Biosynthesis of Defense-Related Proteins in Transformed Root Cultures of Trichosanthes kirilowii Maxim. var japonicum (Kitam.)

Abstract

We have established transformed ("hairy") root cultures from Trichosanthes kirilowii Maxim. var japonicum Kitam. (Cucurbitaceae) and four related species to study the biosynthesis of the ribosome-inactivating protein trichosanthin (TCN) and other rootspecific defense-related plant proteins. Stable, fast-growing root clones were obtained for each species by infecting in vitro grown plantlets with Agrobacferium rhizogenes American Type Culture Collection strain 15834. Each species accumulated reproducibly a discrete protein pattern in the culture medium. Analysis of the extracellular proteins from T. kirilowii var japonicum root cultures showed differential protein accumulation in the medium during the time course of growth in batch cultures. Maximum protein accumulation, approaching 20 pg/mL, was observed at mid-exponential phase, followed by a degradation of a specific protein subset that coincided with the onset of stationary phase. Two major extracellular proteins and one intracellular protein, purified by ion-exchange and reverse-phase high-performance liquid chromatography, were identified as class 111 chitinases (EC 3.2.1.14) based on N-terminal amino acid sequence and amino acid composition homologies with other class 111 chitinases. The Trichosanthes chitinases also showed reactivity with a cucumber class 111 chitinase antiserum and chitinolytic activity in a glycol chitin gel assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis of intracellular proteins showed that normal and transformed 7. kirilowii var japonicum roots accumulated only low levels of TCN (approximately 0.5% total soluble protein). Storage roots from the plant displayed protein and antigen patterns different from root cultures and produced TCN as the dominant protein. Roots undergoing secondary growth and differentiation exhibited patterns similar to those of storage roots, including increased TCN levels, indicating that high production of TCN is associated with induction of secondary growth in roots.Transformed ("hairy") root 'cultures have facilitated elucidation of root-specific metabolism in plants (Flores and Filner, 1985; Flores, 1987;Rhodes et al., 1990). Transformed root cultures stably express root-specific biosynthetic pathways and have been used to study extensively the biosynthesis of a wide range of low mol wt metabolites such as alkaloids, flavonoids, and polyacetylene and sesquiterpene phytoalexins (Rhodes et al., 1990;Flores and Curtis, 1992;Saito et al.,

Basic Proteins Produced by Hairy Root Cultures of Trichosanthes Kiriliowii var. japonica.

“…The major protein in the basic protein fraction was tentatively identified as a class III chitinase based on the N-terminal amino acid sequence. This is consistent with the report by Savary and Flores [14], who identified two major extracellular basic proteins and one intracellular basic protein produced by T kirilowii var. japonica hairy roots as class III chitinases.…”

“…However, the N-terminal sequence of HR-PB 1 was very similar to but not identical with the sequence of any of these proteins. The present result, together with the report describing very low level production of trichosanthin in the hairy roots [14] and transformed callus tissues [15], suggests that production of karasurin or trichosanthin may be associated with induction of secondary growth of roots. Chemical treatments, including addition of sucrose or jasmonates, have been shown to induce accumulation of sporamine in the leaf-petiole region of sweet potatoes [16] and expression of vegetative storage protein genes in soybean [17].…”

“…japonica [14]. Trichosanthin was also identified in cell extracts of the transformed callus tissues resulting from infection by Agrobacterium rhizogenes but not in the untransformed callus of T kirilowii [15].…”

Isolation and Characterization of a Novel Ribosome-Inactivating Protein from Root Cultures of Pokeweed and Its Mechanism of Secretion from Roots

“…A. rhizogenes-transformed hairy roots, in general, exhibit morphological and genetic similarity with fibrous primary roots (for review, see Vivanco and Flores, 2000;Bais et al, 2001). Savary and Flores (1994) have shown that secondary growth induction leading to storage root formation shifts the protein production and accumulation patterns of RIPs. Therefore, PAP-H may be considered a biochemically identical isoform to the protein found in the primary roots.…”

“…Hairy roots show stable expression of root-specific biosynthetic pathways, and thus have been used as an experimental system to study the biology and biochemistry of underground organs (Flores and Curtis, 1992;Flores et al, 1999). Plant roots synthesize and store various macromolecules, including storage and defense-related proteins such as chitinase and ␤-1,3-glucanase, to cope with pathogenic challenge (Mauch et al, 1988;Linthorst, 1991;Savary and Flores, 1994;Savary et al, 1997). Here, we report the identification of a novel RIP, termed PAP-H, located in the cell walls of hairy roots and root border cells of pokeweed.…”