Doctorate thesis of National Institute of Further Education in Agricultural Science

Tuesday, December 6, 2016

 

Study of gene networks involved in the regulation of iron homeostasis in Arabidopsis thaliana

Nicolas Tissot
BPMP, équipe FeROS

 

Jury :
Christian DUBOS, Chargé de recherche, INRA/SupAgro, Biochimie et physiologie moléculaire des plantes, Directeur de thèse
Sébastien THOMINE, Directeur de recherche, CNRS, Institut de Biologie Intégrative de la Cellule, Rapporteur
Sébastien BAUD, Directeur de recherche, CNRS, Institut Jean-Pierre Bourgin, Rapporteur
Nicolas ROUHIER, Professeur, Interactions Arbres/Micro-organismes, Université de Lorraine, Examinateur
Jacqueline GRIMA-PETTENATI, Directeur de recherche, CNRS, Laboratoire de Recherche en Sciences Végétales (LRSV), Examinateur
Bruno TOURAINE, Professeur, INRA/SupAgro, Biochimie et physiologie moléculaire des plantes, Université de montpellier, Examinateur

 

Abstract :
Iron (Fe) is an essential micronutrient required for life. Since it can transfer electrons, Fe is a crucial cofactor for several enzymatic reactions such as photosynthesis, DNA synthesis or respiration. However, Fe is potentially toxic for the cells as it can react with oxygen and generate ROS (Reactive Oxygen Species). Therefore plants have evolved robust strategies to monitor Fe homeostasis in order to avoid Fe deficiency or excess that could be detrimental for their growth and development. Among the molecular actors involved in Fe homeostasis sensing, ferritins are central actors. In plants, ferritins are mainly transcriptionally regulated. AtFER1 (model of ferritin genes in Arabidopsis thaliana) is regulated by at least three independent environmental pathways (Fe excess, phosphate deficiency and diurnal/circadian rhythms). However, how these environmental signals are integrated at AtFER1 promoter remains elusive.
During my PhD, I have functionally characterized the AtFER1 promoter in different growth conditions (i.e. Fe availability), using GUS as a reporter gene. This approach leads to the identification of specific cis-regulatory sequences within the AtFER1 promoter. Yeast one-hybrid screens using these cis-regulatory elements allowed the identification of the transcription factor bHLH105/ILR3 as putative transcriptional regulator of AtFER1 expression. In addition, molecular and physiological characterization of ilr3 mutants (gain- and loss-of-function mutations) brought out the involvement of ILR3 in plant responses to Fe excess and confirmed that ILR3 is a central integrator of Fe homeostasis in plants. I have also investigated the potential role of a long non-coding RNA in controlling AtFER1 expression. A deep characterization of the mechanisms potentially involved in this process demonstrated that this long non-coding RNA is most probably not involved in the control of AtFER1 expression.
The molecular mechanisms by which plants face and adapt against Fe deficiency are well documented, however, very few data are available with regard to Fe excess. In this context, we set up a transcriptome analysis (microarrays) aiming at deciphering the dynamics of the early response (i.e. prior AtFER1 expression is induced) to an excess of Fe in A. thaliana. An analysis of variance was performed on the expression data generated in order to identify genes whose expression is affected by the treatment. We particularly focused on the identification of transcription factors that are major players in the regulation of gene expression in response to Fe and ROS excess. Among them, WRKY33, WRKY40, ZAT10 and MYB51 have been identified.
Finally, I have been investigating the mode of action of PDR9, a transporter involved in the secretion of phenolic compounds in response to Fe deficiency. Through the characterization of pdr9 mutants, I have shown that the secreted phenolic compounds (i) allow the entrance of Fe via the FRO2 / IRT1 mechanism and (ii) that these compounds are stored in the vacuoles of the root cells before secretion.
In conclusion, my PhD brings new elements on the molecular and physiological mechanisms involved in maintaining Fe homeostasis in Arabidopsis.


Full text (pdf – in French)