Plants are sessile and hence must face the real challenges of nature in terms of biotic and abiotic stresses. These inevitable environmental factors often profoundly affect plant metabolism and photosynthesis, leading to a significant decline in crop yields and productivity. Plants have adapted different strategies to cope with the ever-changing climate during evolution. Plants use carbohydrates or their derivatives as stress-sensing and signalling molecules (Cummings, 2019) for coordinating metabolism with developmental features, plant growth and responses to external stimuli (Rolland et al., 2006). Furthermore, low-molecular weight soluble sugars, amino acids, and amines accumulate in the cytosol or vacuoles and help in the cell’s osmotic adjustment and also protect the cell membrane and other cell components from reactive oxygen species (ROS). Raffinose family oligosaccharides (RFOs) and Fructooligosaccharides (FOS) are one such class of soluble sugars which play an important role in abiotic stress response.

RFOs, such as raffinose, stachyose, and verbascose, are the non-reducing carbohydrates formed by α-1,6-galactosyl extensions onto the glucose moiety of sucrose. These compounds are nearly ubiquitous in crops (Minorsky, 2003), with contents varying from species to species and even from plant to plant, depending on the growing conditions. RFOs act as reserve carbohydrates (Gangl and Tenhaken, 2016) and are reported as compatible solutes that function like antioxidants, are a component of carbon partitioning strategies and may act as stress signals (Elsayed et al., 2014). RFOs benefit plants, but humans and other monogastric animals find them difficult to digest due to the absence of the enzyme required for their hydrolysis. Food containing higher RFOs takes a shorter time to pass through the digestive tract, causing reduced absorption of beneficial nutrients. The lack of hydrolysis in the small intestine and subsequent fermentation by the gut flora in the colon results in flatulence (Valentine et al., 2017). This limits the consumption of crops with higher RFOs (Kannan et al., 2018, Kasprowicz-Potocka et al., 2022). The balance between crop quality improvement and plant metabolism/immunity makes RFOs manipulation challenging for researchers.

This review highlights the structural chemistry of RFOs leading to their biosynthesis and subsequent degradation. The latest information on the genetic control of RFOs, their distribution, transport and physiological significance are also discussed. Recent studies targeting the manipulation of RFOs biosynthesis and transport to minimise their content in the human diet have also been highlighted, paving the way for producing low antinutrient, consumer-preferred food crops.

Raffinose family oligosaccharides (RFOs) are formed when galactose units are attached to the glucose moiety of sucrose via α-1,6-galactosidic linkages ( Figure 1A ). Galactinol serves as the galactose donor to sucrose, producing raffinose (trisaccharide), the first member of the RFOs family (Elango et al., 2022). Further addition of galactosyl residues forms stachyose (tetrasaccharide) and verbascose (pentasaccharide), which accumulate primarily in dicotyledonous seeds (Sengupta et al., 2015). Oligosaccharides with a higher degree of polymerization (DP) include ajugose (hexasaccharide), which is limited to some species of the Lamiaceae family, particularly Ajuga reptans (Haab and Keller, 2002). Higher plants seldom produce RFOs isomers with galactosidic linkages at other carbons of glucose (such as umbelliferose), fructose (such as Planteose and Sesamose), or both glucose and fructose moieties concurrently (such as Lychnose and Isolychnose) (Vanhaecke et al., 2010). However, these classes do not fall under the RFOs ( Figure 1B ). Unlike RFOs, sugars like lychnose/isolychnose are exclusively produced by the Caryophyllaceae family, acting as chemotaxic markers of this family (Madore, 2001).

RFOs biosynthesis begins with galactinol synthase (GolS), catalysing the galactosyl transfer to myo-inositol from UDP-D-galactose, synthesizing galactinol (dos Santos and Vieira, 2020). A higher concentration of galactinol than UDP-D-galactose in the developing seed suggests the function of galactinol as a transient galactosyl store, separated from primary carbohydrate metabolism (Peterbauer and Richter, 2001). Raffinose synthase (RS) catalyzes raffinose synthesis by a galactose-transfer (from galactinol) to glucose moiety of sucrose (Li et al., 2020). Myoinositol released in this process returns to the myoinositol pool. Raffinose synthase is said to be the most unstable enzyme in this pathway (Tian et al., 2019). Raffinose can cause a product inhibition effect on RS. So, it is rapidly converted to stachyose by stachyose synthase (SS) (Tian et al., 2019). Further, galactose transfer from galactinol to stachyose is catalyzed by verbascose synthase (VS), yielding verbascose ( Figure 2 ). Unlike RS, SS exhibits a broad substrate specificity, using a range of galactosyl cyclitols (galactosyl ononitol, galactopinitol A, galactinol) and methylated inositols (ononitol and pinitol) or myoinositol as galactosyl donors and acceptors, respectively. (Hoch et al., 1999; Peterbauer and Richter, 2001). Galactosylation of pinitol yields galactopinitol A, which acts as a galactosyl donor (to raffinose) and acceptor (yielding ciceritol). A strong negative correlation between digalactosyl cyclitol (ciceritol) and verbascose was found in two lentil cultivars (Frias et al., 1999), as the two pathways are linked via STS, inhibiting each other.

