Junko Takeda

Somatic variation during long term subculturing of plant cells caused by insertion of a transposable element in a phenylalanine ammonia-lyase (PAL) gene

Abstract We have identified a new En/Spm-like transposable element, Tdc1, in the 5′ flanking region of a phenylalanine ammonia-lyase gene (gDcPAL1) that is normally induced by transferring cells of carrot suspension cultures to fresh liquid medium (transfer or dilution effect). The initial integration into gDcPAL1 occurred more than 4 years after culture initiation. Tdc1 was first detected in gDcPAL1 genomic clones of a genomic library made from cells of the same cultured cell line 7 years after its initiation and thus following repeated subculturing. Twelve years after initiation, about 5–10% of the cells had Tdc1 inserted into the gDcPAL1 gene, indicating that Tdc1 insertion into gDcPAL1 occurred in one (or more) cell(s) during the first 4–7 years of subculturing. These mutant cells did not disappear during numerous passages; instead the proportion of cells having this Tdc1 inserted into gDcPAL1 has been increasing over the last 5 years. The promoter activity and the inducibility by transfer/dilution of the gDcPAL1 gene harboring Tdc1 is reduced relative to wild type. Finally, we show that insertion of a transposable element is one of the mechanisms that can cause variation of plant cell cultures during repeated subculture.

Light-induced synthesis of anthocyanin in carrot cells in suspension. IV: The action spectrum

Abstract— Using carrot cell suspension in 2,4‐dichlorophenoxyacetic acid (2,4‐D)‐depleted culture medium, fluence‐response curves for the formation of anthocyanin were determined at various wavelengths from 250 to 800 nm. In the fluence‐response curves at wavelengths between 260 and 330 nm, the response showed a sharp fluence‐dependent increase after the fluence exceeded threshold level at the respective wavelength. Such a sharp increase in response was not observed by light at 450 nm or longer wavelengths, although the response obtained by higher fluence of such light was always higher than that in the dark control. Action spectra determined at the sharp increasing phase of the response showed the single peak at 280 nm which equals the absorption maximum of UV‐B photoreceptor.

Action Spectrum for Induction of Promoter Activity of Phenylalanine Ammonia‐Lyase Gene by UV in Carrot Suspension Cells

Abstract— The full‐length promoter (−2335) of the carrot (Daucus carota) phenylalanine ammonia‐lyase gene (gDcPALl) fused to the luciferase reporter gene was transiently transformed to carrot protoplasts by electroporation, and the promoter activity induced by monochromatic UV light of various wavelengths was examined. The action spectrum constructed from the fluence–response curves showed a single peak at around 280 nm, suggesting that the activation of the gDcPALl promoter is categorizable as one of the UVB light responses. The same assay system was applied to variously truncated gDcPALl promoters and to CaMV35S promoter fusion with various parts 5′‐upstream of the gDcPALl promoter. The region from ‐396 to ‐190 (relative to the transcription start site) fused to the CaMV35S core (‐90) promoter showed a 280 nm‐dominant response. However, gDcPALl promoters truncated above ‐570 and ‐396, although they contain the region between ‐396 and ‐190, did not show such a typical UVB response, i.e. they responded to 260 nm light as much as to 280 nm light. The promoter truncated to below ‐190 also responded to 260 nm light as much as to 280 nm light. Therefore we assumed that the gDcPALl promoter is composed of three functionally different parts: the upstream above ‐570 (modulator), the region from ‐396 to ‐190 (UVB responsive) and the downstream below ‐190 (UVB and C responsive). The overall UVB response of the gDcPALl full‐length promoter is explained as the result of interaction of these three components.

Assignment of UVB-responsive cis-element and Protoplastization- (Dilution-) and Elicitor-responsive Ones in the Promoter Region of a Carrot Phenylalanine Ammonia-lyase Gene (gDcPAL1)¶

Expression of a carrot phenylalanine ammonia‐lyase (PAL) gene (gDcPAL1) in suspension‐cultured carrot cells is induced by dilution of the culture or by application of a fungal elicitor, as well as by ultraviolet B (UVB) irradiation. We demonstrated that among its upstream cis‐elements (Takeda et al.[1997] Photochem. Photobiol. 66, 464–470), L4 is UVB responsive, and L1 is protoplastization‐ (dilution‐) and elicitor responsive, from studies with transiently transformed mutated or truncated gDcPAL1 promoter–luc constructs. This conclusion is consistent with our observation that PAL activities induced by UVB and by protoplastization (dilution) or elicitor are additive.

Phenylalanine Ammonia-Lyase Genes Involved in Anthocyanin Synthesis and the Regulation of its Expression in Suspension Cultured Carrot Cells

D and is also induced rapidly and transiently by transfer of cells to fresh medium and lowering the cell density. From the carrot genomic library, four clones of PAL genes, gDcPAL1,2,3 and 4, were obtained. Analyses of nucleotide sequences revealed that only the gDcPAL3 gene is responsible for the induction of anthocyanin synthesis by 2,4-D. Several cis-elements, boxes M, P, A, L, and G, exist in the proximal promoter region of gDcPAL3. Transient expression experiments in carrot protoplasts using deletion mutants of the proximal promoter region of gDcPAL3 gene showed that boxes M and L, both of which contain core sequences of the Myb binding sites, might play an important role in gDcPAL3 promoter activity. Four myb cDNAs, Dcmyb8,10,12 and 14 were obtained from a carrot subtracted cDNA library and their structure and expression patterns were analyzed. In addition to the analysis of the proximal region of gDcPAL3 promoter, the possibility of the regulation of gene expression by genomic DNA structure and chromatin modification in metabolic differentiation is discussed.

Regulation of phenylalanine ammonia-lyase genes in carrot suspension cultured cells

Induction of anthocyanin synthesis occurs during metabolic differentiation in carrot suspension cultured cells grown in medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D), and is closely correlated with embryogenesis. Anthocyanin synthesis may also be induced by light-irradiation under different culture conditions. The phenylalanine ammonia-lyase (PAL) gene (TRN-PAL), which was transiently induced by the transfer effect, was also rapidly induced after light-irradiation. However, TRN-PAL was not involved in anthocyanin synthesis. A second PAL gene, ANT-PAL, was involved in anthocyanin synthesis. ANT-PAL was induced during metabolic differentiation in medium lacking 2,4-D parallel with the induction of chalcone synthase (CHS). PAL genes in the carrot genome are expressed differentially depending on the nature of the environmental stimulus, e.g. transfer effect and light, and other parameters which also affect anthocyanin synthesis.