A new model for chloroplast-to-nucleus communication during seedling development

Lead Research Organisation: University of Southampton
Department Name: School of Biological Sciences

Abstract

The ability of plants to use sunlight for photosynthesis is an essential process that supports life on Earth. Photosynthesis represents the most significant source of new energy to the planet and is therefore central to our considerations on future energy needs. Much of our food is also derived from plants, either directly from vegetables, cereals etc, or indirectly as a source of animal food. In plants (and algae), photosynthesis takes place in organelles called chloroplasts. Most of the 2000-3000 proteins contained in the chloroplast are synthesized from DNA present in another organelle called the nucleus, although the chloroplast can make about 80 of its own proteins. When a new chloroplast is made (chloroplast biogenesis), the important role played by the nucleus means that the two organelles need to communicate. We know quite a lot about how the nucleus sends information to chloroplasts, but the mechanisms by which chloroplasts communicate with the nucleus have remained poorly understood despite over 30 years of research in this area. In this proposal we have taken a large body of published information and used it to develop a model for chloroplast-to-nucleus communication. The principal aim of this proposal is to robustly test this model to see if it is correct.

The model proposes two pathways: a promotive pathway in which chloroplasts signal to the nucleus that all is well; and a second, inhibitory pathway that is activated when things go wrong. Specifically, the accumulation of intermediates in the synthesis of the green, photosynthetic pigment, chlorophyll, activates the inhibitory pathway when these pigments are excited by light. The inhibitory pathway then reduces the amount of chlorophyll being made. Such an inhibitory pathway would be important as most chloroplasts are made during early seedling development and too many chlorophyll intermediates would be lethal to a seedling because in the light they are photo-toxic.

The promotive pathway is proposed to be mediated by heme, a molecule related to chlorophyll. We will test whether heme is involved by making plants that contain excess of the heme biosynthesis enzyme ferrochelatase or the heme-degrading enzyme heme oxygenase. We will ascertain whether these plants are still able to communicate between chloroplasts and the nucleus by using assays that measure the expression of specific nuclear genes under a range of different conditions. The second way we will test the model is to examine the role of an important protein in chloroplast-to-nucleus communication called GUN1. This is a chloroplast protein that has been proposed by others to be important in the signaling pathway between chloroplasts and the nucleus. In our model we propose that this is incorrect, and that instead the GUN1 protein has a role in chloroplast biogenesis itself that affects the making of the chloroplast signal. We will test whether this is the case by looking carefully at processes involved in chloroplast biogenesis in mutants lacking GUN1 and conversely in plants that contain excess GUN1 protein. We will determine whether these plants lack the ability to make the chloroplast promotive signal required to maintain nuclear gene expression.

Finally, we will investigate the inhibitory pathway. In a previous study we isolated mutants that were unable to use the inhibitory pathway to reduce nuclear gene expression. In this proposal we will determine which genes are affected in these mutants and use this information to better understand how this pathway might work.

Signals from chloroplasts to the nucleus have been implicated in all sorts of responses to changing environments such as to cold and drought. Our results may have important implications for understanding how plants interact with their changing environment, information that may be important in the future for producing better food and energy crops.

