- Semi-quantitative RT-PCR indicated that both OsICE1 and OsICE2 were constantly expressed, but that cold stress sequentially upregulated OsDREB1B, rice heat shock factor A3 (OsHsfA3), and trehalose-6- phosphate phosphatase (OsTPP1). It still remains uncertain whether OsDREB1B is involved directly or indirectly in the induction of OsHsfA3 or OsTPP1, leading to cold acclimation via trehalose synthesis.
- Expression analysis demonstrated that OsTPP1 was initially and transiently up-regulated after salt, osmotic and abscisic acid(ABA) treatments but slowly up-regulated under cold stress. OsTPP1 overexpression in rice enhanced tolerance to salt and cold stress. OsTPP1 plays an important role in salt or cold stress. The current study revealed the mechanism of an OsTPP gene involved in stress tolerance in rice and also suggested the use of OsTPP1 in abiotic stress engineering of crops.
- OsTPS1 was possibly activated under a positive feedback mechanism regulated by OsTTP1, and in vivo, OsTPS1 might function as trehalose-6-phosphatesynthase. OsTPP1 plays a role in the activation of stress response genes, which might be the main reason for the enhanced tolerance to abiotic stress of OsTPP1 overexpression lines.
- Northern blots revealed that the OsTPP1 transcript levels were fairly low or under detectable limits in most of the tissues under ambient conditions but were highly induced within 1–2 h of chilling stress (12°C) in both root and shoot tissues of seedlings. This induction was transient and disappeared after 6 h of the chilling stress. Transient expression of OsTPP1 was also induced under severe chilling stress (4°C) as well as salinity and drought stresses at ambient temperatures.
- Application of exogenous ABA (50 lM) resulted in a transient increase of OsTPP1 expression within 20 min of the treatment, thereby suggesting involvement of ABA in OsTPP1 gene regulation. Measurements of total cellular TPP activity and trehalose content in roots indicated that both TPP activity and trehalose levels were transiently increased after chilling (12°C) stress. Collectively, the data indicate that transient activation of trehalose biosynthesis is involved in early chilling stress response in rice.
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.
three transgenic lines:
While in salt solution, the seed germination rate was obviously decreased. However, compared with the vector line, the germination rate of overexpression lines OX5 and OX8 remained relative high at both 60 and 100 mM salt environments. In contrast, the empty vector line exhibited almost the same germination rate with the overexpression lines when immersed in water, but when in a salt environment, the germination rate was significantly lower than the overexpression lines, which suggesting that overexpression of OsTPP1 in rice enhanced seed germination rate under salt stress.
- One of the TPP homologues, OsTPP1 was quickly induced under salt stress. OsTPP1 is of particular interest due to its initial transiently up-regulated pattern and different responses to salt stress. Though the content before or after stress in the overexpression lines seemed higher than that in vector line, the difference was not significant.
After prolonged salt treatment, overexpression lines were more vigorous, and more leaves of them remained green and alive compared with the control. No difierences between overexpression lines and the control were noted following osmotic stress treatment, with wilting and death of all seedlings(data not shown).
- Old treatment resulted in dehydration and wilting of control plants, however OsTPP1 overexpression lines were less damaged and hydrated.
overexpression lines were mostly able to recuperate and grow during the restorative post-cold period while control lines almost died. OsTPP1 was induced by salt, osmotic and cold stress, as well as exogenous ABA. Although overexpression of OsTPP1 improved salt and cold tolerance, the trehalose content was not significantly increased compared with the vector line.
- 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.
- OsTPP1 belongs to Class III. AtTPPA and OsTPP1 were closely allied.
- 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.
Labs working on this gene
- National Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, 200032 Shanghai, China
- Graduate School of the Chinese Academy of Sciences (D.C.), 19 Yuquan Road, 100039 Beijing, China
- 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
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