Immunochemical Studies on Arachis hypogaea Proteins With Particular Reference to the Reserve Proteins. I. Characterization, Distribution, and Properties of alpha-Arachin and alpha-Conarachin

Fourteen antigenic constituents have been detected in Arachis hypogaea seeds. The major proteins of the classic arachin and conarachin fractions have been identified. Arachin contains 4 antigens (the major one called α-arachin) and conarachin contains 2 which have been called α₁, and α₂-conarachin. Structural differences between α-arachin, α₁ and α₂-conarachin are indicated by their different antigenic specificities. α-Arachin precipitates as a separate entity at low temperature. The action of trypsin on this protein induces an increase in electrophoretic mobility and prevents precipitation at low temperature. This enzyme has no detectable effect on α₁ and α₂-conarachin.     

beta-Amylases in Cereals : A Study of the Maize beta-Amylase System

β-Amylase of maize (Zea mays L.) caryopses was studied during development and germination by means of enzymic, electrophoretic, and immunochemical techniques. β-Amylase activity increased during caryopsis development to a maximum value at the beginning of the water content plateau (at this stage the enzyme was located primarily within the pericarp) and then decreased. Almost no β-amylase (activity or antigen) was found in either free or bound forms in the mature maize caryopsis. The activity increased again during seedling growth and reached much higher values. Both the aleurone layer (to a major extent) and the scutellum produced and secreted β-amylase during germination, the secretion being stimulated by Ca²⁺. No posttranslational modification of the enzyme was detected during germination. The molecular specific activity of the enzyme remained unchanged during the observed periods, indicating that the regulation of the activity is based essentially on protein turnover. The enzyme from developing and germinating caryopses was found to be identical in terms of antigenicity, isoelectric point, and molecular mass to the β-amylases extracted from the roots and the leaves of the maize seedling. The maize β-amylase resembles in all respects the ubiquitous β-amylase described for rye and wheat, whereas the major β-amylase of those cereals appears to be lacking in the maize caryopsis.     

The Guanine Nucleotide Exchange Factor RIC8 Regulates Conidial Germination through Gα Proteins in Neurospora crassa

Heterotrimeric G protein signaling is essential for normal hyphal growth in the filamentous fungus Neurospora crassa. We have previously demonstrated that the non-receptor guanine nucleotide exchange factor RIC8 acts upstream of the Gα proteins GNA-1 and GNA-3 to regulate hyphal extension. Here we demonstrate that regulation of hyphal extension results at least in part, from an important role in control of asexual spore (conidia) germination. Loss of GNA-3 leads to a drastic reduction in conidial germination, which is exacerbated in the absence of GNA-1. Mutation of RIC8 leads to a reduction in germination similar to that in the Δgna-1, Δgna-3 double mutant, suggesting that RIC8 regulates conidial germination through both GNA-1 and GNA-3. Support for a more significant role for GNA-3 is indicated by the observation that expression of a GTPase-deficient, constitutively active gna-3 allele in the Δric8 mutant leads to a significant increase in conidial germination. Localization of the three Gα proteins during conidial germination was probed through analysis of cells expressing fluorescently tagged proteins. Functional TagRFP fusions of each of the three Gα subunits were constructed through insertion of TagRFP in a conserved loop region of the Gα subunits. The results demonstrated that GNA-1 localizes to the plasma membrane and vacuoles, and also to septa throughout conidial germination. GNA-2 and GNA-3 localize to both the plasma membrane and vacuoles during early germination, but are then found in intracellular vacuoles later during hyphal outgrowth.     

