Trehalose Biosynthesis in Response to Abiotic Stresses
Mihaela Iordachescu and Ryozo Imai*
Crop Cold Tolerance Research Team, National Agricultural Research Center
for Hokkaido Region, National Agriculture and Food Research Organization,
Hitsujigaoka 1, Toyohira-ku, Sapporo, 062-8555, Japan.
*Author for correspondence
Ryozo Imai
Crop Cold Tolerance Research Team, National Agricultural Research Center
for Hokkaido Region, National Agriculture and Food Research Organization,
Hitsujigaoka 1, Toyohira-ku, Sapporo, 062-8555, Japan.
Tel/FAX: +81 11 857 9382
E-mail: [email protected]
Running Title: Trehalose for Abiotic Stress Responses
1
Abstract
Trehalose is a nonreducing 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-6phosphate 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 response.
Key words: abiotic stress, Arabidopsis, cold stress, drought stress, rice,
stress, tobacco, trehalose, trehalose biosynthesis.
Abbreviations: TPS, trehalose-6-phosphate synthase; TPP, trehalose-6phosphate phosphatase
2
Increasing food production in the conditions of an increasing world
population is of a major concern. Enhancing the capability of crop plants to
withstand different abiotic stresses, such as salt, drought or cold, will lead to
higher yields by either increasing the crop set and/or by being able to extend
crop cultivation in the areas previously denied due to stress intolerance.
Trehalose, a nonreducing disaccharide consisting of two units of glucose
(-D-glucopyranosyl-1,1--D-glucopyranoside) is widely spread in a variety of
organisms: bacteria, yeast, fungi, lower and higher plants, as well as insects
and other invertebrates (Elbein et al, 2003). In insects, trehalose is a blood
sugar and serves as energy source during flight (Elbein et al, 2003).
Trehalose is known to accumulate to high concentrations to survive complete
dehydration in anhydrobiotic organisms (Drennan et al., 1993), by preserving
the membranes during drought period (Crowe et al, 1984). In yeast trehalose
plays a role in osmotic stress tolerance (Hounsa et al., 1998), heat and
desiccation tolerance (Hottinger et al., 1987). Bacteria also accumulate
trehalose in response to osmotic stress (Styrvold and Strom, 1991).
The main pathway by which trehalose is synthesized in microorganisms is
composed of two steps. First, trehalose 6-phosphate synthase (TPS)
catalyzes the synthesis of trehalose-6-phosphase from glucose-6-phosphate
and UDP-glucose, and then trehalose 6-phosphate phosphatase (TPP)
catalyzes the dephosphorylation of trehalose-6-phosphate to trehalose (Fig.
1). In yeast, both TPS (TPS1) and TPP (TPS2) are part of a complex that
contains two other regulatory subunits, TPS3 and TSL1 (Bell et al., 1998).
Until recently, there were few reports regarding the presence of trehalose
in higher, vascular plants. Most of those referred to a few desiccation-tolerant
plants (Drennan et al., 1993; Bianchi et al., 1993; Albini et al., 1994). However,
reports are emerging in multiple species about transcripts encoding putative
trehalose biosynthetic genes. For instance, eleven TPSs and ten TPPs are
present in Arabidopsis, and rice genome was found to contain nine TPSs and
nine TPPs.
Even though some of the plant trehalose biosynthesis genes have been
isolated and functionally characterized, we are still far from a thorough
understanding of the roles and the impact of trehalose/trehalose-6-phosphate
3
has on plant growth and development. This review describes recent
advancement in research on trehalose biosynthesis and the works linking
trehalose biosynthesis process and products to abiotic stresses. In addition, a
comprehensive study of the abiotic stress responsiveness of Arabidopsis
trehalose biosynthesis genes is presented.
