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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - LUPINROOTS (Unravelling cluster root development in white lupin)

Teaser

Summary

Plant development is continuous throughout their lifetime and reflects their ability to adapt to their environment. This developmental plasticity is very obvious in the development of the root system. Surprisingly, the fundamental mechanisms of root development have been studied into great detail but the effect of the environment on its plasticity is still largely unknown. I will use phosphate, since this nutrient has a very low mobility in soil, as a mean to study plant developmental adaptation in white lupin.
This species has developed extreme adaptive mechanism to improve phosphate uptake by producing structures called â€œcluster rootsâ€. They are dense clusters of lateral roots with determinate development and highly specific physiology. I will develop new tools to identify cluster root mutants in white lupin, sequence white lupin genome, perform tissues specific transcriptomics and perform full molecular characterization of selected genes. This project will also lead me to compare adaptive mechanisms between white lupin and narrow-leafed lupin, a closely related species that does not produce cluster roots. We will also test whether it is possible to transfer the ability to form cluster roots in this species. Altogether, this project will lead to a major advance in our capacity to understand how plants are able to sense and respond to their environment and how evolution has selected adaptive developmental mechanisms to improve their capacity to use limited resources.
This project focuses on the most extreme developmental adaptation produced in response to phosphate starvation. It is ambitious, as it will necessitate the development of several tools. However, it is highly feasible since it builds on my previous experience and important outcome can be expected in term of crop improvement and means to reduce the use of phosphate fertilizers.

Work performed

â€¢ From a global point of view, the LUPIN ROOTS project is taking place as planned. There have been some adaptations; a major one is that I decided to move to a different Research Unit located in Montpellier (remaining with the same Host Institution CNRS). This has only positively affected the project by creating a more suitable research environment. The main equipment has been installed without delay and is fully operational. There has been some delay in recruiting personnel but this is only because some of them first joined the team as students, resulting in a positive impact on the project. A CNRS personnel (Fanchon Divol) has joined the team and is working part time on the LUPIN ROOTS project, therefore reinforcing the Work Package 1 (Task 2 Genetic screen to identify white lupin mutants). This additional task force is a great addition to the project and gives me more time to focus on the other aspects that are very time consuming. Indeed, I am devoting more time to the project than expected (73%). To date our main achievements are on Work Package 1 (as planned for this reporting period). The WP2 will start in the next reporting period.
WP1: Explore cluster root formation in white lupin
Task 1
-Full description of cluster root development, setting up histological protocols to image lupin roots. We are now generating more detailed analysis to generate an atlas. (Fanchon Divol, CÃ©lia Casset)
-Setting up white genetic transformation using hairy root, tested several transformation protocols, set up leaf agroinfiltration, produced various plasmids. We are still working on developing a stable transformation system. (CÃ©lia Casset)
Task 2
-Production of an EMS-mutagenized population (sub-contracting). This was performed without delays and the population demonstrated to be of high-quality. (Cost was reduced to 14kâ‚¬)
-Genetic screen started dec 2016 and has now identified several mutants including 3 showing a very strong phenotype. We are now proceeding to genetic analysis and continue screening. (Fanny Garcia, Fanchon Divol)
-Genome sequencing will be performed in 2017 but I have already identified collaborators to perform the high quality DNA production, optical maps, bioinformatics analysis (genome assembly and annotation) and we will soon launch a call for offers for the sequencing itself (PacBio long read sequencing or equivalent). (Partners: CNRGV Toulouse and LIPM Toulouse). Post-doc 2 (Barbara Hufnagel) is leading this task.
-Travels: Benjamin PÃ©ret (PI) Participation to a Lateral Root Workshop (Heidelberg, Germany), visit to CNRGV and LIPM Toulouse with Barbara Hufnagel (setting up collaboration for genome sequencing)
-This ERC project benefitted from additional funding for the purchase of major equipment (500kâ‚¬ including overheads). The equipment was acquired in respect with the time frame (10 months after the starting date) and without technical difficulties.
Details: Installing growth chambers facilities (budget 400kâ‚¬). Growth chambers are fully functional and are used full-time for the genetic screen and other plants exclusively on the LUPIN ROOTS project (100%). Due to change of Research Unit, an additional growth chamber was bought (cost 16,9kâ‚¬) that is also used exclusively on the LUPIN ROOTS project (100%). (Benjamin PÃ©ret)

Final results

We have already identified several mutants affected in cluster root development and have described the development of this new organ. We have now sequenced and annotated the white lupin genome. This represents an invaluable tool for this project. We have now several transcriptomics experiments that have been produced, based on a careful examination of the root system. We should receive and start analyzing the data soon (March 2018). We generated a detailed anatomical description of cluster root development (published in Gallardo et al., 2018).