Trehalose Biosynthesis in Response to Abiotic Stresses

Trehalose is a non‐reducing disaccharide that is present in diverse organisms ranging from bacteria and fungi to invertebrates, in which it serves as an energy source, osmolyte or protein/membrane protectant. The occurrence of trehalose and trehalose biosynthesis pathway in plants has been discovered recently. Multiple studies have revealed regulatory roles of trehalose‐6‐phosphate, a precursor of trehalose, in sugar metabolism, growth and development in plants. Trehalose levels are generally quite low in plants but may alter in response to environmental stresses. Transgenic plants overexpressing microbial trehalose biosynthesis genes have been shown to contain increased levels of trehalose and display drought, salt and cold tolerance. In‐silico expression profiling of all Arabidopsis trehalose‐6‐phosphate synthases (TPSs) and trehalose‐6‐phosphate phosphatases (TPPs) revealed that certain classes of TPS and TPP genes are differentially regulated in response to a variety of abiotic stresses. These studies point to the importance of trehalose biosynthesis in stress responses.     

Trehalose‐6‐phosphate phosphatases from Arabidopsis thaliana: identification by functional complementation of the yeast tps2 mutant

It is currently thought that most flowering plants lack the capacity to synthesize trehalose, a common disaccharide of bacteria, fungi and invertebrates that appears to play a major role in desiccation tolerance. Attempts have therefore been made to render plants more drought-resistant by the expression of microbial genes for trehalose synthesis. It is demonstrated here that Arabidopsis thaliana itself possesses genes for at least one of the enzymes required for trehalose synthesis, trehalose-6-phosphate phosphatase. The yeast tps2 mutant, which lacks this enzyme, is heat-sensitive, and Arabidopsis cDNA able to complement this effect has been screened for. Half of the yeast transformants that grew at 38.6°C were also able to produce trehalose. All of these expressed one of two Arabidopsis cDNA, either AtTPPA or AtTPPB, which are both homologous to the C-terminal part of the yeast TPS2 gene and other microbial trehalose-6-phosphate phosphatases. Yeast tps2 mutants expressing AtTPPA or AtTPPB contained trehalose-6-phosphate phosphatase activity that could be measured both in vivo and in vitro. The enzyme dephosphorylated trehalose-6-phosphate but not glucose-6-phosphate or sucrose-6-phosphate. Both genes are expressed in flowers and young developing tissue of Arabidopsis. The finding of these novel Arabidopsis genes for trehalose-6-phosphate phosphatase strongly indicates that a pathway for trehalose biosynthesis exists in plants.     

Effect of nitrogen limitation and surplus upon trehalose metabolism in wine yeast

Trehalose metabolism in yeast has been related to stress and could be used as a stress indicator. Winemaking conditions are stressful for yeast and understanding trehalose metabolism under these conditions could be useful for controlling alcoholic fermentation. In this study, we analysed trehalose metabolism of a commercial wine yeast strain during alcoholic fermentation by varying the nitrogen levels from low (below adequate) to high (excess). We determined trehalose, nitrogen, sugar consumption and expression of NTH1, NTH2 and TPS1. Our results show that trehalose metabolism is slightly affected by nitrogen availability and that the main consumption of nitrogen occurs in the first 24 h. After this period, nitrogen is hardly taken up by the yeast cells. Although nitrogen and sugar are still available, no further growth is observed in high concentrations of nitrogen. Increased expression of genes involved in trehalose metabolism occurs mainly at the end of the growth period. This could be related to an adaptive mechanism for fine tuning of glycolysis during alcoholic tumultuous fermentation, as both anabolic and catabolic pathways are affected by such expression.     

