Purification and characterization of three phytases from germinated lupine seeds (Lupinus albus var. amiga)

Three phytases were purified about 14200-fold (LP11), 16000-fold (LP12), and 13100-fold (LP2) from germinated 4-day-old lupine seedlings to apparent homogeneity with recoveries of 13% (LP11), 8% (LP12), and 9% (LP2) referred to the phytase activity in the crude extract. They behave as monomeric proteins of a molecular mass of about 57 kDa (LP11 and LP12) and 64 kDa (LP2), respectively. The purified proteins belong to the acid phytases. They exhibit a single pH optimum at 5.0. Optimal temperature for the degradation of sodium phytate is 50 degrees C. Kinetic parameters for the hydrolysis of sodium phytate are K(M) = 80 microM (LP11), 300 microM (LP12), and 130 microM (LP2) and k(cat) = 523 s(-1) (LP11), 589 s(-1) (LP12), and 533 s(-1) (LP2) at pH 5.0 and 35 degrees C. The phytases from lupine seeds exhibit a broad affinity for various phosphorylated compounds and hydrolyze phytate in a stepwise manner.     

Root turnover of faba beans (Vicia faba L.) and its interaction with P and K supply

An increased root turnover can be a mechanism of improved nutrient‐uptake efficiency. The objectives of this study were to investigate P and K efficiency of faba beans (Vicia faba L.), to determine their root growth and root turnover, and to assess the relevance of root turnover on P and K uptake at limited supply. Faba beans were grown as part of a long‐term fertilization experiment on fertilized plots (control) and plots that had not received any P or K fertilizer for 16 years (P0, K0). Although the unfertilized soils were low and very low in their P‐ and K‐supply level, respectively, no differences in shoot‐dry‐matter production occurred compared to the control. However, relative K concentration in dry matter of the K0 plants (control plants = 100) decreased during the experiment and was only 60% of the control at the final harvest. This indicated a high K‐utilization efficiency of faba bean. Relative phosphorus concentration increased in the P0 treatment and was not different from the control at the last harvest, indicating an improvement in P‐uptake efficiency with time. The size of the standing root system determined by sequential auger sampling (net development) was not influenced by P and K supply. Total root production as measured by the ingrowth‐core method was about 6 times higher than the average size of the standing root system and even increased under low‐K conditions. This indicated a fast root turnover. Modeling soil nutrient transport and uptake revealed that calculated uptake of the control was up to 48% higher when root turnover was taken into account, compared to calculations based on the net development of the root systems. This is due to a better soil exploitation. Under K shortage, root turnover resulted in a 117% higher calculated uptake, which was close to measured K uptake. Root turnover was also of benefit for P uptake, but calculated P uptake was significantly less than measured, indicating that root turnover was of little importance for P uptake of faba beans.     

Purification and partial characterization of broccoli (Brassica oleracea Var. Italica) peroxidases

Three peroxidase (POD) isoenzymes were purified from a soluble extract of broccoli stems. The acidic and neutral PODs were purified to homogeneity by using ion exchange and hydrophobic chromatography. The basic POD was purified by cation exchange and gel filtration chromatography. The neutral and basic PODs had molecular masses of approximately 43 kDa, and the acidic POD had a molecular mass of 48 kDa by SDS-PAGE. pI was approximately 4, 5, and 8 for acidic, neutral, and basic PODs, respectively. Optimum activity using guaiacol as the H donor was obtained at pH approximately 6 for both neutral and basic PODs and at pH approximately 4 for acidic POD. All three of the purified isoenzymes are glycosylated. Reaction rates with various substrates including guaiacol, guaiacol/MBTH, DMAB/MBTH, and ferulic acid/MBTH were different among the isoenzymes. K(m) and amino acid composition were also determined.     

