The effect of ethylene on the cell-type-specific and intracellular localization of β-1,3-glucanase and chitinase in tobacco leaves

We have studied the effect of ethylene on the localization of the basic isoforms of glucan endo-1,3--glucosidase (-1,3-glucanase, EC 3.2.1.39) and endo-chitinase (chitinase, EC 3.2.1.14) in leaves of Nicotiana tabacum L. cv. Havana 425. Comparisons of the enzyme contents of the lower epidermis of the leaf, leaf expiants with the lower epidermis removed, and intercellular wash fluid indicate that both enzymes are localized inside epidermal cells of untreated leaves. Ethylene treatment (20 l·l^-1, 4d) induced a marked -10- to 30-fold-coordinated accumulation of the enzymes. This was due primarily to induction of the basic isoforms inside chlorenchyma cells of the leaf interior. The localization of basic -1,3-glucanase was confirmed by immunofluorescence histochemistry and immunogold cytochemistry. Immunolabelling was confined to electron-dense bodies of the cell vacuole. No extracellular immunolabelling was detected in control or ethylene-treated leaves. We conclude that ethylene changes the cell-type-specific distribution but not the intracellular compartmentation of the two enzymes. These results support the generalization that basic isoforms of chitinase and -1,3-glucanase are intracellular whereas the acidic isoforms are secreted into the extracellular space.Abbreviations IgG immunoglobulin G - IWF intercellular wash fluid - PBS 0.14 M NaCl, 0.1 M K₂HPO₄, pH 7.5 - TMV tobacco mosaic virus We thank Monique Seldran and Alfred Milani for expert technical help, Patricia Ahl-Goy, Ciba-Geigy, AG, Basel for supplying IWF from TMV-infected leaves, and our colleagues Thomas Boller and Lilian Sticher for their comments and criticism.     

Selenium modulates the activities of antioxidant enzymes,osmotic homeostasis and promotes the growth of sorrel seedlings under salt stress

Using physiological assays coupled with ultrathin tissue sections, we investigated the impacts of exogenous selenium (Se) on the growth, antioxidant enzymes, osmotic regulation and ultrastructural modifications of leaf mesophyll and root tip cells of 100 mM NaCl-stressed sorrel (Rumex patientia × R. tianshanicus) seedlings. At low concentrations (1–5 M), Se tended to stimulate the growth, the activities of superoxide dismutase and peroxidase enzymes, as well as the accumulation of water-soluble sugar in leaves of sorrel seedlings. At higher concentrations (10–30 M), Se exerted diminished beneficial effects on growth and enzyme activities. CAT activity did not change with Se addition (1–30 M). Electrolyte leakage of leaf cells declined, and K⁺ and Na⁺ ions increased in leaves with Se treatment, notably at 5 M of Se. TEM observations revealed that treatment with 5 M of Se positively promoted the integrity of membrane systems and cellular organelles, such as chloroplasts and mitochondria in leaf mesophyll and root tip cells. These results strongly suggest that an appropriate concentration of exogenous Se functions positively to promote the antioxidative and osmoregulatory capacity, and enhance the salt-resistance in sorrel seedlings.     

Production and characterization of β-glucosidases from different Aspergillus strains

-D-Glucosidase enzymes (-D-glucoside glucohydrolase, EC 3.2.1.21) from different Aspergillus strains (Aspergillus phoenicis, A. niger and A. carbonarius) were examined with respect to the enzyme production of the different strains using different carbon sources and to the effect of the pH and temperature on the enzyme activity and stability. An efficient and rapid purification procedure was used for purifying the enzymes. Kinetic experiments were carried out using p-nitrophenyl -D-glucopyranoside (pNPG) and cellobiose as substrates. Two different fermentation methods were employed in which the carbon source was glucose or wheat bran. Aspergillus carbonarius proved to be the less effective strain in -glucosidase production. Aspergillus phoenicis produced the highest amount of -glucosidase on glucose as carbon source however on wheat bran A. niger was the best enzyme producer. Each Aspergillus strain produced one single acidic -glucosidase with pI values in the range of pH 3.52–4.2. There was no significant difference considering the effect of the pH and temperature on the activity and stability among the enzymes from different origins. The enzymes examined have only -glucosidase activity. The kinetic parameters showed that all enzymes hydrolysed pNPG with higher efficiency than cellobiose. This shows that hydrophobic interaction plays an important role in substrate binding. The kinetic parameters demonstrated that there was no significant difference among the enzymes from different origins in hydrolysing pNPG and cellobiose as the substrates.     

