In vitro antioxidative and binding properties of phenolics in traditional, citrus and exotic fruits.

Many polyphenols bind proteins, therefore our research was focused on the potential of protein binding to polyphenols of investigated fruits and their health-related effects. The contents of polyphenols and related antioxidant activities of traditional, citrus and exotic fruits were compared. The presence of polyphenols (flavonoids and phenolic acids) in the investigated samples and their interaction with human serum albumin (HSA) was studied by HPLC, Fourier Transform Infrared (FT-IR) and three dimensional fluorescence spectroscopy (3D-FL). The highest levels of polyphenols, antioxidant and binding capacities were found in red and blond grapefruits (citrus group), followed by strawberries and apples (traditional group) and mangosteen and kiwi fruit (exotic fruit), which also contained the highest levels of protocatechuic, p-coumaric, ferulic acids and quercetin. In conclusion, for the first time, the interaction of the polyphenols with human serum albumin was evaluated by fluorometry/FTIR. The obtained binding profiles allowed the comparison of three different groups of fruits. A mixture of these fruits can be recommended for consumption.

Effect of drought and combined drought and heat stress on polyamine metabolism in proline-over-producing tobacco plants.

The roles of proline and polyamines (PAs) in the drought stress responses of tobacco plants were investigated by comparing the responses to drought alone and drought in combination with heat in the upper and lower leaves and roots of wild-type tobacco plants and transformants that constitutively over-express a modified gene for the proline biosynthetic enzyme Δ1-pyrroline-5-carboxylate synthetase (P5CSF129A; EC 2.7.2.11/1.2.1.41). In both genotypes, drought stress coincided with a decrease in relative water content (RWC) that was much less severe in the upper leaves than elsewhere in the plant. The drought also increased proline levels in both genotypes. A brief period of heat stress (2 h at 40 °C) at the end of the drought period did not significantly influence the proline levels in the upper leaves and roots but caused a further increase in the lower leaves of both genotypes. The rate at which these elevated proline levels returned to normal during the post-stress recovery period was slower in the transformants and plants that had been subjected to the combined stress. In both genotypes, drought stress significantly reduced the levels of spermidine (Spd) and putrescine (Put) in the leaves and roots relative to those for controls, and increased the levels of spermine (Spm) and diaminopropane (Dap, formed by the oxidative deamination of Spd and Spm). Spd levels may have declined due to its consumption in Spm biosynthesis and/or oxidation by polyamine oxidase (PAO; EC 1.5.3.11) to form Dap, which became more abundant during drought stress. During the rewatering period, the plants' Put and Spd levels recovered quickly and the activity of the PA biosynthesis enzymes in their leaves and roots increased substantially; this increase was more pronounced in transformants than WT plants. The high levels of Spm observed in drought stressed plants persisted even after the 24 h recovery and rewatering phase. The malondialdehyde (MDA) contents of the lower leaves of WTs increased substantially during the drought stress period; a less pronounced increase occurred in the transformants and after the application of the combined stress. After the post-stress recovery period, the MDA contents in the leaves of both genotypes were higher than those in the corresponding controls. The MDA contents of the upper leaves in plants of both genotypes remained relatively constant throughout, indicating that these leaves are preferentially protected against the adverse effects of oxidative stress and demonstrating the efficiency of the plants' induced antioxidative defense mechanisms.

Effect of heat stress on polyamine metabolism in proline-over-producing tobacco plants.