Increasing root and leaf growth and yield in Mg‐deficient faba beans (Vicia faba) by MgSO4 foliar fertilization

Magnesium (Mg) deficiency reduces yield and quality of crops as Mg is involved in carbohydrate and protein anabolism. Foliar application of MgSO₄ represents a means of ameliorating the nutritional status of crops growing under conditions of deficiency. Foliar Mg application has been demonstrated to increase the chlorophyll concentration and vegetative yield of plants. However, in crop plants used for seed production, the limited number of available studies provides only conflicting results in terms of the efficiency of Mg leaf application on yield and quality. We have examined the effect of increasing Mg foliar application rates on differentially developed leaves and leaf stages with regards to Mg concentration, chlorophyll concentration and biomass formation. Our data reveal that in order to obtain significant effects MgSO₄ concentrations in the spraying solution should be higher than 50 mM, as lower concentrations did not lead to significant changes in the vegetative or generative material. Positive effects of the foliar application on chlorophyll concentration and leaf area were only measured in leaves treated with 200 mM foliar MgSO₄. The application of Mg did not affect the parameters chlorophyll, ion concentration and leaf area in the newly developing leaves, but it had an important influence with regard to yield traits, especially on the formation of pods. The quality of the seeds with regard to the protein concentration was reduced in response to the magnesium deficiency treatment, but the application of foliar magnesium prevented a significant loss of protein.     

Purification and characterization of a lipoxygenase enzyme from durum wheat semolina.

Purification of a lipoxygenase enzyme from the cultivar Tresor of durum wheat semolina (Triticum turgidum var. durum Desf) was reinvestigated furnishing a new procedure. The 895-fold purified homogeneous enzyme showed a monomeric structure with a molecular mass of 95 +/- 5 kDa. Among the substrates tested, linoleic acid showed the highest k(cat)/K(m) value; a beta-carotene bleaching activity was also detected. The enzyme optimal activity was at pH 6. 8 on linoleic acid as substrate and at pH 5.2 for the bleaching activity on beta-carotene, both assayed at 25 degrees C. The dependence of lipoxygenase activity on temperature showed a maximum at 40 degrees C for linoleic acid and at 60 degrees C for bleaching activity on beta-carotene. The amino acid composition showed the presence of only one tryptophan residue per monomer. Far-UV circular dichroism studies carried out at 25 degrees C in acidic, neutral, and basic regions revealed that the protein possesses a secondary structure content with a high percentage of alpha- and beta-structures. Near-UV circular dichroism, at 25 degrees C and at the same pH values, pointed out a strong perturbation of the tertiary structure in the acidic and basic regions compared to the neutral pH condition. Moreover, far-UV CD spectra studying the effects of the temperature on alpha-helix content revealed that the melting point of the alpha-helix is at 60 degrees C at pH 5.0, whereas it was at 50 degrees C at pH 6.8 and 9.0. The NH(2)-terminal sequence allowed a homology comparison with other lipoxygenase sequences from mammalian and vegetable sources.     

Purification and characterization of a hexanol-degrading enzyme extracted from apple

An enzyme having activity toward n-hexanol was purified from apple, and its biochemical characteristics were analyzed. The purification steps consisted of sedimentation with ammonium sulfate, DEAE Sepharose Fast Flow ion exchange chromatography, and Sephadex G-100 column. The obtained enzyme had a yield of 16.00% with a specific activity of 18879.20 U/mg protein and overall purification of 142.77-fold. The enzyme showed activity to isoamylol, 1-propanol, n-hexanol, and isobutanol but not toward methanol and ethanol. With n-hexanol as a substrate, the optimum conditions were pH 4.0 and 30 °C for enzyme activity and pH 3.0-4.0 and temperatures below 40 °C for enzyme stability. The enzyme activity was increased significantly by adding l-cysteine and Fe(2+) at all tested concentrations and slightly by Zn(2+) at a high concentration but decreased by additions of EDTA, Ga(2+), K(+), Mg(2+), sodium dodecyl sulfate (SDS), sodium aluminum sulfate (SAS), dithiothreitol (DTT), and glutathione (GSH). The enzyme activities toward n-hexanol and n-hexanal were increased by NADH but decreased by NAD(+), in contrast to a decrease toward n-hexane by addition of both NAD(+) and NADH.     

Influence of sulphur supply on glucose and ATP concentrations of inoculated broad beans (Vicia faba minor L.)

