Elicitation of H2O2 production in cucumber hypocotyl segments by oligo-1,4-α-D-galacturonides and an oligo-β-glucan preparation from cell walls of Phythophthora megasperma f. sp. glycinea

The production of H₂O₂ by cucumber hypocotyl segments ( Cucumis sativus L. cv. Wisconsin SMR 58) in response to α-1,4-linked oligomers of galacturonic acid and oligo-β-glucans from the cell walls of Phytophthora megasperma f. sp. glycinea was studied. Oligogalacturonides with degrees of polymerization of 9 to 13 elicited H₂O₂ production, the most effective being the deca-, undeca- and dodecamers. A similar relationship between size and effect was previously obtained when oligogalacturonides were tested for their ability to elicit lignification in cucumber hypocotyls. The oligogalacturonide-induced increase in H₂O₂ concentration was detected after 4 h, reaching a maximum after 10 h of incubation. The glucan elicitor induced lignification at a 100-fold lower concentration than the oligogalacturonides, but yielded only 10% of the maximum H₂O₂ accumulation seen with oligogalacturonides. The glucan elicitor-induced H₂O₂ production was detectable after 2 h, and reached a maximum after 4 to 6 h. Catalase abolished the elicitation of both phenol red oxidation and lignification in cucumber hypocotyls. At least part of the oligogalacturonide-induced H₂O₂ production appeared to be dependent upon de novo protein synthesis.     

Lignin deposition induced by aluminum in wheat (Triticum aestivum) roots

We investigated the relation between the toxic effect of aluminum (Al) on root growth and the lignin deposition in wheat ( Triticum aestivum L. cvs Atlas 66 and Scout 66). In the Al-tolerant cultivar Atlas 66, control treatment without AlCl₃ at pH 4.75, cell length increased dramatically in the portion of the root that was 0.6 to 3.2 mm from the root cap junction (approximately 1.0 to 3.6 mm from the root tip). However, treatment with 20 μ M AlCl₃ for 24 and 48 h completely inhibited root elongation and markedly decreased the length and increased the diameter of the cells in the same portion of the root. Moreover, marked deposition of lignin was observed in the cells that corresponded to the portion 1.5 to 4.5 mm from the root tip in Atlas 66 roots treated with 20 μ M AlCl₃, while no deposition of lignin was detected in control roots. Treatment with 5 μ M AlCl₃ slightly inhibited root growth and there was no deposition of lignin in the root. On the other hand, in roots of the Al-sensitive cultivar Scout 66, treatment with 5 μ M AlCl₃ completely inhibited root growth and markedly induced deposition of lignin. These results suggest that lignification in the elongating region coincided with the extent of inhibition of root growth by Al in two wheat cultivars that differed in their sensitivity to Al.     

Lignification and lignin heterogeneity for various age classes of bamboo (Phyllostachys pubescens) stems

The lignification process and lignin heterogeneity of fibre, vessel and parenchyma cell walls for various age classes of bamboo stems of Phyllostachys pubescens Mazel were investigated. It was shown that protoxylem vessels lignified in the early stage of vascular bundle differentiation, metaxylem vessel and fibre walls initiated lignification from the middle lamella and cell corners after the completion of vascular bundle differentiation. Most of the parenchyma cell walls lignified after the stem reached its full height, while a few parenchyma cells remained non-lignified even in the mature culm. The cell walls of fibres and most parenchyma cells thickened further during the stem growth to form polylamellate structure and the lignification process of these cells may last even up to 7 years. The fibre walls were rich in guaiacyl lignin in the early stage of lignification, and lignin rich in syringyl units were deposited in the later stage. Vessel walls mainly contained guaiacyl lignin, while both guaiacyl and syringyl lignin were present in the fibre and parenchyma cell walls.     