Technical Summary

The development of photosynthetically-active chloroplasts is a critical phase during seedling development and requires co-ordination between the nucleus and chloroplasts. Chloroplasts are able to signal the nucleus to regulate gene expression, but this pathway is poorly understood and represents a major gap in our knowledge. In this proposal we have developed a new model for chloroplast-to-nucleus communication that is consistent with published data in the field and comprises both a promotive pathway from developing chloroplasts mediated by a specific heme pool, and an inhibitory pathway when changes in environmental conditions leads to the accumulation of photo-toxic chlorophyll intermediates. We will test this model robustly in three ways. Firstly we will manipulate heme levels by overexpression of the heme biosynthesis enzyme ferrochelatase and the heme-degrading enzyme heme oxygenase and follow the effect of this on nuclear gene expression. We will also develop new methodology to measure heme pools accurately within seedlings subjected to treatments affecting chloroplast signaling. Secondly, we will determine whether the chloroplast protein GUN1 functions as a repressor of chloroplast development, as we hypothesize, rather than as a central integrator of various chloroplast signaling pathways as generally proposed. We will do this by examining chloroplast development in new gun1 mutants and a GUN1 overexpressor, and testing whether GUN1 affects accumulation of the specific heme pool that functions as the promotive signal. Thirdly, we will identify components of the inhibitory pathway by undertaking a molecular characterization of five mutants unable to downregulate nuclear gene expression following accumulation of chlorophyll intermediates in the light. These mutants have already been isolated and represent a significant resource to this project.

Planned Impact

Plants are central both to agriculture, and to conservation of the natural environment. As well as food, they offer resources for construction, natural fibre, and fuel, and contribute to mitigation of climate change. Given these key roles, it is becoming increasingly recognised that we need to understand better the factors that ensure plant productivity so that we can safeguard our food and, increasingly, fuel supply. The most vulnerable stage of a plant's life is during seedling establishment as it becomes photosynthetically competent. This project will study how the photosynthetic apparatus is assembled efficiently and safely in the early stages of seedling development, and how this may be affected by environmental conditions. Our research will have academic impact, and will provide indicators that may be useful in crop plant management and chloroplast biotechnology.

For the academic community, the topic of chloroplast-to-nucleus signaling has been the subject of considerable research effort, but is still poorly understood. As well as researchers in chloroplast biogenesis, a better understanding of how chloroplasts communicate will also benefit those in the related fields of photosynthesis, photomorphogenesis and stress signaling. The methodology that we will develop during the project to measure heme and other tetrapyrroles will be applicable to many analytical projects both academically and in the medical/industrial arenas. The technician employed on the grant will become highly skilled in the use of sophisticated analytical equipment and methodology, enabling her to progress further in a scientific career. The PDRA will benefit from the opportunity to collaborate with and visit Professor Terauchi at Iwate Biotechnology Research Center in Japan.

In terms of the economic impact, our work on chloroplast development in seedlings will impact on our understanding of seedling establishment, an essential factor in plant survival and therefore crop productivity. Moreover, information about how chloroplasts interact with the environment to provide information to the cell is of considerable interest and likely to be important in designing crop plants that will be able to withstand future environmental change scenarios. Future industrial biotechnological approaches using plants will increasingly focus on strategies to harness the biosynthetic potential of chloroplasts, so a better understanding of the interactions of these organelles with the rest of the cell will support these endeavours. To ensure information from this project has significant impact in these commercial sectors we will continue with links that have been developed with technology transfer enterprises and networks, as well as commercial partners, to exploit opportunities arising from this work. Both applicants (independently) are engaged in more targeted research related to the development of algal biofuels and information and skills arising from the current proposal will be beneficial in supporting this work

Both applicants will utilize ongoing and new public outreach activities to explain the role of light and photosynthesis in plant biology through both school activities and to the general public. The current project has important implications for all those interested in plant productivity whether for agriculture, forestry, conservation, bioenergy or the use of plants for the expression of high value products. The work in the current study will also utilise the model plant Arabidopsis to address a fundamental cellular process, namely communication between organelles. Basic research undertaken with Arabidopsis has been shown to translate not only into crop plants but more broadly to be better understanding of fundamental cellular processes that can impact on human health (Jones et al, 2008, Cell 133, 939-43). These are important broad messages about the potential impact of this work that we intend to communicate.
 
