History and traditional cultivation ofLathyrus sativus L. andLathyrus citera L. in the Iberian peninsula

Recent ethnographic research carried out in Spain has allowed the cultivation ofLathyrus sativus L. andL. cicera L. to be documented. Both species already appear in the Spanish archaeological record. Grown under traditional farming systems,L. sativus L. andL. cicera L. are still used for human consumption (onlyL. sativus L.) and both are used for fodder. Data on cultivation history, fanning practices, crop processing sequence and uses are here presented, which will enable a better understanding of their possible roles in prehistoric times.     

Diamine oxidase from Lens esculenta seedlings: Purification and properties

A diamine oxidase (DAO) (EC 1.4.3.6) has been purified to homogeneity from lentil seedlings. The purified protein has a MW of 154 000 and is composed of two apparently identical subunits. It contains two CU²⁺ atoms and one carbonyl-like group per mol. The purified enzyme is pink-red in concentrated solution and shows a broad, well-defined, absorption band in the visible region centered at 498 nm. The ESR spectrum is typical of Cu²⁺ in a tetragonal symmetry. The enzyme oxidizes only aliphatic diamines and spermidine with formation of the corresponding aldehydes, hydrogen peroxide and ammonia. Putrescine and cadaverine are oxidized most rapidly and the oxidation rate decreases when longer diamines are tested.     

Diamine oxidase from millet catalyzes the oxidation of 1, 3-diaminopropane

Diamine oxidase was purified sixty-fold from millet shoots. The partially purified enzyme of 150 kDa oxidized 1, 3-diaminopropane (1, 3-DAP) to 3-aminopropionaldehyde. The K_m values were 9.1×10⁻⁵M for 1, 3-DAP and 6.3×10⁻⁴M for putrescine. Extracts of shoots of prosomillet, maize and barley also contained an activity that oxidized 1, 3-DAP.     

Chromosomal instability in Lathyrus sativus L.

Summary In Lathyrus sativus (2n=14), variety LSD-1 shows an instability of somatic chromosome number which can be observed in root tip and shoot tip mitoses. In this variety, approximately 54% of the seedlings showed intra-individual variation in chromosome number ranging from 2n=14–3. This variability in chromosome number was recorded in approximately 60% of the dividing cells. Two seedlings were triploid with 21 chromosomes. Variation in chromosome number in somatic cells within individual plants is possibly controlled by genetic factors, which result in spindle abnormalities, chromosome degradation and minute chromosomes. The variation in chromosome number is probably responsible for the pollen polymorphism noted in this particular strain. The possible mechanism of intra-individual variability and the occurrence of the phenomenon vis-a-vis its applications are discussed.     

Purification of glycollate oxidase from greening cucumber cotyledons

Glycollate oxidase (glycollate: oxygen oxidoreductase, EC 1.1.3.1) was purified to apparent homogeneity from crude extracts of greening cucumber cotyledons (Cucumis sat vus). Molecular sieving and chromatofocusing resulted in 700-fold purification and specific activity of 1 kat mg^-1 protein. The enzyme exhibited a M_r of 180,000, or 700,000, respectively, and is a tetramer or 16-mer made of identical subunits of M_r 43,000. Monospecific antibodies were raised against the homogeneous protein.     

Plant regeneration from stem-derived callus of the seed legume Lathyrus sativus L.

Callus cultures from stem explants of six Lathyrus sativus L. cultivars were tested for their morphogenic capacity. Shoot-buds were formed in calli of only one cultivar. Maximum response was observed in the medium containing 10^-8 M picloram and 10^-6 M benzylaminopurin. Supplementation with adenine sulphate was required for shoot-bud formation. The greatest frequency of shoot-bud formation was detected at the second passage and complete lack of regeneration capacity was observed after the 8th passage. Cell regenerates were diploid.     