In the present study, the influence of S supply on S concentrations, N₂ fixation, available amounts of glucose and adenylates of Vicia faba minor L. were weekly investigated, starting 6 weeks after sowing. Glucose was determined photometrically in shoots, roots and nodules and in ATP, ADP and AMP by bioluminescence in roots, mitochondria and bacteroids. Sulphur deficiency resulted in significantly lower S concentrations of shoots, roots and nodules, in a reduced N₂ fixation as well as in significantly lower amounts of glucose in shoots and nodules. In roots and bacteroids, S deficiency resulted in lower ATP concentrations, while the influence of S supply on ADP and AMP was less pronounced. With optimum S supply, the available amounts of glucose and ATP were strongly influenced by flower formation and seed development. Dedicated to Prof. Dr. Dr. h.c. W. Werner on the occasion of his 75th birthday.     

Mechanical weeding effects on soil structure under field carrots (Daucus carota L.) and beans (Vicia faba L.)

The successful production of organic vegetables relies heavily on mechanical weeding, flame weeding and stale seedbeds. These operations involve repeated passes by tractors. Mechanical weeding also involves regular tillage. This combination of repeated tillage and compaction changes soil structure. We studied these structural changes in two fields of organic carrots and one field of beans in eastern Scotland. Structure was described by measuring soil strength with a vane shear tester and a cone penetrometer, by measuring bulk density and by visual assessment. Under beans, vane shear strength below the growing root zone was highly variable and in some areas was high enough to restrict root growth (>50 kPa). The carrots were grown in beds containing crop rows separated by bare soil. The bare soil was regularly weeded mechanically. The structure of this weeded soil in the top 10 cm layer of a loam eventually became disrupted and compacted enough to deter root growth (vane shear strength of 70 kPa). In addition the topsoil and subsoil in the wheel-tracks between the beds became very compact with little distinguishable structure. This compaction extended to the subsoil and persisted into the next cropping season (cone resistance >3 MPa at 35–50 cm depth). Reduced tillage by discing without ploughing was used to incorporate the straw used to protect the carrots overwinter and prepare the soil for the next crop. The resulting topsoil quality was poor leading to anaerobic growing conditions which restricted growth of the following crop and led to losses of the greenhouse gas nitrous oxide. The greatest threat to soil quality posed by mechanical weeding was subsoil compaction by tractor wheeling.     

Purification and characterization of a methylene urea-hydrolyzing enzyme from Rhizobium radiobacter (Agrobacterium tumefaciens)

Slow-release fertilizers are gaining acceptance to increase fertilizer use efficiency and reduce environmental impact. The release of nitrogen from methylene urea, a common slow release N fertilizer, is controlled by microbial decomposition. An enzyme hydrolyzing slow-release nitrogen fertilizer, methylene urea, was purified from Rhizobium radiobacter (Agrobacterium tumefaciens) to homogeneity using a four-step purification procedure with an overall yield of 3%. The active enzyme has a molecular mass of approximately 180 kDa determined by size exclusion chromatography, and the SDS page of the purified protein indicated three subunits of different sizes (62, 34 and 32 kDa). The N-terminal amino acid sequence of the 62 kDa fragment indicates identity with urease subunits from Mycobacterium tuberculosis (73%) and Helicobacter pylori (71%). However, for the internal amino acid sequences of the 62 kDa fragment no matches with known proteins were found. Some internal peptides in the smaller subunits (32 and 34 kDa) are homologous to urease subunits and unknown proteins in Agrobacterium tumefaciens. Based on the kinetic properties, substrate selectivity, and inhibition characteristics, the novel enzyme (MUase) is an intracellular enzyme complex with urease activity. The enzymatic mechanism of methylene urea breakdown was studied using a novel LC-MS method for MU analysis, which indicates that all cold-water soluble nitrogen forms of methylene urea are subjected to hydrolysis, and the hydrolysis proceeds via methylurea, urea and other yet unidentified hydrolysis-products, suggesting that the isolated enzyme complex performs a multistep hydrolysis. The microbiological and molecular data is useful in determining the soil factors affecting the efficacy of methylene urea as a slow release fertilizer in agricultural production systems.     

