**5. Raffinose Family Oligosaccharides (RFOs) and plant strategy to address stresses**

RFOs are a class of compatible solutes coming from Ins metabolism ramifications. As men‐ tioned before, the enzyme GolS (EC 2.4.1.123, highlighted in red in Figure 2) connects the metabolism of these compounds, producing galactinol (highlighted in red in Figure 2), which serves as galactose donor for further synthesis of RFO members [8, 9]. To date, structural genomics data and global transcriptome analysis concerning RFOs are only available for corn [46]. For this crop, the authors have performed a genomic identification of genes associated with raffinose metabolism, together with an expression analysis using data-mining from GEO (http://www.ncbi.nlm.nih.gov/geo) and PLEXdb databases (http://www.plexdb.org). Addi‐ tionally some transgenic lines overexpressing specifics gene isoforms related to RFO pathway, under particular growth conditions, are available for some species. These studies showed a positive impact in some crops.

Taji et al. [47], for example, analyzed the expression of seven genes encoding GoIS in *A. thaliana* under different stresses. From those genes, only three (AtGolS1, 2 and 3) were stressresponsive. AtGolS1 and AtGolS2 were induced by drought and high salt (250 mM NaCl), but not by low temperature (4°C). On the other hand, AtGolS3 was induced by low temperatures, but not high salinity or drought. The AtGolS2 overexpression in *A. thaliana* was associated with an increase of raffinose and galactinol and resulted in a reduction of leaf transpiration with a greater drought tolerance. In turn, Dos Santos et al. [48] analyzing three *Coffea arabica* galactinol synthase isoforms (CaGolS1, CaGolS2, CaGolS3) observed a mainly tissue-specific expression but differentiated regulation depending on the applied stress (drought, heat, and high salinity). This reinforces the observation by Taji et al. [47], indicating that different galactinol sets can be necessary for response to various stresses. The GOLS (BnGOLS-1) activity was also positively correlated with desiccation tolerance in cabbage seeds (*Brassica napus*) during the vegetative growth period [8]. The tolerance was observed around 21-24 days after flowering cabbage, coinciding with the accumulation of raffinose and stachyose. The BnGOLS-1 transcripts accumulation was concomitant with the formation of these two RFO members [8].

transgenic lines, transcription levels significantly induced for genes coding enzymes associ‐

Moreover, Alford et al. [45] reported MIOX enzymes responding to growing conditions of *A. thaliana* in low energy/nutrient environment. Their data supported the hypothesis that MIOX2 and MIOX4 enzymes would be encoded by multiple genes. Furthermore, on plants growing in low energy conditions/nutrients environments, MIOX2 enzyme would have a significant role in providing Ins to many different tissues, whereas MIOX4 would act in supplemental form at some tissues. An analysis of promoters was presented, considering multiple lines of MIOX2p:GUS and MIOX4p:GUS seedlings growing on: (I) no nutrients (agar); (II) low nutrients (agar + 0.5× MS salts); and (III) optimal nutrients (agar + 0.5× MS salts + 3% glucose) in low light (40 μE) during seven days. As a result, only MIOX2p:GUS was expressed abun‐ dantly in condition I, whereas in condition II, both MIOX2p:GUS and MIOX4p:GUS showed

**5. Raffinose Family Oligosaccharides (RFOs) and plant strategy to address**

RFOs are a class of compatible solutes coming from Ins metabolism ramifications. As men‐ tioned before, the enzyme GolS (EC 2.4.1.123, highlighted in red in Figure 2) connects the metabolism of these compounds, producing galactinol (highlighted in red in Figure 2), which serves as galactose donor for further synthesis of RFO members [8, 9]. To date, structural genomics data and global transcriptome analysis concerning RFOs are only available for corn [46]. For this crop, the authors have performed a genomic identification of genes associated with raffinose metabolism, together with an expression analysis using data-mining from GEO (http://www.ncbi.nlm.nih.gov/geo) and PLEXdb databases (http://www.plexdb.org). Addi‐ tionally some transgenic lines overexpressing specifics gene isoforms related to RFO pathway, under particular growth conditions, are available for some species. These studies showed a

Taji et al. [47], for example, analyzed the expression of seven genes encoding GoIS in *A. thaliana* under different stresses. From those genes, only three (AtGolS1, 2 and 3) were stressresponsive. AtGolS1 and AtGolS2 were induced by drought and high salt (250 mM NaCl), but not by low temperature (4°C). On the other hand, AtGolS3 was induced by low temperatures, but not high salinity or drought. The AtGolS2 overexpression in *A. thaliana* was associated with an increase of raffinose and galactinol and resulted in a reduction of leaf transpiration with a greater drought tolerance. In turn, Dos Santos et al. [48] analyzing three *Coffea arabica* galactinol synthase isoforms (CaGolS1, CaGolS2, CaGolS3) observed a mainly tissue-specific expression but differentiated regulation depending on the applied stress (drought, heat, and high salinity). This reinforces the observation by Taji et al. [47], indicating that different galactinol sets can be necessary for response to various stresses. The GOLS (BnGOLS-1) activity was also positively correlated with desiccation tolerance in cabbage seeds (*Brassica napus*) during the vegetative growth period [8]. The tolerance was observed around 21-24 days after flowering cabbage, coinciding with the accumulation of raffinose and stachyose. The

ated with ROS scavenging, suggesting an MIOX activity in reducing oxidative stress.

moderate expression, having been more expressed previously.

90 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

**stresses**

positive impact in some crops.

In *Arabidopsis thaliana* leaves overexpressing HsfA2 (Heat-Shock Transcription Factor A2; [49]) was also found highly induced GolS1, -2, -4 and Raffinose Syntase 2 (RS2; EC 2.4.1.82, highlighted in red in Figure 2) transcriptions. The galactinol and raffinose levels in the transgenic plants were higher compared to the wild-type lines, both in the control condition. These higher levels were positively correlated with an increase in plant tolerance when exposed to the studied stresses [mevalonate (50 mM), high salt (100 mM NaCl) and low temperatures (4°C)].

Latter, Pennycooke et al. [50] studied the expression of α-galactosidase gene (EC 3.1.2.22 highlighted in red in Figure 2) from petunia (Petunia x hibrida "Mitchell"), monitoring acclimated plants to low temperatures (4°C) and in response to increasing temperature (25°C). Transcripts induction were observed after one hour of desacclimation occurring together with an increase in enzymatic activity and decreased raffinose content, suggesting that the rise in temperature can regulate the RFO catabolism of certain members, through gene regulation that encoding α-galactosidase.

Thus, the diversity of functions performed by compounds presenting in the Ins metabolism was shown in the described works. Also, studies of distinct isoforms showed positive corre‐ lations with plant responses to various abiotic stresses. In this way, the identification of new transcripts, as well as the understanding of its regulation (spatial and temporal) in plants under unfavorable conditions for the development may lead to the discovery of new genes with biotechnological potential.

According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway database (http://www.genome.jp/kegg/pathway.html), which provides diagrams of various metabolic processes, at least 45 enzymes are associated with the metabolic pathways described here. Of these enzymes, 21 (highlighted in green boxes in Figures 3A and 3B) have been studied in previous works addressing the transcriptional expression of their genes or effects on plants under stress. Therefore, there are at least 24 enzymes (in red boxes in Figures 3A and 3B) that have not been targets of these analyses, with significant potential for further research in biotechnology.
