N-terminal acetylation as a signal for protein degradation controlling plant development and stress responses

Unlike animals, plants cannot move, and have therefore evolved to grow and survive in constantly changing environments. Understanding the mechanisms that plants use to achieve this is critical if we are to develop superior crops to produce enough food to support a growing global population in the face of climate change. One way in which plants control their growth and respond to the environment is by regulating the stability of the proteins in their cells - plants need to precisely control when to get rid of a protein in order to successfully and rapidly respond to a wide range of signals. Protein degradation (proteolysis) in plants is important for controlling almost all aspects of plant life - for example, the sensing of and response to most plant hormones and a large number of external and internal signals (e.g. light and oxygen) is reliant on protein degradation. Therefore, increasing our understanding of the signals and mechanisms regulating protein stability is a major focus for plant science in order to identify targets that plant breeders and biotechnologists can focus on to develop improved crop varieties.

This work will identify and characterize a new pathway for targeted protein degradation in plants. In this pathway, which was recently identified for the first time in yeast, degradation is initiated through the addition of a small molecule (acetyl) at the beginning (N-terminus) of a protein. Once a protein has been N-terminally acetylated, it can then be recognised by another type of protein that adds a second marker (ubiquitin), which acts as a signal for degradation by the cell. Our initial studies suggest that protein degradation via this pathway plays important roles during plant development and stress response (including the control of seed germination, drought response and chlorophyll content). This pathway therefore represents a promising new system for understanding and manipulating plant growth and survival, a key focus for future food security.

We will investigate in detail how this pathway functions and what important aspects of plant life it controls. Studies will be carried out in the plant Arabidopsis - the 'lab rat' of the plant world - since it is much easier to grow and study compared to crop species, yet has all the same genes and mechanisms. We will develop and analyse Arabidopsis plants that have had the key components of this pathway removed (mutants) and ones which 'over produce' them, in order to understand what roles these factors play during normal growth and development. We will also perform studies to see where this pathway is working in the plant, both spatially (i.e. leaves vs roots?) and over time during the life cycle. Collectively this will allow us to dissect where and when this pathway is functional, and identify what key aspects of plant life it regulates. We will also perform biochemical analyses on protein 'targets' of the pathway, to show that their degradation is dependent on Nt-acetylation and subsequent addition of ubiquitin, which will provide important insight into the mechanisms and signals underpinning proteolysis via this pathway, and help guide future studies into identifying natural protein targets.

Functional characterization of this novel pathway will greatly enhance our understanding of plant signalling and behaviour. Since these genes are conserved in important crop species - from barley to broccoli - this research will therefore help inform future studies into creating better, more efficient crop varieties. As well as uncovering an entirely new mechanism for regulating protein stability in plants, this work will also provide new insight into why some proteins are acetylated at their N-terminus. This modification is widely conserved in plants and animals, and was recently linked to human disease, but its functions are largely unknown. Thus our detailed studies will provide scientific insight that may also benefit human and medical research.

The N-end rule pathway of targeted proteolysis is a highly conserved component of the ubiquitin proteasome system (UPS) that degrades proteins based on the nature of their N-terminal (Nt-) amino acid. This pathway has emerged as a critical regulator of development and environmental signal sensing in plants. Recently, studies in yeast have identified a novel branch of the pathway that specifically degrades Nt-acetylated proteins (the 'Ac/N-end rule pathway'). The key enzymes involved - Nt-acetyltransferases and specific E3 ligases - are all present in Arabidopsis and crop plant genomes, but have not been studied in detail previously. We found that knockouts of these genes in Arabidopsis have several shared growth and stress-related phenotypes - including altered germination, ABA and drought responses as well as growth and chlorophyll defects - and that artificial Ac/N-end rule protein reporters accumulate to higher levels in Ac/N-end rule-defective mutants than in wild type plants. These findings suggest that Nt-acetylation can act as a signal for protein degradation via the previously uncharacterised plant Ac/N-end rule, and that this pathway controls a range of important processes. The main focus of this work is to functionally characterize the structural and enzymatic components of the pathway in Arabidopsis, linking their activity to growth, development and stress responses, and to demonstrate that proteolytic targeting via these components is dependent on Nt-acetylation, thus uncovering a novel cellular function for this co-translational protein modification in plants. Furthermore, putative physiological substrates of the pathway will be investigated. Collectively these studies will identify and characterize a new proteolytic pathway regulating processes of agronomic importance and provide a molecular framework for the future identification of protein targets, thus opening up a new area of research into plant proteolysis and signal transduction.

There are a number of key societal and economic impacts that could eventually arise from this research. A growing human population is placing great demands on world agricultural productivity. However, the associated need for improved crop yields is hindered by increasingly extreme and unpredictable weather conditions linked to climate change. Enhancing stress tolerance and productivity in crop species is therefore a key strategic target for agricultural sustainability. Due to the central role targeted proteolysis plays in plant development and environmental interactions, enzymatic components and substrates of protein degradation pathways are prime targets to manipulate in order to modulate plant growth and stress responses.

Our preliminary work suggests that the previously uncharacterised plant Ac/N-end rule pathway for proteolysis plays an essential role during normal plant development, controlling a range of plant responses including seed germination, drought tolerance and chlorophyll content. The work proposed here therefore has the potential to inform future breeding and biotechnological efforts to develop crops with enhanced survivability and improved yields in both marginal lands and areas where harsh environmental conditions can place constraints on productivity, since it will identify new molecular targets and cellular mechanisms associated with diverse agriculturally important plant responses. Targeted manipulation of plant Ac/N-end rule components could ultimately lead to improvements in crop resource utility and food production, which will eventually benefit farmers, consumers and businesses who are reliant upon agriculture (e.g. the bioenergy and brewing industries), both nationally and internationally. This work therefore aligns strongly with the BBSRC's strategic priority area 'sustainably enhancing agricultural productivity'. The Plant Genetics and Cell Biology theme in the School of Biosciences has a formal collaborative agreement with the National Institute of Agricultural Botany (NIAB) in Cambridge, who will enable key research outputs from this work to be presented to relevant breeders and industry.

During the lifetime of this project there will be many opportunities to present this research to non-academic audiences. Impact in this area will be delivered through a number of educational outreach and public engagement approaches. From an educational perspective, aspects of this work will be presented to visiting students (generally aged 16-18) as part of a departmental master class, and will also be promoted to visiting applicants and their parents on University open days. The University of Birmingham is also the subregional partner for STEMNET, a national scheme which aims to stimulate scientific interest in young people; as part of this scheme we will initiate visits to local schools to present our research to younger children. Public engagement will include participation in the recently initiated School of Bioscience's public lecture series, as well as through poster and display contributions at the annual university community day and exhibits at Birmingham's Science museum - the Thinktank - which has successfully hosted plant-specific outreach events from the School of Biosciences in recent years.

This work will also contribute to individual's career development, since it will result in the training of a PDRA who will develop a range of desirable and widely applicable molecular biology skills. This will result in a highly qualified research scientist who will contribute to competitive national and international research, and be prepared for future employment in academic or commercial environments. The associated technician will also obtain a range of scientific skills and research experience that will advance their career development. In addition, it is expected that PhD and MSc student training will be carried out on related aspects of this work alongside the PDRA and technician.