Purification of beta-amylase from alfalfa (Medicago sativa L.) seeds

An amylase from alfalfa (Medicago sativa L. c.v. Moapa) seeds was purified by column chromatography and gel filtration, followed by chromatofocusing on Mono P HR 5/20. The last step was effective for separation of the alfalfa amylase to a homogeneous state. The purified amylase was identified as beta-amylase from the fact that only beta-maltose was formed by the enzymatic degradation of soluble starch. The molecular weight and specific activity of the beta-amylase (E1%(280 nm) = 18.3) were determined to be 61,000 and 1,077 A.U./mg, respectively. The beta-amylase activity was inhibited by the modification of sulfhydryl groups with p-chloromercuribenzoic acid. The optimum pH and isoelectric point of alfalfa beta-amylase were 7.0 and 4.8, respectively, which were different from other plant beta-amylases.     

Purification and characterization of two leaf polypeptide inhibitors of leaf protease from alfalfa (Medicago sativa)

Two polypeptides with antiproteolytic activities have been isolated from alfalfa leaves. Polypeptide I resembles the previously described plant protease inhibitors in both structural and functional features; it has a molecular weight of 15,000, a random coil secondary structure, and inhibits exogenous protease as well as alfalfa leaf protease. Polypeptide II is a novel type of plant inhibitor with a molecular weight of 6300 and a highly organized structure with a high (40-50%) alpha-helix content. It only inhibits endogenous protease with a molar stoichiometry polypeptide/enzyme protein of 1.     

Adenylate cyclase activity in a higher plant, alfalfa (Medicago sativa).

An adenylate cyclase activity in Medicago sativa L. (alfalfa) roots was partially characterized. The enzyme activity remains in the supernatant fluid after centrifugation at 105,000 g and shows in crude extracts an apparent Mr of about 84,000. The enzyme is active with Mg2+ and Ca2+ as bivalent cations, and is inhibited by EGTA and by chlorpromazine. Calmodulin from bovine brain or spinach leaves activates this adenylate cyclase.     

Liming of alfalfa (Medicago sativa L.)

Summary A growth-chamber experiment was conducted to study the effect of liming upon growth of alfalfa. The beneficial effects observed were related to changes in soil properties brought about by lime application. Reductions of aluminum and manganese toxicities were the major factors responsible for the increased yields and the decreased growth period required to reach harvest stage. Significant correlations between plant growth parameters and various measures of extractable aluminum were found.     

Features of the beta-amylase isoform system in dry and germinating seeds of alfalfa (Medicago sativa L.)

Five isoforms of beta-amylase were purified to homogeneity from alfalfa seeds (Medicago sativa L.) by chromatofocusing and cation-exchange chromatography. These isoforms were identified as beta-amylase based on their catalytic mode to the substrates. These isoforms of beta-amylase were also found in germinating seeds of alfalfa. All the isoforms existed in free form, because they could be extracted without reducing agent. The five isoforms had different isoelectric points (5.05, 4.97, 4.85, 4.82 and 4.77), but their Mr was the same (61 kDa) on SDS-polyacrylamide gels. The amino acid compositions were similar, but not identical, to each other. An antiserum raised against one of the five isoforms cross-reacted with all of other isoforms, but did not recognize the component 2 of soybean beta-amylase. The amounts of five isoforms increased during seed germination, which was responsible for significant increase of the beta-amylase activity in germinating seeds.     

Artificial seeds of alfalfa (Medicago sativa L.). Induction of desiccation tolerance in somatic embryos

Summary The use of somatic embryos from cell culture systems in the clonal propagation of plants would be greatly facilitated if the somatic embryos could be dried and stored in a dormant state similar to true seeds. A cell culture system was developed for alfalfa (Medicago sativa L.) line RL34 which gave high yields of somatic embryos in an approximately synchronized pattern. These somatic embryos were treated with abscisic acid (ABA) at the cotyledonary stage of development to induce desiccation tolerance. With no visual preselection, approximately 60% of the dried embryos converted into plants upon reimbibition. When high quality embryos were selected prior to drying, 90 to 100% conversion rates were observed. The timing of the application of ABA in terms of embryo development was critical with an optimum being at cotyledonary stage spanning approximately 4 days; thus, synchronized embryo development is required for optimal expression in bulk samples. The vigor of the seedlings from dried somatic embryos was greater than those from embryos which had not been dried, but remained substantially lower than those from true seeds.     

