Any non-living factors that affect negatively living organisms are reunited under the general term “abiotic stress” and its effects can be and are mitigated by a variety of defense mechanisms developed by the different biological systems in existence. Examples of abiotic stress are desiccation, salinity, high and low temperature. There are two general mechanisms used to counteract abiotic stress: avoidance and adaptation. In the case of avoidance, organisms migrate to deeper soil layers where temperatures are within tolerable range (Roelofs et al., 2008). Adaptation to stress is based on activation of stress defense gene pathways, which results in the production of heat shock proteins, LEA proteins, redox regulating proteins, different compatible solutes, cytochrome P450s (Roelofs et al., 2008).
Trehalose, also known as tremalose or mycose, a non-reducing disaccharide, is widely spread in biological systems: bacteria, yeast, fungi, lower and higher plants, as well as insects and invertebrates (Elbein, 2003), and its function is associated with tolerance against multiple abiotic stresses. It was discovered in 1832 by H.A. Wiggers in the ergot of rye and later isolated from mooshrooms by Mitscherlich in 1858, who called it mycose (Richards et al., 2002). In the same year Berthelot isolated a novel sugar from trehala-manna, a secretion left by different insects on leaves on Middle East. He named this new sugar ‘‘trehalique glucose’’ or trehalose (Richards et al., 2002). Initially trehalose was considered to be a rare sugar because it could only be extracted from trehala manna or the resurrection plant. Later Koch and Koch in 1925 discovered it in yeast and established basic protocols for trehalose isolation from yeast (Richards et al., 2002). Still, the cost of trehalose production was high enough so that it limited its use for commercial exploitation. In 1990’s Hayashibara company in Japan discovered a method to mass-produce trehalose inexpensively from starch. The enzymes used in the process, maltooligosyl–trehalose synthase (MTSase) and maltooligosyl–trehalose trehalohydrolase (MTHase), are derived from a non-pathogenic soil bacteria,
Trehalose has multiple functions, and some of them are species specific. In microorganisms trehalose appears to act as an energy source, during certain stages of development such as spore germination (Elbein, 2003). In anhydrobiotic organisms, trehalose is known to accumulate to high concentrations to survive complete dehydration (Drennan et al., 1993), by preserving the membranes during drought period (Crowe et al., 1984). Trehalose acts as a structural component in mycobacteria, being incorporated into glycolipids (Elbein, 1974). In
2. Trehalose biosynthesis
There are five known pathways reported in living organisms for trehalose biosynthesis (Fig.1). Some organisms possess only one pathway, whereas others use multiple pathways, which are used depending on the stress affecting the organism (Paul et al., 2008).
2.1. TPS-TPP (OtsA-OtsB) pathway
TPS-TPP (OtsA-OtsB) pathway is a two steps process and it is the most common pathway for trehalose biosynthesis. It is present in both prokaryotes and eukaryotes (archaea, bacteria, fungi, plants and arthropods) (Paul et al, 2008). In plants, trehalose 6-phosphate synthase (TPS) catalyzes the synthesis of the intermediate trehalose-6-phosphase from glucose-6-phosphate and Uridine Diphosphate (UDP)-glucose, and then trehalose-6-phosphate phosphatase (TPP) catalyzes the dephosphorylation of trehalose-6-phosphate to trehalose. In bacteria OtsA and OtsB enzymes catalyze the conversion of glucose-6-phosphate and UDP-glucose to trehalose, whereas in yeast TPS1 and TPS2 (homologues of TPS and TPP, respectively) are catalyzing the process. If in plants and bacteria the two trehalose biosynthesis enzymes are separate entities, in yeast the TPS1 (TPS homologue) and TPS2 (TPP homologue) are part of a complex that contains two other regulatory subunits, TPS3 and TSL1 (Bell et al., 1998).
