Spatial distribution of apoplastic antioxidative constituents in maize root.

Apoplastic antioxidative constituents (enzymes, primary and secondary metabolites, ROS) from different root zones of hydroponically grown maize (Zea mays L.) were investigated using a noninvasive isolation procedure: filter strip method. Filter strips were placed at specific positions on the root surface: apical zone (tip) and basal zone (base) to absorb apoplastic fluid. Three major classes of low-weight metabolites (organic acids, sugars, and phenolics) have been identified by HPLC-ECD. The longitudinal distribution of sugars and organic acids had the same pattern: higher concentration in the tip than the base, while it was vice versa for phenolics. The specific activities of guaiacol peroxidase, superoxide dismutase, and ascorbate peroxidase were higher in the apoplastic fluid from the root base than the tip, and their different isoforms were separated by isoelectric focusing. Electron paramagnetic resonance (EPR) spectroscopy coupled with the spin-trapping method using DEPMPO showed a persistent generation of hydroxyl radical in the root tip. In vivo EPR imaging of the whole maize root with membrane-permeable and impermeable aminoxyl spin-probes, enabling real-time detection of ROS formation within and outside the membranes, demonstrated ROS accumulation on the root surface, while endodermis and central cylinder were ROS free. For the first time in plant research, 2D EPR images enabled the direct demonstration of site-specific free radical production along the root. Highly sensitive analytical techniques combined with the filter strips, as a non-invasive tool, have increased our knowledge of metabolic processes occurring in the apoplast and their spatial-temporal changes in small regions of the intact root tissue.

Variations in polyamine conjugates in maize (Zea mays L.) seeds contaminated with aflatoxin B1: a dose-response relationship.

BACKGROUND: Cereal seeds, such as maize seeds, are frequently contaminated with aflatoxin B1 (AFB1), one of the most dangerous naturally occurring carcinogens. In plants, phenolamides are involved in biotic stress response. The data on variations of phenolamides in AFB1-containing seeds are limited. RESULTS: Five polyamine conjugates, including two spermidine and three putrescine conjugates, were tentatively identified in methanolic extracts, using HPLC-DAD-MS. The ratio of putrescine to spermidine conjugates changed with increasing AFB1 concentration in a logistic dose-response manner, with a ratio of below 1 up to a concentration of 51.51 µg kg-1 , and approximately 2.54 and 3 at higher concentrations of 177.4 and 308.13 µg kg-1 , respectively. The observed variations of the total antioxidant activity and the total phenolic content may support this biphasic behaviour of the seeds against AFB1 stress. CONCLUSIONS: The obtained data are a contribution to the understanding of the roles of polyamine conjugates in seed defence to increasing AFB1 concentrations. According to our knowledge, this study reports for the first time the biphasic response of maize seeds to increasing AFB1 contamination level, comprising the induction of polyamine conjugate accumulation and variation in the ratio of conjugates. This dose-response relationship may provide useful information in the field of agricultural and food chemistry as an indicator of AFB1 contamination level and, hence, for selecting an appropriate seed quality. © 2020 Society of Chemical Industry.

Revealing mechanisms of salinity tissue tolerance in succulent halophytes: A case study for Carpobrotus rossi.

Efforts to breed salt tolerant crops could benefit from investigating previously unexplored traits. One of them is a tissue succulency. In this work, we have undertaken an electrophysiological and biochemical comparison of properties of mesophyll and storage parenchyma leaf tissues of a succulent halophyte species Carpobrotus rosii ("pigface"). We show that storage parenchyma cells of C. rossii act as Na+ sink and possessed both higher Na+ sequestration (298 vs. 215 mM NaCl in mesophyll) and better K+ retention ability. The latter traits was determined by the higher rate of H+ -ATPase operation and higher nonenzymatic antioxidant activity in this tissue. Na+ uptake in both tissues was insensitive to either Gd3+ or elevated Ca2+ ruling out involvement of nonselective cation channels as a major path for Na+ entry. Patch-clamp experiments have revealed that Caprobrotus plants were capable to downregulate activity of fast vacuolar channels when exposed to saline environment; this ability was higher in the storage parenchyma cells compared with mesophyll. Also, storage parenchyma cells have constitutively lower number of open slow vacuolar channels, whereas in mesophyll, this suppression was inducible by salt. Taken together, these results provide a mechanistic basis for efficient Na+ sequestration in the succulent leaf tissues.

