Trehalose is a non-reducing disaccharide present in diverse organisms ranging from bacteria and fungi to invertebrates, in which serves as an energy source as well as an osmolyte and/or protein/membrane protectant. Until recently, trehalose was thought to be of any real significance in plants, although genetic studies have confirmed the existence of surprising abundance of genes for trehalose metabolism in plants which have lead to propose trehalose pathway as a central metabolic regulator. Multiple studies have linked trehalose to abiotic stress tolerance in plants and different research groups have attempted to create stress tolerant plants by introducing trehalose biosynthetic genes in important crops such as rice, tomato and potato. Particular cases of the trehalose metabolism are plant symbiotic interactions such as the Rhizobium-legume symbiosis, where trehalose has been described as a major carbohydrate in root nodules of some species. The discovery of trehalose metabolism in the recent years has pointed to the importance of trehalose biosynthesis in stress responses in plants.
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ROLE OF NONREDUCING DISACCHARIDES IN PLANTS
Nonreducing disaccharides provide a soluble energy source in the form of a stable molecule that can also function as a protectant compound under stress conditions in all organisms except vertebrates. Trehalose and sucrose are the two sugars that perform this role.
The component of reducing sugars, glucose in the case of trehalose and glucose and fructose in the case of sucrose, are linked at their reducing ends. In naturally occurring trehalose the two glucose units are linked in a Î±,Î±-1,1 configuration. Isomers include neotrehalose with an Î±,Î² link, and isotrehalose, which has a Î², Î² link. In sucrose, fructose and glucose are linked in a Î±,Î²-1,2 configuration. Both configurations produce stable energy molecules (Figure 1).
Plants are unique in that they can synthesize both non-reducing disaccharides, but sucrose performs the main role of translocated sugar in plants. Trehalose is found in milimolar amounts in only a few plants, namely resurrection species, where it is though to protect against desiccation. In the vast majority of plants, trehalose is only present in trace amounts.
The different chemistries of trehalose and sucrose dictate their biology, the functions they perform, and the mechanisms that determine the concentration of these compounds in vivo. Several arguments can be put forward to explain the prevalence of sucrose as translocated sugar in plants:
1. Sucrose is more soluble than trehalose, particularly at low temperatures, and hence may be more suited as a transport sugar in plant phloem at concentrations as high as 1M.
2. Sucrose can be cleaved by invertase into glucose and fructose, and by sucrose synthase into uridine diphosphoglucose (UDPG) and fructose, preserving energy as UDPG.
3. Cell wall polysaccharides are synthesized from UDPG, thus the ability to liberate UDPG directly from sucrose to synthesize cell wall polysaccharides may be the main reason sucrose dominates in plants.
The importance of trehalose in stress conditions compared with other sugars can be explained by its protective ability, stability and low reactivity:
1. X-ray diffraction studies show that trehalose fits well between the polar head groups with multiple sites of interaction which suggest the strong stabilizing effect of trehalose (Rudolph et al. 1990).
2. Trehalose possesses several unique physical properties which include high hydrophilicity, chemical stability and the absence of internal hydrogen bond formation that account for the principal ability of trehalose for protein stabilization. It has been proposed that in the absence of water, trehalose preserves membrane or protein structures by forming an amorphous glass structure and interacting through hydrogen bonds with polar phospholipids head groups or with amino acids (Crowe et al. 1984). Thereby trehalose is helping the protein to keep in shape and concentrate the remaining water next to the protein (Schiraldi et al. 2002). Trehalose is among the most chemically unreactive sugars and its strong stability is result of the very low energy (1 kcal mol-1) of the glycoside oxygen bond joining the two hexose rings. In comparison, sucrose, has an energy bond of 27 kcal mol-1 (Paiva and Panek 1996). Therefore, trehalose does not dissociate into two reducing monosaccharidic constituents unless exposed to extreme hydrolytic conditions or to the action of trehalase.
OCCURRENCE OF TREHALOSE IN DIFFERENT ORGANISMS
Trehalose was first identified as a constituent of the ergot fungus of rye in 1832. The name trehalose was introduced in 1858 when it was found in the concoons or "trehala" of the desert beetles of the Middle East, Laurinus nidificans and L. maculates. These secretions, known by native peoples to be edible and sweet, were called "trehala manna".
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The occurrence of trehalose has been described in all kingdoms where it is assumed to play a similar role in vivo as its demonstrated properties in vitro. With the exception of plants, trehalose has a central role as an energy source and stress response molecule in microorganisms and invertebrates and as a starting point for chitin synthesis in fungi.
Trehalose has been found in a number of different bacteria, including Streptomyces hygroscopicus and other species of streptomyces (Martin et al. 1986), various mycobacteria, including Mycobacterium smegmatis and tuberculosis (Elbein and Mitchell 1973) and corynebacteria (Shimakata and Minatagawa 2000) where this disaccharide plays a structural role as a cell wall component. It is also present in Escherichia coli (Kaasen et al. 1994) and a number of other bacteria, such as Rhizobium sp. (Maruta et al. 1996a), Sulfdolobus acidocaldarius (Maruta et al. 1996b), Pimelobacter sp. R48 (Nishimoto et al. 1995) and Arthrobacter sp. Q36 (Maruta et al. 1996c). In many of these organisms, trehalose is used as the sole carbon source, compatible osmolyte, and as a part of the cell wall structure. Several of the organisms listed appear to have rather unusual biosynthetic pathways for synthesizing trehalose.