Trehalose Biosynthesis in Plants
Plants synthesize trehalose in a pathway that is common to most
organisms, through the production of the intermediate trehalose-6-phosphate
(Fig.1). Major advances in the study of trehalose biosynthesis in plants were
made in the past decade. Following whole genomes sequencing, eleven
TPSs and ten TPPs were identified in Arabidopsis and rice genome was
found to contain nine TPSs and nine TPPs. Among these, functionality have
been proven by yeast complementation for Arabidopsis TPS1, TPS6, TPPA,
and TPPB, rice OsTPP1 and OsTPP2, and maize RAMOSA3 (RA3). However,
not all TPSs studied to date have catabolic activity. Although AtTPS7 and
AtTPS8 have both synthase and phosphatase domains, they failed the yeast
complementation assays (Vogel et al., 2001).
In 1998, Blazquez et al. and Vogel et al. reported cloning and
characterization of Arabidopsis TPS1, TPPA and TPPB, respectively. All three
proteins were proved to be functional synthase and phosphatases in vivo, as
demonstrated by complementation of yeast mutants defective in trehalose
biosynthetic genes. Later on, the finding that Arabidopsis homozygous tps1
mutant is embryo lethal (Eastmond et al., 2002) established the vital role of
plant trehalose metabolism. Furthermore, Schluepmann et al. (2003)
demonstrated that trehalose-6-phosphate is an absolute requisite for plant
development. Complementation with yeast TPS1 rescued the tps1 mutant
plants, whereas external application of trehalose did not. In addition, the study
of AtTPS1 role throughout plant development using a dexamethasoneinducible AtTPS1 system revealed that AtTPS1 is important for vegetative
growth and transition to flowering (van Dijken et al., 2004).
4
A genetic study has shown that Arabidopsis AtTPS6 controls cell
morphology, trichome branching, and stem branching (Chary et al., 2008).
AtTPS6 encodes a class II TPS that contains both synthase and phosphatase
domains and can complement both yeast TPS and TPP mutants. A point
mutation (csp-1) in the synthase domain of AtTPS6 resulted in reduced
trichome branching, distorted stem branching and increased number of
stomata, while a T-DNA insertion knock-out mutant (csp-2) displayed only
abnormal cell shapes. As AtTPS6 transcript still accumulates in csp-1 plants,
the protein might also be produced, and a functional phosphatase domain
coupled with a defective synthase domain may account for the differences
between the two mutants. Overexpression of the gene resulted in plants with
increased size and number of rosette leaves, as well as an increased number
of trichome branches.
Satoh-Nagasawa et al. (2006) reported that maize RAMOSA (RA) genes
are involved in the control of inflorescence architecture. Transcripts of RA3, a
functional TPP, accumulated mostly in young inflorescences at the stage of
axillary meristem primordia initiation. The expression of close homologues of
RA3 in sorghum and rice revealed a similar pattern of expression, implying
that the RA3 function might be conserved in other grasses.
Study of ra
mutants revealed that RA3 acts upstream of transcriptional regulator RA1 to
regulate
maize
inflorescence
meristem
identity
and
determinacy.
Consequently, RA3 may control inflorescence architecture either through
trehalose/trehalose-6-phosphate, or through RA3 itself (Satoh-Nagasawa et
al., 2006).
Trehalose biosynthesis also appears to be linked to storage carbohydrates
biosynthesis. External trehalose induced the expression of ApL3, which
encodes the large subunit of ADP-glucose pyrophosphorylase in Arabidopsis
(Wingler et al, 2000). Furthermore, trehalose-6-phosphate promoted the redox
activation of ADP-glucose pyrophosphorylase and subsequently starch
biosynthesis (Lunn et al. 2006). Additionally, trehalose induces expression
and
activity
of
Suc:Suc-1-fructosyltransferase
and
Suc:fructan-6-
fructosyltransferase, the enzymes involved in fructan biosynthesis, in barley,
although glucose or mannitol is required in the presence of trehalose to
induce fructan accumulation (Wagner et al., 1986; Müller et al., 2000).
5
Yet another function for trehalose biosynthesis is its control over
photosynthesis. Enhanced trehalose-6-phosphate levels following plant
transformation with E. coli TPS resulted in increased photosynthetic capacity
per unit leaf area due to increased Rubisco content (Pellny et al., 2004).
