Components of organic phosphorus in soil extracts that are hydrolysed by phytase and acid phosphatase

 Extracts were prepared from soil using water, 50 mM citric acid (pH ∼2.3) or 0.5 M NaHCO₃ (pH 8.5), and were incubated with excess phytase from Aspergillus niger to determine the amounts of labile P. Two A. niger phytase preparations were used: (1) a purified form which exhibited a narrow substrate specificity and high specific activity against phytate; and (2) a commercial preparation (Sigma) with activity against a broad range of P compounds. A comparatively large proportion (up to 79%, or 5.7 μg g^–1 soil) of the organic P (P_o) extracted with citric acid was hydrolysed by the commercial phytase, while between 28% and 40% (up to 3.1 μg g^–1 soil) was hydrolysed using purified phytase. By comparison, only small quantities of the P_o in water and NaHCO₃ soil extracts were enzyme labile. While extractable P_o was increased both with increasing concentrations of citric acid (up to 50 mM) and increasing pH (pH 2.3–6.0), enzyme-labile P increased only with citric acid concentration. The labile component of P_o in citric acid extracts from soils with contrasting fertiliser histories indicated that enzyme-labile P_o is a relatively large soil P pool and is potentially an important source of P for plants. Received: 29 October 1999     

Comparison of Characteristics of Acid Phosphatases Secreted from Roots of Lupin and Tomato

In this study we purified both acid phosphatases (Apase) secreted from tomato and lupin roots, and compared the properties of these two enzymes. The secretory Apases from tomato and lupin showed the following similar properties. 1) Both enzymes were homodimers consisting of two identical subunits, each with a molecular weight of approximately 68 kilodaltons (kD) in tomato and 72 kD in lupin. 2) The enzymes were stable in the pH range of 4–9. 3) The enzymes showed an optimum temperature of 37–40°C for their activity and were stable at temperatures below 60°C. 4) The enzymes exhibited a comparable affinity for p-nitrophenyl phosphate (the apparent K _m values were 2.7–3.0 × 10^-5 M). On the other hand, there were some slight differences in the isoelectric point, optimum pH, specific activity, substrate specificity, and inhibitory effect of metal ions between the two enzymes. Therefore, it was considered that the most important difference in the root ability of the two plants to hydrolyze organic phosphorus may depend mainly on the amount of Apase secreted from a unit amount of roots.     

Effect of difference between day and night temperature on tomato (Lycopersicon esculentum Mill.) root activity and low molecular weight organic acid secretion

The difference between day and night temperature (DIF) is a major environmental factor affecting crop growth, but the mechanisms are not fully understood. We investigated crop performance, root activity and concentrations of low molecular weight organic acids (LMWOAs) secreted by tomato (Lycopersicon esculentum Mill.) root under different DIF conditions. A fixed daily temperature of 25°C and five DIF treatments (?12, ?6, 0, 6 and 12°C) were used to grow tomato in a climate chamber. Root/shoot ratio; leaf maximum photosynthetic rate (P_max); root activity; total nitrogen (N), phosphorus (P) and potassium (K) concentrations in roots; and types and concentrations of LMWOAs were measured at different growth stages. Results showed that positive and negative DIFs inhibited the dry matter accumulation of aerial parts, while 0°C DIF was conducive to the accumulation. Compared to 0°C DIF, positive DIFs significantly increased root dry weight, P_max, root activity and total N, P and K concentrations in roots, while negative DIFs had contrary effects. During the whole growth period, tomato root activity decreased in the order of fruit setting stage, mature stage and flowering stage. Tomato roots secreted oxalic acid, formic acid, malic acid, malonic acid, lactic acid, acetic acid, citric acid, succinic acid and propionic acid under positive DIFs, while acetic acid was not detected in the negative DIF treatments. Oxalic acid concentration was significantly higher than other LMWOAs. Furthermore, in the same growth stage, positive DIFs caused more LMWOA secretion than negative DIFs and 0°C DIF. There were significant positive correlations between the total LMWOA concentration and root activity, root/shoot ratio, P_max and total N, P and K concentrations in roots. Based on the results, more attention should be paid to the potential effect on tomato growth posed by DIFs, positive DIFs have higher positive influence than negative DIFs, and 6°C DIF is best for greenhouse tomato growth.     