Uptake of vanadium and its intracellular metabolism by Coprinellus truncorum mycelial biomass.

BACKGROUND: Fungi absorb and solubilize a broad spectrum of heavy metals such as vanadium (V), which makes them a main route of its entry into the biosphere. V as vanadate (V5+) is a potential medical agent due to its many metabolic actions such as interaction with phosphates in the cell, and especially its insulin-mimetic activity. Antidiabetic activity of V-enriched fungi has been studied in recent years, but the biological and chemical bases of vanadium action and status in fungi in general are poorly understood, with almost no information on edible fungi. METHODS: This manuscript gives a deeper insight into the interaction of V5+ with Coprinellus truncorum, an edible autochthonous species widely distributed in Europe and North America. Vanadium uptake and accumulation as V5+ was studied by 51V NMR, while the reducing abilities of the mycelium were determined by EPR. 31P NMR was used to determine its effects on the metabolism of phosphate compounds, with particular focus on phosphate sugars identified using HPLC. RESULTS: Vanadate enters the mycelium in monomeric form and shows no immediate detrimental effects on intracellular pH or polyphosphate (PPc) levels, even when applied at physiologically high concentrations (20 mM Na3VO4). Once absorbed, it is partially reduced to less toxic vanadyl (V4+) with notable unreduced portion, which leads to a large increase in phosphorylated sugar levels, especially glucose-1-phosphate (G1P) and fructose-6-phosphate (F6P). CONCLUSIONS: Preservation of pH and especially PPc reflects maintenance of the energy status of the mycelium, i.e., its tolerance to high V5+ concentrations. Rise in G1P and F6P levels implies that the main targets of V5+ are most likely phosphoglucomutase and phosphoglucokinase(s), enzymes involved in early stages of G6P transformation in glycolysis and glycogen metabolism. This study recommends C. truncorum for further investigation as a potential antidiabetic agent.

Silicon alleviates iron deficiency in cucumber by promoting mobilization of iron in the root apoplast.

· Root responses to lack of iron (Fe) have mainly been studied in nutrient solution experiments devoid of silicon (Si). Here we investigated how Si ameliorates Fe deficiency in cucumber (Cucumis sativus) with focus on the storage and utilization of Fe in the root apoplast. · A combined approach was performed including analyses of apoplastic Fe, reduction-based Fe acquisition and Fe-mobilizing compounds in roots along with the expression of related genes. · Si-treated plants accumulated higher concentrations of root apoplastic Fe, which rapidly decreased when Fe was withheld from the nutrient solution. Under Fe-deficient conditions, Si also increased the accumulation of Fe-mobilizing compounds in roots. Si supply stimulated root activity of Fe acquisition at the early stage of Fe deficiency stress through regulation of gene expression levels of proteins involved in Fe acquisition. However, when the period of Fe deprivation was extended, these reactions further decreased as a consequence of Si-induced enhancement of the Fe status of the plants. · This work provides new evidence for the beneficial role of Si in plant nutrition and clearly indicates that Si-mediated alleviation of Fe deficiency includes an increase of the apoplastic Fe pool in roots and an enhancement of Fe acquisition.

Possible health impacts of naturally occurring uptake of aristolochic acids by maize and cucumber roots: links to the etiology of endemic (Balkan) nephropathy.

Aristolochic acids (AAs) are nephrotoxic and carcinogenic derivatives found in several Aristolochia species. To date, the toxicity of AAs has been inferred only from the effects observed in patients suffering from a kidney disease called "aristolochic acid nephropathy" (AAN, formerly known as "Chinese herbs nephropathy"). More recently, the chronic poisoning with Aristolochia seeds has been considered to be the main cause of Balkan endemic nephropathy, another form of chronic renal failure resembling AAN. So far, it was assumed that AAs can enter the human food chain only through ethnobotanical use (intentional or accidental) of herbs containing self-produced AAs. We hypothesized that the roots of some crops growing in fields where Aristolochia species grew over several seasons may take up certain amounts of AAs from the soil, and thus become a secondary source of food poisoning. To verify this possibility, maize plant (Zea mays) and cucumber (Cucumis sativus) were used as a model to substantiate the possible significance of naturally occurring AAs' root uptake in food chain contamination. This study showed that the roots of maize plant and cucumber are capable of absorbing AAs from nutrient solution, consequently producing strong peaks on ultraviolet HPLC chromatograms of plant extracts. This uptake resulted in even higher concentrations of AAs in the roots compared to the nutrient solutions. To further validate the measurement of AA content in the root material, we also measured their concentrations in nutrient solutions before and after the plant treatment. Decreased concentrations of both AAI and AAII were found in nutrient solutions after plant growth. During this short-term experiment, there were much lower concentrations of AAs in the leaves than in the roots. The question is whether these plants are capable of transferring significant amounts of AAs from the roots into edible parts of the plant during prolonged experiments.