Two members of the Lamiaceae family (Ajuga reptans and Coleus blumei) exhibit a galactinol-independent pathway, where the galactan: galactan galactosyltransferase (GGT) enzyme catalyzes the galactosyl transfer from one RFO to another (Haab and Keller, 2002) ( Figure 3 ). Stachyose synthesized in the cytoplasm can be transported via the stachyose/H⁺ antiporter to the vacuole to participate in the galactinol-independent pathway (Greutert et al., 1998). GolS, RS, STS being extravacuolar and stachyose, verbascose and ajugose being exclusively vacuolar, suggests the synthesis of higher-order RFOs via a galactinol-independent pathway in the vacuole (Peterbauer and Richter, 2001). However, in seeds, the alkaline pH (pH 6.7) of vacuoles creates an unfavorable condition for acidic GGT enzyme, facilitating RFOs biosynthesis in the conventional galactinol-dependent pathway (Herman and Larkins, 1999; Peterbauer and Richter, 2001; Haab and Keller, 2002).

The RFOs and phytic acid biosynthetic pathways share a common intermediate (myoinositol). Low phytic acid mutants have increased myoinositol levels, possibly contributing to RFOs accumulation (Zhawar et al., 2011; Redekar et al., 2020). Higher sucrose concentrations also increase the accumulation of raffinose (Borisjuk et al., 2002). From this, it is difficult to say which substrate (myoinositol or sucrose) is more important. Since monocots predominantly accumulate raffinose, sucrose may play an important role, but for dicots, which mainly synthesize stachyose or verbascose, galactinol (from myoinositol) play a critical role rather than sucrose. This implies that all the metabolites are tightly regulated and that any changes can markedly affect RFOs deposition in seeds (Karner et al., 2004). Further study can be conducted to establish the differences between the dicot and monocot raffinose synthase genes, causing differential accumulation of RFOs in such plants.

Genes encoding enzymes involved in RFOs biosynthesis primarily have either seed-specific or phloem tissue-specific expression. Seed-specific GolS expression was reported to confer seed desiccation tolerance in tomatoes (Downie et al., 2003). No GolS expression in flowers, fruits, roots and endosperm, but a very high expression in leaves was observed when coffee plants were exposed to stress (drought/salt/heat) (dos Santos et al., 2011). The spatio-temporal expression patterns of hybrid poplar GolS homologues (Unda et al., 2012) and leaf-specific or anther-specific expression of cloned GolS genes from cotton (Zhou et al., 2012) suggest GolS expression is highly tissue-specific. GolS1 was also reported to facilitate raffinose synthesis in the storage pool of the common bugle (Ajuga reptans). At the same time, GolS2 plays a central role in synthesizing galactinol to transport the RFOs pool (Sprenger and Keller, 2000). Transcriptomic analysis of water stress-treated peanuts identified AdGolS3 as a candidate gene for drought tolerance (Vinson et al., 2020). Similar studies on kiwifruit (Actinidia chinensis) identified AcRS4 as critical during salt stress (Yang et al., 2022). The leaf and latex-specific HbGolS1 and latex-specific expression of HbRS1 were also reported in rubber (Hevea brasiliensis) (Lu et al., 2022). Among six GolS genes and the three RS genes in soybean, GmGolS1_A, GmRS2_A, and GmRS2_B form attractive gene targets because of their seed-specific expression patterns (de Koning et al., 2021). GmGolS1_A expression was highest during seed maturity, whereas soybean vegetative tissues primarily showed GmGolS1_B expression (Le et al., 2020). In Arabidopsis thaliana, among seven GolS isoforms, three isoforms, AtGolS1/AtGolS2 and AtGolS3 were induced during drought/salt/heat stress and cold stress, respectively (Taji et al., 2002). The inability of galactinol and raffinose accumulation in AtGolS1 mutants also suggests AtGolS1 as the major GolS isoform facilitating RFOs accumulation under heat stress (Panikulangara et al., 2004). Among six putative RS genes in Arabidopsis, overexpression of two RS genes showed oxidative stress tolerance in tobacco (Nishizawa et al., 2008). High seed-specific expression of PvGolS1 and PvRS2 in common beans (Phaseolus vulgaris) (de Koning et al., 2021) and AhRS14 and AhSS7 in peanuts (Sanyal et al., 2023) made them good candidates to knock out for low RFOs cultivar development just like the low raffinose soybean cultivars with mutated RS2 (Dierking and Bilyeu, 2008; Bilyeu and Wiebold, 2016).

