Amylose starch with no detectable branching developed through DNA-free CRISPR-Cas9 mediated mutagenesis of two starch branching enzymes in potato

DNA-free genome editing was used to induce mutations in one or two branching enzyme genes (Sbe) in tetraploid potato to develop starch with an increased amylose ratio and elongated amylopectin chains. By using ribonucleoprotein (RNP) transfection of potato protoplasts, a mutation frequency up to 72% was achieved. The large variation of mutations was grouped as follows: Group 1 lines with all alleles of Sbe1 mutated, Group 2 lines with all alleles of Sbe1 as well as two to three alleles of Sbe2 mutated and Group 3 lines having all alleles of both genes mutated. Starch from lines in Group 3 was found to be essentially free of amylopectin with no detectable branching and a chain length (CL) distribution where not only the major amylopectin fraction but also the shortest amylose chains were lost. Surprisingly, the starch still formed granules in a low-ordered crystalline structure. Starch from lines of Group 2 had an increased CL with a higher proportion of intermediate-sized chains, an altered granule phenotype but a crystalline structure in the granules similar to wild-type starch. Minor changes in CL could also be detected for the Group 1 starches when studied at a higher resolution.EEA BalcarceFil: Zhao, Xue. Swedish University of Agricultural Sciences. Department of Molecular Sciences; Suecia.Fil: Jayarathna, Shishanthi. Swedish University of Agricultural Sciences. Department of Molecular Sciences; Suecia.Fil: Turesson, Helle. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.Fil: Fält , Ann Sofie. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.Fil: Nestor, Gustav. Swedish University of Agricultural Sciences. Department of Molecular Sciences; Suecia.Fil: González, Matías Nicolás. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.Fil: Olsson, Niklas. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.Fil: Beganovic, Mirela. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.Fil: Hofvander, Per. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.Fil: Andersson, Roger. Swedish University of Agricultural Sciences. Department of Molecular Sciences; Suecia.Fil: Anderson, Mariette. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia

Cloning of glycerophosphocholine acyltransferase (GPCAT) from fungi and plants: A novel enzyme in phosphatidylcholine synthesis

Glycero-3-phosphocholine (GPC), the product of the complete deacylation of phosphatidylcholine (PC), was long thought to not be a substrate for reacylation. However, it was recently shown that cell-free extracts from yeast and plants could acylate GPC with acyl groups from acyl-CoA. By screening enzyme activities of extracts derived from a yeast knock-out collection, we were able to identify and clone the yeast gene (GPC1) encoding the enzyme, named glycerophosphocholine acyltransferase (GPCAT). By homology search, we also identified and cloned GPCAT genes from three plant species. All enzymes utilize acyl-CoA to acylate GPC, forming lyso-PC, and they show broad acyl specificities in both yeast and plants. In addition to acyl-CoA, GPCAT efficiently utilizes LPC and lysophosphatidylethanolamine as acyl donors in the acylation of GPC. GPCAT homologues were found in the major eukaryotic organism groups but not in prokaryotes or chordates. The enzyme forms its own protein family and does not contain any of the acyl binding or lipase motifs that are present in other studied acyltransferases and transacylases. In vivo labeling studies confirm a role for Gpc1p in PC biosynthesis in yeast. It is postulated that GPCATs contribute to the maintenance of PC homeostasis and also have specific functions in acyl editing of PC (e.g. in transferring acyl groups modified at the sn-2 position of PC to the sn-1 position of this molecule in plant cells)