Nitrate reductase is inhibited in leaves of Triticum aestivum treated with high levels of copper

Copper is a potent sulfhydryl reagent which can also catalyse the generation of active oxygen. Since nitrate reductase (EC 1.6.6.1) is an SH-enzyme sensitive to oxidative environments, the relations among copper, active oxygen species and nitrate reductase (NR) activity are of interest. Foliar segments of wheat ( Triticum aestivum cv. Oasis) were floated on CuSO₄ solutions (up to 250 μ M ) for 24 h under continuous light. Copper decreased NR activity before affecting active oxygen generation as estimated by changes in oxidative parameters, including malondialdehyde, K⁺ leakage and chlorophyll degradation. Cysteine and Na-benzoate counteracted this decrease, suggesting an oxidative damage of the enzyme in leaves exposed to high copper levels. Copper-induced NR inactivation was further studied in the partially purified enzyme. Preincubation with CuSO₄ inhibited NR. Copper inhibition was reversed by subsequent incubation with EDTA, indicating that the metal bonded to key -SH groups of the enzyme. In addition, an ^˙OH-generating system (composed of CuSO₄, ascorbate and H₂O₂) irreversibly decreased the activity of purified NR to a greater extent than copper alone. Our results show that copper affects nitrogen metabolism by diminishing NR activity, involving a direct effect on key SH-groups and an indirect effect via attack by active oxygen species induced by the metal.     

Effects of the Application of Different Concentrations of NaN3 for Different Times on the Morphological and Cytogenetic Characteristics of Barley （Hordeum vulgate L.） Seedlings

Sodium azide （NAN3） has been used in many biological studies as a mutagen. In the present study, the morphological and cytogenetic effects of NaN3 on barley （Hordeum vulgare L.） seedlings were investigated. Seeds of barley were treated with different concentrations of NaN3 （0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mmol/L） and applied for different periods of time （3 and 4 h）. Parameters investigated, except for the mitotic index, were determined on Days 7 and 14. Increasing concentrations of NaN3 affected germination rates on Days 7 and 14 following application for 3 and 4 h. Although the length of the roots and leaves was affected by treatment with NaN3 on Day 14 of the germination period, coleoptile length was affected by NaN3 treatment on Day 7. Increasing concentrations of NaN3 and increased treatment period decreased the mitotic index compared with the untreated control. The inhibitory effects of NaN3 on the mitotic index indicate that NaN3 can have genotoxic and mutagenic effects on barley seedlings.     

Distinct responses to copper stress in the halophyte Mesembryanthemum crystallinum

Selective gene expression allows the halophyte Mesembryanthemum crystallinum to survive a salt stress. To broaden our understanding of the environmental cues initiating diverse stress responses in this higher plant, unstressed and 0.4 M NaCl‐stressed plants were compared to plants treated with several concentrations of copper (CuSO₄), an increasingly relevant environmental heavy metal pollutant. Comparisons of control and copper‐stressed plants included germination, chlorophyll content, accumulation of proline, heat shock protein (HSP) 60 and a Crassulacean acid metabolism (CAM)‐specific marker enzyme, phospho enol pyruvate carboxylase (PEPCase). In germination and whole plant tests, M. crystallinum was significantly more tolerant to copper than Arabidopsis thaliana. Mature M. crystallinum plants stressed with 50 ppm CuSO₄ for 48 h became dehydrated. These plants produced a 4‐fold increase in proline concentration and accumulated both the CAM‐specific PEPCase and HSP 60 compared to controls. Higher levels of copper stress resulted in a 10‐fold increase in leaf proline content, 10‐fold HSP 60 accumulation but no detectable PEPCase protein compared to unstressed controls. HSP 60 did not accumulate under NaCl stress. Concurrent with copper‐induced genetic responses to stress, copper was accumulated and concentrated in leaves (3 500 ppm). Together, these results suggest that this halophyte copes with copper metal exposure through distinct genetic mechanisms.     

