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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

The Seeds of Lotus japonicus Lines Transformed with Sense, Antisense, and Sense/Antisense Galactomannan Galactosyltransferase Constructs Have Structurally Altered Galactomannans in Their Endosperm Cell Walls¹

School of Biological and Environmental Sciences, University of Stirling, Stirling FK9 4LA, United Kingdom; and Unilever Research Laboratory, Colworth House, Sharnbrook, Bedford MK44 1LQ, United Kingdom

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 (16)--galactose (Gal) substitution of the (14)--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 (T₁ generation) seeds, at frequencies that were consistent with posttranscriptional silencing of GMGT. For T₁ 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 T₂ 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.

¹ This work was supported by the Biotechnology and Biological Sciences Research Council (UK; research grant).

² Present address: Centre for Nutrition and Food Sciences, University of Queensland, Brisbane, 4072, Australia

^* Corresponding author; e-mail j.s.g.reid{at}stir.ac.uk; fax 44-1786-464994.

Received July 9, 2003; returned for revision August 10, 2003; accepted November 13, 2003.

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