Regulation of gene expression in plants

Our group investigates the regulation of gene expression in plants. Differential expression of gene subsets in specific cell types underpins proper growth, development, and stress responses. The lab has traditionally focused on epigenetic regulation, transcriptional gene silencing and the specific process of RNA-directed DNA methylation. Recently, our findings have led us into a new area, namely alternative splicing of pre-mRNA, which involves regulation at the co- and posttranscriptional levels.

The experimental pipeline employs classical genetic screens to identify putative regulatory proteins, followed by genomics approaches to identify their genomic targets, and molecular/biochemical methodologies to understand their mechanism of action. One current project centers on a system we developed that allows genetic analysis of pre-mRNA splicing in the model plant Arabidopsis thaliana. Mutants retrieved in this screen display either reduced or enhanced expression of an alternatively-spliced GFP reporter gene (Figure 1). So far, the screen has identified several previously uncharacterized factors, some of which may be important for stress responses and hence of interest for agricultural biotechnology. Further analysis of the mutants is likely to uncover additional novel factors that will expand knowledge of alternative splicing and its role in stress adaptation.

We also investigate interphase chromosome organization, which influences gene expression in ways not yet fully understood. Current work involves determining gene expression profiles and DNA methylation patterns in plants containing abnormal numbers of chromosomes (aneuploids), and examining biophysical organizing principles acting on chromosomes, particularly electrical gradients at nuclear membranes. We are presently adapting genetically-encoded fluorescent voltage indicators (GEVIs), developed by neurobiologists to map neural circuits in animals, for ‘light-based’ electrophysiology in plants. GEVIs are advantageous for electrophysiological studies because they can be targeted to internal membranes, such as the nuclear membrane (Figure 2), which are inaccessible in intact cells to classical tools for measuring membrane voltage, such as microelectrodes.

Figure 1. GFP phenotypes of mutant seedlings: left, wild-type T line; middle, GFPweak mutant (reduced expression); right, HyperGFP mutant (enhanced expression)

Figure 2. GEVI localized to inner nuclear membrane of Arabidopsis root cells