Solid Phase Microextraction-Multicapillary Column-Ion Mobility Spectrometry (SPME-MCC-IMS) for Detection of Methyl Salicylate in Tomato Leaves.

Methyl salicylate (MeSA) is a plant-signaling molecule that plays an essential role in the regulation of plant responses to biotic and abiotic pathogens. In this work, solid phase microextraction (SPME) and a multicapillary column (MCC) are coupled to ion mobility spectrometry (IMS) to detect MeSA in tomato leaves. The SPME-MCC-IMS method provides two-dimensional (2D) separation by both MCC and IMS, based on the retention and drift times. The effect of the IMS polarity on the separation efficiency of MCCs was also investigated. In the positive polarity, ionization of MeSA resulted in [MeSA + H]+ formation while, in the negative, deprotonated ions, [MeSA - H]-, and the O2- adduct ion, [MeSA + O2]-, were formed. In the real sample analysis, the negative polarity operation resulted in the suppression of many matrix molecules and thus in the reduction of interferences. Four different SPME fibers were used for head space analysis, and four MCC columns were investigated. In the negative polarity, complete separation was achieved for all of the MCCs columns. The limits of detection (LODs) of 0.1 µg mL-1 and linear range of 0.25-12 µg mL-1 were obtained for the measurement of MeSA in a standard solution (H2O/CH3OH, 50:50) by the SPME-IMS method with a 5 min extraction time using an SPME with a PDMS fiber, in the negative mode of IMS. The MeSA contents of fresh tomato leaves were determined as 1.5-9.8 µg g-1, 24-96 h after inoculation by tomato mosaic ringspot virus (ToRSV).

Effect of silicon on root growth, ionomics and antioxidant performance of maize roots exposed to As toxicity.

Nowadays, one of the biggest challenges of plant physiology is to find out the ways how to mitigate negative impacts of abiotic stress on plants. It is the pollution of groundwater or soil by various metals and metalloids that significantly affects the quality of life. Both arsenic (As) and silicon (Si) are metalloids - while the first one is toxic in general, the latter one is considered as beneficial for plants suffering from various kinds of stresses. The aim of our work was to elucidate the growth and development of young maize (Zea mays L.) plants exposed to both of these metalloids simultaneously. Experiments were focused on the comparison of root growth and biomass allocation, changes in uptake of macro- and micronutrients, visualisation of free radicals along with monitoring of the dynamics of main antioxidant enzymes activity in roots. The results showed that increasing concentration of As (75 and 150 µM As) severely inhibited root length and the amount of biomass, and addition of Si (2.5 mM) to the medium containing As did not have a significant effect on root growth. Similarly, the application of Si did not influence the uptake of macro- and microelements into the roots (mainly Ca, P, K, Mo, Cu, Zn and Ni) which was mostly decreased due to As. On the other hand, Si significantly decreased the presence of both superoxide and hydrogen peroxide in roots that suffered from As toxicity. Although the overall growth of maize plants was not improved by Si amendment, we assume that Si might affect the functionality of key antioxidant enzymes in time, and in this way at least partially help to overcome negative effects of As on maize roots.

Cold Atmospheric Pressure Plasma Treatment of Maize Grains-Induction of Growth, Enzyme Activities and Heat Shock Proteins.

Zea mays L. is one of the most produced crops, and there are still parts of the world where maize is the basic staple food. To improve agriculture, mankind always looks for new, better methods of growing crops, especially in the current changing climatic conditions. Cold atmospheric pressure plasma (CAPP) has already showed its potential to enhance the culturing of crops, but it still needs more research for safe implementation into agriculture. In this work, it was shown that short CAPP treatment of maize grains had a positive effect on the vitality of grains and young seedlings, which may be connected to stimulation of antioxidant and lytic enzyme activities by short CAPP treatment. However, the prolonged treatment had a negative impact on the germination, growth, and production indexes. CAPP treatment caused the increased expression of genes for heat shock proteins HSP101 and HSP70 in the first two days after sowing. Using comet assay it was observed that shorter treatment times (30-120 s) did not cause DNA damage. Surface diagnostics of plasma-treated grains showed that plasma increases the hydrophilicity of the surface but does not damage the chemical bonds on the surface.

The Effects of Cold Atmospheric Pressure Plasma on Germination Parameters, Enzyme Activities and Induction of DNA Damage in Barley.

Climate change, environmental pollution and pathogen resistance to available chemical agents are part of the problems that the food industry has to face in order to ensure healthy food for people and livestock. One of the promising solutions to these problems is the use of cold atmospheric pressure plasma (CAPP). Plasma is suitable for efficient surface decontamination of seeds and food products, germination enhancement and obtaining higher yields in agricultural production. However, the plasma effects vary due to plasma source, treatment conditions and seed type. In our study, we tried to find the proper conditions for treatment of barley grains by diffuse coplanar surface barrier discharge, in which positive effects of CAPP, such as enhanced germination or decontamination effects, would be maximized and harmful effects, such as oxidation and genotoxic potential, minimized. Besides germination parameters, we evaluated DNA damage and activities of various germination and antioxidant enzymes in barley seedlings. Plasma exposure resulted in changes in germination parameters and enzyme activities. Longer exposures had also genotoxic effects. As such, our findings indicate that appropriate plasma exposure conditions need to be carefully optimized in order to preserve germination, oxidation balance and genome stability, should CAPP be used in agricultural practice.