Three transcription factors (TFs) can regulate GolS gene expression: heat shock factors (HSFs), DREB1A/CBF3, and WRKY transcription factors (Elsayed et al., 2014). HSFs and DREB1A/CBF3 are reported in Arabidopsis (Panikulangara et al., 2004; Maruyama et al., 2009), while WRKY regulates both GolS and RS via W-box cis-elements present in their promoters, as explained in Boea hygrometrica (Wang et al., 2009). GolS1 and GolS2 expression is regulated by HSFs like HsfA1a, HsfA1b, and HsfA2 in Arabidopsis (Nishizawa et al., 2008), HsfA4a in mustard (Lang et al., 2017) and HsfA2 in maize (Gu et al., 2019). HsfA2 and heat shock binding protein (HSBP) physically interact with each other and antagonistically modulate GolS expression. Overexpression of maize ZmDREB1A in the leaf also showed upregulation of ZmRS by binding to the DRE motif in the ZmRS promoter, enhancing raffinose synthesis and chilling stress tolerance (Han et al., 2020). A MYB-like transcription factor (AQUILO) isolated from Amur grapes (Vitis amurensis) improved cold tolerance through the upregulation of GolS and RS and osmoprotectant accumulation (Sun et al., 2018). Ethylene-responsive factors (ERFs) (PtrERF108) from trifoliate orange (Poncirus trifoliata) also target raffinose synthase (PtrRafS) directly, modulating raffinose levels in response to cold stress (Khan et al., 2021). Most TFs regulating RFOs in response to cold stress have been reported, while limited information is available for TF-mediated RFOs modulation in other stress situations.

The interplay of RFOs and phytohormones is also tightly controlled. Studies suggest ABA-induced RFOs accumulation in alfalfa somatic embryos (Blöchl et al., 2005) and regulation of maize GolS2 expression by VIVIPAROUS1- ABA INSENSITIVE5 (ZmVP1- ZmABI5) interaction (Zhang et al., 2019). The studies observed an increase in GolS activity and raffinose accumulation, revealing an ABA-RFOs crosstalk, the mechanism of which is yet to be identified. Promoter-GUS study by Salvi et al., 2020, demonstrated the positive influence of ABA and dehydration stress on chickpea GolS (CaGolS1 and CaGolS2) gene. Improved chlorophyll retention, relative water content and lower H₂O₂, malondialdehyde (MDA) content, and ion-leakage in transgenic lines suggested the potential role of GolS in modulating ROS and alleviating dehydration stress. OsPP65 (a type 2C protein phosphatase) knockout rice plants showed significant expression of ABA and jasmonic acid biosynthetic genes as well as their high endogenous levels during osmotic (salt) stress (Liu Q. et al., 2022). Metabolomics analysis showed higher endogenous galactose and galactinol content but a lower raffinose content in the transgenic rice suggesting negative regulation of OsPP65 through ABA and JA-mediated modulation of RFOs during salt stress tolerance. The role of Brassinosteroid (24-epibrassinolide/EBR) in the positive regulation of tea GolS gene (CsGolS2) and enhancement in ABA signal transduction (Zhang et al., 2022) also suggests the possible regulation of the RFOs gene by phytohormones.

α-Galactosidases (AGAL) are activated during germination and hydrolyze RFOs into simpler molecules, i.e., sucrose and galactose ( Figure 3 ). The galactokinases act upon the galactose removed during this process, forming D-galactose-1-phosphate. This compound is further metabolized by UDP-D-glucose-hexose-1-phosphate uridyltransferase (via the Leloir pathway) or UDP-D-galactose pyrophosphorylase (via the pyrophosphorylase pathway) (Peterbauer and Richter, 2001). RFOs and AGALs co-occur in protein storage vacuoles, but simultaneous synthesis and degradation are prevented due to the vacuole’s high pH during the reserve deposition and storage phase (Keller and Pharr, 1996). Evidence also negates the function of the protein storage vacuole as a lytic compartment (Jauh et al., 1999; Peterbauer and Richter, 2001). AGAL synthesised de novo (Group II AGAL) plays a role in galactomannan degradation, while pre-existing AGAL (Group I AGAL) appears to be responsible for RFOs hydrolysis (Katrolia et al., 2014). AGALs with acidic pH optima are also present in extracytoplasmic or vacuolar regions, but they are not effective at hydrolyzing larger RFOs, such as stachyose, and generally show a preference for small oligosaccharides (Peterbauer and Richter, 2001).

