Light dependent differentiation of outdoors developed purple eggplant (Solanum melongena L.) pericarp layers: Leaf chlorenchyma characteristics of the pericarp layers dissected in the dark.

To interpret the final steps of chlorophyll biosynthesis, detailed knowledge of etiolation symptoms is necessary. Most of our knowledge originates from studies on plant materials grown in complete darkness. Hardly any information is available about the plastid development in internal parenchyma cells of fleshy fruits in which the food supply is almost unlimited. In this work, etiolation symptoms were studied in pericarp layers of purple eggplant (Solanum melongena L.). Tissue layers of fruits developed under open-air conditions and of etiolated fruits were dissected in a dark room. Transmission and 77 K fluorescence spectroscopy and ultrastructural studies were performed. Photosynthetic activities were measured and pigment contents were determined in light-grown fruits. The purple exocarp and a 1-1.5 cm wide green mesocarp layer of large fruits fully shade the internal pericarp layers, thus protochloropyll (ide) accumulated, flash-photoactive 644 and 655 nm emitting protochlorophyllide complexes, and only small amounts of chlorophylls were found. Photosynthetic activity was detected only in the external, green layer, which had fully developed chloroplasts, and showed 77 K fluorescence emission spectra characteristic for green leaves. The innermost endocarp regions and the etiolated fruits contained mainly protochlorophyll (ide), proplastids, and etioplasts, i.e. they showed etiolation symptoms. These symptoms correspond to those of leaves of dark-grown seedlings but are stable for long periods due to the almost unlimited nourishment supply from storage parenchyma cells. These results prove that the laboratory works with artificially dark-developed plant materials are good models of natural chlorophyll biosynthesis and plastid development.

Ionic, not the osmotic component, is responsible for the salinity-induced inhibition of greening in etiolated wheat (Triticum aestivum L. cv. Mv Béres) leaves: a comparative study.

MAIN CONCLUSION: Greening was partially (in 300 mM NaCl, CaCl2, 600 mM KNO3 or KCl) or fully inhibited (in 600 mM NaCl, NaNO3 or NaCl:KCl) by the ionic and not the osmotic component of salinity. Although high soil salinity is an increasing global problem, not much is known about how direct exposure to salinity affects etiolated leaves of seedlings germinating in the soil and then reaching the surface. We investigated the effect of various salt treatments on the greening process of leaves in 8- to 11-day-old etiolated wheat (Triticum aestivum L. Mv. Béres) seedlings. Etiolated leaf segments pre-treated on different salt (600 mM NaCl:KCl 1:1, 600 mM NaCl, 600 mM KCl, 600 mM NaNO3, 600 mM KNO3, 300 mM KCl, 300 mM NaCl or 300 mM CaCl2) or isosmotic polyethylene glycol 6000 (PEG) solutions for 1.5 h in the dark and then greened for 16 h on the same solutions were studied. Leaf segments greened on PEG (osmotic stress) or on 300 mM KCl had similar chloroplasts compared to control samples greened on Hoagland solution. Slightly slower development of chloroplast structure and function (photosynthetic activity) was observed in segments greened on 300 mM NaCl or CaCl2, 600 mM KNO3 or KCl. However, etioplast-to-chloroplast transformation and chlorophyll accumulation were fully inhibited and peculiar prothylakoid swelling occurred in segments greened on 600 mM NaCl, NaNO3 or NaCl:KCl (1:1) solutions. The data indicate that not the high osmolarity of the used salt solution, but its ions, especially Na+, had the strongest negative impact on these processes.

Micro-scale Experimental System Coupled with Fluorescence-based Estimation of Fungal Biomass to Study Utilisation of Plant Substrates.