Evaluation of the Impact of Cold Atmospheric Pressure Plasma on Soybean Seed Germination.

The present study aims to define the effects of Cold Atmospheric Pressure Plasma (CAPP) exposure on seed germination of an agriculturally important crop, soybean. Seed treatment with lower doses of CAPP generated in ambient air and oxygen significantly increased the activity of succinate dehydrogenase (Krebs cycle enzyme), proving the switching of the germinating seed metabolism from anoxygenic to oxygenic. In these treatments, a positive effect on seed germination was documented (the percentage of germination increased by almost 20% compared to the untreated control), while the seed and seedling vigour was also positively affected. On the other hand, higher exposure times of CAPP generated in a nitrogen atmosphere significantly inhibited succinate dehydrogenase activity, but stimulated lactate and alcohol dehydrogenase activities, suggesting anoxygenic metabolism. It was also found that plasma exposure caused a slight increment in the level of primary DNA damage in ambient air- and oxygen-CAPP treatments, and more significant DNA damage was found in nitrogen-CAPP treatments. Although a higher level of DNA damage was also detected in the negative control (untreated seeds), this might be associated with the age of seeds followed by their lower germination capacity (with the germination percentage reaching only about 60%).

Abundance of cysteine endopeptidase dionain in digestive fluid of Venus flytrap (Dionaea muscipula Ellis) is regulated by different stimuli from prey through jasmonates.

The trap of the carnivorous plant Venus flytrap (Dionaea muscipula) catches prey by very rapid closure of its modified leaves. After the rapid closure secures the prey, repeated mechanical stimulation of trigger hairs by struggling prey and the generation of action potentials (APs) result in secretion of digestive fluid. Once the prey's movement stops, the secretion is maintained by chemical stimuli released from digested prey. We investigated the effect of mechanical and chemical stimulation (NH4Cl, KH2PO4, further N(Cl) and P(K) stimulation) on enzyme activities in digestive fluid. Activities of ß-D-glucosidases and N-acetyl-ß-D-glucosaminidases were not detected. Acid phosphatase activity was higher in N(Cl) stimulated traps while proteolytic activity was higher in both chemically induced traps in comparison to mechanical stimulation. This is in accordance with higher abundance of recently described enzyme cysteine endopeptidase dionain in digestive fluid of chemically induced traps. Mechanical stimulation induced high levels of cis-12-oxophytodienoic acid (cis-OPDA) but jasmonic acid (JA) and its isoleucine conjugate (JA-Ile) accumulated to higher level after chemical stimulation. The concentration of indole-3-acetic acid (IAA), salicylic acid (SA) and abscisic acid (ABA) did not change significantly. The external application of JA bypassed the mechanical and chemical stimulation and induced a high abundance of dionain and proteolytic activity in digestive fluid. These results document the role of jasmonates in regulation of proteolytic activity in response to different stimuli from captured prey. The double trigger mechanism in protein digestion is proposed.

Cuscuta europaea plastid apparatus in various developmental stages: localization of THF1 protein.

It was generally accepted that Cuscuta europaea is mostly adapted to a parasitic lifestyle with no detectable levels of chlorophylls. We found out relatively high level of chlorophylls (Chls a+b) in young developmental stages of dodder. Significant lowering of Chls (a+b) content and increase of carotenoid concentration was typical only for ontogenetically more developed stages. Lower content of photosynthesis-related proteins involved in Chls biosynthesis and in photosystem formation as well as low photochemical activity of PSII indicate that photosynthesis is not the main activity of C. europaea plastids. Previously, it has been shown in other species that the Thylakoid Formation Protein 1 (THF1) is involved in thylakoid membrane differentiation, plant-fungal and plant-bacterial interactions and in sugar signaling with its preferential localization to plastids. Our immunofluorescence localization studies and analyses of haustorial plasma membrane fractions revealed that in addition to plastids, the THF1 protein localizes also to the plasma membrane and plasmodesmata in developing C. europaea haustorium, most abundantly in the digitate cells of the endophyte primordium. These results are supported by western blot analysis, documenting the highest levels of the THF1 protein in "get together" tissues of dodder and tobacco. Based on the fact that photosynthesis is not a typical process in the C. europaea haustorium and on the extra-plastidial localization pattern of the THF1, our data support rather other functions of this protein in the complex relationship between C. europaea and its host.

Nutritional benefit from leaf litter utilization in the pitcher plant Nepenthes ampullaria.

