Root Growth-promoting Activity of Unsaturated Oligomeric Uronates from Alginate on Carrot and Rice Plants

The root elongation activity of unsaturated oligomeric uronates from alginate on carrot and rice plants was investigated. Unsaturated oligomeric uronates were prepared by digesting polymannuronate (PM) and polyguluronate (PG) with an alginate lyase purified from Pseudoalteromonas sp. strain No. 272. The root elongation activity was measured by elongation in length of carrot- and rice-excised root incubated in the B5-medium containing 0.8% agar in the dark. PM and PG showed no activity, but the enzymatic digestion mixtures of PG had promoting activity on roots of both plants at a final concentration of 0.5 mg/ml. The maximum activity was obtained at 0.75 mg/ml. The dependence of activity on degree of polymerization of the uronates was tested and the pentamer was most active, but the mechanism of the action of unsaturated uronates on the cells remains to be solved.     

Purification of alginate oligosaccharides with root growth-promoting activity toward lettuce

Sodium alginate was degraded by alginate lyase from Corynebacterium sp., and the product was purified by an ultrafiltration (UF) membrane module. The UF treatment was carried out at a transmembrane pressure of 0.15 MPa and a flow velocity of 0.6 m/s in the cross-flow mode, and non-degraded alginate was almost completely removed. The alginate oligosaccharide obtained was a mixture of di- to octasaccharides and had promoting activity toward lettuce root elongation (about 2-fold compared with the control) in the concentration range of 200-3000 microg/ml. The effect of the degree of polymerization on this activity was examined by using each oligosaccharide fractionated by gel chromatography. The tri-, tetra-, penta- and hexasaccharides were each found to have root growth-promoting activity in a lettuce bioassay.     

Structure-activity relationship of alginate oligosaccharides in the induction of cytokine production from RAW264.7 cells

Guluronate and mannuronate oligomers with various degree of polymerization were prepared from polyguluronate (PG) and polymannuronate (PM) with an alginate lyase from a Pseudoalteromonas sp., and their activities to induce cytokine secretion from mouse macrophage cell line RAW264.7 cells were examined. Enzymatically depolymerized unsaturated alginate oligomers induced tumor necrosis factor (TNF)-alpha secretion from RAW264.7 cells in a structure-depending manner, while the activities of saturated alginate oligomers prepared by acid hydrolysis were fairly low or only trace levels. These results suggest that unsaturated end-structure of alginate oligomers was important for the TNF-alpha-inducing activity. Among the unsaturated guluronate (G3-G9) and mannuronate (M3-M9) oligomers, G8 and M7 showed the most potent activity, respectively. Bio-Plex assay revealed that interleukin (IL)-1alpha, IL-1beta, and IL-6 secretion from RAW264.7 cells were also induced by unsaturated alginate oligomers with similar structure-activity relationship profiles as seen in TNF-alpha, and the most potent activities were observed with G8 and M7. These results suggest that G8 and M7 may have the most suitable molecular size or entire structural conformation as stimulant for cytokine secretion. Since antibodies to Toll-like receptor (TLR)2 and TLR4 effectively inhibited the G8- and M7-induced production of TNF-alpha, these alginate oligomers may stimulate innate immunity through the pattern recognition receptors on macrophages similar to microbial products.     

Aluminum inhibits the H(+)-ATPase activity by permanently altering the plasma membrane surface potentials in squash roots

Although aluminum (AL) toxicity has been widely studied in monocotyledonous crop plants, the mechanism of Al impact on economically important dicotyledonous plants is poorly understood. Here, we report the spatial pattern of Al-induced root growth inhibition, which is closely associated with inhibition of H(+)-ATPase activity coupled with decreased surface negativity of plasma membrane (PM) vesicles isolated from apical 5-mm root segments of squash (Cucurbita pepo L. cv Tetsukabuto) plants. High-sensitivity growth measurements indicated that the central elongation zone, located 2 to 4 mm from the tip, was preferentially inhibited where high Al accumulation was found. The highest positive shifts (depolarization) in zeta potential of the isolated PM vesicles from 0- to 5-mm regions of Al-treated roots were corresponded to pronounced inhibition of H(+)-ATPase activity. The depolarization of PM vesicles isolated from Al-treated roots in response to added Al in vitro was less than that of control roots, suggesting, particularly in the first 5-mm root apex, a tight Al binding to PM target sites or irreversible alteration of PM properties upon Al treatment to intact plants. In line with these data, immunolocalization of H(+)-ATPase revealed decreases in tissue-specific H(+)-ATPase in the epidermal and cortex cells (2--3 mm from tip) following Al treatments. Our report provides the first circumstantial evidence for a zone-specific depolarization of PM surface potential coupled with inhibition of H(+)-ATPase activity. These effects may indicate a direct Al interaction with H(+)-ATPase from the cytoplasmic side of the PM.     

