No abstract

Two ␣-amylase inhibitors, called ␣AI-1 and ␣AI-2, that share 78% amino acid sequence identity and have a differential specificity toward mammalian and insect ␣-amylases are present in different accessions of the common bean (Phaseolus vulgaris). Using greenhouse-grown transgenic peas (Pisum sativum), we have shown previously that expression of ␣AI-1 in pea seeds can provide complete protection against the pea weevil (Bruchus pisorum). Here, we report that ␣AI-1 also protects peas from the weevil under field conditions. The high degree of protection is explained by our finding that ␣AI-1 inhibits pea bruchid ␣-amylase by 80% over a broad pH range (pH 4.5-6.5). ␣AI-2, on the other hand, is a much less effective inhibitor of pea bruchid ␣-amylase, inhibiting the enzyme by only 40%, and only in the pH 4.0 -4.5 range. Nevertheless, this inhibitor was still partially effective in protecting field-grown transgenic peas against pea weevils. The primary effect of ␣AI-2 appeared to be a delay in the maturation of the larvae. This contrasts with the effect of ␣AI-1, which results in larval mortality at the first or second instar. These results are discussed in relationship to the use of amylase inhibitors with different specificities to bring about protection of crops from their insect pests or to decrease insect pest populations below the economic injury level.

The pea (Pisum sativum) is an important grain legume crop plant that has gained worldwide economic importance as a source of protein for animal and human nutrition. In addition, it has well-defined genetics, and it has been commonly used as a model plant for research in plant physiology and biochemistry. The productivity and value of peas could be greatly increased by the introduction of stably inherited traits such as pest and disease resistance, herbicide resistance, and improved protein quality. These traits are not available in natural populations of near relatives of cultivated peas, but current advances in plant genetic engineering provide a potentially powerful tool for achieving these goals by another means.The prerequisites for the transfer of foreign genes into any plant species by genetic engineering are an efficient gene delivery system, such as Agrobacterium-mediated DNA transfer, an effective selectable marker for transformed cells, and the ability to regenerate mature, fertile, transgenic plants from transformed tissue in culture.Regeneration via embryogenesis or organogenesis has been described for a variety of pea explants, e.g. from immature leaflets (Mroginski and Kartha, 1981;Rubluo et al., 1984), from cotyledonary node (Jackson and Hobbs, 1990), from hypocotyls (Nielsen et al., 1991), from embryos (Kysely et * Corresponding author; fax 61-6-246-5000. 75 1 Natali and Cavallini, 1987;Tetu et al., 1990), from various organs of seedlings (Malmberg, 1979;Hussey and Gunn, 1984;Ezhova et al., 1985), and from protoplast cultures (Jacobsen and Kysely, 1984; Puonti-Kaerlas and Eriksson, 1988; Lehminger-Mertens and Jacobsen, 1989). Agrobacterium-mediated transfonnation of various pea explants has also been reported, e.g. stem explants (Lulsdorf et al., 199 l), embryonic axis and epicotyl segments (Filippone and Lurquin, 1989;Puonti-Kaerlas et al., 1989), nodus explants (De Kathen and Jacobsen, 1990; Nauerby et al., 1991), and root explants and protoplasts (Schaerer and Pilet, 1991). Tumors were induced in young pea plants by wild-type Agrobacterium . However, no mature transgenic pea plants were regenerated from any of the above transformation systems. The only report to date of stable transformation of peas and the production of mature, flowering, transgenic pea plants is by Puonti-Kaerlas et al. (1990), who achieved regeneration by organogenesis via callus formation using a gene encoding hygromycin phosphotransferase as a selectable marker.In this paper we report the development of a routine, reliable transformation and regeneration system for peas. The procedure has been used to introduce herbicide resistance and the expression of an antibiotic resistance gene into two cultivars of peas using an Agrobacterium tumefaciens-mediated delivery system. Integration of the two traits was stable, and their frequency in the first generation progeny followed the Mendelian pattem. MATERIALS AND METHODS Plant Material and Transformation ProcedurePea (Pisum sativum L.) cv Greenfeast and cv Rondo were grown in t...

Bruchid larvae cause major losses of grain legume crops throughout the world. Some bruchid species, such as the cowpea weevil and the azuki bean weevil, are pests that damage stored seeds. Others, such as the pea weevil (Bruchus pisorum), attack the crop growing i n the field. We transferred the cDNA encoding the a-amylase inhibitor (a-AI) found in the seeds of the common bean (Phaseolus vulgaris) into pea (Pisum sativum) using Agrobacferium-mediated transformation. Expression was driven by the promoter of phytohemagglutinin, another bean seed protein. The a-amylase inhibitor gene was stably expressed in the transgenic pea seeds at least to the T, seed generation, and a-AI accumulated i n the seeds up to 3% of soluble protein. This level is somewhat higher than that normally found in beans, which contain 1 to 2 % a-AI. In the 1 , seed generation the development of pea weevil larvae was blocked at an early stage. Seed damage was minimal and seed yield was not significantly reduced in the transgenic plants. These results confirm the feasibility of protecting other grain legumes such as lentils, mungbean, groundnuts, and chickpeas against a variety of bruchids using the same approach. Although a-AI also inhibits human a-amylase, cooked peas should not have a negative impact on human energy metabolism.The common bean (Phaseolus vulgaris L.) contains a family of structurally related seed proteins: PHA-E and -L, arcelin, and a-AI. PHA-E and PHA-L are strong agglutinins, i.e. classical lectins that bind carbohydrate, and arcelin, which is found only in certain wild accessions of the common bean, may be a weak agglutinin (Hartweck et al., 1991). The bean a-AI has 65 to 70% amino acid sequence identity with the other three but lacks at least one of the conserved residues needed for lectin activity. Its biochemical mode of action is to form a one-to-one complex with certain amylases (for reviews, see Chrispeels and Raikhel, 1991;Rouge et al., 1993).

