Changes In Phenolic Metabolism Research Articles

Impact of Elicitation on Plant Antioxidants Production in Taxus Cell Cultures.

Elicited cell cultures of Taxus spp. are successfully used as sustainable biotechnological production systems of the anticancer drug paclitaxel, but the effect of the induced metabolomic changes on the synthesis of other bioactive compounds by elicitation has been scarcely studied. In this work, a powerful combinatorial approach based on elicitation and untargeted metabolomics was applied to unravel and characterize the effects of the elicitors 1 µM of coronatine (COR) or 150 µM of salicylic acid (SA) on phenolic biosynthesis in Taxus baccata cell suspensions. Differential effects on cell growth and the phenylpropanoid biosynthetic pathway were observed. Untargeted metabolomics analysis revealed a total of 83 phenolic compounds, mainly flavonoids, phenolic acids, lignans, and stilbenes. The application of multivariate statistics identified the metabolite markers attributed to elicitation over time: up to 34 compounds at 8 days, 41 for 16 days, and 36 after 24 days of culture. The most notable metabolic changes in phenolic metabolism occurred after 8 days of COR and 16 days of SA elicitation. Besides demonstrating the significant and differential impact of elicitation treatments on the metabolic fingerprint of T. baccata cell suspensions, the results indicate that Taxus ssp. biofactories may potentially supply not only taxanes but also valuable phenolic antioxidants, in an efficient optimization of resources.

<b>The effects of storage temperature on the quality and phenolic metabolism of whole and minimally processed kale leaves

We studied the effects of storage temperature on the activities of phenylalanine ammonia- lyase (PAL), peroxidase (POD) and polyphenol oxidase (PPO) in minimally processed kale (Brassica oleracea var. acephala) that was stored for 15 and 9 days at 5 ± 1oC and 10 ± 1oC, respectively. The main visual evidence for quality loss in whole leaves was yellowing and loss of turgescence. Minimally processed leaves presented significant browning, indicating increased POD and PPO activities. The PAL activity in minimally processed leaves stored at 5oC was fourfold higher than that of whole leaves after two days of storage. We showed that minimal processing influenced PAL, POD and PPO activities. The activity of all enzymes studied increased during storage, indicating that changes in phenolic metabolism play an important role in the decline of kale quality. PAL activity increased rapidly at the beginning of storage and exhibited a reduced rate of increase over time, while the PPO and POD activities increased continuously over time. The storage at 5 ° C was a great ally in delaying changes in phenolic metabolism; however, the absolute PAL activity was higher at 5 than at 10oC.

Changes in phenolic metabolism in salicylic acid-treated shoots of Cistus heterophyllus

The exogenous application of salicylic acid (SA) not only protects plants against stress, but also enhances their growth and productivity. In this study, proliferating shoots of Cistus heterophyllus subsp. carthaginensis, an endangered plant species, were incubated in the presence of 0, 10, 100, and 1,000 μM SA for a period of 2 months. Overall growth, phenylpropanoid metabolism and antioxidant capacity were then determined. At low SA concentration, the efficiency of photosystem II (PSII) and shoot growth remained stable, while chlorophyll and carotenoid levels increased. Furthermore, there were no major changes in the levels of H2O2 in the different treatments (less than 10 % compared with the control), but an increase in lipid peroxidation, proline content and free and bound SA concentrations was observed in 100 μM SA-treated shoots. SA treatments resulted in increased activities of phenylalanine ammonia lyase (EC 4.3.1.24) and soluble peroxidases (EC 1.11.1.7), which strongly correlated with the decrease in soluble flavanols and the increase of proanthocyanidins, whereas cell wall-bound peroxidases exhibited a SA-concentration-dependent down-regulation. The results provided evidence that the differences in SA-induced changes in phenolic metabolism, especially the oxidation of flavanols by soluble peroxidases, could serve as a backup defence system contributing to a reduction in oxidative cellular damage, as suggested by the high anti-lipid oxidation activity displayed by Cistus extracts.

