Cloning and functional characterization of two abiotic stress-responsive Jerusalem artichoke (Helianthus tuberosus) fructan 1-exohydrolases (1-FEHs)

Abstract

Two fructan hydrolases were previously reported to exist in Jerusalem artichoke (Helianthus tuberosus) and one native fructan-β-fructosidase (1-FEH) was purified to homogeneity by SDS-PAGE, but no corresponding cDNA was cloned. Here, we cloned two full-length 1-FEH cDNA sequences from Jerusalem artichoke, named Ht1-FEH I and Ht1-FEH II, which showed high levels of identity with chicory 1-FEH I and 1-FEH II. Functional characterization of the corresponding recombinant proteins in Pichia pastoris X-33 demonstrated that both Ht1-FEHs had high levels of hydrolase activity towards β(2,1)-linked fructans, but low or no activity towards β(2,6)-linked levan and sucrose. Like other plant FEHs, the activities of the recombinant Ht1-FEHs were greatly inhibited by sucrose. Real-time quantitative PCR analysis showed that Ht1-FEH I transcripts accumulated to high levels in the developing leaves and stems of artichoke, whereas the expression levels of Ht1-FEH II increased in tubers during tuber sprouting, which implies that the two Ht1-FEHs play different roles. The levels of both Ht1-FEH I and II transcript were significantly increased in the stems of NaCl-treated plants. NaCl treatment also induced transcription of both Ht1-FEHs in the tubers, while PEG treatments slightly inhibited the expression of Ht1-FEH II in tubers. Analysis of sugar-metabolizing enzyme activities and carbohydrate concentration via HPLC showed that the enzyme activities of 1-FEHs were increased but the fructose content was decreased under NaCl and PEG treatments. Given that FEH hydrolyzes fructan to yield Fru, we discuss possible explanations for the inconsistency between 1-FEH activity and fructan dynamics in artichokes subjected to abiotic stress.

Development of rubber‐enriched dandelion varieties by metabolic engineering of the inulin pathway

“…Whereas 27% of the inulin (DP ≈ 21) and ~8% of the nystose and 1‐kestose were converted by Tk1‐FEH, sucrose was unaffected (Figure 3c). Furthermore, less inulin was converted when inulin and sucrose were used as combined substrates, suggesting that sucrose acted as a dose‐dependent inhibitor as shown for other plant 1‐FEHs (Lothier et al ., 2007; Xu et al ., 2015) (Figure 3d). Compared to the activity displayed with inulin alone as the substrate, the presence of 8.8 m m sucrose reduced the conversion efficiency to 65%, and the presence of 88 m m sucrose reduced the conversion efficiency to 15%.…”

“…This host species does not express any fructosyltransferases, making it highly suitable for the production of recombinant 1‐FEHs (Xu et al ., 2015). The native signal peptide was removed and replaced with an N‐terminal α‐mating factor signal peptide from Saccharomyces cerevisiae to ensure the secretion of Tk1‐FEH into the culture supernatant.…”

“…Mass spectrometry revealed Tk1‐FEH‐specific peptide sequences covering 76.42% of the protein compared to the full‐length Tk1‐FEH used as target sequence (Table S1). Because glycosylation has previously been reported for heterologous 1‐FEH expression in yeast (Ueno et al ., 2011; Xu et al ., 2015), the glycosylation status of the recombinant Tk1‐FEH was checked by digestion with PNGase F. The enzyme removes N ‐linked glycans and reduced the molecular mass of the recombinant Tk1‐FEH to the in silico predicted value of ~63 kDa without the native signal peptide (Figure S2).…”

Expression of Jerusalem artichoke (Helianthus tuberosus L.) fructosyltransferases, and high fructan accumulation in potato tubers

“…Depolymerization or hydrolysis of fructans is mainly caused by fructan exohydrolase, which sequentially releases fructose units from the fructan polymer, eventually leaving sucrose [36]. The 1-FFT enzyme also contributes to the de-polymerization of fructans under unfavorable conditions [36,37]. Therefore, we speculate that the accumulation of low DP inulin-type fructans in transgenic potato tubers was caused by 1-FFT postharvest, despite its role in fructan biosynthesis during tuber formation and maturation.…”

“…Additionally, storage conditions (prolonged storage and temperature, 18 °C, 4 °C and 4 °C under polypropylene film packing) may cause a substantial decrease in the average DP of inulin-type fructans because of depolymerization or degradation of higher molecular weight carbohydrates [35]. Depolymerization or hydrolysis of fructans is mainly caused by fructan exohydrolase, which sequentially releases fructose units from the fructan polymer, eventually leaving sucrose [36]. The 1-FFT enzyme also contributes to the de-polymerization of fructans under unfavorable conditions [36,37].…”