Root elongation in saline solution related to calcium binding to root cell plasma membranes

To gain a better understanding of the relations between root elongation and the amount of Ca²⁺ bound to the plasma membrane (PM), melon plants were grown in aerated solutions containing different concentrations of CaCl₂ with various concentrations of NaCl or mannitol. With increasing external concentrations of NaCl or mannitol, root elongation was suppressed. Addition of CaCl₂ to the external medium alleviated the inhibition of root elongation by high concentrations of Na⁺, but not of mannitol. Root elongation in media containing high concentrations of NaCl was correlated with the computed amount of Ca²⁺ bound to the PM. A model describing relative root elongation (RRL) under salt stress was developed. This model takes into account the osmotic potential in the growing solution (based on the mannitol experiments) and the computed amount of Ca²⁺ bound to the PM. Calcium binding was calculated by applying a Gouy-Chapman-Stern sorption model using the same parameters deduced from studies on PM vesicles. This model combines electrostatic theory with competitive binding at the PM surface. The model for RRL allowed the computation of a critical value for the fraction of negative sites binding Ca²⁺ on the PM needed for nearly optimal (95%) root elongation. Any decrease below this critical value decreased the RRL. Root elongation of Honey Dew (salt-resistant cv.) was greater than that of Eshkolit Ha'Amaqim (salt-sensitive cv.) under NaCl stress. Nearly optimal root growth for Honey Dew and Eshkolit Ha'Amaqim occurred when 40% and 51% of total membrane charged sites were bound by Ca²⁺, respectively. The effect of osmotic potential on the suppression of root elongation was the same for the two cultivars. To our knowledge, this report provides the first fully quantitative estimates of PM-bound Ca²⁺ relative to salt toxicity.     

Toxicity of arsenic (III) and (V) on plant growth, element uptake, and total amylolytic activity of mesquite (Prosopis juliflora x P. velutina)

The effects of arsenite [As(III)] and arsenate [As(V)] on the growth of roots, stems, and leaves and the uptake of arsenic (As), micro- and macronutrients, and total amylolytic activity were investigated to elucidate the phytotoxicity of As to the mesquite plant (Prosopis juliflora x P. velutina). The plant growth was evaluated by measuring the root and shoot length, and the element uptake was determined using inductively coupled plasma optical emission spectroscopy. The root and leaf elongation decreased significantly with increasing As(III) and As(V) concentrations; whereas, stem elongation remained unchanged. The As uptake increased with increasing As(III) or As(V) concentrations in the medium. Plants treated with 50 mg/L As(III) accumulated up to 920 mg/kg dry weight (d wt) in roots and 522 mg/kg d wt in leaves, while plants exposed to 50 mg/L As(V) accumulated 1980 and 210 mg/kg d wt in roots and leaves, respectively. Increasing the As(V) concentration up to 20 mg/L resulted in a decrease in the total amylolytic activity. On the contrary, total amylolytic activity in As(III)-treated plants increased with increasing As concentration up to 20 mg/L. The macro- and micronutrient concentrations changed in As-treated plants. In shoots, Mo and K were reduced but Ca was increased, while in roots Fe and Ca were increased but K was reduced. These changes reduced the size of the plants, mainly in the As(III)-treated plants; however, there were no visible sign of As toxicity.     