Reinforcement of cell wall in roots of Lycopersicon esculentum through induction of phenolic compounds and lignin by elicitors

Changes in phenolic metabolism and lignin deposition have been studied in roots of tomato plants after elicitation with four elicitors which are Fusarium mycelium extract (FME), chitosan (CHT), Fusarium culture filtrate (FCF) and Trichoderma mycelium extract (TME). Most profound effect of elicitors was observed on ferulic acid among the phenolic compounds. After 24 h elicitation, the increase in ferulic acid content of root cell wall was 3.71 and 3.30 times by FME and CHT, respectively. The increase of 4-hydroxybenzoic acid was 2.71 and 2.16 times by these two elicitors. The level of 4-coumaric acid was little more than double by these two elicitors after 24 h elicitation. Most pronounced increase in lignin synthesis was also effected by FME followed by CHT. Lignin deposition in the root cell wall was increased 3.6, 5.4 and 7.1 times by FME during 12, 24 and 36 h after elicitation, respectively. Similarly, CHT increased lignin deposition by 2.8, 5.1 and 6.8 times at 12, 24 and 36 h after elicitation, respectively. FCF and TME also increased lignin deposition significantly in the cell walls of tomato roots during the above time periods of elicitation. Activity of phenylalanine ammonia lyase reached highest level at 24 h post elicitation under the influence of the elicitors. Peroxidase activity registered a sharp increase at 24 h post elicitation. Markedly increased level of polyphenol oxidase activity was found at 12 h post elicitation. Cinnamyl alcohol dehydrogenase activity was observed to reach highest level at 48 h post elicitation. Cell wall strengthening, through the deposition of lignin, preceded by the induction of the synthesizing enzymes appears to play an important role in the defense response of Lycopersicon esculentum in reaction to elicitors, including one derived from Fusarium oxysporum f. sp. lycopersici, the causal organism of Fusarium wilt of tomato.

Effect of cadmium on the phenolic compounds formation in the callus cultures derived from various organs of the tea plant

The effects of cadmium (6.3 × 10−5 M or 10.6 × 10−5 M) on the growth of tea plant (Camellia sinensis L.) callus cultures derived from leaves, stems, and roots and on the formation, in these cultures, of phenolic compounds, including flavans and lignin, which are characteristic of the tea plant, were investigated. In the calli derived from leaves and stems, cadmium treatment decreased the biomass increment, while in the calli derived from roots, growth characteristics remained at the control level. Under the effect of cadmium, the content of phenolic compounds, including flavans, in the leaf calli decreased, while in the stem and root calli, it either increased (at the cadmium concentration of 6.3 × 10−5 M), or was close to a control one (at the cadmium concentration of 10.6 × 10−5 M). The lignin content in the root and stem calli increased, but it did not change in the leaf calli. All this data demonstrate that the cadmium-induced changes in phenolic metabolism of the tea plant callus culture depended both on the cadmium concentration in the medium and on the origin of calli.

Induction of phenolic compounds in Hypericum perforatum L. cells by Colletotrichum gloeosporioides elicitation

Changes in phenolic metabolism after elicitation with Colletotrichum gloeosporioides (CG) has been studied in Hypericum perforatum L. (HP) cell suspension cultures. Soluble phenolics were analysed by HPLC–DAD and HPLC–DAD–MS/MS. HP cultures elicited with the CG elicitor showed a significant increase in xanthone accumulation. Xanthone accumulation increased twelve fold when the cells were primed with methyl-jasmonate (MeJ) or salicylic acid (SA), before elicitation. HP cultures exposed only to MeJ produced a set of flavonoids, the flavones which represent a substantial part (approx. 40%) of the total flavonoids accumulated in these cells. The possible importance of xanthones as a component of defence mechanism of HP against biotic stress is discussed.

Biochemical Response of Soybean Roots to Fusarium solani f. sp. glycines Infection

The soil‐borne fungus Fusarium solani (Mart.) Sacc. f. sp. glycines (FSG) infects soybean [Glycine max (L.) Merr.] roots and causes the disease sudden death syndrome (SDS). The biochemical response of soybean roots to FSG infection, which has not been studied before, was investigated by comparing FSG‐inoculated and noninoculated roots of two partially resistant (‘PI 520733’ and ‘PI 567374’) and susceptible (‘Spencer’) genotypes. Activity of phenylalanine ammonia‐lyase (PAL), the first enzyme in the phenylpropanoid biosynthetic pathway, was increased in inoculated roots of all three genotypes. The phytoalexin glyceollin increased to much higher levels in inoculated roots of the partially resistant cultivars PI 520733 and PI 567374 than in the susceptible Spencer. The changes in phenolic metabolism were localized in lesion‐containing areas of roots rather than in the new portion growing under the FSG inoculum. No clear correlation was found between the glyceollin precursor daidzein (4′,7‐dihydroxyisoflavone) and its conjugates and glyceollin levels in root tissues; however, isoflavone levels increased only in roots of inoculated plants of partially resistant lines, even though constitutive isoflavone levels were higher in the susceptible control. The FSG growth on potato dextrose agar medium was inhibited by increasing concentrations of glyceollin. Induction of lignin synthesis was found in the inoculated roots of all three lines, with the highest rate of lignification observed in roots of the partially resistant genotypes, especially PI 567374. These studies show for the first time that FSG inoculation of soybean roots in soil induces the phenylpropanoid pathway to synthesize isoflavones, the phytoalexin glyceollin, and lignin, indicating that these compounds may be involved in the partial resistance response.