In animals, due to the lack of α-galactosidase enzyme, RFOs cannot be utilized. It passes to the lower gut and gets fermented by the gut microbiota (Arunraj et al., 2020). Out of thousands of bacteria in the human gut, about 10-15% have the potential to utilize raffinose as their substrate (Mao et al., 2018). Bacteria that prefer galactose to glucose or fructose as an energy source metabolize stachyose better than raffinose, while most bacteria commonly metabolize raffinose (Zartl et al., 2018). All bacteria that utilize raffinose do not necessarily have AGAL activity but still manage to degrade raffinose by using enzymes like β-fructofuranosidases (BFLUCT, hydrolase class) or levansucrases (transferase class). BFLUCT removes the fructosyl moiety of raffinose (yielding melibiose) and stachyose (yielding manninotriose). Bacteria producing both AGAL and BFLUCT can hydrolyze raffinose into galactose, glucose, and fructose. Two types of hydrolysis generally occur: intracellular and extracellular. In the case of intracellular hydrolysis, raffinose hydrolysis occurs inside the cell after transporting it via raffinose transporters. In contrast, for extracellular hydrolysis, raffinose can be hydrolyzed into fructose and melibiose by levansucrases ( Figure 3 ). The hydrolysis products are transported inside the cell and metabolized for energy supply. Hence, glycosidases and transporters play a vital role, enabling gut bacteria to utilize galactosides differently (Teixeira et al., 2012; Mao et al., 2018). However, most studies used raffinose as the substrate for gut bacteria, while the effect of stachyose and verbascose as a substrate needs further validation.

The content and composition of RFOs vary across the genotypes and environmental conditions ( Table 1 ) (Kumar et al., 2010; Redekar et al., 2020). Seeds are the primary storage site for RFOs. Plants may store RFOs in tubers or mesophyll cells of photosynthesizing leaves, sometimes reaching even 25-80% of dry weight (Keller and Matile, 1985). All seed parts, viz., embryo, endosperm or seed coat, can retain α-galactosides at varying levels (Martínez-Villaluenga et al., 2008; Sengupta et al., 2015). Reports suggest that lupin seeds have the highest RFOs, followed by soybean (Ruiz-López et al., 2000; Martínez-Villaluenga et al., 2008). Stachyose seems to be the predominant RFO in dicot crops. However, monocot seeds such as barley and wheat primarily accumulate raffinose (Yan et al., 2022). Among the commonly cultivated crops, ajugose was found exclusively in lupin seeds. In crop plants, the reports suggest that ciceritol, a pinitol digalactoside, is found only in chickpeas and lentils, with chickpeas accumulating the maximum amount (1.2-3.1%). Among legumes, groundnut and faba bean have been reported to have lower amounts of RFOs (0.12-.076% and 1.0-4.5%, respectively) (Bishi et al., 2015; Kannan et al., 2018; Sanyal et al., 2023). Analysis of RFOs in Brassica, barley (Andersen et al., 2005), and wheat (Huynh et al., 2008) suggested that non-legumes contain comparatively lower amounts of these oligosaccharides. Although various studies discussed the variations in RFOs content/composition, the evolutionary reason behind the accumulation of high DP compounds (verbascose, ajugose, ciceritol), having higher energy costs, is still unexplored. Moreover, there is significant research on RFO concentrations in legume crops, while it is still scanty for non-legumes.

Plants belonging to Gamalei’s “Type 1 category” (such as cucurbits) have a high plasmodesmata abundance between companion cells and mesophyll cells and assimilate is loaded via a polymer trap mechanism in a symplastic route (Gamalei, 1989). RFOs are predominantly transported in such plants. On the other hand, “Type 2 plants” with lower plasmodesmata frequency (such as potato and Arabidopsis) primarily transport sucrose via proton symport in an apoplastic route (Turgeon, 1996; Haritatos et al., 2017). The polymer trap model states that the specialized companion cells (intermediary cells) in the minor veins are where RFOs biosynthetic enzymes transform the sucrose generated by photosynthesis in mesophyll cells (source) into RFOs (mainly raffinose and stachyose) (Cao et al., 2013). The plasmodesmata in RFOs-utilizing plants are characteristically branched on the side of companion cells, significantly reducing the plasmodesmatal pore size. The RFOs cannot diffuse back to the mesophyll (source) because they are larger and are trapped in the intermediary cells. Conversion of sucrose into RFOs favors passive entry of sucrose, while RFOs accumulation increases osmotic pressure. This makes it easier for the RFOs to migrate toward the sieve elements, followed by transportation to other areas of the plant (sinks), where AGAL may break them down (Turgeon and Medville, 2004). Plants can maintain a high phloem sugar concentration by producing RFOs in the intermediate cells. Although species-specific, this paradigm is primarily observed in the Cucurbitaceae family. Meagre amounts of RFOs are transported by Type 1 plant species lacking intermediate cells, and they mostly load assimilates via the apoplastic pathway (Hannah et al., 2006).

