Fructan 1-Exohydrolases. β-(2,1)-Trimmers during Graminan Biosynthesis in Stems of Wheat? Purification, Characterization, Mass Mapping, and Cloning of Two Fructan 1-Exohydrolase Isoforms

• Published in: Plant physiology (Bethesda) 2003-02, Vol.131 (2), p.621-631
• Main Authors: Van den Ende, Wim, Clerens, Stefan, Vergauwen, Rudy, Liesbet Van Riet, Van Laere, André, Yoshida, Midori, Kawakami, Akira
• Format: Article
• Language: English
• Subjects: Agronomy. Soil science and plant productions; Amino Acid Sequence; Amino acids; Barley; beta-Fructofuranosidase; Biological and medical sciences; Biosynthesis; Cell Wall - enzymology; Cell walls; Classical and quantitative genetics. Population genetics. Molecular genetics; Cloning, Molecular; Complementary DNA; DNA, Complementary - chemistry; DNA, Complementary - genetics; Economic plant physiology; Enzymes; Flowering; Fructans; Fundamental and applied biological sciences. Psychology; Generalities. Genetics. Plant material; Genes. Genome; Genetics and breeding of economic plants; Glycoside Hydrolases - genetics; Glycoside Hydrolases - isolation & purification; Glycoside Hydrolases - metabolism; Hydrogen-Ion Concentration; Isoenzymes - genetics; Isoenzymes - metabolism; Mass Spectrometry; Metabolism; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Net assimilation, photosynthesis, carbon metabolism. Photorespiration, respiration, fermentation (anoxia, hypoxia); Nutrition. Photosynthesis. Respiration. Metabolism; Photosynthesis, respiration. Anabolism, catabolism; Phylogeny; Plant physiology and development; Plant Stems - enzymology; Plant Stems - genetics; Plant Stems - growth & development; Plants; Protein isoforms; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Substrate Specificity; Triticum - enzymology; Triticum - genetics; Triticum - growth & development; Whole Plant and Ecophysiology
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.015305

Graminan-type fructans are temporarily stored in wheat (Triticum aestivum) stems. Two phases can be distinguished: a phase of fructan biosynthesis (green stems) followed by a breakdown phase (stems turning yellow). So far, no plant fructan exohydrolase enzymes have been cloned from a monocotyledonous species. Here, we report on the cloning, purification, and characterization of two fructan 1-exohydrolase cDNAs (1-FEH w1 and w2) from winter wheat stems. Similar to dicot plant 1-FEHs, they are derived from a special group within the cell wall-type invertases characterized by their low isoelectric points. The corresponding isoenzymes were purified to electrophoretic homogeneity, and their mass spectra were determined by quadrupole-time-of-flight mass spectrometry. Characterization of the purified enzymes revealed that inulin-type fructans [β-(2,1)] are much better substrates than levan-type fructans [β-(2,6)]. Although both enzymes are highly identical (98% identity), they showed different substrate specificity toward branched wheat stem fructans. Although 1-FEH activities were found to be considerably higher during the fructan breakdown phase, it was possible to purify substantial amounts of 1-FEH w2 from young, fructan biosynthesizing wheat stems, suggesting that this isoenzyme might play a role as a β-(2,1)-trimmer throughout the period of active graminan biosynthesis. In this way, the species and developmental stage-specific complex fructan patterns found in monocots might be determined by the relative proportions and specificities of both fructan biosynthetic and breakdown enzymes.