Histone variants and acetylated species from the alfalfa plant Medicago sativa

The histones from the alfalfa plant Medicago sativa have been characterized in terms of type variants and levels of acetylation. Histones were isolated directly from total plant tissue (callus), eliminating the need to develop methods for nuclear isolation. An acid-urea-polyacrylamide gel with a transverse Triton X-100 gradient resolved and identified in a single gel at least one type of histone H4, two variant forms of histone H2B, two variant forms of histone H3, and four variant forms of histone H2A from a crude histone preparation. Histone H4 was present 25% in an unmodified state and 75% as monomodified, presumably as monoacetylated histone. Both histone H3 variants displayed five bands, consistent with up to four internal sites of acetylation. The two H3 variants differed in their steady-state level of acetylation, suggesting that they may reside in different chromatin environments. Several histone H1 species were identified by solubility and cross-reactivity with antiserum raised against the globular part of bovine H1(0), indicating conservation of epitopes between histone H1 of mammals and higher plants.     

Crystal structure of isoflavone reductase from alfalfa (Medicago sativa L.)

Isoflavonoids play important roles in plant defense and exhibit a range of mammalian health-promoting activities. Isoflavone reductase (IFR) specifically recognizes isoflavones and catalyzes a stereospecific NADPH-dependent reduction to (3R)-isoflavanone. The crystal structure of Medicago sativa IFR with deletion of residues 39-47 has been determined at 1.6A resolution. Structural analysis, molecular modeling and docking, and comparison with the structures of other NADPH-dependent enzymes, defined the putative binding sites for co-factor and substrate and potential key residues for enzyme activity and substrate specificity. Further mutagenesis has confirmed the role of Lys144 as a catalytic residue. This study provides a structural basis for understanding the enzymatic mechanism and substrate specificity of IFRs as well as the functions of IFR-like proteins.     

Crystal structure of vestitone reductase from alfalfa (Medicago sativa L.)

Isoflavonoids are commonly found in leguminous plants, where they play important roles in plant defense and have significant health benefits for animals and humans. Vestitone reductase catalyzes a stereospecific NADPH-dependent reduction of (3R)-vestitone in the biosynthesis of the antimicrobial isoflavonoid phytoalexin medicarpin. The crystal structure of alfalfa (Medicago sativa L.) vestitone reductase has been determined at 1.4 A resolution. The structure contains a classic Rossmann fold domain in the N terminus and a small C-terminal domain. Sequence and structural analysis showed that vestitone reductase is a member of the short-chain dehydrogenase/reductase (SDR) superfamily despite the low levels of sequence identity, and the prominent structural differences from other SDR enzymes with known structures. The putative binding sites for the co-factor NADPH and the substrate (3R)-vestitone were defined and located in a large cleft formed between the N and C-terminal domains of enzyme. Potential key residues for enzyme activity were also identified, including the catalytic triad Ser129-Tyr164-Lys168. A molecular docking study showed that (3R)-vestitone, but not the (3S) isomer, forms favored interactions with the co-factor and catalytic triad, thus providing an explanation for the enzyme's strict substrate stereo-specificity.     

Purification and some properties of an aminopeptidase from the seeds of Cannabis sativa

An aminopeptidase (HSA) with a molecular mass of 78 kDa was purified from hemp (Cannabis sativa) seeds. The activity was inhibited by monoiodeacetic acid, p-chloromercuri-phenylsulfonic acid, and Zn2+ ion. The specificity of HSA was similar to that of a leucyl aminopeptidase [EC 3.4.11.1] from mammalian cytosol. However, other enzyme properties were different from these of leucyl aminopeptidase.     

