The Hot and the Classic

RAFFINOSE OLIGOSACCHARIDES
Raffinose family oligosaccharides (RFOs) are -galactosyl derivatives of Suc. The most common RFOs are the trisaccharide raffinose, the tetrasaccharide stachyose, and the pentasaccharide verbascose. RFOs are nearly ubiquitous in the plant kingdom and are found in a large variety of seeds from many different families. They are components of the carbohydrate reserves of many seed types, ranking second only to Suc in abundance as soluble carbohydrates. RFOs accumulate during seed development and disappear rapidly during germination. Because -galactosidic linkages are not cleaved by human digestion, flatulence often results after the consumption of RFO-rich seeds. Because legume seeds are particularly rich sources of RFOs, RFO-induced flatulence reduces the acceptance and nutritional benefits of grain legumes. RFOs may also play a role in desiccation tolerance during seed maturation. More recently, there has been accumulating evidence that RFOs may play a role in protecting vegetative plant parts to dehydrating stresses. eating more legumes (Delumen, 1992). The problem of legume seed-induced flatulence affects not just humans, but all monogastric animals. Because of RFOs, the quantity of soybean meal, for example, must be limited in animal feeds to avoid flatulence in dogs (Canis familiaris) and digestive disturbances in baby pigs (Sus scrofa) and chicks (Gallus domesticus) (Hartwig et al., 1997). Various methods have been recommended for the removal of flatulence-inducing RFOs, including dehulling, soaking and/or cooking in water and in buffer solutions, irradiation, enzymatic treatment, germination, and solvent extraction (Naczk et al., 1997). Although a reduction in RFOs is desirable from a nutritional perspective, it is possible that if they become too low, the seed may have reduced desiccation tolerance and storability. Consequently, plant biologists have been searching for other desiccation-protecting factors such as galactosyl cyclitols that might substitute for RFOs in promoting desiccation tolerance but without having the unwanted anti-nutritional side effects of RFOs (Horbowicz and Obendorf, 1994). RFOs, in particular, have been shown to have an excellent ability to form stable glasses, and therefore have been considered to be major determinants of seed storability (Koster and Leopold, 1988). Indeed, the presence of glasses has been associated with improved seed storage stability (Sun and Leopold, 1997), and the content of raffinose in maize (Zea mays) seeds is positively correlated with both storage stability and the magnitude of the glassy state (Bernal-Lugo and Leopold, 1995). Nevertheless, a causal role, if any, for RFOs, in contributing universally to desiccation tolerance is unclear, and several recent studies have clouded the issue even more. To examine the role of RFOs in promoting the vitreous state and seed storage stability, Buitink et al. (2000) took advantage of the fact that osmopriming (the pre-imbibition of seeds in osmotic solution) changes the oligosaccharide composition of seeds. Seed priming improves seed quality by enhancing germination rates and seedling uniformity, but has the drawback of reducing seed longevity by unknown means. The authors used a spin probe technique to measure the molecular mobility and glass transition temperature of the cytoplasm of impatiens (Impatiens walleriana) and bell pepper (Capsicum annuum) seeds that had been osmo-primed or not. They found that the rotational correlation time of the polar spin probe in the cytoplasm decreased, together with seed longevity, as a function of increasing seed water content, suggesting that longevity may indeed be regulated by cytoplasmic mobility. As expected, osmo-priming of the seeds resulted in considerable decreases in longevity and the oligosaccharide content. There was no difference, however, in the rotational motion of the spin probe in the cytoplasm between control and primed impatiens and bell pepper seeds. They concluded, therefore, that oligosaccharides in seeds do not affect the stability of the intracellular glassy state. Based on their studies of a desiccation-intolerant, ABA-deficient Arabidopsis mutant, Ooms, Wilmer, and Karssen (1994) also concluded that RFOs are not the primary factor
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Flatulence Promoters
RFOS are found at especially high concentrations in legume seeds. Due to the absence of -galactosidase activity in human and animal intestine mucosa, RFOs escape digestion and are metabolized by bacteria to hydrogen, carbon dioxide, and methane. Thus, RFOs have been identified as the principal flatulence-causing factors present in legumes and other seeds (Naczk et al., 1997). The commercial product Beano (GlaxoSmithKline, Middlesex, UK) advertised as a social and scientific breakthrough) supplies -galactosidase and sucrase and helps prevent intestinal gas by catalyzing the hydrolysis of these complex sugars. Flatulence is no laughing matter: this problem is the single most important factor that deters people from
www.plantphysiol.org/cgi/doi/10.1104/ pp.900066.

Seed Desiccation and Storability

RFOs have been implicated in the protection of seeds against damage during seed dehydration and aging, and therefore in seed survival and storability (Obendorf, 1997). RFOs may protect membranes, proteins, and nucleic acids against the damage that occurs during and upon the withdrawal of water in the drying seeds. This protective role of oligosaccharides has been explained mainly by their capacity to retain the integrity of membranes through their interaction with the phospholipid headgroups, thus replacing water during dehydration (Bentsink et al., 2000). It has also been suggested, however, that RFOs may form a viscous glassy state (a thermodynamically unstable solid state with an extremely high viscosity) during seed dehydration. It has been hypothesized that this glassy state may serve as a physical stabilizer protecting against deteriorative reactions.