Description The ability of plants to use sunlight for photosynthesis is an essential process that supports life on Earth. Photosynthesis represents the most significant source of new energy to the planet and is therefore central to our considerations on future energy needs. Much of our food is also derived from plants, either directly from vegetables, cereals etc, or indirectly as a source of animal food. In plants (and algae), photosynthesis takes place in organelles called chloroplasts, but most of the 2000-3000 proteins contained in the chloroplast are synthesized from DNA present in another organelle called the nucleus. When a new chloroplast is made (chloroplast biogenesis), the important role played by the nucleus means that the two organelles need to communicate. We know quite a lot about how the nucleus sends information to chloroplasts, but the mechanisms by which chloroplasts communicate with the nucleus have remained poorly understood. We previously developed a model to explain how the chloroplast communicates with the nucleus and the principal aim of this proposal was to robustly test this model to see if it is correct. To do this required the production of numerous transgenic and mutant Arabidopsis lines in which proposed components of this communication pathway are affected. These transgenic and mutant Arabidopsis lines were successfully produced and were used to test our model. Our data support a model in which two signals from the chloroplast to the nucleus operate during chloroplast biogenesis: a positive signal related to the production of the a heme molecule and an inhibitory signal generated by a reactive oxygen species called singlet oxygen. We have also identified mutant plants that are unable to respond to the singlet oxygen signal and identified one of the underlying genes.
Exploitation Route In addition to having an important role in building the photosynthetic apparatus required for energy harvesting, signals from chloroplasts to the nucleus have been implicated in all sorts of responses to changing environments such as to cold and drought. Our results may have important implications for understanding how plants interact with their changing environment, information that may be important in the future for producing better food and energy crops. In particular we have identified a potential interaction between different biogenic retrograde pathways and drought and heat stress responses.
Sectors Agriculture, Food and Drink,Energy,Environment

 
Description Advances Life Sciences Research Technology Initiative (ALERT 14)
Amount £448,654 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2014 
End 11/2015
 
Description Research grant (responsive mode)
Amount £453,248 (GBP)
Funding ID BB/P019331/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2018 
End 12/2020
 
Description SOS for plant stress: singlet oxygen signalling pathways mediating stress acclimation in plants
Amount £9,530 (GBP)
Funding ID IEC\R2\181029 
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 03/2019 
End 03/2021
 
Description SOS for plant stress: singlet oxygen signalling pathways mediating stress acclimation in plants Co-I with Dr H Okamoto
Amount £35,070 (GBP)
Organisation The Gerald Kerkut Charitable Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2017 
End 09/2020
 
Description Sainsbury PhD studentship
Amount £108,000 (GBP)
Organisation Gatsby Charitable Foundation 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2013 
End 09/2017
 
Title Overexpressor lines for Arabidopsis tetrapyrrole-related proteins 
Description We have made a series of transgenic lines to manipulate levels of tetrapyrroles and the plastid signalling protein GUN1 in plant cells. 35S::RecA:GFP (for green fluorescent protein) 35S::CoxIV:GFP 35S::RecA:FC1:GFP 35S::CoxIV:FC1:GFP 35S::RecA:FC1:GFP and 35S::CoxIV:FC1:GFP 35S::RecA:HO1:GFP 35S::RecA:HO1:GFP in a hy1 (Col-0) background 35S::CoxIV:HO1:GFP 35S::(no transit peptide)HO1:GFP 35S::RecA:FC2:GFP 35S::CoxIV:FC2:GFP iBE::GUN1:GFP iBE::GFP iBE::GUN1 35S::GUN1 35S::GUN1:GFP in a gun1 background Once published these lines will be made available to the research community on request. 
Type Of Material Cell line 
Provided To Others? No  
Impact No impact to date. 
 
