Raffinose Family Oligosaccharides Act As Galactose Stores in Seeds and Are Required for Rapid Germination of Arabidopsis in the Dark.

Roman Gangl,Raimund Tenhaken

doi:10.3389/fpls.2016.01115

Abstract

Raffinose synthase 5 (AtRS5, At5g40390) was characterized from Arabidopsis as a recombinant enzyme. It has a far higher affinity for the substrates galactinol and sucrose than any other raffinose synthase previously reported. In addition raffinose synthase 5 is also working as a galactosylhydrolase, degrading galactinol, and raffinose under certain conditions. Together with raffinose synthase 4, which is predominantly a stachyose synthase, both enzymes contribute to the raffinose family oligosaccharide (RFO) accumulation in seeds. A double knockout in raffinose synthase 4 and raffinose synthase 5 (ΔAtRS4,5) was generated, which is devoid of RFOs in seeds. Unstressed leaves of 4 week old ΔAtRS4,5 plants showed drastically 23.8-fold increased concentrations of galactinol. Unexpectedly, raffinose appeared again in drought stressed ΔAtRS4,5 plants, but not under other abiotic stress conditions. Drought stress leads to novel transcripts of raffinose synthase 6 suggesting that this isoform is a further stress inducible raffinose synthase in Arabidopsis. ΔAtRS4,5 seeds showed a 5 days delayed germination phenotype in darkness and an elevated expression of the transcription factor phytochrome interacting factor 1 (AtPIF1) target gene AtPIF6, being a repressor of germination. This prolonged dormancy is not seen during germination in the light. Exogenous galactose partially promotes germination of ΔAtRS4,5 seeds in the dark suggesting that RFOs act as a galactose store and repress AtPIF6 transcripts.

Highlights

Seeds of higher plants store sugars presumably as carbon reserves for metabolism, as storage molecules for germination and as structural components for growth
Since we were searching for the second seed-specific RafS next to AtRS5 as suggested by Egert et al (2013), we performed a sequence alignment (Carmi et al, 2003) with
In different enzyme assays we demonstrate that AtRS5 is a functional high affinity RafS (Figures 3C,D) as well as a Raf (Figure 3E) and Gol specific galactosylhydrolase (Figure 3F)

Summary

Introduction

Seeds of higher plants store sugars presumably as carbon reserves for metabolism, as storage molecules for germination and as structural components for growth. During exposure of Arabidopsis to abiotic stress, Raf is the only RFO to accumulate (Taji et al, 2002; Nishizawa et al, 2008; ElSayed et al, 2014), whereas. RFO biosynthesis proceeds by stepwise transfer of galactosyl units (Sengupta et al, 2015) involving raffinose synthase (RafS, AtRS5, EC 2.4.1.82) (Lehle et al, 1970; Lehle and Tanner, 1973) and stachyose synthase (StaS, AtRS4, EC 2.4.1.67) (Tanner and Kandler, 1968; Lehle and Tanner, 1973; Holthaus and Schmitz, 1991; Hoch et al, 1999; Peterbauer and Richter, 2001)

Methods

Results

Conclusion