Thèse Charcaterisation Fonctionneles des Protéines Trms Impliquées dans la Différenciation du Protoxylème chez Arabidopsis H/F

Doctorat.Gouv.Fr

Établissement : Université Paris-Saclay GS Biosphera - Biologie, Société, Ecologie & Environnement, Ressources, Agriculture & Alimentation École doctorale : Sciences du Végétal : du gène à l'écosystème Laboratoire de recherche : IJPB - Institut Jean-Pierre Bourgin-Sciences du Végétal Direction de la thèse : Magalie UYTTEWAAL ORCID 0000000328816637 Début de la thèse : 2026-10-01 Date limite de candidature : 2026-05-06T23:59:59 La différenciation du protoxylème est une étape critique du développement des plantes au cours de laquelle les cellules du xylème se préparent au transport de l'eau dans diverses conditions environnementales. Au cours de cette transition, les vaisseaux du protoxylème développent des parois cellulaires secondaires (SCW) organisées en bandes et en spirale. L'organisation de ces motifs est dépendante des microtubules (MT) corticaux qui servent de voies de guidage aux complexe cellulose synthases. L'organisation précise des MT est donc essentielle pour établir et renforcer ces parois secondaires. Certaines protéines TRMs, membre du complexe TON1-TRM-PP2A (TTP) un régulateur clé de l'organisation des microtubules (MT) corticaux, sont spécifiquement exprimées dans ce tissu.
L'objectif principal de ce projet de doctorat est donc d'élucider le rôle de TRM dans la différenciation du protoxylème.
Au cours de la première phase, le doctorant confirmera et précisera les profils d'expression de gènes TRM candidats. La deuxième phase évaluera les rôles fonctionnels de ces protéines TRM à l'aide d'analyses génétiques et phénotypiques. Les anomalies du développement du protoxylème et du xylème, de la formation de la paroi cellulaire secondaire et de l'organisation des MT corticaux en bandes régulièrement espacées seront caractérisées chez les mutants trm. La phase finale, plus exploratoire, se concentrera sur la caractérisation de la fonction moléculaire des TRM sélectionnées et des voies de régulation dans lesquelles elles sont impliquées. À cette fin, une résolution spatio-temporelle élevée des microtubules et de protéines fusion TRM-YFP, ainsi que des approches biochimiques seront développées.
Developing xylem vessels comprise two cell types: protoxylem (PX), with spiral or banded secondary cell walls (SCWs), and metaxylem (MX), featuring thicker walls with regularly spaced pits [1]. These patterns arise from cellulose synthase complexes (CSCs) that deposit cellulose along cortical microtubules (MTs), whose organization into band-gap arrays is essential for precise SCW patterning [2-5]. The organization of the microtubules into interspaced band-gap arrays (annular and spiral configurations) is thus crucial for controlling how and where the cellulose microfibrils are deposited.
To support PX band patterning, microtubules undergo, simultaneously across the entire cell surface, a transition from a diffuse array with variable microtubule orientations into a band-gap array where microtubule orientations are homogeneous [6,7]. The principles by which the dynamic microtubule arrays are re-organized during the transition remain elusive. Several microtubule-associated proteins involved in distinct aspects of microtubule (MT) array reorganization into banded patterns have been identified [8]. These include MIDD1 and KINESIN13-A, which are recruited by the Rho-of-plants GTPase ROP11 and likely other ROPs to promote MT depolymerization in gap regions [9-12]; -tubulin complex proteins (GCPs), which drive MT nucleation and ensure tubulin accumulation within bands [13]; and the MT-severing protein KATANIN, which promotes MT bundle formation and reinforcement of banded arrays [8]. In addition, Cellulose Synthase Interacting 1 (CSI1/POM2), which tethers cellulose synthase complexes (CSCs) to cortical MTs, represents a key component linking MT organization to secondary cell wall deposition [14,15]. Despite these detailed insights into individual molecular functions, current models remain fragmented and lack an integrated biophysical framework explaining how robust banded MT patterns emerge.
Under ectopic overexpression of the master regulator gene Vascular-related NAC Domain7 (VND7), inducing PX differentiation [16,17], several TRM genes are upregulated (data from René Schneider's lab). This analysis is in agreement with TRM expression profiles in the PX and MX that we can extract from data mining of single-cell root tip transcriptomics [18-20]. Among them, TRM7 and TRM30 exhibited the strongest and/or most PX-specific expression, making them prime candidates for further investigation. Using the SCW pattern phenotyping tool developed by René Schneider [6], significantly larger band gaps and SCW band orientation irregularities were detected in the trm30 mutant background.
TRM proteins are components of the TON1-TRM-PP2A (TTP) complex, which regulates MT organization across development [21-24]. While core TTP components are essential, individual TRMs confer specificity to MT array formation. Given its role in SCW band regularity, TRM30 emerges as a key factor for understanding the robustness and mechanics of protoxylem cell wall patterning.
The aim of this thesis is to elucidate the role of the TRM30 gene (and potentially TRM7) in protoxylem differentiation and to determine how its function is integrated into existing biophysical and molecular models of xylem development. To this end, the project will address three main questions:

1-What is the expression pattern of TRM30 (and TRM7) during xylem differentiation? 2-What is the role of the TRM30 (and TRM7) protein in xylem differentiation and secondary cell wall formation? Does TRM30 (and TRM7) contribute to the organization and dynamics of microtubule arrays during protoxylem differentiation? 3-Does TRM30 act in coordination with other microtubule-regulatory proteins and with other TRM family members (such as TRM7) to control protoxylem patterning?
The models used for this study will be the root protoxylem of Arabidopsis thaliana which exhibits predominantly annular patterns with a minor proportion of mixed spirals, as well as the inducible VND7 system whose overexpression induces the ectopic formation of PX-like SCWs in cell layers more easily accessible for microscopy such as the epidermis of the hypocotyl [6,16]. which allows to induce differentiation in cells located in the epidermis and thus accessible for high-resolution dynamic imaging approaches

