Pilar Lara

Plant proteinaceous inhibitors of hydrolases from heterologous systems have been implied in plant defense and have been extensively studied at the molecular level. In wheat and barley, a substantial fraction of the total endosperm protein content is represented by toxins and inhibitors that are active against heterologous systems. More than 20 different members from a single multigene family of alpha-amylase/trypsin inhibitors, specifically expressed in endosperm, have been characterised. Their apparent molecular weigth are in the 12-16,000 range. The alpha-amylase inhibitors can be classified, according to their degree of aggregation into monomeric (as the trypsin inhibitors), dimeric and tetrameric forms. During kernel development, their synthesis precedes that of the storage proteins and they are rapidly degraded upon germination. Genes encoding these inhibitors are scattered on the Triticeae chromosomes: at least five out of the seven homeologous groups carry genes encoding different family members. Structure-Functional relationships have been pursued through site-directed mutagenesis of the wheat monomeric alpha-amylase inhibitor. In this molecule, both the carboxy- and amino-terminal ends, that are proximal in the 3D-structure due to the position of the disulphide-bridges, have been shown to be important for activity against the Tenebrio molitor amylase. NMR-structural studies in the finger-millet bifunctional inhibitor have shown that the reactive-site for trypsin is located in a loop opposite to the reactive site for alpha-amylase (in the C- and Nterminal ends of the molecule), strongly suggesting that the activity of these inhibitors can be improved by domain-swapping among different members of the family. More recently, transgenic tobacco and wheat plants have being obtained, expressing the gene encoding barley trypsin inhibitor BTI-Cme, and these where shown to be more resistant to lepidopterous Agrotis ipsilon and Sitotroga cerealella pests, respectively, than non-transformed controls. Preliminary data with other transgenically expressed members of the family confirmed the potential of these inhibitors for increasing insect resistance in genetically modified plants (GMP). This methodology is aimed at complementing an integrated pest management system, by amplifying the natural variability outside the species barrier, in a more environmental friendly Agriculture less dependant on chemical pesticides

Plant proteinaceous inhibitors of hydrolases from heterologous systems have been implied in plant defense and have been extensively studied at the molecular level. In wheat and barley, a substantial fraction of the total endosperm protein content is represented by toxins and inhibitors that are active against heterologous systems. More than 20 different members from a single multigene family of alpha-amylase/trypsin inhibitors, specifically expressed in endosperm, have been characterised. Their apparent molecular weigth are in the 12-16, 000 range. The alpha-amylase inhibitors can be classified, according to their degree of aggregation into monomeric (as the trypsin inhibitors), dimeric and tetrameric forms. During kernel development, their synthesis precedes that of the storage proteins and they are rapidly degraded upon germination. Genes encoding these inhibitors are scattered on the Triticeae chromosomes: at least five out of the seven homeologous groups carry genes encoding different family members. Structure-Functional relationships have been pursued through site-directed mutagenesis of the wheat monomeric alpha-amylase inhibitor. In this molecule, both the carboxy- and amino-terminal ends, that are proximal in the 3D-structure due to the position of the disulphide-bridges, have been shown to be important for activity against the Tenebrio molitor amylase. NMR-structural studies in the finger-millet bifunctional inhibitor have shown that the reactive-site for trypsin is located in a loop opposite to the reactive site for alpha-amylase (in the C- and Nterminal ends of the molecule), strongly suggesting that the activity of these inhibitors can be improved by domain-swapping among different members of the family. More recently, transgenic tobacco and wheat plants have being obtained, expressing the gene encoding barley trypsin inhibitor BTI-Cme, and these where shown to be more resistant to lepidopterous Agrotis ipsilon and Sitotroga cerealella pests, respectively, than non-transformed controls. Preliminary data with other transgenically expressed members of the family confirmed the potential of these inhibitors for increasing insect resistance in genetically modified plants (GMP). This methodology is aimed at complementing an integrated pest management system, by amplifying the natural variability outside the species barrier, in a more environmental friendly Agriculture less dependant on chemical pesticides.