Annamária Kósa

Reactive oxygen species from type‐I photosensitized reactions contribute to the light‐induced wilting of dark‐grown pea (Pisum sativum) epicotyls

Type-II, singlet oxygen-mediated photosensitized damage has already been shown to occur in epicotyls of dark-germinated pea (Pisum sativum L.) seedlings upon illumination, resulting in fast turgor loss and wilting. In this study we show evidence that the palette of reactive oxygen species (ROS) is more complex. Hydrogen peroxide, superoxide and hydroxyl radicals are also formed, suggesting the occurrence of type-I reactions as well. Moreover, hydrogen peroxide injection into the epicotyls in the dark was able to provoke wilting directly. Formation of hydroxyl radicals could also be triggered by the addition of hydrogen peroxide in the dark, preferentially in the mid-sections where wilting occurs, showing that potential mediators of a Fenton reaction are present in the epicotyls, but unevenly distributed. Localization of light-inducible ROS formation fully (hydrogen peroxide) or partially (superoxide radicals) overlaps with the distribution of monomer protochlorophyllide complexes, showing that these pigment forms are capable of provoking both type-I and type-II reactions.

Comparative evaluation of the effect of cyclodextrins and pH on aqueous solubility of apigenin

The aqueous solubility of a flavonoid, apigenin, was studied in the presence of first generation cyclodextrins (α-CyD, β-CyD, γ-CyD), ionic and nonionic synthetic derivatives of β-CyD, namely SBE-β-CyD, HP-β-CyD and RM-β-CyD at various physiological pH. The order of solubility enhancement was as follows: RM-β-CyD>SBE-β-CyD>γ-CyD>HP-β-CyD>β-CyD>α-CyD. The phase solubility diagrams of HP-β-CyD and SBE-β-CyD indicated Higuchi AL subtype behavior, suggesting 1:1 stoichiometry of the complex. In contrast, AP subtype, so higher order complex formation can be assumed in the case of RM-β-CyD and γ-CyD. The formation of inclusion complexes has been confirmed by absorption and fluorescence spectroscopic measurements. Increased antioxidant activity was observed due to the inclusion complexes. These results prove that synthetic derivatives of β-CyD will be potentially useful excipients in the development of drug delivery systems for healthcare products containing flavonoids.

Artificial Tripartite Symbiosis Involving a Green Alga (Chlamydomonas), a Bacterium (Azotobacter) and a Fungus (Alternaria): Morphological and Physiological Characterization

A long-living artificial tripartite symbiosis involving a green alga (Chlamydomonas), a bacterium (Azotobacter) and a fungus (Alternaria) was established on carbon- and nitrogen-free medium. The basis of the interdependence is the complementation of photosynthetic CO2 assimilation and atmospheric nitrogen fixation. Green color of the colonies indicated that the algal cells had enough nitrogen to synthesize chlorophylls. The chlorophyll content was nearly 40 % of the control cells. The relatively high rate of photosynthetic oxygen evolution proved that nitrogen was effectively used for building up a well functioning photosynthetic apparatus. This was supported by the analysis of photosystems and ultrastructural investigations. In comparison with degreened algae cultured on nitrogen-free medium, the chloroplasts in the symbiont algal cells contained a well-developed, stacked thylakoid membrane system without extreme starch or lipid accumulation. The occurrence of the fungus in the association greatly increased the chlorophyll content. Far fewer types of amino acids were excreted by the tripartite cultures than by pure cultures. Cystathionine, which is a common intermediate in the sulfur-containing amino acid metabolism, was produced in high quantities by the tripartite symbiosis. This can mostly be attributed to the activity of the fungus.

Simultaneous specific in planta visualization of root-colonizing fungi using fluorescence in situ hybridization (FISH)

In planta detection of mutualistic, endophytic, and pathogenic fungi commonly colonizing roots and other plant organs is not a routine task. We aimed to use fluorescence in situ hybridization (FISH) for simultaneous specific detection of different fungi colonizing the same tissue. We have adapted ribosomal RNA (rRNA) FISH for visualization of common mycorrhizal (arbuscular- and ectomycorrhiza) and endophytic fungi within roots of different plant species. Beside general probes, we designed and used specific ones hybridizing to the large subunit of rRNA with fluorescent dyes chosen to avoid or reduce the interference with the autofluorescence of plant tissues. We report here an optimized efficient protocol of rRNA FISH and the use of both epifluorescence and confocal laser scanning microscopy for simultaneous specific differential detection of those fungi colonizing the same root. The method could be applied for the characterization of other plant–fungal interactions, too. In planta FISH with specific probes labeled with appropriate fluorescent dyes could be used not only in basic research but to detect plant colonizing pathogenic fungi in their latent life-period.