Influence of bicarbonate and metal ions on the development of root Fe(III) reducing capacity by Fe-deficient cucumber (Cucumis sativus) plants

The effect of bicarbonate and selected metal ions on the development of enhanced root Fe(III) reducing capacity (a response to Fe deficiency of dicotyledons) was studied in young plants of cucumber ( Cucumis sativus L. cv. Ashley) grown in nutrient solution. Pretreatment of 11-day-old Fe-deficient cucumber plants with 20 m M NaHCO₃, for at least 23 h prior to determination of root Fe(III) reducing capacity, markedly inhibited this response. The inhibitory effect of bicarbonate could be partly reversed by a 4- to 8-h treatment with either 10 μ M MnSO₄, 10 μ M FeEDDHA, 2 μ M ZnSO₄, 0.5 μ M NiCl₂, or 0.25 μ M , or CoSO₄ (final concentrations), added to the nutrient solution. By contrast, the addition of other salts of metal ions, like CuSO₄ and Cd(NO₃)₂, at 0.25, 0.5 or 1 μ M , or MgSO₄, at 0.5, 1 or 2 m M (final concentrations), had no beneficial effect. The results suggest that bicarbonate may inhibit the development of root Fe(III) reducing capacity by diminishing the availability of certain metal ions required for this response.     

Role of endogenous gibberellins in germination of melon (Cucumis melo) seeds

The effect of the plant growth retardants ancymidol. mefluidide and uniconazole on germination of two melon accessions differing in their ability to germinate at 14°C was examined. The accessions were the cold sensitive Noy Yizre'el and the cold tolerant Persia 202. The three growth retardants were able to delay the germination of intact Noy Yizre'el seeds, but did not affect that of intact Persia 202 seeds. On the other hand germination of decoated seeds of both accessions was unaffected by these inhibitors at normal oxygen concentration, but was inhibited at 5% oxygen. When gibberellin-like activity was measured by a dwarf rice biological assay following HPLC fractionation, it was found that seeds of Persia 202 contained much more gibberellin-like activity than Noy Yizre'el seeds. Among the extracted compounds several endogenous gibberellins were identified by combined gas chromatography-mass spectrometry (GC-MS). They included GA₄, GA₂₀, GA₁ and GA₃ in Noy Yizre'el and GA₃₄, GA₂₀, GA₁ and GA₈ in Persia 202. It is suggested that the better germination of intact Persia 202 seeds, compared to Noy Yizre'el seeds at low temperature and low oxygen concentration, is due to a higher endogenous level of GA and a better seed coat permeability to oxygen.     

Seed dormancy in Avena fatua: Interacting effects of nitrate, water and seed coat injury

In experiments conducted under controlled conditions. KNO₃ (50 or 100 m M ) promoted germination of a dormant strain (AN 474) of Avena fatua when either one or two holes were pierced in the lower (adaxial) surface of the caryopsis in contact with the nitrate solution. Germination was increased by increasing either the KNO₃ concentration or the number of holes in the seed coat. The germination response induced by the application of water to a hole pierced in the upper surface of the caryopsis was. increased by pre-treatment of the intact caryopsis with KNO₃. Treatment with either 50 or 100 m M KNO₃ caused a transient reduction in embryo water content of intact cary-opses, but increased the nitrate and amino- N content of pierced caryopses prior to germination. Supplying a 100 m M solution of KNO₃ to pierced caryopses reduced the total water potential and osmotic potential of the embryo, and increased its pressure potential by the same amount as an equimolar solution of KC1; however, while both treatments promoted germination, the KNO₃ induced more rapid germination than the KCI. Both treatments also increased the K⁺ content of the embryo, the KNO₃ again having the greater effect. These results are consistent with the hypothesis, based on our previous investigations, that KNO₃ promotes germination of dormant caryopses by accumulating in the embryo where it acts osmotically to increase water uptake. It is also postulated, that, in contrast to KCI, KNG₃ may combine an osmotic effect on water uptake with a nutritional effect on protein synthesis.     

Cambial activity,annual rhythm of xylem production in relation to phenology and climatic factors and lignification pattern during xylogenesis in drum-stick tree (Moringa oleifera)

The interrelationship among seasonality of cambium, wood formation, cell size variation, lignification, tree phenology and climatic factors has been examined in Moringa oleifera, a tropical evergreen tree. The vascular cambium in Moringa is a storied with a distinct seasonal variation in its structure due to dimensional changes in rays. Though cambium remains active throughout the year it is sensitive to water availability. Peak cambial cell division and rate of xylem differentiation are influenced by average rainfall during the monsoon period. Cambial cell division reaches higher up in the tree trunk when it is supporting a high number of branches and leaves. Statistical analysis of cell size variation and climate factors revealed that xylem cell development is greatly influenced by rainfall and rarely by temperature. Lengths of fusiform initials and vessel elements are positively correlated. The pattern of lignification during xylogenesis shows that the vessels are the first element to develop lignified walls and ray cells are the last elements to become lignified. Fiber cell walls show more syringyl lignin, while the cell walls of other xylem elements are characterized by relatively more guaiacyl lignin units.     

