Trehalose 6-phosphate is indispensable for carbohydrate utilization and growth in Arabidopsis thaliana

Abstract

Genes for trehalose metabolism are widespread in higher plants. Insight into the physiological role of the trehalose pathway outside of resurrection plant species is lacking. To address this lack of insight, we express Escherichia coli genes for trehalose metabolism in Arabidopsis thaliana, which manipulates trehalose 6-phosphate (T6P) contents in the transgenic plants. Plants expressing otsA [encoding trehalose phosphate synthase (TPS)] accumulate T6P whereas those expressing either otsB [encoding trehalose phosphate phosphatase (TPP)] or treC [encoding trehalose phosphate hydrolase (TPH)] contain low levels of T6P. Expression of treF (encoding trehalase) yields plants with unaltered T6P content and a phenotype not distinguishable from wild type when grown on soil. The marked phenotype obtained of plants accumulating T6P is opposite to that of plants with low T6P levels obtained by expressing either TPP or TPH and consistent with a critical role for T6P in growth and development. Supplied sugar strongly inhibits growth of plants with reduced T6P content and leads to accumulation of respiratory intermediates. Remarkably, sugar improves growth of TPS expressors over wild type, a feat not previously accomplished by manipulation of metabolism. The data indicate that the T6P intermediate of the trehalose pathway controls carbohydrate utilization and thence growth via control of glycolysis in a manner analogous to that in yeast. Furthermore, embryolethal A. thaliana tps1 mutants are rescued by expression of E. coli TPS, but not by supply of trehalose, suggesting that T6P control over primary metabolism is indispensable for development.

Fig. 1.

Reactions catalyzed by the E. coli enzymes used for this study. otsA encodes a trehalose phosphate synthase (TPS), otsB a trehalose phosphate phosphatase (TPP), treC a trehalose phosphate hydrolase (TPH), and treF a trehalase.

Fig. 2.

Typical seedling and mature plant phenotypes of Arabidopsis Col.0 expressing E. coli TPS, TPP, THP, and trehalase using the CaMV35S promoter. (A) Seedlings grown for 5 days on 1/2 MS medium. (B) Plants grown for 6 wk in long day conditions. (C) Leaves from rosettes 1 wk after bolting, wt, TPS line A19, and TPP line B12. (D) Seedlings grown for 7 days on 1/2 MS with 100 mM trehalose. wt, trehalase expressor lines 42 and 34.

Fig. 3.

T6P content in lines expressing TPS (line A19), TPP (lines 7 and 12), TPH, and trehalase. The mean content of three independent lines is shown in the case of TPH and trehalase expressors. Plants were grown on soil for 4 wk, and leaf material was harvested as described in Methods. A minimum of three independent determinations were carried out for each line. *, Significantly different from wt (P < 0.05).

Fig. 5.

Profiles of metabolites in seedlings without or with sugars in the medium at 7 days. Seedlings were grown on 1/2 MS without or with 100 mM glucose (gluc), fructose (fruc), sucrose (suc), or sorbitol (sorb). Data shown are typical for a series of experiments. TPS, and TPP were as in Fig. 4. *, Significantly different from wt under the same growth conditions (P < 0.05).

Fig. 4.

Growth of seedlings expressing TPS (line A19), TPP (line B12), TPH (line C10.10), and trehalase (line F12.1) on media with or without sugars. (A) Seedling responses to 100 mM sorbitol and sucrose. Seedlings were grown for 7 days in plates as described in Methods.(B) Fresh weight (FW) and dry weight (DW) of 7-day seedlings on 1/2 MS (MS) without or with 100 mM sorbitol, glucose, fructose, and sucrose. TPSdry, wtdry, and TPPdry correspond to DW determinations. Differences between TPS, wt, and TPP are significant for all FW determinations, except those from seedlings grown in 1/2 MS, and for DW determinations on sucrose (P < 0.005; n = 6). (C) Seedling responses at 14 days to high concentrations of sorbitol and metabolizable sugars. TPH seedlings are not shown because they resemble TPP seedlings.

Fig. 6.

Complementation of tps1-2 with AtTPS1promoter::otsA. (A) Segregation of TPS1 and tps1-2 in the T₂ generation containing tps1-2 and AtTPS1promoter::otsA. Heterozygous tps1-2 plants were transformed with the AtTPS1promoter::otsA construct described in Methods on a T-DNA with a hygromycin selectable marker. The first transgenic generation, T₁, was selected hygromycin, and 42 T₁ were chosen randomly for analysis of their offspring, the T₂ generation. T₂ plants resistant to hygromycin and phosphinotricin [the bar resistance gene is linked to the transposon insertion in tps1-2 (7)] were then grown from each of the T₁ and DNA was extracted from the individual T2 plants and analyzed by PCR for presence of tps1-2 and TPS1 genes as described in Methods. A 570-bp fragment is amplified from AtTPS1 but not from the gene disrupted by transposon insertion in tps1-2 whereas a 1056-bp fragment is amplified from the transposon border present in tps1-2. MW is pst1-digested lambda phage; NoDNA is PCR without template DNA; then PCR with DNA from wt Col.0 (wt), tps1-2 (tps1-2), and from six offspring of the T₁ plant 201-1, from eight offspring of the T₁ plant 201-8, and from three offspring of the T₁ plant 201-19 (T₂ generation). (B) Examples of late-flowering T₂ plants obtained from tps1-2 complementation with AtTPS1promoter::otsA. T₂ offspring were grown until flowering, which, for 8 of 18 otsA complemented homozygous tps1-2, was late. Three examples of these late-flowering plants (, , and 3) are compared with a wt plant.