Functional characterization and vacuolar localization of fructan exohydrolase derived from onion (Allium cepa)

Abstract

Fructans such as inulin and levan accumulate in certain taxonomic groups of plants and are a reserve carbohydrate alternative to starch. Onion (Allium cepa L.) is a typical plant species that accumulates fructans, and it synthesizes inulin-type and inulin-neoseries-type fructans in bulb. Although genes for fructan biosynthesis in onion were identified so far, no genes for fructan degradation had been found. In this study, phylogenetic analysis predicted that we isolated a putative vacuolar invertase gene (AcpVI1), but our functional analyses demonstrated that it encoded a fructan 1-exohydrolase (1-FEH) instead. Assessments of recombinant proteins and purified native protein showed that the protein had 1-FEH activity, hydrolyzing the β-(2,1) fructosyl linkage in inulin-type fructans. Interestingly, AcpVI1 had an amino acid sequence close to those of vacuolar invertases and fructosyltransferases, unlike all other FEHs previously found in plants. We showed that AcpVI1 was localized in the vacuole, as are onion fructosyltransferases Ac1-SST and Ac6G-FFT. These results indicate that fructan-synthesizing and -degrading enzymes are both localized in the vacuole. Contrary to previously reported FEHs, our data suggest that onion 1-FEH evolved from a vacuolar invertase and not from a cell-wall invertase. This demonstrates that classic phylogenetic analysis on its own is insufficient to discriminate between invertases and FEHs, highlighting the importance of functional markers in the near active region.

Fructan exohydrolases (FEHs) are upregulated by salicylic acid together with defense‐related genes in non‐fructan accumulating plants

“…This could be due to differences in the kinetics of protein synthesis and/or to post‐translational regulation. Indeed, FEH proteins are close to invertases (Lammens et al, 2009; Oku et al, 2022), which are known to be regulated by protein inhibitors (Hothorn et al, 2010). In addition, the decoupling between transcript levels and activities could be due to partial protein extraction.…”

“…Plant FEHs are mainly found in the vacuole (Frehner et al, 1984; Oku et al, 2022), where they are involved in the degradation of fructans during periods of reserve mobilization (Morvan‐Bertrand et al, 2001), in fructan trimming during fructan biosynthesis (Lothier et al, 2007; Van den Ende, Clerens, et al, 2003) and in stress tolerance (Yoshida, 2021). Under stress, FEH activity was also detected in the apoplast together with fructans in response to cold in oat ( Avena sativa ; Livingston & Henson, 1998) and wheat ( Triticum aestivum ; Van den Ende et al, 2005).…”

Different evolutionary pathways to generate plant fructan exohydrolases

“…Also, during the last stages of onion bulb development, 1-FEH activities remained significantly higher in this variety, suggesting that a continuous trimming by 1-FEH inhibits overall FOS accumulation. Therefore, it was decided to search for the 1-FEH gene and purify the native 1-FEH enzyme from developing onion bulbs ( Oku et al , 2022 ).…”

“…However, it was found that FEHs resembled cell wall invertases instead. Oku et al (2022 ) have now characterized a novel fructan 1-exohydrolase (1-FEH) from onion, an economically important species. This enzyme fits well within the vacuolar invertase subgroup, demonstrating that plant FEHs may be recruited from different types of ancestral invertases.…”

“…In the last two decades, all characterized plant FEHs were found to fit well within the CWI/FEH subgroup. Oku et al (2022) have now demonstrated that evolution followed a different path to generate a novel 1-FEH in onion with a vacuolar localization, fitting into the VI/FT subgroup instead.…”