The Antirrhinum majus transposon Tam3 undergoes low temperature-dependent transposition (LTDT). Growth at 158C permits transposition, whereas growth at 258C strongly suppresses it. The degree of Tam3 DNA methylation is altered somatically and positively correlated with growth temperature, an exceptional epigenetic system in plants. Using a Tam3-inactive line, we show that methylation change depends on Tam3 activity. Random binding site selection analysis and electrophoretic mobility shift assays revealed that the Tam3 transposase (TPase) binds to the major repeat in the subterminal regions of Tam3, the site showing the biggest temperature-dependent change in methylation state. Methylcytosines in the motif impair the binding ability of the TPase. Proteins in a nuclear extract from plants grown at 158C but not 258C bind to this motif in Tam3. The decrease in Tam3 DNA methylation at low temperature also requires cell division. Thus, TPase binding to Tam3 occurs only during growth at low temperature and immediately after DNA replication, resulting in a Tam3-specific decrease in methylation of transposon DNA. Consequently, the Tam3 methylation level in LTDT is regulated by Tam3 activity, which is dependent on the ability of its TPase to bind DNA and affected by growth temperature. Thus, the methylation/demethylation of Tam3 is the consequence, not the cause, of LTDT.

SUMMARYIt has been proposed that environmental stimuli can activate transposable elements (TEs), whereas few substantial mechanisms have been shown so far. The class-II element Tam3 from Antirrhinum majus exhibits a unique property of low-temperature-dependent transposition (LTDT). LTDT has proved invaluable in developing the gene isolation technologies that have underpinned much of modern plant developmental biology. Here, we reveal that LTDT involves differential subcellular localization of the Tam3 transposase (TPase) in cells grown at low (15°C) and high (25°C) temperatures. The mechanism is associated with the nuclear import of Tam3 TPase in Antirrhinum cells. At high temperature, the nuclear import of Tam3 TPase is severely restricted in Antirrhinum cells, whereas at low temperature, the nuclear localization of Tam3 TPase is observed in about 20% of the cells. However, in tobacco BY-2 and Allium cepa (onion) cells, Tam3 TPase is transported into most nuclei. In addition to three nuclear localization signals (NLSs), the Tam3 TPase is equipped with a nuclear localization inhibitory domain (NLID), which functions to abolish nuclear import of the TPase at high temperature in Antirrhinum. NLID in Tam3 TPase is considered to interact with Antirrhinumspecific factor(s). The host-specific regulation of the nuclear localization of transposase represents a new repertoire controlling class-II TEs.

Transposon insertions occasionally occur in the promoter regions of plant genes, many of which are still capable of being transcribed. However, it remains unclear how transcription of such promoters is able to occur. Insertion of the Tam3 transposon into various genes of Antirrhinum majus can confer leaky phenotypes without its excision. These genes, named Tam3-permissible alleles, often contain Tam3 in their promoter regions. Two alleles at different anthocyanin biosynthesis loci, nivea recurrens::Tam3 (niv rec ) and pallida recurrens::Tam3 (pal rec ), both contain Tam3 at a similar position immediately upstream of the promoter TATA-box; however, these insertions had different phenotypic consequences. Under conditions where the inserted Tam3 is immobilized, the niv rec line produces pale red petals, whereas the pal rec line produces no pigment. These pigmentation patterns are correlated with the level of transcripts from the niv rec or pal rec alleles, and these transcriptional activities are independent of DNA methylation in their promoter regions. In niv rec , Tam3 is inserted in an orientation that results in the 3# end of Tam3 adjacent to the 5# region of the gene coding sequence. In contrast, the pal rec allele contains a Tam3 insertion in the opposite orientation. Four of five different nonrelated genes that are also Tam3-permissible alleles and contain Tam3 within the promoter region share the same Tam3 orientation as niv rec . The different transcriptional activities dependent on Tam3 orientation in the Antirrhinum promoters were consistent with expression of luciferase reporter constructs introduced into yeast chromosomes but not with transient expression of these constructs in Antirrhinum cells. These results suggest that for Tam3 to sustain stable transcriptional activity in various promoters it must be embedded in chromatin.

Summary• In Antirrhinum, several unique regulations of the transposon, Tam3, have been described. Tam3 activity in Antirrhinum is strictly controlled by the growing temperature of plants (low-temperature-dependent transposition: LTDT), by chromosomal position of Tam3 copy and by two specific repressor genes Stabiliser (St) and New Stabiliser (NSt).• Here, the effects of the St and NSt loci on Tam3 transposition are compared. In cotyledons and hypocotyls, Tam3 is active even at high growing temperatures, indicating that LTDT does not operate when these organs are developing. This developmental regulation of Tam3 activity is differentially influenced by the St and NSt loci: St permits Tam3 transposition in cotyledons and hypocotyls, whereas NSt suppresses it in these organs.• The effects of these host genes on Tam3 activity at the molecular level were examined. It was found that neither of these genes inhibits the transcription of the Tam3 transposase gene nor its translation, and that the Tam3 transposase has the potential to catalyze transposition in the St and NSt lines.• The differences between the effects of St and NSt imply that they regulate Tam3 activity independently. Our molecular data indicate that their influence on Tam3 transposition seems to be nonepigenetic; possible mechanisms for their activity are discussed.

SummaryOur knowledge is limited regarding mechanisms by which transposable elements control host gene expression. Two Antirrhinum lines, HAM2 and HAM5, show different petal colors, pale-red and white, respectively, although these lines contain the same insertion of transposon Tam3 in the promoter region of the nivea (niv) locus encoding chalcone synthase.Among 1000 progeny from HAM5 grown under the preferred conditions for the Tam3 transposition, a few showed an intermediate petal color between HAM2 and HAM5. Transposon tagging using these progeny identified a causative insertion of Tam3 for the HAM5 type (white) petal color, which was found 1.6 kb downstream of the niv gene.Insertion of Tam3 at the position 1.6 kb downstream of niv alone showed nearly wildtype petal pigmentation, and the niv expression reduced by only 50%. Severe suppression of niv observed in HAM5 required interaction of two Tam3 copies on either side of the niv coding sequence. DNA methylation and small interfering RNAs (siRNAs) were not associated with the suppression of niv expression in HAM5.Insertion of a pair of transposons in close proximity can interfere with the expression of gene located between the two copies, and also provide evidence that this interference is not directly associated with pathways mediated by siRNAs.