Fig. 14.3 Summary of the role of trehalose metabolism in plants. Trehalose-6-phosphate (T6P) act as a signal metabolite because of its close proximity to the pool size of hexose phosphates and uridine diphosphoglucose (UDPG), which are the starting point of different basic processes in the plant cell metabolism. G6P glucose 6-phosphate, F6P fructose 6-phosphate, TPS trehalose phosphate synthase, TPP trehalose phosphate phosphatase an inhibitor of the trehalose-degrading enzyme, trehaiase. More recently, trehalose has been detected in crops, such as rice (Oryza sativa) (Garg et al. 2002) and tobacco (Nicotiana tabacum) (Karim et al. 2007) . The publication of the full genomic sequence database for A. thaliana confirmed the existence of a surprising abundance of genes for the trehalose synthesis (Leyman et al. 2001) and trehalose and trehalose 6-phosphate (T6P) were subsequently detected in this specie (Schluepmann et al. 2003; Vogel et al. 2001).
Eastmond et al. (2002) were the first to demonstrate the indispensability of a plant trehalose pathway gene, AtTPS1, which codify for the enzyme trehalose phosphate synthase (TPS) involved in the synthesis of T6P from glucose 6-phosphate (G6P) and UDP-glucose (UDPG) in A. thaliana. Numerous effects of altering the tre-halose pathway on metabolism and development (Ramon and Rolland 2007). possibly all due to modification of T6P content, have been reported. These include embryo (Eastmond et al. 2002) and leaf (Pellny et al. 2004) development, cell division, and cell wall synthesis (Gomez et al. 2006), inflorescence architecture (Satoh-Nagasawa et al. 2006) , seedling biomass (Schluepmann et al. 2003), adult plant biomass and photosynthesis (Pellny et al. 2004), sucrose utilization (Schluepmann et al. 2003), starch metabolism (Kolbe et al. 2005), and tolerance to abiotic stresses, particularly drought (Almeida et al. 2007; Garg et al. 2002; Karim et al. 2007; Miranda et al. 2007; Pilon-Smits et al. 1998).
In plants the major and possibly only role of the trehalose pathway, except in specialized resurrection plants, is as a central metabolic regulator. This regulatory function is performed, at least in part, by T6P (Kolbe et al. 2005; Lunn et al. 2006; Schluepmann et al. 2003).
The initial discovery that the trehalose pathway and T6P in particular, has a powerful function in metabolic regulation is, on reflection, perhaps not surprising. T6P and trehalose are made from UDPG and G6P, so T6P and trehalose synthesis is drawn from a metabolite pool at the center of metabolism, but because trehalose is not a major end product in plants, it is removed from major metabolic flux (Fig. 14.3). This means that the synthesis of T6P and trehalose can act as an effective indicator of G6P and UDPG pool size without compromising any other function of the trehalose pathway. T6P is a low-abundance molecule and responds rapidly to the sucrose supply (Lunn et al. 2006) . This rapid and large response is a consequence of a yet not fully elucidated control features (transcriptional and post-translational control, including phosphorylation) that regulate T6P synthesis and breakdown. However, T6P synthesis mediated through constitutive TPS1 expression likely reflects the availability of hexose phosphates, UDPG, and sucrose, which feeds into this pool. T6P, therefore, has all the characteristics of a signaling molecule.
The low abundance and dynamic response of T6P could potentiate specific and rapid communication of metabolic status that reflects pool sizes of G6P, UDPG, and sucrose and hence provide a different and specific kind of signaling to that of other sugars. TPS1 expression appears to be constitutive, but other trehalose pathway enzymes are regulated developmentally and by stress, providing the basis for a regulatory system linking the hexose phosphate pool and UDPG with development and the environment.
The wide range of phenotypes observed in transgenic plants with a modified trehalose pathway clearly suggests that T6P is involved in coordinating UDPG and G6P with growth and development in different tissues.
T6P is an essential factor in embryo maturation based on studies performed with a transposon insertion mutant of Arabidopsis AtTPSl gene (Schluepmann et al. 2004). Modifications of T6P levels cause dramatic effects on carbohydrate metabolism and partitioning as well as on morphogenesis and development in Arabidopsis (Schluepmann et al. 2003). Interestingly, AtTPSl is an essential gene, and knocking the gene out results in an embryo lethal phenotype (Eastmond et al. 2002). In vegetative stage, AtTPSl is essential for normal growth in particular for the development in the flowers, buds, and ripening fruits (Van Dijken et al. 2004).
In transgenic plants expressing E. coli TPS and TPP genes, researchers observe large changes in vegetative development, which correlate with T6P content (Paul 2007; Paul et al. 2001; Pellny et al. 2004). Quite remarkably, photosynthetic capacity per unit leaf area is enhanced in trans-genic plants expressing TPS. This enhancement is due to a specific increase in Rubisco activity, because of increased amounts of Rubisco protein, chlorophyll, and the light-harvesting apparatus. Nevertheless, targeting the trehalose pathway provides another means to alter plant photosynthesis for improved yield.
