Molecular cloning of AtRS4, a seed specific multifunctional RFO synthase/galactosylhydrolase in Arabidopsis thaliana

Abstract

Stachyose is among the raffinose family oligosaccharides (RFOs) one of the major water-soluble carbohydrates next to sucrose in seeds of a number of plant species. Especially in leguminous seeds, e.g. chickpea, stachyose is reported as the major component. In contrast to their ambiguous potential as essential source of carbon for germination, RFOs are indigestible for humans and can contribute to diverse abdominal disorders. In the genome of Arabidopsis thaliana, six putative raffinose synthase genes are reported, whereas little is known about these putative raffinose synthases and their biochemical characteristics or their contribution to the RFO physiology in A. thaliana. In this paper, we report on the molecular cloning, functional expression in Escherichia coli and purification of recombinant AtRS4 from A. thaliana and the biochemical characterisation of the putative stachyose synthase (AtSTS, At4g01970) as a raffinose and high affinity stachyose synthase (Km for raffinose 259.2 ± 21.15 μM) as well as stachyose and galactinol specific galactosylhydrolase. A T-DNA insertional mutant in the AtRS4 gene was isolated. Only semi-quantitative PCR from WT siliques showed a specific transcriptional AtRS4 PCR product. Metabolite measurements in seeds of ΔAtRS4 mutant plants revealed a total loss of stachyose in ΔAtRS4 mutant seeds. We conclude that AtRS4 is the only stachyose synthase in the genome of A. thaliana that AtRS4 represents a key regulation mechanism in the RFO physiology of A. thaliana due to its multifunctional enzyme activity and that AtRS4 is possibly the second seed specific raffinose synthase beside AtRS5, which is responsible for Raf accumulation under abiotic stress.

Regulation of Seed Vigor by Manipulation of Raffinose Family Oligosaccharides in Maize and Arabidopsis thaliana

“…The atrs4 mutant was devoid of stachyose, but had greater raffinose in the seeds, while the doubleknockout atrs4 atrs5 mutant seeds lacked both raffinose and stachyose but had much more galactinol (Gangl and Tenhaken, 2016). In keeping with a role for RFOs in seed vigor, the A. thaliana atrs4 mutant seeds (no stachyose; increased raffinose) completed germination faster than control seeds (Gangl et al, 2015), while the atrs4 atrs5 double-mutant seeds exhibited a 5-day delayed completion of germination in darkness, which was alleviated in the light or partially alleviated by the addition of galactose to the medium in darkness, both novel findings. To our knowledge, the atrs4 mutant in A. thaliana is the sole example of the recovery and characterization of a mutation in a STACHYOSE SYNTHASE (Gangl et al, 2015) while no reports have explored the mutation of enzymes involved in RFOs with a DP greater than that of stachyose.…”

“…RFOs with a higher degree of polymerization (DP) exceeding three monosaccharides are synthesized by the further addition of galactose moieties, donated by galactinol, to lower DP RFOs. For example, stachyose is synthesized by STACHYOSE SYNTHASE (STS; galactinol-raffinose galactosyltransferase; EC 2.4.1.67) using the substrates galactinol and raffinose (Gangl et al, 2015). Alternatively, the higher DP RFOs can also be produced by the action of a GALACTAN:GALACTAN GALACTOSYL TRANSFERASE (GGT) in those species in which this enzyme exists (Bachmann and Keller, 1995;Peterbauer and Richter, 1998;Tapernoux-Luthi et al, 2004); the terminal galactosyl residue is transferred from one RFO member to another to form one higher DP (HDP) and one lower DP RFOs.…”

“…Compared with the well-studied GOLS, little is known about RS, which shares high sequence homology with ALKALINEa-GALACTOSIDASE (AGA; EC 3.2.1.22) (Peters et al, 2010). To date, while several putative RS genes have been reported, few of these have been confirmed to have the ability to synthesize raffinose in vitro or in vivo (Peterbauer et al, 2002;Li et al, 2007;Sui et al, 2012;Egert et al, 2013;Lahuta et al, 2014;Gangl et al, 2015). In maize there were 10 RS genes predicted but none have been characterized for RS activity (Zhou et al, 2012).…”

“…In maize there were 10 RS genes predicted but none have been characterized for RS activity (Zhou et al, 2012). In the model plant, A. thaliana, there are six putative RS genes (AtRS1-6) (Nishizawa et al, 2008) but to date, only AtRS4, identified as a STACHYOSE SYNTHASE (AtSTS) with some raffinose synthetic capacity (Gangl et al, 2015), and AtRS5, a RAFFINOSE SYNTHASE without the capacity to synthesize HDP RFOs (Egert et al, 2013), have been biochemically verified to produce raffinose. The atrs5 mutation abolished raffinose in the leaves but only reduced the raffinose content in seeds, leading to speculation that a second RS exists in this species (Egert et al, 2013), a role filled by AtRS4.…”

“…A positive correlation between raffinose content in embryos and seed vigor was declared in maize seeds (Bernal-Lugo and Leopold, 1992). Despite these studies, the knockout mutants in genes involved in RFO biosynthesis in A. thaliana have failed to show any phenotypic changes during normal growth other than perturbations in soluble sugar quantities (Egert et al, 2013;Gangl et al, 2015). In addition, the function of galactinol and each individual member of RFOs, such as raffinose, stachyose, or verbascose, has not been specifically investigated or has not been distinguished from one another.…”