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

Lead Research Organisation: University of Birmingham
Department Name: Sch of Biosciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description What were the most significant achievements from the award?

We have now characterised the NOT4 E3 ligases, which were putative N-recognins of the Ac/N-end rule. Our data indicate that these are, biochemically, functionally equivalent to NOT4 in yeast, and that they can degrade N-terminally acetylated targets. We have however found that their overall function in plants is related to the co-translational quality control of protein synthesis, which seems distinct from yeast. For one of the isoforms, we have connected its activity in the cytosol to the biogenesis and function of chloroplasts, through controlling production and stability of key regulatory proteins that are nuclear encoded but translocate to the chloroplast. This work is currently being written up for submission in the next 2 months, and will represent the first main output form this grant. Other achievements associated with this award are the identification and characterisation of VRN2 as an oxygen regulated N-end rule target in plants (Gibbs et al 2018 Nature communications amd Labandera et al 2020 New Phytologist), and we also contributed to the understanding of N-end rule regulated flooding tolerance in rice (Li et al 2019 PNAS).


To what extent were the award objectives met?

Overall a majority of research objectives were met. We developed and characterised all the lines that were outlined in the grant, and have uncovered novel new functions for these Ac/N-end rule regulated N-recognins in the control of various aspects of plant development and stress response. We envisage at least two further papers will directly arise from this work.
Exploitation Route Some of the findings from this work have already been used to secure a follow on grant (KatNat, an ERACAPs grant). We now are in the process of developing an application for further funding where we will seek to delineate the mechanisms through which the three NOT4 ligases in plants coordinate and control protein synthesis and translation rates to ensure proteostasis under stress. Ultimately, our increased understanding of the regulation of proteostasis, and particularly its control of chloroplast function, might be of use for mainpulation plant productivity in crop species.
Sectors Agriculture, Food and Drink

 
Description ERC-STG
Amount € 1,500,000 (EUR)
Funding ID 715441 
Organisation European Research Council (ERC) 
Sector Public
Country Belgium
Start 03/2017 
End 02/2022
 
Description Investigating the cytosolic NOT4 E3 ligase as a regulator of chloroplast function in Arabidopsis
Amount £2,950 (GBP)
Funding ID BB/T004002/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2019 
End 04/2019
 
Description KatNat 
Organisation Heidelberg University
Country Germany 
Sector Academic/University 
PI Contribution My group at the University of Birmingham, together with research groups from the four institutes listed above, is part of the multi-partner EU-wide grant "KatNat - Elucidating the multifaceted functions of protein acetyltransferases in plant stress response and regulation of metabolism". This grant has been recently funded by the DFG and ANR as part of the ERA-CAPs third call. Unfortunately, due to BBSRC budget restraints in this particular call, i will be self-financed in this consortium. Nontheless, my participation in this new network stems from significant progress we have made during the current BBSRC grant, where we have uncovered a direct link between N-terminal protein acetylation and protein stability. The data and tools we have generated (yet to be published) mean we are in a position to directly interact with experts at these other institutes (e.g. proteomics experts at I2BC, and biochemistry experts at Heidelberg), to fully characterise this novel protein degradation system. This will being added value to current research funded by the BBSRC, and strengthen links between UK science and that being conducted in other European countries. This new collaboration and its anticipated outputs would not have been possible without BBSRC funding.
Collaborator Contribution This partnership has just commenced, with the recent announcement that funding was approved (except for the UK component, see prevous section). The abstract for the grant, which highlights key aims for the next three years, is copied here: Plants must constantly respond to a wide range of signals, including stresses, in order to coordinate their development and survival within a dynamic environment. One way in which this is achieved is through chemical modifications of proteins, allowing flexible and rapid changes of the proteome to alter cellular and physiological outputs. Protein acetylation is one such modification, which occurs on the N-termini (Nt) and internal lysines (K) of many proteins. Despite its prevalence, and in contrast to other well-studied modifications (e.g., phosphorylation), our knowledge of: (i) the regulation, specificity and plasticity of protein acetylation, and (ii) its downstream functional consequences on protein activity and physiology are severely lacking. It is therefore extremely timely to elucidate the multifaceted functions of protein acetylation and open up this new area of plant molecular biology, in which Europe has the capacity to take a world lead through strategic ERA-CAPS funding. The overarching aim of the KatNat project is to provide a mechanistic understanding of protein acetylation in plants, with a particular focus on investigating the enzymes that catalyze this modification (Nt- and K-acetyltransferases) and the resultant effects on proteostasis, photosynthesis, and metabolism. Crucially, this work will be carried out within the context of agronomically relevant stresses. KatNat consists of four interrelated objectives that will answer broad questions: (1) How does abiotic stress regulate the global Nt- and K-acetylome?; (2) What are the specificities, targets and stress-responsive dynamics of the acetyltransferases?; (3) How does protein acetylation impact protein stability and turnover?; (4) How does protein acetylation in plastids regulate photosynthesis and metabolism? By answering these connected questions, KatNat will not only shift the forefront of the field but will provide regulatory mechanisms and fundamental insight into how plants sense and respond to environmental changes. Last but not least, the obtained information will identify key new targets for the future development of superior crops. The KatNat consortium brings together five European groups, who all have a significant, demonstrable interest in the study of protein acetylation, and who have the highly complementary expertise in mass spectrometry, protein biochemistry and molecular plant biology required to carry out this original research at the highest international level. Consortium members already have a world lead in this field, and several members currently collaborate informally. The synergistic value of our collaboration will be the development and exploitation of an understudied area of in plant science, with key importance to agriculture. The proposed research is highly innovative, aligns closely with ERA-CAPS priority themes and has measurable and impactful outcomes that will shed light onto this emerging, exciting and important new area of plant biology
Impact n/a
Start Year 2018
 
