The Influence of Lead and Acyrthosiphon pisum (Harris) on Generation of Pisum sativum Defense Signaling Molecules and Expression of Genes Involved in Their Biosynthesis.

The main aim of this study was to understand the regulation of the biosynthesis of phytohormones as signaling molecules in the defense mechanisms of pea seedlings during the application of abiotic and biotic stress factors. It was important to identify this regulation at the molecular level in Pisum sativum L. seedlings under the influence of various concentrations of lead-i.e., a low concentration increasing plant metabolism, causing a hormetic effect, and a high dose causing a sublethal effect-and during feeding of a phytophagous insect with a piercing-sucking mouthpart-i.e., pea aphid (Acyrthosiphon pisum (Harris)). The aim of the study was to determine the expression level of genes encoding enzymes of the biosynthesis of signaling molecules such as phytohormones-i.e., jasmonates (JA/MeJA), ethylene (ET) and abscisic acid (ABA). Real-time qPCR was applied to analyze the expression of genes encoding enzymes involved in the regulation of the biosynthesis of JA/MeJA (lipoxygenase 1 (LOX1), lipoxygenase 2 (LOX2), 12-oxophytodienoate reductase 1 (OPR1) and jasmonic acid-amido synthetase (JAR1)), ET (1-aminocyclopropane-1-carboxylate synthase 3 (ACS3)) and ABA (9-cis-epoxycarotenoid dioxygenase (NCED) and aldehyde oxidase 1 (AO1)). In response to the abovementioned stress factors-i.e., abiotic and biotic stressors acting independently or simultaneously-the expression of the LOX1, LOX2, OPR1, JAR1, ACS3, NCED and AO1 genes at both sublethal and hormetic doses increased. Particularly high levels of the relative expression of the tested genes in pea seedlings growing at sublethal doses of lead and colonized by A. pisum compared to the control were noticeable. A hormetic dose of lead induced high expression levels of the JAR1, OPR1 and ACS3 genes, especially in leaves. Moreover, an increase in the concentration of phytohormones such as jasmonates (JA and MeJA) and aminococyclopropane-1-carboxylic acid (ACC)-ethylene (ET) precursor was observed. The results of this study indicate that the response of pea seedlings to lead and A. pisum aphid infestation differed greatly at both the gene expression and metabolic levels. The intensity of these defense responses depended on the organ, the metal dose and direct contact of the stress factor with the organ.

Probing behavior of Adelges laricis Vallot (Hemiptera: Adelgidae) on Larix decidua Mill: Description and analysis of EPG waveforms.

Adelgidae are a sister group of Aphididae and Phylloxeridae within Hemiptera, Aphidoidea and occur exclusively on Pinaceae. The piercing-sucking mouthparts of Adelgidae are similar to those of aphids and it is believed that adelgids ingest sap from both the non-vascular and vascular (phloem) tissues. The aim of the present study was to identify and characterize the adelgid stylet activities during their penetration in plant tissues. The probing behavior of Adelges laricis Vallot (Hemiptera: Adelgidae) on European larch Larix decidua Mill. and sucrose diets was monitored using the DC-EPG (Electrical Penetration Graph technique = electropenetrography). The EPG waveforms were described based on amplitude, frequency, voltage level, and electrical origin of the observed traces, and associated with putative behavioral activities based on analogy with aphid activities. Waveform frequency, duration, and sequence were analysed as well. A. laricis generated EPG signals at two clearly distinct voltage levels positive and negative, suggesting extracellular and intracellular stylet penetration, respectively. The adelgid EPG patterns were ascribed to four behavioral phases, which were non-probing, pathway, phloem, and xylem phases. Non-probing referred to the position of the stylets outside the plant tissues. Pathway phase was represented by three waveform patterns that visualized extracellular stylet penetration in non-vascular tissues without potential drops (AC1), with serial short (1.2-1.5 s) potential drops (AC2), and with 'aphid-like' (5-10 s) potential drops (AC3). Phloem phase comprised three waveform patterns at intracellular level, which in all probability represented phloem salivation (AE1), and phloem sap passive (AE2) and active ingestion (AE3). AE3 was a newly described waveform, previously unreported from Hemiptera. Waveform AG represented the ingestion of xylem sap. The comparative analysis demonstrated that the gymnosperm-associated adelgids show certain similarities in probing behavior typical of aphids and phylloxerids on angiosperm plants. The present work is the first detailed analysis of specific adelgid stylet activities on gymnosperms.

