High-Nitrate-Supply-Induced Transcriptional Upregulation of Ascorbic Acid Biosynthetic and Recycling Pathways in Cucumber.

Nowadays open field and protected vegetable cultivation practices require and use genotypes which are precisely tailored to their intended growth environments. Variability of this kind provides a rich source of material to uncover molecular mechanisms supporting the necessarily divergent physiological traits. In this study, typical field-optimized and glasshouse-cultivated cucumber F1 hybrids were investigated, and displayed slower growth ('Joker') and faster growth ('Oitol') in seedlings. Antioxidant capacity was lower in 'Joker' and higher in 'Oitol', pointing to a potential redox regulation of growth. The growth response of seedlings to paraquat treatment indicated stronger oxidative stress tolerance in the fast-growing 'Oitol'. To test whether protection against nitrate-induced oxidative stress was also different, fertigation with increasing potassium nitrate content was applied. This treatment did not change growth but decreased the antioxidant capacities of both hybrids. Bioluminescence emission revealed stronger lipid peroxidation triggered by high nitrate fertigation in the leaves of 'Joker' seedlings. To explore the background of the more effective antioxidant protection of 'Oitol', levels of ascorbic acid (AsA), as well as transcriptional regulation of relevant genes of the Smirnoff-Wheeler biosynthetic pathway and ascorbate recycling, were investigated. Genes related to AsA biosynthesis were strongly upregulated at an elevated nitrate supply in 'Oitol' leaves only, but this was only reflected in a small increase in total AsA content. High nitrate provision also triggered expression of ascorbate-glutathion cycle genes with stronger or exclusive induction in 'Oitol'. AsA/dehydro-ascorbate ratios were higher in 'Oitol' for all treatments, with a more pronounced difference at high nitrate levels. Despite strong transcriptional upregulation of ascorbate peroxidase genes (APX) in 'Oitol', APX activity only increased significantly in 'Joker'. This suggests potential inhibition of APX enzyme activity specifically in 'Oitol' at a high nitrate supply. Our results uncover an unexpected variability in redox stress management in cucumbers, including nitrate inducibility of AsA biosynthetic and recycling pathways in certain genotypes. Possible connections between AsA biosynthesis, recycling and nitro-oxidative stress protection are discussed. Cucumber hybrids emerge as an excellent model system for studying the regulation of AsA metabolism and the roles of AsA in growth and stress tolerance.

Modelling biophoton emission kinetics based on the initial intensity value in Helianthus annuus plants exposed to different types of stress.

Biophoton radiation also referred to as ultra-weak photon emission (UPE) is used to denote a spontaneous and permanent photon emission associated with oxidative processes in cells and seems to universally occur in all living systems as a result of the generation of reactive oxygen species (ROS) that are produced under stress conditions. The measurement of this biophoton emission allows for a non-invasive approach in monitoring phenological stages throughout plant development which has direct relevance in agriculture research. In this study, the emission of photons emanating from sunflower (Helianthus annuus, L.) plants exposed to biotic and abiotic stress has been investigated. In healthy plants raised under controlled growth conditions UPE was low whereas in stressed individuals it considerably increased; particularly upon water stress. The kinetics of the signal is shown to reveal an exponential decay with characteristic dynamics, which appears to reflect different physiological states concomitantly setting in upon stress. The dynamics of the signal decay is shown to vary according to the type of stress applied (biotic vs. abiotic) hence suggesting a putative relationship between the kinetic traits of change in the signal intensity-decay and stress. Intriguingly, the determination of the change in the intensity of biophoton emission that ensued in a short time course was possible by using the initial biophoton emission intensity. The predictability level of the equations demonstrated the applicability of the model in a corroborative manner when employing it in independent UPE-measurements, thus permitting to forecast the intensity change in a very accurate way over a short time course. Our findings allow the notion that albeit stress confers complex and complicated changes on oxidative metabolism in biological systems, the employment of biophoton imaging offers a feasible method making it possible to monitor oxidative processes triggered by stress in a non-invasive and label-free way which has versatile applications especially in precision agriculture.

Co-encapsulation of probiotic Lactobacillus acidophilus and Reishi medicinal mushroom (Ganoderma lingzhi) extract in moist calcium alginate beads.

