme activity by a new in vitro assay method in berry fruits.

An HPLC method for the determination of phenylalanine ammonia-lyase, flavanone 3ß-hydroxylase and flavonol synthase enzyme activity is proposed. This method is based on the determination of the compounds produced and consumed on the enzymatic reaction in just one chromatographic analysis. Optimisation of the method considered kinetic studies to establish the incubation time to perform the assay. The method here described proved to be an interesting approach to measure the activities of the three enzymes simultaneously increasing the rapidity, selectivity and sensitivity over other exiting methods. The enzyme activity method developed was applied to strawberry, raspberry, blackberry, redcurrant and blackcurrant fruits.

Browning inhibition and quality preservation of button mushroom (Agaricus bisporus) by essential oils fumigation treatment.

The effect of essential oil fumigation treatment on browning and postharvest quality of button mushrooms (Agaricus bisporus) was evaluated upon 16 days cold storage. Button mushrooms were fumigated with essential oils, including clove, cinnamaldehyde, and thyme. Changes in the browning index (BI), weight loss, firmness, percentage of open caps, total phenolics, ascorbic acid, microbial activity and activities of polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL), and peroxidase (POD) were measured. The results indicated that all essential oils could inhibit the senescence of mushrooms, and the most effective compound was cinnamaldehyde. Fumigation treatment with 5 µl l⁻¹ cinnamaldehyde decreased BI, delayed cap opening, reduced microorganism counts, promoted the accumulation of phenolics and ascorbic acid. In addition, 5 µl l⁻¹ cinnamaldehyde fumigation treatment inhibited the activities of PPO and POD, and increased PAL activity during the storage period. Thus, postharvest essential oil fumigation treatment has positive effects on improving the quality of button mushrooms.

Design and application of an in vivo reporter assay for phenylalanine ammonia-lyase.

Phenylalanine ammonia-lyase (PAL) is an important enzyme that links primary metabolism to secondary metabolism. Its efficiency is often a critical factor that affects the overall flux of a related metabolic pathway, the titer of the final products, and the efficacy of PAL-based therapies. Thus, PAL is a common target for metabolic engineering, and it is of significant interest to screen efficient PALs for industrial and medical applications. In this study, a novel and efficient visible reporter assay for screening of PAL efficiency in Escherichia coli was established based on a plant type III polyketide biosynthetic pathway. The candidate PALs were co-expressed with a 4-coumarate:CoA ligase 4CL1 from Arabidopsis thaliana and curcuminoid synthase (CUS) from Oryza sativa in E. coli BL21(DE3) to form a dicinnamoylmethane biosynthetic pathway. Taking advantage of the yellow color of the product, a microplate-based assay was designed to measure the titer of dicinnamoylmethane, which was validated by HPLC analysis. The different titers of the product reflect the overall performance (expression level and enzymatic activity) of the individual PALs in E. coli. Using this system, we have screened three PALs (PAL1, PAL3, and PAL4) from Trifolium pratense, among which PAL1 showed the best performance in E. coli. The engineered E. coli strain containing PAL1, 4CL1, and CUS led to the production of dicinnamoylmethane at a high level of 0.36 g/l. Supplement of 2-fluoro-phenylalanine yielded two fluorinated dicinnamoylmethane derivatives, 6,6'-difluoro-dicinnamoylmethane and 6-fluoro-dicinnamoylmethane, of which the latter is a new curcuminoid.

Molecular cloning and sequence analysis of a phenylalanine ammonia-lyase gene from dendrobium.

In this study, a phenylalanine ammonia-lyase (PAL) gene was cloned from Dendrobium candidum using homology cloning and RACE. The full-length sequence and catalytic active sites that appear in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum are also found: PAL cDNA of D. candidum (designated Dc-PAL1, GenBank No. JQ765748) has 2,458 bps and contains a complete open reading frame (ORF) of 2,142 bps, which encodes 713 amino acid residues. The amino acid sequence of DcPAL1 has more than 80% sequence identity with the PAL genes of other plants, as indicated by multiple alignments. The dominant sites and catalytic active sites, which are similar to that showing in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum, are also found in DcPAL1. Phylogenetic tree analysis revealed that DcPAL is more closely related to PALs from orchidaceae plants than to those of other plants. The differential expression patterns of PAL in protocorm-like body, leaf, stem, and root, suggest that the PAL gene performs multiple physiological functions in Dendrobium candidum.

Effect of natamycin in combination with pure oxygen treatment on postharvest quality and selected enzyme activities of button mushroom (Agaricus bisporus).

The combined effects of natamycin (NA) and pure oxygen (PO) treatment on microbial and physicochemical characteristics of button mushroom ( Agaricus bisporus ) stored at 4 ± 1 °C for 16 days was investigated. Mushroom respiration rate, weight loss, firmness, color, percent open caps, total soluble solids, microbial and activities of polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL), and peroxidase (POD) were measured. The results indicate that treatment with natamycin + pure oxygen (NAPO) maintained tissue firmness, inhibited increase of respiration rate, delayed browning and cap opening, and reduced microorganism counts of yeasts and molds compared to control treatment. The efficiency was better than that of NA or PO treatment. Furthermore, NAPO inhibited the activities of PPO, PAL, and POD throughout the storage period. Our study suggests that NAPO treatment has the potential to improve the quality of button mushroom and extend the shelf life.

