Chicory R2R3-MYB transcription factors CiMYB5 and CiMYB3 regulate fructan 1-exohydrolase expression in response to abiotic stress and hormonal cues.

In the biennial Cichorium intybus, inulin-type fructans accumulate in the taproot during the first year. Upon cold or drought exposure, fructans are degraded by fructan exohydrolases, affecting inulin yield and degree of polymerization. While stress-induced expression of 1-FEH genes has been thoroughly explored, the transcriptional network mediating these responses has remained unknown. In this study, several R2R3-MYB transcriptional regulators were analysed for their possible involvement in 1-FEH regulation via transient transactivation of 1-FEH target promoters and for in vivo co-expression with target genes under different stress and hormone treatments. CiMYB3 and CiMYB5 selectively enhanced promoter activities of 1-FEH1, 1-FEH2a, and 1-FEH2b genes, without affecting promoter activities of fructosyltransferase genes. Both factors recognized the MYB-core motifs (C/TNGTTA/G) that are abundantly present in 1-FEH promoters. In chicory hairy root cultures, CiMYB5 displayed co-expression with its target genes in response to different abiotic stress and phytohormone treatments, whereas correlations with CiMYB3 expression were less consistent. Oligofructan levels indicated that the metabolic response, while depending on the balance of the relative expression levels of fructan exohydrolases and fructosyltransferases, could be also affected by differential subcellular localization of different FEH isoforms. The results indicate that in chicory hairy root cultures CiMYB5 and CiMYB3 act as positive regulators of the fructan degradation pathway.

CiMYB17, a stress-induced chicory R2R3-MYB transcription factor, activates promoters of genes involved in fructan synthesis and degradation.

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.

Linking Expression of Fructan Active Enzymes, Cell Wall Invertases and Sucrose Transporters with Fructan Profiles in Growing Taproot of Chicory (Cichorium intybus): Impact of Hormonal and Environmental Cues.

In chicory taproot, the inulin-type fructans serve as carbohydrate reserve. Inulin metabolism is mediated by fructan active enzymes (FAZYs): sucrose:sucrose 1-fructosyltransferase (1-SST; fructan synthesis), fructan:fructan-1-fructosyltransferase (1-FFT; fructan synthesis and degradation), and fructan 1-exohydrolases (1-FEH1/2a/2b; fructan degradation). In developing taproot, fructan synthesis is affected by source-to-sink sucrose transport and sink unloading. In the present study, expression of FAZYs, sucrose transporter and CWI isoforms, vacuolar invertase and sucrose synthase was determined in leaf blade, petiole and taproot of young chicory plants (taproot diameter: 2 cm) and compared with taproot fructan profiles for the following scenarios: (i) N-starvation, (ii) abscisic acid (ABA) treatment, (iii) ethylene treatment (via 1-aminoyclopropane-1-carboxylic acid [ACC]), and (iv) cold treatment. Both N-starvation and ABA treatment induced an increase in taproot oligofructans. However, while under N-starvation this increase reflected de novo synthesis, under ABA treatment gene expression profiles indicated a role for both de novo synthesis and degradation of long-chain fructans. Conversely, under ACC and cold treatment oligofructans slightly decreased, correlating with reduced expression of 1-SST and 1-FFT and increased expression of FEHs and VI. Distinct SUT and CWI expression profiles were observed, indicating a functional alignment of SUT and CWI expression with taproot fructan metabolism under different source-sink scenarios.