In vivo modification of the cell wall polysaccharide galactomannan of guar transformed with a α-galactosidase gene cloned from senna

Abstract

Galactomannans are composed of a β 1to4 mannan backbone with varying degrees of α 1to6 galactose substitutions and are found in the cell walls of legume endosperm. Like other cell wall polysaccharides, many factors controlling the metabolism of galactomannans remain to be elucidated. In the endospermous legume senna (Senna occidentalis) increased α-galactosidase activity has previously been observed to coincide temporarily with a decrease of the galactose content of the galactomannan. To evaluate the potential role of α-galactosidase for the control of the final galactose content, a α-galactosidase gene expressed in immature senna seeds was cloned and transformed into the related high-yielding species guar (Cyamopsis tetragonoloba). The isolated cDNA encoded a 406 amino acid protein with a calculated molecular mass of 44 313 Da. The amino acid sequence was 75% identical to the galactomannan hydrolysing α-galactosidases from germinating guar and coffee bean. The senna α-galactosidase gene was inserted behind a wheat high-molecular-weight glutenin promoter in the vector employed for transformation of guar by Agrobacterium tumefaciens-mediated gene transfer. About 30% of the guar transformants produced endosperm with galactomannans where the galactose content was significantly reduced. After self-fertilization of primary transformants displaying the highest galactose reduction of the galactomannan, endosperms of R₁ plants were analysed demonstrating that this property was inherited stably to the progeny and that it was 100% coupled to the presence of the senna α-galactosidase gene. This suggests that α-galactosidases can be involved in the determination of the final galactose content of endosperm galactomannans, demonstrating that cell wall polysaccharide biosynthesis can be modified in vivo.