College of Resources and Environmental Sciences, Nanjing Agricultural University, Tongwei Road 6, Xuanwu District, Nanjing, 210095, Jiangsu Province, People's Republic of China.
Plant Mol Biol. 2015 Jan;87(1-2):81-98. doi: 10.1007/s11103-014-0262-1. Epub 2014 Oct 22.
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.
先前有报道称，菊芋（Helianthus tuberosus）中存在两种果聚糖水解酶，一种天然的果聚糖-β-果聚糖酶（1-FEH）通过 SDS-PAGE 被纯化至均一性，但没有相应的 cDNA 被克隆。在这里，我们从菊芋中克隆了两个全长的 1-FEH cDNA 序列，命名为 Ht1-FEH I 和 Ht1-FEH II，它们与菊苣 1-FEH I 和 1-FEH II 具有高度的同源性。在毕赤酵母 X-33 中对相应重组蛋白的功能特征进行了表征，结果表明，两种 Ht1-FEH 对β（2,1）-连接的果聚糖均具有高水平的水解酶活性，但对β（2,6）-连接的蔗果聚糖和蔗糖几乎没有活性。与其他植物 FEHs 一样，重组 Ht1-FEH 的活性受到蔗糖的极大抑制。实时定量 PCR 分析表明，Ht1-FEH I 转录本在菊芋发育叶片和茎中大量积累，而 Ht1-FEH II 的表达水平在块茎萌芽期间在块茎中增加，这表明这两种 Ht1-FEHs 发挥不同的作用。在 NaCl 处理的植物茎中，Ht1-FEH I 和 II 的转录本水平均显著增加。NaCl 处理还诱导了 Ht1-FEH 在块茎中的转录，而 PEG 处理则轻微抑制了 Ht1-FEH II 在块茎中的表达。通过 HPLC 分析糖代谢酶活性和碳水化合物浓度表明，在 NaCl 和 PEG 处理下，1-FEH 的酶活性增加，但果糖含量降低。鉴于 FEH 将果聚糖水解为 Fru，我们讨论了在非生物胁迫下菊芋中 1-FEH 活性和果聚糖动态之间不一致的可能解释。