Biochemical Response of Soybean Roots to f. sp. Infection

The soil-borne fungus Fusarium solani (Mart.) Sacc. f. sp. glycines (FSG) infects soybean [Glycine max (L.) Merr.] roots and causes the disease sudden death syndrome (SDS). The biochemical response of soybean roots to FSG infection, which has not been studied before, was investigated by comparing FSG-inoculated and noninoculated roots of two partially resistant (‘PI 520733’ and ‘PI 567374’) and susceptible (‘Spencer’) genotypes. Activity of phenylalanine ammonia-lyase (PAL), the first enzyme in the phenylpropanoid biosynthetic pathway, was increased in inoculated roots of all three genotypes. The phytoalexin glyceollin increased to much higher levels in inoculated roots of the partially resistant cultivars PI 520733 and PI 567374 than in the susceptible Spencer. The changes in phenolic metabolism were localized in lesion-containing areas of roots rather than in the new portion growing under the FSG inoculum. No clear correlation was found between the glyceollin precursor daidzein (4′,7-dihydroxyisoflavone) and its conjugates and glyceollin levels in root tissues; however, isoflavone levels increased only in roots of inoculated plants of partially resistant lines, even though constitutive isoflavone levels were higher in the susceptible control. The FSG growth on potato dextrose agar medium was inhibited by increasing concentrations of glyceollin. Induction of lignin synthesis was found in the inoculated roots of all three lines, with the highest rate of lignification observed in roots of the partially resistant genotypes, especially PI 567374. These studies show for the first time that FSG inoculation of soybean roots in soil induces the phenylpropanoid pathway to synthesize isoflavones, the phytoalexin glyceollin, and lignin, indicating that these compounds may be involved in the partial resistance response.

Changes in phenolic metabolism of tobacco plants during short-term boron deficiency

The effects of boron (B) deficiency on several phenolics and enzyme activities involved in the biosynthesis of these compounds were investigated in tobacco plants ( Nicotiana tabacum L. cv. Gatersleben). The levels of phenylpropanoids (mainly the caffeic acid esters, chlorogenic acid and its isomers) as well as phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and polyphenoloxidase (PPO, EC 1.14.18.1) activities were determined in plants subjected to B starvation for 1–7 d. The results presented here show that a short-term B deficiency causes both quantitative and qualitative changes in the phenolic metabolism of tobacco plants, which are especially evident after 3 d of B starvation. Although the concentration of B decreased from the onset of B starvation, root B level was less affected than leaf B by a short-term B deficiency. The concentration of phenylpropanoids as well as PAL and PPO activities increased mainly in the leaves of tobacco plants during B starvation. Moreover, leaves starved of B for 7 d showed the accumulation of new compounds, one of which was identified as caffeoylputrescine. In addition, a positive correlation between PAL activity and phenylpropanoid concentration was observed in tobacco leaves, especially after 5–7 d of B starvation, suggesting that an increase in PAL activity during B starvation could be responsible for the enhancement in the levels of phenylpropanoids.

Kinetin regulation of growth and secondary metabolism in waterlogging and salinity treated Vigna sinensis and Zea mays

Waterlogging mostly increased fresh weight and water content in shoots and roots of Vigna sinensis and Zea mays while salinity seemed to have a decreasing effect. There was a marked induction of proline in shoots and roots of both plants by salinity with lower values in logged plants. In addition, anthocyanin content was increased in Vigna sinensis by both treatments and in Zea mays only by salinity. Meanwhile the treatments significantly accumulated phenolic compounds in plant shoots. Also there were increased activities of phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) in shoots and roots of both plants. Foliar application of kinetin equilibrated, if any, the effects of both treatments on contents of proline, anthocyanin and phenolic compounds as well as activities of PAL and TAL in shoots and roots of treated plants. These findings reveal that kinetin alleviates the stress symptoms and regulates the changes in phenolic metabolism of waterlogged or salinity treated Vigna sinensis and Zea mays.

