Trehalose 6-Phosphate Synthase and Trehalose 6-Phosphate Phosphatase from Nicotiana tabacum Function in Trehalose Biosynthesis and Environmental Stress Tolerance of Yeast

To investigate functions of trehalose 6-phosphate synthase (TPS) and trehalose 6?phosphate phosphatase (TPP) from a tobacco plant, Nicotiana tabacum, the corresponding cDNA clones were isolated. Those genes were designated NtTPS and NtTPP, respectively. NtTPS included a N-terminal extension according to comparison of deduced amino acid sequence of NtTPS with those of TPSs from Escherichia coli and yeast. The NtTPS was genetically modified to lack a region for the N-terminal extension and the modified gene was designated ΔNNtTPS. The genes were expressed in yeast tps1 mutant as two separate proteins and as a NtTPS (or ΔNNtTPS)-NtTPP fusion protein. Western blot analysis showed that the NtTPS, NtTPP, and NtTPS-NtTPP were expressed abundantly in yeast, while the ΔNNtTPS and ΔNNtTPS-NtTPP were not detected. Interestingly, high levels of trehalose were accumulated in yeast expressing ΔNNtTPS and ΔNNtTPS-NtTPP in spite of their low-level expressions. Furthermore, stress tolerances of yeast against osmotic, freezing-thawing, and heat stresses were significantly improved by the expression of the tobacco gene, and the increased levels in tolerance were proportional to their trehalose levels. Our results showed that NtTPS and NtTPP functioned in trehalose synthesis by the removal of N-terminal extension of NtTPS and several environmental stress tolerances