Oxidative stress links response to lead and Acyrthosiphon pisum in Pisum sativum L.

This study demonstrates the impact of lead at hormetic (0.075 mM Pb(NO3)2) and sublethal (0.5 mM Pb(NO3)2) doses on the intensity of oxidative stress in pea seedlings (Pisum sativum L. cv. 'Cysterski'). Our first objective was to determine how exposure of pea seedlings to Pb alters the plant defence responses to pea aphid (Acyrthosiphon pisum Harris), and whether these responses could indirectly affect A. pisum. The second objective was to investigate the effects of various Pb concentrations in the medium on demographic parameters of pea aphid population and the process of its feeding on edible pea. We found that the dose of Pb sublethal for pea seedlings strongly reduced net reproductive rate and limited the number of A. pisum individuals reaching the phloem. An important defence line of pea seedlings growing on Pb-supplemented medium and next during combinatory effect of the two stressors Pb and A. pisum was a high generation of superoxide anion (O2-). This was accompanied by a considerable reduction in superoxide dismutase (SOD) activity, and a decrease in the level of Mn2+ ions. A the same time, weak activity of Mn-SOD was detected in the roots of the seedlings exposed to the sublethal dose of Pb and during Pb and aphid interaction. Apart from the marked increase in O2-, an increase in semiquinone radicals occurred, especially in the roots of the seedlings treated with the sublethal dose of Pb and both infested and non-infested with aphids. Also, hydrogen peroxide (H2O2) generation markedly intensified in aphid-infested leaves. It reached the highest level 24 h post infestation (hpi), mainly in the cell wall of leaf epidermis. This may be related to the function of H2O2 as a signalling molecule that triggers defence mechanisms. The activity of peroxidase (POX), an important enzyme involved in scavenging H2O2, was also high at 24 hpi and at subsequent time points. Moreover, the contents of thiobarbituric acid reactive substances (TBARS), products of lipid peroxidation, rose but to a small degree thanks to an efficient antioxidant system. Total antioxidant capacity (TAC) dependent on the pool of fast antioxidants, both in infested and non-infested and leaves was higher than in the control. In conclusion, the reaction of pea seedlings to low and sublethal doses of Pb and then A. pisum infestation differed substantially and depended on a direct contact of the stress factor with the organ (Pb with roots and A. pisum with leaves). The probing behavior of A. pisum also depended on Pb concentration in the plant tissues.

HBcAg produced in transgenic tobacco triggers Th1 and Th2 response when intramuscularly delivered.

Hepatitis B core Antigen (HBcAg) assembled into Capsid-Like Particles (CLPs) is investigated as a therapeutic vaccine in treatment of chronic hepatitis B (CHB) and in diagnostic tests or as a carrier for various epitopes. While the expression of HBcAg has been thoroughly clarified in E. coli and yeast, it has also been investigated in other expression systems. Stably transformed tobacco expressed HBcAg at a level of 110-250µg/g fresh weight, therefore in view of its large leaf biomass it offers a production platform comparable with transient expression systems regarding the final yield of HBcAg. Several extraction and purification methods were tested and finally the antigen was purified up to 43% using sucrose density gradient centrifugation. The purified HBcAg retained its antigenicity, as confirmed by ELISA and western blot, while maintaining its CLP-structure as observed in TEM. In mice HBcAg intramuscularly delivered at 2×10µg triggered a significant response (serum anti-HBc titre around 150,000), being statistically equivalent to that induced by the reference antigen. Among anti-HBc IgG isotypes, IgG2a and then IgG1 were increasing during immune response. However IgG2b and IgG3 were also induced, especially in mice immunised with the plant-derived antigen. Analysis of the isotype profile indicates mainly Th1 polarisation, but completed with Th2 response. Obtained results indicate a considerable potential of plant-derived HBcAg as a therapeutic vaccine, since a mixed immune response with a stronger Th1 component is particularly required for treatment of CHB.