Partial characterization of chitin degrading enzymes from two euphausiids,Euphausia superba and Meganyctiphanes norvegica

Summary Chitinase and N-acetyl--D-glucosaminidase have been demonstrated in Meganyctiphanes norvegica and in Euphausia superba and partly characterized. The enzymes from both species have broad pH-optima (maxima around pH 5.0) and temperature optima between 40 and 50°C. The enzymes are relatively stable; even at about 45°C half of the enzyme activity is retained after 30 min incubation. The presence of fluoride does not affeet enzymatic activity. Chitinase activity appears in three different molecular masses, N-acetyl--D-glucosaminidases in two different forms. pH and temperature optima, thermal stability and kinetic properties of the two enzymes are strikingly similar in the polar E. superba versus the boreal euphausiid M. norvegica. Enzyme activity in the lower temperature range is still high, whereas activation energies are low in both euphausiids. This suggests a functional adaptation to a low temperature range in seawater.     

An alkalophilic thermostable lipase produced by a new isolate of Bacillus alcalophilus

An extremophilic bacterium, isolated from mangrove detritus, produced an extracellular alkaline-thermostable lipase. The bacterium was identified on the basis of cell morphology, growth characteristics, G+C molar ratio and DNA/DNA hybridization as a strain of Bacillus alcalophilus. The bacterium grew optimally at pH 10.6, 60°C with NaCl tolerance up to 7.5% (w/v). Carbonates and/or bicarbonates enhanced lipase production, while NaCl had an inhibitory effect. Maximum lipase activity was at 60°C at pH 10.6, with approx. 60% of its activity being retained at 80°C after 20min and 80% of its activity was retained at pH 11 after incubation at 60°C. A partially purified lipase had similar stabilities to the crude enzyme.     

Hydrolysis of lactose by β-galactosidase immobilized in polyvinylalcohol

Summary Fungal -galactosidase was immobilized in polyvinylalcohol gel formed in pores of contton material. Temperature and pH effects on the activity of free and immobilized enzymes were studied. The optimum temperatures of free and immobilized enzymes were 60° C and 55° C respectively. The pH optimum ranged from 4.5 to 5.0 for both enzymes. The thermal stability of the immobilized -galactosidase was slightly higher. The K_m values for soluble and immobilized enzymes were respectively 1.9 mM and 2.5 mM. The optimization of conditions for a highly effective hydrolysis of 4% lactose solution and reusability of the immobilized enzyme resulted in 75% hydrolysis after 5–6 h. The degree of conversion decreased to 50% after 30 repeated runs. The capacity of the immobilized enzyme to hydrolyze lactose in whey was also studied.     

Controlling simultaneous production of endoglucanase and beta-glucosidase by Fusarium oxysporum in submerged culture

Summary The simultaneous production of endoglucanase and -glucosidase by Fusarium oxysporum was investigated in submerged culture. Consecutive optimization of growth conditions resulted in the correction of large activity differences, observed during production of enzymes, and substantially enhanced low enzyme yields. At optimum growth conditions yields as high as 1650 and 232 U per g of carbon source of endoglucanase and -glucosidase were obtained respectively competing favourably with those reported for microorganisms grown on the same carbon source. The most important kinetic characteristics of the enzymes were the high temperature optima of endoglucanase (60°C) and -glucosidase (65°C) and the exceptionally high thermostability of endoglucanase. The latter enzyme retained 50% of the activity at pH 5.0 after approximately 6.5 h at 70°C     

Aluminium toxicity expression nutrient uptake,growth and root morphology ofTrifolium repens L. cv. ‘Grasslands Huia’