Two-way reaction of versatile peroxidase with artificial lignin enhances low-molecular weight fractions.

In recent years, versatile peroxidase (VP) has emerged as a promising enzyme for biotechnological applications, as it can oxidize lignin without the external mediators. To gain insights into the breakdown process of artificial lignin by VP, reaction between the two was studied. Degradation products were fractionated using ultrafiltration and analyzed by RP- high performance liquid chromatography with mass detection (HPLC-MS) chromatography. Four fractions were obtained based on their molecular sizes: >10, 3-10, 1-3, and <1 kDa. Interestingly, while VP did not significantly alter the yields of these fractions, the chromatograms revealed the presence of oligomers with different molecular weights (MWs) resulting from the enzymatic activity. The VP exhibits a dual role in its enzymatic activity: both degrading and synthesizing these oligomers. This was confirmed by principal component analysis (PCA). The positive correlations were found between certain oligomers (D1 and D2, D5 and D6, as well as between D7, D10, T2, and T4), suggesting their simultaneous degradation. On the other hand, a negative correlation was found between the monomer and some oligomers (D7, D10, T2, and T4), indicating the decomposition of these oligomers into monomers. These findings shed light on the intricate interplay between VP and artificial lignin, offering valuable insights for potential applications in lignin valorization.

Phenolics and Sesquiterpene Lactones Profile of Red and Green Lettuce: Combined Effect of Cultivar, Microbiological Fertiliser, and Season.

The main goal of our study was to find an optimal combination of tested factors to achieve lettuce rich in bioactive compounds sustaining its pleasant taste. We examined three red and three green cultivars in a greenhouse using two microbiological fertilisers (EM Aktiv and Vital Tricho), and their combination. Plants were grown in three consecutive growing seasons (autumn, winter, and spring). Lactones accumulated in autumn, whereas phenolics' concentration rose during winter. Red cultivars showed higher phenolics and lactone content, where chicoric acid and luteolin-7-glucoside were the most abundant in the 'Gaugin' winter trial. Lactucopicrin was the predominant lactone among tested cultivars with the highest value in the red cultivar 'Carmesi'. Solely applicated, the fertiliser EM Aktiv and Vital Tricho led to significantly higher phenolic acid and dihydrolactucopicrin content, while combined, there were notably increased levels of all detected lactones. Application of single fertilisers had no effect on flavonoid content, while the combination even reduced it. A sensory analysis showed a negative correlation between overall taste and total sesquiterpene lactones, lactucopicrin, caffeoylmalic, and chlorogenic acid, indicating a less bitter taste with decreasing content of these compounds. Our findings indicate that the cultivar, fertiliser, and growing season jointly affected all of the tested parameters, highlighting the differences in the application of EM Aktiv, Vital Tricho, and their combination.

Sugars and Organic Acids in 25 Strawberry Cultivars: Qualitative and Quantitative Evaluation.

(1) The nutritional quality of strawberry (Fragaria × ananassa Duch) fruits, among others, is largely maintained by the presence of soluble sugars and organic acids. As the primary products of photosynthesis, they are energy depots in plants, necessary for the construction of cell constituents, but also serve as precursors of aromatic compounds and signaling molecules. (2) In this study, fruits of 25 strawberry cultivars were qualitatively and quantitatively characterized concerning individual sugars and organic acids by HPLC, FT-ICR-MS, and MS imaging analysis. In addition, the total quality index (TQI), as a novel mathematical model, was used to compare all individual parameters evaluated to obtain a quantitative single score, as an indicator of overall fruit quality. (3) Regardless of a large number of cultivars and monitored parameters that were studded, several cultivars stood out in terms of selected primary metabolites, such as 'Rumba', 'Jeny', and 'Sandra', while the latter had the best TQI score. (4) Intercultivar variations in sugars and organic acids profiles, along with other bioactive compounds, should be considered for selection of promising cultivars with improved naturally occurring nutraceutical traits. Besides the search for a pleasant taste, increased awareness of healthy nutrition resulted in heightening consumer demand for high-quality fruit.

