Endosperm Enzyme Research Articles

Identification of four starch-branching enzymes in barley endosperm: partial purification of forms I, IIa and IIb.

Developing caryopses of barley (Hardeum vulgare L. ev. Golf) were monitored for starch-branching enzyme activity for 1 month after anthesis. Homogenized caryopses from grains with the highest specific activity were used as starting material for purification of branching enzyme by FPLC chromatography. Several branching enzyme activity fractions were resolved. We have previously described the isolation of a 51/50 kDa protein with starch branching activity from one of these fractions. From three other fractions, we have now partially purified starch branching enzymes corresponding to forms I, IIa and IIb.

The Miniature1 Seed Locus of Maize Encodes a Cell Wall Invertase Required for Normal Development of Endosperm and Maternal Cells in the Pedicel.

Collective evidence demonstrates that the Miniature1 (Mn1) seed locus in maize encodes an endosperm-specific isozyme of cell wall Invertase, CWI-2. The evidence includes (1) isolation and characterization of ethyl methanesulfonate-induced mn1 mutants with altered enzyme activity and (2) a near-linear relationship between gene/dose and invertase activity and the CWI-2 protein. In addition, molecular analyses showed that the cDNA clone incw2 maps to the Mn1 locus and differentiates the six ethyl methanesulfonate-induced mn1 mutants of independent origin into two classes when RNA gel blot analyses were used. We also report two unexpected observations that provide significant new insight into the physiological role of invertase and its regulation in a developing seed. First, a large proportion of total enzyme activity (~90%) was dispensable (i.e., nonlimiting). However, below the threshold level of ~6% of wild-type activity, the endosperm enzyme controlled both the sink strength of the developing endosperm as well as the developmental stability of maternal cells in the pedicel in a rate-limiting manner. Our data also suggest an unusually tight coordinate control between the cell wall-bound and the soluble forms of invertase, which are most likely encoded by two separate genes, presumably through metabolic controls mediated by the sugars.

Phospholipase D from Soybean (Glycine max L.) Suspension-Cultured Cells: Purification, Structural and Enzymatic Properties

Phospholipase D (phosphatidylcholine phosphatidohydrolase EC 3.1.4.4) from soybean (Glycine max L.) suspension-cultured cell was purified around 1,200-fold to homogeneity by acetone precipitation, Macro-Prep High Q anion exchange, and octyl-Sepharose CL-4B affinity chromatography. The purified enzyme released 1,600 mumol of choline per min per mg of protein. The enzyme is monomeric with a molecular mass of 92 kDa, as estimated by SDS-PAGE. One of the most interesting characteristics of the purified soybean phospholipase D was the dependence of the pH optimum on the Ca2+ ion concentration in the assay. With 10 mM, 20 mM and 40 mM Ca2+ ions, the optima were at pH 7.5, 6 and 5.5, respectively. The specific adsorption of phospholipase D onto octyl-Sepharose gel suggests that the molecule becomes more hydrophobic in the presence of Ca2+ ions. The amino acid sequence of the first 18 N-terminal residues of soybean phospholipase D revealed a high degree of homology with those previously published for cabbage leaf and castor bean endosperm enzymes. Western blots of the soybean phospholipase D showed an immunoreactivity with antibodies raised against a synthetic peptide corresponding to the 15 N-terminal amino acid residues of phospholipase D from cabbage leaves.