Studies on the influence of host plants and effect of chemical stimulants on the feeding behavior in the muga silkworm, Antheraea assamensis.

The feeding habits of Antheraea assamensis, Helfer (Lepidoptera: Saturniidae) larvae towards the leaves of its four different host plants, Persea bombycina King ex. Hook (Laurales: Lauraceae), Litsea polhantha Jussieu, L. salicifolia Roxburgh ex. Nees and L. citrata Blume, and the chemical basis of feeding preference were investigated. Nutritional superiority of young and medium leaves with respect to soluble protein, total phenol and phenylalanine ammonia lyase activity was observed in the leaves of P. bombycina compared to other host plants. Attraction and feeding tests with detached leaves and artificial diet with different chemical stimulants revealed that a mixture of the flavonoids, myrcetin, and 7, 2', 4' trimethoxy dihydroxy flavone with sterol compound ß-sitosterol elicited the most biting behavior by A. assamensis larvae. While linalyl acetate alone attracted larvae towards the leaves of the host plants, a mixture of caryophyllene, decyl aldehyde and dodecyl aldehyde was found to both attract them to the host leaves and cause biting behavior. Azaindole was found to deter them from the host plants.

Immunohistochemical localization of enzymes that catalyze the long sequential pathways of lignin biosynthesis during differentiation of secondary xylem tissues of hybrid aspen (Populus sieboldii x Populus grandidentata).

We have investigated the spatial localization of enzymes that catalyze the sequential pathways of lignin biosynthesis in developing secondary xylem tissues of hybrid aspen (Populus sieboldii Miq. x Populus grandidentata Michx.) using immunohistochemical techniques. The enzymes phenylalanine ammonia-lyase, caffeic acid 3-O-methyltransferase and 4-coumarate:CoA ligase in the common phenylpropanoid pathway, cinnamyl-alcohol dehydrogenase (CAD) and peroxidase in the specific lignin pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) in the shikimate pathway and glutamine synthetase (GS) in the nitrogen reassimilation system were abundantly localized in the 6th to 9th wood fibers away from cambium; these wood fibers are likely undergoing the most intense lignification. Only weak immunolabeling of enzymes involved in the general phenylpropanoid and specific lignin pathways was detected in the cells near the cambium; lignification of these cells has likely been initiated after primary cell wall formation. In contrast, distinct localization of DAHPS and GS was observed around the cambium, which may be involved not only in lignin biosynthesis, but also in amino acid and protein synthesis, which are essential for cell survival. Our observations suggest that co-localization of enzymes related to the sequential shikimate, general phenylpropanoid and specific lignin branch pathways and to the nitrogen recycling system is associated with cell wall lignification of wood fibers during secondary xylem development.

Exogenous systemin has a contrasting effect on disease resistance in mycorrhizal tomato (Solanum lycopersicum) plants infected with necrotrophic or hemibiotrophic pathogens.

A study was performed to determine the effect of the systemin polypeptide on the bio-protective effect of arbuscular mycorrhizal fungi (AMF) in tomato plants infected with Alternaria solani, Phytophthora infestans or P. parasitica. Before infection, tomato plants were colonized with two different AMF, Glomus fasciculatum or G. clarum. In addition, a group of inoculated plants was treated with systemin, just after emergence. The exogenous application of systemin marginally suppressed the resistance against A. solani leaf blight observed in G. fasciculatum mycorrhizal plants but significantly enhanced it in plants colonized with G. clarum. Systemin induced resistance to P. parasitica in leaves of G. fasciculatum mycorrhizal plants, in which AMF colonization alone was shown to have no protective effect. Conversely, none of the treatments led to resistance to root or stem rots caused by P. infestans or P. parasitica. The above effects did not correlate with changes in the activity levels of beta-1,3-glucanase (BG), chitinase (CHI), peroxidase (PRX), and phenylalanine ammonium lyase (PAL) in leaves of infected plants. However, they corroborated previous reports showing that colonization by AMF can lead to a systemic resistance response against A. solani. Systemic resistance to A. solani was similarly observed in non-mycorrhizal systemin-treated plants, which, in contrast, showed increased susceptibility to P. infestans and P. parasitica. The results indicated that the pattern of systemic disease resistance conferred by mycorrhizal colonization was dependent on the AMF employed and could be altered by the exogenous application of systemin, by means of a still undefined mechanism.

Changes and subcellular localizations of the enzymes involved in phenylpropanoid metabolism during grape berry development.

