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We have previously demonstrated that cutting induces the rapid response of genes for ethylene-responsive transcription factors (ERFs) in leaf strips of tobacco, and that the induction was not interfered but enhanced in the presence of the protein synthesis inhibitor cycloheximide (CHX). In this study, we analyzed the expression of genes for ERFs in tobacco plants by injuring leaf tissues with a hemostat. The results verified that mechanical damage is a trigger for rapid and concurrent induction of both the local and the systemic expression of genes for ERFs in tobacco plants. Further studies on systemic response of ERF genes in response to different severity and position of the wound on a leaf suggested that a threshold value might exist for the magnitude of wound signal to induce systemic activation of these genes. Then, we examined expression of genes for ERFs by analysis in transgenic tobacco plants that harbored reporter genes in which the promoter of the gene for NsERF2, NsERF3 or NsERF4 was fused to a gene for beta-glucuronidase. The results suggested that the local and systemic accumulation of ERF mRNAs after wounding was primarily mediated by the rapid activation of transcription of the respective genes. In addition, we found that cycloheximide triggered rapid activation of genes for ERFs which might be mediated via activation of transcription of the genes for ERFs.

The Chn48 gene is a representative of a family of tobacco class I basic chitinase genes, and the expression is induced by the stress hormone ethylene. To investigate the molecular basis for transcriptional regulation by ethylene we have examined the Chn48 promoter to identify cis-elements and trans-acting factors that are involved in the chitinase gene expression. In transgenic tobacco plants, a chimeric gene construct containing a 2 kb Chn48 promoter fused to a beta-glucuronidase reporter gene was induced by ethylene in leaf tissues. Deletion analysis indicated that a positive ethylene-responsive region is located between nucleotides -503 and -358 relative to the transcription initiation site. This 146 bp sequence was found to confer ethylene-responsive reporter gene expression when inserted in either orientation upstream of the heterologous promoter, indicating that the sequence functions as a regulatory enhancer. The ethylene-responsive region contains two copies of a GCC-box (TAAGAGCCGCC), which is conserved in a number of ethylene-responsive defense genes. The sequences within this ethylene-responsive region that are necessary for ethylene-responsive transcription were further localized to the 71 bp sequence between positions -480 and -410 containing two copies of the GCC-box by loss-of-function analysis. Gel mobility-shift experiments showed the presence of leaf nuclear factors that interact with the DNA sequences included in the ethylene-responsive region.

The expression of tobacco class I chitinase gene is effectively induced by a fungal elicitor in suspension-cultured tobacco cells. To identify cis-acting DNA elements that respond to the elicitor, a series of promoter constructs of the chitinase gene CHN50 fused to beta-glucuronidase gene was introduced into tobacco cultured cells. Promoter deletion analysis of the chitinase gene CHN50 in transgenic tobacco calli indicated that the DNA region between positions -788 and -345 from the start site of transcription is required for inducibility by the elicitor. A gel mobility shift assay revealed that nuclear factor(s) specifically interacted with the DNA region between positions -574 and -476. Moreover, this novel DNA-binding activity was present in nuclear extracts prepared from elicitor-treated cultured cells but not in extracts from untreated cells. Competitive binding assays and methylation interference experiments showed that the nuclear factor(s) bound specifically to a sequence of 22 bp that extended from positions -539 to -518 and contained a direct repeat of GTCAG spaced by three nucleotides. This motif is a candidate for a cis-acting elicitor-responsive element (ElRE) that is involved in the transcription of the class I chitinase gene.

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.

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.

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.