Probiotic L. acidophilus La-14 cells were co-encapsulated with Ganoderma lingzhi extract to prolong the viability of the cells under simulated gastrointestinal (SGI) condition and to protect the active ingredients of Reishi mushroom during the storage period. Combinations of distinctive reagents (sodium alginate, chitosan, maltose, Hydroxyethyl-cellulose (HEC), hydroxypropyl methylcellulose (HPMC), and calcium lactate) were tested. Optimal double layer Ca-alginate hydrogel beads were fabricated with significantly improved characteristics. The incorporation of maltose significantly decreases the release rate of mushrooms' phenolics, antioxidants, and ß-glucan during the storage time. Significant improvement in probiotic cells viability under SGI condition has been found and confirmed by confocal laser microscopy in maltose containing double layer coated calcium alginate beads variants. The encapsulation of newly formulated prebiotic Reishi extract and probiotic L. acidophilus is creating a new potential food application for such medicinal mushrooms and natural products with unpleasant taste upon oral consumption.

A Non-Invasive Approach in the Assessment of Stress Phenomena and Impairment Values in Pea Seeds Caused by Pea Weevil.

Pea (Pisum sativum L.) is an important leguminous plant worldwide, in which pests trigger significant damage every year. One of the most important pest is pea weevil (Bruchus pisorum, L) which causes covert damage in crops. In the present study, our aim was to obtain precise information pertaining to the extent and the nature of damage in pea caused by B. pisorum by means of non-invasive imaging methods. The infested pea samples were analysed by an infrared thermometer and a bioluminescence plant imaging system as well as a computer tomograph under laboratory conditions. The calculated weight of organic matter destroyed by the developing larvae was 36.46%. The changing of RGB (red, blue, green) codes obtained through thermal imaging and the CPS (counts per second) values originating from bioluminescence imaging in infested samples were statistically verifiable. According to our CT assay, the damage caused by B. pisorum changed the tissue density, volume and shape of the pea seeds by the end of the development of the pest. The results of thermal and bioluminescence imaging contribute to a better understanding of the internal chemical processes and the CT analysis helps to understand the alteration trends of the inner structure of seeds caused by this pest.

Downregulation of Polyamine and Diamine Oxidases in Silicon-Treated Cucumber.

Silicon (Si) is a ubiquitous element in soil with well-known beneficial effects under certain conditions, in several plant species, if supplied in available form for uptake. It may alleviate damage in various stress situations and may also promote growth when no obvious stressors are applied. Effects of Si are often linked to mitigation of oxidative stress, in particular to the induction of antioxidant defense mechanisms. In the work presented, the impact of silicon provision on pro-oxidant systems was investigated in cucumber. Plants of the F1 cultivar hybrid 'Joker' were grown under in vitro conditions in the absence of any applied external stressor. Silicon provision decreased H2O2 content and lowered lipid peroxidation in the leaves of the treated plants. This was paralleled by declining polyamine oxidase (PAO) and diamine oxidase (DAO) activities. Several PAO as well as lipoxygenase (LOX) genes were coordinately downregulated in Si-treated plants. Unlike in similar systems studied earlier, the Si effect was not associated with an increased transcript level of gene coding for antioxidant enzymes. These results suggest an inhibitory effect of Si provision on pro-oxidant amine oxidases, which may decrease the level of reactive oxygen species by retarding their production. This extends the molecular mechanisms linked to silicon effects onto redox balance in plants.

Effect of cadmium stress on certain physiological parameters, antioxidative enzyme activities and biophoton emission of leaves in barley (Hordeum vulgare L.) seedlings.

Biophoton emission is a well-known phenomenon in living organisms, including plant species; however, the underlying mechanisms are not yet well elucidated. Nevertheless, non-invasive stress detection is of high importance when in plant production and plant research. Therefore, the aim of our work was to investigate, whether biophoton emission is suitable for the detection of cadmium stress in the early phase of stress evolution and to identify certain stress-related events that occur rapidly upon cadmium exposure of barley seedlings parallel to biophoton emission measurements. Changes of biophoton emission, chlorophyll content estimation index, ascorbate level, the activity of ascorbate- and guaiacol peroxidase enzymes and lipid oxidation were measured during seven days of cadmium treatment in barley (Hordeum vulgare L.) seedlings. The results indicate that the antioxidant enzyme system responded the most rapidly to the stress caused by cadmium and the lipid oxidation-related emission of photons was detected in cadmium-treated samples as early as one day after cadmium exposure. Furthermore, a concentration-dependent increase in biophoton emission signals indicating an increased rate of antioxidative enzymes and lipid oxidation was also possible to determine. Our work shows evidence that biophoton emission is suitable to identify the initial phase of cadmium stress effectively and non-invasively.

