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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 (K m  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.

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