Transport of Foliar Applied Iron (59Fe) in Vicia faba

Abstract

Radioactively labeled iron (⁵⁹Fe) was used to study iron retranslocation from mature leaves of Broad bean (Vicia faba L. var. Scirocco). Our experiments offered the possibility to detect and quantify the translocation of foliar applied iron by imaging technique in combination with tissue analysis. ⁵⁹Fe labeled solution was placed as a droplet onto the leafs upper surface of intact plants. Distribution of ⁵⁹Fe was analyzed after 0.5 h up to 2 days. Iron was translocated acropetally (towards the tip of the treated leaf) as well as basipetally. Movement in the apical direction was predominant, amounting to about 65% of ⁵⁹Fe translocated from the application site. About 35% of ⁵⁹Fe were transported basipetally, corresponding to absolute amounts of 2.8–53.6 pmol h^?1. After 30 min, it was detectable in the petiole, which included a translocation of 20 mm basipetal from the application site. A mean of 15% of the iron retranslocated from a leaflet was detected in non‐treated leaflets of the same leaf. This iron was supposed to have been exchanged from the phloem into the xylem pathway, probably within the petiole. When the loading rate into the phloem was estimated on basis of the sum of retranslocated ⁵⁹Fe per time and per area of the leaf treated, a range of 0.031–2.21 pmol h^?1 mm^?2 (mean: 0.62 pmol h^?1 mm^?2) was obtained. This was not sufficient to meet an estimated demand for iron in the growing terminal bud, but could cover about 25% of it. In conclusion, average iron retranslocation from leaves of Fe‐sufficient plants was not large enough to meet the iron demand of the growing shoot. This was not due to a limitation in iron availability for transport, as an excess amount of iron was supplied which was not biologically bound, but a limitation due to transport facilities, probably in the phloem, seemed to be more likely in this case.     

Purification and properties of a neutral peroxidase isozyme from turnip (Brassica napus L. Var. Purple Top White Globe) roots

A neutral peroxidase isozyme (pI 7.2) from turnip roots (TNP) was purified to homogeneity and partially characterized. TNP is a monomeric glycoprotein with 9.1% carbohydrate content and a molecular weight of 36 kDa. Optimum pH values for activity using 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and guaiacol as H donors were 4.5 and 5.5, whereas the K(m) values were 0.7 and 3.7 mM, respectively. The ABTS K(m) was approximately 7 times higher than that reported for basic commercial horseradish peroxidase (HRP-C). TNP retained approximately 70% activity after 11 min of heating at 65 degrees C, whereas the activation energy for inactivation (132 kJ/mol) was higher than or comparable to that of other peroxidases. The low ABTS K(m) and high specific activity (1930 units/mg) gave a high catalytic efficiency (500 M(-1) s(-1)). These properties make TNP an enzyme with a high potential as an alternative to HRP in various applications.     

Characterization of Greek populations of faba bean (Vicia faba L.) and their evaluation using a new parameter

Using data from four environments of a two-year experiment, a collection of 55 Greek faba bean populations wascharacterized on the basis of ten morphological and ten agronomical traits. The collection can be described ashaving flowers with medium intensity of streaks on standard petal, two to three flowers per inflorescence, leaveswith six leaflets per leaf, sub-elliptic leaflet shape and small or medium leaflet size. The stems had weak ormedium pigmentation at flowering time, were of low or medium thickness, had medium height and mediumbranching from basal nodes and high resistance to lodging. Pods were mainly basal with pendent attitude, flattenedin shape, had dark colour at maturity and were small with few ovules and seeds. Most seeds had testa of light greencolour. This characterization makes the present collection similar to other South European faba bean populations.Apart from characterization, collections should also be evaluated from the breeding point of view. Since themost desirable populations within a collection are those with valuable but 'low frequency' levels of traits, theparameter R (rareness) was proposed. For each trait, a partial rareness (R ) parameter is calculated using only ithose trait levels that can be considered as having 'low frequency'. The parameter R is the sum of the 'partialrarenesses'. Populations with high frequencies of the 'lowest frequency' levels of traits in the collection arecharacterized by high values of R and can, therefore, be more easily identified.     