Oxidative stress level and dehydrin gene expression pattern differentiate two contrasting cucumber F1 hybrids under high fertigation treatment.

Two cucumber F1 cultivar hybrids were investigated for stress tolerance markers upon application of different strength of Hoagland fertigation solutions (HG). 'Joker' and 'Oitol' cultivar hybrids were studied, representing typically field grown and greenhouse cultivated genotypes, respectively. At standard fertigation level (0.5 × HG) in controlled environment young 'Joker' plants displayed slower growth than 'Oitol' based on total leaf area. At this basal nutrient concentration leaves of 'Joker' plants had significantly lower antioxidant capacity and higher malondialdehyde (MDA, an indicator of lipid peroxidation) level than 'Oitol'. According to RT-qPCR transcript levels of several antioxidant enzymes' genes (ascorbate peroxidase, glutathione reductase and glutathione peroxidase) were significantly higher in 'Joker' compared to 'Oitol'. At increased HG concentrations (1.0, 1.5, 2.0, and 2.5 × HG) growth didn't change significantly in either hybrid. Osmotic potential declined at higher fertigation levels. Antioxidant capacity increased in both hybrids with strong characteristic differences favouring 'Oitol' plants. Higher MDA content of leaves testified more oxidative burden in 'Joker' plants at all and especially at the more concentrated HG treatments. This trend was also approved by results of bio photon emission imaging, which is a powerful method to quantify stress level in living tissues with autoluminescence detection technology. Gene expression for antioxidant enzymes followed HG concentration-dependent increase in both hybrids, at a substantially higher level in 'Joker'. Expression of the dehydrin gene DHN3 was preferentially induced at elevated fertigation levels in 'Oitol' plants, which could contribute to the lower oxidative stress detected in this hybrid. Results presented in this report demonstrate differences in shoot growth, antioxidant capacity, level of oxidative stress and antioxidant gene expression in two contrasting cucumber hybrids at basal fertigation. Furthermore, excessive HG fertigation was found to increase oxidative stress in a genotype-specific way. This effect may be due to different antioxidant capacity and differential expression of stress protective genes, such as the DHN3 dehydrin.

When isolated at full receptivity, in vitro fertilized wheat (Triticum aestivum, L.) egg cells reveal [Ca2+]cyt oscillation of intracellular origin.

During in vitro fertilization of wheat (Triticum aestivum, L.) in egg cells isolated at various developmental stages, changes in cytosolic free calcium ([Ca2+]cyt) were observed. The dynamics of [Ca2+]cyt elevation varied, reflecting the difference in the developmental stage of the eggs used. [Ca2+]cyt oscillation was exclusively observed in fertile, mature egg cells fused with the sperm cell. To determine how [Ca2+]cyt oscillation in mature egg cells is generated, egg cells were incubated in thapsigargin, which proved to be a specific inhibitor of the endoplasmic reticulum (ER) Ca2+-ATPase in wheat egg cells. In unfertilized egg cells, the addition of thapsigargin caused an abrupt transient increase in [Ca2+]cyt in the absence of extracellular Ca2+, suggesting that an influx pathway for Ca2+ is activated by thapsigargin. The [Ca2+]cyt oscillation seemed to require the filling of an intracellular calcium store for the onset of which, calcium influx through the plasma membrane appeared essential. This was demonstrated by omitting extracellular calcium from (or adding GdCl3 to) the fusion medium, which prevented [Ca2+]cyt oscillation in mature egg cells fused with the sperm. Combined, these data permit the hypothesis that the first sperm-induced transient increase in [Ca2+]cyt depletes an intracellular Ca2+ store, triggering an increase in plasma membrane Ca2+ permeability, and this enhanced Ca2+ influx results in [Ca2+]cyt oscillation.