Cloning And Functional Characterization Of Fructan 1-exohydrolases (1-FEHs) In Jerusalem Artichoke (Helianthus Tuberosus)

Two fructan hydrolases were previously reported to exist in Jerusalem artichoke(Helianthus tuberosus)and one native fructan-?-fructosidase(1-FEH)was purifed to homogeneity by SDS-PAGE,but no corresponding cDNA was cloned.Here,we cloned two full-length 1-FEH cDNA sequences from Jerusalem artichoke(NY-1),named Ht1-FEH ?and Ht1-FEH ?.The Ht1-FEHs were expressed in the methylotrophic yeast Pichia pastoris X-33 with the secretory expression vector pPICZaC and the corresponding recombinant proteins was used to analyze the properties of enzymes to preliminary determine the function of the genes.Moreover,the relative expression levels of Ht1-FEH ? and Ht1-FEH? in diverse tissues at different development stages or under different stresses,such as salt and drought,were carried out to explore the gene expression patterns.Then,the activities of fructan metabolism key enzymes and sugar contents in the stress of Jerusalem artichoke tubers were determined in order to elucidate the regulation mechanism of fructan metabolism under abiotic stress.The results obtained were listed as follows:1.The previously reported coding sequences of chicory and Vernonia herbacea 1-FEHs were used to BLASTN the Jerusalem artichoke EST database.Based on these,two genes were isolated from Jerusalem artichoke,named Ht1-FEH ? and Ht1-FEH ?,which showed high levels of identity with chicory 1-FEH ? and 1-FEH ?.The accession numbers for Ht1-FEH ? and Ht1-FEH ? in the genebank are KJ946352 and KJ946353,respectively.Both Ht1-FEH ? and Ht1-FEH ? contained three conserved amino acid motifs(NDPNG,RDP,and EC)that are essential for ?-fructosidase activity in FEH family.2.The Htl-FEHs were succesfully over-expressed in P.pastoris X-33 by heterologous expression and the corresponding recombinant proteins were determined to be the recombinant Htl-FEHs by SDS-PAGE and LC-MS/MS analysis.Functional characterization of the recombinant proteins demonstrated that both Htl-FEHs had high levels of hydrolase activity towards ?(2,1)-linked fructans,but low or no activity towards sucrose and ?(2,6)-linked levan.The optium pH and temperation of purified recombinant Ht1-FEHs are 6.0 and 35?,respectively.Like other plant FEHs,the activities of the recombinant Ht1-FEHs were greatly inhibited by sucrose.3.In the whole growth period of Jerusalem artichoke,real-time quantitative PCR analysis showed that Ht1-FEH ? transcripts accumulated to high levels in the developing leaves and stems of artichoke,whereas the expression levels of Ht1-FEH ? increased intubers during tuber sprouting.The levels of both Htl-FEH ? and ? transcript were significantly increased in the stems of NaCl-treated plants.NaCl treatments also induced transcription of both Htl-FEHs in the tubers,while PEG treatments slightly inhibited the expression of Htl-FEH ? in tubers,which implies that the two Htl-FEHs play different roles.4.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 fructose,we discuss possible explanations for the inconsistency between 1-FEH activity and fructan dynamics in artichokes subjected to abiotic stress.