Purification and characterization of a carboxypeptidase from squid hepatopancreas (Illex illecebrosus)

The hepatopancreas of squid (Illex illecebrosus) extract contains a wide range of carboxypeptidase (CP) activities based on hydrolysis of N-CBZ-dipeptide substrates. SDS-PAGE zymograms with N-CBZ-Phe-Leu substrate revealed three activity zones (CP-I, 23 kDa; CP-II, 29 kDa; CP-III, 42 kDa). CP-I was purified 225-fold with 86.20% recovery based on N-CBZ-Ala-Phe activity by chromatography on DEAE-cellulose, gel filtration, and chromatofocusing. The purified enzyme had broad specificity toward N-CBZ-dipeptides; however, it preferred substrates with a hydrophobic amino acid at the C terminus. CP-I had greatest activity with N-CBZ-Ala-Phe (specific activity = 7104 units/mg of protein, K(m) = 0.40 mM, and physiological efficiency = 22863). CP-I had a pI of 3.4 and is a metalloprotease that is activated by Co(2+) and partially inhibited by Pefabloc, a serine protease inhibitor. With N-CBZ-Ala-Phe and Gly-Phe, it had optimum activity at pH 8 and 70 degrees C. The amino acid composition of squid CP-I is similar to that of CP A from other species.     

Collection, evaluation and classification of Greek populations of faba bean (Vicia faba L.)

Fifty-five Greek Vicia faba L. populations, collected from diverse areas, were planted at two dry and low fertility sites for evaluation and classification. Yield evaluation, which was carried out by Principal Component Analysis (PCA) on the basis of seven yield traits, showed the number of pods per plant, number of ovules and seeds per pod, and branching from the basal nodes to be the most important traits for population evaluation regarding yield. For population classification, four dissimilarity coefficients (Manhattan, Average Taxonomic Distance, Euclidean distance and squared Euclidean distance) and four multivariate methods (PCA, UPGMA, Neighbor-joining and Principal Coordinate Analysis) were evaluated using fifteen morphological and seven yield traits. Neighbor-joining was chosen as the most suitable multivariate method. This method combined with PCA for the seven yield traits, placed the populations into six groups. As revealed by the application of PCA on all twenty-two traits the grouping was based mainly on pod characteristics, stem thickness, plant height, 1000 seed weight and branching from basal nodes. Based on the results of the present study, a model is proposed for conserving cross-pollinated species, such as faba bean.     

Uptake and distribution of manganese applied to leaves of Vicia faba (cv. Herzfreya) and Zea mays (cv. Regent) plants

Uptake and transport of Mn applied to leaves was studied in maize (Zea mays cv. Regent) and horse bean (Vicia faba cv. Herzfreya), two important crops in Egypt. Under controlled conditions in a growth chamber, maize and horse bean plants were grown in solution culture without Mn. After 20 days, Mn was applied to one older leaf by submerging part of the leaf blade into 0.1 mM MnSO₄ or MnEDTA solution for 48 h. At harvest (24 h later), plants were divided into fractions and Mn concentrations and contents determined. Plants without Mn application served as control. Only 1% of the Mn supply was taken up. Most of it remained at the application zone. However, part of the Mn moved out of the leaf of application and was preferentially transported to the shoot apex. This was indicated by a up to 2 times higher Mn concentration of the youngest leaf. When Mn was applied as MnEDTA, Mn uptake was lower but translocation enhanced compared to MnSO₄. There were no consistent differences between the plant species although mobility of Mn seemed to be higher in maize. Although the amounts of Mn taken up and translocated were low, the results suggest that in these plant species, leaf-applied Mn may contribute to the Mn nutrition of new growth.     

Phosphorus use efficiency of improved faba bean (Vicia faba) varieties in low‐input agro‐ecosystems

The use of phosphorus (P)‐efficient legumes is a prerequisite for sustainable intensification of low‐input agro‐ecosystems. A study was undertaken in a farmer's field in the tropical highlands of Ethiopia to assess the agronomic performance, P acquisition efficiency (PAE), and P utilization efficiency (PUE) of six improved faba bean varieties (Vicia faba L. var. CS‐20DK, Degaga, Gebelcho, Moti, Obse, Walki) without and with P application. Varieties showed significant variations in PUE, but P application had no significant effect on PUE. Variety Moti demonstrated highest PUE of 272 kg grain kg^?1 P, which was 1.6‐fold higher than the lowest PUE (164 kg grain kg^?1 P) of Gebelcho. PUE was significantly and positively correlated with grain yield (r = 0.542) and negatively correlated with shoot PAE (r = –0.541), indicating that PUE is important for grain yield. The results demonstrate that variations in grain and biomass yield of faba beans were largely due to differences in PUE and not due to PAE. Therefore, we argue that genetic resources of faba bean varieties showing optimal agronomic performance and high PUE in low‐input agro‐ecosystems should be better explored. Introduction of such varieties in low‐input cereal‐based cropping systems could improve and enhance P use efficiency at the system level.     

