MONITORING THE PRE-GERMINATION GENE EXPRESSION OF INDIVIDUAL ARABIDOPSIS THALIANA SEEDS USING A REAL-TIME LUCIFERASE REPORTER SYSTEM TO ADDRESS SEED VARIABILITY
Mitchell, J. and Bassel, G.W.
School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK
Contact: Jack Mitchell, firstname.lastname@example.org
Variability is present in biology across many scales, from populations, individuals and cells down to the molecular events within cells. Variability within seeds impacts seed behaviour and adaptations to the environment, consequently this can pose many problems to agriculture where crop uniformity is desired. We propose that understanding this variability is critical in enhancing strategies to increase crop uniformity and maximize agricultural output across varying climatic conditions. Individual seeds vary in absolute hormone concentrations as well as hormone sensitivity thresholds, both of which play a role in the developmental mechanisms during seed development. The antagonistic relationship between the hormones absissic acid (ABA) and gibberellin (GA) is known to control the physiological state of developing seeds, ABA induces dormancy and prevents germination, whereas GA stimulates germination and cell expansion. Although this relationship is widely accepted the exact mechanisms and roles that ABA and GA play during seed development are not understood in depth. A major obstacle to monitoring gene expression patterns during seed development is the destructive nature of sampling and measurement methods which involve the need to kill samples. As a result tracking the dynamics of expression over developmental time is currently not possible. We have developed a method to monitor gene expression in Arabidopsis thaliana seeds in real-time using a luciferase based reporter system. ABA and GA-responsive genes were monitored in real-time by inserting promoters upstream of a luciferase reporter and transformed into the Arabidopsis thaliana transparent testa mutant (tt4-1). When these seeds are exposed to luciferin the gene expression patterns of the selected genes can be monitored non-invasively from initial imbibition to germination providing an insight into the changes taking place during seed development. In the future dual-luciferase reporters are to be developed to enable the monitoring of multiple expression patterns from different combinations of genes to further develop current understanding of seed behavioural changes pre-germination.