Analysis of the Raffinose Family Oligosaccharide Pathway in Pea Seeds with Contrasting Carbohydrate Composition

Abstract

(J.M.) Raffinose family oligosaccharides (RFOs) are synthesized by a set of galactosyltransferases, which sequentially add galactose units from galactinol to sucrose. The accumulation of RFOs was studied in maturing seeds of two pea (Pisum sativum) lines with contrasting RFO composition. Seeds of the line SD1 accumulated stachyose as the predominant RFO, whereas verbascose, the next higher homolog of stachyose, was almost absent. In seeds of the line RRRbRb, a high level of verbascose was accumulated alongside with stachyose. The increase in verbascose in developing RRRbRb seeds was associated with galactinol-dependent verbascose synthase activity. In addition, a galactinol-independent enzyme activity was detected, which catalyzed transfer of a galactose residue from one stachyose molecule to another. The two enzyme activities synthesizing verbascose showed an optimum at pH 7.0. Both activities were almost undetectable in SD1. Maximum activity of stachyose synthase was about 4-fold higher in RRRbRb compared with SD1, whereas the activities of galactinol synthase and raffinose synthase were only about 1.5-fold higher in RRRbRb. The levels of galactinol synthase and stachyose synthase activity were reflected by steady-state levels of corresponding mRNAs. We suggest that the accumulation of verbascose in RRRbRb was controlled by a coordinated up-regulation of the last steps of verbascose biosynthesis. Many higher plants accumulate raffinose family oligosaccharides (RFOs) during seed maturation.

Chain Elongation of Raffinose in Pea Seeds

“…Likewise, the rate of self-transfer from stachyose to stachyose was highest at pH 7.0, as previously shown for a crude enzyme preparation from pea seeds (16).…”

“…Then, self-transfer from stachyose simply represents the combination of reversal of the second half-reaction of stachyose synthase activity with the second half-reaction of verbascose synthase activity. The ratio V/K for stachyose as a donor was considerably lower than that for galactinol, suggesting that the contribution of the galactinol-independent pathway to the overall synthesis of verbascose in vivo is low unless the ratio of stachyose to galactinol is very high, which is only observed during late stages of seed development (16). It should also be noted that the water content of pea seeds progressively declines toward maturation, reaching a final value of as little as 0.1 g/g dry matter.…”

“…We have previously detected a galactinol-dependent verbascose synthase activity as well as a GGT activity in crude enzyme extracts from pea seeds (16). In contrast to GGT activity from A. reptans, highest activity was observed at pH 7.0.…”

Differences in accumulation of soluble α-galactosides during seed maturation of several Vicia species

“…Ob served pat terns of RFOs ac cu mu la tion dur ing mat u ra tion of Vicia seeds are sim i lar to other legumes like: soy bean (Lowell and Kuo 1989, Blackman et al 1992, Kuo et al 1997, Obendorf et al 1998a, Modi et al 2000, lu pin (Górecki et al 1996, Górecki et al 1997, pea (Górecki et al 2000, Peterbauer et al 2001, and field bean . Ac cu mu la tion of raffinose and stachyose takes place at the stage of seed fi nal matu ra tion and des ic ca tion, and dur ing that time sucrose con cen tra tion de clined (Obendorf et al 1998b).…”

“…α-Galactosides of su crose and cyclitols were an alyzed by high res o lu tion gas chro ma tog ra phy on a DB-1 cap il lary col umn (J&W Sci en tific, USA; 15 m length, 0.25 mm in ter nal di am e ter and 0.1 µm film thick ness) ac cord ing to method de scribed earlier (Górecki et al 1997, Peterbauer et al 2001. Each car bo hy drate was quan ti fied by us ing au thentic stan dards pur chased from Sigma (raffinose, stachyose, myo-inositol, D-pinitol) and Megazyme, Aus tra lia (verbascose).…”