Amylase synthesis and stability in crested wheatgrass seeds at low water potentials

Drying of seeds of Agropyron desertorum (Fisch. ex Link) Schult. did not result in breakdown of α-amylase nor impair the ability of seeds to resume its synthesis when moistened again. β-Amylase activity did not change during 5 days of germination at a water potential of 0 atmosphere nor during 40 days of incubation at −40 atmospheres. Seeds synthesized α-amylase at 0, −20, and −40 atmospheres, but not at −60 atmospheres. At 0 and −20 atmospheres, the log of α-amylase activity was linearly related to hastening of germination. But at −40 atmospheres, seeds synthesized α-amylase during a time when there was little hastening of germination. Thus, it appears that other biochemical reactions are less drought-tolerant than synthesis of α-amylase. It is concluded that inhibition of α-amylase synthesis is not a controlling factor in the germination of these seeds at low water potentials.     

Absence of Transient Elevated UV Resistance during Germination of Bacillus subtilis Spores Lacking Small, Acid-Soluble Spore Proteins α and β

Germinating spores of Bacillus subtilis mutants which lack small, acid-soluble spore proteins α and β did not exhibit the transient elevated UV resistance seen during germination of wild-type spores.     

Physicochemical studies on the proteins of the peanut cotyledon and embryonic axis

Subcellular fractions from the cotyledon obtained by differential and density gradient centrifugation, and extracts of total proteins from both cotyledon and axial tissues were analyzed by diethylaminoethyl cellulose chromatography, zone electrophoresis, ultracentrifugation, immunodiffusion, and immunoelectrophoresis. Fractionation and characterization of proteins in subcellular organelles of the peanut reaffirm that α-arachin is located in the protein bodies of the cells. Results obtained by diethylaminoethyl cellulose chromatography of subcellular fractions suggest that some of the conarachin proteins are cytoplasmic. α₁-Conarachin is cytoplasmic, and α₂-conarachin is particle-bound. α-Arachin and α₂-conarachin predominate in the cotyledon. Quantitative differences for other proteins were also observed. Although qualitative similarities are apparent by immunoelectrophoresis, major differences were observed in the sedimentation patterns, zone electrophoreograms, and in the diethylaminoethyl cellulose chromatograms of total protein extracts from the cotyledon and the axis.     

Enzymic mechanism of starch breakdown in germinating rice seeds : 15. Immunochemical study on multiple forms of amylase

The formation of multiple forms of amylases in germinating rice (Oryza sativa L. cv Kimmaze) grains was examined by means of isoelectric focusing, cross-immunoelectrophoresis, and rocket-line immunoelectrophoresis followed by a reaction of enzymic characterization by using β-limit dextrin or starch as substrate. The constituents detected by isoelectric focusing were identified as three electrophoretically heterogeneous antigens. The major α-amylase bands A and B corresponded to a same antigen, the main portion of which was produced within 2 days' germination. The bulk of α-amylase D appeared between 2 and 4 days' germination. Component E, a debranching enzyme according to its action on the β-limit dextrin, already exists in the ungerminated seeds; its amount decreases within the first 2 days of germination and increases again thereafter.

Evidence showing that β-amylase (band C) is produced by the scutellum at an early stage of germination was provided. The enzyme appeared in a suspension of the scutellum after a prolonged incubation.

     

Small, Acid-Soluble Spore Proteins of the α/β Type Do Not Protect the DNA in Bacillus subtilis Spores against Base Alkylation

Ethyl methanesulfonate (EMS) killed wild-type Bacillus subtilis spores as rapidly as spores lacking small, acid-soluble proteins (SASP) of the α/β type (α⁻β⁻ spores), and 20% of the survivors had obvious mutations. A recA mutation increased the EMS sensitivity of wild-type and α⁻β⁻ spores similarly but reduced their mutagenesis; EMS treatment of dormant spores also resulted in the induction of RecA synthesis during spore germination. EMS generated similar levels of alkylated bases in wild-type and α⁻β⁻ spore DNAs, in purified DNA, or in DNA saturated with α/β-type SASP. Ethylene oxide (EtO) also generated similar levels of base alkylation in wild-type and α⁻β⁻ spore DNAs. These data indicate that EMS and EtO kill spores at least in part by DNA damage but that α/β-type SASP, which protect DNA against many types of damage, do not protect spore DNA from base alkylation.     

