In this thesis we have examined the spatial and temporal aspects of gene expression and the position induced differences in transgene expression between individual transformants. For this purpose we imaged luciferase ( luc ) gene expression driven by three different promoters that are active throughout a leaf tissue of transgenic petunia plants. The Cauliflower Mosaic Virus (CaMV) 35S promoter, a modified CaMV 35S promoter (m35S) and an Arabidopsis thaliana lipid transfer protein (LTP) were studied. We observed differences in in planta luciferase activity between individual transformants, but also between leaves within a plant and within a single leaf. The luciferase activity within a leaf of a single transformant could differ up to a factor 300. Moreover, the way in which in planta luciferase activity varies within a plant is different in independent transformants expressing the same transgene.
The local differences in inplanta luciferase activity cannot be attributed to possible artefacts of the reporter system. Local limitations of luciferin, ATP or oxygen, which are all required for the luciferase reaction, might contribute to these artefacts. Pre-spraying the plants three to five times with luciferin is sufficient to obtain a steady state light production in "undisturbed" luciferase reporter plants, similar to feeding luciferin through the vascular system. We calculated that the ATP consumed by the luciferase reaction in planta is very small compared to the ATP steady state levels within leaf cells, while the cellular ATP concentration is far above the Km of luciferase for ATP. Furthermore, it was shown that oxygen levels could not be limited physiologically, resulting in changes in the local differences in luciferase activity within a leaf. We showed that the in planta activity of accumulated luciferase is rapidly declining after the first application of luciferin, indicating that in planta either no or very little regeneration of luciferase (activity) occurs due to a possible co-reaction with CoA. We also showed that the half-life of the luciferase protein itself is 2.5 hours in planta and that the half life of the active luciferase protein in the presence of luciferin is only 15 minutes or less. We thus conclude that, under the condition of repeated pre-treatment with luciferin, the in planta luciferase activity is closely related to on-going promoter and translational activity. The differences in luciferase activity that we observed within a plant thus truly reflect differences in luciferase gene expression.
We thoroughly characterised the spatial differences of luciferase gene expression within a leaf and show that the variegated luciferase activity within a leaf may also vary from day to day. Both the type of spatial expression pattern and the type of temporal expression pattern differed between independent transformants with the same promoter-luciferase construct. This indicates that these differences are related to the different sites of integration in independent transformants. Analysis of local luciferase mRNA levels showed a good correlation with local in planta luciferase activity, indicating that in planta luciferase activity is directly related to luciferase transgene expression.
The influence of matrix-associated-regions (MAR elements) on reducing the position induced variation was already known from literature. In plants it had been shown that MAR elements can reduce the variation in activity of the stable ß-glucuronidase (GUS) in a population of independent transformants. The luciferase reporter gene allowed a much more precise study of the effect of MAR elements, especially on the spatial and temporal aspects of transgene expression. We analysed the effect of MAR elements on position induced differences in luciferase transgene expression. We noted no change in the level of variegation within leaves, in a population of transformants with a luciferase reporter gene flanked by MAR elements compared to a population of transformants with a luciferase reporter gene not flanked by MAR elements. Also, the presence of MAR elements did not seem to result in a reduction in the variation of in planta luciferase activity within this plant population. The effect of MAR elements on in planta luciferase activity is thus different from that on GUS activity. We concluded that MAR elements probably reduce the position effect of stable gene products by reducing the long-term temporal variation of gene expression. This effect is not visible when real-time reporter genes like luciferase are used. The complicated relation between gene product stability, a reduction in temporal regulation in gene expression by MAR elements, and the potential effects on reducing variation in gene expression were discussed using a model. Analysis of the model system shows that indeed some changes in gene regulation would affect the variance in stable transgene expression with no effect on the variance in unstable transgene expression.
Apart from the differences in day-to-day temporal variations in luciferase activity between independent transformants, the variation in short-term temporal regulation of luciferase activity was also investigated. We investigated whether and how the position effect influences the changes in transgene expression in response to wounding. Each of the three promoters that were tested (35S, m35S and LTP) showed a different response to wounding. Although the overall response per promoter type was characteristic, there were also differences in wound response dynamics between independent transformants, carrying the same luciferase construct. This indicated that also the short-term dynamics in transgene expression, like that during the wound response, is affected by the site of integration of the transgene. As it was known from literature that ethylene and jasmonic acid play a role in the wound response, we investigated the effects of inhibition of ethylene synthesis, blocking of ethylene receptors, addition of ethylene, or addition of jasmonic acid on the wound response. None of these treatments showed a consistent and dramatic effect on the wound response.
The central question now is: What is the origin of the variegated expression patterns that were observed with in planta activity of luciferase reporter genes? We showed that MAR elements did not reduce the level of variegation of 35S-luciferase activity in leaves, indicating that the variegated luciferase activity is not influenced by the chromosomal organisation of transgene DNA in defined loops of transcriptional activity. The question remained whether the spatial and temporal variation of transgene expression, as shown throughout this thesis, are specific for just the luciferase transgene, or also apply to some endogenous plant genes. We speculated on the possible causes for variegated transgene activity: cell-to-cell differences in chromatin structure, local differences in the level of transcription factor activity or in sensitivity to transcription factors. We showed that the expression of some endogenous plant genes is also variegated in leaves, albeit this variegated activity was different from or opposite to the luciferase expression. As endogenous gene expression does show signs of variegated activity it is not likely that variegated transgene activity is caused by local differences in transgene accessibility for transcription factors and that this is specific for the transgene only. One might further hypothesise, assuming that hormones can influence the level of transcription factor activity, that local differences within a leaf in level or in sensitivity to hormones can result in variegated patterns of gene expression. However, equalising hormone signalling, either by blocking of ethylene receptors, or exogenous application of ethylene or jasmonic acid, had no dramatic effect on either the 35S-LUC, m35S-LUC or LTP-LUC activity in leaves. These hormone treatments neither resulted in a reduction in the level of variegated in planta luciferase activity, indicating that at least these hormones are not (solely) responsible for the observed variegated patterns.
The results presented in this thesis show that the luciferase reporter gene allows a much broader analysis of the position induced differences in gene expression than previously shown with more stable reporter genes, like GUS. The results also show that gene expression in a tissue or in a whole plant is more complex than we anticipated, i.e. there are very complex patterns in local promoter activity, both in space and in time.
|Qualification||Doctor of Philosophy|
|Award date||21 Mar 2001|
|Place of Publication||S.l.|
|Publication status||Published - 2001|
- petunia hybrida
- transgenic plants
- gene expression
- cauliflower mosaic virus
- reporter genes