Sugar signaling and growth control
Sugars are essential in metabolism but are signaling molecules as well and this function is comparable to the signaling function of hormones. Sugar receptors perceive the presence of sugar and initiate downstream signaling events. We are investigating how sugar signals affect gene expression, metabolism and development in plants. Our goal is to understand the signal transduction networks in which sugars participate in molecular detail. We focus on a group of bZIP transcription factors that link sugar availability to growth.
bZIP transcription factors are translationally regulated in response to sugar signals.
Five Arabidopsis transcription factors (collectively names the S1 group) are regulated on the mRNA translation level in response to sucrose concentrations. Interestingly, this effect is specific for sucrose and thereby we have the possibility to investigate how sucrose signaling is wired in plants without interference by the many other signaling pathways present in plants. We have shown that sucrose mediated repression of translation depends on upstream open reading frames (uORFs) present in the 5’leaders of the S1 group mRNAs. We are now identifying the factors that are involved in the regulation and thereby gain information on the signaling pathway regulating this process.
We use biochemistry, genetics and molecular genetics. For example, we use yeast two hybrid analysis to detect peptide interacting proteins, immuno-precipitations combined with label free LC-MS (proteomics), expression analysis in protoplasts. Moreover, we combine this approach with the analysis of transgenic and mutant plants.
The S1 group sucrose regulated transcription factors described above activate the transcription of genes encoding enzymes involved in central metabolism. Changed levels of these transcription factors result in major changes in metabolite levels and thereby growth of the plants.
Analysis of mRNA expression levels is important in this project. We study expression levels by using Real Time Quantitative PCR and whole genome microarrays. Much of our activities are focused on array analysis and the bioinformatic procedures that follow a successful experiment. We are as well testing the results by DNA-Protein interaction studies and genetic studies using mutated or transgenic Arabidopsis plants. To test the effect of specific dimers we express the proteins transiently in isolated leaf protoplasts.
bZIP proteins bind DNA as dimers consisting of two identical bZIP proteins or two distinct bZIP proteins. We have shown the sugar-regulated bZIPs bind DNA as a dimer with another type of bZIP. Depending on dimers formed, different target genes are activated.
Analysis of plants with modified levels or activities of specific proteins or genes are as well important. Changed development or physiology of a plant in which a single gene is affected in activity indicates the function of that particular gene. To further characterize these types of mutant or transgenic plants we use a plethora of tools ranging form microscopy to whole plant analysis. As the transcription factors studied regulate metabolite levels and metabolic profiling using mass spectrometry techniques is important. We use GC-MS based method to simultaneously assay the levels of hundreds of metabolites in the plant. The analyses of changed metabolites levels is tightly connected to the analysis of mRNA levels and thereby can we get an understanding of the genes that regulate metabolite levels and in this way we can build regulatory networks describing the sugar regulated processes.
Fructose is a very abundant sugar like glucose and sucrose in plants. Using a natural variation based screen we were able to show that also fructose is sensed independently from glucose and sucrose in plants. We identified the NAC89 gene from the Arabidopsis Cvi accession as a mediator of fructose specific seedling establishment. Using this genetic material we are investigating fructose specific signaling in Arabidopsis using various techniques. Importantly, we are investigating the target genes regulated by the NAC89 transcription factor and how the protein interacts with other signaling components.
In this project we take advantage of the rich genetic material existing in Arabidopsis. By crossing NAC89 mutant and transgenes to other mutants with known involvement in sugar signaling we are able to reconstruct the genetic pathway regulating seedling establishment in response to sugars. We are as well using molecular methods like micro arrays to understand what genes are regulated by the NAC89 protein and thereby gain knowledge of the fructose-signaling pathway.
Several signaling system are responsive to energy and primary metabolism of which some are well conserved among all eukaryotes. These signaling pathways are of interest to the Molecular Plant Physiology group and all link sugar status to the control of growth. To get a better view over the relative importance of the different signaling pathways we are approaching these in parallel using global transcriptional profiling (microarrays and massive sequencing). We use genetic material in which one or more of the signaling pathways are modified (either reduced or overactive). By comparing the resulting changes on the global transcriptome we gain information on the relative importance of the individual pathways. Based on the data we create networks to generate hypothesis on regulatory hubs and cross regulation of the different pathways. These models can be tested experimentally.