Two novel stilbene phytoalexins from Arachis hypogaea

Three phytoalexins were isolated from groundnut seeds which had been sliced and incubated for 48 hr at 252. Two were novel isoprenylated stilbene der     

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.     

Heme enzymes in a chlorina hybrid of ground nut (Arachis hypogaea) and its parents

Chlorophyll biosynthesis in the Chlorina hybrid was affected due to the lower levels of the enzyme δ-amino levulinate dehydratase responsible for the synthesis of porphobilinogen. A comparison of the amounts of different heme containing enzymes from the etiolated and green seedlings of the Chlorina and its parents suggested that the chlorophyll and heme moiety of catalase share the same pool of porphobilinogen and that this pool is different to the one shared by peroxidase and indole acetic acid oxidase. The enzyme δ-amino levulinate dehydratase possesses two isoenzyme bands. These isoenzymes may be spatially separated and responsible for the synthesis of two pools of porphobilinogen.     

Purification and properties of agmatine iminohydrolase from groundnut cotyledons

Agmatine iminohydrolase was purified ca 375-fold from groundnut cotyledons. The enzyme exhibited an optimum pH between 5.5 and 8.5 and the energy of activation was 22 kcal/mol. The K_m for agmatine was (7.57 ± 0.77) × 10^?4 M. The enzyme was inhibited by tryptamine, putrescine, cadaverine, spermidine and spermine. Inhibition by cadaverine and spermidine was competitive. The K_i values for cadaverine and spermidine were 4.1 × 10^?3 and 7.5 × 10^?4 M, respectively.     

Purification and properties of sucrose-6-phosphatase from Pisum sativum shoots

Sucrose-6-phosphatase from pea shoots, which was purified to homogeneity, consists of two similar sub-units each with an MW of about 55 000. The pH optimum was at 6.8; the K_m for sucrose-6-phosphate was 250 μM and the K_m for magnesium was 175 μM. The enzyme was specific for sucrose-6-phosphate and was not inhibited by sucrose except at very high concentrations.     

Purification of acyl hydrolase enzymes from the leaves of Phaseolus multiflorus

Acyl hydrolase activities have been purified from the leaves of Phaseolus multiflorus. The purification procedure involved heat treatment, DEAE-cellulose chromatography, Sephadex G-100 filtration and hexyl agarose chromatography. The elution pattern from hexyl agarose columns together with substrate competition experiments indicated the presence of two hydrolase enzymes. The first could hydrolyse oleoylglycerol and phosphatidylcholine while the second would deacylate glycosylglycerides and oleoylglycerol. Overall purification of both enzymes was ca 70-fold and the MW of the glycosylglyceride-hydrolysing enzyme was in the range 70–78000.     

Ectopic over-expression of peroxisomal ascorbate peroxidase (SbpAPX) gene confers salt stress tolerance in transgenic peanut (Arachis hypogaea)

Peroxisomal ascorbate peroxidase gene (SbpAPX) of an extreme halophyte Salicornia brachiata imparts abiotic stress endurance and plays a key role in the protection against oxidative stress. The cloned SbpAPX gene was transformed to local variety of peanut and about 100 transgenic plants were developed using optimized in vitro regeneration and Agrobacterium mediated genetic transformation method. The T₀ transgenic plants were confirmed for the gene integration; grown under controlled condition in containment green house facility; seeds were harvested and T₁ plants were raised. Transgenic plants (T₁) were further confirmed by PCR using gene specific primers and histochemical GUS assay. About 40 transgenic plants (T₁) were selected randomly and subjected for salt stress tolerance study. Transgenic plants remained green however non-transgenic plants showed bleaching and yellowish leaves under salt stress conditions. Under stress condition, transgenic plants continued normal growth and completed their life cycle. Transgenic peanut plants exhibited adequate tolerance under salt stress condition and thus could be explored for the cultivation in salt affected areas for the sustainable agriculture.     

Purification and properties of carbonic anhydrase from Chlamydomonas reinhardii

Carbonic anhydrase (CA) was purified from the unicellular green alga Chlamydomonas reinhardii, and the purity of the preparation was established by gradient gel electrophoresis. The purified enzyme exhibited a MW of 165 000 and contained 6 atoms of Zn. The subunit MW, as determined by dodecyl sulfate electrophoresis, was 27 000. These results are consistent with a quarternary structure which is hexameric, each monomer containing 1 g atom of Zn. Like spinach CA, and in contrast to other oligomeric plant CAs, a sulfhydryl reducing agent is not needed to stabilize the enzyme. CO₂-hydrase activity was inhibited by both acetazolamide (I₅₀ = 7.8 × 10^?9M) and sulfanilamide (I₅₀ = 1.3 × 10^?5M), as well as by certain inorganic anions. The purified enzyme showed relatively weak esterase activity with p-nitrophenyl acetate but was an extremely effective esterase with 2-hydroxy-5-nitro-α-toluenesulfonic acid sultone as the substrate. Both esterase activities could be completely inhibited by adding acetazolamide. In its gross structural characteristics, the C. reinhardii enzyme resembles the CAs from higher plants. However, in its esterase activity and the inhibition by sulfonamides it is markedly different from plant CAs and bears more resemblance to erythrocyte CAs.     