The degradation capacity and utilisation of complex plant substrates are crucial for the functioning of saprobic fungi and different plant symbionts with fundamental functions in ecosystems. Measuring the growth capacity and biomass of fungi on such systems is a challenging task. We established a new micro-scale experimental setup using substrates made of different plant species and organs as media for fungal growth. We adopted and tested a reliable and simple titration-based method for the estimation of total fungal biomass within the substrates using fluorescence-labelled lectin. We found that the relationship between fluorescence intensity and fungal dry weight was strong and linear but differed among fungi. The effect of the plant organ (i.e. root vs. shoot) used as substrate on fungal growth differed among plant species and between root endophytic fungal species. The novel microscale experimental system is useful for screening the utilisation of different substrates, which can provide insight into the ecological roles and functions of fungi. Furthermore, our fungal biomass estimation method has applications in various fields. As the estimation is based on the fungal cell wall, it measures the total cumulative biomass produced in a certain environment.

Distinct UV-A or UV-B irradiation induces protochlorophyllide photoreduction and bleaching in dark-grown pea (Pisum sativum L.) epicotyls.

The effects of distinct UV-A and UV-B radiations were studied on etiolated pea (Pisum sativum L.) epicotyls. Emission spectra of the native protochlorophyll and protochlorophyllide forms were measured when epicotyls were excited with 360 or 300 nm light. The UV-A (360 nm) excited mainly the non-enzyme-bound monomers of protochlorophyll and protochlorophyllide and the UV-B (300 nm) excited preferentially the flash-photoactive protochlorophyllide complexes. These latter complexes converted into short- and long-wavelength chlorophyllide forms at 10-s illumination with both wavelength irradiations. As the spectral changes were very small, the effects of longer illumination periods were studied. Room temperature fluorescence emission spectra were measured from the same epicotyl spots before and after irradiation with various wavelengths between 280 and 360 nm for 15 min and the "illuminated" minus "dark" difference spectra were calculated. Both the UV-A and the UV-B irradiations caused photoreduction of protochlorophyllide into chlorophyllide. At 10 µmol photons m-2 s-1, the photoreduction rates were similar, however, at 60 µmol photons m-2 s-1, the UV-B irradiation was more effective in inducing chlorophyllide formation than the UV-A. The action spectra of protochlorophyllide plus protochlorophyll loss and chlorophyllide production showed that the radiation around 290 nm was the most effective in provoking protochlorophyllide photoreduction and the UV light above 320 nm caused strong bleaching. These results show that the effect of the UV radiation should be considered when discussing the protochlorophyllide-chlorophyllide photoreduction during germination and as a part of the regeneration of the photosynthetic apparatus proceeding in the daily run of photosynthesis.

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.

Wavelength-dependent photooxidation and photoreduction of protochlorophyllide and protochlorophyll in the innermost leaves of cabbage (Brassica oleracea var. capitata L.).

The photoreduction and photooxidation processes of different protochlorophyll(ide) forms were studied in the innermost leaves of cabbage (Brassica oleracea var. capitata L.) under monochromatic irradiations. Room-temperature fluorescence emission spectra were measured from the same leaf spots before and after illumination to follow the wavelength dependence of the photochemical reactions. Short-wavelength light of 7 µmol photons m(-2) s(-1) (625-630 nm) provoked mainly bleaching, and longer wavelengths (630-640 nm) caused both bleaching and photoreduction, while above 640 nm resulted in basically photoreduction. When bleached leaves were kept in darkness at room temperature, all protochlorophyll(ide) forms regenerated during 72 h. Oxygen-reduced environment decreased the extent of bleaching suggesting the involvement of reactive oxygen species. These results confirm that the short-wavelength, 628 nm absorbing, and 633 nm emitting protochlorophyll(ide) form in etiolated cabbage leaves sensibilizes photooxidation. However, the 628 nm light at low intensities stimulates the photoreduction of the longer wavelength protochlorophyllide forms. Kinetic measurements showed that photoreduction saturates at a low PFD (photon flux density) compared to bleaching, suggesting that the quantum yield of photoreduction is higher than that of bleaching.