Brachycorynella asparagi (Mordv.) Induced-Oxidative Stress and Antioxidative Defenses of Asparagus officinalis L.

The aim of this study was to investigate whether and to what extent oxidative stress is induced in leaves of one- and two-month-old plants of Asparagus officinalis L. cv. Argenteuil infested by Brachycorynella asparagi (Mordvilko) at a varied population size. The pest B. asparagi has been described as the most damaging species feeding on asparagus. Analyses using electron paramagnetic resonance (EPR) demonstrated generally higher concentrations of semiquinone radicals with g-values of 2.0045 ± 0.0005 and 2.0026 ± 0.0005 in Asparagus officinalis (A. officinalis) leaves after Brachycorynella asparagi (B. asparagi) infestation than in the control. Observations of leaves under a confocal microscope showed a post-infestation enhanced generation of the superoxide anion radical (O2•-) and hydrogen peroxide (H2O2) in comparison to the control. Strong fluctuations in Mn2+ ion levels detected by EPR spectroscopy versus time were detected in leaves infested by aphids, which may indicate the involvement of these ions in the control of O2•- production. An enhanced superoxide dismutase activity is an important element in leaf defense against oxidative stress. Visible symptoms were found in aphid-infested A. officinalis. Damage to leaves of one- and two-month-old A. officinalis plants by the aphid B. asparagi was dependent on the intensity, duration of infestation and plant age.

Pectinous cell wall thickenings formation - A common defense strategy of plants to cope with Pb.

Lead, one of the most abundant and hazardous trace metals affecting living organisms, has been commonly detected in plant cell walls including some tolerant plants, mining ecotypes and hyperaccumulators. We have previously shown that in tip growing Funaria sp. protonemata cell wall is remodeled in response to lead by formation of thickenings rich in low-methylesterified pectins (pectin epitope JIM5 - JIM5-P) able to bind metal ions, which accumulate large amounts of Pb. Hence, it leads to the increase of cell wall capacity for Pb compartmentalization. Here we show that diverse plant species belonging to different phyla (Arabidopsis, hybrid aspen, star duckweed), form similar cell wall thickenings in response to Pb. These thickenings are formed in tip growing cells such as the root hairs, and in diffuse growing cells such as meristematic and root cap columella cells of root apices in hybrid aspen and Arabidopsis and in mesophyll cells in star duckweed fronds. Notably, all analyzed cell wall thickenings were abundant in JIM5-P and accumulated high amounts of Pb. In addition, the co-localization of JIM5-P and Pb commonly occurred in these cells. Hence, cell wall thickenings formed the extra compartment for Pb accumulation. In this way plant cells increased cell wall capacity for compartmentalization of this toxic metal, protecting protoplast from its toxicity. As cell wall thickenings occurred in diverse plant species and cell types differing in the type of growth we may conclude that pectinous cell wall thickenings formation is a widespread defense strategy of plants to cope with Pb. Moreover, detection of natural defense strategy, increasing plant cell walls capacity for metal accumulation, reveals a promising direction for enhancing plant efficiency in phytoremediation.

Blue light-dependent changes in loosely bound calcium in Arabidopsis mesophyll cells: an X-ray microanalysis study.

Calcium is involved in the signal transduction pathway from phototropins, the blue light photoreceptor kinases which mediate chloroplast movements. The chloroplast accumulation response in low light is controlled by both phot1 and phot2, while only phot2 is involved in avoidance movement induced by strong light. Phototropins elevate cytosolic Ca(2+) after activation by blue light. In higher plants, both types of chloroplast responses depend on Ca(2+), and internal calcium stores seem to be crucial for these processes. Yet, the calcium signatures generated after the perception of blue light by phototropins are not well understood. To characterize the localization of calcium in Arabidopsis mesophyll cells, loosely bound (exchangeable) Ca(2+) was precipitated with potassium pyroantimonate and analyzed by transmission electron microscopy followed by energy-dispersive X-ray microanalysis. In dark-adapted wild-type Arabidopsis leaves, calcium precipitates were observed at the cell wall, where they formed spherical structures. After strong blue light irradiation, calcium at the apoplast prevailed, and bigger, multilayer precipitates were found. Spherical calcium precipitates were also detected at the tonoplast. After red light treatment as a control, the precipitates at the cell wall were smaller and less numerous. In the phot2 and phot1phot2 mutants, calcium patterns were different from those of wild-type plants. In both mutants, no elevation of calcium after blue light treatment was observed at the cell periphery (including the cell wall and a fragment of cytoplasm). This result confirms the involvement of phototropin2 in the regulation of Ca(2+) homeostasis in mesophyll cells.