Efficiency of Bacillus coagulans as P biofertilizer to mobilize native soil organic and poorly soluble phosphates and increase crop yield

Bacillus coagulans, a phosphatase- and phytase-producing bacterium was isolated and tested under greenhouse conditions and in the field in a loamy sand soil. Bacterial population build-up and efficiency was compared under sterilized and non-sterilized soil conditions. Exploitation of plant unavailable (poorly soluble) P was higher in sterilized soil, mainly due to an increased bacteria population. A gradual increase in microbial build-up of up to 21 times the inoculated population was observed over a 4-week period under the sterilized soil condition. Clusterbean influenced acid phosphatase and phytase activity. The depletion of organic P was much higher than the depletion of mineral and phytin P. The microbial contribution to the hydrolysis of the different P fractions was significantly higher than the plant contribution. The maximum effect of inoculation on different enzyme activities (acid phosphatase, alkaline phosphatase, phytase and dehydrogenase) was observed in pants between 5 and 8 weeks of age. A significant improvement in plant biomass (25%), root length (28%), plant P concentration (22%), seed (19%) and straw yield (28%) resulted from inoculation. The results suggested that B. coagulans produces phosphatases and phytase, which mobilized P from unavailable native P sources and enhanced the production of clusterbean.     

Effects of fractions of coal-derived humic substances on seed germination and growth of seedlings (Lactuga sativa and Lycopersicum esculentum)

Summary Lettuce and tomato seeds were treated in Petri dishes with a humic acid derived from an oxidized coal and with fractions of the same humic acid obtained by ultrafiltration through membranes of known molecular cutoff and by extracting with buffers set at pH 4 and pH 5. The unfractionated humic acid was applied at 40, 100, 1000, and 5000 mg l^-1 whereas the humic fractions were applied at 40, 100, and 200 mg l^-1. Germination parameters such as the number of total germinated seeds, the velocity of seed germination, the fresh weight and dry weight of total seedlings were measured and related to the chemical and physicochemical properties of the humic material. No increase in the germination percentage or the germination rate was observed for either lettuce or tomato seeds. The fresh weight of total seedlings and per seedling increased in treatments with unfractionated humic acid with increasing concentrations for both lettuce and tomato plants without showing signs of growth inhibition up to 5000 mg l^-1. This was attributed to cell elongation and more efficient water uptake. For the lettuce, the fresh weight both of total seedlings and per seedling was enhanced by treating the seeds with fractions of low molecular weight and high content of acidic functions, whereas the dry weight both of total seedlings and per seedling did not change with the humic fraction used. For the tomato seeds in contrast, the dry weight both of total seedlings and per seedling was increased by the use of unfractionated humic acid and by some of the humic fractions. An uptake of humic material by growing tomato seedlings was inferred.     

Relationship between viscosity and molecular weight in an andosol humic acid

Viscosity and molecular weight of particle size fractions obtained from an Andosol humic acid (HA) were determined. Viscosity was determined both in solutions with and without the addition of 0.1 M NaCl (Cs 0.1 and Cs 0, respectively) at pH 7.0. Polyelectrolytic character was observed in the particle size fractions based on the changes in the concentration dependence of reduced viscosity with NaCI concentrations. The use of a Cs 0.1 solution was suitable for the determination of the values of intrinsic viscosity ([η]) of the particle size fractions. The [η] value increased with increasing weight average molecular weight ( M _w) determined by gel permeation chromatography (GPC), and ranged from 4.3 to 12.9 X 10-3 (L g^-1) in the Cs 0 solution and from 3.5 to 6.6 × 10^-3 (L g^-1) in the Cs 0.1 solution. A linear relationship between [η] in the Cs 0.1 solution and M _w on a logarithmic scale, which was similar to the MarkHouwink equation, was observed. The value of the constant a calculated from the relationship, which reflected the polymer morphology, was 0.75 in the Cs 0.1 solution, and it corresponded to a polymer with flexible chains.     

