Thèse Rôles de la Synthèse de la Cellulose et de l'Homogalacturonane dans la Morphogenèse et la Différenciation Cellulaires 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 : Alexis PEAUCELLE ORCID 0000000285826500 Début de la thèse : 2026-10-01 Date limite de candidature : 2026-05-06T23:59:59 Les microfibrilles de cellulose sont connues pour être un élément clé de la croissance anisotrope orientée des cellules végétales, car elles sont souvent organisées perpendiculairement à l'axe d'élongation. La perturbation de cette organisation par des traitements pharmacologiques conduit à une croissance cellulaire isotrope. Les nanofilaments d'homogalacturonane (HG), également organisés perpendiculairement à l'axe de croissance, sont de même impliqués dans l'élongation anisotrope. Leur modification chimique (déméthylation) est à la fois nécessaire et suffisante pour l'expansion cellulaire et la morphogenèse.
Nous cherchons à élucider les fonctions relatives, complémentaires ou potentiellement opposées de ces deux composants de la paroi cellulaire dans la croissance anisotrope et la morphogenèse, en induisant des mutations tissus spécifiques dans GAUT1 et CESA3, impliqués respectivement dans la synthèse de l'HG et des fibrilles de cellulose. Comme les mutants nuls de ces gènes sont létaux au stade embryonnaire, nous avons généré des lignées CRISPR inductibles à l'éthanol et spécifiques de l'épiderme, ciblant l'un ou l'autre de ces gènes, produisant soit des mutants épidermiques complets, soit des plantes mosaïques.
L'étudiant(e) étudiera les effets de ces perturbations aux niveaux tissulaire, cellulaire et moléculaire afin de révéler les relations structure-fonction de la cellulose et de l'HG. Les rôles dépendants du tissu versus autonomes à la cellule de ces polymères dans la morphogenèse seront examinés à l'aide d'analyses en time-lapse. Enfin, grâce à un contrôle photo-inductible de l'activité enzymatique, nous visons à atteindre une résolution subcellulaire et à l'échelle de la minute des variations de la synthèse de l'HG.
À l'aide de ces approches, nous espérons distinguer les conséquences directes des modifications structurelles de la paroi cellulaire des effets indirects de signalisation déclenchés par ces modifications. Plant cell growth relies on the controlled expansion of the cell wall. The most widely
accepted model proposes that plant cell growth is driven by the high turgor pressure
present in plant cells, which places the cell wall under tension. The organization of loadbearing
cellulose polymers perpendicular to the growth axis leads to anisotropic
expansion. Disruption of this organization, through drugs affecting cellulose deposition or
microtubule organization, results in a transition from anisotropic to isotropic cell
expansion. In the model organ hypocotyl, this transition can be observed as rapidly as 15
minutes following treatment.
In addition to cellulose, some homogalacturonan (HG) polymers also display an organized
filamentous arrangement perpendicular to the growth axis. Chemical modification of their
methylesterification status is necessary and sufficient for proper cell morphogenesis and
growth.
Cellulose and HG nanofilaments are tightly linked, suggesting a common, redundant, or
possibly opposing function in oriented growth. To dissect the respective roles of these
polymers, it is necessary to control their synthesis in a precise manner, both spatially and
temporally.
Indeed, loss-of-function mutants of GAUT1 (Galacturonosyltransferase 1), which is
involved in the synthesis of cellulose-associated HG nanofilaments, are embryo lethal.
Similarly, although most cellulose synthase knockout mutants are viable-suggesting
functional redundancy-loss of CESA3 is male gametophyte lethal, indicating that its role
in cell wall biosynthesis cannot be compensated by other members of the CESA family.
Therefore, alternatives to the classical null-mutant approach are required. In addition,
4
signaling feedback between the cell wall and cell differentiation requires tight spatial and
temporal control of changes in cell wall chemical composition in order to disentangle
signaling effects from the functional role of a given cell wall polymer in cell growth. The objective of this thesis proposal is to study the role of the biosynthesis and delivery of
key cell wall components involved in anisotropic growth and morphogenesis in Arabidopsis
thaliana. To achieve this, the student will use tools recently developed in the host laboratory
to disrupt, in a spatially and temporally controlled manner, biosynthetic enzymes involved in
cellulose and HG nanofilament synthesis respectively, in order to analyze the resulting effects
on macroscopic phenotypes and nanoscopic cell wall architecture in order to establish
structure-function relationships underlying anisotropic growth. First objective: Dissecting the role of cellulose and pectin HG in anisotropic growth
Genetic analyses of constitutive cell wall mutants often fail to establish direct links between
mutations (e.g. in polysaccharide biosynthetic enzymes) and chemical or macroscopic
phenotypes, due to the high plasticity of cell wall composition. To overcome this limitation,
the student will use a cell type-specific, ethanol-inducible CRISPR-Cas9 system to
inactivate, specifically in the hypocotyl epidermis, key enzymes involved in the synthesis of
cellulose and the cellulose-associated pectin nanofilament homogalacturonan (HG), which
play critical roles in polar growth.
