Wound-induced Phenylalanine Ammonia-lyase Research Articles

Quality Changes and Respiration Rates of Fresh-Cut Sunchoke Tubers (Helianthus tuberosus L.)

Changes in quality attributes (discoloration, surface dehydration, translucency, color values, texture), respiration, phenolics and phenylalanine ammonia lyase (PAL) and polyphenol oxidase activities of sunchoke tuber slices were studied at 0, 5 and 10C. Cleanly cut smooth surfaces had less wound-induced discoloration and surface drying than rougher cut surfaces. The main quality defect was red discoloration, and a* and hue color values were highly correlated to this defect. The time course of discoloration was related to wound-induced respiration and PAL activity but not polyphenol oxidase activity or concentrations of soluble phenolics. Respiration rates of slices at 0, 5 and 10C averaged 5, 14 and 26 μL CO2/g-h, respectively, during 7 days. No decay was observed under any condition. Storage at 0 and 5C effectively retarded surface discoloration, but 0C was clearly better. A shelf life of 9–12 and 6–8 days was obtained with sunchoke slices at 0 and 5C, respectively, while shelf life was 3 days at 10C. Practical Applications Sunchoke tubers contain indigestible inulins and have potential as a fresh-cut product or a component in fresh-cut mixtures to be consumed raw or cooked. Sliced tubers retain quality when held at 0–5C, but discoloration, the main defect limiting shelf life, will occur more rapidly at 5C. The rate and severity of discoloration was related to storage temperature and to wound-induced increases in respiration and PAL activity. With this information, strategies to effectively control discoloration can be implemented.

Impact of different washing procedures on quality of fresh-cut iceberg lettuce (Lactuca sativa var. capitata L.) and endive (Cichorium endivia L.)

The objective of this study was to determine the effects of alternative washing procedures on fresh-cut iceberg lettuce and endive, applying warm water (45 °C), warm water with CaCl2 (1 g/L), and chlorinated water (4 °C, 120 ppm), respectively. Processing was conducted on pre-industrial scale, and the produce was subsequently stored in consumer-sized bags (4 °C, 10 days). O2 and CO2 levels in the modified atmosphere, and activities of phenylalanine ammonia-lyase (PAL), peroxidase (POD) and polyphenoloxidase (PPO) of the samples were analyzed. Counts of total aerobic bacteria and pseudomonades were determined on the produce and in the process water. In both commodities, wound-induced PAL activities were significantly reduced by warm water treatment. In iceberg lettuce, adding CaCl2 to warm water resulted in a further reduction of PAL activities. Chlorinated water was less effective than warm water in suppressing wound-induced PAL activity in endive, but proved to be most effective in reducing total microbial cell counts by 1.1 and 2.1 log10 cfu/g in iceberg lettuce and endive, respectively. Warm water treatment exhibited reduction rates of similar magnitudes. For process water, chlorine proved to be the most efficient sanitizer. PPO and POD activities were only marginally affected by the different washing procedures. As indicated by respiration of the fresh-cut produce, warm water treatments applied retained the vitality of both plant species. The results suggest that warm water treatments are suitable measures to improve the quality of iceberg lettuce and endive, but were less efficient in sanitizing the process water.

Age-induced loss of wound-healing ability in potato tubers is partly regulated by ABA

