Metabolism control over growth: a case for trehalose-6-phosphate in plants

How plants relate their requirements for energy with the reducing power necessary to fuel growth is not understood. The activated glucose forms and NADPH are key precursors in pathways yielding, respectively, energy and reducing power for anabolic metabolism. Moreover, they are substrates or allosteric regulators of trehalose-phosphate synthase (TPS1) in fungi and probably also in plants. TPS1 synthesizes the signalling metabolite trehalose-6-phosphate (T6P) and, therefore, has the potential to relate reducing power with energy metabolism to fuel growth. A working model is discussed where trehalose-6-phosphate (T6P) inhibition of SnRK1 is part of a growth-regulating loop in young and metabolically active heterotrophic plant tissues. SnRK1 is the Snf1 Related Kinase 1 and the plant homologue of the AMP-dependent protein kinase of animals, a central energy gauge. T6P accumulation in response to high sucrose levels in a cell inhibits SnRK1 activity, thus promoting anabolic processes and growth. When T6P levels drop due to low glucose-6-phosphate, uridine-diphosphoglucose, and altered NADPH or due to restricted TPS1 activity, active SnRK1 promotes catabolic processes required to respond to energy and carbon deprivation. The model explains why too little or too much T6P has been found to be growth inhibitory: Arabidopsis thaliana embryos and seedlings without TPS1 are growth arrested and Arabidopsis seedlings accumulating T6P on a trehalose medium are growth arrested. Finally, the insight gained with respect to the possible role of T6P metabolism, where it is known to alter developmental and environmental responses of plants, is discussed.     

Regulation of growth by the trehalose pathway: Relationship to temperature and sucrose

Carbon signaling can override carbon supply in the regulation of growth. At least some of this regulation is imparted by the sugar signal trehalose 6-phosphate (T6P) through the protein kinase, SnRK1. This signaling pathway regulates biosynthetic processes involved in growth under optimal growing conditions. Recently, using a seedling system we showed that under sub-optimal conditions, such as cold, carbon signaling by T6P/ SnRK1 enables recovery of growth following relief of the stress. The T6P/ SnRK1 mechanism thus could be selected as a means of improving low temperature tolerance. High-throughput automated Fv/Fm measurements provide a potential means to screen for T6P/ SnRK1, and here we confirm through measurements of Fv/Fm in rosettes that T6P promotes low temperature tolerance and recovery during cold to warm transfer. Further, to better understand the coordination between sugars, trehalose pathway, and temperature-dependent growth, we examine the interrelationship between sugars, trehalose phosphate synthase (TPS), and trehalose phosphate phosphatase (TPP) gene expression and T6P content in seedlings. Sucrose, particularly when fed exogenously, correlated well with TPS1 and TPPB gene expression, suggesting that these enzymes are involved in maintaining carbon flux through the pathway in relation to sucrose supply. However, when sucrose accumulated to higher levels under low temperature and low N, TPS1 and TPPB expression were less directly related to sucrose; other factors may also contribute to regulation of TPS1 and TPPB expression under these conditions. TPPA expression was not related to sucrose content and all genes were not well correlated with endogenous glucose. Our work has implications for understanding acclimation to sink-limited growth conditions such as low temperature and for screening cold-tolerant genotypes with altered T6P/ SnRK1 signaling.     

植物海藻糖代谢及海藻糖-6-磷酸信号研究进展

海藻糖代谢和海藻糖-6-磷酸（T6P）信号途径在植物生长和发育过程中具有重要的调控作用。T6P是海藻糖的代谢前体,是植物响应碳元素可用性、调控生长发育的关键信号分子。植物体中除了自身的海藻糖合成途径外,由病原菌产生的海藻糖或T6P能够导致植物代谢和发育的重新编程。植物不同阶段的生长发育,包括胚胎发育、幼苗生长、成花诱导及叶片衰老等,都受T6P的调控。T6P信号的一个关键互作因子是蔗糖非发酵相关激酶1（SnRKl）,T6P能够抑制SnRK1的催化活性,进而调控植物的生长和发育过程。     

An unexpected plethora of trehalose biosynthesis genes in Arabidopsis thaliana.