The effect of heat stress on the accumulation of proline and on the level of polyamines (PAs) in tobacco plants was investigated. Responses to heat stress were compared in the upper and lower leaves and roots of tobacco plants that constitutively over-express a modified gene for the proline biosynthetic enzyme Δ1-pyrroline-5-carboxylate synthetase (P5CSF129A) and in the corresponding wild-type. In the initial phases of heat stress (after 2h at 40°C), the accumulation of proline increased in the wild type but slightly decreased in the transformants. The response to heat stress in proline-over-producing tobacco plants involved a transient increase in the levels of free and conjugated putrescine (Put) and in the levels of free spermidine (Spd), norspermidine (N-Spd) and spermine (Spm) after a 2-h lag phase, which correlated with stimulation of the activity of the corresponding biosynthetic enzymes. Diamine oxidase (DAO) activity increased in both plant genotypes, most significantly in the leaves of WT plants. Polyamine oxidase (PAO) activity increased in the roots of WT plants and decreased in the leaves and roots of the transformants. After 6h of heat stress, proline accumulation was observed in the transformants, especially in the lower leaves; much more modest increase was observed in the WT plants. A decrease in the levels of free and conjugated Put coincided with down-regulation of the activity of ornithine decarboxylase and marked stimulation of DAO activity in the leaves and roots of the transformants. PAO activity increased in the roots of the transformants but decreased in the leaves. Conversely, in WT tobacco subjected to 6h of heat stress, slight increases in free and conjugated PA levels were observed and the activity of DAO only increased in the roots; PAO activity did not change from the value observed during the initial phase of heat stress. 6 Hours' heat stress had no effect on the level of malondialdehyde (MDA; a product of lipid peroxidation), in the upper leaves of either genotype. After a recovery period (2h at 25°C), most of the studied parameters exhibited values comparable to those observed in untreated plants. The coordination of the proline and polyamine biosynthetic pathways during heat stress conditions is discussed.

Cryotolerance in Norway spruce and its association with growth rates, anatomical features and polyamines of embryogenic cultures.

Our study focused on the possible association between the cryotolerance of Norway spruce (Picea abies (L.) Karst.) embryogenic cultures and the anatomical structures of their embryogenic suspensor mass (ESM), their growth rate and their content of endogenous polyamines (PAs). The anatomical characteristics and PA content during cryopreservation and regrowth were studied in the ESMs of AFO 541 and C110 cultures, which have comparable ESM anatomy but diverse growth rates, PA content and regeneration abilities after cryopreservation. Different levels of tolerance to exogenous treatment were already apparent after transfer of the ESMs to liquid media. The endogenous free PAs were maintained at high levels, with spermidine being the predominant PA in the ESM of AFO 541, while in the ESM of C110 the content of putrescine and spermidine was almost identical and rather low, the content of spermidine being approximately one-third that in the ESM of AFO 541. Osmotic pretreatment, using a double application of sorbitol followed by an application of dimethyl sulfoxide (DMSO) resulted in the continual disintegration of polyembryogenic centers and suspensors in both cell lines. A continual decrease in the level of PAs was observed during the cell osmotic pretreatment. The cells that retained their viability and regrowth ability after cryopreservation were the meristematic cells inside the embryonal heads and the cells in the intermediate area between suspensor and meristems. Restoration of AFO 541 growth after cryopreservation was almost immediate; however, the C110 ESM culture regrew with difficulty, often exhibiting callogenesis. High levels of PA-soluble conjugates and an increase in the amount of PAs bound to high-molecular-mass substances was observed in cells of AFO 541 on Day 6 after thawing and also to some extent on Day 11. On Day 21 after thawing, the amount of free putrescine and spermidine in the AFO 541 cells reached the level observed in the suspension culture before the cryotreatment. The extremely low level of PAs determined in the ESM of C110 3 weeks after thawing agreed with the cell viability and rate of regrowth observed in this culture. The possible role of PAs in the process of cryopreservation of Norway spruce cultures is discussed.

Horseweed with reduced susceptibility to glyphosate found in the czech republic.