Task 1: Characterize TRM30 (and TRM7) expression patterns during xylem differentiation
Characterizing the spatial and temporal patterns of TRM30 (and TRM7) expression and protein accumulation is a critical prerequisite for developing mechanistic hypotheses on how and when this TRM contributes to the progressive establishment of protoxylem, and possibly metaxylem, secondary cell walls.
We will use native promoter-driven transcriptional reporters in fusion with reporter fragment (erVENUS for endoplasmic reticulum targeting or, alternatively, -glucuronidase for GUS staining), allowing straight forward detection of cells expressing the construct. Transformations will be performed in col-0 wild type and in plasma membrane reporter line (pUB10::mScarlet-LTI6B) to assess cell-type specificity. After selection of the lines, confocal imaging will be used to reveal where and when TRM30 (and TRM7) genes are active during xylem formation, distinguishing xylem cell-type specificity, PX exclusivity, organ specificity, and assess their timing of expression during differentiation. The promoter-reporters will also be introduced in the VND7-inducible background to assess and confirm their induced expression in the context of the VND7-system.
In parallel to these investigations, we will use the translational reporters pTRM30::TRM30-GFP (and pTRM7::TRM7-3xYFP) to consolidate the above expected results by analyzing the accumulation of the whole proteins in PX cells specifically and within the vascular cylinder more generally. The expression and accumulation patterns of the TRM7 protein, will be a first critical point in deciding whether to continue the analysis by including TRM7 or not to this study.

Task 2: Characterize how TRMs contribute to xylem differentiation via MT and cell wall patterning
First, the PhD student will confirm and precise the endogenous protoxylem SCW phenotypes in the roots of two trm30 independent mutant alleles (trm30-1 and trm30-2), as well as describe those of trm7 and the double trm7trm30 mutants. Characteristic properties of SCW patterns, such as band width and band order (anisotropy) will be quantified. Briefly, five-day-old seedlings will be fixed, cleared and stained with cellulose- and lignin-specific dyes, and imaged via confocal microscopy. A computer script then segments the xylem cells, extracts their shape, and unrolls the cylindrical SCWs into a 2D maps for the SCW analysis. This step will also be the second opportunity to conclude whether the TRM7 gene has activity during protoxylem differentiation and whether or not it is redundant with that of TRM30. Finally, SCW profiles will also be analyzed in mutant backgrounds combined to VND7-inducible system, and thus in induced hypocotyl cells to further confirm if this inducible system can be used as a tool for functional study of the TRM proteins.
In a second time, the student will quantify MT patterns (anisotropy, periodicity, orientation) and their divergence to the wild-type ones in PX cells of trm30 mutants. Since PX cells are difficult to access for high-resolution microscopy, we will combine several approaches: immunolocalization mastered by our team, the use of MT markers exclusively expressed in the root PX (pCESA7::mCherry-TUA5), and finally the induction of the differentiation of PX cells in the hypocotyl epidermis using the inducible VND7 system. This will allow us to confirm whether the TRM30 protein contributes to microtubule patterning during protoxylem differentiation, and identify the output of this contribution. Will the MT patterns in band and gaps be more spaced and irregular, similar to SCWs?

Task 3: Characterize the molecular function of TRM30 proteins related to protoxylem differentiation
The re-organization of MTs into organized bands at the subcellular scale requires fine spatial and temporal adjustment of MT dynamic instability parameters and MT nucleation. We will therefore characterize its protein localization on microtubules and quantify the effects of its loss of function at high resolution (Spinning-Disk microscopy), by systematically using the inducible VND7 system.
So, first, the student will perform high-resolution and dynamic imaging of the translational reporter pTRM30::TRM30-EGFP to characterize the spatial and temporal localization of TRM30 on MTs. This will either exclude or provide some options. For example, if there is no TRM30 localization on MT overlaps, the hypothesis of TRM30 regulation of nucleation could be eliminate. Similarly, if TRM30 proteins are uniformly present across MTs, no huge changes in MT tip dynamics will be expected. Thus, accurately describing TRM localization on MTs will help determine which MT properties to analyze.
Then, depending on the observed subcellular localization of TRM30 on MTs, and MT array dis-/organization in the trm30 mutant, the student will analyze in the trm30 mutant background i) MT de-/polymerization parameters, ii) MT flexibility measurements, and iii) MT nucleation and severing rates. MT nucleation complexes will be imaged thanks to the GIP1a-GFP marker. These analyses will allow us to develop hypotheses about TRM30 functions, their molecular environment, and their activity.
A third part of this task is to complete the characterization of TRM30 protein environments using biochemistry. The student will employ TURBO-ID [25] (mastered at the IJPB), in combination with the VND7-inducible system to amplify the number of cells at the relevant developmental stage. If interactions are identified, they will obviously be confirmed by other approaches (BIFC, etc.).
Finally, we suggest a very optional part which will be considered depending on the progress and the results of the other tasks. In vitro polymerization tests of MTs (adapted from [26]) in the presence and absence of TRM30 proteins purified (by pulldown) from VND7-induced samples will be proposed. These in vitro tests would allow to quantify the instability dynamics of synthetic MTs and their assembly into bundles in the presence and absence of TRM proteins and their putative co-purified protein partners.