Specific expression of the tobacco Tnt1 retrotransposon in protoplasts.

Abstract

The Tnt1 transposable element of tobacco belongs to the retrotransposon family and shares the structural features of viral retroelements including two long terminal repeats (LTRs) which are known to contain promoter regions. We show that two Tnt1 RNAs of 5.2 and 6.5 kb can be found. The 5.2 kb RNA matches with the size of the Tnt1 elements so far isolated (5.3 kb), whilst the evidence suggests that the 6.5 kb RNA could be a chimaeric RNA initiated in a gene in which Tnt1 has inserted. The Tnt1 5.2 kb RNA starts in the LTR, and the LTR can promote the expression of a translational LTR‐beta‐glucuronidase (GUS) fusion at a high level in transient expression assays. The Tnt1 5.2 kb RNA and the LTR‐GUS fusion of transgenic tobacco plants are specifically expressed in leaf‐derived protoplasts whereas they are not expressed in leaf tissue. The 5.2 kb RNA is also transcribed at low levels in roots. This RNA is induced after 2 h of maceration in the protoplast isolation medium, and its level declines rapidly after protoplast isolation. The induction requires only the presence of cell wall hydrolases, and is independent of wounding and plasmolysis. The induction of Tnt1 expression is not mediated by typical oligosaccharide elicitors released from the cell wall known to mediate defense gene responses. Tnt1 transcription features provide a first example of tissue culture‐induced mutagenesis in plants and a molecular basis for some of the somaclonal variation events.

Characterisation of LTR sequences involved in the protoplast specific expression of the tobacco Tnt1 retrotransposon

“…Deletion of an internal HindMEcoRV region containing the major transcriptional start site and its putative TATA-box (9), giving the 7011 construct, results in a substantial decrease of the activity. However, the activity of this construct, that contains most of the LTR U3 region and the minor transcriptional start site (9), is still high, being similar to that of the control plasmid pBI221, wich contains the GUS reporter gene under the control of the complete CaMV 35S promoter, a strong constitutive promoter in plant systems. In order to determine if the LTR upstream sequences were sufficient to confer protoplast-specific expression, we have developed a transient expression assay based on particle bombardment.…”

“…Protoplasts were isolated from fully expanded young leaves according to Chupeau et al (12). Cell wall enzymatic digestions Constructs The 2120 plasmid (pBMCV 102120) (Fig 1) containing a translational LTR-GUS fusion (containing 110 bp of tobacco nitrate reductase genomic sequences, the entire Tntl 5' LTR, and the first 160 bp of the internal Tntl sequences) in a pBluescript KS plasmid has been previously described (9). The 2146 plasmid (pBMCV 102146), in which the LTR The EcoRI-BgllI fragment of the EMBLGUS46 plasmid (14), containing the minimal (-46,+8) CaMV 35S promoter fused to the reporter gene GUS and the 3C terminator from the pea rbc SE9 gene, was subcloned in the EcoRI-BamHI sites of a pUC18 plasmid, to give the construct pG46.…”

“…This induction was shown to be associated with the presence of cell wall hydrolases (9). Tntl transcription is initiated in the 5' LTR and a major transcriptional start site, as well as a minor transcriptional start site, have been determined (9). Furthermore, transgenic plants transformed with translational fusions between the 5' LTR and the ,B-glucuronidase reporter gene (10) (LTR-GUS), showed a pattern of GUS activity similar to the pattern found for Tntl expression (9), demonstrating that LTR sequences are sufficient to mediate protoplast-specific expression.…”

“…Tntl expression is low in adult roots and almost absent in leaves, but is strongly induced in leaf-derived protoplasts (9). This induction was shown to be associated with the presence of cell wall hydrolases (9).…”

“…This induction was shown to be associated with the presence of cell wall hydrolases (9). Tntl transcription is initiated in the 5' LTR and a major transcriptional start site, as well as a minor transcriptional start site, have been determined (9).…”