ABSTRACT
Steroidal glycoalkaloids (SGAs) such as α-solanine found in solanaceous food plants--as, for example, potato--are antinutritional factors for humans. Comparative coexpression analysis between tomato and potato coupled with chemical profiling revealed an array of 10 genes that partake in SGA biosynthesis. We discovered that six of them exist as a cluster on chromosome 7, whereas an additional two are adjacent in a duplicated genomic region on chromosome 12. Following systematic functional analysis, we suggest a revised SGA biosynthetic pathway starting from cholesterol up to the tetrasaccharide moiety linked to the tomato SGA aglycone. Silencing GLYCOALKALOID METABOLISM 4 prevented accumulation of SGAs in potato tubers and tomato fruit. This may provide a means for removal of unsafe, antinutritional substances present in these widely used food crops.
Subject(s)
Crops, Agricultural/genetics , Multigene Family , Nutritive Value/genetics , Solanaceous Alkaloids/biosynthesis , Solanaceous Alkaloids/genetics , Solanum lycopersicum/genetics , Solanum tuberosum/genetics , Gene Expression Profiling , Gene Expression Regulation, Plant , Gene Silencing , Genes, Plant , Solanaceous Alkaloids/toxicityABSTRACT
The mustard trypsin inhibitor MTI-2 is a potential tool in the study of interactions between pest insects and plants. It can be applied to study the adaptations of digestive proteases in pest insects. Phage display allows a rapid and exhaustive system for the selection of heterologous protein variants with novel specificities. Here we describe a bacteriophage expression system which permits functional expression of MTI-2 variants. Active and inactive mutants of MTI-2 are constructed and displayed on phage. These are used to demonstrate that an active variant can be selected from a background of 10,000 inactive mutants in four rounds of selection and amplification.
Subject(s)
Mustard Plant/genetics , Plant Proteins/genetics , Plants, Medicinal , Trypsin Inhibitors/genetics , Animals , Base Sequence , DNA Primers/genetics , Escherichia coli/genetics , Gene Expression , Genetic Variation , Peptide Library , Pichia/genetics , Plant Proteins/metabolism , Protein Engineering , Trypsin Inhibitors/metabolismABSTRACT
Potato proteinase inhibitor II (PI-2) is composed of two sequence repeats. It contains two reactive site domains. We developed an improved protocol for the production of PI-2 using the yeast Pichia pastoris as the expression host. We then assessed the role of its two reactive sites in the inhibition of trypsin and chymotrypsin by mutating each of the two reactive sites in various ways. From these studies it appears that the second reactive site strongly inhibits both trypsin (Ki = 0.4 nM) and chymotrypsin (Ki = 0.9 nM), and is quite robust towards mutations at positions P2 or P1'. In contrast, the first reactive site inhibits only chymotrypsin (Ki = 2 nM), and this activity is very sensitive to mutations. Remarkably, replacing the reactive site amino acids of domain I with those of domain II did not result in inhibitory activities similar to domain II. The fitness for protein engineering of each domain is discussed.