Trehalose Biosynthesis and Abiotic Stress
Since trehalose is an osmoprotectant (Müller et al, 1995) and membrane
and protein integrity protectant (Crowe et al, 1984) in other organisms,
different groups attempted to create stress-tolerant plants by introducing
microbial trehalose biosynthetic genes in tobacco (Holmström et al., 1996;
Romero et al., 1997; Goddijn 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 et al., 2005), potato (Goddijn et al., 1997) and
Arabidopsis (Karim et al., 2007; Miranda et al., 2007)(Table 1). First trials with
yeast TPS1 or E. coli OtsA were successful, in the sense that trehalose did
accumulate, although at a low level, and plants were stress tolerant. However,
the plants displayed abnormal phenotypes due to trehalose-6-phosphate
accumulation (Goddijn et al., 1997; Romero et al., 1997; Pilon-Smits et al.,
1998; Cortina et al., 2005). Nevertheless, subsequent studies overcame this
problem by using a microbial TPS-TPP fusion gene together with a stress
inducible promoter, directing the gene construct to chloroplast (Karim et al.,
2007, Garg et al. 2002; Jang et al., 2003, Miranda et al., 2007), or using a
different type of trehalose biosynthetic gene (trehalose phosphorylase) that
bypasses the trehalose-6-phosphate (Han et al., 2005). These improved
methods had increased trehalose content as a result, and stress tolerance
without the phenotypic alterations. Since the levels of trehalose accumulated
in these transgenic plants are still low and do not correlate well with the level
of tolerance, it is considered that trehalose may have a function other than
direct protective roles in plants.
Multiple studies have linked trehalose to stress tolerance in plants.
Fougere et al. (1991) reported that in alfalfa (Medicago sativa L.) upon salt
stress trehalose concentrations in roots and bacteroids increased significantly
(3.5 fold and 4.4 fold respectively), but they were still too low to account for an
6
osmoprotectant role for trehalose. Garcia et al. (1997) showed that trehalose
accumulated in small amounts in rice roots 3 days following salt stress. While
salt stress inhibited plant growth, external applications of low concentrations
(up to 5 mM) of trehalose reduced Na+ accumulation and growth inhibition,
and higher concentrations (10 mM) prevented chlorophyll loss in leaf blades
and preserved root integrity.
Transgenic plants overexpressing AtTPS1 (Avonce et al., 2004) did not
show any phenotypic alterations, with the exception of delayed flowering.
Mutant plants exhibited drought stress tolerance, but not salt tolerance and
glucose and ABA insensitive phenotypes. The altered regulation of genes
involved in ABA and glucose signaling during seedling vegetative growth may
account for the insensitivity, pointing out AtTPS1 and/or trehalose-6phosphate as a major player in gene regulation and signaling during seedling
development. Arabidopsis csp-1 mutant plants, with a point mutation in the
synthase domain of another Arabidopsis TPS, AtTPS6, apart from the altered
cell shape and plant architecture phenotype, is also drought tolerant (Chary et
al., 2008).
The recent study by Suzuki et al. (2008) revealed the involvement of yet
another TPS, AtTPS5, in plant stress tolerance. AtTPS5 interacts with MBF1c,
a transcriptional activator that is a key regulator of thermotolerance. AtTPS5
was briefly induced within 20 min of heat treatment, and tps5 mutants were
deficient in basal thermotolerance, although their acquired thermotolerance
was similar to that of the wild type plants. Trehalose accumulated in wild type
plants during heat stress and exogenously applied trehalose could rescue not
only tps5 mutants, but also mbf1c and mutants deficient in ethylene (ein2-1)
and salicylic acid (sid2) signaling, indicating trehalose biosynthesis pathway
interaction with plant hormone pathways.
Rice genome was found to contain nine TPSs and nine TPPs. Pramanik
and Imai (2005) and Shima et al. (2007) isolated and characterized two rice
TPPs (OsTPP1 and OsTPP2). Both rice TPPs were induced transiently by
cold, salt and drought stress, as well as exogenous ABA applications,
however OsTPP1 was induced generally earlier than OsTPP2 suggesting a
tight regulation of trehalose biosynthesis in response to multiple abiotic
stresses (Shima et al, 2007). Trehalose was also transiently induced following
7
chilling stress, its increase being correlated with the increase of OsTPP1
transcript and OsTPP1 activity (Pramanik and Imai, 2005). Moreover,
accumulation of trehalose in response to cold stress coincided with the phase
change of glucose and fructose levels (Pramanik and Imai, 2005).
