Two genes for prepro glucan endo-1,3-beta-glucosidase (1,3-beta-glucanase; 1,3-beta-D-glucan glucanohydrolase, EC 3.2.1.39) of tobacco were cloned and their sequences were compared with cDNA clones. Southern analysis indicates that the genomic clones represent genes derived from ancestral parents of tobacco similar to the present day species Nicotiana sylvestris and Nicotiana tomentosiformis, whereas the genes represented by two of the cDNA clones appear to be unique to tobacco. The coding sequences of the genomic clones and cDNA clones differed at less than 2.2% of the positions, indicating that the tobacco 1,3-beta-glucanase gene family is highly conserved. Alternating blocks of sequence in the cDNA clones were identical to the coding sequence of the two genomic clones. These results and an analysis of evolutionary distances for nucleotide substitution are consistent with the hypothesis that the evolution of the tobacco 1,3-beta-glucanase gene family has involved exchange of DNA between members of the tomentosiformis and sylvestris subgenomes by recombination or gene conversion.

We determined the primary structure of a tobacco beta-1,3-glucanase gene. The beta-1,3-glucanase gene has a single large intron, and the intron separates coding regions of the signal peptide and the mature enzyme. Analysis of the 5'-flanking region sequence revealed an 11 bp GC-rich element with perfect homology to the putative regulatory sequence of tobacco chitinase genes. RNA blot analysis showed that levels of mRNAs of beta-1,3-glucanase and chitinase are coordinately increased in response to ethylene and salicylic acid. Accumulation of beta-1,3-glucanase mRNA in suspension-cultured cells is rapidly induced at late logarithmic growth phase. Members of the tobacco beta-1,3-glucanase gene families are classified into two subfamilies. One of the subfamilies appeared to be transcriptionally inactive.

Sequence analysis of five gene families that were isolated from tobacco thin cell layer explants initiating floral development [Meeks-Wagner et al. (1989). Plant Cell 1, 25-35] showed that two encode the pathogenesis-related proteins basic chitinase and basic beta-1,3-glucanase, while a third encodes the cell wall protein extensin, which also accumulates during pathogen attack. Another sequence family encodes the water stress-induced protein osmotin [Singh et al. (1989). Plant Physiol. 90, 1096-1101]. We found that osmotin was also induced by viral infection and wounding and, hence, could be considered a pathogenesis-related protein. These genes, which were highly expressed in explants during de novo flower formation but not in explants forming vegetative shoots [Meeks-Wagner et al. (1989). Plant Cell 1, 25-35], were also regulated developmentally in day-neutral and photoresponsive tobacco plants with high expression levels in the roots and moderate- to low-level expression in other plant organs including flowers. An unidentified gene family, FB7-4, had its highest level of expression in the basal internodes. Our findings indicate that these genes, some of which are conventionally considered to encode pathogen-related proteins, also have a complex association with normal developmental processes, including the floral response, in healthy plants.

Healthy tobacco plants accumulate beta-1,3-glucanases (glucan endo-1,3-beta-glucosidase; EC 3.2.1.39) in their roots and in specific parts of the flowers. After infection with tobacco mosaic virus, acidic and basic beta-1,3-glucanases are induced in the inoculated and virus-free leaves of the plant. An analysis of cDNA clones demonstrated that at least five genes for acidic beta-1,3-glucanases are induced after tobacco mosaic virus infection. Southern blot analysis indicated that the tobacco genome contains approximately eight genes for acidic beta-1,3-glucanases and a smaller number of genes encoding basic beta-1,3-glucanases. Genes from both gene families were cloned and sequenced. The basic isozymes contain a C-terminal extension that is cleaved off during their targeting to the vacuoles. This extension is absent in the acidic isozymes, which accumulate extracellularly. Northern blot hybridization showed that genes encoding acidic and basic beta-1,3-glucanases are strongly induced after tobacco mosaic virus infection or salicylate treatment of tobacco. The cloning of these genes is a first step toward the identification of regulatory elements involved in their coordinate induction.