Immobilization in alginate as a technique for the preservation of Bacillus thuringiensis var. israelensis for long-term preservation

Technique for immobilization using sodium alginate as the matrix to preserve Bacillus thuringiensis var. israelensis isolates for long time storage was developed. Two strains of B. thuringiensis var. israelensis viz., VCRC B-17 and WHO standard strain IPS-82 were immobilized in alginate matrix and preserved at 4 degrees C and when tested both were found to have maintained excellent viability and mosquito larvicidal activity for 10 years. Mosquito larvicidal activity of B-17 and IPS-82 alginate beads, in term of LC(50) values before storage was 72.07 ng/ml and 47.07 ng/ml, respectively and after storage at 4 degrees C for a period of 1 to 10 years the values ranged from 69.88 to 73.86 ng/ml with a mean of 72.38 ng/ml and 45.32 to 48.60 ng/ml with a mean of 47.49 ng/ml, respectively. Similarly spore count of the beads of the respective strains was 4.37 x 10(8) and 3.33 x 10(10) CFU/mg before storage. After storage at 4 degrees C for a period of 1 to 10 years the counts of the beads of the respective strains ranged from 4.23 x 10(8) to 4.83 x 10(8) CFU/mg (mean of 4.49 x 10(8) CFU/mg) and 3.2 x 10(10) to 3.87 x 10(10) CFU/mg (mean of 3.54 x 10(10) CFU/mg). The alginate matrix immobilization technique has many advantages over free cells are that they enhance the stability of both spores and toxin against several physicochemical conditions and confer reduced susceptibility to contamination.     

Overexpression of formate dehydrogenase inArabidopsis thaliana resulted in plants tolerant to high concentrations of formate

The potential role played by formate dehydrogenase (FDH) in formate metabolism has been examined by the overexpression of FDH in Arabidopsis thaliana. Three independent transgenic lines were selected and shown to produce elevated amounts of FDH protein with a corresponding elevated FDH activity (2.5-5 fold) over wild-type (WT) plants. Under normal growth conditions, no altered phenotype was observed in these transgenic plants; in growth media supplied with formate, however, significant differences in shoot and root growth, compared to that of WT plants, were observed. WT plants were severely injured if grown in the presence of 16 mmol/L formate, while the transgenic plants were able to grow well. Formate delayed germination of both WT and transgenic seeds at concentrations above 4 mmol/L, but both types of seeds were eventually able to complete more than 95 % germination even at 32 mmol/L formate. Formate markedly inhibited primary root elongation, and its inhibitory action on WT was much stronger than on transgenic plants. Different formate salts affected root elongation similarly, indicating that the formate ion was the major factor inhibiting root growth. Sodium acetate (NaAc), an analogue of formate, also inhibited root elongation, but its action on WT and transgenic plants was the same, indicating that tolerance of transgenic plants to formate toxicity was specific. Transgenic plants showed no significant tolerance to the toxicity of two other one-carbon metabolites, methanol and formaldehyde. A role for FDH in detoxifying formate is proposed.     

Plasma membrane-bound NADH: Fe3+-EDTA reductase and iron deficiency in tomato (Lycopersicon esculentum). Is there a Turbo reductase?

The properties of NADH-dependent Fe³⁺-EDTA reductase in plasma membranes (PM) from roots of iron-deficient and -sufficient tomato plants [Lycopersicon esculentum L. (Mill.) cv. Abunda] were examined. Iron deficiency resulted in a 3-fold increase of in vivo root iron-chelate reductase activity with a K_m (Fe³⁺-EDTA) of 230 μM. In purified root PM, average specific activities of ferric chelate reductase of 410 and 254 nmol Fe (mg protein)^?1 min^?1 were obtained for iron-deficient and -sufficient plants, respectively. In both cases, the PM-bound activity showed a pH optimum at pH 6.8. Activity depended on NADH and not on NADPH and on the presence of detergent. The activity was inhibited 40-50% by superoxide dismutase (EC 1.15.1.1) and ca 30% by oxygen. Kinetic analysis of the membrane-bound enzyme revealed a K_m (Fe³⁺-EDTA) of ca 200 μM for both iron-stressed and -sufficient plants. For NADH, K_m values around 230 μM were obtained. The ferric chelate reductase could be solubilised from salt-washed PM with Triton X-100 at a protein:detergent ratio of 1:2.8 (w/w). The Triton-soluble fraction revealed one enzyme-stained band in native polyacrylamide electrophoresis. Although the membranes showed no nitrate reductase (NR; EC 1.6.6.1) activity, anti-spinach NR immunoglobulin G (IgG) recognized a 54 kDa band both in the PM and the Triton-soluble fraction, but not in the enzymatically active material obtained from the native gel. No evidence could be found for the synthesis of a new, biochemically distinct PM-bound ferric chelate reductase under iron deficiency, which might be identified as the so-called Turbo reductase. It is concluded that iron deficiency in tomato induces increased expression of a ferric chelate reductase in root PM, which is already present in iron-sufficient plants and probably also in plants, which do not contain the Turbo reductase, like the grasses. The iron reductase is not identical with the recently reported PM-associated nitrate reductase.     