Trehalose accumulation has been documented in many organisms, such as bacteria and fungi, where it serves a storage and stress-protection role. Although conspicuously absent in most plants, trehalose biosynthesis genes were discovered recently in higher plants. We have uncovered a family of 11 TPS genes in Arabidopsis thaliana, one of which encodes a trehalose-6-phosphate (Tre6P) synthase, and a subfamily of which might encode the still elusive Tre6P phosphatases. A regulatory role in carbon metabolism is likely but might not be restricted to the TPS control of hexokinase activity as documented for yeast. Incompatibility between high trehalose levels and chaperone-assisted protein folding might be a reason why plants have evolved to accumulate some alternative stress-protection compounds to trehalose.     

Trehalose 6‐phosphate is involved in triggering axillary bud outgrowth in garden pea (Pisum sativum L.)

Trehalose 6‐phosphate (Tre6P) is a signal of sucrose availability in plants, and has been implicated in the regulation of shoot branching by the abnormal branching phenotypes of Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) mutants with altered Tre6P metabolism. Decapitation of garden pea (Pisum sativum) plants has been proposed to release the dormancy of axillary buds lower down the stem due to changes in sucrose supply, and we hypothesized that this response is mediated by Tre6P. Decapitation led to a rapid and sustained rise in Tre6P levels in axillary buds, coinciding with the onset of bud outgrowth. This response was suppressed by simultaneous defoliation that restricts the supply of sucrose to axillary buds in decapitated plants. Decapitation also led to a rise in amino acid levels in buds, but a fall in phosphoenolpyruvate and 2‐oxoglutarate. Supplying sucrose to stem node explants in vitro triggered a concentration‐dependent increase in the Tre6P content of the buds that was highly correlated with their rate of outgrowth. These data show that changes in bud Tre6P levels are correlated with initiation of bud outgrowth following decapitation, suggesting that Tre6P is involved in the release of bud dormancy by sucrose. Tre6P might also be linked to a reconfiguration of carbon and nitrogen metabolism to support the subsequent growth of the bud into a new shoot.     

Sugar-induced increases in trehalose 6-phosphate are correlated with redox activation of ADPglucose pyrophosphorylase and higher rates of starch synthesis in Arabidopsis thaliana

Tre6P (trehalose 6-phosphate) is implicated in sugar-signalling pathways in plants, but its exact functions in vivo are uncertain. One of the main obstacles to discovering these functions is the difficulty of measuring the amount of Tre6P in plant tissues. We have developed a highly specific assay, using liquid chromatography coupled to MS-Q3 (triple quadrupole MS), to measure Tre6P in the femto-picomole range. The Tre6P content of sucrose-starved Arabidopsis thaliana seedlings in axenic culture increased from 18 to 482 pmol x g(-1) FW (fresh weight) after adding sucrose. Leaves from soil-grown plants contained 67 pmol x g(-1) FW at the end of the night, which rose to 108 pmol x g(-1)FW after 4 h of illumination. Even greater changes in Tre6P content were seen after a 6 h extension of the dark period, and in the starchless mutant, pgm. The intracellular concentration of Tre6P in wild-type leaves was estimated to range from 1 to 15 microM. It has recently been reported that the addition of Tre6P to isolated chloroplasts leads to redox activation of AGPase (ADPglucose pyrophosphorylase) [Kolbe, Tiessen, Schluepmann, Paul, Ulrich and Geigenberger (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 11118-11123]. Using the new assay for Tre6P, we found that rising sugar levels in plants are accompanied by increases in the level of Tre6P, redox activation of AGPase and the stimulation of starch synthesis in vivo. These results indicate that Tre6P acts as a signalling metabolite of sugar status in plants, and support the proposal that Tre6P mediates sucrose-induced changes in the rate of starch synthesis.     