In sacco degradation kinetics of fresh and field-cured peanut (Arachis hypogaea L.) forage harvested at different maturities

There is interest in growing peanut (Arachis hypogaea L.) for forage, but little is known about the nutritive value and forage quality of modern cultivars. The objective of this study was to compare the chemical composition and in sacco degradation kinetics of three cultivars of peanuts (cv. ‘C99-R’, ‘Georgia-01R’, and ‘York’) at either stage 2 or 8 maturities when fresh and field-cured. Herbage yield was at least 3000 kg DM/ha for all cultivars at both maturities. Crude protein (CP) was greater (P < 0.0001) at R2 stage than at R8 stage; whereas, neutral detergent fiber (aNDF), acid detergent fiber, and Lignin (sa) were greater (P < 0.01) at R8 than R2 maturity stages. Water soluble carbohydrate and acid detergent insoluble nitrogen was not different (P > 0.07) among cultivars, maturity stage, or harvest forms. In vitro true digestibility was greatest (P < 0.02) for C99-R and least for York. Undegradable intake protein concentration was greatest (P < 0.04) in York and least for C99-R. Maturity had a greater effect on the degradation kinetics than harvest form or cultivar. The dry matter (DM) and CP in the soluble wash fraction (A) and insoluble but degradable fraction (B) and the effective ruminal degradability were greater among all cultivars and both harvest forms of the R2 maturity stage than the R8. The undegradable DM, aNDF, and CP in the undegradable fraction were greatest (P < 0.002) for all three cultivars at R8 maturity. The rate of degradation of DM and CP in the B fraction was faster (P < 0.001) at R2 stage than at R8 stage; whereas, rate of aNDF degradation was not different (P > 0.09) among treatments. Lag of DM, aNDF, or CP degradation was not different (P > 0.1) among treatments. The cultivars C99-R and Georgia-01R are recommended for further feeding trials.     

RFLP AND CYTOGENETIC EVIDENCE ON THE ORIGIN AND EVOLUTION OF ALLOTETRAPLOID DOMESTICATED peanut,Arachis hypogaea (Leguminosae)

Nuclear restriction fragment length polymorphism (RFLP) analysis was used to determine the wild diploid Arachis species that hybridized to form tetraploid domesticated peanut. Results using 20 previously mapped cDNA clones strongly indicated A. duranensis as the progenitor of the A genome of domesticated peanut and A. ipaensis as the B genome parent. A large amount of RFLP variability was found among the various accessions of A. duranensis, and accessions most similar to the A genome of cultivated peanut were identified. Chloroplast DNA RFLP analysis determined that A. duranensis was the female parent of the original hybridization event. Domesticated peanut is known to have one genome with a distinctly smaller pair of chromosomes (“A”), and one genome that lacks this pair. Cytogenetic analysis demonstrated that A. duranensis has a pair of “A” chromosomes, and A. ipaensis does not. The cytogenetic evidence is thus consistent with the RFLP evidence concerning the identity of the progenitors. RFLP and cytogenetic evidence indicate a single origin for domesticated peanut in Northern Argentina or Southern Bolivia, followed by diversification under the influence of cultivation.     

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.     

Chloroplast cysteine synthases of Trifolium repens and Pisum sativum

About 68–86% of the cysteine synthase activity in leaf tissue of white clover (Trifolium repens) and peas (Pisum sativum cultivar Massey Gem) was associated with chloroplasts. The enzymes from white clover and peas were purified ca 66 and 12-fold respectively. For clover, the K_m values determined by calorimetric and S^2? ion electrode methods were: S^2? 0.51 and 0.13 mM; O-acetylserine (OAS), 3.5 and 2.O mM respectively. The analogous values for the pea enzyme were: S^2?, 0.24 and 0.06 mM; OAS, 3.1 and 0.24 mM. Both enzymes were inhibited by cystathionine and cysteine. Pretreatment with cysteine inactivated the enzyme, but addition of pyridoxal phosphate caused partial reactivation. Isolated pea chloroplasts (70–75 % intact) catalysed OAS-dependent assimilation of sulphide at a mean rate of 88 μmol/mg Chl/hr. About 85 % of the OAS-dependent sulphide assimilated was recovered as cysteine. The rates were unaffected by light and 2 μM DCMU. Sonicating the chloroplasts enhanced the rate by 1.3–2 fold. Cysteine synthase activity was associated with the chloroplast stroma. Similar results were obtained for clover chloroplasts except that both the intactness and the rates were lower.     