Silicon ameliorates manganese toxicity in cucumber by decreasing hydroxyl radical accumulation in the leaf apoplast.

This work was focused on the role of silicon (Si) in amelioration of manganese (Mn) toxicity caused by elevated production of hydroxyl radicals (·OH) in the leaf apoplast of cucumber (Cucumis sativus L.). The plants were grown in nutrient solutions with adequate (0.5 µM) or excessive (100 µM) Mn concentrations with or without Si being supplied. The symptoms of Mn toxicity were absent in the leaves of Si-treated plants subjected to excess Mn, although the leaf Mn concentration remained extremely high. The apoplastic concentration of free Mn(2+) and H(2)O(2) of high Mn-treated plants was significantly decreased by Si treatment. Si supply suppressed the Mn-induced increased abundance of peroxidase (POD) isoforms in the leaf apoplastic fluid, and led to a rapid suppression of guaiacol-POD activity under excess Mn. The spin-trapping reagent 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide was used to detect ·OH by electron paramagnetic resonance spectroscopy. Although supplying Si markedly decreased the accumulation of ·OH in the leaf apoplast with excess Mn, adding monosilicic acid to the Mn(2+)/H(2)O(2) reaction mixture did not directly affect the Fenton reaction in vitro. The results indicate that Si contributes indirectly to a decrease in ·OH in the leaf apoplast by decreasing the free apoplastic Mn(2+), thus regulating the Fenton reaction. A direct inhibitory effect of Si on guaiacol-POD activity (demonstrated in vitro) may also contribute to decreasing the POD-mediated generation of ·OH.

The effects of iron deficiency on lead accumulation in Ailanthus altissima (Mill.) Swingle seedlings.

Understanding the effects of root Fe deficiency on the uptake and translocation of toxic metals can be important for improving the phytoremediation strategies of polluted soils. The present study investigated how Fe nutritional status affects the uptake and root-to-shoot-translocation of Pb in hydroponically grown seedlings of (Mill.) Swingle. The interactions of Fe deficiency and Pb were assessed by measuring the root Fe(III) reductase activity, carboxylic acids concentration in root exudates, root and shoot biomass, and accumulation of Pb and other metals (Fe, Zn, Mn, and Cu) in roots and leaves of Fe-sufficient (+Fe) and Fe-deficient (-Fe) plants. The results indicate that Fe deficiency induced 18-fold higher Fe(III) reductase activity in roots compared with +Fe plants, which was followed by increased root exudation of citric acid (28.2 ± 1.39 in +Fe and 498 ± 256.4 µmol g DW 2 h in -Fe plants). Iron deficiency also induces a significant decrease of root and shoot dry weight compared with the control +Fe plants, whereas 2-wk Pb (20 µM) treatment did not influence root and shoot growth. Iron-sufficient plants accumulated more Pb (56.8 ± 17.29 µg g) in leaves than -Fe plants (21.5 ± 8.10 µg g). Two weeks of exposure to Pb significantly decreased Fe(III) reductase activity and accumulation of Fe, Zn, and Mn in the roots of -Fe plants. It is hypothesized that 2 wk of root exposure to Pb blocks functioning of a specific Fe transport system activated under Fe deficiency.

Influence of silicon on polymerization process during lignin synthesis. Implications for cell wall properties.

Silicon (Si) is considered a beneficial element for plants, mostly accumulating in cell walls, where its location and content are primed by the chemistry and structure of lignin. It is unrevealed how Si interacts with the process of lignin formation in the CWs. We studied, in an in vitro system, the interaction of SiO2 with the peroxidase-catalyzed polymerization of a lignin monomer into the lignin model compound, imitating conditions of the last step of lignin formation. FTIR and fluorescence spectroscopy and microscopy showed that Si is bound to the final polymer, and the structure of the Si-DHP differs from pure DHP. Fluorescence spectroscopy showed that Si does not bind to the monomers, so Si probably inhibits the formation of the larger lignin fragments, as evidenced by HPLC-DAD, by binding to dimmers formed during DHP synthesis. The structural changes of the polymer are related to the changed proportion of the fractions of various MW. The enzyme catalyzing DHP synthesis was not inhibited by Si. HRP activity was increased in presence of Si except for 6 mM Si. This may indicate that the complex formed with Si and short oligomers activates the enzyme, and prevents the formation of the large fragments.