Purification and characterization of an endoamylase from tulip (Tulipa gesneriana) bulbs

Amylase activity extracted from tulip ( Tulipa gesneriana L. cv. Apeldoorn) bulbs that had been stored for 6 weeks at 4°C was resolved to 3 peaks by anion-exchange chromatography on diethylaminoethyl-Sephacel. These 3 amylases exhibited different relative mobilities during non-denaturing polyacrylamide gel electrophoresis (PAGE). The most abundant amylase form (amylase I) was purified to apparent homogeneity using hydrophobic interaction chromatography, gel filtration and chromatofocusing. The apparent molecular mass of the purified amylase was estimated to be 51 kDa by sodium dodecyl sulfate-PAGE and 45 kDa by gel filtration chromatography. The purified amylase was determined to be an endoamylase (EC 3.2.1.1) based on substrate specificity and end-product analysis. The enzyme had a pH optimum of 6.0 and a temperature optimum of 55°C. The apparent K_m value with soluble starch (potato) was 1.28 mg ml⁻¹. The presence of Ca²⁺ increased the activity and thermal stability of the enzyme. The presence of dithiothreitol enhanced the activity, while β -mercaptoethanol and reduced glutathione had no significant effect. When pre-incubated in the absence of the substrate, N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid) partially inhibited the enzyme. α -cyclodextrins or β -cyclodextrins had no effect on enzyme activity up to 10 m M . In addition to CaCl₂, CoCl₂ slightly enhanced activity, while MgCl₂ and MnCl₂ had no significant effect at a concentration of 2 m M . ZnCl₂, CuSO₄, AgNO₃ and EDTA partially inhibited enzyme activity, while AgNO₃ and HgCl₂ completely inhibited it at 2.0 m M .     

Elevated atmospheric CO2 alters wood production, wood quality and wood strength of Scots pine (Pinus sylvestris L) after three years of enrichment

Scots pine ( Pinus sylvestris L.) trees were grown in open top chambers for three years under ambient and elevated CO₂ concentrations. The trees were aged 3 y at the beginning of the CO₂ exposure, and the effects of the treatment on total stem volume, stem wood biomass, wood quality and wood anatomy were examined at the end of the exposure. The elevated CO₂ treatment lead to a 49% and 38% increase in stem biomass and stem wood volume, respectively. However, no significant effects of the elevated CO₂ treatment on wood density were observed, neither when green wood density was estimated from stem biomass and stem volume, nor when oven-dry wood density was measured on small wood samples. Under elevated CO₂ significantly wider growth rings were observed. The effect of elevated CO₂ on growth ring width was primarily the result of an increase in earlywood width. Wood compression strength decreased under elevated CO₂ conditions, which could be explained by significantly larger tracheids and the increased earlywood band, that has thinner walls and larger cavities. A significant decrease of the number of resin canals in the third growth ring was observed under the elevated treatment; this might indicate that trees produced and contained less resin, which has implications for disease and pest resistance. So, although wood volume yield in Scots pine increased significantly with elevated CO₂ after three years of treatment, wood density remained unchanged, while wood strength decreased. Whilst wood volume and stem biomass production may increase in this major boreal forest tree species, wood quality and resin production might decrease under future elevated CO₂ conditions.     