Wounding of potato (Solanum tuberosum L.) tubers induces the development of a suberized closing layer and wound periderm that resists desiccation and microbial invasion. Wound-healing ability declines with tuber age (storage period). The mechanism of loss in healing capacity with age is not known; however, upregulation of superoxide production, increased ABA biosynthesis and phenylalanine ammonia lyase (PAL) activity in response to wounding are processes critical to the development of a suberized closing layer and wound periderm. Therefore, the role of ABA in modulating the age-induced loss of wound-healing ability of tubers was examined. Non-wounded older tubers had 86% less ABA (dry matter basis) than younger tubers. PAL transcript increased in younger tubers within 24h of wounding, but transcription was delayed by 5days in older tubers. Wound-induced PAL activity increased more rapidly in younger than older tubers. ABA treatment increased PAL expression and activity in tissue from both ages of tubers and restored the 24h transcription time line in older tubers. Moreover, ABA treatment of wounded older tubers enhanced their resistance to water vapor loss following a 6-day wound-healing period. Wound-induced accumulation of suberin poly(phenolic(s)) (SPP) and suberin poly(aliphatic(s)) (SPA) was measurably slower in older versus younger tubers. ABA treatment hastened SPP accumulation in older tubers to match that in younger tubers, but only enhanced SPA accumulations over the initial 4days of healing. Age-induced loss of wound-healing ability is thus partly due to reduced ability to accumulate ABA and modulate the production of SPP through PAL in response to wounding and to dysfunction in the downstream signaling events that couple SPA biosynthesis and/or deposition to ABA. ABA treatment partly restored the healing ability of older tubers by enhancing the accumulation of SPP without restoring wound-induced superoxide forming ability to the level of younger tubers. The coupling of phenolic monomers into the poly(phenolic) domain of suberin was therefore not limited by the diminished wound-induced superoxide production of older tubers.

Regulatory involvement of abscisic acid in potato tuber wound-healing

Rapid wound-healing is crucial in protecting potato tubers from infection and dehydration. Wound-induced suberization and the accumulation of hydrophobic barriers to reduce water vapour conductance/loss are principal protective wound-healing processes. However, little is known about the cognate mechanisms that effect or regulate these processes. The objective of this research was to determine the involvement of abscisic acid (ABA) in the regulation of wound-induced suberization and tuber water vapour loss (dehydration). Analysis by liquid chromatography-mass spectrometry showed that ABA concentrations varied little throughout the tuber, but were slightly higher near the periderm and lowest in the pith. ABA concentrations increase then decrease during tuber storage. Tuber wounding induced changes in ABA content. ABA content in wound-healing tuber discs decreased after wounding, reached a minimum by 24 h, and then increased from the 3rd to the 7th day after wounding. Wound-induced ABA accumulations were reduced by fluridone (FLD); an inhibitor of de novo ABA biosynthesis. Wound-induced phenylalanine ammonia lyase activity was slightly reduced and the accumulation of suberin poly(phenolics) and poly(aliphatics) noticeably reduced in FLD-treated tissues. Addition of ABA to the FLD treatment restored phenylalanine ammonia lyase activity and suberization, unequivocally indicating that endogenous ABA is involved in the regulation of these wound-healing processes. Similar experiments showed that endogenous ABA is involved in the regulation of water vapour loss, a process linked to wax accumulation in wound-healing tubers. Rapid reduction of water vapour loss across the wound surface is essential in preventing desiccation and death of cells at the wound site; live cells are required for suberization. These results unequivocally show that endogenous ABA is involved in the regulation of wound-induced suberization and the processes that protect surface cells from water vapour loss and death by dehydration.

Effect of temperature and chlorination of pre-washing water on shelf-life and physiological properties of ready-to-use iceberg lettuce

The effects of cold and warm, chlorinated water as well as warm water without chlorination for pre-washing trimmed, cored iceberg lettuce heads was assessed regarding the shelf-life and physiological properties of the resulting ready-to-use (RTU) produce. On a pilot-plant scale, lettuce heads were shredded with or without pre-washing (50 °C, no chlorine; 4 and 50 °C, 200 mg/l free chlorine; 60 s). After shredding, lettuce was washed for 90 s (tap water, 4 °C), spin-dried, packaged in 200 g consumer-sized bags, and stored at 4 °C for ≤ 9 days. Samples were analyzed for their headspace O 2 and CO 2 levels, sensory properties, microbiological status, and phenylalanine ammonia-lyase (PAL) activity at regular intervals throughout processing and storage. Parallelly, instrumental color and texture measurements were performed. All pre-washing procedures of lettuce heads lowered the initial counts of total aerobic bacteria, pseudomonades, and Enterobacteriaceae by ≥ 1.63, ≥ 1.55, and ≥ 1.85 log 10 cfu/g, respectively, while the single-wash of shredded lettuce in tap water without any pre-washing resulted in significantly smaller reduction rates (0.23, 0.11, and 0.71 log 10 cfu/g, respectively). During storage, all pre-washing procedures had a persistent effect on bacterial counts of the RTU lettuce. Furthermore, all pre-washing procedures delayed vascular tissue browning at cut edges and retarded the decline in the overall visual quality of the samples. Whereas wound-induced PAL synthesis was reduced by the use of warm water (± chlorine), O 2 depletion and CO 2 evolution in the corresponding bags were slightly enhanced, thus proving that processing kept the food as a living tissue. Industrial relevance Ready to use fresh like produce is getting increasing attention and industrial relevance. The aim of this study was to specifically overcome problems associated with product heterogeneity. Interestingly, the use of warm tap water without chlorination as a pre-washing step proved as effective alternative to the application of cold chlorinated water without substantially compromising vitality and freshness of the product.