Trehalose and its applications in plant biotechnology

Trehalose, a nonreducing disaccharide of glucose, is one of the most effective osmoprotectants. Several strategies leading to its accumulation have been envisaged in both model and crop plants using genes of bacterial, yeast and, more recently, plant origin. Significant levels of trehalose accumulation have been shown to cause abiotic stress tolerance in transgenic plants. In this review, we describe the most biologically relevant features of trehalose: chemical and biological properties; occurrence and metabolism in organisms with special reference to plants; protective role in stabilizing molecules; physiological role in plants with special reference to carbohydrate metabolism. The emphasis of this review, however, will be on manipulation of trehalose metabolism to improve abiotic stress tolerance in plants.     

Molecular characterization of a novel,nuclear-encoded,NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase in plastids of the gymnosperm Pinus sylvestris L.

Angiosperms and algae possess two distinct glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzymes, an NAD⁺-dependent tetramer involved in cytosolic glycolysis and an NADP⁺-dependent enzyme of the Calvin cycle in chloroplasts. We have found that the gymnosperm Pinus sylvestris possesses, in addition to these, a nuclear-encoded, plastid-specific, NAD⁺-dependent GAPDH, designated GapCp, which has not previously been described from any plant. Several independent full-size cDNAs for this enzyme were isolated which encode a functional transit peptide and mature subunit very similar to that of cytosolic GAPDH of angiosperms and algae. A molecular phylogeny reveals that chloroplast GapCp and cytosolic GapC arose through gene duplication early in chlorophyte evolution. The GapCp gene is expressed as highly as that for GapC in light-grown pine seedlings. These findings suggest that aspects of compartmentalized sugar phosphate metabolism may differ in angiosperms and gymnosperms and furthermore underscore the contributions of endosymbiotic gene transfer and gene duplication to the nuclear complement of genes for enzymes of plant primary metabolism.     

Two inositol hexakisphosphate kinases drive inositol pyrophosphate synthesis in plants

Inositol pyrophosphates are unique cellular signaling molecules with recently discovered roles in energy sensing and metabolism. Studies in eukaryotes have revealed that these compounds have a rapid turnover, and thus only small amounts accumulate. Inositol pyrophosphates have not been the subject of investigation in plants even though seeds produce large amounts of their precursor, myo‐inositol hexakisphosphate (InsP₆). Here, we report that Arabidopsis and maize InsP₆ transporter mutants have elevated levels of inositol pyrophosphates in their seed, providing unequivocal identification of their presence in plant tissues. We also show that plant seeds store a little over 1% of their inositol phosphate pool as InsP₇ and InsP₈. Many tissues, including, seed, seedlings, roots and leaves accumulate InsP₇ and InsP₈, thus synthesis is not confined to tissues with high InsP₆. We have identified two highly similar Arabidopsis genes, AtVip1 and AtVip2, which are orthologous to the yeast and mammalian VIP kinases. Both AtVip1 and AtVip2 encode proteins capable of restoring InsP₇ synthesis in yeast mutants, thus AtVip1 and AtVip2 can function as bonafide InsP₆ kinases. AtVip1 and AtVip2 are differentially expressed in plant tissues, suggesting non‐redundant or non‐overlapping functions in plants. These results contribute to our knowledge of inositol phosphate metabolism and will lay a foundation for understanding the role of InsP₇ and InsP₈ in plants.     

Overexpression of the trehalose-6-phosphate synthase gene <Emphasis Type="Italic">OsTPS1</Emphasis> enhances abiotic stress tolerance in rice

Trehalose plays an important role in metabolic regulation and abiotic stress tolerance in a variety of organisms. In plants, its biosynthesis is catalyzed by two key enzymes: trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). The genome of rice (Oryza sativa) contains 11 OsTPS genes, and only OsTPS1 shows TPS activity. To demonstrate the physiological function of OsTPS1, we introduced it into rice and found that OsTPS1 overexpression improved the tolerance of rice seedling to cold, high salinity and drought treatments without other significant phenotypic changes. In transgenic lines overexpressing OsTPS1, trehalose and proline concentrations were higher than in the wild type and some stress-related genes were up-regulated, including WSI18, RAB16C, HSP70, and ELIP. These results demonstrate that OsTPS1 may enhance the abiotic stress tolerance of plants by increasing the amount of trehalose and proline, and regulating the expression of stress-related genes. Furthermore, we found that overexpression of some Class II TPSs also enhanced plant tolerance of abiotic stress. This work will help to clarify the role of trehalose metabolism in abiotic stress response in higher plants.     

