Re-engineering photosynthesis: targeted genome editing to replace nuclear-encoded Rubisco in higher plants

Lead Research Organisation: University of Edinburgh
Department Name: Sch of Biological Sciences

Abstract

Rubisco is the enzyme responsible for CO2 assimilation in the vast majority of photosynthetic organisms, including higher plants. It is relatively inefficient, and decades of research have gone into attempting to improve its affinity and specificity for CO2. Success has been very limited, in large measure because of a dearth of tools with which to manipulate Rubisco properties in planta (1).

A growing set of synthetic biology-based tools now enable plant genomes to be edited with unprecedented precision. The aim of this project is to develop a means of replacing the family of nuclear-encoded small subunits (SSUs) of the Rubisco enzyme in Arabidopsis with heterologous SSUs of choice. This approach will provide an unprecedented capacity to evaluate the role of Rubisco SSU properties in determining photosynthetic efficiency in a range of conditions (2). It opens up the prospect of enhancing crop yields in the future through a novel genetic tailoring strategy.

Training: The project is an outstanding training opportunity for developing a multidisciplinary approach to plant science. You will develop expertise in a wide range of molecular and whole plant analysis skills, including DNA, RNA and protein analyses (e.g. qRT-PCR, Western blot, enzyme activity assays), and photosynthetic physiology using leaf gas exchange and fluorescence techniques. You will gain experience in synthetic biology-based approaches by working with transgenic Arabidopsis lines using the CRISPR/cas9 system (3).

These lines will be used to directly and unambiguously test if substitution of native SSUs can lead to i) the assembly of functional Rubisco holoenzymes, ii) improved Rubisco performance, and iii) increased plant growth rates. This work will provide important new information for both fundamental and applied research about the regulation and makeup of Rubisco in plants and the role of the SSU in growth and development. You will interact with crop scientists to develop strategies to take important findings through to application in crops. There will also abundant opportunity for you to present your research at regular meetings, including national and international conferences.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M010996/1 30/09/2015 31/03/2024
1645210 Studentship BB/M010996/1 30/09/2015 29/09/2019
 
Description One of the first stages of photosynthesis in plants is the capture of CO2 from the atmosphere. A protein called Rubisco captures CO2 and converts it into a molecule that is later used to synthesise sugar; however, it is inefficient in crops such as rice and wheat. To understand why Rubisco proteins from some species are more efficient than others it is important to understand the composition of the Rubisco protein. In particular, we are interested in a part of the protein called the small subunit (SSU). First, we characterised the family of SSU proteins in a model crop species for Rubisco engineering (tobacco (Nicotiana tabacum)). Second, we used a genetic engineering method, called CRISPR/Cas9, to remove most of the native SSUs from tobacco and replace them with non-native SSUs. The main outcome of this work was the development of a variety of tobacco that has ca. 7% of wild-type Rubisco content. This Rubisco mutant offers a useful platform to test the effect of non-native SSUs on Rubisco activity, plant photosynthesis, and growth.
Exploitation Route The tobacco Rubisco mutant is currently being used to test the effect of non-native SSUs on Rubisco activity, plant photosynthesis, and growth. This future work has implications outside of academia for strategies to engineer Rubisco to improve photosynthesis and growth in crops.
Sectors Agriculture, Food and Drink

 
Title Rubisco small subunit mutant 
Description CRISPR/Cas9 was used to mutate two Rubisco small subunit (SSU) genes in tobacco to produce a line that has a ca. 93% reduction in Rubisco content. We have obtained mutants lacking the CRISPR/Cas9 transgene that can be used as a model for SSU engineering strategies. 
Type Of Material Biological samples 
Year Produced 2019 
Provided To Others? No  
Impact The Rubisco small subunit mutant is currently being used by our group to test the effect of non-native SSUs on plant growth and photosynthesis, which contributes to strategies to engineer Rubisco in crop species. 
 
Description Midlothian Science Festival Activity 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact A virtual reality game was made to communicate basic principles of photosynthesis to children (target age <10). As well as engaging with the children we also had the opportunity to discuss the importance of our research with adults that were also attending. We received excellent feedback from the public and organizers.
Year(s) Of Engagement Activity 2017