发芽蚕豆左旋多巴超声强化提取及其动力学过程

为了充分提取和利用高生物活性的天然左旋多巴(L-DOPA),研究了超声波强化提取发芽蚕豆L-DOPA的工艺条件,并初步探讨了超声浸提动力学过程。基于单因素试验,以超声功率、液固比、萃取时间为考察因素,采用Box-Behnken试验设计进行了工艺参数优化,结果表明,超声功率对发芽蚕豆L-DOPA得率的影响较大;原料用量2.0g,采用含30%乙醇的0.1mol/L醋酸溶液为提取溶剂,其较佳提取工艺条件为:超声功率257W、液固比31mL/g、萃取时间为37.4min。在此条件下,发芽蚕豆L-DOPA的平均得率为1.47%,较未发芽蚕豆增加0.58倍,略高于传统提取法,且超声强化提取显著缩短了浸提时间。对最优提取条件下L-DOPA得率随时间变化的动力学分别用Film模型、非稳态扩散理论和Ponomaryov经验方程进行拟合,其中Film理论模型的拟合度较好,其决定系数R2值最大,为0.9928。研究结果为天然L-DOPA制备提供了参考。     

Effectiveness of native Rhizobium on nodulation and yield of faba bean (Vicia faba L.) in Eastern Ethiopia

This study was initiated to evaluate the effect of locally isolated Rhizobium on nodulation and yield of faba bean at Haramaya, Ethiopia for three consecutive years. Ten treatments comprising of eight effective isolates of rhizobia, uninoculated, and N-fertilized (20 kg N ha^?1) were laid out in a randomized complete block design with three replications. The result of the experiment indicated that all inoculation treatments increased nodule number and dry weight over the control check in all cropping seasons. The result, however, showed the non-significant effect of Rhizobium inoculation on shoot length, number of tiller per plant and 100 seed weight in all cropping season. Inoculating Haramaya University Faba Bean Rhizobium (HUFBR)-15 in 2011 and National Soil Faba Bean Rhizobium (NSFBR)-30 in 2012 and 2013 gave the highest grain yields (4330, 5267 and 4608 kg ha^?1), respectively. These records were 75%, 48%, and 5% over the uninoculated treatment of respective years. Over the season, NSCBR-30 inoculation resulted in the highest nodulation and grain yield production as compared to the other treatments. In general, isolates from central Ethiopia were better than those isolated from eastern Ethiopia and Tropical Agricultural Legume (TAL)-1035 in enhancing faba bean production at Haramaya site. Therefore, NSFBR-30 is recommended as a candidate isolate for faba bean biofertilizer production in eastern Ethiopia soils.     

Effects of intercropping and Rhizobium inoculation on yield and rhizosphere bacterial community of faba bean (Vicia faba L.)

The effects of intercropping with maize and Rhizobium inoculation on the yield of faba bean and rhizosphere bacterial diversity were analyzed by terminal restriction fragment length polymorphism, amplified 16S rDNA restriction analysis (ARDRA), and 16S rDNA sequencing. The results showed that intercropping but not Rhizobium inoculation significantly increased the faba bean yield. Probably the relatively high level of native rhizobia in soil annulled the effect of rhizobia inoculation. ARDRA results showed that intercropping did not affect bacterial diversity whereas Rhizobium inoculation decreased bacterial diversity. The canonical correspondence analysis showed that the composition of bacterial community was changed apparently by intercropping, and there was a positive correlation (P = 0.724) between faba bean yields and intercropping and an apparent correlation (P = 0.648) between intercropping and total N. The available content of K and P had a lower effect on the bacterial community composition than did the total N content, Rhizobium inoculation, and microbial biomass C. Rhizobium inoculation negatively correlated with microbial biomass C (P = −0.827). These results revealed a complex interaction among the intercropped crops, inoculation with rhizobia, and indigenous bacteria and implied that the increase of faba bean production in intercropping might be related to the modification of rhizosphere bacterial community.