Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - SSPinROOTS (Small Signalling Peptides for Stimulating Root Growth - a Novel Approach to Improve Nutrient Use Efficiency)

Teaser

Application of nitrogen (N) and phosphorus (P) fertilizers is done to increase crop yields. However, production of N-fertilizers is associated with greenhouse gas emission and P-fertilizers constitute a finite natural resource. Thus, future sustainable agricultural systems...

Summary

Application of nitrogen (N) and phosphorus (P) fertilizers is done to increase crop yields. However, production of N-fertilizers is associated with greenhouse gas emission and P-fertilizers constitute a finite natural resource. Thus, future sustainable agricultural systems rely on efficient utilization of applied N- and P-fertilizers in order to limit environmental impacts, while sustaining agricultural production. Legumes are able to fix N from the atmosphere, thereby reducing the need for fertilization, but only if the plant can take up sufficient amounts of P in order to run various physiological processes.

Small signaling peptides (SSPs) have emerged as important regulators of developmental processes in response to environmental stimuli, including root development, an important trait for nutrient uptake. SSPs interact with specific receptors to initiate signaling pathways that regulate gene expression and concurrent identification of peptides and their receptors is therefore important.

In this project, SSPs produced by the plant in response to N and P availability have been identified in the legume model plant Medicago truncatula (Gaertn.). Application of synthetically produced peptides to roots has been shown to change root architecture and development of root nodules in which N-fixation takes place. More detailed studies of selected SSPs and their corresponding receptors are currently undertaken in Medicago to unravel the mechanisms controlling the observed root and nodule developmental phenotypes.

Work performed

The overall objectives of the SSPinROOTS project are divided into four work packages (WP), and the work carried out within each of them is described below.

WP1 and WP2: Identification of N- and P-responsive SSPs in roots and nodules of Medicago truncatula
In Medicago truncatula only a small fraction of known SSP families had been annotated upon commencing the project. Therefore, a large effort has been put into identifying Medicago SSPs on a genome-wide scale. This work resulted in the identification of nearly 2,000 SSPs belonging to 46 established families, as well as the identification more than 2,000 potential SSP genes.

With this data in hand, RNA-sequencing data sets from plant tissue deficient in N and P, as well as tissue re-supplied with N and P after deficiency was produced and analyzed for SSPs responding to these conditions. In addition, RNA-sequencing data from roots inoculated with rhizobia and different nodule developmental stages were collected and analyzed. Hundreds of SSP genes were found to significantly change expression in response to nodulation and N- and P-availability. This number of SSPs by far exceeds what was previously known, and is as such a main outcome of the SSPinROOTS project. Based on RNA-sequencing expression profiles, a panel of SSPs for further studies was selected. At this stage, more than 20 different synthetic peptides have been tested for their effect on root growth and nodulation. As a main result of the peptide screening, a set of peptides from the PIP family promoting lateral root development has been identified. Treatment of seedlings with PIP peptides resulted in increased total root length and as such this family of peptides have the potential to improve nutrient uptake in plants.

WP3: Identification of SSP-receptor modules and down-stream target genes
Based on the results from the RNA-sequencing data and the effects of peptide addition on root and nodule development, three Medicago receptors have been selected for further analysis. The first receptor analyzed is the one expected to bind PIP peptides described in WP1. Analyzing the roots of the PIP receptor mutant revealed that lateral root growth is delayed, a phenotype which is opposite to the one observed when adding synthetic PIP peptides seedlings. Hence, these findings indicate that the discovered PIP-receptor signaling module is important for controlling root growth in response to nutrient availability, and thus represents a novel finding of agronomic importance.

Deviations from the original research plan
After a thorough homology analysis in Medicago, it was not possible to identify homologs to the P-responsive Arabidopsis peptide initially intended to be the starting point for receptor studies within this project.

WP4: Postdoctoral training in transferable skills
One of the main goals of WP4 was to improve my skills within project management and project leadership, of which of have gained a lot of experience up until this point. During the first four months, I worked independently with most aspects of the project, while at the same time receiving advice and supervision from my peers at the Noble Foundation. In this period, I made detailed project plans, produced sample material and developed laboratory protocols. In the following period of 7 months, I had the daily responsibility of laboratory technicians who conducted experiments under my supervision. This experience I am now utilizing at University of Copenhagen in terms of student supervision, e.g. I supervise a master student who is studying a nodule-specific SSP, as well as experimental planning with new collaborators from Institute of Plant Sciences Paris-Saclay (IPS2) at Université Paris Sur. Lastly, the part of the project related to bioinformatics is being written together by myself as the primary author, and it is expected to be published in one of the very best journals within plant science. In this process, I am receiving valuable inputs to improve the

Final results

Summary of main results:
i) Known and novel SSP families have been identified in Medicago truncatula, and expression profiles obtained from RNA-sequencing data has clearly shown that SSPs are important signals in response to plant nutrient status. The current project findings have significantly broadened the implications of SSPs in these processes important for agriculture.

ii) In response to N addition, members of a particular SSP family were upregulated (PIP). Addition of synthetic peptides resulted in promotion of root growth. The corresponding receptor displays the opposite root phenotype, and thus this peptide-receptor signaling module constitutes a so far unknown mechanism to control root growth in response to N status. The agronomic importance of this finding will be further tested in a second project.

iii) In response to P availability, a handful peptides from the CLE familiy has been identified, and their role in P-signaling and regulation of symbiosis with mycorrhizal fungi has been studied.

iii) I have significantly improved my skills within teaching, supervision, project management and paper writing. Specifically in the second part of the project, I have increased my teaching activity to graduate, undergraduate and PhD students, and also project-based teaching.

In addition, the project findings have already enabled me to engage with new collaborators from academia which will be beneficial for pursuing the findings after the project ends. This includes researchers from both Denmark, France, England, USA and Australia.