Raffinose family oligosaccharides (RFOs) are putative abiotic stress protectants: case studies on frost tolerance and water deficit in Ajuga reptans and Arabidopsis thaliana

Abstract

Raffinose family oligosaccharides (RFOs) are α1,6 galactosyl extensions of sucrose, occurring exclusively in plants and some photoautotrophic algae. They have important roles as phloem translocates and carbon stores in plants. Additionally, correlative mass increases in RFOs during abiotic stress events have been observed. Despite such correlations, an in vivo functional role for RFOs in abiotic stress tolerance remains speculative as the stress tolerance response represents a concerted multigenic phenomenon that results in the accumulation of a number of other molecules with predicted roles in abiotic stress protection (e.g. sucrose and proline). As part of a multipronged strategy to further examine the role/s of RFOs in abiotic stress tolerance, we performed the following experiments. A previously described excised leaf system from the frost hardy evergreen labiate, Ajuga reptans, which effectively uncouples RFO accumulation from low temperature-acclimation was refined by physiologically validating that the basic carbohydrate metabolism of excised leaves is similar to that of whole plants. Using 14 classical C photosynthetic pulse/chase experiments, we demonstrated that excised 14 leaves operate within normal photosynthetic parameters producing C-sucrose as 14 the major photosynthate and transporting C-stachyose as the main phloem translocate, comparable to previous published data obtained with whole plants. Furthermore, in excised leaves, RFOs increased in both concentration and oligomer length over time. Using excised leaves grown in the warm with low, intermediate and high RFO concentrations, we demonstrated that frost tolerance (as determined by EL50, the temperature at which 50% electrolyte leakage occurred) increased from about -10.5 ℃ to -24.5 ℃ as RFO concentrations increased. Using Arabidopsis atgols1 and atgols1 atgols2 T-DNA loss-of-function mutants [galactinol synthases (GolS) are responsible for the first committed step in RFO biosynthesis; AtGolS-1 and -2 are water deficit-induced], we demonstrated that both soil-droughted mutants underwent a loss of leaf relative water content (RWC) more rapidly than wild type (Col-0) plants. The mutants showed a visible loss of leaf turgor at 53-60% RWC when Col-0 plants remained fully turgid (80% RWC). The typical water deficit-induced increase in GolS activity was absent in both atgols2 and atgols1 atgols2 plants. Galactinol and raffinose accumulation decreased strongly in stressed leaves of the mutant plants. Surprisingly, in stressed mutant leaves, sucrose accumulation decreased to less than 50% of stressed Col-0 leaves. An Arabidopsis raffinose synthase T-DNA loss-of-function mutant was also reported to display attenuated sucrose accumulation during cold acclimation. These data suggest that water-soluble carbohydrate accumulation in Arabidopsis during abiotic stress events is a complex process, possibly a homeostatic balance between a number of osmolytes. Whilst our data clearly demonstrates that GolS activity is a prerequisite for water deficit-induced accumulation of galactinol and raffinose, it is unclear whether the hypersensitive phenotype is the result of perturbation of raffinose accumulation alone or a combined effect of both sucrose and raffinose accumulation. To manipulate RFO concentrations in Arabidopsis while keeping the RFO biosynthetic pathway intact, the sequences of known and functionally expressed alkaline α-galactosidases (-Gals, enzymes involved in the first step of RFO hydrolysis) were compared against the Arabidopsis gene database. Two candidate genes (ATSIP1 and -2) were found. Since ATSIP1 had been previously characterised and predicted to be involved in RFO biosynthesis, we focused on ATSIP2 which we predicted to possibly encode an α-Gal. When ATSIP2 was heterologously expressed in Sf9 insect cells, crude cell lysates hydrolysed raffinose, contrary to uninfected control cells. Recombinant ATSIP2 enzyme hydrolysed, from the artificial substrate, p-nitrophenyl-D-galactopyranoside, only the α- and not the β-form, displayed a pH optimum of 7.5-8.0 and was inhibited by deoxygalactonojirimycin (a classical α-Gal inhibitor) as well as high galactose concentrations. No raffinose synthase activity was observed. Collectively, these results unequivocally demonstrate that ATSIP2 is an alkaline α-Gal. This finding is important in the context of recent publications which present real time PCR data for ATSIP2 during abiotic stress and define it as a raffinose synthase. Using this newly discovered α-Gal we created an overexpression vector (pMDC32::ATSIP2) and transformed Arabidopsis with it. We provide preliminary and partial proof-of-concept data demonstrating that, in independent T1 transgenic lines, ATSIP2 is overexpressed and alkaline α-Gal activity is higher than in untransformed wild type plants.