Effect of light and 2,4‐dichlorophenoxyacetic acid on the level of mRNAs for phenylalanine ammonia‐lyase and chalcone synthase in carrot cells cultured in suspension

Abstract

Changes in the amount of mRNA for phenylalanine ammonia‐lyase (PAL, EC 4. 3. 1. 5) and chalcone synthase (CHS, EC 2. 3. 1. 74) induced by light were investigated in carrot (Daucus carota L. cv. Kurodagosun) cells cultured in suspension. Cells were cultured for 5 days in 2,4‐dichlorophenoxy acetic acid (2,4‐D)‐free medium and absolute darkness. Transcription of pal and chs genes was induced by irradiation at day 5, resulting in the induction of anthocyanin synthesis. PAL mRNA transcripts were rapidly and transiently up‐regulated within 6 h after onset of irradiation, after which they were immediately down‐regulated. Then, PAL mRNA increased again slowly, the time course being coordinated with induction of CHS transcription and anthocyanin synthesis. The semi‐quantitative polymerase chain reaction (PCR) and primer extension using gene specific oligonucleotides for pal genes revealed that two different pal genes were induced sequentially by light. The rapid and transient induction of the pal gene was not related to anthocyanin synthesis, but was identical to that induced by dilution or transfer effect. This slowly‐induced pal gene, which was induced in parallel to the chs gene, corresponded to the pal gene which was induced for anthocyanin synthesis. Anthocyanin synthesis was induced by simultaneous activation of both ant‐pal and chs genes, and both mRNAs remained at a high level during anthocyanin synthesis. Expression of ant‐pal and chs was partially repressed by either depletion of light or addition of 2,4‐D. These genes were completely repressed by addition of 2,4‐D in darkness. Therefore, the two signals may act at different points. Trn‐pal was not repressed by depletion of light or addition of 2,4‐D.