Lead-stress induced changes in the content of free, thylakoid- and chromatin-bound polyamines, photosynthetic parameters and ultrastructure in greening barley leaves.

The aim of this study was to determine the impact of lead (Pb) stress as 0.6mM Pb(NO3)2 on the content of free, thylakoid- and chromatin-bound polyamines (PAs) and diamine oxidase (DAO) activity in detached greening barley leaves. Additionally, photosynthetic-related parameters, generation of hydrogen peroxide (H2O2) and malondialdehyde (MDA) content and ultrastructural changes under Pb-stress were studied. The level of putrescine (Put) was reduced progressively to 56% at 24h of Pb stress, and it was correlated with 38% increase of DAO activity. Spermidine (Spd) content was not affected by Pb-stress, while the free spermine (Spm) level significantly increased by about 83% at 6h, and in that time the lowest level of H2O2 was observed. The exogenous applied Spm to Pb-treated leaves caused a decrease in the content of H2O2. In greening leaves exposed to Pb an accumulation of chlorophylls a and b was inhibited by about 39 and 47%, respectively, and photosynthetic parameters of efficiency of electron transport and photochemical reaction in chloroplasts as ΦPSII, ETR and RFd were lowered by about 23-32%. The level of thylakoid-bound Put decreased by about 22%. Moreover, thylakoids isolated from chloroplasts of Pb-treated leaves were characterized with lower Put/Spm ratio as compared to control leaves. In the presence of Pb the significant decrease in the number of thylakoids per granum and cap-shape invaginations of cytoplasmic material were noticed. In Pb-stressed leaves the level of chromatin-bound Spm increased by about 48% and sometimes condensed chromatin in nuclei was observed. We conclude that in greening barley leaves exposed to Pb-stress changes in free, thylakoid- and chromatin-bound PAs play some role in the functioning of leaves or plants in heavy metal stress conditions.

Pb-induced avoidance-like chloroplast movements in fronds of Lemna trisulca L.

Lead ions are particularly dangerous to the photosynthetic apparatus, but little is known about the effects of trace metals, including Pb, on regulation of chloroplast redistribution. In this study a new effect of lead on chloroplast distribution patterns and movements was demonstrated in mesophyll cells of a small-sized aquatic angiosperm Lemna trisulca L. (star duckweed). An analysis of confocal microscopy images of L. trisulca fronds treated with lead (15 µM Pb2+, 24 h) in darkness or in weak white light revealed an enhanced accumulation of chloroplasts in the profile position along the anticlinal cell walls, in comparison to untreated plants. The rearrangement of chloroplasts in their response to lead ions in darkness was similar to the avoidance response of chloroplasts in plants treated with strong white light. Transmission electron microscopy X-ray microanalysis showed that intracellular chloroplast arrangement was independent of the location of Pb deposits, suggesting that lead causes redistribution of chloroplasts, which looks like a light-induced avoidance response, but is not a real avoidance response to the metal. Furthermore, a similar redistribution of chloroplasts in L. trisulca cells in darkness was observed also under the influence of exogenously applied hydrogen peroxide (H2O2). In addition, we detected an enhanced accumulation of endogenous H2O2 after treatment of plants with lead. Interestingly, H2O2-specific scavenger catalase partly abolished the Pb-induced chloroplast response. These results suggest that H2O2 can be involved in the avoidance-like movement of chloroplasts induced by lead. Analysis of photometric measurements revealed also strong inhibition (but not complete) of blue-light-induced chloroplast movements by lead. This inhibition may result from disturbances in the actin cytoskeleton, as we observed fragmentation and disappearance of actin filaments around chloroplasts. Results of this study show that the mechanisms of the toxic effect of lead on chloroplasts can include disturbances in their movement and distribution pattern.