Contribution of trehalose biosynthetic pathway to drought stress tolerance of Capparis ovata Desf

Trehalose and the trehalose biosynthetic pathway are important contributors and regulators of stress responses in plants. Among recent findings for trehalose and its metabolism, the role of signalling in the regulation of growth and development and its potential for use as a storage energy source can be listed. The xerophytic plant Capparis ovata (caper) is well adapted to drought and high temperature stress in arid and semi‐arid regions of the Mediterranean. The contribution of trehalose and the trehalose biosynthetic pathway to drought stress responses and tolerance in C. ovata are not known. We investigated the effects of PEG‐mediated drought stress in caper plants and analysed physiological parameters and trehalose biosynthetic pathway components, trehalose‐6‐phosphate synthase (TPS), trehalose‐6‐phosphate phosphatase (TPP), trehalase activity, trehalose and proline content in drought stress‐treated and untreated plants. Our results indicated that trehalose and the trehalose biosynthetic pathway contributed to drought stress tolerance of C. ovata. Overall growth and leaf water status were not dramatically affected by drought, as both high relative growth rate and relative water content were recorded even after 14 days of drought stress. Trehalose accumulation increased in parallel to induced TPS and TPP activities and decreased trehalase activity in caper plants on day 14. Constitutive trehalose levels were 28.75 to 74.75 μg·g·FW^?1, and drought stress significantly induced trehalose accumulation (385.25 μg·g·FW^?1 on day 14) in leaves of caper. On day 14 of drought, proline levels were lower than on day 7. Under drought stress the discrepancy between trehalose and proline accumulation trends might result from the mode of action of these osmoprotectant molecules in C. ovata.     

Arabidopsis trehalose-6-phosphate synthase 1 is essential for normal vegetative growth and transition to flowering

In resurrection plants and yeast, trehalose has a function in stress protection, but the absence of measurable amounts of trehalose in other plants precludes such a function. The identification of a trehalose biosynthetic pathway in angiosperms raises questions on the function of trehalose metabolism in nonresurrection plants. We previously identified a mutant in the Arabidopsis trehalose biosynthesis gene AtTPS1. Plants homozygous for the tps1 mutation do not develop mature seeds (Eastmond et al., 2002). AtTPS1 expression analysis and the spatial and temporal activity of its promoter suggest that this gene is active outside the seed-filling stage of development as well. A generally low expression is observed in all organs analyzed, peaking in metabolic sinks such as flower buds, ripening siliques, and young rosette leaves. The arrested tps1/tps1 embryonic state could be rescued using a dexamethasone-inducible AtTPS1 expression system enabling generation of homozygous mutant plants. When depleted in AtTPS1 expression, such mutant plants show reduced root growth, which is correlated with a reduced root meristematic region. Moreover, tps1/tps1 plants are retarded in growth and remain generative during their lifetime. Absence of Trehalose-6-Phosphate Synthase 1 in Arabidopsis plants precludes transition to flowering.     

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.     

Plant homologue of human excision repair gene ERCC1 points to conservation of DNA repair mechanisms

Nucleotide excision repair (NER), a highly versatile DNA repair mechanism, is capable of removing various types of DNA damage including those induced by UV radiation and chemical mutagens. NER has been well characterized in yeast and mammalian systems but its presence in plants has not been reported. Here it is reported that a plant gene isolated from male germline cells of lily (Lilium longiflorum) shows a striking amino acid sequence similarity to the DNA excision repair proteins human ERCC1 and yeast RAD10. Homologous genes are also shown to be present in a number of taxonomically diverse plant genera tested, suggesting that this gene may have a conserved function in plants. The protein encoded by this gene is able to correct significantly the sensitivity to the cross-linking agent mitomycin C in ERCC1-deficient Chinese hamster ovary (CHO) cells. These findings suggest that the NER mechanism is conserved in yeast, animals and higher plants.     