Etioplasts with protochlorophyll and protochlorophyllide forms in the under-soil epicotyl segments of pea (Pisum sativum) seedlings grown under natural light conditions†

To study if etiolation symptoms exist in plants grown under natural illumination conditions, under-soil epicotyl segments of light-grown pea (Pisum sativum) plants were examined and compared to those of hydroponically dark-grown plants. Light-, fluorescence- and electron microscopy, 77 K fluorescence spectroscopy, pigment extraction and pigment content determination methods were used. Etioplasts with prolamellar bodies and/or prothylakoids, protochlorophyll (Pchl) and protochlorophyllide (Pchlide) forms (including the flash-photoactive 655 nm emitting form) were found in the (pro)chlorenchyma of epicotyl segments under 3 cm soil depth; their spectral properties were similar to those of hydroponically grown seedlings. However, differences were found in etioplast sizes and Pchlide:Pchl molar ratios, which indicate differences in the developmental rates of the under-soil and of hydroponically developed cells. Tissue regions closer to the soil surface showed gradual accumulation of chlorophyll, and in parallel, decrease of Pchl and Pchlide. These results proved that etioplasts and Pchlide exist in soil-covered parts of seedlings even if they have a 3-4-cm long photosynthetically active shoot above the soil surface. This underlines that etiolation symptoms do develop under natural growing conditions, so they are not merely artificial, laboratory phenomena. Consequently, dark-grown laboratory plants are good models to study the early stages of etioplast differentiation and the Pchlide-chlorophyllide phototransformation.

Structural and functional changes in the photosynthetic apparatus of Chlamydomonas reinhardtii during nitrogen deprivation and replenishment

Nitrogen is an essential factor for normal plant and algal development. As a component of nucleic acids, proteins, and chlorophyll (Chl) molecules, it has a crucial role in the organization of a functioning photosynthetic apparatus. Our aim was to study the effects of nitrogen starvation in cultures of the unicellular green alga, Chlamydomonas reinhardtii, maintained on nitrogen-free, and then on nitrogen-containing medium. During the three-week-long degreening process, considerable changes were observed in the Chl content, the ratio of Chl-protein complexes, and photosynthetic activity of the cultures as well as in the ultrastructure of single chloroplasts. The regreening process was much faster then the degradation; total greening of the cells occurred within four days. The rate of regeneration depended on the nitrogen content. At least 50% of the normal nitrogen content of Tris-Acetate-Phosphate (TAP) medium was required in the medium for the complete regreening of the cells and regeneration of chloroplasts.

Preferential regeneration of the NADPH: protochlorophyllide oxidoreductase oligomer complexes in pea epicotyls after bleaching

The regeneration and stability of the NADPH:protochlorophyllide oxidoreductase (POR, EC 1.3.1.33) enzyme complexes were studied in bleached epicotyls of 9-day-old dark-germinated pea (Pisum sativum L. cv. Zsuzsi) seedlings. Middle segments were illuminated with 1300 micromol m(-2) s(-1) photon flux density (PFD) white light and subsequently incubated in total darkness for 4-24 h at 24 degrees C. Almost the full amount of protochlorophyllide (Pchlide) was degraded after 60 min illumination. The preferential regeneration of the 655 nm emitting Pchlide form was observed after 4 h dark incubation; the accumulation of the short-wavelength Pchlide form-dominating in epicotyls of dark-grown seedling-required 18-24 h dark. The Pchlide content of bleached samples was around 2.5% of that of the etiolated samples; after 4 h of dark incubation this value increased to 4-7%. Polyacrylamide gel electrophoresis and western blot showed that the amount of the POR protein decreased to about 50% during bleaching; after 4 h regeneration it reached almost the same level as that of dark-grown samples. We concluded that much more POR protein compared with Pchlide pigment remained stable during bleaching and the non-destroyed POR units were able to form preferentially oligomers during the dark-regeneration which could collect de novo synthesized Pchlide into 655 nm emitting complexes. These data indicate the high stability of the POR protein in pea epicotyls and the importance of the molecular environment in stimulating the aggregation of POR units.