Effects of endogenous signals and Fusarium oxysporum on the mechanism regulating genistein synthesis and accumulation in yellow lupine and their impact on plant cell cytoskeleton.

The aim of the study was to examine cross-talk interactions of soluble sugars (sucrose, glucose and fructose) and infection caused by Fusarium oxysporum f.sp. lupini on the synthesis of genistein in embryo axes of Lupinus luteus L.cv. Juno. Genistein is a free aglycone, highly reactive and with the potential to inhibit fungal infection and development of plant diseases. As signal molecules, sugars strongly stimulated accumulation of isoflavones, including genistein, and the expression of the isoflavonoid biosynthetic genes. Infection significantly enhanced the synthesis of genistein and other isoflavone aglycones in cells of embryo axes of yellow lupine with high endogenous sugar levels. The activity of ß-glucosidase, the enzyme that releases free aglycones from their glucoside bindings, was higher in the infected tissues than in the control ones. At the same time, a very strong generation of the superoxide anion radical was observed in tissues with high sugar contents already in the initial stage of infection. During later stages after inoculation, a strong generation of semiquinone radicals was observed, which level was relatively higher in tissues deficient in sugars than in those with high sugar levels. Observations of actin and tubulin cytoskeletons in cells of infected embryo axes cultured on the medium with sucrose, as well as the medium without sugar, showed significant differences in their organization.

Oxidative stress in pea seedling leaves in response to Acyrthosiphon pisum infestation.

In this study we examined whether and to what extent oxidative stress is induced in seedling leaves of Pisum sativum L. cv. Cysterski in response to pea aphid (Acyrthosiphon pisum Harris) infestation. A. pisum caused oxidative stress conditions in pea leaves through enhanced production of the reactive oxygen species (ROS) hydrogen peroxide (H2O2) and superoxide anion radical (O2(·-)). Early, strong generation of H2O2 was observed at 24h in aphid-infested leaves. The highest level of H2O2 at this time point may be related to the functioning of H2O2 as a signaling molecule, triggering defense mechanisms in pea leaves against A. pisum. Additionally, the strong generation and continuous increase of O2(·-) production in aphid-infested leaves from 0 to 96 h enhanced the defense potential to protect against aphid herbivory. Also in the study cytochemical localization of H2O2 and O2(·-) in pea leaves after aphid infestation was determined using the confocal microscope. Relative release of H2O2 and O2(·-) was estimated by staining leaves with specific fluorochromes, i.e. dichlorodihydro-fluorescein diacetate (DCFH-DA) and dihydroethidium (DHE), respectively. DCFH-DA and DHE derived fluorescence was observed to cover a much larger tissue area in aphid-infested leaves, whereas little or no fluorescence was observed in the control leaves. Enhanced activity of the antioxidant enzymes superoxide dismutase (SOD, 1.15.1.1) and catalase (CAT, 1.11.1.6) is one of the most essential elements of defense responses in pea seedling leaves to oxidative stress. Additionally, generation of semiquinones, stable free radicals with g-values of 2.0020 and 2.0035, detected by electron paramagnetic resonance spectroscopy (EPR), was suggested as a protective action of pea that may contribute to build-up of a defensive barrier or activate other defense mechanisms. Concentrations of semiquinone radicals in aphid-infested seedling leaves not only were generally higher than in the control plants but also significantly increased with cultivation time. On the other hand, the small increase in content of thiobarbituric acid reactive substances (TBARS), a product of lipid peroxidation, and the percentage of injury (3-8%) indicated that the cellular damage was caused by oxidative stress. The induced changes in levels of H2O2, O2(·-) and semiquinone radicals as well as activities of antioxidant enzymes in the pea defense responses were proportional to the population size of A. pisum. These findings indicate that the defensive strategies against A. pisum infestation were stimulated in seedling leaves of P. sativum L. cv. Cysterski. Our observations of the enhanced defense responses of P. sativum to infestation by A. pisum reveal some aspects and contribute to current knowledge of regulatory mechanisms in plant-aphid interactions.