Rich in Phenolics-Strong Antioxidant Fruit? Comparative Study of 25 Strawberry Cultivars.

Phenolic compounds of 25 newly introduced strawberry cultivars were profiled using spectrophotometry, electron paramagnetic resonance (EPR) spectroscopy, and high-performance liquid chromatography-mass spectrometry. Total phenolic and anthocyanin content (TPC and TACY, respectively), as well as vitamin C, and concentrations of individual phenolic compounds in fruits were evaluated to identify the most promising cultivars according to their phenolic profile. The highest values of TPC, TACY, and vitamin C were recorded in 'Premy' (1.53 mg eq GA g-1 FW), 'Sandra' (30.60 mg eq Pg-3-g 100 g-1 FW), and 'Laetitia' (56.32 mg 100 g-1 FW), respectively. The DPPH and •OH radicals scavenging activity of fruit methanolic extracts was estimated using EPR spectroscopy. All cultivars are almost uniformly effective in the scavenging of •OH radical, while 'Tea', 'Premy', and 'Joly' were marked as highly potent cultivars (over 70%) in terms of DPPH-antiradical activity. Specific peroxidase activities were the highest in 'Garda', 'Federica', and 'Rumba' (0.11, 0.08, and 0.06 U mg-1 prot, respectively). 'Laetitia', 'Joly', 'Arianna', 'Tea', and 'Mila' cultivars were distinguished from others as the richest concerning almost all flavonoids and phenolic acids, including some other parameters of bioactivity. These cultivars could be recommended to consumers as functional fruit foods.

A comparative assessment of the potential of polysaccharide production and intracellular sugar composition within Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (W.Curt.:Fr.)P. Karst. (Aphyllophoromycetideae).

Ganoderma lucidum is a well-known medicinal mushroom species in which polysaccharides are one of the major sources of biological activity. The species was considered as a species-complex due to significant variations in morphological, biochemical, and genetic features among populations with a worldwide distribution. This fact was the basis for setting the aim of this research: to study intraspecific diversity in polysaccharide production and intracellular sugar composition among selected G. lucidum strains. The presence ofintraspecific diversity among 10 G. lucidum strains, from different areas worldwide, was noted. Values of produced mycelia biomass and intracellular polysaccharides were found in wide ranges (3.1 - 28.2 g L(-1) and 20.0 - 53.3 mg g(-1), respectively), while differences in extracellular polysaccharide amounts were minor (0.2 - 1.5 mg mL(-1)). The significant quantitative and qualitative differences in intracellular sugar composition were noted. Glucose was the predominant sugar in almost all strains except one (HAI 447), where sucrose was dominant. The potential of polysaccharide production and intracellular sugar composition could be one more taxonomic criterion for strain characterization within G. lucidum. The differences in intracellular sugar composition and proportions could be reflected in features of produced polysaccharides and also in their biological activities.

Antagonistic Interaction between Phosphinothricin and Nepeta rtanjensis Essential Oil Affected Ammonium Metabolism and Antioxidant Defense of Arabidopsis Grown In Vitro.

Phosphinothricin (PPT) is one of the most widely used herbicides. PTT targets glutamine synthetase (GS) activity in plants, and its phytotoxicity is ascribed to ammonium accumulation and reactive oxygen species bursts, which drives rapid lipid peroxidation of cell membranes. In agricultural fields, PPT is extensively sprayed on plant foliage; however, a portion of the herbicide reaches the soil. According to the present study, PPT absorbed via roots can be phytotoxic to Arabidopsis, inducing more adverse effects in roots than in shoots. Alterations in plant physiology caused by 10 days exposure to herbicide via roots are reflected through growth suppression, reduced chlorophyll content, perturbations in the sugar and organic acid metabolism, modifications in the activities and abundances of GS, catalase, peroxidase, and superoxide dismutase. Antagonistic interaction of Nepeta rtanjensis essential oil (NrEO) and PPT, emphasizes the existence of complex control mechanisms at the transcriptional and posttranslational level, which result in the mitigation of PPT-induced ammonium toxicity and in providing more efficient antioxidant defense of plants. Simultaneous application of the two agents in the field cannot be recommended; however, NrEO might be considered as the PPT post-treatment for reducing harmful effects of herbicide residues in the soil on non-target plants.