Crystallization of single and binary iron‐ and aluminum hydroxides affect phosphorus desorption

In acidic soils, phosphorus availability is affected by its strong affinity for mineral surfaces, especially Fe‐ and Al‐hydroxides. Plant roots have developed adaptive strategies to enhance the availability of phosphorus, including producing and exuding low molecular weight organic acids with a high affinity for phosphorus that competes with high molecular weight organic ligands formed during humification and mineralization. The aim of this study was to characterize the kinetics and mechanism of phosphorus desorption from Fe‐ and Al‐hydroxides of variable crystallinity, as well as binary Fe:Al‐hydroxide mixtures. Long‐term desorption experiments (56 days) were conducted with CaCl₂, CaSO₄, citric acid, and humic acid as competitive sorptives. CaCl₂ and CaSO₄ were selected as general inorganic sorptives and citric and humic acids were selected as organic ligands produced by organisms in the rhizosphere or following humification. The cumulative phosphorus desorption increased following the order CaCl₂ < CaSO₄ < humic acid < citric acid. Amorphous ferrihydrite and Fe‐rich Fe:Al‐hydroxides exhibited much less desorption when exposed to inorganic solutions than the crystalline and Al‐rich Fe:Al‐hydroxide mixtures. Models of the desorption data suggest phosphorus desorption with citric acid is diffusion‐controlled for ferrihydrite and Fe‐rich amorphous Fe:Al‐hydroxides. When humic acid was the sorptive, metal‐organic complexes accumulated in the solution. The results suggest organic compounds, especially citric acid, are more important for liberating phosphorus from Fe‐ and Al‐minerals than inorganic ions present in the soil solution.     

Characterization of a metalloproteinase component extracted from soil

Summary A metalloproteinase was found to be the main component of a protease in the extract from an Andosol collected from a tomato field. The protease has a pH optimum of 7 for benzyloxycarbonyl-L-phenylalanyl-L-tyrosyl-L-leucine with tyrosylleucine as the main reaction product. The K_m value for the substrate was 0.4 mM. Activity was inhibited by EDTA but not by pepstatin, p-chloromercuribenzoate and phenylmethanesulfonyl fluoride. After the removal of EDTA from the inactivated enzyme by dialysis and the addition of metal ions (Zn²⁺, Mn²⁺ and Fe³⁺), the enzyme activity could be recovered. The apparent isoelectric points of the metalloproteinase components were estimated to be 4.9, 4.5 and 4.1 by isoelectric focusing. A fraction with an apparent isoelectric point of 4.9 was the main component. The apparent molecular weight of the main protease component was estimated to be 4.7 × 10⁴ by gel filtration of Sephadex G-100. The enzyme hydrolyzed a natural polypeptide, angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu). Main split sites in the peptide were -Tyr⁷-Ile⁵- and -Pro⁷-Phe⁸-. The former was the most sensitive site to the soil metalloproteinase concerned.     

Effects of nitrogen form,nighttime nutrient solution strength,and cultivar on greenhouse tomato production

Higher greenhouse tomato (Lycopersicon esculentum Mill.) yield is obtained by using 25% of NH₄‐N in solution compared to using NO₃‐N as the sole nitrogen (N) source. However, blossom‐end rot (BER) may occur in tomato fruit when NH₄‐N was present in nutrient solutions. High nutrient solution strengths improve tomato fruit quality, but can also increase BER. Two NH₄‐N concentrations in solution (0 and 25%), and two nighttime solution strengths (NSS) (1X and 4X Steiner solution strength applied at 7 p.m.) were used to grow five indeterminate type greenhouse tomato cultivars: Caruso, Jumbo, Match, Max, and Trust. A significant interaction occurred between NH₄‐N concentration and NSS factors: 0% NH₄‐N and high NSS increased marketable yield and fruit:whole plant ratio, and reduced BER. In contrast, a concentration of 25% NH₄‐N and high NSS reduced marketable yield and the fruit:whole plant ratio, and increased BER incidence. Max, Match, and Trust tomato cultivars produced high marketable yield and high dry weight of stem and leaves, but were susceptible to BER. Use of NH₄‐N in solution reduced vegetative growth, and high NSS increased stem and leaf dry weight of the tomato plants. Fruit firmness was greater for the Max cultivar, and was unaffected by NH₄‐N and NSS at the mature green, breaker, and red ripe fruit development stages. However, at the fully ripe stage, fruit firmness was higher with high NSS and with 25% NH₄‐N.     

