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Post-translational modifications during floral initiation: molecular dissection of the UFO-dependent LFY activation

Periodic Reporting for period 1 - PFIMDULA (Post-translational modifications during floral initiation: molecular dissection of the UFO-dependent LFY activation )

Reporting period: 2017-06-08 to 2019-06-07

Issue: Why LEAFY (LFY), a transcriptional factor, activity require interaction with unusual floral organ (UFO), a Skp1-Cullin-F-box E3 ubiquitin ligase complex, to build flowers?

Importance for society: Provide insights on the mechanism of LFY activation by UFO, a key step in the making of flowers and hence the continuation of life in flowering plants. Project sheds light on the molecular mechanism of flower formation setting the means to modify at will the architecture of plant inflorescences. The outputs have application in agriculture and potentially impacting social needs on food security.

Overall Objectives:
WP1. To identify LFY post-translational modifications (PTMs)
WP2. To validate ubiquitinated LFY lysine residues and characterize their functional importance
WP3. To map LFY-UFO interacting domains and the role of PTMs
WP4. To determine LFY interactome in planta
Month 1-12
WP1 and 4. To isolate LFY and identify PTMs
Immunoprecipitation (IP) of LFY in planta was challenging due to antibody leakage increasing non-specificity.

WP3. To determine the structure of LFY-UFO complex
Task 3.1: Isolating and purifying LFY and UFO proteins
His tagged LFY∆40 (lminus 40 amino acids) and truncated MBP-tagged UFO were purified from bacterial expression system aiming to isolate soluble and non-aggregating proteins (Figure 1).
Figure 1. Expressed UFO constructs (A) Five UFO constructs and (B) representative image showing isolation of UFO construct recombinantly expressed in bacterial cells.
Task 3.2: Determining interacting domains of UFO and LFY using LC-MS/MS
(a) Co-purification: Co-purifications of MBP-UFO and HIS-LFY showed that the two could interact (WB; Figure 2).
Figure 2. Western blots to visualize MBP-UFO (CM04) and HIS-LFY following co-purification. (A) panel for anti-MBP (B) anti-LFY. M- marker; FT-flow-through; W-wash; E-elution.
(b) Yeast-2 hybrid assay: Various LFY constructs were used to test which region of LFY can interact with UFO. We show that UFO could interact with the C-term of LFY (Figure 3).
Figure 3. Yeast-2 hybrid assays. Panel 1 shows truncations of LFY and panel 2 yeast colonies visualized after 6 days of incubation.
(c) Chemical crosslinking and Mass spectrometry (MS)
An MS cleavable cross-linker, disuccinimidyl sulfoxide was used to map the interacting domains between LFY and UFO and analyzed by MS. We identified 6 peptides, three from LFY and three from UFO (Table 1, Figure 4). Using LFY and UFO structural models the identified peptides are well exposed for interaction.
Table 1. Peptides of UFO and LFY obtained after chemical crosslinking and MS.
Figure 4. Pictorial view showing the location of the peptides identified in Table 1. (A) Peptides of LFY, (B) peptides of UFO and (C) predicted structural model of UFO indicating the peptide loops on UFO that crosslinked with LFY.
Task 3.3: Determine the crystal structure of LFY-UFO
This ambitious task could not be performed due to aggregation of UFO.

Month 13-18
WP1 and 4. To isolate and identify LFY interactome and PTMs
LFY IP from in planta was successful using dynabeads conjugated with antiLFY antibody (Figure 5A). PTMs were assessed by WB (Figure 5B and C).
Figure 5. WB depicting LFY and PTMs. IP of LFY from in planta samples using antiLFY antibody conjugated dynabeads. WB were performed against (A) antiLFY, (B) anti-Ser/Thr and antiUbiSite.
In vitro phosphorylation: In collaborated with Dr. Claude Cochet, in vitro LFY phosphorylation using casein kinase (CK)II was done and CKII could phosphorylate LFY (Figure 6). LFY phosphopeptides were identified by MS.
Figure 6. In vitro casein kinase II assay (A) with radiolabeled ATP. (B) different reactions of ATP with alpha subunit, alpha+LFY, and alpha-beta+LFY. (C) shows the samples in the gel after coomassie staining.

Month 19-24
WP2. To characterize ubiquitinated lysines
Tasks could not be executed since ubiquitinated residues were not identified by MS. However, in vitro ubiquitination assay will be performed in the future.
WP3. To determine the structure of LFY-UFO complex
Synthesized LFY and UFO peptides (Figure 4) were used in fluorescent anisotropy. LFY peptides showed no reaction with UFO while UFO peptides showed potential interaction. This work require further investigation.
WP4. To identify LFY interactome and associated PTMs
Data is currently under analysis.

WP5. Training and dissemination of deliverables
1. Plant regeneration, transformation and genome editing, 14-20 April 2018, Institut Jean-Pierre Bourgin, Versailles
2. French course level A1-A2, May-August 2017, IFRA, Valence; level A2-B1+, January-July 2018, CCI, Grenoble
1. Marondedze C, Rieu P, Thevenon E, Le Masson M, Tichtinsky G, Dumas R, Parcy F. Molecular mechanism of LEAFY activation by UFO, Swiss Proteomics Meeting, Montreux, 23-24 May 2019 (Oral)
2. Marondedze C, Rieu P, Thevenon E, Le Masson M, Tichtinsky G, Vachon G, Dumas R, Parcy F. PTMs during floral initiation: molecular dissection of the UFO-dependent LFY activation. IPMB, Montpellier, 5-10 August 2018 (poster).
Invited talks/seminars
1. Marondedze C. mRNA interactomics reveal novel RNA-binding proteins involved in abiotic stress responses. LGDP, University of Perpignan, France, 18 September 2018.
2. Marondedze C. Arabidopsis thaliana mRNA-binding proteins: In vivo determination of the repertoire and stress response. LPCV/IRIG/CEA, Grenoble, France, 15 June 2017.
3. Weekly group meetings in the floral regulators and structural biology teams at LPCV/CEA, Grenoble.
Book chapter “Flower development in Arabidopsis” submitted 28 June 2019:
Chahtane H, Lai X, Tichtinsky G, Rieu P, Cancé C, Marondedze C and Parcy F.
Flower development in Arabidopsis.
Published 4 research articles and a review from my previous work. In two of these, H2020 was acknowledged:
1. Marondedze C et al. (2018) Towards a tailored indoor horticulture: A functional genomics guided phenotypic approach. Nature Horticulture Research. 5, 68.
2. Wong A, Tian X, Gehring C, Marondedze C. (2018) Discovery of novel functional centers with rationally designed amino acid motifs. Computational and Structural Biotechnology Journal. 16, 70-76.

Public engagement on research
- Plant Science International masters program, University of Grenoble, 11 September 2018
- Co-supervised a M1 master student (March- September 2018)
- (Co-) Supervised M1 and M2 students (January- June 2019)
- Taught BIO101, University of Grenoble, September-December 2018
- Workshop/Seminar on plants and germination, St Joseph school, Valence (May-June 2018)
Progress was slower than expected. However, state of the art technologies applied paved way for follow up experiments. E.g. a combination of chemical crosslinking and MS would be used to confirm LFY and UFO interacting residues. Also, interactome MS data will shed light on the interactors of LFY and in deducing a network of events critical in flower formation. This research sheds light on key mechanisms in flower formation, which will allow modifications on the architecture of plant inflorescences. This has high potential in agriculture through breeding to tackle the social need of food security. So far, our project has caught attention of young generations at primary school through discussions and practicals that allowed them to gain basic understanding on plant growth and flowering. This forms the basics in nurturing love for science to the young generation.