Iron-Stress Induced Redox Activity in Tomato (Lycopersicum esculentum Mill.) Is Localized on the Plasma Membrane

Tomato plants (Lycopersicum esculentum Mill.) were grown for 21-days in a complete hydroponic nutrient solution including Fe³⁺-ethylenediamine-di(o-hydroxyphenylacetate) and subsequently switched to nutrient solution withholding Fe for 8 days to induce Fe stress. The roots of Fe-stressed plants reduced chelated Fe at rates sevenfold higher than roots of plants grown under Fe-sufficient conditions. The response in intact Fe-deficient roots was localized to root hairs, which developed on secondary roots during the period of Fe stress. Plasma membranes (PM) isolated by aqueous two-phase partitioning from tomato roots grown under Fe stress exhibited a 94% increase in rates of NADH-dependent Fe³⁺-citrate reduction compared to PM isolated from roots of Fe-sufficient plants. Optimal detection of the reductase activity required the presence of detergent indicating structural latency. In contrast, NADPH-dependent Fe³⁺-citrate reduction was not significantly different in root PM isolated from Fe-deficient versus Fe-sufficient plants and proceeded at substantially lower rates than NADH-dependent reduction. Mg²⁺-ATPase activity was increased 22% in PM from roots of Fe-deficient plants compared to PM isolated from roots of Fe-sufficient plants. The results localized the increase in Fe reductase activity in roots grown under Fe stress to the PM.     

Characterization of dextransucrase immobilized on calcium alginate beads from Leuconostoc mesenteroides PCSIR-4

Immobilization of dextransucrase from Leuconostoc mesenteroides PCSIR-4 on alginate is optimized for application in the production of dextran from sucrose. Dextransucrase was partially purified by ethanol upto 2.5 fold. Properties of dextransucrase were less affected by immobilization on alginate beads from soluble enzyme. Highest activities of both soluble and immobilized dextransucrase found to be at 35 degrees C and optimum pH for activity remain 5.00. Substrate maxima for immobilized enzyme changed from 125 mg/ml to 200 mg/ml. Incubation time for enzyme-substrate reaction for maximum enzyme activity was increased from 15 minutes to 60 minutes in case of immobilized enzyme. Maximum stability of immobilized dextransucrase was achieved at 25 degrees C with respect to time.     

Correlation between Long-Day Flowering and Root Elongation in Pharbitis nil, Sfrain Kidachi

Long-day flowering of Pharbitis nil, dwarf strain Kidachi, at20?C was greatly influenced by the size of the culture vesseland the number of plants per vessel. The smaller the vessel,the greater the flowering response. The volume of nutrient solutionper plant was not decisive for long-day flowering. For instance,plants cultured singly in 200-ml beakers flowered, but thosecultured in 5,000-ml vessels (33?26?11.5 cm, 48 plants per vessel)did not, even though there was only about 100 ml of nutrientsolution per plant. Long-day flowering was always accompaniedby the suppression of root elongation, but not by a decreasein the dry weight of roots or shoots, or in the rate at whichthe leaf primordia appeared (plastochrone). Aeration of thenutrient solution or culture in vermiculite promoted root elongationeven in small vessels, thereby inhibiting long-day flowering.Thus the suppression of root elongation seems to be necessaryfor long-day flowering. Removal of the roots or cotyledons;however, suppressed long-day flowering even when root elongationwas inhibited by culture in small vessels. When plants werecultured at 24?C, suppression of root elongation (culture ina small vessel) did not induce long-day flowering; but, short-daytreatment induced flowering without suppressing root elongation. (Received April 19, 1982; Accepted June 24, 1982)     

Cold-active polygalacturonase from psychrophilic-basidiomycetous yeast Cystofilobasidium capitatum strain PPY-1

We purified and characterized a cold-active polygalacturonase (PG) from the extracellular fraction of Cystofilobasidium capitatum strain PPY-1. The purified PG from strain PPY-1 has a molecular mass of about 44 kDa, and exhibited high activity at 0 degrees C, although its optimum temperature was 45 degrees C. Although the Km value for polygalacturonate as a substrate at 45 degrees C was found to be 11.2 mg/ml, it decreased gradually with decreasing temperature, and it was 0.66 mg/ml at 0 degrees C. Moreover, its cleavage pattern was of the endo-type. These findings might indicate that PG from strain PPY-1 is a novel type of cold-active endo-PG that is able to degrade pectin compounds at low temperatures.     

