Molecular characterization of a bean α-amylase inhibitor that inhibits the α-amylase of the Mexican bean weevil Zabrotes subfasciatus

Cultivated varieties of the common bean (Phaseolus vulgaris L.) contain an α-amylase inhibitor (αAI-1) that inhibits porcine pancreatic α-amylase (PPA; EC 3.2.1.1) and the amylases of certain seed weevils, but not that of the Mexican bean weevil, Zabrotes subfasciatus. A variant of αAI-1, called αAI-2, is found in certain arcelin-containing wild accessions of the common bean. The variant αAI-2 inhibits Z. subfasciatus α-amylase (ZSA), but not PPA. We purified αAI-2 and studied its interaction with ZSA. The formation of the αAI-2-ZSA complex is time-dependent and occurs maximally at pH 5.0 or below. When a previously isolated cDNA assumed to encode αAI-2 was expressed in transgenic tobacco seeds, the seeds contained inhibitory activity toward ZSA but not toward PPA, confirming that the cDNA encodes αAI-2. The inhibitors αAI-1 and αAI-2 share 78% sequence identity at the amino acid level and they differ in an important region that is part of the site where the enzyme binds the inhibitor. The swap of a tripeptide in this region was not sufficient to change the specificity of the two inhibitors towards their respective enzymes. The three-dimensional structure of the αAI-1/PPA complex has just been solved and we recently obtained the derived amino acid sequence of ZSA. This additional information allows us to discuss the results described here in the framework of the amino acid residues of both proteins involved in the formation of the enzyme-inhibitor complex and to pinpoint the amino acids responsible for the specificity of the interaction. Received: 14 April 1997 / Accepted: 10 May 1997     

Hydroxyproline-rich cell wall protein (extensin): role in the cessation of elongation in excised pea epicotyls

The synthesis and accumulation of cell wall hydroxyproline increases coincident with the cessation of elongation growth in pea epicotyls. We examined the relationship between these biochemical and physiological events by using epicotyl sections challenged with α, α′-dipyridyl. This chelator blocked hydroxyproline biosynthesis without affecting overall protein synthesis. Epicotyl sections mimicked elongation growth in situ when placed in indoleacetic acid. Elongation was blocked by the addition of benzimidazole or Ethrel. These latter compounds acted independently as judged by their kinetics of action and the inhibition of Ethrel's effect only by CO₂.During rapid elongation growth in indoleacetic acid, there was no increase in cell wall hydroxyproline. However, incubation in either growth-inhibitory agent increased hydroxyproline 3-fold. When this increase was blocked by dipyridyl incubation, growth was not inhibited in benzimidazole or Ethrel, but proceeded at the maximal rate. During long-term incubations in buffer, cell wall hydroxyproline increased and the sections eventually became unable to grow. However, if dipyridyl was added to block the hydroxyproline increase, growth potential remained. Elongation was inhibited by supraoptimal concentrations of indoleacetic acid. However, such inhibition did not occur in the presence of dipyridyl.These results indicate that an hydroxyproline-containing component is necessary in rendering the cell wall inextensible when elongation growth ceases.     

Synthesis and Secretion of Hypoxyproline-containing Macromolecules in Carrots: III. Metabolic Requirements for Secretion

Using pulse-chase experiments with radioactive proline, it is possible to study the rapid transfer from the cytoplasm to the cell wall of the hydroxyproline-rich protein found in the cell walls of higher plants. The secretion of this protein is not obligatorily coupled to protein synthesis. Secretion is completely inhibited by uncouplers of oxidative phosphorylation and strongly inhibited by the inhibitors of electron transport, cyanide and azide. It is concluded that the transfer of proteins from the cytoplasm to the cell wall is an energy-requiring step.     