Summary Hydroponic experiments were undertaken to examine the effect of increasing aluminium levels on the mineral nutrition and root morphology ofT. repens growing in nutrient solution. Toxicity symptoms appear between 27.8 and 47.5 M Al³⁺ activity (148 to 297 M total aluminium). The threshold level corresponding to a 10% reduction in leaf fresh weight is estimated to be approximately 20 M Al³⁺ activity.The concentration of aluminium in the leaves of white clover increases exponentially with aluminium activity in the nutrient solution. The uptake of divalent cations was inhibited but aluminium enhanced potassium and nitrogen concentrations in both leaves and roots.At high pH (pH 6.0) the speciation of aluminium is controlled by the formation of solid aluminium phosphate and aluminium hydroxide except at the lowest aluminium level (37 M) where 99.9 per cent is present as the DTPA complex. As the concentration of total aluminium increases, the percentage of Al-DTPA and soluble aluminium hydroxide decreases whilst solid Al(OH)₃ increases rapidly to reach a maximum of 91.6 percent (of the total aluminium) in the 1180 M aluminium treatment. At pH 4.5 the dominant forms of aluminium are free aluminium ion Al-DTPA, AlSO ₄ ⁺ and AlOH²⁺.The roots of aluminium stressed plants showed symptoms typical of aluminium toxicity.     

Correlation between loss of turgor and accumulation of abscisic acid in detached leaves

Mature leaves of Phaseolus vulgaris L. (red kidney bean), Xanthium strumarium L. (cocklebur), and Gossypium hirsutum L. (cotton) were used to study accumulation of abscisic acid (ABA) during water stress. The water status of individual, detached leaves was monitored while the leaves slowly wilted, and samples were cut from the leaves as they lost water. The leaf sections were incubated at their respecitive water contents to allow ABA to build up or not. At least 8 h were required for a new steady-state level of ABA to be established. The samples from any one leaf covered a range of known water potentials (), osmotic pressures (), and turgor pressures (p). The and p values were calculated from pressure-volume curves, using a pressure bomb to measure the water potentials. Decreasing water potential had little effect on ABA levels in leaves at high turgor. Sensitivity of the production of ABA to changes in progressively increased as turgor approached zero. At p=1 bar, ABA content averaged 4 times the level found in fully turgid samples. Below p=1 bar, ABA content increased sharply to as much as 40 times the level found in unstressed samples. ABA levels rose steeply at different water potentials for different leaves, according to the at which turgor became zero. These differences were caused by the different osmotic pressures of the leaves that were used; must cqual - for turgor to be zero. Leaves vary in , not only among species, but also between plants of one and the same species depending on the growing conditions. A difference of 6 bars (calculated at =0) was found between the osmotic pressures of leaves from two groups of G. hirsutum plants; one group had previously experienced periodic water stress, and the other group had never been stressed. When individual leaves were subsequently wilted, the leaves from stress-conditioned plants required a lower water potential in order to accumulate ABA than did leaves from previously unstressed plants. On the basis of these results we suggest that turgor is the critical parameter of plant water relations which controls ABA production in water-stressed leaves.Abbreviations ABA abscisic acid - me-ABA abscisic-acid methyl ester - leaf water potential - osmotic pressure - p volumeaveraged turgor - volumetric modulus of elasticity     

Pathway of assimilate transfer between mesophyll cells and minor veins in leaves of Cucumis melo L.

Photoassimilating mature leaves of Cucumis melo exported carbon at a rate of 1.7 mg C·dm^-2·h^-1. Radiolabeling with ¹⁴C showed that stachyose and raffinose are the main carbohydrates translocated. Autoradiograms indicated that sieve elements of the abaxial phloem of minor veins are the sole conduits for carbon export from mature leaves and carbon import into immature leaflets. Sieve elements of the abaxial phloem are associated with intermediary cells which are intimately connected with the surrounding mesophyll cells by numerous plasmodesmata. Photoassimilate, labeled with ¹⁴C, was released into the leaf apoplast and could be trapped in a buffer solution circulating over the abraded adaxial epidermis. Carbon efflux was 1% of the carbon-export rate. A comparable distribution of ¹⁴C among the sugars, amino acids and organic acids, recovered from the free space and from leaf extracts, was recorded. The composition of released ¹⁴C-labeled carbohydrates in the free space resembled the pattern of photoassimilate, but differed clearly from the translocate. Release of organic compounds into the leaf apoplast was stimulated by chelating agents like Na-ATP, ethylenediaminetetraacetic acid and ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid; a correlation between carbon efflux into the apoplast and carbon export from the leaf was not detected. It is suggested that the release of organic compounds into the leaf apoplast of Cucumis melo is the consequence of a general leakage from mesophyll and vascular parenchyma cells. A selective release of transport oligosaccharides was not observed. The experimental results presented here do not preclude a symplastic transfer of assimilates in mature leaves.Abbreviations EDTA ethylenediaminetetraacetate - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetate - PCMBS p-chloromercuribenzenesulfonic acid     