Fatty Acid Inheritance in Wide Reciprocal Oilseed Crosses (Brassica rapa and B. napus)

Abstract

Erucic acid content and inheritance were studied in Brassica alboglabra, B. rapa and resynthesized B. napus from these parental species. Although significant variation in erucic acid content was found between the 20 B. alboglabra lines, all of them had to be classified as high erucic types. No major erucic acid gene polymorphism was identified.

In B. rapa the erucic acid content was regulated by one major gene, which showed partial dominance in three of the four crosses analysed. The high erucic gene seemed to function more efficiently in the Yellow Sarson cytoplasm, resulting in maternal differences when reciprocal crosses were compared. In crosses where modern low erucic acid lines were used as female parents the zero erucic acid F₂-individuals occurred more frequently than expected.

Segregation of the erucic acid content in crosses involving resynthesized B. napus showed a good fit to a two locus model. No maternal effects were found, but deviations from mid-parent values in the high erucic acid direction occurred in most of the F₁-hybrids, indicating partial dominance. Crosses between resynthesized oilseed rape and natural oilseed rape resulted in oleic acid transgressive F₅-lines.     

Effects of CaCN2 treatment of seeds of Inubie (Echinochloa oryzicola) on synthesis of protein and RNA during germination

Protein and RNA induction during the germination of Inubie (Echinochloa oryzicola) seeds pretreated with CaCN₂ was studied. It was observed that the germination of Echinochloa oryzicola seeds was delayed after pretreatment with a high concentration of CaCN₂ for 18 h. A considerable difference in the protein patterns was detected in the SDS-PAGE gel between the control (H₂O) and CaCN₂ treatment, particularly a 48 kilodalton (kD) protein band disappeared in CaCN₂ treatment. At the tested three levels of CaCN₂, the induction of this 48 kD protein was apparently inhibited during the germination process even when the duration of the pretreatment was as short as 2 h. When imbibed with water, the amount of the 48 kD protein increased rapidly within 1 h from the trace level in the dry seeds, and reached the maximum level after about 1–2 d. The results showed that the CaCN₂ treatment also decreased the total RNA level in the germinating seeds. The relation of this protein induction and RNA decline with the beginning of Echinochloa oryzicola germination was discussed.     

电荷辅助氢键对腐殖酸分子量和溶解性的影响

采用体积排阻色谱和总有机碳分析法,分别测定腐殖酸体系中加入盐酸、甲酸、乙酸和丙酸后其分子量和溶解性发生的变化;通过分析抗坏血酸、苯甲酸、苯酚和邻苯二酚4种模型化合物在甲酸影响下的紫外光谱,以验证电荷辅助氢键的存在。结果表明:小分子有机酸能够明显降低腐殖酸的分子量;此外,当腐殖酸体系pH接近小分子有机酸的pKa时(DpKa<0.5),腐殖酸溶解性明显增大。小分子有机酸可能与腐殖酸之间形成电荷辅助氢键,从而打破弱作用力维持的腐殖酸超分子的稳态结构,导致腐殖酸分子量的降低和溶解性的增加;且弱酸与有机物之间的pKa相差越小,形成的电荷辅助氢键能量越高,腐殖酸分子结构受到的扰动程度越大。一维紫外光谱和同步二维相关紫外光谱分析进一步表明,小分子有机酸可能与pKa接近的化合物之间形成电荷辅助氢键,整体跃迁所需能量提高,造成低波长吸收增大而高波长吸收减小的结果。     