Purification and characterization of oxalate oxidase from wheat seedlings

Oxalate oxidase (OxO, EC 1.2.3.4.) was purified to homogeneity from wheat (Triticum aestivum) seedlings by sequential thermal treatment, ultrafiltration, Sephadex G-100 gel filtration and affinity chromatography with concanavalin A. The enzyme was purified 66.11-fold with a recovery of 21.97%. It showed a subunit molecular mass of 32.6 kDa on SDS-PAGE and a native molecular mass of 170 kDa on Sephadex G-150 filtration, suggesting that it is a pentamer. The wheat OxO had a maximum activity at pH 3.5. Its K _m for oxalate was 0.21 mM. Chemical modification revealed that cysteine, lysine and carboxylate residues were essential for OxO activity, whereas arginine, serine, threonine and tryptophane residues were not essential.     

Transgenic grasspea (Lathyrus sativus L.): Factors influencing Agrobacterium-mediated transformation and regeneration

A reproducible procedure was developed for genetic transformation of grasspea using epicotyl segment co-cultivation with Agrobacterium. Two disarmed Agrobacterium tumefaciens strains, EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT with the neomycin phosphotransferase II (nptII) gene and the -glucuronidase (gus)-intron, were studied as vector systems. The latter was found to have a higher transforming ability. Several key factors modifying the transformation rate were optimized. The highest transformation rate was achieved using hand-pricked explants for infection with an Agrobacterium culture corresponding to OD₆₀₀0.6 and diluted to a cell density of 10⁹ cells ml^–1 for 10 min, followed by co-cultivation for 4 days in a medium maintained at pH 5.6. Putative transformed explants capable of forming shoots were selected on regeneration medium containing kanamycin (100 g ml^–1). We achieved up to 36% transient expression based on the GUS histochemical assay. Southern hybridization of genomic DNA of the kanamycin-resistant GUS-expressive shoots to a gus-intron probe substantiated the integration of the transgene. Transformed shoots were rooted on half-strength MS containing 0.5 mg l^–1 indole-3-acetic acid, acclimated in vermi-compost and established in the experimental field. Germ-line transformation was evident through progeny analysis. Among T₁ seedlings of most transgenic plant lines, kanamycin-resistant and -sensitive plants segregated in a ratio close to 3:1.     

Inheritance of seed weight in Cucumis sativus (L.) var. sativus and var. hardwickii (Royle) Kitamura

Summary A series of experiments was conducted to determine the inheritance of seed weight in cucumber. Matings between a Cucumis sativus var. sativus (Cs) L. inbred line (USDA WI 1606; P₁) and a C. sativus var. hardwickii (Royle) Kitamura (Ch) collection (PI 215589; P₂) were made to produce seed of reciprocal F₁, F₂, and BC₁ families. Families were grown under field and greenhouse conditions, and seeds were extracted from fruit 55 to 60 days post-pollination. Seed of F₁ and F₂ families was obtained using the Cs inbred WI2808 (P₁₂) and the Ch collection LJ 90430 (P₁₀), and seed of F₁ families were produced using a North Carolina Design II mating scheme in which three Cs (P₃= GY-14; P₄=WI 1379; P₅=WI 1909) inbreds were used as maternal parents and seven Ch collections (P₂; P₆= PI462369; P₇=486336; P₈=LJ91176; P₉=273469; P₁₀= 2590430; P₁₁=PI187367) were used as paternal parents. Mean seed weights of F₁ progeny reflected the dominance of genes of the C. sativus var. sativus parent. Transformation to number of seeds per unit weight resulted in increased variance homogeneity within generations and a broad-sense heritability ranging between 26% to 56%. Additive and dominance effects were important in the expression of seed weight in P₁×P₂ progeny produced in the greenhouse and additive effects were important in field grown progeny resulting from P₁×P₂ and P₁₀×P₁₂ matings. The estimated number of factors or loci involved ranged between 10 to 13, depending on the method of calculation.     

Immunoaffinity purification and characterization of diamine oxidase from Cicer

Antiserum specific for diamine oxidase (DAO;EC 1.4.3.6) from Lens culinaris cross-reacted with DAO from several other members of the Leguminosae when tested by agar double diffusion. Antibodies purified by affinity chromatography were used to make an immunoadsorbent for the one-step purification of DAO from various species of the Leguminosae. This technique has made it possible to purify in one step the already characterized DAO from pea and lentil, and the unknown diamine oxidase from Cicer arietinum. This enzyme was partially characterized; it showed a pH optimum of 7.5 with putrescine as substrate and followed typical Michaelis-Menten kinetics with a K_m of 2.4 × 10^?4 M. Copper ligands and carbonyl group-directed reagents inhibited the enzyme.     