An anatomical study of the haustoria of Rhinanthus minor attached to roots of different hosts

Roots of a range of potential hosts responded differently when Rhinanthus minor attempted to form haustoria. Roots of Fabaceae show the weakest reaction as apart from slight lignification, no reaction was observed at the interface between the endophyte and the cortical tissue of the host root. Grass roots react with strong lignification of all cells within the stele with the exception of a small number of phloem cells whilst the endodermis fully enters the tertiary stage. In the case of Phleum bertolonii the cortical cells also become lignified. The lignification is even observed in the host root tissue in a distance of about 1 mm from the haustorium (both apically and basipetally). In the case of Leucanthemum vulgare, strong suberisation can be observed in the cell walls of the interface between endophyte (tip of the sucker) and host. Plantago lanceolata exhibits the strongest reactions against the haustorial tissues. Cells of the interface between the endophyte and the host cortex are completely destroyed, as well as a few cell layers outside the central xylem cylinder, even in some distance from the haustorium. Thus, host xylem is completely isolated from the haustorium in this case. Extraction of sap from xylem vessels is likely to be drastically impaired in such a situation.     

Infection biology and defence responses in sorghum against Colletotrichum sublineolum

Aims:  To investigate the infection biology of Colletotrichum sublineolum (isolate CP2126) and defence responses in leaves of resistant (SC146), intermediately resistant (SC326) and susceptible (BTx623) sorghum genotypes.
Methods and Results:  Infection biology and defence responses were studied quantitatively by light microscopy, H₂O₂ accumulation by DAB staining and HRGP accumulation by immunological methods. Inhibition of conidial germination and appressorium formation may represent prepenetration defence responses on the leaf surface. Inducible defence responses in the resistant genotypes included decreases in formation of appressoria as well as accumulation of H₂O₂, HRGPs and phytoalexins. Concomitant with these inducible responses, fungal growth was stopped during or just after penetration in genotypes SC146 and SC326. High levels of H₂O₂ accumulating at late infection stages (5 days after inoculation) in the susceptible genotype BTx623 correlated with necrosis and tissue degeneration.
Conclusions:  The early accumulation of H₂O₂ and HRGPs indicates roles in defence whereas the late accumulation in genotype BTx623 correlated with successful pathogenesis.
Significance and Impact of the Study:  The fact that there is a significant correlation between induced accumulation of H₂O₂, papilla formation and cell wall cross-linking, as evidenced by HRGP accumulation, and cessation of pathogen growth in resistant genotypes may help exploit host resistance in sorghum.     

Effect of enhanced atmospheric CO2 on mycorrhizal colonization by Glomus mosseae in Plantago lanceolata and Trifolium repens

Plantago lanceolata L. and Trifolium repens L. were grown for 16 wk in ambient (360 μmol mol⁻¹) and elevated (610 μmol mol⁻¹) atmospheric CO₂. Plants were inoculated with the arbuscular mycorrhizal (AM) fungus Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe and given a phosphorus supply in the form of bonemeal, which would not be immediately available to the plants. Seven sequential harvests were taken to determine whether the effect of elevated CO₂ on mycorrhizal colonization was independent of the effect of CO₂ on plant growth. Plant growth analysis showed that both species grew faster in elevated CO₂ and that P. lanceolata had increased carbon allocation towards the roots. Elevated CO₂ did not affect the percentage of root length colonized (RLC); although total colonized root length was greater, when plant size was taken into account this effect disappeared. This finding was also true for root length colonized by arbuscules. No CO₂ effect was found on hyphal density (colonization intensity) in roots. The P content of plants was increased at elevated CO₂, although both shoot and root tissue P concentration were unchanged. This was again as a result of bigger plants at elevated CO₂. Phosphorus inflow was unaffected by CO₂ concentrations. It is concluded that there is no direct permanent effect of elevated CO₂ on mycorrhizal functioning, as internal mycorrhizal development and the mycorrhizal P uptake mechanism are unaffected. The importance of sequential harvests in experiments is discussed. The direction for future research is highlighted, especially in relation to C storage in the soil.     

Factors Affecting Germination and the Mode of Germination of Zygospores of Choanephora cucurbitarum

Treatment of zygospores of Choanephora cucurbitarum with KMnO₄, NaClO or H₂O₂ effectively activated the spores and induced their germination. The optimum concentration of KMnO₄ for activation of zygospores was 0.25 to 0.5%. Zygospores were not able to germinate in darkness even after activation by KMnO₄. When zygospores from 40 to 50-day-old cultures were treated with 0.5% KMnO₄ solution for 60 min before incubation on water agar at 24% under light, about 50% germinated in 10 days. KMnO₄ treatment killed more than 99% of residual mycelial fragments, sporangiospores and sporangiola in the zygospore suspension. During germination disappearance of oil droplets in zygospores occurred prior to the cracking on zygospore wall. Both sporangial germination and mycelial germination were found. Moreover, sporangiole germination was observed for the first time.     