Wound induced changes in phenolic metabolism and tissue browning are altered by heat shock

Wounding is one of many abiotic stresses that produce signals that propagate from injured into adjacent non-injured tissues and induce the de novo synthesis of specific wound-induced proteins. Some of these induced proteins are enzymes of phenolic metabolism, such as phenylalanine ammonia-lyase (PAL), whose increased activity leads to the accumulation of phenolic compounds (e.g., chlorogenic acid, dicaffeoyl tartaric acid and isochlorogenic acid) and tissue browning. Wounding of iceberg lettuce leaves increases PAL activity six- to 12-fold over 24 h at 10°C and leads to a three-fold increase in the total phenolic content within 3 days. There may be a hierarchical order to the plant's response to different abiotic stresses. Plant tissue simultaneously exposed to a heat shock and wounding responds to the heat shock in preference to wounding by producing heat shock proteins instead of PAL. A 90 s, 45°C heat shock prevents an increase in PAL activity if administered either 4 h before or 2 h after wounding. This diversion of wound-induced protein synthesis by heat shock might be used to prevent browning in other crops that normally have low phenolic content; e.g., celery and lettuce. The persistence of the ability of a heat shock to preferentially induce the synthesis of heat shock proteins (hsps) in place of wound-induced enzymes of phenylpropanoid metabolism offers a new way to control browning in lightly processed fruits and vegetables. The design of processing lines using a heat shock to extend the shelf-life of fresh-cut lettuce will need to be modified from existing designs to take full advantage of the effect of the heat treatment.

473 Beneficial Effect of Heat-shock Treatments on Lettuce Applied before and after Wounding

Changes in phenolic metabolism are induced by minimally processing, which ultimately leads to the browning of lettuce tissue. Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5.) is greatly influenced by storage temperature. Evaluation of PAL activity at temperatures going from 0 to 25 °C showed that peaks occurred sooner at higher temperatures but at lower levels. Heat-shock treatments (50 °C, 90 s) have a protective effect against browning, help to retain greenness of tissue, and decrease the production of phenolics when applied either after or before wounding. To achieve a considerable, beneficial effect from hot water treatments applied after wounding these should not be delayed more than 36 h. The best results for heat-shock treatments before wounding occurred when applied at ≈12 h before cutting the tissue. Although cycloheximide did reduce PAL activity in a similar pattern as heat-shock treatments, it did not prevent browning itself. Cycloheximide seems to cause some sort of chemical damage that promotes the browning of lettuce tissue. When cycloheximide was applied in combination with heat-shock treatments browning did not occur.

Effect of Selected Browning Inhibitors on Phenolic Metabolism in Stem Tissue of Harvested Lettuce

Wound-induced changes in phenolic metabolism causes stem browning (butt discoloration) in harvested lettuce. Stem tissue near the harvesting cut exhibited increased phenylalanine ammonia-lyase (PAL) activity and accumulation of caffeic acid derivatives. These o-diphenols can be oxidized by the enzyme polyphenol oxidase (PPO) to produce brown pigments. This browning reaction can readily be followed by measuring a* values. Browning was reduced by washing stem disks with solutions of 0.3 M calcium chloride, 1.0 mM 2,4-dichlorophenoxyacetic acid (2,4-D), or 0.5 M acetic acid. These browning inhibitors appear to act in different ways. Calcium chloride decreased PAL activity to 60% of the control, but did not substantially affect the accumulation of phenolic compounds. The mechanism of calcium action could be to decrease PPO activity or to preserve membrane structure. PAL activity was inhibited 60% by 2,4-D, and the biosynthesis of phenolic compounds was strongly inhibited but not suppressed. Acetic acid completely inhibited PAL activity and the production of wound-induced phenolics. PAL was irreversibly inhibited by acetic acid, and this may explain its role as a browning inhibitor. Keywords: Lettuce; Lactuca sativa; postharvest storage; butt discoloration; phenolic metabolism; PAL; browning inhibition

Association between increased phenylalanine ammonialyase activity and cinnamic acid synthesis and the induction of temporary inaccessibility caused by Erysiphe graminis primary germ tube penetration of the barley leaf

The interactions between barley leaves and compatible or incompatible races of Erysiphe graminis whose primary germ tubes (PGTs) had penetrated the host prior to appressorial penetration and E. pisi (which has no PGTs) were investigated for changes in phenolic metabolism during the early stages of the infection process. Cinnamic acid synthesis and the elevation of phenylalanine ammonia-lyase (PAL) activity were examined over the period during which PGT penetration of the leaves occurred. Increased synthesis of cinnamic acid began 3–4 h after inoculation with E. graminis, reached a maximum at 4–5 h, and then decreased. PAL activity began to increase 2 h after inoculation, reached a maximum at 4 h, and then declined in leaves inoculated with E. graminis. Neither the synthesis of cinnamic acid nor the activity of PAL was significantly affected by inoculation with E. pisi, or by treatment with heat-killed conidia of E. graminis. The fact that the state of temporary inaccessibility of host cells which is produced by PGT penetration coincides with the time of the increase of PAL activity and the increased synthesis of cinnamic acid suggests that both phenomena are closely associated.