- Substantial levels of trehalose accumulate in bacteria, fungi, and invertebrates, where it serves as a storage carbohydrate or as a protectant against environmental stresses. In higher plants, trehalose is detected at fairly low levels; therefore, a regulatory or signaling function has been proposed for this molecule. In many organisms, trehalose-6-phosphate phosphatase is the enzyme governing the final step of trehalose biosynthesis. Here we report that OsTPP1 and OsTPP2 are the two major trehalose-6-phosphate phosphatase genes expressed in vegetative tissues of rice. Similar to results obtained from our previous OsTPP1 study, complementation analysis of a yeast trehalose-6-phosphate phosphatase mutant and activity measurement of the recombinant protein demonstrated that OsTPP2 encodes a functional trehalose-6-phosphate phosphatase enzyme.
- Trehalose is a nonreducing disaccharide in which two glucose units are linked by an a,a-1,1-glycosidic linkage. The prevalent pathway for trehalose synthesis includes two enzymatic reactions. Trehalose 6-phos-phate (Tre6P) is generated from UDP-glucose and glucose 6-phosphate (Glc6P) in a reaction catalyzed by trehalose-6-phosphate synthase (TPS). Tre6P is then dephosphorylated to form trehalose via trehalose-6-phosphate phosphatase (TPP). In yeast, trehalose synthesis is carried out by a large enzyme complex that is composed of four subunits, including TPS1, TPS2, and regulatory subunits TSL1 and TPS3.
- Although trehalose biosynthesis in higher plants has been demonstrated, details of both the physiologic functions and regulation of this pathway remain largely unknown. Genome sequencing of Arabidopsis and rice has revealed complex genomic organization of plant trehalose biosynthesis genes. Eleven putative TPS and 10 putative TPP genes were identified within the Arabidopsis genome, and nine putative TPS and nine putative TPP genes were found within the rice genome. Genetic studies have revealed that trehalose biosynthesis genes function specifically in regulating plant growth and development.
- OsTPP2 encodes a functional trehalose-6-phosphate phosphatase enzyme. A full-length OsTPP2 cDNA was then isolated from root tissue by RT-PCR. The OsTPP2 gene contained an ORF encoding a 42.6 kDa protein with 382 amino acid residues.
- Expression of OsTPP2 was regulated by multiple stress factors, such as chilling, drought, and salt stresses, as well as ABA treatment. Enzymatic characterization of recombinant OsTPP1 and OsTPP2 revealed stringent substrate specificity for trehalose 6-phosphate and about 10 times lower Km values for trehalose 6-phosphate as compared with trehalose-6-phosphate phosphatase enzymes from microorganisms.
OsTPP1 and OsTPP2
- Enzymatic characterization of recombinant OsTPP1 and OsTPP2 revealed stringent substrate specificity for trehalose 6-phosphate and about 10 times lower Km values for trehalose 6-phosphate as compared with trehalose-6-phosphate phosphatase enzymes from microorganisms.
- OsTPP1 and OsTPP2 also clearly contrasted with microbial enzymes, in that they are generally unstable, almost completely losing activity when subjected to heat treatment at 50°C for 4 min. These characteristics of rice trehalose-6-phosphate phosphatase enzymes are consistent with very low cellular substrate concentration and tightly regulated gene expression.
- OsTPP1 and OsTPP2 were the major TPP genes expressed in rice seedlings.
- Both OsTPP1 and OsTPP2 exhibited strong phosphatase activity upon Tre6P, but almost no activity (less than 1% relative to Tre6P) was detected with any of the other sugar phosphates tested (data not shown).
- The pH optima of OsTPP1 and OsTPP2 were approximately 7.0 and 6.5, respectively, whereas the enzymes had almost no activity at pH 5.5 or 9.
- Heat treatment at 50°C or higher for 4 min nearly eliminated both OsTPP1 and OsTPP2 activity, indicating that both enzymes are heat-labile.
- yeast △tps2 mutant:
Whereas wild-type cells grow at both 30°C and 36°C, growth of the △tps2 mutant at 36°C was inhibited because of its inability to synthesize trehalose. The same mutant yeast strain transformed with OsTPP2 recovered wild-type levels of growth at 36°C, suggesting that OsTPP2 is a functional TPP enzyme in yeast cells.
- OsTPP2 mRNA levels were detectable prior to stress treatments, and transiently increased in response to low temperature, peaking at 10 h after the initiation of treatment in both shoot and root tissues. This expression pattern contrasted with the observed rapid induction of OsTPP1 and gradual induction of OsMEK1 in response to low temperature treatment.
- Drought stress transiently induced OsTPP2 expression, which peaked at 6 h in shoots and 2 h in roots. The induction of OsTPP2 expression occurs earlier during stress treatment compared with expression of another drought-induced gene (salT) . Treatment with 150 mm NaCl also induced OsTPP2 expression in roots, suggesting that stresses associated with water deficit similarly affect expression of this gene.
- However, in contrast to chilling and drought stress treatments, clear induction of OsTPP2 was not observed in shoots, whereas the salt treatment effectively induced salT in both roots and shoots. Slight and transient induction of OsTPP2 was observed in roots and shoots in response to exogenous ABA. Together, these expression analyses indicated involvement of OsTPP2 in multiple stress responses.