First, the student will study the transition from isotropic to anisotropic growth in hypocotyl
cells using ethanol-inducible CRISPR lines targeting the HG biosynthetic enzyme GAUT1,
together with complemented control lines.
Second, the student will use ethanol-inducible CRISPR lines targeting the cellulose synthase
CESA3 to achieve temporal control of cellulose biosynthesis inhibition. This will allow
investigation of growth transitions in the absence of cellulose synthesis in the epidermis.
In both cases, comparisons of cell elongation with mutants affected in other cell wall
polymers-such as the quasimodo1 mutant, with reduced amorphous HG levels, or MUR
mutants affected in rhamnogalacturonan I synthesis that display near-normal anisotropic cell
expansion-will help disentangle structure-function relationships of individual polymers.
The student will combine his lines with lines carrying reporters for cell polarity (microtubules
and actin) as well as signaling reporters (calcium and auxin response), to follow in real time
the effects of cell wall disruption on cell polarity and cell signaling. Finally, if time permits,
fluorescent reporter lines and in situ hybridization will be used to monitor changes in cell
differentiation, using transcription factors involved in cell fate specification, such as
SPEECHLESS in guard cell development.
5
Risk management
No major risks are anticipated for this objective. Inducible CRISPR lines affecting GAUT1
expression have already been generated, and preliminary phenotypic analyses confirm the
specific role of GAUT1 in anisotropic cell elongation and cell morphogenesis. Two lines have
been selected for further analysis: one in which all epidermal cells show a GAUT1 phenotype
following induction, and another in which only a subset of the cells display altered expansion.
Detailed comparison of these phenotypes will allow discrimination between tissue-level and
cell-autonomous functions of HG in hypocotyl anisotropic expansion.
Similarly, at the start of the PhD, the preliminary characterization of inducible CESA3
CRISPR lines will be carried out.
Second objective: Relating cell wall structure to function in plant growth and development
The student will examine the effects of GAUT1 and CESA3 loss-of-function on the fine
structure of the cell wall using 3D-dSTORM super-resolution microscopy. Antibodies
targeting highly methylesterified HG (LM20), low- or non-esterified HG (2F4), and
carbohydrate-binding modules specific for cellulose (CBM3) will be used to unravel cell wall
molecular architecture before, during, and after the growth transition. This will clarify the
relationship between HG, cellulose, and changes in anticlinal wall structure preceding
anisotropic growth.
Initially, the student will use dSTORM microscopy to analyze cell wall nanoarchitecture
following inducible genetic perturbation of GAUT1 and CESA3. Dark-grown Arabidopsis
thaliana hypocotyls will be used as a model system. These studies will allow the
establishment of structure-function relationships underlying anisotropic growth.
Risk management
dSTORM analysis of the gaut1 mutant cell wall has already been performed in the laboratory
and confirmed the absence of HG nanofilaments.
Alternative approaches
In parallel, we will explore the effects of altered cell polarity-through pharmacological
inhibition of cellulose synthesis and microtubule organization-on cell wall nanostructure.
Preliminary analyses indicate that, HG nanofilament reorganization can be detected as early
as 20 minutes after treatment.
Third objective: Optogenetic degradation of biosynthetic enzymes (optoGAUT)
To study the role of key HG biosynthetic enzymes in plant cell growth, optogenetic actuators
will be used to trigger light-induced aggregation of Golgi-localized type II membrane proteins
and their transfer to early endosomes for degradation. This aggregation is expected to induce
rapid, local, and reversible arrest of HG synthesis. This approach will allow real-time
monitoring of how targeted perturbations of specific cell wall components affect growth,
enabling direct causal links between growth anisotropy and cell wall composition to be
established at subcellular resolution and minute timescales.
6
First, the student will test optogenetic tools in Nicotiana benthamiana using fluorescently
tagged GAUT1 constructs. Aggregation kinetics following light induction will be monitored
across different genetic constructs currently developed in the laboratory.
Thene the student will transform Arabidopsis thaliana. Transformant lines will be selected for
GAUT1 mutant phenotypes under light induction condition.
Finally, the student will locally activate GAUT1 disruption in specific cellular regions, such
as the growing root tip or lateral walls of hypocotyl cells, and monitor effects on cell
elongation over minutes to hours.
Risk management
We understand that this part of the project is high risk, high gain. However, the lightinducible
aggregation systems have already been tested in the lab on tobacco leaves and have
been demonstrated to be functional.