Effects of water stress on the gas exchange,the activities of some enzymes of carbon and nitrogen metabolism,and on the pool sizes of some organic acids in maize leaves

Water-stressed maize (Zea mays L.) leaves showed a large decrease in leaf conductance during photosynthesis. Net CO₂ uptake and evaporation declined fast at mild stress (=–0.6 to –1.0 MPa) and slower at more severe stress (=–1.0 to -1.2 MPa), whereas the CO₂ concentration in the intercellular spaces (C_i) did not drop to the CO₂ compensation point. The activities of the enzymes of photosynthetic carbon metabolism tested in this study dropped by approx. 30% at =-1.2 MPa. Glutamine synthetase activity was unaffected by water stress, whereas the activity of nitrate reductase was almost completely inhibited. The decline of enzyme activities in relation to was correlated with a concomitant decrease in the content of total soluble protein of the stressed leaves. The total leaf pools of malate, pyruvate and oxaloacetate decreased almost linearly in relation to , thus obviously contradicting the almost constant C_i. In comparison to the controls (=0.6 MPa) the content of citrate and isocitrate increaed markedly at =-0.9 MPa and decreased again at =-1.2 MPa.Abbreviations PCR photosynthetic carbon reduction cycle - PCO photosynthetic carbon oxidation cycle - PEP phosphoenolypyruvate - RuBP ribulose-1,5-bisphosphate     

Distribution of magnesium in wheat (Triticum aestivum L.) in relation to supply

The aims of this study were to describe the distribution of magnesium (Mg) and its retranslocation within wheat, in order to develop diagnostic procedures for Mg deficiency. Plants were grown in solution culture with both constant supply (0, 5, 10, 20, 40, 80, 160 MMg) and discontinued supply (40 M and 160 M decreased to nil).Magnesium was depleted from old leaves when Mg supply to the roots was halted. However, initial deficiency symptoms occurred on young leaves under constant but inadequate supply, contrasting with previous reports. Magnesium concentrations were also lower in young leaves compared to old leaves. Symptoms of yellowing and necrosis occurred if the leaf tissue contained <1194 gg^–1, irrespective of leaf age. The minimum Mg concentration in whole shoots associated with maximum shoot weight was 932 gg^–1; for the youngest emerged blade (YEB) it was 861 gg^–1. Symptoms were apparent on the young leaf before a reduction in shoot weight was measurable. The concentration of Mg in the YEB and whole shoot were better related to solution Mg concentration than was the Mg concentration in the old leaf.     

Pattern of Phyllogenesis in the neonatal and regenerating Pongamia glabra L.

The seedling of Pongamia glabra L. generates some simple leaves before producing the first compound leaf. The regenerates arising from decapitated adult plants also show the same sequence of phyllogenesis, i.e. generation of an initial instalment of simple leaves followed by compound-leaf-production.     

Relationship of photosynthetic acclimation to changes of Rubisco activity in field-grown winter wheat and barley during growth in elevated carbon dioxide

