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).

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.

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.

Fermentation of pectin and glucose, and activity of pectin-degrading enzymes in the rabbit caecal bacterium Bacteroides caccae.

AIMS: To compare fermentation pattern in cultures of Bacteroides caccae supplied with pectin and glucose, and identify enzymes involved in metabolism of pectin. METHODS AND RESULTS: A strain KWN isolated from the rabbit caecum was used. Fermentation pattern, changes of viscosity and enzyme reactions products were determined. Cultures grown on pectin produced significantly more acetate and less formate, lactate, fumarate and succinate than cultures grown on glucose. Production of cell dry matter and protein per gram of substrate used was the same in pectin- and glucose-grown cultures. The principal enzymes that participated in the metabolism of pectin were extracellular exopectate hydrolase (EC 3.2.1.67), extracellular endopectate lyase (EC 4.2.2.2) and cell-associated 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (EC 4.1.2.14). The latter enzyme is unique to the Entner-Doudoroff pathway. Activities of pectinolytic enzymes in cultures grown on glucose were low. Activity of KDPG aldolase was similar in pectin- and glucose-grown cells. CONCLUSIONS: Metabolites and activities of pectin-degrading enzymes differed in cultures of B. caccae KWN grown on pectin and glucose. Yields of dry matter and protein were the same on both substrates. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on metabolism of pectin in animal strains of Bacteroides is incomplete. This study extends the knowledge on metabolism in bacteria from the rabbit caecum.

Fermentation of pectin and glucose, and activity of pectin-degrading enzymes in the rabbit caecal bacterium Bifidobacterium pseudolongum.

AIMS: In a rabbit caecal bacterium Bifidobacterium pseudolongum, metabolites of pectin and glucose, and activities of enzymes involved in the degradation of pectin were assayed. Simultaneously, activities of these enzymes were assayed in a rumen pectinolytic strain of Streptococcus bovis. METHODS AND RESULTS: A strain B. pseudolongum P6 which grew best on pectin was selected among bifidobacteria isolated from the rabbit caecum. Cultures of B. pseudolongum P6 grown on pectin produced significantly less formate, lactate and ethanol, and more acetate and succinate than cultures grown on glucose. No CO2 production on pectin was observed. Pectin macromolecule was degraded by extracellular pectinase (EC 3.2.1.15). Cell extracts possessed the activity of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (EC 4.1.2.14). Streptococcus bovis X4, possessed activity of exopectate lyase and pectinase, but not that of KDPG aldolase. CONCLUSIONS: Our results are consistent with the assumption that in B. pseudolongum P6 acidic products of pectin degradation are catabolized via a modified Entner-Doudoroff pathway, as shown previously in rumen pectin-utilizing bacteria. The missing KDPG aldolase activity in Strep. bovis X4 seems to be the reason for the absence of growth of this bacterium on pectin. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on polysaccharide metabolism in bifidobacteria is fragmentary. This study extends the knowledge on pectin metabolism in intestinal bacteria.

Relationship between concentration of plum pox virus and content of pigments in virus-infected symptomatic apricot leaves.

Besides other factors, occurrence of plum pox virus (PPV)-caused spots and mosaic symptoms on leaves of stone fruits are known to influence important physiological functions including production of assimilates. Apricot (Prunus armeniaca L.) seedlings were used as a test material for typical manifestation of symptoms of the disease on the foliage. The relations of the abovementioned visual symptoms to the virus and pigments concentrations in leaves have so far not been known sufficiently. We detected PPV only in symptomatic leaf tissue of the infected apricot. In the green tissue of the same leaves, the double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) of the virus was negative. The content of pigments was changed by PPV infection. In the symptomatic leaf tissues, the content of chlorophylls "a" and "b" was lower, and the content of carotenoids was higher in comparison to the respective controls. We conclude that the PPV infection could cause the change in the content of particular leaf pigments leading to the decreased yield of photosynthesis which in turn could influence the sugar metabolism in the infected trees.