2.2. TreY-TreZ pathway
Initially this pathway was discovered in
2.3. TreS pathway
The TreS pathway is a reversible transglycosylation reaction in which trehalose synthase (TreS) converts maltose, a disaccharide with ,-1,4 linkage between the two glucose molecules, to trehalose. Trehalose synthase was first cloned from
2.4. TreT pathway
TreT pathway involves the reversible formation of trehalose from ADP-glucose and glucose and it is catalyzed by trehalose glycosyltransferase (TreT) (Qu et al., 2004). TreT appears both in archaea and bacteria (Paul et al., 2008), and it was first reported in the hyperthermophilic archaea
2.5. TreP pathway
TreP is the second trehalose synthesis pathway found in both prokaryotes and eukaryotes. It is a potential reversible reaction that converts glucose-1-phosphate (G1P) and glucose into trehalose, catalyzed by trehalose phosphorylase. The pathway was first discovered in
3. Trehalose roles in abiotic stresses
Trehalose involvement in tolerance to abiotic stress has been documented in numerous organisms, both prokaryotes and eukaryotes. The effects of desiccation, salt, high and low temperature stresses have been shown to be averted by trehalose. The information is by no means complete and future studies may reveal trehalose implication in the fight against abiotic stresses in additional species.
3.1. Desiccation stress
Water is essential to the existence of life. Not only it is a basic component of living organisms, but also it is critical for their survival. However, there are organisms that can forgo water for extended periods of times, even for decades or centuries. Anhydrobiotic organisms can survive almost complete dehydration, the term anhydrobiosis literally meaning in Greek “life without water”. Such organisms are the invertebrates rotifers, tardigrades, brine shrimp and nematodes, but also certain resurrection plants, and microorganisms like baker's yeast (
Many of the anhydrobiotic organisms accumulate high concentrations of trehalose during drought stress. In the case of desiccation, water loss can be extremely high, as much as 99% (Strom et al., 1993). Among the protecting disaccharides that accumulate during drought, trehalose is the most effective in stabilizing dry membranes (Crowe et al., 1992). During dehydration, membranes are destabilized because of lipid phase transitions and vesicle fusion (Crowe & Crowe, 1990). Trehalose, even in small quantities, inhibits vesicles fusion completely and depresses the phase transition temperature of dry lipids, maintaining them in liquid crystalline phase in the absence of water (Crowe et al., 1992). It appears that during dehydration or freezing trehalose molecules replace bound water normally associated with biological structures (Donnamaria et al., 1994). Because of its high hydration potential, trehalose may stabilize dry biological membranes and proteins by hydrogen bonding of its hydroxyl groups to the polar groups of proteins and phosphate groups of membranes (Kawai et al., 1992).
Another mechanism by which trehalose protects against desiccation stress is vitrification. Trehalose has the tendency to form a protective glass-like structure that has a low reactivity, making it more stable than other disaccharides due to its non-reducing character. In this hygroscopic glass-like structure, trehalose is extremely stable both at high temperature and when completely desiccated and may hold biomolecules in a form that allows them to return to their native structure and function following rehydration (Crowe & Crowe, 2000). Trehalose glass is suggested to have such a great stability because a small addition of water may form trehalose dihydrate on the outer surface of the glass, which may result in a structure that encloses the inner glass, isolating it (Richards et al., 2002).
Trehalose involvement in bacteria resistance to desiccation stress has been studied extensively.
Rhizobia, soil bacteria that live in symbiosis with legumes are important to agriculture because of their biological nitrogen fixation capacity.
Trehalose is widely distributed in fungi and it accumulates in both vegetative and reproductive stages, and at particularly high concentrations in periods with reduced growth rates and during starvation (Thevelein, 1984). Trehalose is also present in the extra-radical mycelium as well as in spores of arbuscular mycorrhizal fungi (Becard et al., 1991).
Log-phase cultures of yeast have low concentrations of trehalose and are quite susceptible to dehydration, but as they enter the stationary phase of growth the levels of trehalose increase (Elbein, 2003). A study of desiccation tolerance of yeast cells subjected to temperature shifts revealed a clear correlation between the cells trehalose content and the changes in desiccation tolerance, demonstrating the trehalose function as a protectant against desiccation (Hottiger et al., 1987).
The presence of trehalose in higher plants was discovered relative recently, and most of the reports were referring to a few desiccation tolerant plants (Bianchi et al., 1993, Drennan et al., 1993, Albini et al., 1994). Following whole genome sequencing,
Typically, trehalose does not accumulate in plants in quantities high enough to directly protect against abiotic stress as a compatible solute, the way it accumulates in other organisms. Transgenic plants engineered to overexpress microbial trehalose biosynthesis genes accumulated low levels of trehalose and still became tolerant to abiotic stresses, specifically to drought stress. Tobacco and tomato plants transformed with yeast
Tardigrades are microscopic animals, also known as water bears, which show an extraordinary tolerance to variety of extreme environmental conditions, particularly anhydrobiosis (Welnicz et al, 2011). In some species, the trehalose levels are increased during the induction of anhydrobiosis, but in others there was no difference in trehalose levels between the desiccated and hydrated specimens. In addition, the absolute levels of trehalose detected in tardigrades are much lower than those detected in other anhydrobiotic organisms, indicating that trehalose may have a specific function connected to desiccation, but the nature of that function is not currently known (Welnicz et al., 2011).
