CiDREB Regulates Chicory Fructan 1-Exohydrolases Under Cold Stress

Chicory is a main commercial source of inulin extraction and a model plant for inulin metabolism research.As the simplest fructan,inulin is used for human consumption in manifold food and non-food applications.The two primary goals for breeders and planters are to maintain the quantity and quality of inulin in chicory grown in the fields.The degradation of inulin in chicory taproot occurring in the late harvest season is largely caused by the expression of fructan 1-exohydrolases(1-FEH)due to the low temperature.Although three isoforms of 1-FEH(1-FEH1,1-FEH2 a and 1-FEH2b)and their activities have been rigorously characterized,the transcriptional network orchestrating their development-and stress-related genes expression has remained unclear.In order to study the transcriptional regulation of 1-FEH,in this thesis,we performed the following strategies:(1)RT-q PCR analysis has been used to figure out the putative transcription factors,which could co-express with their target genes(FEHs)under cold treatment.Although the previous research suggested that in chicory hairy root system,transcription factor Ci MYB3 and Ci MYB5 co-expressed with their target genes 1-FEHs in response to biotic and abiotic stress,however,our results showed that the corresponding expressions of Ci MYB3 and Ci MYB5 were not found in the mature taproot tissue.Other transcription factors Ci DREB1 A and Ci DREB1 C were found that their expression significantly increased and peaked right before 1-FEH2 a was up-regulated.(2)In vivo transient transactivation assays of FEHs promoters via dual luciferase assay(DLA)following particle bombardment of grapevine suspension cells.The result demonstrated that Ci DREBs activated the promoters with 5-7 fold-induction.Site-directed mutation of the putative DRE/CRT element on the promoter of 1-FEH2 a did not eliminate the induction,whereas the complete deletion of this motif erased the effect.(3)The gel electrophoresis mobility shift assay(EMSA)is used to detect whether Ci DREB protein to combine with its target nucleic acids.Unexpectedly,no direct binding event was observed between Ci DREB1 C and the putative DRE/CRT binding motif on the promoter of 1-FEH2 a.Taking all into consideration,Ci DREBs upstream regulate the expression of 1-FEHs in response to low temperature,and other factor may be involved in the transcriptional regulation.