Is callose a barrier for lead ions entering Lemna minor L. root cells?

Plants have developed a range of strategies for resisting environmental stresses. One of the most common is the synthesis and deposition of callose, which functions as a barrier against stress factor penetration. The aim of our study was to examine whether callose forms an efficient barrier against Pb penetration in the roots of Lemna minor L. exposed to this metal. The obtained results showed that Pb induced callose synthesis in L. minor roots, but it was not deposited regularly in all tissues and cells. Callose occurred mainly in the protoderm and in the centre of the root tip (procambial central cylinder). Moreover, continuous callose bands, which could form an efficient barrier for Pb penetration, were formed only in the newly formed and anticlinal cell walls (CWs); while in other CWs, callose formed only small clusters or incomplete bands. Such an arrangement of callose within root CWs inefficiently protected the protoplast from Pb penetration. As a result, Pb was commonly present inside the root cells. In the light of the results, the barrier role of callose against metal ion penetration appears to be less obvious than previously believed. It was indicated that induction of callose synthesis is not enough for a successful blockade of the stress factor penetration. Furthermore, it would appear that the pattern of callose distribution has an important role in this defence strategy.

The thylakoid protease Deg2 is involved in stress-related degradation of the photosystem II light-harvesting protein Lhcb6 in Arabidopsis thaliana.

• The thylakoid protease Deg2 is a serine-type protease peripherally attached to the stromal side of the thylakoid membrane. Given the lack of knowledge concerning its function, two T-DNA insertion lines devoid of Deg2 were prepared to study the functional importance of this protease in Arabidopsis thaliana. • The phenotypic appearance of deg2 mutants was studied using a combination of stereo and transmission electron microscopy, and short-stress-mediated degradation of apoproteins of minor light-harvesting antennae of photosystem II (PSII) was analysed by immunoblotting in the mutants in comparison with wild-type plants. • Deg2 repression produced a phenotype in which reduced leaf area and modified chloroplast ultrastructure of older leaves were the most prominent features. In contrast to the wild type, the chloroplasts of second-whorl leaves of 4-wk-old deg2 mutants did not display features typical of the early senescence phase, such as undulation of the chloroplast envelope and thylakoids. The ability to degrade the photosystem II light-harvesting protein Lhcb6 apoprotein in response to brief high-salt, wounding, high-temperature and high-irradiance stress was demonstrated to be impaired in deg2 mutants. • Our results suggest that Deg2 is required for normal plant development, including the chloroplast life cycle, and has an important function in the degradation of Lhcb6 in response to short-duration stresses.

Involvement of Deg5 protease in wounding-related disposal of PsbF apoprotein.

Deg5 is a serine-type protease peripherally attached to luminal side of thylakoid membrane. Given the lack of knowledge concerning its function homozygous T-DNA insertion line depleted in Deg5 was prepared to study the functional importance of this protease in Arabidopsis thaliana. deg5 mutants displayed a pleiotropic phenotype with regard to fourth whorl leaves of four-weeks old plants. The alterations involved an increased leaf area, reduced leaf thickness, reduced cross-sectional area of palisade mesophyll cells as well as changed chloroplast ultrastructure including lack of signs of entering the senescence phase (e.g. presence of much smaller plastoglobules) and the accumulation of large starch grains at the end of the dark period. It was shown that whereas PsbA, C and F apoproteins of photosystem II reaction center undergo an extensive disappearance in response to a set of brief stresses deg5 mutant was fully resistant to the disappearance of PsbF apoprotein which follows an exposition of leaves to wounding. Our results demonstrate that Deg5 is of seminal importance for normal plant development and degradation of PsbF which occurs following brief wounding.

Cross-talk interactions of sucrose and Fusarium oxysporum in the phenylpropanoid pathway and the accumulation and localization of flavonoids in embryo axes of yellow lupine.