3.2. Salt stress
Salt stress affects organisms in two major ways. Low water potential causes loss of water and turgor pressure, whereas the high ionic strength of the surrounding environment creates a continuous flux of inorganic ions into the living cells. Living organisms maintain their turgor pressure and cell volumes within acceptable limits by accumulating organic osmolytes, called compatible solutes (sugars, polyols, free aminoacids and their derivatives, quaternary amines and their sulfonium analogues, sulfate esters and small peptides (Kempf et al., 1998). Compatible solutes participate in alleviating salt stress in two major ways. First, they lower the osmotic potential of the cytoplasm, maintaining the normal turgor pressurte of the cells (Kempf et al., 1998). Second, they serve as stabilizers of proteins and cell components against the denaturing effects of high ionic strength (Hincha & Hagemann, 2004). Among the compatible solutes, trehalose occupies an important place, acting as a stress protectant in both prokaryotes and eukaryotes.
Most of cyanobacteria are living in waters of different or changing salinities. Those who dwell in fresh water habitats are adapted to a low osmotic strength environment. Nevertheless, most freshwater cyanobacteria are able to withstand at least partially increasing salt concentrations. Among the compatible solutes that are mostly induced to accumulate in response to low osmotic stress are trehalose and sucrose (Klahn et al., 2011). Trehalose accumulation in cyanobacteria was first demonstrated in
The soil bacterium
Salinity stress affects negatively the symbiotic interactions between rhizobia and legume plants, limiting nitrogen fixation and reducing crop yields. Trehalose accumulates in rhizobium
Arbuscular mycorrhizal fungi colonize plant root systems of over 80% of terrestrial plant species and have been shown to promote plant growth and salinity tolerance by numerous studies (Evelin et al., 2009).
Tomatoes engineered to overexpress yeast trehalose synthesis genes are resistant not only to drought, but also to salt and oxidative stresses (Cortina & Culianez-Macia, 2005). Rice plants overexpressing bacterial fused trehalose synthesis genes under the control of tissue-specific or stress-dependent promoters accumulate trehalose and are salt, drought, and low temperature stress tolerant, without showing growth abnormalities (Garg et al., 2002).
3.3. Low and high temperature stress
Biological membranes are affected during low temperature stress, as their fluidity decreases. Protein denaturation and aggregation happen both under low and high temperature stresses. As a compatible solute trehalose, can prevent protein degradation and aggregation. It can also stabilize biological membranes, by hydrogen bonding with the phosphate groups.
Kandror et al. (2002) reported a protective role of trehalose in cold adaptation of
The most studied abiotic stress that induces trehalose in fungi is heat stress. The high level of trehalose in fungal spores increases their resistance to heat stress. Trehalose biosynthesis genes as well as trehalase, responsible for trehalose breakdown are up regulated, resulting in trehalose accumulation. In the yeast
Trehalose appears to also have a role in low temperature stress. Hino et al. (1990) reported a correlation between trehalose intracellular accumulation and freeze tolerance of
In nematodes, trehalose induces thermotolerance by preventing damage under heat stress. In addition, trehalose extends the nematodes life-span, possibly by protecting against heat-stress associated damage (Honda et al., 2010). Arctic nematode
Exposure to a mild cold stress over a period of few minutes to a few hours can increase the cold tolerance of some insects.
4. Trehalose uses in relation to abiotic stress
Due to the fact that trehalose proved to be a protectant compound under abiotic stress conditions, it has been and may be used in countless applications in pharmaceutical industry, agriculture, food industry, cosmetics industry, medicine, and research.