The physiological and molecular basis of apparent resistance to glyphosate in horseweed (Conyza canadensis L. Cronq.) plants that had survived being sprayed with the herbicide at Prague-Bubny railway station in the Czech Republic was investigated. For the sake of comparison, plants expected to be susceptible were collected in areas where no herbicides had been used. Plants of both sets were treated, at the rosette stage (10-25 leaves, diameter of 3-5 cm), with herbicide at the rate recommended for use in the Czech Republic to control horseweed (960 g of glyphosate-IPA/ha; Roundup Klasik, Monsanto, 480 g of glyphosate-IPA ae L(-1)). Phytotoxic symptoms of the treated plants varied substantially, both between and within these sets of plants. Leaves of susceptible (S) plants wilted and turned yellow, and the plants subsequently died; leaves of plants with reduced susceptibility (RS) remained green, or new leaves were created in the center of their rosettes a few weeks after glyphosate application. There were no significant differences in the accumulation of shikimate between S and RS plants 3 days after treatment (DAT). However, the time course of changes in shikimic acid contents differed between the two biotypes; from 3 to 10 DAT, they decreased more than 4-fold in RS plants, while in S plants, they increased (3-fold, on average) from 3 to 7 DAT. A conserved region of the epsps gene, in which mutations are known to confer resistance in several plant species, was amplified from samples of both S and RS plants and sequenced, but no changes in the encoded amino acid sequence were found, indicating that mutations at another epsps site were responsible for the observed resistance, or that the mechanism may be at least partially non-target-based. Our results suggest that the reduced susceptibility to glyphosate may be due to impaired herbicide translocation, as previously found in studies of horseweed in the United States.

Polyamine profiles and biosynthesis in somatic embryo development and comparison of germinating somatic and zygotic embryos of Norway spruce.

The polyamine (PA) contents and activities of PA biosynthetic enzymes in Norway spruce somatic embryos [Picea abies L. (Karst.), genotype AFO 541] were studied in relation to anatomical changes during their development, from proliferation to germination, and changes in these variables associated with the germination of mature somatic and zygotic embryos were compared. Activities of PA biosynthetic enzymes steadily increased during the development of somatic embryos, from embryogenic suspensor mass until early cotyledonary stages. In these stages, the spermidine (Spd) level was significantly higher than the putrescine (Put) level, and the increases coincided with the sharp increases in S-adenosylmethionine decarboxylase activity in the embryos. The biosynthetic enzyme activity subsequently declined in mature cotyledonary embryos, accompanied by sharp reductions in PA contents, especially in cellular Put contents in embryos from 6 weeks old through the desiccation phase (although the spermine level significantly increased during the desiccation phase), resulting in a shift in the Spd/Put ratio from ca. 2 in early cotyledonary embryos to around 10 after 3 weeks of desiccation. In mature zygotic embryos, Spd contents were twofold lower, but Put levels were higher, than in mature somatic embryos, hence their Spd/Put ratio was substantially lower (ca. 2, in both embryos and megagametophytes). In addition, the PA synthesis activity profiles in the embryos differed (ornithine decarboxylase and arginine decarboxylase activities predominating in mature somatic and zygotic embryos, respectively). The start of germination was associated with a rise in PA biosynthetic activity in the embryos of both origins, which was accompanied by a marked increase in Put contents in somatic embryos, resulting in the decline of Spd/Put ratio to about 2, similar to the ratio in mature and germinating zygotic embryos. The accumulation of high levels of PAs in somatic embryos may be causally linked to their lower germinability than in zygotic embryos.

Comparison of the main bioactive compounds and antioxidant activities in garlic and white and red onions after treatment protocols.

Polish garlic and white and red onions were subjected to blanching, boiling, frying, and microwaving for different periods of time, and then their bioactive compounds (polyphenols, flavonoids, flavanols, anthocyanins, tannins, and ascorbic acid) and antioxidant activities were determined. It was found that blanching and frying and then microwaving of garlic and onions did not decrease significantly the amounts of their bioactive compounds and the level of antioxidant activities ( P > 0.05). The HPLC profiles of free and soluble ester- and glycoside-bound phenolic acids showed that trans-hydroxycinnamic acids (caffeic, p-coumaric, ferulic, and sinapic) were as much as twice higher in garlic than in onions. Quercetin quantity was the highest in red onion among the studied vegetables. The electrophoretic separation of nonreduced garlic and onion proteins after boiling demonstrated their degradation in the range from 50 to 112 kDa.