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.