来自Yeosuana marina sp.JLT21内切型海藻酸裂解酶的异源表达及酶学表征

褐藻寡糖有着丰富的生物学功能,酶法制备功能性褐藻寡糖具有重要实践应用价值.为发掘高活性及稳定性的褐藻寡糖制备酶,对浅海热液嗜热菌Yeosuana marina sp.JLT21中的海藻酸裂解酶YMA-1的基因在大肠杆菌中进行表达、纯化及酶活鉴定.结果发现YMA-1由306个氨基酸残基构成,是多糖裂解酶家族7(PL7)新...     

Purification and characterization of an extracellular polygalacturonase from Lactobacillus plantarum strain BA 11

Lactobacillus plantarum produced extracellular polygalacturonase in a medium containing 1.5% low methyl-pectin (w/v) and 0.5% glucose (w/v) as inducers. The enzyme was purified (approximately 70-fold) by ammonium sulphate fractionation, Sephadex G-100 gel filtration and DEAE-cellulose ion exchange chromatography. Two peaks (PG I and PG II) of enzymic activity were obtained from the DEAE-cellulose column. The molecular mass of PG I was similar to that of PG II (32 000 Da). The K _m values of PG I and PG II for sodium polypectate were calculated to be 1.63 mg/ml and 1.78 mg/ml respectively. Their isoelectric points were about pH 5.5. The pH optimum was 4.5, while the optimum temperature was 35°C for both PG I and PG II. The two purified enzymes had similar endo modes of action on polygalacturonic acid, as determined by comparison of viscosity reduction and reducing group release.     

Sugarcane Straw and its Phytochemicals as Growth Regulators of Weed and Crop Plants

The present study investigates the effect of soil amended with sugarcane straw leachate and its constituents on root elongation of weed and crop plants. The influence of soil biotic and abiotic factors on plant growth were also evaluated through assays in both autoclaved soil and sand. In unsterile soil, straw leachates stimulated root growth of some test plants at 6 g dry straw ls⁻¹ and inhibited root elongation at higher concentrations. A bioassay guided process of the bioactive leachate constituents led to the isolation of vanillic, syringic and ferulic acids. These compounds were also assayed on the test plants in the three mentioned substrates. In unsterile soil, phenolics stimulated the growth of some species at 19 mg l⁻¹. Higher phenolics concentrations were inhibitory. The concentration needed to inhibit 25% root elongation (EC₂₅) was calculated from the dose–response curves. The concentration of phenolics in the leachate (64 g dry straw l⁻¹) was estimated to be 187 mg l⁻¹ (ferulic acid), 131 mg l⁻¹ (vanillic acid) and 20 mg l⁻¹ (Syringic acid). In general, these concentrations were below the EC₂₅ values determined in unsterile soil indicating that these compounds cannot completely explain the strong inhibitory activity of sugarcane straw leachates. The role of soil factors on phytotoxicity of sugarcane straw leachate and its identified growth regulators is also discussed.     

Aluminum-induced citric acid secretion is not the sole mechanism of Al-resistance in maize