4.1. Agriculture and food industry
Since trehalose has been found to participate in increasing tolerance to abiotic stresses in other organisms, many attempts have been made to engineer plants with microbial trehalose biosynthetic genes from OtsA-OtsB pathway in order to create stress tolerant plants: tobacco (Holmstrom et al., 1996, Goddijn et al., 1997, Romero et al., 1997, Pilon-Smits et al., 1998, Lee et al., 2003, Han et al., 2005, Karim et al., 2007), rice (Garg et al., 2002; Jang et al., 2003), tomato (Cortina & Culianez-Macia, 2005), potato (Goddijn et al., 1997) and
The symbiotic relationship between rhizobia and legumes has a considerable impact not only on the legumes yields but also on the significant amount of the fixed nitrogen that remains in the soil for future crops use. A way to encourage the formation of the legume-rhizobia symbiosis could be the application of rhizobia to legume seeds prior to planting in the field. However, the percent of survival of rhizobia applied in this manner is very low, less than 5%, because of rapid desiccation (Roughley et al., 1993). By supplying external trehalose (3 mmol l-1) to
Trehalose role in protecting agricultural products may not be resumed only in helping the direct preservation. For instance, the yeast
The ability of trehalose to protect protein structure suggested its use in food packaging and preservation. Fresh fruits, herbs, and vegetables are preserving their color, taste and flavors when dried after a brief immersion in trehalose solution (Iturriaga et al., 2009). Superoxide dismutase (SOD) in plants acts as an antioxidant and protects cell components against oxidative damage by reactive oxygen species (Alscher et al., 2002). SOD-like activity of vegetables (carrots, cucumber, spinach onion) was preserved upon drying fresh vegetables with trehalose (Ohtake & Wang, 2011). Trehalose in its glass form encases and protects biomolecules, permitting them to return to their native structure and function upon rehydration (Crowe & Crowe, 2000). Fresh banana, strawberry, mango, avocado, apple, and raspberry, pureed in the presence of trehalose and dried at 25–50C, kept their color and aroma during prolonged storage. Volatile aromatics are trapped within the trehalose nonpermeable glass and released only after reconstitution of the product (Ohtake & Wang, 2011).
Because of its shielding properties, trehalose has been included as a food additive in japanese rice cake for protection against low temperature and freezing stresses (Ohtake & Wang, 2011). Freeze-tolerant yeast strains, accumulate trehalose when exposed to freezing (Hino et al., 1990). These strains retain their fermentative ability and bread leavening activity up to six weeks of frozen storage, maintaining their ability to produce good quality bread following freezing (Hino et al., 1987). As trehalose had been found to be able to suppress fatty acid degradation from heat and also from free radical oxidation (Higashiyama, 2002), it is effective in suppressing the formation of unpleasant odors associated with cooking fish (Ohtake & Wang, 2011).
4.2. Cosmetics industry
Trehalose has been found to suppress the unpleasant odors emitted by human skin by up to 70%, which makes is a very good candidate for use in facial and body creams and body deodorants (Higashiyama, 2002). In addition, trehalose incorporated in cosmetic products may enhance their shelf life and mask the odor of active ingredients and their degradation products, due to its antioxidation properties (Ohtake & Wang, 2011).
The high water retention capabilities of trehalose make it useful as a moisturizer in cosmetics (Hyde et al., 2010)
4.3. Medicine and pharmacy
As trehalose proved to be able to suppress peptide aggregation (Singer & Lindquist, 1998), it has been used to reduce the symptoms of Huntington’s disease. In the cells of patients suffering of this disease, the mutant protein huntingtin forms insoluble aggregates that are thought to produce the disease. Oral administration of trehalose inhibited the formation of huntingtin aggregates and improved the associated motor dysfunction in a transgenic mouse model of Huntington disease (Tanaka et al., 2004).
Researchers at Tokyo University developed an organ preservation solution, extracellular-type trehalose-containing Kyoto (ET-Kyoto) solution, that was successfully used in clinical lung transplantation (Ohtake & Wang, 2011).
Another application for trehalose use is the formulations of heat stable vaccines. An optimized
Unstable molecules such as antibodies can be dehydrated at room temperature or 37 C in the presence of trehalose, maintaining their activity after months in storage (Iturriaga et al., 2009). Dried proteins adopt the same configuration as they do in hydrated state when they are stabilized with trehalose, because trehalose prevents degradation by deamidation, oxidation, and aggregation (Ohtake & Wang, 2011).
In plant research, Arabidopsis AtTPS1 gene can be employed as a selectable marker gene during the process of plant transformation, using glucose as a selective agent Iturriaga et al., 2009). The AtTPS1 gene of A. thaliana encodes the TPS1 enzyme, which confers glucose insensibility to seeds and tissues of plants overexpressing this gene when cultivated under tissue-culture conditions (Avonce et al., 2004).