Biological variability in the ratios of protochlorophyllide forms in leaves and epicotyls of dark-grown pea (Pisum sativum L.) seedlings (A statistical method to resolve complex spectra)

Low-temperature (77K) fluorescence emission spectra of 100 dark-grown pea (Pisum sativum L.) seedlings of various ages were measured. The spectra of the 100 leaf samples were collected into a separate data group and those of epicotyls formed another one. This group was divided into three sub-groups as spectra of uppermost, middle and lowermost 3 cm sections. Further sub-groups were formed on the basis of the ages of the plants. The spectra were normalized to their total integral values (within the 580-780 nm region) then the AVERAGE (arithmetic mean function) and AVEDEV (average of the absolute deviations of data points of their mean function) spectra were calculated. Very sharp bands were found in the AVEDEV spectra. Even the strongly overlapped 629 and 636 nm emission bands appeared as separate peaks, due to the decrease of their half-bandwidth values in the AVEDEV function. Both types of spectra were resolved into Gaussian components. The results showed that the variabilities of the 633 and 655 nm protochlorophyllide forms were similar in the leaves. In epicotyls, the protochlorophyllide forms had different variabilities, especially in the middle sections. The most variable was the amplitude of the 636 nm band and the variabilities of the 629 and 655 nm bands were smaller but still remarkable. The calculation of AVEDEV spectra is an effective method to study the biological variability and spectral resolution of biological samples containing chromophores with multiple spectral properties.

Study on the Pulmonary Delivery System of Apigenin-Loaded Albumin Nanocarriers with Antioxidant Activity

BACKGROUND Respiratory diseases are mainly derived from acute and chronic inflammation of the alveoli and bronchi. The pathophysiological mechanisms of pulmonary inflammation mainly arise from oxidative damage that could ultimately lead to acute lung injury. Apigenin (Api) is a natural polyphenol with prominent antioxidant and anti-inflammatory properties in the lung. Inhalable formulations that consist of nanoparticles (NPs) have several advantages over other administration routes, and therefore, this study investigated the application of apigenin-loaded bovine serum albumin nanoparticles (BSA-Api-NPs) for pulmonary delivery. METHODS Dry powder formulations of BSA-Api-NPs were prepared by spray drying and characterized by laser diffraction particle sizing, scanning electron microscopy, differential scanning calorimetry, and powder X-ray diffraction. The influence of dispersibility enhancers (lactose monohydrate and l-leucine) on the in vitro aerosol deposition using a next-generation impactor was investigated in comparison to excipient-free formulation. The dissolution of Api was determined in simulated lung fluid by using the Franz cell apparatus. The antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH⋅) free radical scavenging assay. RESULTS The encapsulation efficiency and the drug loading were measured to be 82.61% ± 4.56% and 7.51% ± 0.415%. The optimized spray drying conditions were suitable to produce particles with low residual moisture content. The spray-dried BSA-Api-NPs possessed good aerodynamic properties due to small and wrinkled particles with low mass median aerodynamic diameter, high emitted dose, and fine particle fraction. The aerodynamic properties were enhanced by leucine and decreased by lactose, however, the dissolution was reversely affected. The DPPH⋅ assay confirmed that the antioxidant activity of encapsulated Api was preserved. CONCLUSION This study provides evidence to support that albumin nanoparticles are suitable carriers of Api and the use of traditional or novel excipients should be taken into consideration. The developed BSA-Api-NPs are a novel delivery system against lung injury with potential antioxidant activity.

Nitrogen deficiency hinders etioplast development in stems of dark-grown pea (Pisum sativum) shoot cultures

The effects of nitrogen (N) deprivation were studied in etiolated pea plants (Pisum sativum cv. Zsuzsi) grown in shoot cultures. The average shoot lengths decreased and the stems significantly altered considering their pigment contents, 77 K fluorescence spectra and ultrastructural properties. The protochlorophyllide (Pchlide) content and the relative contribution of the 654-655 nm emitting flash-photoactive Pchlide form significantly decreased. The etioplast inner membrane structure characteristically changed: N deprivation correlated with a decrease in the size and number of prolamellar bodies (PLBs). These results show that N deficiency directly hinders the pigment production, as well as the synthesis of other etioplast inner membrane components in etiolated pea stems.