Plant Physiology, March 2003, Vol. 131, pp. 11591160, www.plantphysiol.org 2003 American Society of Plant Biologists

determining desiccation tolerance in Arabidopsis seeds. Desiccation tolerance can be induced in this ABAdeficient mutant in vivo by supplying an ABA-analog to the plant root system, but this increase in desiccation tolerance is not accompanied by significant changes in the carbohydrate composition of the seeds. The research of Bentsink et al. (2000) also casts doubt on the importance of RFOs in seed desiccation in Arabidopsis. These authors analyzed the soluble oligosaccharides (Suc, raffinose, and stachyose) content in the seeds of several Arabidopsis accessions and identified a genotype that had a very low content of these carbohydrates. By performing (QTL) mapping in a recombinant inbred line population, they identified one major QTL responsible for the near monogenic segregation of seed stachyose content. This locus also affected the content of Suc and raffinose. A comparison of the QTL genetic positions revealed that the genomic region containing the major oligosaccharide locus did not significantly affect seed storability. They concluded that in Arabidopsis neither RFOs nor Suc content has a major effect on seed storability.

RFOs in Drought- and Cold-Stress in Vegetative Plant Parts

RFOs have been proposed to serve as osmoprotectants when plants are exposed to environmental water deficit stresses, such as cold and desiccation. Taji et al. (2002) found that drought-, high salinity-, and coldtreated Arabidopsis plants accumulate a large amount of raffinose and galactinol, but not stachyose. Raffinose and galactinol were not detected in unstressed plants. This suggests that raffinose and galactinol are involved in tolerance to drought, high salinity, and cold stresses. They identified

three stress-responsive galactinol synthase (GolS) genes (AtGolS1, 2 and 3) among seven Arabidopsis GolS genes. (GolS catalyzes the first step in the biosynthesis of RFOs from UDP-Gal). AtGolS1 and 2 were induced by drought and high-salinity stresses, but not by cold stress, whereas AtGolS3 was induced by cold stress but not by drought or salt stress. The overexpression of AtGolS2 caused an increase in endogenous galactinol and raffinose, and reduced transpiration. These results show that stress-inducible GolS plays a key role in the accumulation of galactinol and raffinose under abiotic stress conditions, and that galactinol and raffinose may function as osmoprotectants during drought-stress in plants. Liu et al. (1998) also presented several diverse lines of evidence that suggest a role for RFOs in drought- and cold-tolerance. They report that GolS activity increased in kidney bean (Phaseolus vulgaris) seeds upon exposure of plants to cold. Moreover, GolS mRNA levels in the vegetative tissues of Arabidopsis increased significantly upon cold exposure, and these transcripts diminished upon return of the plants to room temperature. Finally, they established by protein sequence comparison that a previously unidentified gene belonging to a group of ABAindependent, desiccation stress inducible genes isolated from rice (Oryza sativa) encodes the rice homolog the GolS gene. LITERATURE CITED Bentsink L, Alonso-Blanco C, Vreugdenhil D, Tesnier K, Groot SPC, Koornneef M (2000) Genetic analysis of seed-soluble oligosaccharides in relation to seed storability of Arabidopsis. Plant Physiol 124: 15951604 Bernal-Lugo I, Leopold AC 1995 Seed stability during storage - raffinose content and seed glassy state. Seed Sci Res 5: 7580

Buitink J, Hemminga MA, Hoekstra FA (2000). Is there a role for oligosaccharides in seed longevity? An assessment of intracellular glass stability. Plant Physiol 122: 12171224 Delumen BO (1992) Molecular strategies to improve protein-quality and reduce flatulence in legumes - a review. Food Struct 11: 3346 Hartwig EE, Kuo TM, Kenty MM (1997) Seed protein and its relationship to soluble sugars in soybean. Crop Sci 37: 770773 Horbowicz M, Obendorf RL (1994) Seed desiccation tolerance and storability: dependence on flatulenceproducing oligosaccharides and cyclitols. Seed Sci Res 4: 385405 Koster KL, Leopold AC (1988) Sugars and desiccation tolerance in seeds. Plant Physiol 88: 829832 Liu JJJ, Krenz DC, Galvez AF, de Lumen BO (1998) Galactinol synthase (GS): increased enzyme activity and levels of mRNA due to cold and desiccation. Plant Sci 134: 1120 Naczk M, Amarowicz R, Shahidi F (1997) alpha-galactosides of sucrose in foods: Composition, flatulencecausing effects, and removal. ACS Symp Ser 662: 127151 Obendorf RL (1997) Oligosaccharides and galactosyl cyclitols in seed desiccation tolerance. Seed Sci Res 7: 6374 Ooms JJJ, Wilmer JA, Karssen CM (1994) Carbohydrates are not the sole factor determining desiccation tolerance in seeds of Arabidopsis thaliana. Plant Physiol 90: 431436 Sun WQ, Leopold AC (1997) Cytoplasmic vitrification acid survival of anhydrobiotic organisms. Comp Biochem Physiol A-Physiol 117: 327333 Taji T, Ohsumi C, Iuchi S, Seki M, Kasuga M, Kobayashi M, Yamaguchi-Shinozaki K, Shinozaki K (2002) Important roles of droughtand cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana. Plant J 29: 417426

Peter V. Minorsky Department of Natural Sciences Mercy College Dobbs Ferry, NY 10522

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