In Vitro Synthesis of the alpha and alpha' Subunits of the 7S Storage Proteins (Conglycinin) of Soybean Seeds

Messenger RNAs (mRNAs), isolated from immature soybean (Glycine max L., Merr.) seeds, that bound to oligo(dT)-cellulose were fractionated by centrifugation in sucrose density gradients containing dimethyl sulfoxide. mRNAs with sedimentation values between 21S and 25S coded for the in vitro translation of polypeptides with electrophoretic mobilities similar to those of the α′ and α subunits of the 7S seed storage protein. High pressure liquid chromatographic analyses of the trypsin-induced fragments (“column fingerprinting”) verified that the polypeptides produced in vitro were closely related to authentic α′ and α subunits.     

Independent Regulatory Aspects and Posttranslational Modifications of Two beta-Amylases of Rye : Use of a Mutant Inbred Line

We have examined the occurrence/disappearance, tissue location, and posttranslational modification of β-amylase proteins in rye (Secale cereale L.) kernels at three physiological stages (development, maturity, germination) with a normal inbred line and a mutant line exhibiting a high but incomplete β-amylase deficiency. This deficiency corresponds to a lack of accumulation of β-amylase activity in the endosperm and does not affect the level of activity in the outer pericarp and green tissues as compared to the normal line. Two antigenically related but distinct β-amylases (I and II) were detected in the normal line (II being the major constituent) and only one (I) in the mutant line. I and II display very similar electrophoretic polymorphism. In both lines, I appears to be ubiquitous, although it disappears from the outer pericarp during ripening. Antigen II was present only in the normal line and appears to be specific for the endosperm and perhaps for the maternal green tissues of the seed. Posttranslational modifications occurring during germination, which are mimicked by the action of papain, affect II but not I. The two groups of β-amylases are discussed in relation to recent reports indicating the presence of two types of β-amylase with different functions and gene loci in barley and wheat.     

Occurrence of alpha-amylase in the axis of germinating peas

α-Amylase was found in the axis portion of ungerminated pea seeds (Pisum sativum var. Alaska). The occurrence of this enzyme was demonstrated with crude homogenates (also containing β-amylase) using three different methods: the hydrolysis of β-limit dextrin, the change in absorption spectra for the iodine-starch complex, and the increase in reducing materials relative to the decrease in starch. The first method was used to quantitate the changes in α-amylase activity during germination. The increase in total amylase activity (primarily β-amylase) paralleled germination; the accumulation of α-amylase activity was not initiated for an additional day. The increased α-amylase activity was related to epicotyl growth. Approximately half of this activity was found in the etiolated stem, the distribution being higher in growing than in nongrowing portions.     

Changes in the Activity of Some Hydrolases, Peroxidase, and Catalase in the Rice Seed during Germination

A study was made of the changes in activity of enzymes involved in the breakdown of stored phytin, lipid, and hemicellulose in the aleurone layer of rice seed (Oryza sativa L., variety IR8) during the 1st week of germination in the light. Enzyme assays were made on crude extracts from degermed seed, and activities were expressed on a per seed basis. Phytase activity increased within the 1st day of germination. The increase in activity of most other enzymes—phosphomonoesterase, phosphodiesterase, esterase, lipase, peroxidase, catalase, β-glucosidase, and α- and β-galactosidase—closely followed the increase in protein content. Their peak activities occurred by the 5th to the 7th day. Some enzymes, such as β-1, 3-glucanase and α-amylase, continued to increase in activity after the 7th day. Phytase, β-1, 3-glucanase, and α-amylase followed a similar sequence of production in embryoless seed halves incubated in 0.12 μM gibberellin A₃, but the production of lipase was delayed.     