Aluminum-induced citric acid (CA) root secretion is a widely accepted mechanism to explain Al-resistance in maize. Nonetheless, several aspects of this mechanism remain controversial. In this study, we used paclobutrazol (PBZ), a plant growth retardant, to gain new insights into the relationship between Δ⁵-sterol composition, membrane permeability, (PM) H⁺-ATPase activity and CA secretion in an Al-sensitive (UFVM-100) and Al-resistant (UFVM-200) maize genotypes challenged with Al. The Al-sensitive genotype displayed greater concentrations of Al in the root tips and greater inhibition of root elongation (RE), which was accompanied by greater electrolyte leakage and greater reduction in the Δ⁵-sterols content after Al treatment. CA secretion by roots increased in both genotypes after Al treatment but to a greater extent in the Al-resistant genotype. The (PM) H⁺-ATPase activity was down-regulated in the sensitive cultivar and up-regulated in its resistant counterpart upon Al treatment. A significant correlation between (PM) H⁺-ATPase activity and CA secretion was observed, but only in the Al-resistant genotype. Upon adding PBZ to the Al-treated plants, differences in the RE and Δ⁵-sterol composition between the maize genotypes were fully abolished, whereas genotypic differences in CA secretion and (PM) H⁺-ATPase activity were reduced but not completely eliminated. Taken together, this information suggests the existence of other processes or mechanisms operating in the Al resistance in these two maize genotypes.     

An assessment of the role of ethylene in mediating lettuce (Lactuca sativa) root growth at high temperatures

Growth of temperate lettuce (Lactuca sativa) plants aeroponically in tropical greenhouses under ambient root-zone temperatures (A-RZTs) exposes roots to temperatures of up to 40 degrees C during the middle of the day, and severely limits root and shoot growth. The role of ethylene in inhibiting growth was investigated with just-germinated (24-h-old) seedlings in vitro, and 10-d-old plants grown aeroponically. Compared with seedlings maintained at 20 degrees C, root elongation in vitro was inhibited by 39% and root diameter increased by 25% under a temperature regime (38 degrees C/24 degrees C for 7 h/17 h) that simulated A-RZT in the greenhouse. The effects on root elongation were partially alleviated by supplying the ethylene biosynthesis inhibitors aminooxyacetic acid (100-500 microM) or aminoisobutyric acid (5-100 microM) to the seedlings. Application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid to seedlings grown at 20 degrees C mimicked the high temperature effects on root elongation (1 microM) and root diameter (1 mM). Compared with plants grown at a constant 20 degrees C root-zone temperature, A-RZT plants showed decreased stomatal conductance, leaf relative water content, photosynthetic CO(2) assimilation, shoot and root biomass, total root length, the number of root tips, and root surface area, but increased average root diameter. Addition of 10 microM ACC to the nutrient solution of plants grown at a constant 20 degrees C root-zone temperature mimicked the effects of A-RZT on these parameters but did not influence relative water content. Addition of 30 microM aminoisobutyric acid or 100 microM aminooxyacetic acid to the nutrient solution of A-RZT plants increased stomatal conductance and relative water content and decreased average root diameter, but had no effect on other root parameters or root and shoot biomass or photosynthetic CO(2) assimilation. Although ethylene is important in regulating root morphology and elongation at A-RZT, the failure of ethylene biosynthesis inhibitors to influence shoot carbon gain limits their use in ameliorating the growth inhibition induced by A-RZT.     

Uronic Acid products release from enzymically active cell wall from tomato fruit and its dependency on enzyme quantity and distribution

Isolated cell wall from tomato (Lycopersicon esculentum Mill. cv Rutgers) fruit released polymeric (degree of polymerization [DP] > 8), oligomeric, and monomeric uronic acids in a reaction mediated by bound polygalacturonase (PG) (EC 3.2.1.15). Wall autolytic capacity increased with ripening, reflecting increased levels of bound PG; however, characteristic oligomeric and monomeric products were recovered from all wall isolates exhibiting net pectin release. The capacity of wall from fruit at early ripening (breaker, turning) to generate oligomeric and monomeric uronic acids was attributed to the nonuniform ripening pattern of the tomato fruit and, consequently, a locally dense distribution of enzyme in wall originating from those fruit portions at more temporally advanced stages of ripening. Artificial autolytically active wall, prepared by permitting solubilized PG to bind to enzymically inactive wall from maturegreen fruit, released products which were similar in size characteristics to those recovered from active wall isolates. Extraction of wall-bound PG using high concentrations of NaCl (1.2 molar) did not attenuate subsequent autolytic activity but greatly suppressed the production of oligomeric and monomeric products. An examination of water-soluble uronic acids recovered from ripe pericarp tissue disclosed the presence of polymeric and monomeric uronic acids but only trace quantities of oligomers. The significance in autolytic reactions of enzyme quantity and distribution and their possible relevance to in vivo pectin degradation will be discussed.