An abundant 17 kDa protein which was isolated and characterized from 10-day old healthy root tissue of white lupin (Lupinus albus) proved to have a high sequence similarity to pathogenesis-related proteins found in other species. Subsequently, a corresponding clone (LaPR-10) was identified in a cDNA library prepared from the same tissue that exhibited a high amino acid sequence similarity to a number of the PR-10 family proteins. The clone contains an open reading frame encoding a polypeptide of 158 amino acids, with a predicted molecular mass of 16,905 Da and an isoelectric point of 4.66. Southern blot analysis indicates that LaPR-10 is likely a single-copy gene, or a member of a small gene family. The clone was expressed in Escherichia coli, and its protein product was purified to near homogeneity. Both the native and the recombinant proteins were immunorecognized by antibodies raised against pea PR-10 proteins, and exhibited a ribonucleolytic activity against several RNA preparations, including lupin root total RNA. Characterization of its enzymatic properties indicates that the LaPR-10 protein belongs to the class II ribonucleases. We present evidence that the white lupin 17 kDa protein is constitutively expressed during all stages of root development and, to a lesser extent, in other plant parts. In addition, we demonstrate the presence, in the LaPR-10 amino acid sequence, of a number of motifs that are common to most PR-10 proteins, as well as a RGD motif that is shared only with the alfalfa SRG1 sequence.
Pulse-labeling experiments of white lupin (Lupinus albus L.) cell cultures with [U-14C1L-phenylalanine for 72 h resulted in the incorporation of the radioactivity into the isoflavone aglucones, glucosides, and prenylated derivatives. Both the aglucones genistein and 2'-hydroxygenistein and their 7-0-glucosides accounted for 85% of the total isoflavonoids identified in the cultured cells and contained 35% of the radioactivity, whereas the prenylated derivatives comprised 15 and 65%, respectively. Almost 20% of the labeled isoflavones of the cellular pool was recovered from the culture medium, 90% of which were monoprenylated and diprenylated derivatives containing 80% of the radioactivity. These results clearly demonstrate the release into the culture medium of a substantial amount of the endogenously synthesized isoflavonoids, especially the prenylated derivatives.With few exceptions, most plant cell cultures fail to produce the secondary metabolites/natural products characteristic of the intact plants from which they were derived (13,17). Furthermore, of the limited variety of natural products that have been produced by cultured cells, flavonoid compounds are the least represented (4, 9). Among the few examples that have been reported in the literature are isoflavonoids formed in response to biotic or abiotic elicitors (1,3,11) or those produced by cell suspension and hairy root cultures transformed with Agrobacterium tumefaciens and A. rhizogenes, respectively (2).Cell cultures of white lupin (Lupinus albus L.) accumulate a complex pattern of isoflavones ( Fig. 1) [3-5, 3a-5a], and 6,3'-diprenyl [6, 6a] The isoflavonoid pattern is quite similar to that of the intact lupin roots (8,10,18,19). However, it was observed that lupin cell cultures release a portion of their metabolites into the culture medium, the latter eventually turning reddishbrown in color. Microscopic examination revealed that this phenomenon was not due to lysis of the cultured cells because the latter remained viable until the end of the culture period. It was considered important, therefore, to investigate the biosynthesis of lupin isoflavones, using "4C-labeled L-phenylalanine as a precursor, and their release into the culture medium. MATERIALS AND METHODS Cell CultureA cell-suspension culture derived from the radicles of white lupin (Lupinus albus L. cv Kievskij Mutant) was maintained on B5 nutrient medium (6) containing 2% sucrose and supplemented with 2,4-D (1 ppm) and kinetin (0.1 ppm). Preparation of Cell and Medium ExtractsAt the end of the metabolic period, the cultured cells were separated from the media by centrifugation at 2000g and washed twice with distilled water. The cells were extracted twice by homogenization with 80% aqueous MeOH4 (5:1, w/ v) at room temperature. The combined extracts were evaporated to an aqueous residue in a rotary evaporator at 300C, and the latter extracted twice with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate before evaporation, and the residue dissolved in 8...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.