The responses of photosynthesis, Rubisco activity, Rubisco protein, leaf carbohydrates and total soluble protein to three carbon dioxide treatments were studied in winter wheat [Triticum aestivum (L.)] and barley [Hordeum vulgare (L.)]. Barley and wheat plants were grown in small field plots during 1995 and 1996 in clear, acrylic chambers (1.2–2.4 m²) and were provided with continuous carbon dioxide fertilization at concentrations of 350, 525 and 700 mol mol^–1. Photosynthetic rates of barley penultimate leaves and wheat flag leaves measured at growth carbon dioxide concentrations decreased with leaf age in all three CO₂ treatments during 1995 and 1996. Photosynthetic acclimation to elevated CO₂ was observed on seven of eight measurement dates for barley and ten of eleven measurement dates for wheat over both years. Initial Rubisco activity, total soluble protein and Rubisco protein in barley penultimate leaves and wheat flag leaves also decreased with leaf age. Total Rubisco activity was not used because of enzyme degradation. There was a significant CO₂ treatment effect on initial Rubisco activity, total soluble protein and Rubisco protein for wheat in 1995 and 1996 and for barley in 1995. Responses of barley penultimate leaf Rubisco activity and leaf protein concentrations to elevated carbon dioxide were nonsignificant in 1996. A significant CO₂ treatment effect also was detected when means of Rubisco activity, soluble protein and Rubisco protein for wheat flag leaves were combined over harvests and years. These three flag leaf parameters were not significantly different in the 350 and 525 mol mol^–1 CO₂ treatments but were decreased during growth in 700 mol mol^–1 CO₂ relative to the other two CO₂ treatments. Ratios of photosynthesis at 700 and 350 mol mol^–1 were compared to ratios of Rubisco activity at 700 and 350 mol mol^–1 using wheat flag leaf data from 1995 and 1996. Regression analysis of these data were linear [y = 0.586 + 1.103t x (r² = 0.432)] and were significant at P 0.05. This result indicated that photosynthetic acclimation was positively correlated with changes of initial Rubisco activity in wheat flag leaves in response to CO₂ enrichment. Effects of elevated CO₂ on wheat leaf proteins during 1995 and 1996 and on barley during 1995 were consistent with an acceleration of senescence.     

Production and localisation of carboxymethylcellulase,xylanase and β-glucosidase fromCellulomonas andMicrococcus spp.

Summary Cellulomonas and Micrococcus spp. grew well at 30°C, pH 7.0, and produced carboxymethylcellulase (CMCase) and xylanase enzymes. Only one species of Micrococcus was able to produce an appreciable amount of -glucosidase. This is the first report where Micrococcus sp., isolated from termite gut, was able to produce all three enzymes (i.e. CMCase, xylanase and -glucosidase) required for degradation of cellulosic and hemicellulosic substrates. Offprint requests to: A. Varma     

Structural and Functional Rearrangements of Mesophyll as a Probable Basis for the Cytokinin-Dependent Assimilate Translocation in Detached Leaves

The effects of benzyladenine (BA) on the mesophyll functioning, such as osmotic potential (), the effect of the inhibitors of ⁺-ATPase on the influx of ¹⁴C-sucrose, the direction of carbon metabolism, and the rate of dark respiration, were followed in the detached leaves of pumpkin (Cucurbita pepo L.) and broad beans (Vicia faba L.). BA elevated and established a gradient of (_p) between the treated and untreated leaf regions. The inhibitors of H⁺-ATPase did not affect the BA-induced influx of ¹⁴C-sucrose. The changes were accompanied with the elevated synthesis of starch and other polymeric compounds and the diminished synthesis of the substances of relatively low molecular weight. The stimulation of dark respiration was short and inconsiderable. The author concludes that the BA-induced transport was a passive process related to a increase. Leaf expansion accompanied by the synthesis of high-molecular-weight substances essential for cell growth and by starch synthesis apparently increased the sink capacity of the BA-treated detached leaves. The diminished efflux from the leaf blade was probably related to a lowered level of the transportable carbon compounds restricting their entry into the phloem. The influx induction could result from the activation of growth and metabolic processes, the decline in the number of organic molecules per cell volume unit, and the development of _p between the source and sink leaf regions.     