TPS1 is also necessary for the normal transition to flowering (Gomez et al. 2006; Van Dijken et al. 2004), probably again through provision of T6P. Ectopic expression of TPS1 also leads to changes in inflorescence development, including increased inflorescence branching. Recently, the genetic basis of ramosa3. a classical mutant of maize, which causes large changes in inflorescence branching, has been determined to be a TPP that metabolizes T6P (Satoh-Nagasawa et al. 2006) . RAMOSA3 is part of a distinct clade of TPPs found in monocots and is expressed in discrete domains subtending axillary inflorescence meristems. Overaccumulation in T6P in these meristems may cause the large change in inflorescence phenotype. Meristems are characterized by the need to coordinate the supply of intermediates, UDPG and G6P, with cell growth and development, and hence the possibility of a crucial role for T6P as in embryos and leaves.
Starch accumulation is one of the most striking examples of metabolic regulation by the treha-lose pathway since it has been shown a strong accumulation of starch in response to trehalose feeding and transcriptional regulation of ADP-glucose pyrophosphorylase (AGPase), the key enzyme of starch synthesis (Wingler et al. 2000). T6P activates AGPase through a thioredoxin-dependent redox activation mechanism (Kolbe et al. 2005). This activation mechanism operates under conditions of high sucrose, which induce high T6P levels (Lunn 2007; Lunn et al. 2006). This finding again supports the concept that T6P reflects conditions of high assimilate supply and in this case communicates these conditions to the chloroplast to activate starch synthesis.
Although five different trehalose synthesis pathways exist in bacteria, fungi, yeast, and algae (Paul et al. 2008; Avonce et al. 2006) , trehalose biosynthesis in higher plants only occurs in the trehalose phosphate synthase (TPS) trehalose phosphate phosphatase (TPP) pathway (also known as OtsA-OtsB pathway) (Fig. 14.2).
The description of trehalose biosynthesis genes in plants began in the late 1990s with the characterization of the A. thaliana TPS (AtTPS1) and TPP encoding genes (AtTPPA and AtTPPB) (Blazquez et al. 1998; Vogel et al. 1998). These three genes encode functional TPS and TPP proteins because they complement tps1 and tps2 yeast mutants, which are deficient in TPS and TPP activities, respectively. A large number of TPS genes have been found in A. thaliana (Leyman et al. 2001). Using in silico analysis, ten homologs of AtTPS1 have been identified; these can be divided into two classes: class I genes (AtTPS1-AtTPS4) encode proteins that have a TPS domain closely related to Saccharomyces cerevisiae ScTPS1; class II genes (AtTPS5-AtTPS11) encode proteins with a TPP domain, exhibiting a strong homology to AtTPPA and AtTPPB, but only 30% homology with class I proteins (AtTPS 1-AtTPS4). The function of these class II genes remains largely unknown.
Ten homologs of TPP have been found in A. thaliana (AtTPPA-AtTPPJ) (Avonce et al. 2006; Leyman et al. 2001). Interestingly, TPS and TPP form multigenic families in other plants. For example, rice (Oryza Sativa) contains at least nine OsTPS and nine OsTPP genes (Avonce et al. 2006) . According to most authors, this redundancy is an indicator that either trehalose or T6P has an important metabolic role. By contrast, tre-halase is encoded by a unique gene in A. thaliana, rice, and soybean (Glycine max) (Frison et al. 2007). The complete trehalose biosynthesis gene expression pattern has been extensively reviewed (Paul et al. 2008).
The presence of trehalase in higher plants has been puzzling because its substrate appeared to be absent although it has been postulated that tre-halases could play a role in defense mechanisms (Fernandez et al. 2010) , In addition, it has been suggested that trehalase could be involved in the degradation of trehalose derived from plant-associated microorganisms, such as rhizobia in root nodules or fungi involved in mycorrhizal symbiosis (Müller et al. 1995a, b).
The role of trehalose in abiotic stress tolerance was first demonstrated in resurrection plants, such as Myrothamnus flabellifolius, Selaginella tamariscina, or Selaginella lepidophylla. These desiccation tolerant plants can withstand almost complete dehydration and upon rehydration regain complete viability (Liu et al. 2008). Interestingly, in these three species, trehalose is the main soluble sugar, with levels reaching 3 mg g-1 FW in M. flabellifolius and 12 mg g-1 FW in S. lepidophylla . During dehydration, tre-halose concentration increases only slightly and it acts as a protector for both proteins and membranes (Liu et al. 2008). Trehalose accumulation under abiotic stresses is not restricted to resurrection plants.