The pitcher plant Nepenthes ampullaria has an unusual growth pattern, which differs markedly from other species in the carnivorous genus Nepenthes. Its pitchers have a reflexed lid and sit above the soil surface in a tighly packed 'carpet'. They contain a significant amount of plant-derived materials, suggesting that this species is partially herbivorous. We tested the hypothesis that the plant benefits from leaf litter utilization by increased photosynthetic efficiency sensu stricto cost/benefit model. Stable nitrogen isotope abundance indicated that N. ampullaria derived around 41.7 ± 5.5% of lamina and 54.8 ± 7.0% of pitcher nitrogen from leaf litter. The concentrations of nitrogen and assimilation pigments, and the rate of net photosynthesis (A(N)), increased in the lamina as a result of feeding, but did not increase in the trap. However, maximal (F(v) /F(m)) and effective photochemical quantum yield of photosystem II (Φ(PSII)) were unaffected. Our data indicate that N. ampullaria benefits from leaf litter utilization and our study provides the first experimental evidence that the unique nitrogen sequestration strategy of N. ampullaria provides benefits in term of photosynthesis and growth.

On the mechanism underlying photosynthetic limitation upon trigger hair irritation in the carnivorous plant Venus flytrap (Dionaea muscipula Ellis).

Mechanical stimulation of trigger hairs on the adaxial surface of the trap of Dionaea muscipula leads to the generation of action potentials and to rapid leaf movement. After rapid closure secures the prey, the struggle against the trigger hairs results in generation of further action potentials which inhibit photosynthesis. A detailed analysis of chlorophyll a fluorescence kinetics and gas exchange measurements in response to generation of action potentials in irritated D. muscipula traps was used to determine the 'site effect' of the electrical signal-induced inhibition of photosynthesis. Irritation of trigger hairs and subsequent generation of action potentials resulted in a decrease in the effective photochemical quantum yield of photosystem II (Φ(PSII)) and the rate of net photosynthesis (A(N)). During the first seconds of irritation, increased excitation pressure in photosystem II (PSII) was the major contributor to the decreased Φ(PSII). Within ∼1 min, non-photochemical quenching (NPQ) released the excitation pressure at PSII. Measurements of the fast chlorophyll a fluorescence transient (O-J-I-P) revealed a direct impact of action potentials on the charge separation-recombination reactions in PSII, although the effect seems to be small rather than substantial. All the data presented here indicate that the main primary target of the electrical signal-induced inhibition of photosynthesis is the dark reaction, whereas the inhibition of electron transport is only a consequence of reduced carboxylation efficiency. In addition, the study also provides valuable data confirming the hypothesis that chlorophyll a fluorescence is under electrochemical control.

Interaction of galactoglucomannan oligosaccharides with auxin in mung bean primary root.

In the present paper timing of galactoglucomannan oligosaccharides (GGMOs) with exogenously added indole-3-butyric acid (IBA) action on early germination stage (24 h) and primary root elongation of mung bean (Vigna radiata (L.) Wilczek) has been studied. GGMOs inhibited primary root elongation induced by low concentration (10(-8) M) of IBA. This inhibition was considerably higher after preincubation with GGMOs compared with other timing experiments. The most intensive inhibition of elongation has been ascertained at the 10(-8) M concentration of GGMOs. On the other hand GGMOs stimulated this elongation inhibited by high IBA concentration (10(-4) M). This stimulation was the most intensive by simultaneous addition of IBA and GGMOs at the beginning of the experiment and subsequent seeds incubation in distilled water. Our results indicate competition between GGMOs and auxin. The root growth inhibition, induced by GGMOs and/or IBA, was accompanied by the increase of cell wall-associated peroxidase activity and by a higher number of peroxidase isoenzymes. The presence of different peroxidase isoenzymes in experiments with distinct treatment of GGMOs and IBA could indicate variations in the mechanism of interaction between GGMOs and IBA.

A novel insight into the regulation of light-independent chlorophyll biosynthesis in Larix decidua and Picea abies seedlings.

Light-independent chlorophyll (Chl) biosynthesis is a prerequisite for the assembly of photosynthetic pigment-protein complexes in the dark. Dark-grown Larix decidua Mill. seedlings synthesize Chl only in the early developmental stages and their Chl level rapidly declines during the subsequent development. Our analysis of the key regulatory steps in Chl biosynthesis revealed that etiolation of initially green dark-grown larch cotyledons is connected with decreasing content of glutamyl-tRNA reductase and reduced 5-aminolevulinic acid synthesizing capacity. The level of the Chl precursor protochlorophyllide also declined in the developing larch cotyledons. Although the genes chlL, chlN and chlB encoding subunits of the light-independent protochlorophyllide oxidoreductase were constitutively expressed in the larch seedlings, the accumulation of the ChlB subunit was developmentally regulated and ChlB content decreased in the fully developed cotyledons. The efficiency of chlB RNA-editing was also reduced in the mature dark-grown larch seedlings. In contrast to larch, dark-grown seedlings of Picea abies (L.) Karst. accumulate Chl throughout their whole development and show a different control of ChlB expression. Analysis of the plastid ultrastructure, photosynthetic proteins by Western blotting and photosynthetic parameters by gas exchange and Chl fluorescence measurements provide additional experimental proofs for differences between dark and light Chl biosynthesis in spruce and larch seedlings.