Selection for low dormancy in annual ryegrass (Lolium rigidum) seeds results in high constitutive expression of a glucose-responsive α-amylase isoform

Background and Aims

α-Amylase in grass caryopses (seeds) is usually expressed upon commencement of germination and is rarely seen in dry, mature seeds. A heat-stable α-amylase activity was unexpectedly selected for expression in dry annual ryegrass (Lolium rigidum) seeds during targeted selection for low primary dormancy. The aim of this study was to characterize this constitutive activity biochemically and determine if its presence conferred insensitivity to the germination inhibitors abscisic acid and benzoxazolinone.

Methods

α-Amylase activity in developing, mature and germinating seeds from the selected (low-dormancy) and a field-collected (dormant) population was characterized by native activity PAGE. The response of seed germination and α-amylase activity to abscisic acid and benzoxazolinone was assessed. Using an alginate affinity matrix, α-amylase was purified from dry and germinating seeds for analysis of its enzymatic properties.

Key Results

The constitutive α-amylase activity appeared late during seed development and was mainly localized in the aleurone; in germinating seeds, this activity was responsive to both glucose and gibberellin. It migrated differently on native PAGE compared with the major activities in germinating seeds of the dormant population, but the enzymatic properties of α-amylase purified from the low-dormancy and dormant seeds were largely indistinguishable. Seed imbibition on benzoxazolinone had little effect on the low-dormancy seeds but greatly inhibited germination and α-amylase activity in the dormant population.

Conclusions

The constitutive α-amylase activity in annual ryegrass seeds selected for low dormancy is electrophoretically different from that in germinating seeds and its presence confers insensitivity to benzoxazolinone. The concurrent selection of low dormancy and constitutive α-amylase activity may help to enhance seedling establishment under competitive conditions.     

Secretion of alpha-amylase by the aleurone layer and the scutellum of germinating barley grain

α-Amylase activities in extracts of different parts of barley grain (Hordeum vulgare L. cv Himalaya) were low after 1 day of germination at 20°C, but they began to increase afterwards. In the scutellum and the aleurone layer, the increases were small, but in the starchy endosperm a great increase took place between days 1 and 6.

When the aleurone layers were separated from germinating whole grains and incubated in 10 millimolar CaCl₂, the α-amylase activity in the medium increased linearly for about 30 to 60 minutes, indicating secretion. The activity inside the aleurone layer decreased only slightly during the incubation, indicating that secretion of α-amylase was accompanied by synthesis. The rates of secretion in vitro by the aleurone layers separated at different stages of germination corresponded rather well to the rate of accumulation of α-amylase activity in the starchy endosperm in a whole grain.

Scutella separated after 1 day of germination released small amounts of α-amylase activity into 10 millimolar CaCl₂. This release was linear for at least 1 hour and did not occur at 0°C; it is therefore likely to be due to secretion. At later stages of germination, the secretion by the scutella was slower than at day 1 and the total secretion accounted for only 5 to 10% of the increase of α-amylase activity in the starchy endosperm in a whole grain.

Since the times from the separation of the parts of the grain to the beginning of the secretion assay (10-40 minutes) as well as the duration of the assay itself (20-60 minutes) were short, the rates of secretion by the separated grain parts are likely to represent those in an intact grain. The results indicate therefore that at least in the conditions used the bulk of the total α-amylase in the starchy endosperm is secreted by the aleurone layer, the contribution by the scutellum being only 5 to 10% of the total activity.

     

Mechanical Resistance in Unstructured Proteins

Single-molecule pulling experiments on unstructured proteins linked to neurodegenerative diseases have measured rupture forces comparable to those for stable folded proteins. To investigate the structural mechanisms of this unexpected force resistance, we perform pulling simulations of the amyloid β-peptide (Aβ) and α-synuclein (αS), starting from simulated conformational ensembles for the free monomers. For both proteins, the simulations yield a set of rupture events that agree well with the experimental data. By analyzing the conformations occurring shortly before rupture in each event, we find that the mechanically resistant structures share a common architecture, with similarities to the folds adopted by Aβ and αS in amyloid fibrils. The disease-linked Arctic mutation of Aβ is found to increase the occurrence of highly force-resistant structures. Our study suggests that the high rupture forces observed in Aβ and αS pulling experiments are caused by structures that might have a key role in amyloid formation.     