Genomic Nucleotide Sequence of a Wild-Type Shrunken-2 Allele of Zea mays

The maize (Zea mays) endosperm enzyme, ADP-glucose pyrophosphorylase, which produces ADP-glucose from ATP and glucose-l-phosphate, is an important enzyme in the synthesis of starch. ADP-glucose arising from the action of this enzyme is the major, if not sole, donor of glucose for starch biosynthesis. The enzyme is composed of two dissimilar subunits encoded by the two unlinked genes, Shrunken-2 (Sh2) and Brittle-2 (Bt2) (1, 2). The enzyme is allosterically activated by 3-phosphoglyceric acid and inhibited by phosphate (3). Although it remains an open question whether these allosteric properties are physiologically relevant or whether they simply reflect the evolutionary history of the two structural genes (4), there does exist, nevertheless, much interest in determining whether genetic modification of this enzyme could lead to increased rates of starch biosynthesis in the maize endosperm. Because endosperm starch content comprises approximately 70% of the dry weight of the seed, alterations in starch biosynthesis would clearly affect corn yield. As a first step toward such experiments, we have isolated and sequenced genomic clones of Sh2. The structure of the gene (Table I, Fig. 1) is based on sequence analysis of three overlapping clones isolated from a Black Mexican Sweet genomic library. Exonic sequences were defined by comparison with the cDNA sequenced previously (2) and further sequencing of cDNAs subsequently isolated. These sequences correct for the fact that the original cDNA contained some non-Sh2 sequences at its extremities. The exonic genomic and cDNA sequences are nearly 100% identical. The few differences may be due to DNA polymorphisms because these clones were isolated from different corn lines. Placement of the start of the first exon is based on primer extension experiments. In the absence of dideoxynucleotides, four major bands, differing by one nucleotide, were observed. In the presence of dideoxynucleotides, the three large bands were seen in all four sequencing tracks. In each case, the largest band occurred at the same distance from the primer. It is unknown whether this reflects heterogeneity within the normal population of Sh2 transcripts or some laboratory artifact. Nevertheless, the start of transcription can be placed within three base pairs.

Enzymic synthesis of 1-O-(indol-3-ylacetyl)-β-d-glucose. Purification of the enzyme from Zea mays, and preparation of antibodies to the enzyme

The enzyme indol-3-ylacetylglucose synthase (UDP-glucose:indol-3-ylacetate beta-D-glucosyltransferase) catalyses the reaction: [formula: see text] This is the first step in the series of reactions leading to the indol-3-ylacetic acid conjugates found in maize. Previous attempts to purify this enzyme from the liquid endosperm of kernels of Zea mays (sweet corn) were not entirely successful owing to the lability of partially purified preparations during column chromatography. Thus this enzyme has not previously been purified to homogeneity. During the present study it was found that retention of enzyme activity required the combined presence of glycerol and dithiothreitol. Adding these requirements permitted purification of the enzyme to homogeneity with retention of catalytic activity. These purified preparations were used for preparation of rabbit polyclonal antibodies to the enzyme. Antibodies to the Zea mays endosperm enzyme cross-react with the enzyme from Zea mays vegetative tissues and with an enzyme from the liquid endosperm of oak acorns (Quercus sp). In this paper we report a simplified purification procedure adaptable to the preparation of milligram amounts of the enzyme.

Regulatory mechanisms involved in the biosynthesis of starch

Abstract

Further Identification of Endogenous Gibberellins in the Shoots of Pea, Line G2

To interpret the metabolism of radiolabeled gibberellins A(12)-aldehyde and A(12) in shoots of pea (Pisum sativum L.), the identity of the radiolabeled peaks has to be determined and the endogenous presence of the gibberellins demonstrated. High specific activity [(14)C]GA(12) and [(14)C]GA(12)-aldehyde were synthesized using a pumpkin endosperm enzyme preparation, and purified by high performance liquid chromatography (HPLC). [(14)C]GA(12) was supplied to upper shoots of pea, line G2, to produce radiolabeled metabolites on the 13-OH pathway. Endogenous compounds copurifying with the [(14)C]GAs on HPLC were analyzed by gas chromatography-mass spectrometry. The endogenous presence of GA(53), GA(44), GA(19) and GA(20) was demonstrated and their HPLC peak identity ascertained. The (14)C was progressively diluted in GAs further down the pathway, proportional to the levels found in the tissue and inversely proportional to the speed of metabolism, ranging from 63% in GA(53) to 4% in GA(20). Calculated levels of GA(20), GA(19), GA(44), and GA(53) were 42, 8, 10, and 0.5 nanograms/gram, respectively.

Comparison of soluble starch synthases and branching enzymes from leaves and kernels of normal andamylose-extender maize

Soluble starch synthases (SS) and branching enzymes (BE) from 20-day-old maize leaves and 22-day-old seeds of normal and amylose-extender (ae) were purified by DEAE-cellulose chromatography. Elution profiles of leaf extracts showed one major SS and two BE fractions from both genotypes. The SS fractions from normal and ae leaf extracts were capable of citrate-stimulated starch synthesis and had different reaction rates with various primers. The two BE fractions from normal leaf extracts differed significantly from each other but not when compared to the same BE from ae. Comparison of BE fractions from ae and normal leaves showed no differences based on chromatographic, kinetic, and immunological properties. Comparison of the leaf enzymes with endosperm enzymes showed major differences. Leaf extracts did not contain SSII or BEIIb observed in endosperm extracts. Developing ae endosperm lacks BEIIb activity and ae is the structural gene for BEIIb. The tissue specific expression of BEIIb in the endosperm provides the basis for explaining the tissue-specific expression of ae. We propose that as BEIIb is expressed in the endosperm, but not leaves, allelic substitution at the ae locus modifies only endosperm starch synthesis.

