Research
Our research aims at understanding how the regulation of gene expression and of protein stability contributes to plant development. Specifically, we are interested in the gene regulatory networks underlying Arabidopsis flower development and in the functions of the so-called N-end rule pathway of protein degradation.
Gene Regulatory Networks
Flower development is an excellent system for studying the molecular mechanisms underlying organogenesis in plants. Extensive genetic analyses have led to the identification of several key regulatory genes of this important biological process. Most of these genes encode transcription factors or other proteins involved in the regulation of transcription, indicating the existence of a complex gene regulatory network that underlies flower development. However, the target genes of these transcriptional regulators, the cis-regulatory elements to which they bind, and their regulatory interactions are still largely unknown. What is more, the vast majority of the known floral regulators act during very early stages of flower development, before floral organs are initiated, and comparatively few genes controlling organ differentiation have been identified so far. Thus, new experimental approaches are needed to obtain a comprehensive view of the developmental mechanisms that control flower formation.
The invention of genomic technologies, such as DNA microarray analysis, has opened the possibility of dissecting the gene regulatory networks underlying development on a global scale. However, the use of these technologies for an in-depth study of flower formation has so far been hampered by difficulties in collecting sufficient amounts of floral buds of distinct developmental stages. To circumvent this problem, we have developed a floral induction system (see pictures on the right), which allows the collection of a large number of synchronized floral buds, and hence permits the analysis of early flower development with a stage-specific resolution. We are currently using this system together with genome-wide analysis methods to dissect the gene regulatory networks underlying early flower develoment on a global scale.
Gene regulatory network controlling early Arabidopsis flower development. Regulatory genes and their known interactions are shown. Upstream inputs and downstream targets are indicated for each gene. Activators are connected to their targets by arrows, repressors by blunted lines. Blue dots underneath gene symbols indicate that direct binding to these genes has been demonstrated. Note their small number in the diagram, indicating the limited knowledge of transcription factor binding sites. White circles represent protein complexes. Dashed lines indicate that gene products do not function as transcriptional regulators.
Protein Degradation
The intracellular concentration of proteins is often essential for their physiological function and thus needs to be tightly regulated. The control of protein stability plays a key role in this process, as indicated by the different half lives of proteins that range from a few seconds to many days. In eukaryotes, the control of protein stability is to a large extent mediated by the ubiquitin system, which catalyzes the conjugation of the small protein ubiquitin to certain other proteins and thus marks them for proteolysis. Ubiquitin is specifically conjugated to lysine residues of these proteins through the action of three enzymes, E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin-protein ligase). E3 ubiquitin ligases confer the specificity to the ubiquitin system, as they recognize and bind certain proteins based on the existence of specific degradation signals, and conjugate ubiquitin. Proteins that have been tagged by multiple ubiquitin molecules are degraded by the 26S proteasome.
The N end rule pathway is a subset of the ubiquitin system and relates the in vivo half life of a protein to the identity of its N terminal residue. It has been shown to play important roles in the regulation of fundamental cellular and developmental processes in yeast and animals. In plants, however, it has been scarcely studied, and its components, organization and physiological functions remain largely unknown. We are currently dissecting the structure of this pathway and its roles in the development of the model plant Arabidopsis thaliana.
The N-end rule pathway in yeast (after Varshavsky, 1996). Blue ovals denote proteins. N terminal residues are indicated by single letter abbreviations. Primary destabilizing residues are recognized by the N-recognin UBR1, while tertiary and secondary destabilizing residues are first modified by a Nt-amidase (NTA1) and the arginine-transferase ATE1, respectively.