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The Hot and the Classic

RAFFINOSEOLIGOSACCHARIDES

Raffinose family oligosaccharides(RFOs) are -galactosyl derivatives of

Suc. The most common RFOs are thetrisaccharide raffinose, the tetrasac-charide stachyose, and the pentasac-charide verbascose. RFOs are nearlyubiquitous in the plant kingdom andare found in a large variety of seedsfrom many different families. They arecomponents of the carbohydrate re-serves of many seed types, rankingsecond only to Suc in abundance assoluble carbohydrates. RFOs accumu-late during seed development and dis-appear rapidly during germination.

Because -galactosidic linkages arenot cleaved by human digestion, flat-ulence often results after the con-sumption of RFO-rich seeds. Becauselegume seeds are particularly richsources of RFOs, RFO-induced flatu-lence reduces the acceptance and nu-tritional benefits of grain legumes.RFOs may also play a role in desicca-tion tolerance during seed maturation.More recently, there has been accumu-lating evidence that RFOs may play arole in protecting vegetative plant

parts to dehydrating stresses.

Flatulence Promoters

RFOS are found at especially highconcentrations in legume seeds. Dueto the absence of -galactosidase ac-tivity in human and animal intestinemucosa, RFOs escape digestion andare metabolized by bacteria to hydro-gen, carbon dioxide, and methane.Thus, RFOs have been identified asthe principal flatulence-causing fac-

tors present in legumes and otherseeds (Naczk et al., 1997). The com-mercial product Beano (GlaxoSmith-Kline, Middlesex, UK) advertised as asocial and scientific breakthrough) sup-plies -galactosidase and sucrase andhelps prevent intestinal gas by cata-lyzing the hydrolysis of these complexsugars. Flatulence is no laughing mat-ter: this problem is the single most im-portant factor that deters people from

eating more legumes (Delumen, 1992).The problem of legume seed-inducedflatulence affects not just humans, butall monogastric animals. Because ofRFOs, the quantity of soybean meal, for

example, must be limited in animalfeeds to avoid flatulence in dogs (Canisfamiliaris) and digestive disturbances inbaby pigs (Sus scrofa) and chicks (Gallusdomesticus) (Hartwig et al., 1997). Vari-ous methods have been recommendedfor the removal of flatulence-inducingRFOs, including dehulling, soakingand/or cooking in water and in buffersolutions, irradiation, enzymatic treat-ment, germination, and solvent extrac-tion (Naczk et al., 1997). Although areduction in RFOs is desirable from a

nutritional perspective, it is possiblethat if they become too low, the seedmay have reduced desiccation toler-ance and storability. Consequently,plant biologists have been searchingfor other desiccation-protecting factorssuch as galactosyl cyclitols that mightsubstitute for RFOs in promoting des-iccation tolerance but without havingthe unwanted anti-nutritional side ef-fects of RFOs (Horbowicz and Oben-dorf, 1994).

Seed Desiccation andStorability

RFOs have been implicated in theprotection of seeds against damageduring seed dehydration and aging,and therefore in seed survival andstorability (Obendorf, 1997). RFOsmay protect membranes, proteins, andnucleic acids against the damage thatoccurs during and upon the with-drawal of water in the drying seeds.This protective role of oligosaccha-rides has been explained mainly bytheir capacity to retain the integrity ofmembranes through their interactionwith the phospholipid headgroups,thus replacing water during dehydra-tion (Bentsink et al., 2000). It has alsobeen suggested, however, that RFOsmay form a viscous glassy state (athermodynamically unstable solidstate with an extremely high viscosity)during seed dehydration. It has beenhypothesized that this glassy statemay serve as a physical stabilizer pro-

tecting against deteriorative reactions.

RFOs, in particular, have been shownto have an excellent ability to formstable glasses, and therefore havebeen considered to be major determi-nants of seed storability (Koster and

Leopold, 1988). Indeed, the presenceof glasses has been associated withimproved seed storage stability (Sunand Leopold, 1997), and the contentof raffinose in maize (Zea mays) seedsis positively correlated with bothstorage stability and the magnitudeof the glassy state (Bernal-Lugo andLeopold, 1995). Nevertheless, a causalrole, if any, for RFOs, in contributinguniversally to desiccation tolerance isunclear, and several recent studieshave clouded the issue even more.

To examine the role of RFOs in pro-moting the vitreous state and seedstorage stability, Buitink et al. (2000)took advantage of the fact that osmo-priming (the pre-imbibition of seedsin osmotic solution) changes the oligo-saccharide composition of seeds. Seedpriming improves seed quality by en-hancing germination rates and seed-ling uniformity, but has the drawbackof reducing seed longevity by un-known means. The authors used aspin probe technique to measure the

molecular mobility and glass transi-tion temperature of the cytoplasm ofimpatiens (Impatiens walleriana) andbell pepper (Capsicum annuum) seedsthat had been osmo-primed or not.They found that the rotational corre-lation time of the polar spin probe inthe cytoplasm decreased, togetherwith seed longevity, as a function ofincreasing seed water content, sug-gesting that longevity may indeed beregulated by cytoplasmic mobility. Asexpected, osmo-priming of the seedsresulted in considerable decreases inlongevity and the oligosaccharide con-tent. There was no difference, how-ever, in the rotational motion of thespin probe in the cytoplasm betweencontrol and primed impatiens and bellpepper seeds. They concluded, there-fore, that oligosaccharides in seeds donot affect the stability of the intracel-lular glassy state.