Description KatNat 
Organisation Institute for Integrative Biology of the Cell (I2BC)
Country France 
Sector Academic/University 
PI Contribution My group at the University of Birmingham, together with research groups from the four institutes listed above, is part of the multi-partner EU-wide grant "KatNat - Elucidating the multifaceted functions of protein acetyltransferases in plant stress response and regulation of metabolism". This grant has been recently funded by the DFG and ANR as part of the ERA-CAPs third call. Unfortunately, due to BBSRC budget restraints in this particular call, i will be self-financed in this consortium. Nontheless, my participation in this new network stems from significant progress we have made during the current BBSRC grant, where we have uncovered a direct link between N-terminal protein acetylation and protein stability. The data and tools we have generated (yet to be published) mean we are in a position to directly interact with experts at these other institutes (e.g. proteomics experts at I2BC, and biochemistry experts at Heidelberg), to fully characterise this novel protein degradation system. This will being added value to current research funded by the BBSRC, and strengthen links between UK science and that being conducted in other European countries. This new collaboration and its anticipated outputs would not have been possible without BBSRC funding.
Collaborator Contribution This partnership has just commenced, with the recent announcement that funding was approved (except for the UK component, see prevous section). The abstract for the grant, which highlights key aims for the next three years, is copied here: Plants must constantly respond to a wide range of signals, including stresses, in order to coordinate their development and survival within a dynamic environment. One way in which this is achieved is through chemical modifications of proteins, allowing flexible and rapid changes of the proteome to alter cellular and physiological outputs. Protein acetylation is one such modification, which occurs on the N-termini (Nt) and internal lysines (K) of many proteins. Despite its prevalence, and in contrast to other well-studied modifications (e.g., phosphorylation), our knowledge of: (i) the regulation, specificity and plasticity of protein acetylation, and (ii) its downstream functional consequences on protein activity and physiology are severely lacking. It is therefore extremely timely to elucidate the multifaceted functions of protein acetylation and open up this new area of plant molecular biology, in which Europe has the capacity to take a world lead through strategic ERA-CAPS funding. The overarching aim of the KatNat project is to provide a mechanistic understanding of protein acetylation in plants, with a particular focus on investigating the enzymes that catalyze this modification (Nt- and K-acetyltransferases) and the resultant effects on proteostasis, photosynthesis, and metabolism. Crucially, this work will be carried out within the context of agronomically relevant stresses. KatNat consists of four interrelated objectives that will answer broad questions: (1) How does abiotic stress regulate the global Nt- and K-acetylome?; (2) What are the specificities, targets and stress-responsive dynamics of the acetyltransferases?; (3) How does protein acetylation impact protein stability and turnover?; (4) How does protein acetylation in plastids regulate photosynthesis and metabolism? By answering these connected questions, KatNat will not only shift the forefront of the field but will provide regulatory mechanisms and fundamental insight into how plants sense and respond to environmental changes. Last but not least, the obtained information will identify key new targets for the future development of superior crops. The KatNat consortium brings together five European groups, who all have a significant, demonstrable interest in the study of protein acetylation, and who have the highly complementary expertise in mass spectrometry, protein biochemistry and molecular plant biology required to carry out this original research at the highest international level. Consortium members already have a world lead in this field, and several members currently collaborate informally. The synergistic value of our collaboration will be the development and exploitation of an understudied area of in plant science, with key importance to agriculture. The proposed research is highly innovative, aligns closely with ERA-CAPS priority themes and has measurable and impactful outcomes that will shed light onto this emerging, exciting and important new area of plant biology
Impact n/a
Start Year 2018
 
