Trehalose‐6‐phosphate phosphatases from Arabidopsis thaliana: identification by functional complementation of the yeast tps2 mutant

Abstract

SummaryIt is currently thought that most flowering plants lack the capacity to synthesize trehalose, a common disaccharide of bacteria, fungi and invertebrates that appears to play a major role in desiccation tolerance. Attempts have therefore been made to render plants more drought-resistant by the expression of microbial genes for trehalose synthesis. It is demonstrated here that Arabidopsis thaliana itself possesses genes for at least one of the enzymes required for trehalose synthesis, trehalose-6-phosphate phosphatase. The yeast tps2 mutant, which lacks this enzyme, is heat-sensitive, and Arabidopsis cDNA able to complement this effect has been screened for. Half of the yeast transformants that grew at 38.6°C were also able to produce trehalose. All of these expressed one of two Arabidopsis cDNA, either AtTPPA or AtTPPB, which are both homologous to the C-terminal part of the yeast TPS2 gene and other microbial trehalose-6-phosphate phosphatases. Yeast tps2 mutants expressing AtTPPA or AtTPPB contained trehalose-6-phosphate phosphatase activity that could be measured both in vivo and in vitro. The enzyme dephosphorylated trehalose-6-phosphate but not glucose-6-phosphate or sucrose-6-phosphate. Both genes are expressed in flowers and young developing tissue of Arabidopsis. The finding of these novel Arabidopsis genes for trehalose-6-phosphate phosphatase strongly indicates that a pathway for trehalose biosynthesis exists in plants.

Gene families and evolution of trehalose metabolism in plants

“…-trehalose levels in untreated plants were below the limits of detection of the assays used (Müller et al 1995;Goddijn et al 1997). Therefore, the discovery of genes encoding trehalose-phosphate synthase (TPS; EC 2.4.1.15) and trehalose-phosphatase (TPP; EC 3.1.3.12) in A. thaliana L. -another desiccation-intolerant species -was rather unexpected (Blázquez et al 1998;Vogel et al 1998). The complete sequencing of the A. thaliana genome (The Arabidopsis Initiative 2000) subsequently revealed a family of 11 genes (AtTPS1-11) encoding TPS or TPS-like proteins, and a family of 10 genes (AtTPPA-J) encoding TPP, whereas trehalase (EC 3.2.1.28) appears to be encoded by a single gene (AtTRE) (Leyman et al 2001).…”

“…The proteins encoded by the 10 A. thaliana TPP genes also contain the three HAD motifs, and two members of the gene family (AtTPPA and AtTPPB) encode active TPP enzymes that complement the yeast tps2∆ mutant (Vogel et al 1998), as do two homologues from rice and maize (Pramanik and Imai 2005;Satoh-Nagasawa et al 2006). The maize TPP (RA3) is of particular interest, because a lesion in the gene encoding this enzyme in the ramosa3 (ra3) mutant leads to increased inflorescence branching (Satoh-Nagasawa et al 2006).…”

“…The AtTPPA and AtTPPB genes can complement the yeast tps2∆ (TPP − ) mutant and have been shown to encode active TPP enzymes (Vogel et al 1998), as have the rice TPP2a and maize RA3 (Pramanik and Imai 2005;Satoh-Nagasawa et al 2006). All of the plant TPP proteins show perfect conservation of the three HAD motifs, and good conservation of the other residues involved in binding the trehalose moiety of Tre6P in the T. acidophilum TPP (Rao et al 2006; Table 2).…”

“…The discovery of TPS and TPP genes in A. thaliana less than 10 years ago (Blázquez et al 1998;Vogel et al 1998) has led to a complete reappraisal of the importance of trehalose metabolism in plants. Previously it was thought to be restricted to just a few specialised resurrection plants, but is now known to be widespread, perhaps universal, in plants.…”

A bifunctional TPS–TPP enzyme from yeast confers tolerance to multiple and extreme abiotic-stress conditions in transgenic Arabidopsis

“…It is known that when the Wrst 504 amino acids are removed from TPS2 encoded protein the truncation allele is still fully active since the remaining 392-amino-acid carboxy-terminus encompasses the homology blocks present in all TPP enzymes (Vogel et al 1998). Therefore, we fused a TPS1 1,526-bp fragment (encoding 493 amino acids plus 2 linker amino acids) to a 1353-bp fragment corresponding to the carboxy-end of TPS2 (encoding 397 amino-acid plus 2 linker amino acids) to yield a 2873-bp DNA fragment fused through the BamHI linker site, which showed a continuous open reading frame encoding the predicted 892 amino-acid sequence after DNA sequencing.…”