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In Cichorium intybus, inulin metabolism is mediated by fructan-active enzymes (FAZYs): sucrose:sucrose 1-fructosyltransferase (1-SST), fructan:fructan 1-fructosyltransferase (1-FFT), and fructan 1-exohydrolases 1, 2a and 2b (1-FEH1, -2a and -2b), respectively. While these enzymes have been rigorously characterized, the transcriptional network orchestrating their development- and stress-related expression has remained largely unknown. Here, the possible role of R2R3-MYB transcription factors in FAZY regulation was explored via bioinformatic identification of R2R3-MYBs (using an RNA sequencing (RNAseq) database), studies of co-expression of these factors with target genes, in vivo transient transactivation assays of FAZY target promoters (dual luciferase assay), and a yeast one-hybrid assay investigating the specificity of the binding of these factors to cis-elements. The chicory MYB transcription factor CiMYB17 specifically activated promoters of 1-SST and 1-FFT by binding to the consensus DNA-motif DTTHGGT. Unexpectedly, CiMYB17 also activated promoters of fructan exohydrolase genes. The stimulatory effect on promoter activities of sucrose transporter and cell wall invertase genes points to a general role in regulating the source-sink relationship. Co-induction of CiMYB17 with 1-SST and 1-FFT (and, less consistently, with 1-FEH1/2) in nitrogen-starved or abscisic acid (ABA)-treated chicory seedlings and in salt-stressed chicory hairy roots supports a role in stress-induced fructan metabolism, including de novo fructan synthesis and trimming of pre-existing fructans, whereas the reduced expression of CiMYB17 in developing taproots excludes a role in fructan accumulation under normal growth conditions., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Two dehydrin genes were identified from a Cichorium intybus EST database. They were among the most abundant sequences obtained from 10 cDNA libraries constructed from chicory roots grown under field conditions. The full length cDNA sequences, designated CiDHN1 and CiDHN2, were 1,176 and 1,055 bp long and encoded predicted polypeptides of 262 and 261 amino acids, respectively. The deduced CiDHN1 protein contains a S-segment and four lysine-rich consensus motifs (K-segments) which represent a typical SK(4) structure of dehydrins. The CiDHN2 sequence contains two Y motifs and two K-segments classifying CiDHN2 as Y(2)K(2)-type dehydrin. Southern-blotting analysis suggested that CiDHN1 and CiDHN2 are single copy genes. Northern-blotting analysis revealed that both CiDHN genes are expressed in roots and leaves, with seasonal variations in transcript accumulation. The effect of cold on the CiDHN1 and CiDHN2 transcript level was demonstrated. CiDHN1 and CiDHN2 promoter analysis revealed the presence of low temperature-responsive and ABA-responsive elements.

Chicory (Cichorium intybus) roots contain high amounts of inulin, a fructose polymer used as a storage carbohydrate by the plant and as a human dietary and prebiotic compound. We performed 2-D electrophoretic analysis of proteins from root material before the first freezing period. The proteins were digested with trypsin and the peptides analyzed by MS (MALDI-TOF/TOF). From the 881 protein spots analyzed, 714 proteins corresponded to a database accession, 619 of which were classified into functional categories. Besides expected proteins (e.g. related to metabolism, energy, protein synthesis, or cell structure), other well-represented categories were proteins related to folding and stability (49 spots), proteolysis (49 spots), and the stress response (67 spots). The importance of abiotic stress response was confirmed by the observation that 7 of the 21 most intense protein spots are known to be involved in cold acclimation. These results suggest a major effect of the low temperature period that preceded root harvesting.