The phenylpropanoid pathway yields a variety of phenolics that are closely associated with fruit qualities in addition to structural and defense-related functions. However, very little has been reported concerning its metabolism in fruit. This experiment was designed to assess changes of eleven phenolic acids in grape berry (Vitis vinifera L. cv. Cabernet Sauvignon) and explore both the activities and amounts of three key enzymes--phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H) and 4-coumarate:coenzyme A ligase (4CL)--catalyzing the biosynthesis of these compounds during berry development. Finally, the subcellular localizations of the enzymes within berry tissues were also investigated using immuno-gold electron microscopic technique. The results indicated that the contents of gallic, protocatechuic, gentisic and caffeic acid all changed drastically during berry development, while other compounds containing p-hydroxybenzoic, vanillic, syringic, chlorogenic, p-coumaric, ferulic and sinapic acid varied only slightly. Activities of PAL, C4H and 4CL showed similar pattern changes with two accumulated peaks throughout berry development. In addition, their activities all showed a highly positive correlation with the total contents of phenolic acids, whereas the immunoblotting analysis showed that changes in enzyme activities were independent of the enzyme amounts. Results from the subcellular-localization study revealed that PAL was mainly present in the cell walls, secondarily thickened walls, and the parenchyma cells of the berry mesocarp cells, C4H was found primarily in the chloroplast (plastid) and nucleus and 4CL predominantly in the secondarily thickened walls and the parenchyma cells of mesocarp vascular tissue.

Epidermis is a pivotal site of at least four secondary metabolic pathways in Catharanthus roseus aerial organs.

Catharanthus roseus produces a wide range of secondary metabolites, some of which present high therapeutic values such as antitumoral monoterpenoid indole alkaloids (MIAs), vinblastine and vincristine, and the hypotensive MIA, ajmalicine. We have recently shown that a complex multicellular organisation of the MIA biosynthetic pathway occurred in C. roseus aerial organs. In particular, the final steps of both the secoiridoid-monoterpene and indole pathways specifically occurred in the epidermis of leaves and petals. Chorismate is the common precursor of indole and phenylpropanoid pathways. In an attempt to better map the spatio-temporal organisation of diverse secondary metabolisms in Catharanthus roseus aerial organs, we studied the expression pattern of genes encoding enzymes of the phenylpropanoid pathway (phenylalanine ammonia-lyase [PAL, E.C. 4.3.1.5], cinnamate 4-hydroxylase [C4H, E.C. 1.14.13.11] and chalcone synthase [CHS, E.C. 2.3.1.74]). In situ hybridisation experiments revealed that CrPAL and CrC4H were specifically localised to lignifying xylem, whereas CrPAL, CrC4H and CrCHS were specifically expressed in the flavonoid-rich upper epidermis. Interestingly, these three genes were co-expressed in the epidermis (at least the upper, adaxial one) together with three MIA-related genes, indicating that single epidermis cells were capable of concomitantly producing a wide range of diverse secondary metabolites (e.g. flavonoïds, indoles, secoiridoid-monoterpenes and MIAs). These results, and data showing co-accumulation of flavonoids and alkaloids in single cells of C. roseus cell lines, indicated the spatio-temporal feasibility of putative common regulation mechanisms for the expression of these genes involved in at least four distinct secondary metabolisms.

Biochemical study of leaf browning in minimally processed leaves of lettuce (Lactuca sativa L. var. acephala).

A series of biochemical parameters, including the concentration of total ascorbic acid (ASA(tot)) and the activities of phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), and peroxidases (PODs), was investigated during cold storage (72 h at 4 degrees C in the dark) in fresh-cut (minimally processed) leaves of two lettuce (Lactuca sativa L. var. acephala) cultivars differing in the susceptibility to tissue browning: Green Salade Bowl (GSB), susceptible, and Red Salade Bowl (RSB), resistant. The two cultivars showed differences also at the biochemical level. The content in ASA(tot) increased in RSB, as a consequence of increased DHA concentration; conversely, ASA(tot) diminished in GSB, in which ASA was not detectable after 72 h of storage, thus suggesting a disappearance of ascorbate (both ASA and DHA) into nonactive forms. The antioxidant capacity (as determined by using FRAP analysis) decreased significantly during storage in RSB, while a strong increase was observed in GSB. PAL activity increased soon after processing reaching a maximum by 3 h, then it declined to a relatively constant value in RSB, while in GSB it showed a tendency to decrease in the first few hours from harvest and processing. POD activity, at least for chlorogenic acid, increased significantly during storage only in GSB.

Biochemical characterization of N-methyl N'-nitro-N-nitrosoguanidine-induced cadmium resistant mutants of Aspergillus niger.

Two cadmium resistant mutants (Cd1 and Cd2) of Aspergillus niger, among the six isolated by mutagenization with N-methyl N'-nitro-N-nitrosoguanidine (MNNG) at pH 6.4 were selected for the study. Analysis of lipid composition of the mutants and the wildtype indicated that total lipid as well as individual lipids of the cadmium resistant mutants were changed as compared with that of the wildtype. The increased activities of metal-lothionein and reduced activities of D-xylose isomerase and L-phenylalanine ammonia lyase in cell free extract of the cadmium resistant mutants suggested that mutants could allow high concentration of cadmium salt as compared with that of the wildtype. The respiratory activity and intracellular as well as extracellular Cd2+ concentration of the mutants reflected the high tolerance of the Cd mutants to cadmium ion.