Inhibition of Bradykinin-Induced Cytosolic Ca2+ Elevation by Muscarinic Stimulation Without Attenuation of Inositol 1,4,5-Trisphosphate Production in Human Neuroblastoma SK-N-BE(2)C Cells

Abstract: Cross talk between two phospholipase C (PLC)-linked receptor signalings was investigated in SK-N-BE(2)C human neuroblastoma cells. Sequential stimulation with two agonists at 5-min intervals was performed to examine the interaction between muscarinic and bradykinin (BK) receptors. Pretreatment of cells with a maximal effective concentration (5 µ M ) of BK did not affect the subsequent carbachol (CCh)-induced [Ca²⁺]_i rise, but CCh (1 m M ) pretreatment completely abolished the BK-induced [Ca²⁺]_i rise without inhibition of BK-induced inositol 1,4,5-trisphosphate (IP₃) generation. Thapsigargin (1 µ M ) pretreatment abolished the subsequent BK- and CCh-induced [Ca²⁺]_i rise, though it did not affect agonist-induced IP₃ generation. However, the addition of atropine at plateau phases of CCh-induced [Ca²⁺]_i rise and IP₃ production caused a rapid decline to the basal levels and then restored the [Ca²⁺]_i rise by BK. Treatment of cells with both CCh and BK at the same time showed additive effects in IP₃ production. However, the [Ca²⁺]_i rise induced by both agonists in the presence or absence of extracellular Ca²⁺ was the same as the responses triggered by CCh alone. The results suggest that each receptor or receptor-linked PLC activity is not influenced by pretreatment with the other agonist but IP₃-sensitive Ca²⁺ stores are shared by signal pathways from both receptors.     

Polygalacturonase biosynthesis by Lactobacillus plantarum: effect of cultural conditions on enzyme production

Lactobacillus plantarum was found to produce extracellular polygalacturonase (EC 3.2.1.15.). Maximum enzyme production was obtained in a medium containing 0.5% glucose and 1.5% low methyl-pectin as inducer at 27°C at an initial pH of 6.8. Enzyme production was strongly inhibited by 5 μmol/l NiCl₂, 5 μmol/l CoCl₂, 5 μmol/l CuSO₄, and 10 μmol/l ZnCl₂. MnSO₄ and MgSO₄ at 200 μmol/l and 50 μmol/l respectively seemed to enhance enzyme biosynthesis. The optimal pH and temperature for enzyme activity were 4.5 and 30°C respectively. Enzyme production in batch culture accompanied growth.     

Highly substituted glucuronoarabinoxylans (hsGAXs) and low-branched xylans show a distinct localization pattern in the tissues of Zea mays L

Polyclonal antibodies which recognized highly substituted glucuronoarabinoxylans (hsGAXs) and low-branched xylans and did not cross-react with each other, were raised in order to examine localization of these epitopes in internodes of maize. Immunofluorescent labeling revealed different pattern between two succeeding developmental stages. The hsGAX epitope was localized evenly in primary walls in all tissue types, and strongly in unlignified secondary walls in phloem. However, lignified secondary walls in protoxylem, parenchyma and a part of fibers were faintly labeled with this epitope. Moreover, the epitope showed limited binding in lignified parenchyma and fiber walls at ultrastructural level. Low-branched xylan epitope was localized evenly throughout lignified walls in all tissue types. This epitope was also localized only in lignified walls of other organs such as leaf, root apex and dark-grown mesocotyl. Low-branched xylans are significantly related to lignification. Localization of hsGAX epitope in their organs was similar to that in internodes. The hsGAX epitope was distributed both in unlignified walls of all tissues and in lignified walls of parenchyma and annular thickening of protoxylem. We propose that hsGAX has separate functions in lignified and unlignified tissues. In conclusion, at tissue level, hsGAX is localized mainly in unlignified walls, and low-branched xylans in lignified walls.