Gene constructs were designed to test the effect of the endoplasmic reticulum (El?)-targeting signal, KDEL, on the level of accumulation of a foreign protein in transgenic plants. The gene for the pea seed protein vicilin was modified by the addition of a sequence coding for this tetrapeptide at its carboxyl terminus. The altered gene was placed under the control of a CaMV 35s promoter and its expression in the leaves of both tobacco and lucerne (alfalfa) was compared with that of an equivalent vicilin construct lacking the KDELcoding sequence. The presence of the ER-targeting signal led to a greatly enhanced accumulation of the heterologous protein. In lucerne and tobacco leaves, the level of vicilin-KDEL protein was 20 and 100 times greater than that of the unmodified vicilin, respectively. These differences in expression level could not be explained by corresponding differences in the steadystate levels or the translatability of the mRNAs. However, when the stability of vicilin and vicilin-KDEL proteins was compared in their respective transgenic hosts, unmodified vicilin was found to be degraded with a half-life of 4.5 h while vicilin-KDEL was much more stable with a half-life of more than 48 h. lmmunogold labelling of leaf tissues from transgenic lucerne and tobacco showed the presence of vicilin associated with large aggregates within the ER lumen of vicilin-KDEL plants. No such aggregates were detected in transgenic plants expressing wild-type vicilin.It is concluded that the carboxy-terminal KDEL caused the retention of the modified vicilin in the ER, and that this retention led to the increased stability and higher level of accumulation of vicilin-KDEL in leaves of transgenic plants.

SummaryGene constructs were designed to test the effect of the endoplasmic reticulum (El?)-targeting signal, KDEL, on the level of accumulation of a foreign protein in transgenic plants. The gene for the pea seed protein vicilin was modified by the addition of a sequence coding for this tetrapeptide at its carboxyl terminus. The altered gene was placed under the control of a CaMV 35s promoter and its expression in the leaves of both tobacco and lucerne (alfalfa) was compared with that of an equivalent vicilin construct lacking the KDELcoding sequence. The presence of the ER-targeting signal led to a greatly enhanced accumulation of the heterologous protein. In lucerne and tobacco leaves, the level of vicilin-KDEL protein was 20 and 100 times greater than that of the unmodified vicilin, respectively. These differences in expression level could not be explained by corresponding differences in the steadystate levels or the translatability of the mRNAs. However, when the stability of vicilin and vicilin-KDEL proteins was compared in their respective transgenic hosts, unmodified vicilin was found to be degraded with a half-life of 4.5 h while vicilin-KDEL was much more stable with a half-life of more than 48 h. lmmunogold labelling of leaf tissues from transgenic lucerne and tobacco showed the presence of vicilin associated with large aggregates within the ER lumen of vicilin-KDEL plants. No such aggregates were detected in transgenic plants expressing wild-type vicilin.It is concluded that the carboxy-terminal KDEL caused the retention of the modified vicilin in the ER, and that this retention led to the increased stability and higher level of accumulation of vicilin-KDEL in leaves of transgenic plants.

SummaryDuring the storage phase, cotyledons of developing pea seeds are nourished by nutrients released to the seed apoplasm by their maternal seed coats. Sucrose is transported into pea cotyledons by sucrose/H + symport mediated by PsSUT1 and possibly other sucrose symporters. PsSUT1 is principally localised to plasma membranes of cotyledon epidermal and subepidermal transfer cells abutting the seed coat. We tested the hypothesis that endogenous sucrose/H + symporter(s) regulate sucrose import into developing pea cotyledons. This was done by supplementing their transport activity with a potato sucrose symporter (StSUT1), selectively expressed in cotyledon storage parenchyma cells under control of a vicilin promoter. In segregating transgenic lines, enhanced [ 14 C]sucrose in¯ux into cotyledons above wild-type levels was found to be dependent on StSUT1 expression. The transgene signi®cantly increased (approximately 2-fold) transport activity of cotyledon storage parenchyma tissues where it was selectively expressed. In contrast, sucrose in¯ux into whole cotyledons through the endogenous epidermal transfer cell pathway was increased by only 23% in cotyledons expressing the transgene. A similar response was found for rates of biomass gain by intact cotyledons and by excised cotyledons cultured on a sucrose medium. These observations demonstrate that transport activities of sucrose symporters in¯uence cotyledon growth rates. The attenuated effect of StSUT1 overexpression on sucrose and dry matter¯uxes by whole cotyledons is consistent with a large proportion of sucrose being taken up at the cotyledonary surface. This indicates that the cellular location of sucrose transporter activity plays a key role in determining rates of sucrose import into cotyledons.

Forty-five lines of peas including primitive or wild forms, field peas, and round and wrinkled garden peas, were grown under uniform conditions and the seeds examined for variation in protein characteristics likely to influence nutritional value. The characters measured were crude protein, extractable protein, globulins and albumins, the percentages of legurnin, total sulphur and protein sulphur, carbon: nitrogen and nitrogen: sulphur ratios. The extractable protein was separated quantitatively into an albumin fraction (20-35 %) and a globulin fraction (legumin and vicilin). Without exception lines high in albumin content were low in legumin content (correlation coefficient r = -0.757). As both the albumin fraction and legumin are rich in sulphur amino acids, this negative correlation has important implications for attempts through plant breeding to improve the nutritional quality of legume seed proteins, by increasing the sulphur amino acid content. Total sulphur was not correlated with any other protein character.