Over-expression of α-galactosidase in pea seeds to reduce raffinose oligosaccharide contentThis paper is one of a selection of papers published in a Special Issue from the National Research Council of Canada – Plant Biotechnology Institute.

Abstract

The raffinose family of oligosaccharides (RFO) is a series of complex carbohydrates stored in seeds of many plant families, especially in legumes. The digestive system of nonruminant animals, including that of humans, cannot break down all of the chemical bonds in these carbohydrates; therefore, catabolism is achieved anaerobically by intestinal flora. The resulting digestive problems reduce acceptance and limit the widespread consumption of these otherwise nutritious seeds. To demonstrate a solution to this problem, transgenic lines of pea ( Pisum sativum L.) expressing the α-galactosidase gene from coffee ( Coffea arabica L.) were developed. Plants with a single copy of the inserted gene were selected, and two of these lines showed significant reductions of up to 40% in oligosaccharide content (raffinose, stachyose). Quantitative RT-PCR confirmed the presence of the α-galactosidase RNA in both leaves and cotyledons. Sugars were analyzed using whole seeds or only a portion of a seed; in the latter case, germination rates for each of the seeds analyzed were determined. The reduced raffinose contents did not affect germination rates, which remained very high (96%). The relative oligosaccharide contents of tissues within a seed also were determined; these were highest in the embryo axis, lower in the cotyledon and lowest in the seed coat.

Optimizing raffinose family oligosaccharides content in plants: A tightrope walk

“…α-Galactosidase hydrolyses the α(1→6) linkage to break RFOs. Overexpression of AGAL from coffee reduced the total RFOs in peas ( Polowick et al., 2009 ). Other RFOs degradative enzymes such as levansucrases and β-fructofuranosidases ( BFLUCT ) can also be targeted.…”

“…10 Strategies to reduce RFOs content in plants for nutritional enhancement 10.1 Upregulation of a-galactosidase and enhancing galactosyl cyclitols synthesis a-Galactosidase hydrolyses the a(1!6) linkage to break RFOs. Overexpression of AGAL from coffee reduced the total RFOs in peas (Polowick et al, 2009). Other RFOs degradative enzymes such as levansucrases and b-fructofuranosidases (BFLUCT) can also be targeted.…”

Properties and applications of α‐galactosidase in agricultural waste processing and secondary agricultural process industries

“…61 Different substrate-specific plant ⊍-galactosidases hydrolyze melibiose, raffinose, stachyose, and galactomannans at acidic or neutral or alkaline pH 48 ; ⊍-galactosidase hydrolyze RFO in yellow lupin (Lupinus luteus), 43 stachyose in (Vigna unguiculata), 43 melibiose, raffinose, and stachyose in Phaseolus coccineus, 44 and both raffinose and stachyose by heterologous expression of coffee ⊍-galactosidase in Pisum sativum. 62 Rice ⊍-galactosidases (⊍-gal I, ⊍-gal II, ⊍-gal III) have different catalytic efficiency with regard to substrates guargum and locust bean gum. 63 Recombinant expression of aglB encoding ⊍-galactosidase of Aspergillus fumigatus in pAN52-4, a fungal expression system, is known to enhance the enzyme yield on simple sugar without an inducer.…”

“… 58 Novel α ‐galactosidases from Mesorhizobium and Streptomyces , 59 thermophilic Talaromyces leycettanus JCM12802, 60 and Lactobacillus amylolyticus L6 also catalyze trans‐galactosylation to produce galacto‐oligosaccharides 61 . Different substrate‐specific plant α ‐galactosidases hydrolyze melibiose, raffinose, stachyose, and galactomannans at acidic or neutral or alkaline pH 48 ; α ‐galactosidase hydrolyze RFO in yellow lupin ( Lupinus luteus ), 43 stachyose in ( Vigna unguiculata ), 43 melibiose, raffinose, and stachyose in Phaseolus coccineus , 44 and both raffinose and stachyose by heterologous expression of coffee α ‐galactosidase in Pisum sativum 62 . Rice α ‐galactosidases ( α ‐gal I, α ‐gal II, α ‐gal III) have different catalytic efficiency with regard to substrates guar‐gum and locust bean gum 63 .…”

Activities of α-galactosidase and polygalacturonase during hydration of Dalbergia nigra ((Vell.) Fr All. ex Benth.) seeds at different temperatures

“…The activity of the α-galactosidase enzyme increased during the germination of Tachigali multijuga seeds, being related to the capacity to hydrolyze oligosaccharides, such as raffinose, stachyose and galactomannan polymers, indicating a special role during germination (Fialho et al, 2008). According to Polowick et al (2009), after super expression of the α-galactosidase gene in Pisum sativum seeds, the lines showed significant reductions in oligosaccharides, specifically raffinose and stachyose, and germination rates were 96%.…”