Cloning and functional characterization of a fructan 1-exohydrolase (1-FEH) in the cold tolerant Patagonian species Bromus pictus.
Fructans are fructose polymers synthesized in a wide range of species such as bacteria, fungi and plants. Fructans are synthesized by fructosyltransferases (FTs) and depolymerized by fructan exohydrolases (FEHs). Bromus pictus is a graminean decaploid species from the Patagonian region of Argentina, which accumulates large amounts of fructans even at temperate temperatures. The first gene isolated from B. pictus fructan metabolism was a putative sucrose:fructan 6-fructosyltransferase (6-SFT). Here, a complete cDNA of the first fructan exohydrolase (FEH) from B. pictus (Bp1-FEHa) was isolated using RT-PCR strategies. The Bp1-FEHa encoding gene is present as a single copy in B. pictus genome. Functional characterization in Pichia pastoris confirmed Bp1-FEHa is a fructan exohydrolase with predominant activity towards beta-(2-1) linkages. Its expression was analyzed in different leaf sections, showing the highest expression levels in the second section of the sheath and the tip of the blade. Bp1-FEHa expression was studied along with FEH and FT activities and fructan accumulation profile in response to chilling conditions during a 7-day time course experiment. Bp1-FEHa expression and FEH activity followed a similar pattern in response to low temperatures, especially in basal sections of the sheaths. In these sections the FEH and FT activities were particularly high and they were significantly correlated to fructan accumulation profile, along with cold treatment.
Instituto de Biotecnología, CICVyA, Instituto Nacional de Tecnología Agropecuaria, INTA, Hurlingham, 1686, Buenos Aires, Argentina.
Florencia
F
del Viso
Andrea F
AF
Puebla
H Esteban
HE
Hopp
Ruth Amelia
RA
Heinz
D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D015139:Blotting, Southern; D031724:Bromus / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D003080:Cold Temperature; D005630:Fructans / Q000378:metabolism; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics*; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D010843:Pichia; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D001426:Bacterial Proteins
D005630:Fructans; D006026:Glycoside Hydrolases; D006602:Hexosyltransferases; D011994:Recombinant Proteins; C021451:inulosucrase; D001426:Bacterial Proteins; C044118:fructan beta-fructosidase