Soil Enzymatic Activity in Eucalyptus Grandis Plantations of Different Ages

Eucalyptus plantations have become increasingly common in Latin America. However, because Eucalyptus is an exotic species, its presence has raised concerns about changes in the environment, especially to soil properties. The objective of this study was to investigate possible changes in selected soil enzyme activity after several years of Eucalyptus cultivation. Soil samples were collected from four locations: a native forest (Atlantic Forest) used as a reference for the original soil conditions and three E. grandis plantations aged 2, 3 and 5 years, established in 2008, 2007 and 2005, respectively. The native vegetation had been removed and the soil graded and ploughed to establish these plantations. We evaluated soil enzymatic activities (β‐glucosidase, acid phosphatase, dehydrogenase, urease and arylsulfatase) at each location. The activity of β‐glucosidase, phosphatase, dehydrogenase and urease was improved after 5 years, whereas arylsulphatase was impacted negatively. The multivariate analysis showed that the majority of enzyme activities reached the values observed in native forest after the third year of reforestation. The activity of β‐glucosidase was crucial in differentiating the area with 2 years of reforestation from the native forest. The removal of native vegetation in order to establish commercial plantations raises concerns about the real impacts of this practice on the soil. In the present study, plantations of Eucalyptus improved most of the selected enzyme activities after the third year of reforestation. Copyright © 2015 John Wiley & Sons, Ltd.     

The maize root system in situ: Evaluation of structure and capability of utilization of phytate and inorganic soil phosphates

• 1 The dependence of the morphology of the maize (Zea mays L.) seminal root system on physical, chemical and biotic parameters was investigated with pot cultures in quartz sand and in a natural loamy sand soil. Low O₂-supply to the soil resulted in a substantially smaller root biomass despite a relative increase in total root length. Reduced N-supply also stimulated root length growth, but also enhanced the formation of laterals. The presence of soil microorganisms, in comparison to sterile cultures, resulted in a reduced length of the main roots, and the production of slender laterals with a decreased root hair density. Generally, the structural variability of laterals in response to different growth conditions was much more pronounced than that of the main roots.
• 2 A major part of the work reported here was dedicated to a detailed study of phosphate (P) acquisition by the maize root system under field conditions. Radioactive labelling of the roots and radioautography of soil cores revealed the in situ distribution pattern of the maize root system. Controlled labelling of the soil with radioactive phosphate allowed the documentation of the development and replenishment of the phosphate depletion zone around roots. Finally, the longevity and phosphate uptake activity of the different parts and tissues of the primary root system of maize was examined by electron microscopy and tracer studies including pulse chase experiments. From these studies the phosphate-acquiring strategy of the maize root system appears as follows: The capability of P uptake decreases in the order: root hairs, 1^st order laterals, 2^nd order laterals, main root. The life-spans of the components of the maize root system increase by the sequence: root hairs, laterals, main root. Inorganic P uptake, therefore, mainly occurs during the first weeks of root development. Dying back of the root occurs in an ordered manner resulting in a relocation of stored P predominantly into the main root cortex. Furthermore, it could be shown that competition for P between roots of the same or of adjacent maize and/or lupin plants virtually does not occur in situ.
• 3 The utilization of phytate-P was studied with ¹⁴C/³²P-labelled Camyo-inositol-hexaphosphate supplied to maize plants grown in sterile quartz sand or in hydroponic cultures. The ratio of P- and C-uptake as well as the incidence of phytate hydrolysis products in the rooting medium indicated the capability of maize roots to acquire P from phytate by enzymatic hydrolysis. This was confirmed by enzyme studies of the root tissues. A specific hydrolyzing enzyme (phytase; molecular weight 51 kD) could be detected in the cell wall of the root, especially in the root tip, which initiates phytate dephosphorylation. Further breakdown is presumably accomplished by monophosphoric phosphohydrolases.