Non-uniformities in the metabolism of excised leaves and leaf discs

Summary In intact tobacco and Chinese-cabbage (Brassica pekinensis) leaves an ageing process begins as soon as the leaves are excised. The terminal few millimetres of the petiole increasingly preempt materials such as phosphate and uracil taken up by the leaf. Actinomycin D treatment blocks this process and leads to increased uptake of such materials into the lamina.Immediately after excision there is a radial geographical gradient, in the ability of excised leaf discs to accumulate phosphate and uracil from solution. Tissue near the cut edge accumulates much more of these materials than that near the centre, and total nucleic acids isolated from the outer zone have a higher specific activity.Over the first day after excision there is a marked increase in this ability of the outer tissue of discs to accumulate labelled precursors but the changes taking place with time are complex and variable. Specific activity of total nucleic acids increases somewhat less than the increased uptake of labelled precursor. Actinomycin D becomes very unevenly distributed in leaf discs floated on solutions of the drug. These various effects are of sufficient magnitude to affect the interpretation of studies using excised leaf tissues.     

Fungal biomass and decomposition in Spartina maritima leaves in the Mondego salt marsh (Portugal)

Spartina maritima (Curtis) Fernald is a dominant species in the Mondego salt marsh on the western coast of Portugal, and it plays a significant role in estuarine productivity. In this work, leaf litter production dynamics and fungal importance for leaf decomposition processes in Spartina maritima were studied. Leaf fall was highly seasonal, being significantly higher during dry months. It ranged from 42 g m^-2 in June to less than 6 g m^-2 during the winter. Fungal biomass, measured as ergosterol content, did not differ significantly between standing-decaying leaves and naturally detached leaves. Fungal biomass increased in wet months, with a maximum of 614 g g^-1 of ergosterol in January in standing-decaying leaves, and 1077 g g^-1 in December, in naturally detached leaves, decreasing greatly in summer. Seasonal pattern of fungal colonization was similar in leaves placed in litterbags on the marsh-sediment surface. However, ergosterol concentrations associated with standing-decaying and naturally detached leaves were always much higher than in litterbagged leaves, suggesting that fungal activity was more important before leaf fall. Dry mass of litterbagged leaves declined rapidly after 1 month (about 50%), mostly due to leaching of soluble organic compounds. After 13 months, Spartina leaves had lost 88% of their original dry weight. The decomposition rate constant (k) for Spartina maritima leaves was 0.151 month^-1.     

Low molecular weight thermostable β-D-glucosidase from Acidothermus cellulolyticus

Summary Industrial use of cellulase enzymes, including -D-glucosidases, can be greatly improved and expanded by the discovery and characterization of more robust, thermotolerant enzymes. A new thermostable -D-glucosidase was isolated from Acidothermus cellulolyticus culture broth. This enzyme has a molecular weight of 52.5 kDa, an isoelectric pI of 4.1, and a pH activity optimum of 4.5. The optimum temperature for activity on p-nitrophenyl--D-glucoside is 63°C. This value is approximately 20°C higher than temperature optima displayed by most previously characterized -D-glucosidases.     

Variation in levels of some leaf enzymes

Summary Several procedures were compared for efficiency in the extraction of certain leaf enzymes (phosphoenolpyruvate carboxylase, ribulose 1,5-diphosphate carboxylase and malate dehydrogenase) in Atriplex hastata (a C₃ species exhibiting conventional photosynthetic metabolism), and in A. spongiosa (a C₄ species in which the initial photosynthetic products are C₄ dicarboxylic acids). Glycolate oxidase was also assayed in some cases, and Atriplex nummularia and Sorghum bicolor were also used as test material. A simple procedure, involving a mortar and pestle grind with carborundum added to the grinding mixture, was found to be as effective as glass bead grind procedures. In addition, it was more rapid and showed less variability with different operations.Using the carborundum grind procedure, sources of variability in enzyme activity in apparently uniform leaves were compared, as were effects of time of day, leaf age and storage procedure. In general, if apparently uniform leaves could be selected, variability in levels of enzyme activity appeared to be relatively small, not exceeding about 12%. Time of day also appeared to be relatively unimportant for the enzymes examined. However, the ontogentic status of the plant was found to be an important source of variability. Leaf age was also a major source of variability where the activity was expressed on a fresh weight basis, but specific activity (i.e. activity expressed on a protein basis) was relatively constant, at least with the range of species and leaf ages examined here.Storage of fresh samples in liquid nitrogen for 24 h, prior to extraction and assay, led to only a small reduction in activity, but substantial changes occurred if storage was in dry ice or in ice and also where extracts were stored in a deep freeze.