Synthesis and release of sucrose by the aleurone layer of barley: Regulation by gibberellic acid

Summary Aleurone layers of barley contain large amounts of a soluble oligosaccharide which was identified as sucrose (30–40 g/mg fresh weight). Treatment of the layers with gibberellic acid (GA₃) causes the release of sucrose from the cells. This release requires the participation of metabolic processes, including protein synthesis. When embryoless half-seeds are incubated sucrose accumulates in the aleurone layers, but when seeds are germinated the sucrose content of the aleurone layers declines. Labeling experiments with radioactive glucose and fructose show that aleurone layers continuously synthesize sucrose and that the release, but not the synthesis of sucrose is enhanced by GA₃.     

Ground wētā in vines of the Awatere Valley,Marlborough: biology,density and distribution

The endemic New Zealand ground wētā (Hemiandrus sp. ‘promontorius’) has a Naturally Uncommon conservation status. This is because of the paucity of information on its density and distribution. Here, the biology, density and distribution of a population of this wētā found in and around vineyards in the Awatere Valley, Marlborough was studied. Wētā density was assessed in vineyards, paddocks and shrublands in this valley. Soil moisture, penetration resistance, pH and organic matter were recorded at locations with and without wētā. Wētā density in vineyards was significantly higher than in either paddocks or shrub habitats. In vineyards, the density of this insect was significantly higher under-vines than in the inter-rows. Higher numbers of this wētā were found in moist soils that required lower force to burrow. Females laid an average of 55 eggs between March and April, which hatched in September. These findings highlight the intersection between agriculture and conservation.     

Isolation and partial characterisation of galactose-specific lectins from African yam beans, Sphenostyles stenocarpa Harms.

A new galactose-specific lectin was isolated from African yam bean (Sphenostyles stenocarpa Harms) by affinity chromatography on galactose-Sepharose 4B. SDS-PAGE analysis resulted in four polypeptide bands of approximately 27, 29, 32 and 34 kDa, respectively. Based on the analysis of carbohydrate content and native PAGE, it is likely that the Sphenostyles lectin is a tetrameric glycoprotein with M(r) of approximately 122 kDa. N-terminal protein sequencing of purified lectins from four different Sphenostyles accessions shows that the four polypeptides have largely identical amino acid sequences. The sequences contain the conserved consensus sequence F-F-LILG characteristic of legume lectins, as well as Phaseolus vulgaris proteins in the arcelin-alpha-amylase inhibitor gene family. The lectin agglutinates both rabbit and human erythrocytes, but with a preference for blood types A and O. Using Western blotting, the lectin was shown to accumulate rapidly during seed development, but levels dropped slightly as seeds attained maturity. This is the first time a lectin has been purified from the genus Sphenostyles. The new lectin was assigned the abbreviation LECp.SphSte.se.Hga1.     

Overexpression of a Gene That Encodes the First Enzyme in the Biosynthesis of Asparagine-Linked Glycans Makes Plants Resistant to Tunicamycin and Obviates the Tunicamycin-Induced Unfolded Protein Response

The cytotoxic drug tunicamycin kills cells because it is a specific inhibitor of UDP-N-acetylglucosamine:dolichol phosphate N-acetylglucosamine-1-P transferase (GPT), an enzyme that catalyzes the initial step of the biosynthesis of dolichol-linked oligosaccharides. In the presence of tunicamycin, asparagine-linked glycoproteins made in the endoplasmic reticulum are not glycosylated with N-linked glycans, and therefore may not fold correctly. Such proteins may be targeted for breakdown. Cells that are treated with tunicamycin normally experience an unfolded protein response and induce genes that encode endoplasmic reticulum chaperones such as the binding protein (BiP). We isolated a cDNA clone for Arabidopsis GPT and overexpressed it in Arabidopsis. The transgenic plants have a 10-fold higher level of GPT activity and are resistant to 1 microg/mL tunicamycin, a concentration that kills control plants. Transgenic plants grown in the presence of tunicamycin have N-glycosylated proteins and the drug does not induce BiP mRNA levels as it does in control plants. BiP mRNA levels are highly induced in both control and GPT-expressing plants by azetidine-2-carboxylate. These observations suggest that excess GPT activity obviates the normal unfolded protein response that cells experience when exposed to tunicamycin.