Description Dr Nobuyoshi Mochizuki - Role of PTM and ABI4 in retrograde signalling 
Organisation University of Kyoto
Country Japan 
Sector Academic/University 
PI Contribution We have performed experiments in parallel to Dr Mochizuki to re-evaluate the role of the PTM and ABI4 proteins in retrograde signalling.
Collaborator Contribution Dr Mochizuki has performed experiments in parallel with us to re-evaluate the role of the PTM and ABI4 proteins in retrograde signalling.
Impact Page MT, Kacprzak SM, Mochizuki N, Okamoto H, Smith AG, Terry MJ (2017) Seedlings lacking the PTM protein do not show a genomes uncoupled (gun) mutant phenotype. Plant Physiol. 174, 21-26.
Start Year 2015
 
Description Dr Ryouichi Tanaka - Understanding the nature of the laf6 mutation 
Organisation Hokkaido University
Department Institute of Low Temperature Science
Country Japan 
Sector Academic/University 
PI Contribution We performed photobiology experiments with mutants and transgenic lines generated by our partner research group.
Collaborator Contribution They generated a series of mutant and transgenic lines with altered levels of SUFB and characterised these with respect to chlorophyll synthesis and chloroplast development.
Impact One paper published: Hu X, Page MT, Sumida A, Tanaka A, Terry MJ, Tanaka R (2017) The iron-sulfur cluster biosynthesis protein SUFB is required for chlorophyll synthesis, but not phytochrome signaling. Plant J. in press, DOI: 10.1111/tpj.13455.
Start Year 2013
 
Description Identification of saf mutations (Terauchi, Japan) 
Organisation Iwate Biotechnology Research Centre
Department Division of Genetics and Genomics
Country Japan 
Sector Public 
PI Contribution We have isolated mutants that are unable to properly communicate between the chloroplast and the nucleus and the collaboration is to identify the causal mutations of these mutants. The mutants have been backcrossed to wild-type plants and mutant and wild-type phenotypes selected in the F2 generation for genomic DNA isolation. We have now identified the nucleotide change responsible for one of the mutants and have narrowed down another to 2 genes.
Collaborator Contribution The collaborator will undertake full genome sequencing of the wild-type and mutant plants and using methods and software he has developed identify the causal mutations based on the pattern of single nucleotide polymorphisms in the sequenced mutant genomes.
Impact We have not yet completed the collaborative activity.
Start Year 2011
 
Description Professor Julian Hibberd - Retrograde signals in the evolution of C4 photosynthesis 
Organisation University of Cambridge
Department Department of Politics and International Studies (POLIS)
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided know-how for experimental design and interpretation of gene expression data for the expression of photosynthesis-related genes in a C4 species.
Collaborator Contribution This work is part of a long term project by the partner laboratory to understand the evolution of C4 photosynthesis. They provided expertise on C4 photosynthesis, high quality experimental data sets and intellectual input in their interpretation.
Impact To date we have one major publication: Burgess SJ, Granero-Moya I, Grangé-Guermente MJ, Boursnell C, Terry MJ, Hibberd JM (2016) Ancestral light and chloroplast regulation form the foundations for C4 gene expression. Nature Plants 2, 16161.
Start Year 2015
 
Description Research project at Peter Symonds College 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Under the Authentic Biology programme we developed a research project that was offered to students at Peter Symonds College in Winchester as part of an extracurricular science club. Matthew Terry gave a short presentation on Arabidopsis photomorphogenesis and chloroplast development and Mike Page supervised a student research project. Students visited the University of Southampton and were given a tour of facilities. The research findings were presented at the Authentic Biology conference in London.
Year(s) Of Engagement Activity 2015
URL http://www.authentic-biology.org/
 
Description School visit (Westgate School, Winchester) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact Students prepared posters that followed on from the discussion on food security and the contribution of current plant research.

Increased interest by the students in the issues around food security and plant research.
Year(s) Of Engagement Activity 2014
 
Description Science and Engineering Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact As part of the University of Southampton exhibits for Science and Engineering Day Matthew Terry (2014) and Mike Page (2015) presented a poster and examples of visually interesting Arabidopsis mutants to stimulate discussion on the relevance of model species to crop research.

No documentable impact realised to date.
Year(s) Of Engagement Activity 2014,2015