Abstract
BackgroundThe sucrosylgalactoside oligosaccharide raffinose (Raf, Suc-Gal1) accumulates in Arabidopsis leaves in response to a myriad of abiotic stresses. Whilst galactinol synthases (GolS), the first committed enzyme in Raf biosynthesis are well characterised in Arabidopsis, little is known of the second biosynthetic gene/enzyme raffinose synthase (RS). Conflicting reports suggest the existence of either one or six abiotic stress-inducible RSs (RS-1 to -6) occurring in Arabidopsis. Indirect evidence points to At5g40390 being responsible for low temperature-induced Raf accumulation in Arabidopsis leaves.ResultsBy heterologously expressing At5g40390 in E.coli, we demonstrate that crude extracts synthesise Raf in vitro, contrary to empty vector controls. Using two independent loss-of-function mutants for At5g40390 (rs 5–1 and 5–2), we confirm that this RS is indeed responsible for Raf accumulation during low temperature-acclimation (4°C), as previously reported. Surprisingly, leaves of mutant plants also fail to accumulate any Raf under diverse abiotic stresses including water-deficit, high salinity, heat shock, and methyl viologen-induced oxidative stress. Correlated to the lack of Raf under these abiotic stress conditions, both mutant plants lack the typical stress-induced RafS activity increase observed in the leaves of wild-type plants.ConclusionsCollectively our findings point to a single abiotic stress-induced RS isoform (RS5, At5g40390) being responsible for Raf biosynthesis in Arabidopsis leaves. However, they do not support a single RS hypothesis since the seeds of both mutant plants still contained Raf, albeit at 0.5-fold lower concentration than seeds from wild-type plants, suggesting the existence of at least one other seed-specific RS. These results also unambiguously discount the existence of six stress-inducible RS isoforms suggested by recent reports.
Highlights
The sucrosylgalactoside oligosaccharide raffinose (Raf, Suc-Gal1) accumulates in Arabidopsis leaves in response to a myriad of abiotic stresses
Raffinose synthase (RS, EC 2.4.1.82) is an important enzyme involved in the biosynthesis of the raffinose family oligosaccharides (RFOs; Suc-[Gal]n, 13 < n ≥ 1) which are α1,6-galactosyl extensions of sucrose (Suc) occurring frequently in higher plants
When incubated with 50 mM Suc and 5 mM Gol at pH 7.5, crude extracts from E. coli cultures induced for recombinant protein expression with isopropyl β-D-1-thiogalactopyranoside (IPTG; 1 mM, 37°C, 4 h) were clearly able to synthesise a compound which eluted at the same retention time as a commercial Raf standard (Figure 1, lower chromatogram)
Summary
The sucrosylgalactoside oligosaccharide raffinose (Raf, Suc-Gal1) accumulates in Arabidopsis leaves in response to a myriad of abiotic stresses. Conflicting reports suggest the existence of either one or six abiotic stress-inducible RSs (RS-1 to −6) occurring in Arabidopsis. Indirect evidence points to At5g40390 being responsible for low temperature-induced Raf accumulation in Arabidopsis leaves. Plant RSs are generally poorly characterised and putative gene sequences have been reported for only a few plants, including pea, cucumber, maize, grape, rice and Arabidopsis [6]. Arabidopsis is reported to contain six putative abiotic stress-inducible RS genes [RS1-6, 10]. We have recently demonstrated that RS2 (At3g57520/ATSIP2), contrary to being reported as an abiotic stress-inducible RS [10,11,12], is a bona fide alkaline α-galactosidase (α-Gal, [13]). In the absence of complete functional characterisation of the six putative RSs, their actual number in Arabidopsis is a matter of speculation
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