Description KatNat 
Organisation University of Münster
Country Germany 
Sector Academic/University 
PI Contribution My group at the University of Birmingham, together with research groups from the four institutes listed above, is part of the multi-partner EU-wide grant "KatNat - Elucidating the multifaceted functions of protein acetyltransferases in plant stress response and regulation of metabolism". This grant has been recently funded by the DFG and ANR as part of the ERA-CAPs third call. Unfortunately, due to BBSRC budget restraints in this particular call, i will be self-financed in this consortium. Nontheless, my participation in this new network stems from significant progress we have made during the current BBSRC grant, where we have uncovered a direct link between N-terminal protein acetylation and protein stability. The data and tools we have generated (yet to be published) mean we are in a position to directly interact with experts at these other institutes (e.g. proteomics experts at I2BC, and biochemistry experts at Heidelberg), to fully characterise this novel protein degradation system. This will being added value to current research funded by the BBSRC, and strengthen links between UK science and that being conducted in other European countries. This new collaboration and its anticipated outputs would not have been possible without BBSRC funding.
Collaborator Contribution This partnership has just commenced, with the recent announcement that funding was approved (except for the UK component, see prevous section). The abstract for the grant, which highlights key aims for the next three years, is copied here: Plants must constantly respond to a wide range of signals, including stresses, in order to coordinate their development and survival within a dynamic environment. One way in which this is achieved is through chemical modifications of proteins, allowing flexible and rapid changes of the proteome to alter cellular and physiological outputs. Protein acetylation is one such modification, which occurs on the N-termini (Nt) and internal lysines (K) of many proteins. Despite its prevalence, and in contrast to other well-studied modifications (e.g., phosphorylation), our knowledge of: (i) the regulation, specificity and plasticity of protein acetylation, and (ii) its downstream functional consequences on protein activity and physiology are severely lacking. It is therefore extremely timely to elucidate the multifaceted functions of protein acetylation and open up this new area of plant molecular biology, in which Europe has the capacity to take a world lead through strategic ERA-CAPS funding. The overarching aim of the KatNat project is to provide a mechanistic understanding of protein acetylation in plants, with a particular focus on investigating the enzymes that catalyze this modification (Nt- and K-acetyltransferases) and the resultant effects on proteostasis, photosynthesis, and metabolism. Crucially, this work will be carried out within the context of agronomically relevant stresses. KatNat consists of four interrelated objectives that will answer broad questions: (1) How does abiotic stress regulate the global Nt- and K-acetylome?; (2) What are the specificities, targets and stress-responsive dynamics of the acetyltransferases?; (3) How does protein acetylation impact protein stability and turnover?; (4) How does protein acetylation in plastids regulate photosynthesis and metabolism? By answering these connected questions, KatNat will not only shift the forefront of the field but will provide regulatory mechanisms and fundamental insight into how plants sense and respond to environmental changes. Last but not least, the obtained information will identify key new targets for the future development of superior crops. The KatNat consortium brings together five European groups, who all have a significant, demonstrable interest in the study of protein acetylation, and who have the highly complementary expertise in mass spectrometry, protein biochemistry and molecular plant biology required to carry out this original research at the highest international level. Consortium members already have a world lead in this field, and several members currently collaborate informally. The synergistic value of our collaboration will be the development and exploitation of an understudied area of in plant science, with key importance to agriculture. The proposed research is highly innovative, aligns closely with ERA-CAPS priority themes and has measurable and impactful outcomes that will shed light onto this emerging, exciting and important new area of plant biology
Impact n/a
Start Year 2018
 