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, ß-CyD, γ-CyD), ionic and nonionic synthetic derivatives of ß-CyD, namely SBE-ß-CyD, HP-ß-CyD and RM-ß-CyD at various physiological pH. The order of solubility enhancement was as follows: RM-ß-CyD>SBE-ß-CyD>γ-CyD>HP-ß-CyD>ß-CyD>α-CyD. The phase solubility diagrams of HP-ß-CyD and SBE-ß-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-ß-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 ß-CyD will be potentially useful excipients in the development of drug delivery systems for healthcare products containing flavonoids.

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.

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.

Dominance of a 675 nm chlorophyll(ide) form upon selective 632.8 or 654 nm laser illumination after partial protochlorophyllide phototransformation.

The phototransformation pathways of protochlorophyllide forms were studied in 8-14-day-old leaves of dark-germinated wheat (Triticum aestivum L.) using white, 632.8 nm He-Ne laser and 654 nm laser diode light. The photon flux density (PFD) values (0.75-360 µmol photons m(-2) s(-1)), the illumination periods (20 ms-10 s) and the temperature of the leaves (between -60 °C and room temperature) were varied. The 77 K fluorescence spectra of partially phototransformed leaves showed gradual accumulation or even the dominance of the 675 nm emitting chlorophyllide or chlorophyll form at room temperature with 632.8 nm of PFD less than 200 µmol photons m(-2) s(-1) or with 654 nm of low PFD (7.5 µmol photons m(-2) s(-1)) up to 1 s. Longer wavelength (685 or 690 nm) emitting chlorophyllide forms appeared at illuminations under -25 °C with both laser lights or at room temperature when the PFD values were higher or the illumination period was longer than above. We concluded that the formation of the 675 nm emitting chlorophyllide form does not indicate the direct photoactivity of the 633 nm emitting protochlorophyllide form; it can derive from 644 and 657 nm forms via instantaneous disaggregation of the newly-produced chlorophyllide complexes. The disaggregation is strongly influenced by the molecular environment and the localization of the complex.

[Pharmaceutical and formulation aspects of Petroselinum crispum extract]. / Petroselinum crispum kivonatának gyógyszerészeti vonatkozásai és formulálási lehetoségei.

Parsley (Petroselinum crispum L.) is a very popular spice and vegetable in Europe, it is widely spread and easy to grow. It's herb and fruits are known to be diuretic, smooth muscle relaxant and hepatoprotective. The most important identified active ingredients are flavonoids, cumarins and vitamin C. Apigenin and its glycosides are the main flavonoids in parsley, it can be found relatively large amounts in the leaves. The bioactive flavonoid apigenin has antiinflammatory, antioxidant and anticancer activities. The objectives of this study were the preparation and detemination of the apigenin content of the parsley extract and the formulation using inert pellets by layering the apigenin in fluid-bed process.

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.

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.

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.

Aggregation of the 636 nm emitting monomeric protochlorophyllide form into flash-photoactive, oligomeric 644 and 655 nm emitting forms in vitro.

Artificial formation of flash-photoactive oligomeric protochlorophyllide complexes was found in etiolated pea (Pisum sativum L. cv. Zsuzsi) epicotyl homogenates containing glycerol (40% v/v) and sucrose (40% m/v). The 77 K fluorescence emission spectra indicated that the ratio of the 644 and 655 nm emitting forms to the 636 nm form increased during 3 to 5-day incubation in the dark at -14 degrees C. Electron micrographs showed the presence of well-organized prolamellar bodies in the homogenates. The same phenomena were found when the homogenates were frozen into liquid nitrogen and thawed to room temperature in several cycles. Similar treatments of intact epicotyl pieces caused significant membrane destructions. In homogenates, the in vitro produced 644 and 655 nm emitting protochlorophyllide forms were flash-photoactive; the extent of phototransformation increased compared to that in native epicotyls. The newly appeared 692 nm chlorophyllide band showed a blue shift (similar to the Shibata shift in leaves), however this process took place only partially due to the effect of the isolation medium. These results prove that the in vitro accumulated 644 and 655 nm protochlorophyllide forms were produced from the flash-photoactive 636 nm emitting monomeric NADPH:protochlorophyllide oxidoreductase units via aggregation, in connection with structure stabilization properties of glycerol and sucrose.