An extracellular, acidic chitinase was purified to homogeneity from tobacco necrosis virus-infected leaves of Cucumis sativis. The amino acid sequences of the intact protein and of peptides isolated following endoproteinase Lys-C digestion, cyanogen bromide cleavage, and trypsin digestion were determined. Oligonucleotide probes derived from this sequence were used to isolate a cDNA clone encoding this protein. No significant homology was found between this chitinase and either the basic chitinase isolated from bean or tobacco or the chitinase isolated from Serratia marcescens; however, strong homology was found between the cucumber chitinase and a lysozyme/chitinase from Parthenocissus quinquifolia. The induction of the protein by tobacco necrosis virus infection or salicylate was found to be at the level of RNA accumulation. Genomic Southern analysis indicates that a single gene in the cucumber genome encodes this protein.Many plants infected with necrotizing pathogens develop local or systemic resistance against subsequent infections (1, 2). This induced resistance is accompanied by a number of biochemical changes in the host plant, including the production of pathogenesis-related (PR) proteins (for review see refs. 3 and 4). The accumulation of these acid-extractable, low molecular weight, protease-resistant proteins correlates with induced resistance, but their function has largely remained elusive. Recently, it has been shown that of the 10 well-characterized PR proteins in tobacco, two (PR-P and -Q) have chitinase activity (5-7), three (PR-O, -N, and -2) have /3-1,3-glucanase activity (8), and two (PR-R and -S) are structurally similar to a maize protease/a-amylase inhibitor (9, 10).From cucumber, we have recently purified one PR protein and identified it as a chitinase (11). After infection with tobacco necrosis virus (TNV), this Mr 28,000 protein accumulates in the intercellular space of the infected, as well as the uninfected, parts of the plant (11, 12). Here, we report the purification of this protein to homogeneity and the determination of 55% of the amino acid sequence (148 of 267 residues) of the mature protein. Oligonucleotide probes were synthesized based on the protein sequence analysis and used to isolate cDNA clones encoding this protein from a library constructed with RNA isolated from TNV-infected cucumber leaves. We have sequenced the cDNA clones and find no significant homology to known chitinase genes. However, striking homology was found between the deduced amino acid sequence and the partial amino acid sequence of a bifunctional lysozyme/chitinase purified from Parthenociccus quinquifolia (13).In preliminary studies on the regulation of chitinase gene expression, we show that there is one gene encoding chitinase in the cucumber genome and the accumulation of this protein after TNV infection or salicylic acid induction correlates with the accumulation of mRNA. MATERIALS AND METHODSProtein Purification and Sequencing. Chitinase protein was isolated from TNV-infected cucumber (Cucu...

A procedure for the regeneration of fertile plants by organogenesis from tissue cultures of soybeans, Glycine max is described. Seeds were germinated on reduced inorganic salt MS medium containing 5μM BA. Cotyledonary nodes were excised and cultured on the same medium. Presence of BA in the medium during seed germination and culture of nodal explants was required for multiple shoot and shoot-bud formation. Histological analyses established the de novo nature of shoot regeneration. Separate reduction of the concentration of inorganic salts or substitution of sucrose for fructose during culture had minimal effects on the regeneration response. Conversely, if the BA was reduced, the inhibition response could not be overcome by increased salt concentration or altered carbon source.

No abstract

No abstract