Cloning of sucrose:sucrose 1-fructosyltransferase from onion and synthesis of structurally defined fructan molecules from sucrose.
Sucrose (Suc):Suc 1-fructosyltransferase (1-SST) is the key enzyme in plant fructan biosynthesis, since it catalyzes de novo fructan synthesis from Suc. We have cloned 1-SST from onion (Allium cepa) by screening a cDNA library using acid invertase from tulip (Tulipa gesneriana) as a probe. Expression assays in tobacco (Nicotiana plumbaginifolia) protoplasts showed the formation of 1-kestose from Suc. In addition, an onion acid invertase clone was isolated from the same cDNA library. Protein extracts of tobacco protoplasts transformed with this clone showed extensive Suc-hydrolyzing activity. Conditions that induced fructan accumulation in onion leaves also induced 1-SST mRNA accumulation, whereas the acid invertase mRNA level decreased. Structurally different fructan molecules could be produced from Suc by a combined incubation of protein extract of protoplasts transformed with 1-SST and protein extract of protoplasts transformed with either the onion fructan:fructan 6G-fructosyltransferase or the barley Suc:fructan 6-fructosyltransferase.
Vijn
I
I
Department of Molecular Cell Biology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands. i.m.c.vijn@bio.uu.nl
van Dijken
A
A
Lüscher
M
M
Bos
A
A
Smeets
E
E
Weisbeek
P
P
Wiemken
A
A
Smeekens
S
S
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005630:Fructans / Q000096:biosynthesis* / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000235:genetics; D006602:Hexosyltransferases / Q000235:genetics*; D008969:Molecular Sequence Data; D019697:Onions / Q000201:enzymology / Q000235:genetics*; D018515:Plant Leaves / Q000201:enzymology; D010947:Plants, Toxic; D011523:Protoplasts / Q000201:enzymology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000201:enzymology; D043324:beta-Fructofuranosidase; D001426:Bacterial Proteins
D018076:DNA, Complementary; D005630:Fructans; D006026:Glycoside Hydrolases; D006602:Hexosyltransferases; D012333:RNA, Messenger; D043324:beta-Fructofuranosidase; C021451:inulosucrase; D001426:Bacterial Proteins