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

Abstract

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 (136)-␣-galactose (Gal) substitution of the (134)-␤-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 ϭ 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.Those leguminous seeds that retain an endosperm in the mature state (the endospermic legumes) always have endosperm cell walls that consist almost entirely of galactomannans. The galactomannans are multifunctional molecules. Before and during germination, their hydrophilic properties enable the endosperm to imbibe water and to deploy it to buffer the embryo against subsequent drought (Reid and Bewley, 1979). After germination, they are mobilized as storage reserves (Reid, 1985). The molecules themselves are composed of a (134)-␤-linked mannan backbone that carries single-unit galactosyl side chains attached (136)-␣. Their hydrophilic properties result from their highly branched structure (Man/Gal between 1.1 and about 3.5; Meier and Reid, 1982;Reid, 1985). Some legume seed galactomannans are used in industry. Once isolated from the seeds, they are water soluble and of high M r , giving viscous solutions that, on drying, form coherent, airtight films that rehydrate only slowly. Their applications exploit these properties (Dea and Morrison, 1975;. In the food context, it is well established that Gal content critically affects the commercial functional...

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Cell‐wall carbohydrates and their modification as a resource for biofuels

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