Based on their studies of adesiccation-intolerant, ABA-deficientArabidopsis mutant, Ooms, Wilmer,and Karssen (1994) also concluded

that RFOs are not the primary factor

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Plant Physiology, March 2003, Vol. 131, pp. 11591160, www.plantphysiol.org 2003 American Society of Plant Biologists 1159

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determining desiccation tolerance inArabidopsis seeds. Desiccation toler-ance can be induced in this ABA-deficient mutant in vivo by supplyingan ABA-analog to the plant root sys-tem, but this increase in desiccationtolerance is not accompanied by sig-nificant changes in the carbohydratecomposition of the seeds. The researchof Bentsink et al. (2000) also castsdoubt on the importance of RFOs inseed desiccation in Arabidopsis. Theseauthors analyzed the soluble oligosac-charides (Suc, raffinose, and stachy-ose) content in the seeds of severalArabidopsis accessions and identifieda genotype that had a very low con-tent of these carbohydrates. By per-forming (QTL) mapping in a recombi-nant inbred line population, theyidentified one major QTL responsiblefor the near monogenic segregation of

seed stachyose content. This locus alsoaffected the content of Suc and raffin-ose. A comparison of the QTL geneticpositions revealed that the genomicregion containing the major oligosac-charide locus did not significantly af-fect seed storability. They concludedthat in Arabidopsis neither RFOs norSuc content has a major effect on seedstorability.

RFOs in Drought- andCold-Stress in VegetativePlant Parts

RFOs have been proposed to serveas osmoprotectants when plants areexposed to environmental water defi-cit stresses, such as cold and desicca-tion. Taji et al. (2002) found thatdrought-, high salinity-, and cold-treated Arabidopsis plants accumulatea large amount of raffinose and ga-lactinol, but not stachyose. Raffinose

and galactinol were not detected inunstressed plants. This suggests thatraffinose and galactinol are involvedin tolerance to drought, high salinity,and cold stresses. They identified

three stress-responsive galactinol syn-thase (GolS) genes (AtGolS1, 2 and 3)among seven Arabidopsis GolS genes.(GolS catalyzes the first step in thebiosynthesis of RFOs from UDP-Gal).AtGolS1 and 2 were induced bydrought and high-salinity stresses, butnot by cold stress, whereas AtGolS3was induced by cold stress but not bydrought or salt stress. The overexpres-sion of AtGolS2 caused an increase inendogenous galactinol and raffinose,and reduced transpiration. These re-sults show that stress-inducible GolSplays a key role in the accumulation ofgalactinol and raffinose under abioticstress conditions, and that galactinoland raffinose may function as osmo-protectants during drought-stress inplants.

Liu et al. (1998) also presented sev-eral diverse lines of evidence that sug-

gest a role for RFOs in drought- andcold-tolerance. They report that GolSactivity increased in kidney bean(Phaseolus vulgaris) seeds upon expo-sure of plants to cold. Moreover, GolSmRNA levels in the vegetative tissuesof Arabidopsis increased significantlyupon cold exposure, and these tran-scripts diminished upon return of theplants to room temperature. Finally,they established by protein sequencecomparison that a previously unidenti-fied gene belonging to a group of ABA-

independent, desiccation stress induc-ible genes isolated from rice (Oryzasativa) encodes the rice homolog theGolS gene.

LITERATURE CITED

Bentsink L, Alonso-Blanco C, Vreug-denhil D, Tesnier K, Groot SPC,Koornneef M (2000) Genetic analy-sis of seed-soluble oligosaccharidesin relation to seed storability ofAra-bidopsis. Plant Physiol 124:

15951604Bernal-Lugo I, Leopold AC 1995 Seedstability during storage - raffinosecontent and seed glassy state. SeedSci Res 5: 7580

Buitink J, Hemminga MA, HoekstraFA (2000). Is there a role for oligo-saccharides in seed longevity? Anassessment of intracellular glass sta-bility. Plant Physiol 122: 12171224

Delumen BO (1992) Molecular strate-gies to improve protein-quality andreduce flatulence in legumes - a re-view. Food Struct 11: 3346

Hartwig EE, Kuo TM, Kenty MM(1997) Seed protein and its relation-ship to soluble sugars in soybean.Crop Sci 37: 770773

Horbowicz M, Obendorf RL (1994)Seed desiccation tolerance and stor-ability: dependence on flatulence-producing oligosaccharides and cy-clitols. Seed Sci Res 4: 385405

Koster KL, Leopold AC (1988) Sugarsand desiccation tolerance in seeds.Plant Physiol 88: 829832

Liu JJJ, Krenz DC, Galvez AF, de Lu-

men BO (1998) Galactinol synthase(GS): increased enzyme activity andlevels of mRNA due to cold anddesiccation. Plant Sci 134: 1120

Naczk M, Amarowicz R, Shahidi F(1997) alpha-galactosides of sucrosein foods: Composition, flatulence-causing effects, and removal. ACSSymp Ser 662: 127151

Obendorf RL (1997) Oligosaccharidesand galactosyl cyclitols in seed des-iccation tolerance. Seed Sci Res 7:6374

Ooms JJJ, Wilmer JA, Karssen CM(1994) Carbohydrates are not thesole factor determining desiccationtolerance in seeds of Arabidopsisthaliana. Plant Physiol 90: 431436

Sun WQ, Leopold AC (1997) Cyto-plasmic vitrification acid survival ofanhydrobiotic organisms. CompBiochem Physiol A-Physiol 117:327333

Taji T, Ohsumi C, Iuchi S, Seki M,Kasuga M, Kobayashi M, Yama-guchi-Shinozaki K, Shinozaki K

(2002) Important roles of drought-and cold-inducible genes for ga-lactinol synthase in stress tolerancein Arabidopsis thaliana. Plant J 29:417426

Peter V. MinorskyDepartment of Natural Sciences

Mercy CollegeDobbs Ferry, NY 10522

1160 Plant Physiol. Vol. 131, 2003