Transcriptome Profiling of the Potato Exposed to French Marigold Essential Oil with a Special Emphasis on Leaf Starch Metabolism and Defense against Colorado Potato Beetle.

Flower strips of French Marigold are commonly used pest repellents in potato fields. However, the effect of French Marigold volatiles on potato metabolism, physiology and induced defense is unknown. Thus, a microarray transcriptome analysis was performed to study the effects of French Marigold essential oil (EO) on laboratory-grown potato. After 8 h of exposure to EO, with gas chromatography/mass spectrometry (GC/MS)-detected terpinolene and limonene as dominant compounds, 2796 transcripts were differentially expressed with fold change >2 compared to expression in controls. A slightly higher number of transcripts had suppressed expression (1493 down- vs. 1303 up-regulated). Since transcripts, annotated to different photosynthesis-related processes, were mostly down-regulated, we selected a set of 10 genes involved in the leaf starch metabolism pathway, and validated microarray patterns using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Except for decreased synthesis and induced decomposition of starch granule in leaves, 8 h long EO exposure slightly elevated the accumulation of sucrose compared to glucose and fructose in subjected potato plants. An in vitro feeding bioassay with Colorado potato beetle showed that EO-induced alternations on transcriptional level and in the sugars' metabolism caused the enhancement of feeding behavior and overall development of the tested larvae. Results of comprehensive analysis of transcriptional responses in potato exposed to French Marigold EO provide a basis for further elucidation of molecular mechanisms underlying eco-physiological interactions in companion planting cropping systems.

Cell wall response to UV radiation in needles of Picea omorika.

The UV-B represents the minor fraction of the solar spectrum, while UV-C is not contained in natural solar radiation, but both radiation types can cause damaging effects in plants. Cell walls (CWs) are one of the targets for external stressors. Juvenile P. omorika trees were treated either with 21 day-high doses UV-B or with 7 day- UV-C in open-top chambers. Using spectroscopic and biochemical techniques, it was shown that the response to UV radiation includes numerous modifications in needle CW structure: relative content of xylan, xyloglucan, lignin and cellulose decreased; cellulose crystallinity changed; yield of lignin monomers with stronger connection of CC in side chain with the ring increased; re-distribution of inter- and intra-polymer H-bonds occurred. The recovery was mediated by an increase in the activities and changes in isoform profiles of CW bound covalent peroxidases (POD) and polyphenol oxidases (PO) (UV-B), and ionic POD and covalent PO (UV-C). A connection between activities of specific POD/PO isoforms and phenolic species (m- and p-coumaric acid, pinoresinol and cinnamic acid derivatives) was demonstrated, and supported by changes in the sRNA profile. In vivo fluorometry showed phenolics accumulation in needle epidermal CWs. These results imply transversal connections between polymers and changed mechanical properties of needle CW as a response to UV. The CW alterations enabled maintenance of physiological functions, as indicated by the preserved chlorophyll content and/or organization. The current study provides evidence that in conifers, needle CW response to both UV-B and UV-C includes biochemical modifications and structural remodeling.

Biochemical and histological characterization of succulent plant Tacitus bellus response to Fusarium verticillioides infection in vitro.

We present changes in Tacitus bellus antioxidative system that specifically correspond to subsequent phases of hemibiotroph Fusarium verticillioides infection revealed by histological analysis. T. bellus response to spore germination 6 h post inoculation (hpi), manifested as first oxidative burst, was characterized by transient decrease in malondialdehyde (MDA) content, transient increase in catalase (CAT), low level of superoxide dismutase (SOD) and peroxidase (POD) activity, as well as with transient decrease in total antioxidant capacity (TAC), total phenol content (TPC) and phenylalanine ammonium lyase activity (PAL), and no changes in polyphenol oxidase (PPO) activity, or phenolic profile. During the biotrophic phase of F. verticillioides infection, characterized by hyphae spread intercellularly in epidermal and mesophyll tissue, the host antioxidative system was suppressed. The transition to necrotrophic phase of F. verticillioides infection (inter- and intracellular colonization and sporulation), occurred 3-4 days post inoculation (dpi). During the necrotrophic phase, 5-7 dpi, slowed progression of colonization of T. bellus mesophyll cells occurred and it coincided with sharp increase in MDA content and CAT, SOD and POD activities, but the drop in TAC, TPC content, and PPO activity, as well as the production of phytotoxin fusaric acid. Presented results add to the knowledge of events and mechanisms related to the transition from biotrophy to necrotrophy in F. verticillioides.

