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Abstract

The preservation of the polyfructan inulin, a valuable commercial product obtained from chicory during the harvest season is severely hampered by its degradation due to low temperatures in late autumn. The degradation process is primarily attributed to the activity of fructan exohydrolases (1-FEH1, 1-FEH2a, and 1-FEH2b). However, the upregulation mechanism of 1-FEH genes in response to cold has remained poorly understood. This thesis endeavors to explore the roles of different transcription factors responsible for regulating the expression of 1-FEH genes and seeks to uncover the underlying transcription regulatory network allowing 1-FEH genes to respond to environmental cues. Thus, the overall aim of this thesis is to shed light on the complex transcriptional regulation of 1-FEH genes and thereby to provide direction for future chicory breeding efforts. The primary objective of the initial part of this thesis was to investigate the variation in expression levels of the three isoforms of 1-FEHs in mature taproots and young seedlings subjected to a detailed time course of cold treatment. The qRT-PCR results indicate that 1-FEH1 exhibited a prolonged and consistent cold-induced up-regulation whereas 1-FEH2a and 1-FEH2b were rapidly but only transiently up-regulated within the initial 24 hours following exposure to low temperatures; interestingly, expression of 1-FEH genes was also affected by heat stress and water deficiency. A co-expression analysis conducted in this study identified a set of cold-inducible transcription factors, namely CiNAC5, CiDREB1A/C/D, and CiDREB2A. Further investigations using the dual luciferase assay, promoter deletion analysis, electrophoretic mobility shift assay (EMSA) and yeast-two-hybrid revealed that i) CiNAC5 specifically activates the promoter region (-353 to ATG) of p1-FEH1; ii) CiDREB2A was identified as a key regulator of 1-FEH2b, which responds to a range of stress conditions by binding to the DRE cis-element located on the 1-FEH2b promoter; iii) CiDREB2B has been found to bind to CiMYB5 (yeast-two-hybrid), resulting in a synergistic upregulation of 1-FEH2b (dual luciferase assay ) and offering an explanation for the strong up-regulation of 1-FEH2b under heat stress; iv) the identification of a single nucleotide polymorphism (SNP) located on the DRE cis-element within the promoter region of p1-FEH2a was observed, which resulted in the inability of the promoter to be recognized and activated by CiDREB1 and CiDREB2. Furthermore, this thesis established a CRISPR RNP delivery system and a chicory protoplast regeneration method, which will facilitate future research on the individual functions of 1-FEH1 and 1-FEH2. By identifying the transcription factors involved in regulating 1-FEH genes, this thesis offers valuable insights into the mechanisms underlying the regulation of 1-FEHs and may have significant implications for the commercial production of inulin in chicory.