Purification and properties of glycollate oxidase from Pisum sativum leaves

A rapid and efficient method for the isolation of glycollate oxidase from pea leaves is described. The method utilizes the unusually high isoelectric point (pH 9·6) which has been determined for the enzyme using isoelectric focusing. The enzyme is apparently homogeneous by polyacrylamide gel electrophoresis and has a MW of ca 100000. Some properties of the enzyme are described.     

In vitro culture of Cucumis sativus L.

Summary The ability to regenerate plants from leaf explants has been tested for three highly inbred cucumber lines (B, G, S), their reciprocal hybrids, F₂ and BC₁ generations. The lines differed from each other in their regenerating ability, which was expressed by the percentage of explants regenerating embryoidal callus and mean number of plantlets per plant. Thus, the lines could be classified as frequently (B), intermediately (G) or occasionally regenerating ones (S). There were no reciprocal cross differences in the regeneration. It was found that the intermediately and intensively regenerating lines contain two pairs of dominant genes responsible for plant regeneration, characterized by complementary and probably additive interaction. The frequently regenerating line differed from the intermediately regenerating in the effect of one gene. It is supposed that the above-mentioned genes belong to three different loci. The ability to regenerate plants from leaf expiants had high heritability.     

Purification,properties and partial amino-acid sequence of 1-aminocyclopropane-1-carboxylic acid oxidase from apple fruits

The enzyme which converts 1-aminocyclo-propane-1-carboxylic acid (ACC) into ethylene, ACC oxidase, has been isolated from apple fruits (Malus x domestica Borkh. cv. Golden Delicious), and for the first time stabilized in vitro by 1,10-phenanthroline and purified 170-fold to homogeneity in a five-step procedure. The sodium dodecyl sulfate-denatured and native proteins have similar molecular weights (approx. 40 kDa) indicating that the enzyme is active in its monomeric form. Antibodies raised against a recombinant ACC oxidase over-produced in Escherichia coli from a tomato cDNA recognise the apple-fruit enzyme with high specificity in both crude extracts and purified form. Glycosylation appears to be absent because of (i) the lack of reactivity towards a mixture of seven different biotinylated lectins and (ii) the absence of N-linked substitution at a potential glycosylation site, in a sequenced peptide. Phenylhydrazine and 2-methyl-1-2-dipyridyl propane do not inhibit activity, indicating that ACC oxidase is not a prosthetic-heme iron protein. The partial amino-acid sequence of the native protein has strong homology to the predicted protein of a tomato fruit cDNA demonstrated to encode ACC oxidase.     

Purification and characterization of arsenite oxidase from Arthrobacter sp.

The chemolithoautotroph, Arthrobacter sp.15b oxidizes arsenite to arsenate using a membrane bound arsenite oxidase. The enzyme arsenite oxidase is purified to its homogeneity and identified using MALDI-TOF MS analysis. Upon further characterization, it was observed that the enzyme is a heterodimer showing native molecular mass as ~100 kDa and appeared as two subunits of ~85 kDa LSU and 14 kDa SSU on SDS–PAGE. The V _max and K _m values of the enzyme was found to be 2.45 μM (AsIII)/min/mg) and 26 μM, respectively. The purified enzyme could withstand wide range of pH and temperature changes. The enzyme, however, gets deactivated in the presence of 1 mM of DEPC suggesting the involvement of histidine at the binding site of the enzyme. The peptide analysis of large sub unit of the enzyme showed close match with the arsenite oxidases of Burkholderia sp. YI019A and arsenite oxidase, Mo-pterin containing subunit of Alcaligenes faecalis. The small subunit, however, differed from other arsenite oxidases and matched only with 2Fe–2S binding protein of Anaplasma phagocytophilum. This indicates that Rieske subunits containing the iron–sulfur clusters present in the large as well as small subunits of the enzyme are integral part of the protein.     

Purification and properties of pea cotyledon and embryo diamine oxidase

Diamine oxidase was purified separately from cotyledon and embryo of pea seedlings germinated for 6 days. The K_m of the cotyledon enzyme for putrescine was 1.6 × 10^?4M while that for the embryo enzyme was 9 × 10^?5M. On heating for 15 min at 70° the embryo enzyme retained about 90% activity whereas the cotyledon enzyme retained only 20% activity. The electrophoretic mobility of the cotyledon enzyme was ca twice that of the enzyme from embryo.     