Isoform-selective Oligomer Formation of Saccharomyces cerevisiae p24 Family Proteins

p24 family proteins are evolutionarily conserved transmembrane proteins involved in the early secretory pathway. Saccharomyces cerevisiae has 8 known p24 proteins that are classified into four subfamilies (p24α, -β, -γ, and -δ). Emp24 and Erv25 are the sole members of p24β and -δ, respectively, and deletion of either destabilizes the remaining p24 proteins, resulting in p24 null phenotype (p24Δ). We studied genetic and physical interactions of p24α (Erp1, -5, and -6) and γ (Erp2, -3, and -4). Deletion of the major p24α (Erp1) partially inhibited p24 activity as reported previously. A second mutation in either Erp5 or Erp6 aggravated the erp1Δ phenotype, and the triple mutation gave a full p24Δ phenotype. Similar genetic interactions were observed among the major p24γ (Erp2) and the other two γ members. All the p24α/γ isoforms interacted with both p24β and -δ. Interaction between p24β and -δ was isoform-selective, and five major α/γ pairs were detected. These results suggest that the yeast p24 proteins form functionally redundant αβγδ complexes. We also identified Rrt6 as a novel p24δ isoform. Rrt6 shows only limited sequence identity (∼15%) to known p24 proteins but was found to have structural properties characteristic of p24. Rrt6 was induced when cells were grown on glycerol and form an additional αβγδ complex with Erp3, Erp5, and Emp24. This complex was mainly localized to the Golgi, whereas the p24 complex containing Erv25, instead of Rrt6 but otherwise with the same isoform composition, was found mostly in the ER.     

Control of alpha-Amylase Development in Cotyledons during and following Germination of Mung Bean Seeds

Developmental patterns of α-amylase in Vigna radiata cotyledons during and following germination were quite different depending on the differences in the treatments of cotyledons during the imbibitional stage. When axis-detached cotyledons were imbibed in water with seed-coats attached, α-amylase activity did not increase and remained low. On the other hand, when the cotyledons were imbibed in water after seed-coat removal, the enzyme activity increased markedly. If the axis was attached to the cotyledons, α-amylase showed a marked development even under the former imbibition conditions. These changes in the enzyme activity were in parallel with those in the enzyme content, and the content, in turn, was dependent upon the availability of mRNA for α-amylase. We propose that the regulation of the development of α-amylase in cotyledons may involve some factor(s) inhibitory to accumulation of α-amylase mRNA, which is present in dry cotyledons and can be removed from cotyledons by leakage or by the presence of the axis.     

The Unique Biosynthetic Route from Lupinus β-Conglutin Gene to Blad

Background

During seed germination, β-conglutin undergoes a major cycle of limited proteolysis in which many of its constituent subunits are processed into a 20 kDa polypeptide termed blad. Blad is the main component of a glycooligomer, accumulating exclusively in the cotyledons of Lupinus species, between days 4 and 12 after the onset of germination.

Principal Findings

The sequence of the gene encoding β-conglutin precursor (1791 nucleotides) is reported. This gene, which shares 44 to 57% similarity and 20 to 37% identity with other vicilin-like protein genes, includes several features in common with these globulins, but also specific hallmarks. Most notable is the presence of an ubiquitin interacting motif (UIM), which possibly links the unique catabolic route of β-conglutin to the ubiquitin/proteasome proteolytic pathway.

Significance

Blad forms through a unique route from and is a stable intermediary product of its precursor, β-conglutin, the major Lupinus seed storage protein. It is composed of 173 amino acid residues, is encoded by an intron-containing, internal fragment of the gene that codes for β-conglutin precursor (nucleotides 394 to 913) and exhibits an isoelectric point of 9.6 and a molecular mass of 20,404.85 Da. Consistent with its role as a storage protein, blad contains an extremely high proportion of the nitrogen-rich amino acids.