Aufklärung des Wirkmechanismus von BROAD LEAF1 bei der Kontrolle des Blattwachstums in Gerste
Genetik und Genomik der Pflanzen
Zusammenfassung der Projektergebnisse
This project studied the mode of action of BROAD LEAF1 (BLF1), a key regulator of leaf growth in barley. BLF1 encodes for an IDD protein, and these have recently been suggested to provide DNA-binding platforms for GRAS-domain transcriptional regulators, such as the DELLA protein SLENDER1 (SLN1) as a key mediator of gibberellic acid (GA) signaling. However, neither direct yeast two-hybrid interaction tests nor genetic and physiological analysis indicated a role for BLF1 in GA signaling, suggesting that it exerts its influence on leaf growth independently of GA. Loss of BLF1 function leads to wider leaves due to increased cell division, while increased BLF1 levels result in narrower leaves than in the wild type. This suggests that control of BLF1 levels is important for leaf-size control. To understand how this is achieved, we searched for BLF1-interacting proteins using a yeast two-hybrid screen. From several candidate interactors, we identified a novel RING/U-BOX protein that specifically binds to BLF1 in yeast and in a heterologous plant system. Co-expression of the RING/U-BOX protein and BLF1 in a heterologous system results in reduced BLF1 levels, and this effect can be blocked by treatment with a proteasome inhibitor. The RING/U-BOX protein can directly ubiquitinate BLF1 in a bacterial reconstitution system, and endogenous BLF1-YFP levels in barley shoot apices can be increased by treating barley seedling with a proteasome inhibitor. We have generated mutants in the RING/U-BOX gene and are analyzing their mutant phenotypes to see whether these mutations result in increased BLF1 levels and as a result in narrower leaves. Thus, this part of the project has demonstrated a role for ubiquitin/proteasome-mediated degradation in maintaining low levels of the key growth repressor BLF1 and has identified a potential E3 ubiquitin ligase mediating this degradation.