Isozymes of the Glycolytic Enzymes in Endosperm from Developing Castor Oil Seeds

Ion filtration chromatography on diethylaminoethyl-Sephadex A-25 has been used to separate two isozymes each of triose phosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, glycerate 3-phosphate kinase, enolase, and phosphoglycerate mutase from homogenates of developing castor oil (Ricinus communis L. cv. Baker 296) seeds. Crude plastid fractions, prepared by differential centrifugation, were enriched in one of the isozymes, whereas the cytosolic fractions were enriched in the other. These data (and data published previously) indicate that plastids from developing castor oil seeds have a complete glycolytic pathway and are capable of conversion of hexose phosphate to pyruvate for fatty acid synthesis. The enzymes of this pathway in the plastids are isozymes of the corresponding enzymes located in the cytosol.

Glutamate Dehydrogenase in Developing Endosperm, Chloroplasts, and Roots of Castor Bean

All the glutamate dehydrogenase activity in developing castor bean endosperm is shown to be located in the mitochondria. The enzyme can not be detected in the plastids, and this is probably not due to the inactivation of an unstable enzyme, since a stable enzyme can be isolated from castor bean leaf chloroplasts. The endosperm mitochondrial glutamate dehydrogenase consists of a series of differently charged forms which stain on polyacrylamide gel electrophoresis with both NAD(+) and NADP(+). The chloroplast and root enzymes differ from the endosperm enzyme on polyacrylamide gel electrophoresis. The amination reaction of all the enzymes is affected by high salt concentrations. For the endosperm enzyme, the ratio of activity with NADH to that with NADPH is 6.3 at 250 millimolar NH(4)Cl and 1.5 at 12.5 millimolar NH(4)Cl. K(m) values for NH(4) (+) and NAD(P)H are reduced at low salt concentrations. The low K(m) values for the nucleotides may favor a role for glutamate dehydrogenase in ammonia assimilation in some situations.

Hexokinase from Maize Endosperm and Scutellum

Hexokinase (EC 2.7.1.1) was isolated from endosperm and scutellum of developing and germinating maize (Zea mays) seeds. With fructose as the variable substate, Michaelis constant values for the scutellum enzyme were about onethird those of the endosperm enzyme (0.05 versus 0.15 mm), and no developmental differences were observed. With glucose as the variable substrate, Michaelis constant values were all in the range 0.1 to 0.2 mm. The enzyme preparation from germinating scutellum was studied further; when glucose was varied over a wide range, a Michaelis constant of 3.4 mm was observed in addition to the much lower Michaelis constant noted above. This low affinity binding of glucose may have regulatory significance and may indicate the presence of a glucokinase in addition to hexokinase.

A Unique Adenosine Diphosphoglucose Pyrophosphorylase Associated with Maize Embryo Tissue

A comparison of heat stabilities and various kinetic properties between the adenosine diphosphoglucose pyrophosphorylases isolated from endosperm and embryo tissues from starchy maize seeds indicates that the adenosine diphosphoglucose pyrophosphorylase associated with the embryo is distinct from the enzyme isolated from the endosperm. The embryo enzyme is more stable to incubation for 5 minutes at 60 C while the endosperm enzyme is labile to this treatment. Both enzymes are activated by glycerate-3-P. The embryo enzyme is more sensitive to inhibition by phosphate than is the endosperm enzyme. Glycerate-3-P, which reverses the inhibition of the endosperm enzyme by phosphate, has little effect on the phosphate inhibition of the embryo enzyme. Other kinetic studies distinguish the two enzymes.

Identification and genetic control of starch-degrading enzymes in maize endosperm.

Three starch-degrading enzymes from liquid endosperm of maize have been separated by means of horizontal acrylamide gel electrophoresis. The three enzymes are tentatively identified as α-amylase (zone 1), β-amylase (zone 2), and α-glucan phosphorylase (zone 3). Electrophoretic variants of these enzymes were found among ten inbred strains examined. Results of genetic crosses with respect to zone 2 amylase show that it is controlled by a pair of alleles (Amy-2 A and Amy-2 B) acting without dominance. It further appears that Amy-2 and Ct (catalase) are linked with 5% recombination frequency.