Studies on the biochemistry and physiology of root-specific ribosome-inactivating proteins (RIPs) in Mirabilis expansa and related species

Mirabilis expansa, a relatively disease-resistant Andean root crop that produces edible storage roots, is one of the most neglected Andean root and tuber crops. The cultivation of this crop has been restricted to three small regions in South America, and its basic biology and agronomy are virtually unknown. To address this lack of biological information, my studies focused on the isolation and characterization of biologically active proteins from the storage roots of this underutilized root crop. Two novel type I ribosome inactivating proteins (RIPs), named ME1 and ME2, were isolated and purified by perfusion and reverse phase chromatography from the storage roots of M. expansa. The two proteins were found to be similar in size (27 and 27.5 kD) by sodium dodecyl sulfato-polyacrylamide gel electrophoresis, and their isoelectric points were determined to be greater than pH 10. Amino acid N-terminal sequencing revealed that both ME1 and ME2 had conserved residues characteristic of RIPs. Amino acid composition and Western blot analysis further suggested a structural similarity between ME1 and ME2.; Western blots and tissue prints using polyclonal monospecific antibodies against ME1/ME2 showed that the expression of RIPs occur only in the storage and primary roots. In both cases, ME1 and ME2 accounted for a major fraction (20%) of the total soluble protein. Tissue prints of root cross sections showed the expression of ME1/ME2 at the periphery of young storage root tissue. As the storage root tissues matured, RIPs accumulated throughout the storage parenchyma. Expression of ME1/ME2 was widespread in the primary roots. ME1 and ME2 were immunolocalized in the amyloplasts (storage and primary roots), cell walls (primary roots) and vascular tissue (storage and primary roots) of M. expansa. Based on their pattern of accumulation and abundance, it was originally proposed that ME1/ME2 could play a role as storage proteins.; The presence of similar RIPs was detected in the storage roots of M. jalapa, M. multiflora and M. longiflora using polyclonal monospecific antibodies against both ME1 and ME2. Intracellular RIPs were found in Agrobacterium-transformed root cultures of M. expansa and M. jalapa. In addition, an extracellular 24 kD RIP was found in the root culture medium of M. expansa. Constitutive RIP production was not detected in root cultures of M. longiflora. However, elicitation with cell wall preparations from Pythium ultimum, P. aphanidermatum, Phytophthora drechsleri, P. cinnamomi and Fusarium oxysporum induced the expression of a 24 kD protein that reacted with ME1/ME2 antibodies. (Abstract shortened by UMI.)...