Study of transgenic tobacco plants containing the ipt-gene under control of a tetracycline-inducible promoter

Abstract

Cytokinins have clearly been shown to have a critical role in plant growth and differentiation (chapter 1). Introduction of the ipt-gene, coding for isopentenyl transferase and first identified in the T-DNA of Agrobacterium tumefaciens, results in a system in which cytokinin levels can be manipulated. When this Agrobacterium gene is put under control of tetracycline-inducible promoter, a very specific system is achieved (chapter 2). The two chloroplast genes psbC and psbD are regulated by a blue light responsive promoter (BLRP) (chapter 3). Moreover, cytokinins are often thought to mimic the effect of light. Therefore, a possible link between cytokinins and the light regulation of psbC/psbD gene expression might exist. Although the regulatory transcription system in our transgenic plants (Tc-inducible ipt-construct) was very promising, several problems and questions arose while looking at the results of our experiments. Since tetracycline (Tc) belongs to the group of antibiotics that have effect on 70S ribosomes, effects of tetracycline on protein synthesis in the chloroplasts and mitochondria may be expected. It is therefore important to induce transgenic plants with a tetracycline concentration that does not affect those processes. Literature survey indicates that 1mg/l tetracycline has no physiological effect on the plants. In this study, several changes at this and even lower concentrations of tetracycline were observed (chapter 5): the length of roots is shortened, total chlorophyll, carotenoid content, and grana stacking is increased at 1mg/l Tc or higher. Since some of these effects are already seen when applying even lower Tc-concentrations, 0.1mg/l and 0.2mg/l tetracycline was used to induce ipt-transgenic plants. Even without tetracycline, some background expression was found in roots and leaves of transgenic plants. When inducing the ipt-gene in transgenic plants by Tc-application through the roots using hydroponics a lot of effects were observed (chapter 6). Upon induction, the ipt-gene is expressed several times higher in roots of transgenic plants, but not in their leaves. Consequently, cytokinin levels (ZRMP, ZR, DHZRMP, Z9G) are also very much elevated in roots. This results in a disturbance of cyt/aux ratio. The remarkable shift in favour of cytokinins results in obstructed root growth. In leaves no significant increase in cytokinins was observed. However, some transport of cytokinins from the roots to the aerial parts of the plants resulting in only a small increase of cytokinins in the leaves, can not be fully excluded, since the large variability in the quantitative results possibly masks such small changes. Indeed, phenotypical and physiological effects, which can be explained as cytokinin-like effects, were observed in leaves of ipt-induced transgenics: leaves are very much wilted, chlorophyll content is elevated and grana stacking is increased. Although the psbD-psbC gene cluster is only present in chloroplasts and no significant increase in cytokinins is found in aerial parts of the plant, it is still interesting to investigate the effect of the hormone on their expression (chapter 7). However, our results do not suggest an effect of cytokinins, present in excess in the roots, on psbC/psbD gene expression in the leaf. Fluorescence techniques enable us to tell more about the physiological state of the plant in a non-destructive way (chapter 8). The steady state fluorescence (S) in induced ipt-transgenic plants is decreased and chlorophyll-a is increased compared with all other plants. Although no increase in physiological active cytokinin in the leaves could be demonstrated, the fluorescence image analysis showed an effect on the photosynthesis process. This could be due to a secondary effect of the increased cytokinin content in the roots.