In leaves that transport, as well as store RFOs, such as Xerosicyos danguyi (Cucurbitaceae) and Ajuga reptans (Lamiaceae), RFOs biosynthetic components are present in both phloem and mesophyll tissues in different isoforms, involving complex cellular partitioning (Madore, 2001). Plants having variegated leaves (such as Coleus blumei Benth) do not use AGAL for RFOs degradation in non-photosynthesizing patches. Instead, they possibly use the reverse (or backward) reaction of RS and SS to degrade RFOs into disaccharides (galactinol and sucrose). The products obtained thereof can support respiration in the absence of photosynthesis (Madore, 2001). ¹⁴C-labelling study in Cucumis blumei indicates limited phloem transport of galactinol and efficient retention and transportation of sucrose, raffinose and stachyose. Studies estimating raffinose and galactinol levels observed 30 times lower raffinose in Cucumis leaves but only two-fold lower raffinose in phloem exudates (Ayre et al., 2003; Hannah et al., 2006), suggesting higher transport efficiency of raffinose as compared to galactinol.

Additionally, raffinose can supplement sucrose as phloem-mobile forms of carbon, delivering 1.5 times more carbon than sucrose at the same osmotic cost. This is often seen to support non-photosynthetic tissues and organs (Madore, 2001). When plants with an apoplastic phloem loading strategy (Type 2 plants) were engineered to follow a symplastic route (polymer trap mechanism) via metabolic engineering of RFOs biosynthetic genes, the synthesis of RFOs and their transportation was deficient despite the high sucrose concentration (Hannah et al., 2006; Cao et al., 2013). Theoretically, apoplasmic loaders should synthesize RFOs efficiently due to ample carbon (reduced form) in the companion cell cytoplasm where RFOs synthesis occurs. Moreover, there are no limitations in the plasmodesmatal pore size. The inability of high RFOs accumulation in companion cells can be a biochemical limitation or a cell biology problem (Yadav et al., 2015). There can be limitations in the flux of early RFOs precursors such as UDP-D-galactose and myoinositol or variations in the internal membrane and vacuoles. “Type 1” plants with numerous, highly branched plasmodesmata generally have small vacuoles, extensive endomembrane systems and companion cells larger than “Type 2” plants. Thus, the internal structure can also contribute to the biosynthesis of RFOs in companion cells (Turgeon et al., 2001). The stability, localization and interaction of enzymes with other cellular components can probably explain the inefficient synthesis and transport of RFOs in “Type 2” plants.

In recent decades, considerable progress has been made in understanding the RFOs structural diversity, biosynthesis, translocation, and catabolism. The varied roles of RFOs in plants and animals ask for the optimization of RFOs level to reduce flatulence production without interfering with the normal metabolism of the crop. Such ideal levels need to be ascertained. In the era of global warming, RFOs have the potential to enhance sugar export to phloem and improve crop performance under elevated carbon dioxide. Superimposition of the polymer trap mechanism on apoplastic phloem loaders or vice versa can be an attractive strategy to increase the economic yield of a crop. Sink-specific expression or catabolism of RFOs can modulate the hydrostatic pressure, allowing for a targeted partitioning of photoassimilates.

In future, the connection of RFOs with phytic acid, fructooligosaccharide phenols and methyl ether derivatives of cyclitols can be studied in detail so that the metabolic shift of high phytate crops into RFOs can be engineered. This will facilitate the reduction of antinutrients in crops and a limited increase in RFOs incapable of causing flatulence. Low RFOs lines with increased protein or oil content can also be prepared by altering central carbon metabolism so that they can be used in the vegetable oil industry. Moreover, using several α-galactosidase crude preparations can enhance the nutritional quality of high RFOs crops and fulfil the protein requirement of the community.

RS and SB compiled and wrote the manuscript. SK, AP, and AK provided revisions of scientific content. All authors contributed to the article and approved the submitted version.