Trehalose 6–phosphate coordinates organic and amino acid metabolism with carbon availability

Trehalose 6–phosphate (Tre6P) is an essential signal metabolite in plants, linking growth and development to carbon metabolism. The sucrose–Tre6P nexus model postulates that Tre6P acts as both a signal and negative feedback regulator of sucrose levels. To test this model, short‐term metabolic responses to induced increases in Tre6P levels were investigated in Arabidopsis thaliana plants expressing the Escherichia coli Tre6P synthase gene (otsA) under the control of an ethanol‐inducible promoter. Increased Tre6P levels led to a transient decrease in sucrose content, post‐translational activation of nitrate reductase and phosphoenolpyruvate carboxylase, and increased levels of organic and amino acids. Radio‐isotope (¹⁴CO₂) and stable isotope (¹³CO₂) labelling experiments showed no change in the rates of photoassimilate export in plants with elevated Tre6P, but increased labelling of organic acids. We conclude that high Tre6P levels decrease sucrose levels by stimulating nitrate assimilation and anaplerotic synthesis of organic acids, thereby diverting photoassimilates away from sucrose to generate carbon skeletons and fixed nitrogen for amino acid synthesis. These results are consistent with the sucrose–Tre6P nexus model, and implicate Tre6P in coordinating carbon and nitrogen metabolism in plants.     

Trehalose-6-phosphate synthase 1, which catalyses the first step in trehalose synthesis, is essential for Arabidopsis embryo maturation

Despite the recent discovery that trehalose synthesis is widespread in higher plants very little is known about its physiological significance. Here we report on an Arabidopsis mutant (tps1), disrupted in a gene encoding the first enzyme of trehalose biosynthesis (trehalose-6-phosphate synthase). The tps1 mutant is a recessive embryo lethal. Embryo morphogenesis is normal but development is retarded and stalls early in the phase of cell expansion and storage reserve accumulation. TPS1 is transiently up-regulated at this same developmental stage and is required for the full expression of seed maturation marker genes (2S2 and OLEOSN2). Sucrose levels also increase rapidly in seeds during the onset of cell expansion. In Saccharomyces cerevisiae trehalose-6-phosphate (T-6-P) is required to regulate sugar influx into glycolysis via the inhibition of hexokinase and a deficiency in TPS1 prevents growth on sugars (Thevelein and Hohmann, 1995). The growth of Arabidopsis tps1-1 embryos can be partially rescued in vitro by reducing the sucrose level. However, T-6-P is not an inhibitor of AtHXK1 or AtHXK2. Nor does reducing hexokinase activity rescue tps1-1 embryo growth. Our data establish for the first time that an enzyme of trehalose metabolism is essential in plants and is implicated in the regulation of sugar metabolism/embryo development via a different mechanism to that reported in S. cerevisiae.     

TREHALOSE PHOSPHATE SYNTHASE11-dependent trehalose metabolism promotes Arabidopsis thaliana defense against the phloem-feeding insect Myzus persicae

Agricultural productivity is limited by the removal of sap, alterations in source-sink patterns, and viral diseases vectored by aphids, which are phloem-feeding pests. Here we show that TREHALOSE PHOSPHATE SYNTHASE11 (TPS11) gene-dependent trehalose metabolism regulates Arabidopsis thaliana defense against Myzus persicae (Sülzer), commonly known as the green peach aphid (GPA). GPA infestation of Arabidopsis resulted in a transient increase in trehalose and expression of the TPS11 gene, which encodes a trehalose-6-phosphate synthase/phosphatase. Knockout of TPS11 function abolished trehalose increases in GPA-infested leaves of the tps11 mutant plant and attenuated defense against GPA. Trehalose application restored resistance in the tps11 mutant, confirming that the lack of trehalose accumulation is associated with the inability of the tps11 mutant to control GPA infestation. Resistance against GPA was also higher in the trehalose hyper-accumulating tre1 mutant and in bacterial otsB gene-expressing plants, further supporting the conclusion that trehalose plays a role in Arabidopsis defense against GPA. Evidence presented here indicates that TPS11-dependent trehalose regulates expression of the PHYTOALEXIN DEFICIENT4 gene, which is a key modulator of defenses against GPA. TPS11 also promotes the re-allocation of carbon into starch at the expense of sucrose, the primary plant-derived carbon and energy source for the insect. Our results provide a framework for the signaling function of TPS11-dependent trehalose in plant stress responses, and also reveal an important contribution of starch in controlling the severity of aphid infestation.     