     

Molecular size distribution and enzymatic degradation of organic phosphorus in root exudates of spring barley

Seeds from two varieties of spring barley (Prisma and Camorgue) were grown axenically in water. After 14 days, the culture solutions contained organic P substances (about 4 g P per plant) derived from root exudation, representing about 3% of the total P found in the seed. Gel filtration, separated the organic P into two well defined peaks, one with a high molecular weight (>45000 daltons) and the other with a low molecular weight (<500 daltons). The bioavailability of the soluble organic P released was assessed enzymatically and chemically. At the optimum pH of 5.0, phytase and acid phosphatase hydrolysed about 80% and 65%, respectively of the organic P in the exudate after 24 h whereas at the optimum pH of 9.8, alkaline phosphatase hydrolysed up to 40% P after the same length of time. In a pH 5.0 buffer, up to 10% of the organic P was hydrolysed compared with up to 45% in a pH 9.8 buffer. The high molecular weight organic P fraction recovered from the G-75 Sephadex behaved similarly.     

Adsorption,activity, and kinetics of acid phosphatase as influenced by selected oxides and clay minerals

Abstrac

The effects of 3 oxides (Fe, Al, and Mn oxides) and 3 clay minerals (kaolin, montmorillonite, and allophane) on the adsorption and subsequent kinetic properties of acid phosphatase were compared. The amount of enzyme adsorbed by the oxides and clay minerals followed the order: montmorillonite ? kaolin > Mn oxide > Fe oxide > Al oxide ? allophane. The adsorption isotherms of the enzyme on the oxides and clay minerals, except for montmorillonite and allophane, fitted the Langmuir equation. The activity of the enzyme immobilized by the inorganic components studied was in the order of allophane > kaolin > Fe oxide > montmorillonite > Al oxide ≒ Mn oxide. Compared to the free enzyme, the V _max, K_m, and V _max / K _m values of the immobilized enzyme decreased, increased, and decreased, respectively. Among the oxides or clay minerals, the higher the ability of the inorganic components to adsorb the enzyme, the lower the value of the V _max / K _m ratio of the immobilized enzyme. These findings suggest that the catalytic efficiency of the enzyme complexes formed is determined by the adsorbability of the inorganic components for the enzyme.     

The Combination of Rhizopus chinensis Lipase and Transglutaminase Affects the Rheology and Glutenin Macropolymer Properties of Frozen Dough

The combination of Rhizopus chinensis lipase (RCL) and transglutaminase (TG) was previously reported to improve the quality of frozen dough bread. In this study, the effects of RCL, TG, and their combination on the modification of glutenin macropolymer (GMP) and rheological properties of dough during frozen storage were investigated. Frozen storage changed both GMP and rheology properties of dough. TG treatment significantly decreased the ratio of high‐molecular‐weight glutenin subunits to low‐molecular‐weight glutenin subunits and GMP content in fresh dough, and GMP particle size increased. The effect of RCL on GMP properties was not significant, but its combination with TG dramatically increased the proportion of the larger particles and weighted average volume (D_4.3) in GMP. The treatment with the enzyme combination could have inhibited the depolymerization of GMP, which slowed down the decrease rate of some parameters such as GMP content, proportion of larger particles, D_4.3, and release of free amino and thiol groups during frozen storage. The modification of GMP properties by enzyme treatment weakened the effect of the freezing process on rheological properties of dough, especially TG treatment and its combination with RCL. Correlation between GMP particle size and dough properties (dough tensile force and elastic modulus) after freezing and enzyme treatment were confirmed.     

Examination of ester sulfates in Podzolic and Regosolic soils using an immobilized arylsulfatase reactor