Phenolic Profiling of 12 Strawberry Cultivars Using Different Spectroscopic Methods.

Phenolic compounds of 12 strawberry cultivars were profiled using spectrophotometry, spectrofluorometry, and high-performance liquid chromatography-mass spectrometry, coupled with multivariate regression analysis. Total phenolic content, total anthocyanin content, and total antioxidant capacity (TPC, TACY, and TAC, respectively) and concentrations of individual phenolics were evaluated, and the multivariate statistic was employed to identify the most promising cultivars based on their phenolic content. According to the principal component analysis, TAC was strongly correlated with the TPC (0.81), pointing out its importance in overall antioxidant activity. 'Joly', 'Laetitia', and 'Asia' cultivars were distinguished from others as the richest in concerning identified anthocyanins, almost all flavonoids and phenolic acids. Multivariate curve resolution with alternating least squares indicated the presence of two main types of fluorophores assigned to anthocyanins and phenolics, in which emission spectral ratios also showed the highest values in the referred cultivars. These cultivars could be recommended to consumers in terms of the health functionality of fruit.

Generation of hydroxyl radical in isolated pea root cell wall, and the role of cell wall-bound peroxidase, Mn-SOD and phenolics in their production.

The hydroxyl radical produced in the apoplast has been demonstrated to facilitate cell wall loosening during cell elongation. Cell wall-bound peroxidases (PODs) have been implicated in hydroxyl radical formation. For this mechanism, the apoplast or cell walls should contain the electron donors for (i) H(2)O(2) formation from dioxygen; and (ii) the POD-catalyzed reduction of H(2)O(2) to the hydroxyl radical. The aim of the work was to identify the electron donors in these reactions. In this report, hydroxyl radical (.OH) generation in the cell wall isolated from pea roots was detected in the absence of any exogenous reductants, suggesting that the plant cell wall possesses the capacity to generate .OH in situ. Distinct POD and Mn-superoxide dismutase (Mn-SOD) isoforms different from other cellular isoforms were shown by native gel electropho-resis to be preferably bound to the cell walls. Electron paramagnetic resonance (EPR) spectroscopy of cell wall isolates containing the spin-trapping reagent, 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO), was used for detection of and differentiation between .OH and the superoxide radical (O(2)(-).). The data obtained using POD inhibitors confirmed that tightly bound cell wall PODs are involved in DEPMPO/OH adduct formation. A decrease in DEPMPO/OH adduct formation in the presence of H(2)O(2) scavengers demonstrated that this hydroxyl radical was derived from H(2)O(2). During the generation of .OH, the concentration of quinhydrone structures (as detected by EPR spectroscopy) increased, suggesting that the H(2)O(2) required for the formation of .OH in isolated cell walls is produced during the reduction of O(2) by hydroxycinnamic acids. Cell wall isolates in which the proteins have been denaturated (including the endogenous POD and SOD) did not produce .OH. Addition of exogenous H(2)O(2) again induced the production of .OH, and these were shown to originate from the Fenton reaction with tightly bound metal ions. However, the appearance of the DEPMPO/OOH adduct could also be observed, due to the production of O(2)(-). when endogenous SOD has been inactivated. Also, O(2)(-). was converted to .OH in an in vitro horseradish peroxidase (HRP)/H(2)O(2) system to which exogenous SOD has been added. Taken together with the discovery of the cell wall-bound Mn-SOD isoform, these results support the role of such a cell wall-bound SOD in the formation of .OH jointly with the cell wall-bound POD. According to the above findings, it seems that the hydroxycinnamic acids from the cell wall, acting as reductants, contribute to the formation of H(2)O(2) in the presence of O(2) in an autocatalytic manner, and that POD and Mn-SOD coupled together generate .OH from such H(2)O(2).