Tomato responds to green peach aphid infestation with the activation of trehalose metabolism and starch accumulation

The disaccharide trehalose and trehalose-6-phosphate that are present in trace amounts are suggested to have a signaling function in plants. Recently, it was demonstrated that trehalose metabolism contributes to Arabidopsis thaliana defense against the green peach aphid (GPA; Myzus persicae Sülzer), an important insect pest of a large variety of plants. TPS11 (TREHALOSE PHOSPHATE SYNTHASE11)-dependent trehalose metabolism was shown to curtail GPA infestation by promoting starch accumulation and expression of the PAD4 (PHYTOALEXIN-DEFICIENT4) gene, which has important roles in regulating antibiosis and antixenosis against GPA. Here we show that trehalose metabolism is similarly activated in leaves of GPA-infested tomato (Solanum lycopersicum) plants and likely contributes to tomato defense against GPA. GPA-infested leaves of tomato accumulated trehalose, which was accompanied by the transient upregulation of SlTPS11, a homolog of the Arabidopsis TPS11. GPA-infestation was also accompanied by starch accumulation and the upregulation of SlPAD4, the tomato homolog of Arabidopsis PAD4. Furthermore, trehalose application induced SlPAD4 expression and starch accumulation, and curtailed GPA infestation, suggesting that like in Arabidopsis trehalose contributes to tomato defense against GPA.     

Trehalose mediated growth inhibition of Arabidopsis seedlings is due to trehalose-6-phosphate accumulation

Trehalose-6-phosphate (T6P) is required for carbon utilization during Arabidopsis development, and its absence is embryo lethal. Here we show that T6P accumulation inhibits seedling growth. Wild-type seedlings grown on 100 mm trehalose rapidly accumulate T6P and stop growing, but seedlings expressing Escherichia coli trehalose phosphate hydrolase develop normally on such medium. T6P accumulation likely results from much-reduced T6P dephosphorylation when trehalose levels are high. Metabolizable sugars added to trehalose medium rescue T6P inhibition of growth. In addition, Suc feeding leads to a progressive increase in T6P concentrations, suggesting that T6P control over carbon utilization is related to available carbon for growth. Expression analysis of genes from the Arabidopsis trehalose metabolism further supports this: Suc rapidly induces expression of trehalose phosphate synthase homolog AtTPS5 to high levels. In contrast, T6P accumulation after feeding trehalose in the absence of available carbon induces repression of genes encoding T6P synthases and expression of T6P phosphatases. To identify processes controlled by T6P, we clustered expression profile data from seedlings with altered T6P content. T6P levels correlate with expression of a specific set of genes, including the S6 ribosomal kinase ATPK19, independently of carbon status. Interestingly, Suc addition represses 15 of these genes, one of which is AtKIN11, encoding a Sucrose Non Fermenting 1 (SNF1)-related kinase known to play a role in Suc utilization.     

The function of trehalose biosynthesis in plants

Trehalose (alpha-D-glucopyranosyl-1,1-alpha-D-glucopyranoside) occurs in a large variety of organisms, ranging from bacteria to invertebrate animals, where it serves as an energy source or stress protectant. Until recently, only few plant species, mainly desiccation-tolerant 'resurrection' plants, were considered to synthesise trehalose. Instead of trehalose, most other plants species accumulate sucrose as major transport sugar and during stress. The ability to synthesize sucrose has probably evolved from the cyanobacterial ancestors of plastids and may be linked to photosynthetic function. Although most plant species do not appear to accumulate easily detectable amounts of trehalose, the discovery of genes for trehalose biosynthesis in Arabidopsis and in a range of crop plants suggests that the ability to synthesise trehalose is widely distributed in the plant kingdom. The apparent lack of trehalose accumulation in these plants is probably due to the presence of trehalase activity. After inhibition of trehalase, trehalose synthesis can be detected in Arabidopsis. Since trehalose induces metabolic changes, such as an accumulation of storage carbohydrates, rapid degradation of trehalose may be required to prevent detrimental effects of trehalose on the regulation of plant metabolism. In addition, the precursor of trehalose, trehalose-6-phosphate, is probably involved in the regulation of developmental and metabolic processes in plants.