In A. thaliana, the trehalose level doubled within 4 h of heat stress (40°C) and increased eightfold 4 days after cold exposure (4°C) (Kaplan et al. 2004). A recent study (Suzuki et al. 2008) revealed the involvement of TPS (AtTPS5) in A. thaliana thermotolerance.
In rice, trehalose accumulation has been shown in roots 3 days following salt stress (García et al. 1997) . and two different TPPs were transiently induced in response to multiple abiotic stresses as well as exogenous ABA applications, which suggest a regulation of trehalose biosynthesis (Pramanik and Imai 2005; Shima et al. 2007).
)n model legumes Medicago truncatula and Lotus japonicus, nodular trehalose content increased by 50 and 100%, respectively, under salt stress conditions (López et al. 2008a). Previously in alfalfa (Medicago sativa L.), it has been also described an increase of trehalose accumulation in roots and bacteroids upon salt stress (Fougère et al. 1991).
Furthermore, a microarray analysis has revealed that a wide range of abiotic stresses, such as cold, salt and UV, cause a response in the genes involved in trehalose metabolism in A. thaliana (Iordachescu and Imai 2008). This finding indicates that trehalose and/or T6P are involved in the response to abiotic environmental fluctuations.
4.3.1 Trehalose: Compatible Solute in Plants?
It has been questioned the role of trehalose as compatible solute in plants under abiotic stress since compatible solutes are nontoxic molecules able to accumulate at high concentrations in the cytoplasm, participating in turgor maintenance and/or the protection of macromolecular structures against the destabilizing effect of anhydro-biotic conditions (Gibon et al. 1997). However, if trehalose concentration in resurrection plants reaches levels consistent with the compatible solutes definition, the concentration of trehalose in other plants is low, which suggests that trehalose is not a compatible solute (Avonce et al. 2004; Schluepmann et al. 2003; Grennan 2007). Furthermore, trehalose genetically engineered plants exhibit altered morphology, possibly caused by toxicity of high trehalose concentrations, indicating that trehalose is a noncompatible solute (Schluepmann et al. 2003; Cortina and Culianez-Macia 2005). In many organisms, tre-halose has been reported as a better stabilizer than other sugars for protecting membranes and biomolecules (Elbein et al. 2003 ; Purvis et al. 2005; Crowe 2007) .
4.4 Engineering Trehalose
The introduction of trehalose biosynthetic genes has been used as an strategy for different research groups to create stress tolerant plants in tobacco (Nicotiana tabaccum) (Romero et al. 1997; Pilon-Smits et al. 1998; Han et al. 2005), rice (Garg et al. 2002), tomato (Solanum lycopersicum) (Cortina and Culianez-Macia 2005) , potato (Stiller et al. 2008), and Arabidopsis (Karim et al. 2007; Miranda et al. 2007). First attempts with yeast TPS1 or E. coli OtsA genes induced treha-lose accumulation, although at a low level. However, T6P accumulation produced abnormal phenotypes (Romero et al. 1997; Pilon-Smits et al. 1998; Cortina and Culianez-Macia 2005). Nevertheless, this problem were overcome directing the gene product into chloroplast by using a TPS-TPP fusion gene together with a stress inducible promoter (Karim et al. 2007; Miranda et al. 2007), or using trehalose biosynthetic genes (trehalose phosphorylase) that avoid the T6P formation (Han et al. 2005) . These improved methods resulted in stress tolerance without the phenotypic alterations.
Drought tolerance was one of the first traits obtained by constitutive overexpression of the yeast ScTPS1 gene in tobacco (Romero et al. 1997) and the AtTPS1 gene in A. thaliana (Avonce et al. 2004). Improved drought tolerance was also achieved with stress inducible and chloroplast-targeted expression of the plastid TPS1 gene in tobacco (Karim et al. 2007) and by expression of bifunctional fusion genes, OtsA-OtsB and ScTPS-ScTPP; in rice and tobacco, respectively (Garg et al. 2002, Karim et al. 2007).
Transgenic tomatoes overexpressing the ScTPS1 gene are more resistant to salt, drought, and oxidative stresses (Cortina and Culianez-Macia 2005). Improved freezing and heat stress tolerance have been obtained in A. thaliana by constitutive or stress-inducible expression of a bifunctional yeast ScTPS1-ScTPS2 gene, leading to a significant accumulation of trehalose (Miranda et al. 2007).
Rice overexpressing the E. coli trehalose synthesis genes (OtsA and OtsB) becomes tolerant to salt and low-temperature stresses. These plants are characterized by trehalose accumulation (increased three- to tenfold, when compared with the nontransgenic controls), stronger photosyn-thetic activity, and global accumulation of carbohydrates (Garg et al. 2002).
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