Osmoregulation in the model organismEscherichia coli: genes governing the synthesis of glycine betaine and trehalose and their use in metabolic engineering of stress tolerance

Glycine betaine is known to be the preferred osmoprotectant in many bacteria, and glycine betaine accumulation has also been correlated with increased cold tolerance. Trehalose is often a minor osmoprotectant in bacteria and it is a major determinant for desiccation tolerance in many so-called anhydrobiotic organisms such as baker's yeast(Saccharomyces cerevisiae). Escherichia coli has two pathways for synthesis of these protective molecules; i.e., a two-step conversion of UDP-glucose and glucose-6-phosphate to trehalose and a two-step oxidation of externally-supplied choline to glycine betaine. The genes governing the choline-to-glycine betaine pathway have been studied inE. coli and several other bacteria and higher plants. The genes governing UDP-glucose-dependent trehalose synthesis have been studied inE. coli andS. cerevisiae. Because of their well-documented function in stress protection, glycine betaine and trehalose have been identified as targets for metabolic engineering of stress tolerance. Examples of this experimental approach include the expression of theE. coli betA andArthrobacter globiformis codA genes for glycine betaine synthesis in plants and distantly related bacteria, and the expression of theE. coli otsA and yeastTPS1 genes for trehalose synthesis in plants. The published data show that glycine betaine synthesis protects transgenic plants and phototrophic bacteria against stress caused by salt and cold. Trehalose synthesis has been reported to confer increased drought tolerance in transgenic plants, but it causes negative side effects which is of concern. Thus, the much-used model organismE. coli has now become a gene resource for metabolic engineering of stress tolerance.     

Gymnosperm Orthologues of Class B Floral Homeotic Genes and Their Impact on Understanding Flower Origin

Class B floral homeotic genes play a key role in specifying the identity of male reproductive organs (stamens) and petals during the development of flowers. Recently, close relatives (orthologues) of these genes have been found in diverse gymnosperms, the sister group of the flowering plants (angiosperms). The fact that such genes have not been found so far, despite considerable efforts, in mosses, ferns or algae, has been taken as evidence to suggest that B genes originated 300–400 million years ago in a lineage that led to extant seed plants. Gymnosperms do not develop petals, and their male reproductive organs deviate considerably from angiosperm stamens. So what is the function of gymnosperm B genes? Recent experiments revealed that B genes from diverse extant gymnosperms are exclusively expressed in male reproductive organs (microsporophylls). At least for some of these genes it has been shown that they can partially substitute for the Arabidopsis B genes AP3 and PI in ectopic expression experiments, or even partially substitute these genes in different class B floral organ identity gene mutants. This functional complementation, however, is restricted to male organ development. These findings strongly suggest that gymnosperm and angiosperm B genes have highly related interaction partners and equivalent functions in the male organs of their different host species. It seems likely that in extant gymnosperms B genes have a function in specifying male reproductive organs. This function was probably established already in the most recent common ancestor of extant gymnosperms and angiosperms (seed plants) 300 million years ago and thus represents the ancestral function of seed plant B genes, from which other functions (e.g., in specifying petal identity) might have been derived. This suggests that the B gene function is part of an ancestral sex determination system in which B gene expression specifies male reproductive organ development, while the absence of B gene expression leads to the formation of female reproductive organs. Such a simple switch mechanism suggests that B genes might have played a central role during the origin of flowers. In the out-of-male and out-of-female hypotheses changes in B gene expression led to the origin of hermaphroditic flower precursors out of male or female gymnosperm reproductive cones, respectively. We compare these hypotheses with other recent molecular hypotheses on the origin of flowers, in which C/D and FLORICAULA/LEAFY-like genes is given a more prominent role, and we suggest how these hypotheses might be tested in the future.     

