Yield improvement of oilseed rape through genetic manipulation of rhizosphere exudation

Plants use photosynthesis to fix CO2 to sugars, which are used for plant growth and development. Up to 50 % of carbon (C) fixed by plants is moved to roots, where it supports growth of root systems through the soil, in order to take up nutrients and water and drive growth. Healthy growing roots pass a large proportion of the C they receive to the soil as 'rhizodeposits'. This includes a range of materials, but soluble exudates, consisting of organic acids (e.g. citric acid), carbohydrates (e.g. glucose) and amino acids (e.g. leucine) comprise the largest rhizodeposit component. The total amount of rhizodeposits, and the specific types lost from roots, vary between plant species and genotypes within species, and is influenced by plant developmental stage and environmental parameters. Because of rhizodeposition and the presence of readily available C, the soil surrounding roots has large populations of microbes, forming a distinct microbial niche, which is termed the rhizoshere. Rhizosphere inhabiting microbes interact with plants in many ways- some are pathogens or beneficial symbionts, and others are involved in cycling and transformation of crop nutrients such as nitrogen (N) and phosphorus (P), altering their availability to plants. These processes determine plant productivity even in agricultural systems in which fertilisers are added to maximise production. The types of organisms which grow in the rhizosphere are determined by the amount and types of materials plants exude.

In the current project we propose to elucidate plant genes involved in determining the amount and types of substrates lost from roots as rhizodeposits, and the response of exudation to plant development. Plants exude a very diverse collection of organic molecules and we will take advantage of cutting edge mass spectrometry techniques to understand the full complexity and composition of rhizodeposits for the first time. Additionally we will use newly emerging high throughput sequencing techniques to profile the genes expressed in roots and their association with release of rhizodeposits.

Furthermore we will investigate the way in which altering the composition of rhizodeposits influences the diversity and composition of microbes inhabiting the rhizosphere, and investigate how the function of these communities is influenced by varying exudate quality and quantity. Soil microbial communities are incredibly abundant and diverse, and we will use the latest high throughput sequencing techniques to characterise the microbial metagenome, allowing us to investigate all the major processes taking place in the rhizosphere simultaneously and the specific components of the community involved. Furthermore we will determine the consequences of rhizodeposition for crop yield, which will provide the first direct quantification of the benefit plants receive from rhizodeposition.

The work will open exciting possibilities to breed new crop varieties in which rhizodeposition is managed to enhance crop yields, increase agricultural sustainability and reduce environmentally damaging inputs. This work could lead to the identification of genes which could be used in breeding programmes for a number of applications, for example 1. Reduction of rhizodeposition could be used to increase C allocated to above ground growth, so that crop yields are enhanced. 2.Managing rhizodeposit quantity and quantity could be used to tailor exudation to promote solubilisation and uptake of growth limiting nutrients such as P, S, K and trace metals, and increase tolerance to harmful soil metals, particularly aluminium 3. The quality and quantity of rhizodeposits could be managed to engineer specific microbial communities in the rhizosphere. This could have many advantages, for instance inhibiting the growth of soil-borne pathogens which reduce crop growth, and stimulating communities which enhance the availability of crop growth limiting nutrients, such as P and N.

Healthy roots pass large amounts of C into soil as rhizodeposits. The function of some rhizodeposit components is known, but that of others is unclear. Rhizodeposits massively stimulate microbial populations in the rhizosphere. The aim of this work is to identify genes controlling rhizodeposition in oilseed rape (OSR) and the effect of developmental stage on rhizodeposition. We will then investigate how genetic control of rhizodeposition could be used as a tool to engineer rhizosphere microbiology to promote functions beneficial to crops.

Genetic variability in rhizodeposition between OSR genotypes at different developmental stages will be determined using 14C pulse-labelling. The chemistry of the major rhizodeposit pool and soluble exudates, will be determined by measuring the secreted metabolome, including organic acid, carbohydrate and amino acid pools using a range of techniques, including the cutting edge technique FTICR-MS which provides ultra-high resolution and accuracy of complex organic mixtures. Exudate chemistry will be analysed in mapping lines with parents shown to have maximum differences in rhizodeposit chemistry, to identify QTL controlling exudate characteristics. High throughput sequencing will be used to investigate the root transcriptome and identify genes associated with rhizodeposition. Using contrasting lines in which genes controlling exudation have been identified, we use cutting edge metagenomic techniques to study the impact of rhizodeposit characteristics on microbial community functioning. We will also investigate how those genetic regions we identify which control rhizodeposition influence crop yield in the field.

The work will identify genes which could be used by OSR breeders to alter rhizodeposition to increase yield, by altering C partitioning within the plant, changing rhizodeposit content to enhance nutrient uptake, or engineering rhizosphere microbes to promote functions beneficial to the plant.

The scientific objectives of this project are given in the case for support; the project also has the following objectives aimed at transferring material and knowledge to industrial members of the CIRC:

Lines possessing beneficial alleles at QTL for rhizodeposition traits (quality and quantity) will be identified for use in OSR breeding and released to breeding companies for crossing with elite lines (WP1 and 2; month 24)

The genetic markers flanking the QTL will be identified and released to breeding companies for use in marker assisted selection in breeding for rhizodeposition traits (WP2; month 24)

Knowledge will be made available as to whether variation in rhizodeposition impacts on yield either directly or via manipulation of the soil microbiota, focussing on microbial functional traits important to supporting crop growth such as nutrient cycling processes (WP 4 and 5; month 36)

Knowledge of the role of genotype x environment on rhizodeposition will be communicated to breeders to allow them to judge the value of breeding for the trait (WP4; month 36)

These objectives will be met by the work under WP1,2,4, and 5 described in the case for support. In addition, if work in WP3 identifies genes which appear to be associated with variation in rhizodeposition gene specific markers can be developed for use in marker assisted selection.
The reporting structure of the CIRC will provide regular fora for exchange of information to club members. In addition we have regular contact with the breeding company members of the club through the project advisory committee, our collaborations in the Oilseed Rape Genetic Improvement Network (OREGIN) and prior to submission of the full proposal have consulted with representatives regarding the commercialisation of the research outputs taking their views into account in writing the full proposal.

Furthermore we have agreement from industry representatives to provide genetic material for use in the proposal. We intend to initiate a project advisory panel which would act as a forum for industry participation in the project. A number of oilseed rape breeders have already agreed to participate in this panel, and we would extend membership to other interested partners from within CIRC.

The results of the research will be communicated through additional routes to increase its impact, including publications in leading peer-reviewed journals specializing in plant science and soil and / or environmental microbiology, presentations at key scientific conferences. Further publicity of the project and scientific progress will be made through dedicated project Web pages of the project's host institutions, the OREGIN and Brassica.info websites and other media as deemed appropriate. Prior to publication of results permission will be sought from the CIRC as specified in the special conditions for this BBSRC initiative.

Although the OSR breeding industry will be the primary route for delivery of the applied outcomes of this research, the impact of the findings will have a much wider scope. For instance, farmers will benefit with improved profitability due to increased yields and more effective use of fertilizer applications. The environment will benefit through reduced pollution. Increasing yield per acre of farmland also contributes to feeding the increasing population without extensive land use change.

Furthermore we have agreement from industry representatives to provide genetic material for use in the proposal (see attached information). We intend to initiate a project advisory panel which would act as a forum for industry participation in the project. A number of oilseed rape breeders have already agreed to participate in this panel, and we would extend membership to other interested partners from within CIRC.