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SSPinROOTS Report Summary

Project ID: 659251
Funded under: H2020-EU.1.3.2.

Periodic Reporting for period 1 - SSPinROOTS (Small Signalling Peptides for Stimulating Root Growth - a Novel Approach to Improve Nutrient Use Efficiency)

Reporting period: 2015-05-01 to 2016-04-30

Summary of the context and overall objectives of the project

Nitrogen (N) and phosphorus (P) fertilizers are used 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 the biochemical processes.

P-uptake is largely dependent on the ability of plant roots to exploit the soil, and therefore identification of genes controlling root development and root branching are intrinsically linked to efficient N-fixation. Small signaling peptides (SSPs) have emerged as important regulators of developmental processes in response to environmental stimuli, including root development. 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. In the second part of the project, pilot experiments to test the ability of synthetic peptides to improve nutrient uptake and N-fixation, and thus the potential of such peptides as bio-fertilizers, will be carried out.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Work carried out by the beneficiary

The overall objectives of the SSPinROOTS project are divided into four work packages (WP). Relevant for this reporting are WP1, WP3 and WP4, and the work carried out within each of them is described below.

WP1: 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, and therefore, a large effort has been put into identifying Medicago SSPs on a genome-wide scale. This work resulted in the identification of 289 SSPs belonging to 15 known families, as well as the identification of 3 novel SSP families. 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. More than 50 SSPs were involved in nodule development, and more than 60 responded to N and P status. This number of SSPs by far exceeds what was previously known. 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, and more peptides are currently being tested. As a main result of the peptide screening, a set of peptides promoting lateral root development has been identified, here coined Promote Lateral Root Growth (PLRG). The PLDG peptides will be further studied for their effects on nutrient uptake in WP2.

Deviations from original research plan:
The bioinformatics work took longer than anticipated due to a much more thorough approach taken, which has resulted in a high quality data set containing N- and P-responsive SSPs. The extra effort put into the bioinformatics part has delayed the selection of peptides for screening assays, but they are currently being conducted and the results will be obtained within the next three months. This will be in due time for testing interesting candidates for their effect on nutrient uptake before ending the project.

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 based on homology to SSP receptors known from Arabidopsis. Mutants in these receptors were obtained from a mutant collection held at the Noble Foundation. The first receptor analyzed is the one expected to bind the PLDG peptides described in WP1. The two other receptors are currently being propagated in the greenhouse in order to identify homozygous lines. Analyzing the roots of the PLRG receptor mutant revealed that lateral root growth is delayed, a phenotype which is opposite to the one observed when adding synthetic PLRG peptides seedlings. Hence, these findings indicate that the discovered PLRG-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. Hence, focus was moved from identifying the receptor of the P-responsive peptide, to work on receptors of nutrient regulated Medicago SSPs. However, a screening assay was set up to identify putative receptor candidates in Arabidopsis. This work revealed a known SSP receptor to take part in the signaling events downstream of the P-responsive SSP, as this mutant was not affected by peptide treatment. But due to the above-mentioned lack of a Medicago homolog the main focus is now on other receptors.

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, 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 quality of the paper.

Deviations from the original plan
WP4 continues into the second half of the project period. Here I will have an increased focus on outreach activities, which includes an oral presentation at the European Peptide Meeting in August 2016. In addition, I will seek to be involved in course activities to promote the most exciting findings achieved within this project.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Summary of main results in the first project period:
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 (PLRG). 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 tested in the second half of the project.

iii) Training in transferable skills has been obtained through the supervision and daily responsibility of technical staff. Moreover, after returning to University of Copenhagen, I have begun student supervision. Valuable skills and advice related to writing excellent papers and successful grant proposals have also been obtained. Furthermore, the project goals have been presented at the European Peptide Meeting 2015, at three research seminars and in the form of a popular science article for the scientific webpage “”.

Overall project progression towards objectives and milestones

The main objective of the SSPinROOTS project was to identify SSPs important for root and nodule development in response to nutrient status, and to gain insight in the underlying signaling pathways. The analyses carried out so far have resulted in an unexpectedly high number of SSPs with interesting expression profiles. In prolongation of these findings, specific SSPs have been tested for their effect on root and nodule development, and furthermore, the roles of the corresponding receptors are under investigation. The results obtained are of high importance with respect to understanding how plants perceive and respond to N and P availability. Further studies to gain insight into the down-stream signaling components are still required and are currently being pursued. Taken together, the achievements obtained in the project so far fully live up to what was anticipated before commencing the project.

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. In the second half of the project, more focus will be given to test the effects of SSPs on nutrient uptake. In relation to this, increased focus will be given to disseminate my results to potential industrial partners in order to have relevant findings tested in a commercial setting in the near future.

Project progression and milestones achieved
Milestones in WP1 included identification (1.1) and testing of SSPs responding to nutrient availability in relation to root development (1.2) and nodule formation, infection and activity (1.3). Milestone 1.1 has been fully achieved, whereas milestones 1.2 and 1.3 have been started and are still ongoing. The bioinformatics work took longer time than anticipated and revealed a much higher number of interesting SSP candidates, which altogether has delayed the subsequent work.

Milestones in WP3 included a list of receptor candidates for the P-responsive peptide in Arabidopsis (3.1). A screening assay was set up which resulted in identification of a known SSP receptor acting downstream of the P-responsive SSP. Homologs to the P-responsive SSP itself could not be identified in Medicago, and therefore focus was changed to Medicago receptors of nutrient-responsive SSPs (3.2). In summary, milestones 3.1 and 3.2 have been achieved, and will enable fulfillment of milestone 3.3.

Milestones in WP4 included project management and project leadership activities, as well as teaching and outreach. These milestones have been fully achieved. Deliverable D4 has also been achieved in the form of a publication at the website “”:
Record Number: 190412 / Last updated on: 2016-11-15
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