Description KatNat 
Organisation University of Turku
Country Finland 
Sector Academic/University 
PI Contribution My group at the University of Birmingham, together with research groups from the four institutes listed above, is part of the multi-partner EU-wide grant "KatNat - Elucidating the multifaceted functions of protein acetyltransferases in plant stress response and regulation of metabolism". This grant has been recently funded by the DFG and ANR as part of the ERA-CAPs third call. Unfortunately, due to BBSRC budget restraints in this particular call, i will be self-financed in this consortium. Nontheless, my participation in this new network stems from significant progress we have made during the current BBSRC grant, where we have uncovered a direct link between N-terminal protein acetylation and protein stability. The data and tools we have generated (yet to be published) mean we are in a position to directly interact with experts at these other institutes (e.g. proteomics experts at I2BC, and biochemistry experts at Heidelberg), to fully characterise this novel protein degradation system. This will being added value to current research funded by the BBSRC, and strengthen links between UK science and that being conducted in other European countries. This new collaboration and its anticipated outputs would not have been possible without BBSRC funding.
Collaborator Contribution This partnership has just commenced, with the recent announcement that funding was approved (except for the UK component, see prevous section). The abstract for the grant, which highlights key aims for the next three years, is copied here: Plants must constantly respond to a wide range of signals, including stresses, in order to coordinate their development and survival within a dynamic environment. One way in which this is achieved is through chemical modifications of proteins, allowing flexible and rapid changes of the proteome to alter cellular and physiological outputs. Protein acetylation is one such modification, which occurs on the N-termini (Nt) and internal lysines (K) of many proteins. Despite its prevalence, and in contrast to other well-studied modifications (e.g., phosphorylation), our knowledge of: (i) the regulation, specificity and plasticity of protein acetylation, and (ii) its downstream functional consequences on protein activity and physiology are severely lacking. It is therefore extremely timely to elucidate the multifaceted functions of protein acetylation and open up this new area of plant molecular biology, in which Europe has the capacity to take a world lead through strategic ERA-CAPS funding. The overarching aim of the KatNat project is to provide a mechanistic understanding of protein acetylation in plants, with a particular focus on investigating the enzymes that catalyze this modification (Nt- and K-acetyltransferases) and the resultant effects on proteostasis, photosynthesis, and metabolism. Crucially, this work will be carried out within the context of agronomically relevant stresses. KatNat consists of four interrelated objectives that will answer broad questions: (1) How does abiotic stress regulate the global Nt- and K-acetylome?; (2) What are the specificities, targets and stress-responsive dynamics of the acetyltransferases?; (3) How does protein acetylation impact protein stability and turnover?; (4) How does protein acetylation in plastids regulate photosynthesis and metabolism? By answering these connected questions, KatNat will not only shift the forefront of the field but will provide regulatory mechanisms and fundamental insight into how plants sense and respond to environmental changes. Last but not least, the obtained information will identify key new targets for the future development of superior crops. The KatNat consortium brings together five European groups, who all have a significant, demonstrable interest in the study of protein acetylation, and who have the highly complementary expertise in mass spectrometry, protein biochemistry and molecular plant biology required to carry out this original research at the highest international level. Consortium members already have a world lead in this field, and several members currently collaborate informally. The synergistic value of our collaboration will be the development and exploitation of an understudied area of in plant science, with key importance to agriculture. The proposed research is highly innovative, aligns closely with ERA-CAPS priority themes and has measurable and impactful outcomes that will shed light onto this emerging, exciting and important new area of plant biology
Impact n/a
Start Year 2018
 
Description MIBTP CASE studentship (Syngenta) 
Organisation Syngenta International AG
Department Syngenta Ltd (Bracknell)
Country United Kingdom 
Sector Private 
PI Contribution We have recently been awarded a BBSRC CASE studentship as part of the Midlands Integrative Biosciences Training Partnership (MIBTP), with Syngenta. This is based off the back of key work undertaken as part of this BBSRC award, which is yet to be published.
Collaborator Contribution We are currently shortlisting to recruit the student who will work on this project, which will commence in September 2020. the project is entitled: Characterisation and chemical inhibition of a novel regulatory module controlling chloroplast protein translation, and will be carried out in collaboration with Syngenta.
Impact n/a
Start Year 2020
 
Description N-term 2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact In the last few years, a vibrant N-end rule research community has formed in Europe. It was the aim of this workshop to bring together researchers from established labs worldwide to exchange ideas about current topics in the fields of the N-end rule pathway and N-terminal protein modifications at a workshop entitled "N-term 2017: Proteostasis via the N-terminus". A key focus was on engaging postgraduate students (primarily PhD students). To our knowledge, this was the the first international meeting focussing on the N-end rule and we believe it is a unique opportunity to bring together this large and diverse community.
Year(s) Of Engagement Activity 2017
URL http://nterm2017.dbg-afgn.de/