1 Scientific RepoRts | 6:35088 | DOI: 10.1038/srep35088 www.nature.com/scientificreports Diferentially expressed galactinol synthase(s) in chickpea are implicated in seed vigor and longevity by limiting the age induced ROS accumulation Prafull Salvi, Saurabh Chandra Saxena, Bhanu Prakash Petla, Nitin Uttam Kamble, Harmeet Kaur, Pooja Verma, Venkateswara Rao, Shraboni Ghosh & Manoj Majee Galactinol synthase (GolS) catalyzes the frst and rate limiting step of Rafnose Family Oligosaccharide (RFO) biosynthetic pathway, which is a highly specialized metabolic event in plants. Increased accumulation of galactinol and RFOs in seeds have been reported in few plant species, however their precise role in seed vigor and longevity remain elusive. In present study, we have shown that galactinol synthase activity as well as galactinol and rafnose content progressively increase as seed development proceeds and become highly abundant in pod and mature dry seeds, which gradually decline as seed germination progresses in chickpea (Cicer arietinum). Furthermore, artifcial aging also stimulates galactinol synthase activity and consequent galactinol and rafnose accumulation in seed. Molecular analysis revealed that GolS in chickpea are encoded by two divergent genes (CaGolS1 and CaGolS2) which potentially encode fve CaGolS isoforms through alternative splicing. Biochemical analysis showed that only two isoforms (CaGolS1 and CaGolS2) are biochemically active with similar yet distinct biochemical properties. CaGolS1 and CaGolS2 are diferentially regulated in diferent organs, during seed development and germination however exhibit similar subcellular localization. Furthermore, seed-specifc overexpression of CaGolS1 and CaGolS2 in Arabidopsis results improved seed vigor and longevity through limiting the age induced excess ROS and consequent lipid peroxidation. Synthesis of Rafnose Family of Oligosaccharides (RFOs) is a highly specialized metabolic event in higher plants where galactinol synthase (GolS; EC: 2.4.1.123) catalyzes the key step in RFO biosynthesis. Tese RFOs partici- pate in many physiological processes like translocation of photoassimilates, abiotic stress tolerance, seed desicca- tion tolerance etc. 1–4 . Apart from these functions, RFOs were recently shown to act as signaling molecules upon pathogen attack and wounding 5–8 . RFOs are generally non-structural, non-reducing but soluble oligosaccha- rides present at high concentrations within the cell. Te RFO biosynthetic pathway is initiated by the synthesis of galactinol (1-O-α-D-galactopyranosyl-L myo-inositol) which subsequently serves as a galactosyl donor and provides galactose moieties to the sucrose for the synthesis of rafnose. Further sequential addition of galactosyl group to the chain leads to the generation of series of RFOs like stachyose, verbascose and ajugose 9,10 . Galactinol is synthesized from UDP galactose and myo-inositol by the action of GolS which is considered as the key regu- latory enzyme of this pathway 11 . GolS is a member of glycosyltransferase 8 (GT8) family and is usually encoded by a small gene family in higher plants. In Arabidopsis, GolS enzymes are encoded by a family of seven distinct genes which are spatially and developmentally regulated 2 . Studies have also shown that disruption of AtGolS1 gene resulted in a decrease in galactinol and rafnose content afer heat stress 12 . Arabidopsis plants overexpressing AtGolS2 exhibited improved tolerance to drought stress 2 . Diferential transcriptional regulation of the members of the galactinol synthase gene family was also observed in several other plant species. An increase in the produc- tion of galactinol and RFOs, as a consequence of coordinated transcriptional induction of the GolS coding genes in response to various abiotic stresses has been reported in several plant species 13–17 . In addition, ZmGolS2 was Lab 203, National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi 110067, India. Correspondence and requests for materials should be addressed to M.M. (email: manojmajee@nipgr.ac.in) Received: 27 July 2016 Accepted: 23 September 2016 Published: 11 October 2016 OPEN