Temporal and genotypic variation of wound-induced gene expression in bark of Populus tremuloides and P. grandidentata

In two related experiments, total RNA was extracted from wounded and unwounded bark of young aspen ramets for Northern and dot blot analyses. Wound-inducible genes isolated from other plant species were hybridized to blots, and mRNA levels were estimated. Analyses of variance (ANOVA) were used to examine the significance of experimental factors (wounding, time after wounding, and genotype) affecting variation in mRNA levels. The first experiment examined the timing (0.5-96 h after wounding) of expression of wound-inducible genes in bark tissue of a single Populus tremuloides Michx. genotype. Wounding and variation among RNA samples significantly (p < 0.05) affected mRNA levels of two chitinases (win6, win8) and phenylalanine ammonia-lyase (PAL). The second experiment examined interclonal variation of wound-induced win6 and PAL expression in aspen bark. Ramets of four P. tremuloides and one Populus grandidentata Michx. genotypes were wounded and bark was collected 4, 8, or 12 h later. Genotype, wounding, and time after wounding all significantly affected win6 and PAL mRNA levels, with levels increasing as a result of wounding.

Wound-induced phenylalanine ammonia lyase activity: factors affecting its induction and correlation with the quality of minimally processed lettuces

Abstract One of the major causes of quality loss in minimally processed lettuce (Lactuca sativa L.) is the browning of the cut pieces. Phenylalanine ammonia lyase (PAL, EC 4.3.1.5) is a key enzyme in phenolic synthesis. PAL activity increased in lettuce midrib tissue with wounding and storage in the presence or absence of ethylene. PAL activity increased 2.5 and 3-fold at 5 and 15 °C, respectively, by reducing midrib size from 2.5 × 15 to 0.5 × 1 cm. Wounding usually induced maximum levels of enzyme activity within 3 days at 5 °C and 1 day at 15 °C. Pre and postharvest factors affected the kinetics of wound-induced PAL (WI-PAL) activity and of subsequent changes in the quality of minimally processed lettuce. The rate at which WI-PAL activity increased and the maximum level attained were influenced by the duration of storage before processing. These parameters were also affected by the cultivar and type of lettuce used. Butterhead and iceberg types had the highest and lowest levels, respectively, of WI-PAL activity; romaine, green leaf and red leaf had intermediate levels. The activity of PAL 1–2 days after processing, and the slope of the induction curve between days 0 and 2, were the enzyme measurements that gave the highest correlations with processed lettuce quality attributes, including overall visual quality, leaf edge browning, and leaf surface browning.