In fungi, apart of the starting point for chitin synthesis, trehalose is synthesized at the onset of reduced growth periods, which protect the cell's integrity against stress damage, an then is rapidly mobilized during recovery and during the early germination of spores (Arguelles 2000). Trehalose is quite common in yeast and fungi where it occurs in spores, fruiting bodies, and vegetative cells (Nwaka and Holzer 1998). Spores of certain species have been reported to contain as much as 10% trehalose on a dry-weight basis. When these spores germinate, the trehalose rapidly disappears suggesting hat this sugar is stored as a source of carbon, and/or energy.
In the animal kingdom, trehalose was first reported in insects, where it is present in hemolymph and thorax muscles (Becker et al. 1996) and also in larvae or pupae (Fairbairn 1958). In the adult insect, the levels of trehalose fall rapidly during certain energy-requiring activities, such as flight indicating a role for this disaccharide as a source of glucose for energy. It has been shown that when the nematode Aphelenchus avenae is slowly dehydrated, it converts as much as 20% of its dry weight into trehalose (Madin and Crowe 1975). The ability of this organism to survive in the absence of water has shown a strong correlation with the synthesis of trehalose.
There are at least five biosynthetic pathways known for trehalose (Figure 2). The first pathway was discovered about 50 years ago (Cabib and Lenoir 1958), is the most widely distributed, and it has been reported in eubacteria, archaea, fungi, insects, and plants. It involves two enzymatic steps catalyzed by trehalose-6-phosphate synthase (TPS) and trehalose-phosphatase (TPP). TPS catalyzes the transfer of glucose from UDP-glucose to glucose 6-phosphate forming trehalose 6-phosphate (T6P) and UDP, while TPP dephosphorylates T6P to trehalose and inorganic phosphate (Elbein et al. 2003, De Smet et al. 2000).
The second pathway involves the conversion of maltodextrines (maltooligosaccharides, glycogen and starch) to trehalose. This pathway was reported in thermophilic archaea of the genus Sulfolobus. These organisms synthesize trehalose in two enzymatic steps catalyzed by maltooligosyl trehalose synthase (TreY), coded by the treY gene, which promotes the transglycosylation of the last glucose moiety at the reduced end of maltodextrins from a Î±1-Î±4 to a Î±1-Î±1 bond leading to maltooligosyltrehalose, which contains a trehalose moiety at the end of the polymer. Next, maltooligosyl trehalose trehalohydrolase (TreZ), coded by the treZ gene, catalyses the hydrolytic release of trehalose (Elbein et al. 2003, Streeter and Bhagwat, 1999).
In the third biosynthetic pathway, the enzyme trehalose synthase (TS) isomerises the Î±1-Î±4 bond of maltose to a Î±1-Î±1 bond, forming trehalose (Elbein et al. 2003, Higashiyama 2002). This enzyme was first reported in Pimelobacter sp. and orthologs of this protein have been found in other eubacteria.
In the fourth pathway, trehalose phosphorylase (TreP), present in some fungi, catalyses the reversible hydrolysis of trehalose in the presence of inorganic phosphate. The transfer of a glucose molecule to a phosphate generates glucose 1-phosphate and releases the other glucose residue. There is uncertainty about the participation of the TreP enzyme in the synthesis or degradation of trehalose, since the biosynthetic reaction has only been shown in vitro (Wannet et al. 1998, Schiraldi et al. 2002).
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A new biosynthetic pathway for trehalose was found in the hyperthermophilic archaeon Thermococcus litoralis, and involves the trehalose glycosyltransferring synthase (TreT), which catalyses the reversible formation of trehalose from ADP-glucose and glucose (Qu et al. 2004, Ryu et al. 2005). It can also use UDP-glucose and GDP-glucose, although it is less efficient with these substrates. The TreT enzyme transfers the glucose moiety from ADP-glucose, and joins it at position 1 of another glucose molecule to form trehalose.
Trehalose catabolism in two glucose molecules is catalyzed by trehalase enzyme activity (Tre) that has been found in a variety of organisms including prokaryotic and eukaryotic (Elbein 1974). Treahalase is ubiquitous in higher plants and it is likely that trehalase is the sole route of trehalose breakdown in plants as trehalose accumulates in the presence of specific trehalase inhibitor validamycin A (Müller et al. 2001).
TREHALOSE IN PLANTS
Until recently, trehalose was no thought to be of any real significance in plants; instead, it was regarded as a rare obscure sugar found in some marginal resurrection species such as Sellaginella species (Anselmino and Gilg 1913) and Myrothamnus flabellifolia (Bianchi et al. 1993) where its accumulation to high levels enables protection for desiccation. The lack of trehalose detection in the majority of plants led to the assumption that trehalose function had died out and had been replaced by sucrose. Later, trehalose was detected in the model plant Arabidopsis thaliana using validamycin A (Goodijn 1997), an inhibitor of the trehalose-degrading enzyme, trehalase. 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 and colleagues (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 trehalose 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).
Importance of Trehalose 6-phosphate
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 draw 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 (Figure 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 as yet not fully elucidated control features (transcriptional and posttranslational 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 signalling 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 signalling 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 AtTPS1 gene (Schluepman et al. 2004). Modifications of T6P levels cause dramatic effects on carbohydrate metabolism and partitioning as well as on morphogenesis and development in Arabidopsis (Schluepman et al. 2003). Interestingly, AtTPS1 is an essential gene, and knocking the gene out results in an embryo lethal phenotype (Eastmond et al. 2002). Embryo development is blocked early in the phase of cell expansion and storage reserve accumulation. In vegetative stage, AtTPS1 is essential for normal growth in particular for the development in the flowers, bunds 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 transgenic 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, was 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, 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 trehalose 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.