Donald Boulter

Legumin and vicilin, storage proteins of legume seeds

Abstract The structure, location in the seed and distribution of the storage protein of legume seeds are described. Methods which have been employed for the extraction, purification and characterisation of seed globulins are reviewed in relation to modern biochemical practice. The physical, chemical and immunological characteristics of the classical legumin and vicilin preparations from Pisum sativum are summarised and the distributions of proteins with sedimentation coefficients and/or immunological determinants similar to those of legumin and vicilin, are tabulated. The structure and composition of various purified legumin and vicilin-type proteins from a variety of legumes, are compared.

Expression of snowdrop lectin in transgenic tobacco plants results in added protection against aphids

The range of sap-sucking insect pests to which GNA, (the mannose specific lectin from snowdrops (Galanthus nivalis) has been shown to be insecticidal in artificial diets has been extended to include the peach potato aphid (Myzus persicae). A gene construct for constitutive expression of GNA from the CaMV35S gene promoter has been introduced into tobacco plants. A transgenic tobacco line which expresses high levels of GNA has been shown to have enhanced resistance toM. persicae in leaf disc and whole plant bioassays,demonstrating the potential for extending transgenic plant technology to the control of sap-sucking insect pests.

Sequences responsible for the tissue specific promoter activity of a pea legumin gene in tobacco

SummaryMaturing pea cotyledons accumulate large quantities of storage proteins at a specific time in seed development. To examine the sequences responsible for this regulated expression, a series of deletion mutants of the legA major seed storage protein gene were made and transferred to tobacco using the Bin19 disarmed Agrobacterium vector system. A promoter sequence of 97 bp including the CAAT and TATA boxes was insufficient for expression. Expression was first detected in a construct with 549 bp of upstream flanking sequence which contained the the leg box element, a 28 bp conserved sequence found in the legumintype genes of several legume species. Constructs containing-833 and-1203 bp of promoter sequence significantly increased levels of expression. All expressing constructs preserved seed specificity and temporal regulation. The results indicate that promoter sequences between positions-97 and-549 bp are responsible for promoter activity, seed specificity, and temporal regulation of the pea legA gene. Sequences between positions-549 and-1203 bp appear to function as enhancer-like elements, to increase expression.

Characterisation of the storage protein subunits synthesised in vitro by polyribosomes and RNA from developing pea (Pisum sativum L.) : II. Vicilin.

Evidence is presented to show that legumin, the major storage protein in Pisum, is synthesised in vitro by the wheat germ and reticulocyte lysate systems, from polyribosomes and mRNA isolated from developing pea seeds. While legumin isolated from mature pea seeds consists of 40,000 and 20,000 MW subunits, the in vitro legumin is synthesised as a 60,000 MW precursor consisting of covalently linked 40,000 and 20,000 MW subunits. The implications of these findings are discussed in relationship to studies with other systems.

Additive protective effects of different plant-derived insect resistance genes in transgenic tobacco plants

Abstract Plant genetic engineering has been proposed as a method of producing crop plants with enhanced resistance to insect pests. One strategy for attaining this goal is to exploit the inherent resistance shown by some plants against insect herbivores, which in some cases has been shown to involve potentially insecticidal proteins. Genes derived from higher plants encoding two such proteins, the cowpea trypsin inhibitor and the pea lectin, have been shown to enhance resistance to Heliothis virescens (tobacco budworm) when expressed in transgenic tobacco plants. Both genes were introduced together into individual plants by cross-breeding the two transgenic lines. Insect bioassays on the plants expressing both genes showed their effects on Heliothis to be additive. Thus, multigene, multimechanistic insect resistance can be built up in genetically engineered plants. Such multiple mechanisms are typical of natural insect resistance in plants and may offer an ecologically acceptable way of protecting crop plants from insect pests.

Genetic engineering of plants for insect resistance

Plants and insects have been co-evolving for at least 300 million years; during this time plant species have been under continuous selection pressure from herbivorous insect predators, and, in the absence of the ability to avoid predators by movement, have been forced to rely on physical and chemical defensive mechanisms. Physical mechanisms include the presence of hairs on the surfaces of plant tissues, spines, exuded gums and toughened surface layers, such as hardened seed coats. Chemical defensive mechanisms include all the cases where a plant metabolite present in the plant tissues is toxic to predators, or interferes with the normal growth and development of predators, or deters predators by its taste or smell (Norris and Kogan, 1980).

The purification and partial amino acid sequence of a polypeptide from the glutelin fraction of rice grains; homology to pea legumin

The glutelin fraction was extracted from grain meals of rice (Oryzea sativa) with 50 mM Tris‐HCl buffer (pH 8.8) containing 6 M urea and 10 mM 2‐mercaptoethanol. Polypeptides of glutelin were separated and purified by ion‐exchange chromatography under denaturing conditions. Analysis by two‐dimensional gel electrophoresis showed that 2 major polypeptides of the rice glutelin fraction, M r 36 000 and 22 000, were linked in disulphide bonded pairs containing one M r 36 000 and one M r 22 000 subunit. A partial amino acid sequence of the purified M r 22 000 glutelin subunit showed it to be homologous to the β‐subunit of pea legumin, a storage protein which also contains disulphide‐linked subunit pairs (M r 38 000 and M r 22 000). It is therefore proposed that the major component of rice glutelin is a legumin‐like protein.

Protein and cDNA sequences of Bowman-Birk protease inhibitors from the cowpea (Vigna unguiculata Walp.)

The protein and gene sequences of the cowpea Bowman-Birk type trypsin inhibitor which confers enhanced insect resistance to transgenic tobacco plants, and of cowpea trypsin/chymotrypsin inhibitors are presented. There are regions of high conservation and high divergence within the 5′ leader, mature protein and 3′ non-coding regions of the Bowman-Birk inhibitors and in the genes which encode them in different members of this family within the Leguminosae. The practical implications of this finding for studies on the evolution of plants and the utilization of these genes for enhancing insect resistance is discussed.

Purification and characterization of the major storage proteins of Phaseolus vulgaris seeds, and their intracellular and cotyledonary distribution

Abstract Several extraction and fractionation procedures have been employed to isolate the major storage proteins of mature seeds of Phaseolus vulgaris cv. “Seafarer”; three proteins which were soluble at pH 4,7, and one that was insoluble at that pH were identified. The characteristic subunits of the three pH 4.7 soluble proteins had MWs 50000 and 47000, 32000, and 23000 respectively; those of the pH 4.7 insoluble fractions had MW 60000 and 20000. Amino acid compositions, N -terminal amino acid residues and the presence of carbohydrate in these proteins have been determined. All these proteins occurred in the protein body fraction and their relative amounts were different in the outer and central parts of the cotyledons.

A phylogeny of higher plants based on the amino acid sequences of cytochrome c and its biological implications

Higher plant phylogenetic trees were constructed from the amino acid sequences of cytochrome c from fifteen plants using the ‘ancestral sequence’ and ‘flexible numerical’ methods. The validity of these methods is discussed and the results obtained are compared with existing phylogenies based mainly on morphological characters.