- Phylogenetic analysis of the TPP domains demonstrated that the whole TPS/TPP gene family in rice was grouped into three classes (Class I/II/III)(Fig. 1). Class I consisted of OsTPS1 which displayed high identity to AtTPS1 and SlTPS1; Class II consisted of all the remaining OsTPSs; and Class III consisted of all independent TPP genes. The phylogenetic tree displayed a similar structure to that in Arabidopsis, suggesting the gene family was highly conserved and formed before the divergence of dicotyledonous and monocotyledonous angiosperms.
- OsTPP2 belongs to Class III.
- Amino acid sequence homology between OsTPP2 and OsTPP1 was 53%. Greater similarity was observed between OsTPP2 and Arabidopsis AtTPPA (57%). OsTPP2 contains two motifs shared by all TPP enzymes, known as phosphatase boxes.
- Although five different trehalose synthesis pathways exist in bacteria, fungi, yeast and algae, trehalose biosynthesis in higher plants only occurs in the trehalose phosphate synthase (TPS)–trehalose phosphate phosphatase(TPP) pathway (also known as OtsA–OtsB pathway)(Figure 2). The first step, catalyzed by TPS, involves the binding of a glucose-6-P to a UDP-glucose to produce T6P, which is cleaved into trehalose by TPP. Trehalase breaks down trehalose to form two glucose residues. This process has been found in all organisms that synthesize trehalose, even when distinct forms of trehalase coexist.
- Trehalose is a disaccharide sugar widely distributed in bacteria, fungi, insects, plants and invertebrate animals. In microbes and yeast, trehalose is produced from glucose by trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP), and serves as a sugar storage, metabolic regulator and protects against abiotic stress. However, its role in plants is not yet fully elucidated.
- Identification and functional characterization of trehalose biosynthesis genes have established trehalose biosynthesis in higher plants. However, low-level accumulation of trehalose in plants suggests a distinctive function of this substance compared with its role in other organisms. Organization of these trehalose biosynthesis genes is also quite unique in higher plants. Only one or two copies of TPS and TPP genes exist in most bacteria, fungi, and insects, whereas these genes constitute a large gene family in higher plants. For example, in Arabidopsis, 11 TPS and 10 TPP genes have been identified from genomic information, and nine TPS and nine TPP homologs are found in the rice genome.
- Using recombinant enzymes, we conducted the first detailed functional characterization of plant TPPs. These rice TPPs displayed three distinct properties compared with the previously characterized microbial TPPs. First, the K m values for the recombinant OsTPP1 and OsTPP2 enzymes are lower than values published for the microbial enzymes. Others have reported that the Tre6P concentration in Arabidopsis is relatively very low (10.1 ± 1.3 lgÆg )1 fresh weight). Therefore, these low K m values for OsTPP1 and OsTPP2 correlate with low concentrations of this substrate in plant cells.
Labs working on this gene
- Crop Cold Tolerance Research Team, National Agricultural Research Center for Hokkaido Region, National Agriculture and Food Research Organization, Hitsujigaoka 1, Toyohira-ku, Sapporo 0628555, Japan
- Department of Crop Botany, Bangladesh Agricultural University, Mymensing, Bangladesh
- Shima S, Matsui H, Tahara S, et al. Biochemical characterization of rice trehalose‐6‐phosphate phosphatases supports distinctive functions of these plant enzymes[J]. FEBS Journal, 2007, 274(5): 1192-1201.
- Pramanik M H R, Imai R. Functional identification of a trehalose 6-phosphate phosphatase gene that is involved in transient induction of trehalose biosynthesis during chilling stress in rice[J]. Plant molecular biology, 2005, 58(6): 751-762.
- Wen J Q, Oono K, Imai R. Two novel mitogen-activated protein signaling components, OsMEK1 and OsMAP1, are involved in a moderate low-temperature signaling pathway in rice[J]. Plant physiology, 2002, 129(4): 1880-1891.
- Ge L F, Chao D Y, Shi M, et al. Overexpression of the trehalose-6-phosphate phosphatase gene OsTPP1 confers stress tolerance in rice and results in the activation of stress responsive genes[J]. Planta, 2008, 228(1): 191-201.
- Blazquez M A, Santos E, Flores C, et al. Isolation and molecular characterization of the Arabidopsis TPS1 gene, encoding trehalose‐6‐phosphate synthase[J]. The Plant Journal, 1998, 13(5): 685-689.
- Zentella R, Mascorro-Gallardo J O, Van Dijck P, et al. A Selaginella lepidophylla Trehalose-6-Phosphate Synthase Complements Growth and Stress-Tolerance Defects in a Yeasttps1 Mutant[J]. Plant Physiology, 1999, 119(4): 1473-1482.
- Leyman B, Van Dijck P, Thevelein J M. An unexpected plethora of trehalose biosynthesis genes in< i> Arabidopsis thaliana</i>[J]. Trends in plant science, 2001, 6(11): 510-513.
- Fernandez O, Béthencourt L, Quero A, et al. Trehalose and plant stress responses: friend or foe?[J]. Trends in plant science, 2010, 15(7): 409-417.
- Paul M J, Primavesi L F, Jhurreea D, et al. Trehalose metabolism and signaling[J]. Annu. Rev. Plant Biol., 2008, 59: 417-441.
- Wiemken A. Trehalose in yeast, stress protectant rather than reserve carbohydrate[J]. Antonie van Leeuwenhoek, 1990, 58(3): 209-217.
- Strom A R, Kaasen I. Trehalose metabolism in Escherichia coli: stress protection and stress regulation of gene expression[J]. Molecular microbiology, 1993, 8(2): 205-210.