This study investigated the effects of cross-talk interactions of sucrose and infection caused by a pathogenic fungus Fusarium oxysporum f.sp. lupini on the regulation of the phenylpropanoid pathway, i.e. the level of expression of genes encoding enzymes participating in flavonoid biosynthesis, as well as cell location and accumulation of these compounds in embryo axes of Lupinus luteus L. cv. Polo. Embryo axes, both non-inoculated and inoculated, were cultured for 96h on Heller medium with 60mM sucrose (+Sn and +Si) or without it (-Sn and -Si). Real-time RT-PCR to assess expression levels of the flavonoid biosynthetic genes, phenylalanine ammonialyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI) and isoflavone synthase (IFS) were used. Sucrose alone strongly stimulated the expression of these genes. There was a very high expression level of these genes in +Si embryo axes in the early phase of infection. Signal amplification by sucrose and the infection was most intense in the 48-h +Si axes, resulting in the highest level of expression of flavonoid biosynthetic genes. In -Si tissues, the expression level of these genes increased at 48 and 72h after inoculation relative to 24h; however, the relative level of expression was much lower than in +Si axes, except at 72h for PAL and CHS.Moreover, at 48h of culture, considerably higher activity of CHI (EC 5.5.1.6) was observed in axes with a high level of sucrose than in those with a sucrose deficit. CHI activity in +Si axes at 48 and 96h post-inoculation was over 1.5 and 2 times higher than that in +Sn axes, as well as higher than in -Si axes.Observations of yellow lupine embryo axes under a confocal microscope showed an increased post-infection accumulation of flavonoids, particularly in cells of embryo axes infected with F. oxysporum and cultured on a medium containing sucrose (+Si). Up to 48h post-infection in +Si axes, a very intensive emission of green fluorescence was observed, indicating high accumulation of these compounds in whole cells. Moreover, a nuclear location of flavonoids was recorded in cells. Strong staining of flavonoid end products in +Si embryo axes was consistent with the expression of PAL, CHS, CHI and IFS.These results indicate that, in the early phase of infection, the flavonoid biosynthesis pathway is considerably enhanced in yellow lupine embryo axes as a strong signal amplification effect of sucrose and the pathogenic fungus F. oxysporum.

Low-dose oral immunization with lyophilized tissue of herbicide-resistant lettuce expressing hepatitis B surface antigen for prototype plant-derived vaccine tablet formulation.

Efficient immunization against hepatitis B virus (HBV) and other pathogens with plant-based oral vaccines requires appropriate plant expressors and the optimization of vaccine compositions and administration protocols. Previous immunization studies were mainly based on a combination of the injection of a small surface antigen of HBV (S-HBsAg) and the feeding with raw tissue containing the antigen, supplemented with an adjuvant, and coming from plants conferring resistance to kanamycin. The objective of this study was to develop a prototype oral vaccine formula suitable for human immunization. Herbicide-resistant lettuce was engineered, stably expressing through progeny generation micrograms of S-HBsAg per g of fresh weight and formed into virus-like particles (VLPs). Lyophilized tissue containing a relatively low, 100-ng VLP-assembled antigen dose, administered only orally to mice with a long, 60-day interval between prime and boost immunizations and without exogenous adjuvant, elicited mucosal and systemic humoral anti-HBs responses at the nominally protective level. Lyophilized tissue was converted into tablets, which preserved S-HBsAg content for at least one year of room temperature storage. The results of the study provide indications on immunization methodology using a durable, efficacious, and convenient plant-derived prototype oral vaccine against hepatitis B.

Lead deposited in the cell wall of Funaria hygrometrica protonemata is not stable--a remobilization can occur.

The hypothesis that lead (Pb) can be uptake or remobilized from the cell wall (CW) by internalization withlow-esterified pectins (up to 40%--JIM5-P), was studied in tip-growing apical cell of Funaria hygrometrica protonemata. Treatment 4h with 1mM PbCl(2) caused marked vesicular traffic intensification and the common internalization of JIM5-P from the CW. Lead bound to JIM5-P was internalized from the CW, together with this compound and entered the protoplast. It showed that Pb deposited in CW is not as safe for plant cell as previously believed. However, pulse-chase experiments (recovering 4 h and 24 h) indicated that CW and its thickenings can function as the final sequestration compartments. In Pb deposition sites, a callose layer occurred. It was localized from the protoplast site, next to Pb deposits separating sequestrated to CW and its thickenings Pb from plasma membrane almost certainly protecting the plant cell from its returning into the protoplast.