The fungicidal class I chitinases are believed to be important in the induced defense response of plants. We isolated and partially characterized genomic clones representing two members, CHN14 and CHN50, of the gene subfamily encoding these enzymes in Nicotiana tabacum L. cv. Havana 425. The coding sequences of genes CHN14, CHN50, and CHN48, which was cloned earlier, are identical at 79-95% of the positions. Tobacco is an amphidiploid species derived from ancestors most closely related to the present-day species N. sylvestris and N. tomentosiformis. Southern analysis of genomic DNA, comparison of deduced amino acid sequences, and partial sequencing of the purified enzymes suggest that the gene pairs CHN48/CHN50 and CHN14/CHN14' are homeologues. Gene CHN48, which encodes chitinase A (Mr ca. 34 kDa), and gene CHN14 are derived from N. tomentosiformis; whereas gene CHN50, which encodes chitinase B (Mr ca. 32 kDa), and gene CHN14' are derived from N. sylvestris. Class I chitinases are induced in leaves of plants treated with ethylene or infected with the fungal pathogen Cercospora nicotianae and in cultured cells transferred to medium without added auxin and cytokinin. RNase protection assays show that under these conditions transcripts encoded by the homeologues CHN48 and CHN50 account for greater than 90% of the total chitinase mRNA. The less abundant transcript, CHN48, consistently showed a greater degree of induction than CHN50. Expression of the homeologues CHN14 and CHN14' represented less than 10% of the total chitinase mRNA. They showed a pattern of hormonal regulation similar to CHN48 and CHN50, but transcripts of these genes were not detected in leaves infected with C. nicotianae. Therefore the two sets of homeologues are regulated in the same way by hormones and respond differently to infection by a pathogen.

We have isolated and characterized the genomic clone lambda CHN50 corresponding to tobacco basic endochitinase (E.C.3.2.1.14). DNA sequence and blotting analysis reveal that the coding sequence of the gene present on lambda CHN50 is identical to that of the cDNA clone pCHN50 and, moreover, the CHN50 gene has its origin in the progenitor of tobacco, Nicotiana sylvestris. Tobacco basic chitinases are encoded by a small gene family that consists of at least two members, the CHN50 gene and a closely related CHN17 gene which was characterized previously. By northern blot analysis, it is shown that the CHN50 gene is highly expressed in suspension-cultured tobacco cells and the mRNA accumulates at late logarithmic growth phase. To identify cis-DNA elements involved in the expression of the CHN50 gene in suspension-cultured cells, the chimeric gene consisting of 1.1 kb CHN50 5' upstream region fused to the coding sequence of beta-glucuronidase (GUS) was introduced by electroporation into protoplasts isolated from suspension-cultured tobacco cells. Transient GUS activity was found to be dependent on the growth phase of the cultured cells, from which protoplasts had been prepared. Functional analysis of 5' deletions suggests that the distal region between -788 and -345 contains sequences that potentiate the high-level expression in tobacco protoplasts and the region (-68 to -47) proximal to the TATA box functions as a putative silencer.

The endochitinases (E.C. 3.2.1.14, chitinase) are a structurally diverse group of enzymes believed to be important in the biochemical defense of plants against potential pathogens. The gene for a chitinase of Nicotiana tabacum L. cv. Havana 425 has been cloned and sequenced. The major transcription start is 11 bp upstream of the ATG codon and 28 bp downstream of the TATA box. The gene contains two introns and encodes a basic chitinase of 329 amino acids with a 23 amino acid N-terminal signal peptide followed by a 43 amino acid, cysteine-rich domain, which is linked by a hinge region to the main structure of the enzyme. This gene appears to be expressed because the exons are identical to the coding sequence of a cDNA which was isolated. Comparison of chitinase amino acid sequences from different plants indicates there are at least three classes of these enzymes: class I, basic chitinases with an N-terminal cysteine-rich domain and a highly conserved main structure; class II, chitinases similar to the main structure of class I chitinases but lacking the cysteine-rich domain; and, class III, chitinases with conserved sequences different from those of the class I and II enzymes. The sequences encoding the cysteine-rich domain in class I chitinases are flanked by 9-10 bp imperfect direct repeats suggesting that these domains arose from a common ancestral gene and were introduced into genes for class I enzymes by transposition events.

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.