The seeds of Lotus japonicus lines transformed with sense, antisense, and sense/antisense galactomannan galactosyltransferase constructs have structurally altered galactomannans in their endosperm cell walls.
Galactomannan biosynthesis in legume seed endosperms involves two Golgi membrane-bound glycosyltransferases, mannan synthase and galactomannan galactosyltransferase (GMGT). GMGT specificity is an important factor regulating the distribution and amount of (1-->6)-alpha-galactose (Gal) substitution of the (1-->4)-beta-linked mannan backbone. The model legume Lotus japonicus is shown now to have endospermic seeds with endosperm cell walls that contain a high-Gal galactomannan (mannose [Man]/Gal = 1.2-1.3). Galactomannan biosynthesis in developing L. japonicus endosperms has been mapped, and a cDNA encoding a functional GMGT has been obtained from L. japonicus endosperms during galactomannan deposition. L. japonicus has been transformed with sense, antisense, and sense/antisense ("hairpin loop") constructs of the GMGT cDNA. Some of the sense, antisense, and sense/antisense transgenic lines exhibited galactomannans with altered (higher) Man/Gal values in their (T(1) generation) seeds, at frequencies that were consistent with posttranscriptional silencing of GMGT. For T(1) generation individuals, transgene inheritance was correlated with galactomannan composition and amount in the endosperm. All the azygous individuals had unchanged galactomannans, whereas those that had inherited a GMGT transgene exhibited a range of Man/Gal values, up to about 6 in some lines. For Man/Gal values up to 4, the results were consistent with lowered Gal substitution of a constant amount of mannan backbone. Further lowering of Gal substitution was accompanied by a slight decrease in the amount of mannan backbone. Microsomal membranes prepared from the developing T(2) generation endosperms of transgenic lines showed reduced GMGT activity relative to mannan synthase. The results demonstrate structural modification of a plant cell wall polysaccharide by designed regulation of a Golgi-bound glycosyltransferase.
School of Biological and Environmental Sciences, University of Stirling, Stirling FK9 4LA, United Kingdom.
Mary E
ME
Edwards
Tze-Siang
TS
Choo
Cathryn A
CA
Dickson
Catherine
C
Scott
Michael J
MJ
Gidley
J S Grant
JS
Reid
D000595:Amino Acid Sequence; D016375:Antisense Elements (Genetics); D001483:Base Sequence; D002473:Cell Wall / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D005690:Galactose / Q000031:analogs & derivatives; D005700:Galactosyltransferases / Q000235:genetics* / Q000378:metabolism*; D000070116:Lotus / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D008351:Mannans / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D030821:Plants, Genetically Modified; D018749:RNA, Plant / Q000737:chemistry / Q000235:genetics; D012639:Seeds / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014170:Transformation, Genetic
D016375:Antisense Elements (Genetics); D018076:DNA, Complementary; D018744:DNA, Plant; D008351:Mannans; D018749:RNA, Plant; C012990:galactomannan; D005700:Galactosyltransferases; D005690:Galactose