Purification and properties of Pinus taeda arginase from germinated seedlings

In germinated loblolly pine (Pinus taeda L.) seeds arginine accumulates in the seedling during its growth immediately following germination. The enzyme arginase (L-arginine amidinohydrolase, EC 3.5.3.1) is responsible for hydrolyzing this arginine into ornithine and urea. Loblolly pine arginase was purified to homogeneity from seedling cotyledons by chromatographic separation on DE-52 cellulose, Matrex Green and arginine-linked Sepharose 4B. The enzyme was purified 148-fold and a single polypeptide band was identified as arginase. The molecular mass was determined to be 140 kDa by FPLC, while the subunit size was shown to be 37 kDa by SDS-PAGE, predicting a homotetramer holoprotein. Removal of manganese from the enzyme abolishes catalytic activity, which can be restored by incubating the protein with Mn²⁺. Antibodies, raised against the arginase subunit, are able to immunotitrate arginase activity and are monospecific for arginase on immunoblots.     

Lathyrus sativus (grass pea) and its neurotoxin ODAP

Lathyrus sativus (grass pea) is a high-yielding, drought-resistant legume consumed as a food in Northern India and neighboring countries as well as in Ethiopia. Its development into an important food legume, however, has been hindered by the presence of the neurotoxin - beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (beta-ODAP) in seeds which, if consumed in large quantities for prolonged periods, can cause irreversible paralysis. Recently, some low-toxin lines have been developed that may prove safe for both animal and human foods. Cultivation of L. sativus should thus be considered in suitable regions because the demand for legume animal feed protein products is expected to increase. This paper addresses advances in understanding L. sativus from the perspective of its taxonomy, genetics, ecology, chemistry, nutrition, medicine, biology and for animal nutrition.     

Extracellular L-glutamate oxidase ofStreptomyces sp. Z-11-6: Obtainment and properties

Mutagenesis induced with nitrous acid and subsequent selection allowed a genetically stable mutant strain,Streptomyces sp. Z-11-6, to be obtained, whose L-glutamate oxidase activity was 40-fold higher than that of the original natural isolate and was as great as 1.6–1.8 units/ml of culture liquid. A procedure for the isolation and purification of the enzyme was developed; the biochemical properties of the enzyme were studied. Out of 20 amino acids tested (including D-glutamate), the glutamate oxidase fromStreptomyces sp. Z-11-6 was active only with L-glutamate. This allows the concentration of L-glutamate to be determined in the presence of other amino acids. Calcium chloride at a concentration of 0.1–0.5% promoted the secretion of the extracellular glutamate oxidase.     

Purification and characterization of anl-amino-acid oxidase fromChlamydomonas reinhardtii

Anl-amino-acid oxidase (EC 1.4.3.1) that catalyzes the oxidative deamination of twelvel-amino acids has been purified 21-fold and with 14% yield to electrophoretic homogeneity fromChlamydomonas reinhardtii cells by ammonium-sulfate fractionation, gel filtration through Sephacryl and Superose, anion-exchange chromatography and preparative electrophoresis in polyacrylamide gels. The native enzyme is a protein of 470 kDa and consists of eight identical or similarsized subunits of 60 kDa each. Optimum pH and temperature were 8.2 and 55° C, respectively, with a Q₁₀ (45–55° C) of 1.7 and an activation energy of 45 kJ · mol^–1. Its absorption spectrum showed, in the visible region, maxima at 360 and 444 nm, characteristic of a flavoprotein with a calculated flavin content of 7.7 mol FAD per mol of native enzyme. ApparentK _m values of the twelvel-amino acids which can act as substrates ofl-amino-acid oxidase ranged between 31 M for phenylalanine and 176 M for methionine. The effect of several specific group reagents, chelating agents and bivalent cations on enzyme activity has also been studied.This work was supported by Grant 780-CO2-01 from CICYT, Spain. The skillful secretarial assistance of C. Santos and I. Molina is gratefully acknowledged.