Investigating the role of DA1 in growth control.

Increasing global demand for food is a major issue facing modern day agriculture. For crops such as wheat and rice, where the seed constitutes the harvestable yield, the engineering of larger seeds provides a possible strategy for yield improvement. A detailed understanding of the growth of plant organs in general is paramount if such advances are to be made. Utilising previously characterised regulators of plant organ growth, this thesis explores the molecular mechanisms involved in the setting of final organ size. This thesis capitalises on previous studies that have identified DA1 as a negative regulator of organ growth; it explores the role of the DA1 protein and investigates its interactions with other proteins. In vitro studies reveal that DA1 forms homo-­‐ and hetero-­‐multimeric complexes with its sister protein DAR1 and in vitro and in yeast assays reveal interactions between DA1 and the transcription factor TCP15 and the growth-­‐regulating receptor-­‐like kinase TMK4. In addition, biochemical assays described in this thesis identify an active ubiquitin interacting motif (UIM) in the N-­‐terminal region of DA1 and an ubiquitin-­‐activated metallopeptidase in its C-­‐terminal region. Further studies reveal that, in addition to being activated by the RING E3 ligases EOD1/BB and DA2, the DA1 peptidase is active towards both EOD1/BB and DA2. In vitro and in vivo studies demonstrate that DA1 cleaves a peptide fragment from the N-­‐terminus of EOD1 and the C-­‐terminus of DA2. Finally, this thesis reports two genetic screens carried out in two separate Arabidopsis mapping populations in order to identify novel regulators of organ growth. Analyses of petal and seed phenotypes in the MAGIC RIL-­type population and in a natural Swedish population identify novel and a priori candidate genes for further characterisation

Ubiquitylation activates a peptidase that promotes cleavage and destabilization of its activating E3 ligases and diverse growth regulatory proteins to limit cell proliferation in Arabidopsis

The characteristic shapes and sizes of organs are established by cell proliferation patterns and final cell sizes, but the underlying molecular mechanisms coordinating these are poorly understood. Here we characterize a ubiquitin-activated peptidase called DA1 that limits the duration of cell proliferation during organ growth in Arabidopsis thaliana. The peptidase is activated by two RING E3 ligases, Big Brother (BB) and DA2, which are subsequently cleaved by the activated peptidase and destabilized. In the case of BB, cleavage leads to destabilization by the RING E3 ligase PROTEOLYSIS 1 (PRT1) of the N-end rule pathway. DA1 peptidase activity also cleaves the deubiquitylase UBP15, which promotes cell proliferation, and the transcription factors TEOSINTE BRANCED 1/ CYCLOIDEA/PCF 15 (TCP15) and TCP22, which promote cell proliferation and repress endoreduplication. We propose that DA1 peptidase activity regulates the duration of cell proliferation and the transition to endoreduplication and differentiation during organ formation in plants by coordinating the destabilization of regulatory proteins