Induced androgenesis in alfalfa (Medicago sativa L.)

Summary Anthers of 10 alfalfa (Medicago sativa L.) lines were used as initial material for the production of androgenic haploids. More than 30 variants of nutrient media were tested. Twenty five different treatments with low temperatures and gamma rays were tried in order to find optimal conditions for callus induction and organogenesis.The genotype, stage of microspore development, phytohormonal composition of the nutrient media and pretreatment with physical agents, alone or in combination, affected the efficiency of organogenesis and regeneration in anther cultures of alfalfa.Plants exhibited a high degree of variability in their chromosome number. Haploids, dihaploids and mixoploids were obtained.Cytological studies of in vitro pollen development revealed the origin of the regenerants from microspores.Abbreviations BAP 6-Benzylaminopurine - 2-ip 6-(,-dimethylallylamino)Purine - IAA Indolylacetic Acid - NAA Naphthaleneacetic Acid - 2,4-D Dichlorophenoxyacetic Acid - CMS Cytoplasmic Male Sterility     

Purification and characterization of S-adenosyl-L-methionine: caffeic acid 3-O-methyltransferase from suspension cultures of alfalfa (Medicago sativa L.)

Caffeic acid O-methyltransferase (COMT) is one of a group of proteins present in alfalfa cell cultures which can be photoaffinity labeled with S-adenosyl-L-[methyl-3H]methionine. The enzyme was purified to homogeneity from elicitor-treated suspension cultures and shown to exist as an active monomer of subunit Mr 41,000. COMT could be separated into two forms on the basis of their isoelectric points and relative affinities for S-adenosyl-methionine and S-adenosylhomocysteine. Both forms had equal affinities for caffeic acid, were highly specific for the 3-hydroxyl group of substituted cinnamic acids, and exhibited negligible activity toward flavonoid substrates. An antiserum raised against COMT from aspen immunoprecipitated alfalfa COMT activity. Peptide mapping studies indicated that the two forms of COMT and an isoflavone O-methyltransferase from alfalfa are closely related proteins. The extractable activity of COMT doubled over a 48-h period following exposure of alfalfa cell suspensions to a yeast elicitor preparation, and this was associated with a small change in the relative proportions of the two forms of the enzyme.     

Embryogenic response of Mexican alfalfa (Medicago sativa) varieties

The in vitro embryogenic response of nine varieties of alfalfa (Medicago sativa L.) grown in México (five Mexican varieties: Puebla 76, Inia 76, Bajío 76, Sintético I and Sintético II and four foreign or introduced varieties: Moapa 69, San Joaquín II, Hairy Peruvian and Valenciana) were tested. We screened 25 genotypes from each variety in four tissue culture protocols. All the varieties, except San Joaquín II, gave a positive response in one or more of the protocols tested. The response in each variety was low; this was also observed in a wider screening performed with the varieties Moapa 69, Hairy Peruvian, Sintético I and Sintético II. Two plants from Moapa 69 were regenerated and appeared normal.     

Direct embryogenesis from single mesophyll protoplasts in alfalfa (Medicago sativa L.)

Summary We used a tetraploid clone derived from an anther culture operation of Ladak alfalfa to study the pathway of direct embryogenesis from leaf-mesophyll protoplasts. About 72% of the protoplasts divided, and 7% of those produced proembryos. Approximately 38% of the proembryos developed into green embryos, and 33% initiated calluses. Other proembryos dedifferentiated into calluses which later redifferentiated embryos. Sixteen percent of the embryos developed directly into plants, whereas 81% produced plants indirectly via secondary embryos. The remaining 3% of the primary embryos failed to develop into plants. The lowest plating efficiency for direct embryogenesis was 0.3%. The high percentage of direct embryogenesis observed was related to the genetic nature of the clone, low density of liquid medium, low protoplast culture density, and the composition of culture media.Contribution no. 90-61-J from the Kansas Agricultural Experiment Station.