The enzyme kinetics of an immobilized arylsulfatase reactor were examined. We found that the optimum operating conditions for the reactor were pH 7.0 and 25°C, using p-nitrophenyl sulfate in acetate buffer. The Michaelis constant (K _m) of immobilized arylsulfatase was 5.29 mM, compared with a K _m of 2.18 mM for soluble arylsulfatase from the same source (Helix pomatia). Since arylsulfatase hydrolyzes organic ester sulfate linkages, the immobilized arylsulfatase reactor was used to examine ester sulfate compounds in two soils subjected to different fertility management schemes. Soil samples were obtained from the Ap horizons of a Podzol from S-amended wheat plots and a Regosol from dykeland hayfield plots which had received additions of NH₄NO₃ and compost. The distribution of S in these soils was examined in the fall of 1993 and the spring of 1994. Soil organic matter was extracted and separated into three molecular weight fractions (<500, 500–10 000, >10 000). There was no difference in the ester sulfate content for the >10 000 fraction of control and S-amended Podzol soils; however, the S-amended samples had significantly higher quantities of hydrolysable ester sulfates than controls for the 500–10 000 range, indicating that S amendments resulted in the incorporation of ester sulfate into this lower molecular weight fraction. Both control and NH₄NO₃ treatments to the Regosol showed significantly higher quantities of hydrolysable ester sulfates in the >10 000 fraction, while compostamended plots showed no difference between the >10 000 and 500–10 000 fractions due to suspected microbial degradation of high molecular weight organic S compounds in the compost. Since there was no significant effect of sampling time, this study indicated that naturally occurring low molecular weight ester sulfate compounds accumulate in soil and persist during storage. Hydrolysable ester sulfates constituted 35–55% of the hydriodic acid-reducible S in these different soils and probably represent an important and easily mineralizable portion of total ester sulfates.     

Characterization of humus-protease complexes extracted from soil

Pyrophosphate (140 mM, pH 7.1) extracts of two arable soils and one pasture soil were ultrafiltrated separating the extracted material into three fractions: A_I with nominal molecular weight (nmw) > 100 kD, A_II with nmw between 10 kD and 100 kD and R with nmw < 10 kD. Protease activity was determined in the fractions by using three different substrates: N-benzoyl-l-argininamide (BAA), specific for trypsin; N-benzyloxy-carbonyl-l-phenylalanyl l-leucine (ZPL), specific for carboxypeptidases; and casein, essentially a non-specific substrate. The derivative fractions were also analysed for their amino acid N and humic (HA) and fulvic (FA) acid contents. The organic matter of extracts and derivative fractions obtained from the pasture soil was analysed by isoelectric focusing (IEF) and that of fractions analysed by pyrolysis gas chromatography (Py-GC). Activities of the extract were monitored for their thermal stability and those of the extract and derivative fractions for their optimal pH.Due to the mechanical disintegrating action of sodium pyrophosphate over the humic substances during the fractionation process the amount of total organic C and FA in the fractions was ranked as R > A_II > A_I. The lowest amino acid N/organic C was found in the R fraction, whereas A_II fraction was rich in humic acids, carbohydrates and amino acid N and A_I fraction showed the lowest carbohydrate content. At least 70% of the total BAA- and ZPL-hydrolysing activity was associated to particles with nmw higher than 10 kD and at least 30% of these activities were present in particles with nmw higher 100 kD. Casein-hydrolysing activity was quite evenly distributed among the three fractions (A_I, A_II and R). The extracted protease-organic complexes were resistant to thermal denaturation and some of them showed optimal activity at pH values higher than 10 as a result of the polyanionic characteristics of the humic material surrounding enzyme molecules and of the presence of alkaline protease. Comparison of data obtained in Py-GC analyses and in protease activity suggests that BAA-hydrolysing activity was associated to a highly condensed humic matter and ZPL-hydrolysing activity to less resistant humic substances, while at least some of the extracted casein-hydrolysing activity was present as glyco-proteins not associated to humus. BAA-hydrolysing activity was probably inhibited by fresh organic matter of carbohydrate origin whereas lignin derived organic matter probably inhibited ZPL- and casein-hydrolysing activity.     

Calcium efficiency among tomato strains depends on nitrogen regimes 1

Two Ca‐efficient and 3 Ca‐inefficient tomato strains were grown for 18 days in nutrient solutions with NH₄‐N:NO₃‐N ratios (%) of 0:100, 50:50, or 90:10 and with 40 mg of total Ca⁺⁺. When NH₄‐N nutrition was used, efficient strains exhibited Ca‐deficiency symptoms sooner or produced less dry matter than inefficient strains. The greatest reductions in dry weight production occurred between 50 and 90% NH₄‐N nutrition. The greatest reduction in solution pH and in % of Ca in plant tissues occurred between 0 and 50% NH₄‐N nutrition. Ratings of Ca efficiency varied according to the proportion of NH₄‐N in the nutrient solutions.