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 (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.

Figure 1.

Dry weights of developing L. japonicus endosperms at various times after anthesis.

Figure 2.

Accumulation of neutral monosaccharide residues in total cell wall material from developing L. japonicus endosperms. □, Man. ▪, Gal. ♦, Glc. ▴, Ara.

Figure 3.

Autoradiogram of thin-layer chromatography (TLC)-separated endo-(1→4)-β-d-mannanase digestion products from radiolabeled polysaccharide products formed on incubating microsomal membranes from 28-DPA L. japonicus endosperms with GDP-(¹⁴C) Man (upper lane) and with GDP-(¹⁴C) Man plus unlabeled UDP-Gal (lower lane). M, Man; M2G, monogalactosylmannobiose; M3G, monogalactosylmannotriose; M5G2, digalactosylmannopentaose.

Figure 4.

Comparison of the protein sequence of fenugreek GMGT (Edwards et al., 1999) with the open reading frame encoded by the putative GMGT cDNA from developing L. japonicus endosperm. The fenugreek sequence and identical amino acid residues in the L. japonicus sequence are boxed. Fgt.pro, Fenugreek GMGT protein sequence. Ljgt.pr, L. japonicus GMGT protein sequence.

Figure 5.

Man/Gal values for endosperm galactomannans in individual seeds (T₁ generation, self-pollination) from various transgenic lines, in relation to transgene segregation in the corresponding embryos. Values marked with a white (□) bar relate to endosperms corresponding to azygous (PCR negative) embryos. Values marked with a filled (▪) bar relate to endosperms corresponding to transgenic (PCR positive) embryos. a, PCR result not available; assumed negative. b, PCR result not available; assumed positive.

Figure 6.

A, Galactomannan amount in relation to Man/Gal values for individual seed endosperms from transgenic and azygous individuals (♦). Superimposed is the theoretical line that would be followed if increasing Man/Gal values were due exclusively to decreasing Gal substitution of a constant amount of mannan backbone. B, Amount of mannan backbone in relation to Man/Gal values for individual seed endosperms from transgenic and azygous individuals. Superimposed is a fitted third order polynomial trend line.