932 PB 530 PAL INDUCTION AND FRESH-CUT LETTUCE QUALITY

Phenylalanine ammonia-lyase (PAL) activity in iceberg lettuce (Lactuca sativa L.) is increased in response to several kinds of stress, including wounding, exposure to ethylene, and fungal infection. Ethylene-induced PAL activity is correlated with the discoloration and shelf-life of fresh cut lettuce (Couture et al. 1993. HortScience 28:723). The objective of this research was to further characterize the kinetics of wound-induced PAL in fresh cut lettuce. Leaves of different cvs were cut into salad pieces (1.5 × 3 cm), rinsed in chlorinated water, centrifuged manually, and placed into containers at 5 or 15C through which humidified air flowed. Samples were evaluated for overall visual quality and specific types of discoloration. Midrib tissue was also finely cut (1 × 0.5 cm) for enhanced wound-induction of PAL, which was assayed spectrophotometrically. The kinetics of PAL in midrib tissue of fine cut and salad cut lettuce were similar, with maximum activities obtained within 12-16 h at 15C and within 40-60 h at 5C. Maximum PAL levels in the fine cut lettuce were 1.5-2.0 times those observed in the salad cut pieces, and similar to those induced by ethylene. The usefulness of PAL as a predictor of the storage life of fresh cut lettuce depends on simplifying and expediting the PAL assay.

Wound-induced phenylalanine ammonia-lyase in potato (Solanum tuberosum) tuber discs. Significance of glycosylation and immunolocalization of enzyme subunits

1. Excised discs of potato (Solanum tuberosum) tuber were incubated with [3H]fucose and extracts were prepared and incubated with an antibody to phenylalanine ammonia-lyase. Analysis of the resulting immunoprecipitated proteins by SDS/PAGE showed [3H]mannose- and [3H]fucose-labelled bands with Mr values corresponding to those of phenylalanine ammonia-lyase subunits. 2. When potato discs were incubated with [3H]sugars in the presence of tunicamycin, an inhibitor of N-linked protein glycosylation, incorporation of radioactivity from [3H]mannose into the immunoprecipitated enzyme subunits was virtually eliminated, whereas that from [3H]fucose was only marginally inhibited. 3. Tunicamycin reduced the level of extractable phenylalanine ammonia-lyase activity induced in excised potato tuber discs. Kinetic analysis revealed that the Vmax value of the enzyme in crude extracts from tunicamycin-treated tissue was reduced, whereas the apparent Km values were unaffected. 4. Immunoprecipitation of the enzyme labelled in vivo with [35S]methionine showed that tunicamycin did not inhibit the synthesis of the enzyme protein per se, nor did it increase the degradation of the enzyme protein. 5. Immunoprecipitation of the enzyme labelled in vitro with [14C]nitromethane showed that tunicamycin did not affect the introduction of the dehydroalanine residue into the active site. 6. These results are consistent with the following hypothesis: tunicamycin inhibits the N-linked glycosylation of phenylalanine ammonia-lyase which, in turn, results in imperfect folding of the enzyme protein. The orientation of the active site is changed in such a way that the affinity of the enzyme for its substrate is unaffected, whereas the catalytic activity of the enzyme is reduced. 7. Both optical- and electron-microscopic immunolocalization studies with antibody to phenylalanine ammonia-lyase showed increased deposition of silver granules in cells in sections of potato discs in which induction of the enzyme was allowed to occur compared with cells from newly wounded tissue. The enzyme was located in the cytoplasm, and was possibly membrane-associated.

Wound-induced phenylalanine ammonia-lyase in potato tuber tissue. Development of enzyme activity and effects of antibiotics.

Phenylalanine ammonia-lyase [EC 4.3.1.5.] activity increased rapidly after a 3-hr lag period in potato tuber (Solanum tuberosum L. cv. May Queen) disks incubated in a suitable medium in the dark at 25 degrees. The activity reached a maxinum after incubation for about 40 hr. The effects of actinomycin D, 6-methylpurine, cycloheximide, chloramphenicol, and mitomycin C on the induction of phenylalanine ammonia-lyase were investigated during incubation of the disks. Actinomycin D, 6-methylpurine, and cycloheximide all inhibited the formation of phenylalanine ammonia-lyase, though cycloheximide was the most effective at low concentrations. Application of actinomycin D for the initial lag period (3 hr) caused strong inhibition; however, if it was supplied later it did not inhibit but actually increased phenylalanine ammonialyase formation. In contrast, cycloheximide was effective over most of the incubation period. Chloramphenicol and mitomycin C did not inhibit phenylalanine phenylalanine ammonialyase induction, but markedly stimulated it. Light was not an essential factor for phenylalanine ammonia-lyase induction in the wounded tissue.