The 2011 ASHS Annual Conference

Increasing resistance of crops to abiotic stresses is one of the primary objectives of plant biotechnology.  Desiccation of plant cells due to drought or salt stress can lead to protein misfolding and inactivation.  Trehalose sugar may act as an osmoprotectant to prevent physical and chemical instability in proteins when exposed to salt or drought stress.  Trehalose is a non-reducing disaccharide sugar and its accumulation is thought to offer osmotic stress protection role in many bacteria and fungi.  Whereas in most plants trehalose levels are low, the concentration of this sugar is high in “resurrection plants” (Selaginella lepidophylla) which recover quickly from drought stress.  Both tomato and rice have been genetically modified with microbial TPS1 increasing their tolerance to salt. With an aim toward overexpressing trehalose-6-phosphate synthase 1 gene to improve salt tolerance in the model floriculture species Petunia x hybrida we cloned the TPS1 gene from Petunia ‘Mitchell Diploid’.  This generated a 2784-bp open reading frame that predicts a 58kDA protein of 927 amino acids. The sequence is available in NCBI database (Accession HQ259080). In Saccharomyces cerevisiae it is thought that the TPS1 exerts an essential control on the influx of glucose into glycolysis.  The deletion of ScTPS1 causes an inability to grow on glucose because of a hyperaccumulation of sugar phosphates and depletion of ATP. To prove the functionality of the petunia TPS1 we carried out yeast complementation experiments to determine whether PhTPS1 would rescue function in mutant yeast (W303-1A background) while growing on glucose as a carbon source. We truncated the first ca. 80 amino acids to increase TPS1 catalytic activity as has been reported for AtTPS1. We cloned the trimmed PhTPS1 (ΔPhTPS1) into pDB20 yeast expression vector in vivo by means of homologous recombination. Eight transformations were carried out using four yeast strains: wild type (WT), knockout for TPS1 (tps1Δ), knockout for the related gene TPS2 (tps2Δ), and a double knockout (tps1Δtps2Δ); they received the pDB20 yeast vector with the overexpressed Petunia ΔTPS1 and with only the empty pDB20 vector. We successfully restored the ability of mutant yeast (tps1Δ, tps1Δtps2Δ) to grow in glucose by the insertion of ΔPhTPS1, indicating that ΔPhTPS1 is a functional gene capable of complementing trehalose biosynthesis. The rate limiting step in trehalose biosynthesis appears to be TPS1 gene since (tps1Δtps2Δ) with the ΔPhTPS1 vector was able to grow in glucose suggesting that the T6P intermediate can be dephosphorylated by nonspecific phosphatases.