Tomato T2 ribonuclease LE is involved in the response to pathogens

T2 ribonucleases (RNases) are RNA-degrading enzymes that function in various cellular processes, mostly via RNA metabolism. T2 RNase-encoding genes have been identified in various organisms, from bacteria to mammals, and are most diverse in plants. The existence of T2 RNase genes in almost every organism suggests an important biological function that has been conserved through evolution. In plants, T2 RNases are suggested to be involved in phosphate scavenging and recycling, and are implicated in defence responses to pathogens. We investigated the function of the tomato T2 RNase LE, known to be induced by phosphate deficiency and wounding. The possible involvement of LE in pathogen responses was examined. Expression analysis showed LE induction during fungal infection and by stimuli known to be associated with pathogen inoculation, including oxalic acid and hydrogen peroxide. Analysis of LE-suppressed transgenic tomato lines revealed higher susceptibility to oxalic acid, a cell death-inducing factor, compared to the wild type. This elevated sensitivity of LE-suppressed lines was evidenced by visual signs of necrosis, and increased ion leakage and reactive oxygen species levels, indicating acceleration of cell death. Challenge of the LE-suppressed lines with the necrotrophic pathogen Botrytis cinerea resulted in accelerated development of disease symptoms compared to the wild type, associated with suppressed expression of pathogenesis-related marker genes. The results suggest a role for plant endogenous T2 RNases in antifungal activity.     

Conservation and diversification of HAIRY MERISTEM gene family in land plants

The shoot apical meristems (SAMs) of land plants are crucial for plant growth and organ formation. In several angiosperms, the HAIRY MERISTEM (HAM) genes function as key regulators that control meristem development and stem cell homeostasis. To date, the origin and evolutionary history of the HAM family in land plants remains unclear. Potentially shared and divergent functions of HAM family members from angiosperms and non-angiosperms are also not known. In constructing a comprehensive phylogeny of the HAM family, we show that HAM proteins are widely present in land plants and that HAM proteins originated prior to the divergence of bryophytes. The HAM family was duplicated in a common ancestor of angiosperms, leading to two distinct groups: type I and type II. Type-II HAM members are widely present in angiosperms, whereas type-I HAM members were independently lost in different orders of monocots. Furthermore, HAM members from angiosperms and non-angiosperms (including bryophytes, lycophytes, ferns and gymnosperms) are able to replace the role of the type-II HAM genes in Arabidopsis, maintaining established SAMs and promoting the initiation of new stem cell niches. Our results uncover the conserved functions of HAM family members and reveal the conserved regulatory mechanisms underlying HAM expression patterning in meristems, providing insight into the evolution of key stem cell regulators in land plants.     

Transport and metabolism of trehalose in Escherichia coli and Salmonella typhimurium

The metabolism of trehalose in wild type cells of Escherichia coli and Salmonella typhimurium has been investigated. Intact cells of Escherichia coli (grown on trehalose) accumulated [¹⁴C]-trehalose as [¹⁴C]-trehalose 6-phosphate. Toluene-treated cells catalyzed the synthesis of the [¹⁴C]-sugar phosphate from [¹⁴C]-trehalose and phosphoenolpyruvate; ATP did not serve as phosphoryl donor. Trehalose 6-phosphate could subsequently be hydrolyzed by trehalose 6-phosphate hydrolase, an enzyme which catalyzes the hydrolysis of the disaccharide phosphate into glucose and glucose 6-phosphate. Both Escherichia coli and Salmonella typhimurium induced this enzyme when they grew on trehalose.These findings suggest that trehalose is transported in these bacteria by an inducible phosphoenolpyruvate:trehalose phosphotransferase system.The presence of a constitutive trehalase was also detected.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanosulfonic acid - PEP phosphoenolpyruvate - PTS phosphoenolpyruvate: glycose phosphotransferase system - O.D. optical density