4-Hydroxyisoleucine from seed of Trigonella foenum-graecum

The principal free amino acid present in seed of Trigonella foenum-graecum has been isolated and identified as (2S, 3R, 4R)-4-hydroxyisoleucine. This compound has not been reported previously as a constituent of higher plants, but it is a component of the toxic peptide, γ-amanitin, produced by Amanita phalloides. The (2S, 3R, 4R)-isomer lactonizes readily under acidic conditions, whilst strong acid causes partial epimerization. The (2R, 3R, 4R)-isomer forms a minor component of Trigonella seed. The 4-hydroxyisoleucine content of fenugreek increases during the growth of seedlings and plants, and ¹⁴C-isoIeucine was used effectively as a biosynthetic precursor.     

Spectral and molecular properties of peanut peroxidase isozymes

Properties of four peroxidase isozymes derived from peanut cells were examined. Electrophoresis on various concentrations of polyacrylamide gel indicated that they had the same molecular size. Filtration on Sephadex G-200 gels indicated the same Stoke's radius for all 4 isozymes. They had the same spectral properties in the oxidized, reduced and CO-reduced the pyridine hemochromogen forms, but they differed with regard to heat stability at 50° and 70° and their substrate specificity.     

Catabolism of sulphoquinovosyl diacylglycerol by an enzyme preparation from Phaseolus multiflorus

An enzyme which will deacylate sulphoquinovosyl diacylglycerol (SQDG) has been partially purified from the leaves of runner bean (Phaseolus multiflorus). No monoacyl intermediate was observed and the acyl hydrolase was more active towards unsaturated molecular species of SQDG than towards saturated species. The major peak of activity of SQDG acyl hydrolase, separated on both DEAE-cellulose and Sephadex columns, also contained galactolipid acyl hydrolase activity. The distribution of these activities together with substrate competition and inhibitor experiments indicated that at least part of the SQDG acyl hydrolase activity was due to an enzyme that also hydrolysed galactolipids.     

Purification and properties of alcohol oxidase from Tanacetum vulgare

Alcohol oxidase (alcohol: O₂ oxidoreductase) from leaves of Tanacetum vulgare has been purified 5150-fold to homogeneity on disc electrophoresis and gel electrofocussing. The enzyme which is probably flavoprotein, has molecular weight 180 000 daltons and is comprised of two sub-units of 94 000 and 75 000 daltons. It is active over a broad range (pH 5–9) and best accepts primary aliphatic alcohols with 6 to 10 carbons, especially those with a 2-ene group. K_m values for hex-trans-2-ene-1-ol, geraniol (3,7-dimethylocta-trans-2,6-dien-1-ol) and n-octanol were 0.19, 1.56 and 0.49 mM respectively. The significance of the enzyme in the formation of leaf aldehyde (hex-trans-2-ene-1-al) and in terpene metabolism is discussed.     

Properties of acyl hydrolase enzymes from Phaseolus multiflorus leaves

The properties of acyl hydrolase enzymes purified from the leaves of Phaseolus multiflorus have been studied. Hydrolase I which deacylates phosphatidylcholine and oleoylglycerol had a pH optimum towards phosphatidylcholine of 5.3. Hydrolase II which deacylates glycosylglycerides and oleoylglycerol showed pH optima of 7.3 (monogalactosyldiglyceride, MGDG) and 4.3 (sulphoquinovosyldiglyceride, SQDG). Both enzymes showed activity peaks towards oleoylglycerol at pH 6.8 and 8.8. Unesterified fatty acids and Triton X-100 inhibited the rate of SQDG hydrolysis while bovine serum albumin increased activity. An apparent K_m for SQDG of 0.15 mM was found. Hydrolase II catalysed transmethylation of liberated fatty acids during the hydrolysis of oleoylglycerol when methanol was included in the assay system. A number of salts inhibited SQDG hydrolysis but their effect on oleoylglycerol was less consistent. The position of ester cleavage of oleoylglycerol was determined by the use of H₂¹⁸O. Cell-free extracts from P. multiflorus leaves degraded SQDG as far as sulphoquinovose.     

Aminopeptidase activity associated with alpha1-conarachin (peanut protein)

Aminopeptidases were investigated in protein extracts of dormant and germinated peanut cotyledons by electrophoretic immunochemical techniques. Considerable activity was observed in protein zones that migrated toward the anode after simple electrophoresis. Of the immunogenic proteins in dormant seed, aminopeptidase activity was associated only with the immunocomplex of α₁-conarachin, a globular protein in peanuts. The specific aminopeptidase activity of total extracts was marginally higher than that of purified α-conarachin. Specific iso-enzyme(s) adsorption on the antigen-antibody complex of α₁-conarachin might be attributed in part, to these phenomena. Reactions of protein extracts from germinated cotyledons with immune sera made against protein in germinated tissues—roots and cotyledons—showed the identical associative interaction. Some of the determinant groups on α₁-conarachin in the germ and cotyledon were apparently maintained during early phases of germination.