Cluster analysis of microarray data obtained by Schluepmann et al. (2004)
following 100 mM trehalose treatment revealed a correlation between
trehalose-6-phosphate levels and the induction of several genes known to be
involved in plant responses to abiotic and biotic stresses. The abiotic
stresses-related genes induced by trehalose were ATPK19, a kinase that is
induced by salt and cold stress, as well as calcium and phosphorylation
signaling proteins.
Bae et al. (2005) studied the transcripts levels following 1-6 h of 30 mM
trehalose together with 1% sucrose treatment using DNA microarray analysis.
As opposed to the results of Schluepmann et al. (2004), in this study more of
genes involved with abiotic stress were repressed than induced by trehalose.
This difference may be due to different DNA microarray providers and/or
because a different concentration of trehalose was used. Among the
transcripts with altered level following trehalose treatment were those involved
in ethylene and jasmonate signaling. Several stress responsive genes were
up-regulated by trehalose treatment: anthocyanidin synthase, aldo-keto
reductase, S-adenosyl-L-methionine:carboxyl methyltransferase. However,
other transcripts, involved in plant defense and abiotic stress were repressed:
peroxidase-2 (PRXR2), basic endochitinase (PR), endo-1,3-endo--glucanase
(BGL1), lipoxygenase 2 (LOX2), chitinase-like protein 1 (CTL1), vacuolar ATP
synthase catalytic subunit A, ferritin (AtFER1), cytosolic glyceraldehyde-3phosphate dehydrogenase (GAPDHc), plasma membrane intrinsic protein
(PIP1C), cysteine proteinase (RD21A), and 5'-adenylsulphate reductase
(APR1).
Stress
Responsiveness
of
Biosynthesis Genes
8
Arabidopsis
Trehalose
We performed a comprehensive in silico analysis of Arabidopsis TPSs and
TPPs gene expression in response to a range of abiotic stresses (cold,
osmotic, salt, drought, genotoxic, oxidative, UV-B, wounding and heat stress)
using the software and microarray data offered by the Botany Array Resource
(Toufighi et al., 2005). For TPPs, we followed the nomenclature proposed by
Schluepmann et al, (2004).
Transcripts Responsive to Abiotic Stresses in Root
Among the genes induced by abiotic stresses in root, AtTPS2 and AtTPS3
appear to be induced by all abiotic stresses under study (Fig.2A). AtTPS3 is
induced early, within 30 min of treatment, and at a high level (16-48 fold),
whereas AtTPS2 is induced much later (12 h) and at lower levels (3-6 fold).
However, during salt stress, following a 20-fold temporary induction within 30
min of treatment, AtTPS3 is also transiently repressed after 3 h to the same
extent.
Following 12 h of cold stress, AtTPPF (At4G12430) is induced 8-fold
whereas AtTPPB is repressed 8-fold. AtTPPD (At1G35910) and AtTPPG
(At4G22590) are both induced by salt stress. The first is induced 32-fold after
6 h of treatment, and the second is induced 11-fold after 24 h of treatment.
AtTPS4, AtTPS6 and AtTPPD are induced to a lesser extent. AtTPS4 is
induced after 24 h of osmotic (8 fold), salt (4 fold), drought (4 fold), UV-B (5.3
fold), wounding (3 fold) and heat (4.3 fold) stresses. AtTPS6 is slightly
induced from 3 to 12 h of osmotic (3 fold) and salt (3.5 fold) stresses. AtTPPD
was induced by 6 h of cold (3 fold), osmotic (4.3 fold) and salt stresses (34.3
fold).
Transcripts Responsive to Abiotic Stresses in Shoots
During cold stress, AtTPS8 and AtTPPD are transiently induced early,
within 1 h (12-fold) and 3 h (6-fold) of treatment, respectively (Fig. 2B). Later
on, following 12 h of treatment AtTPS10, AtTPS11, AtTPPE (At2G22190),
AtTPPI (At5G10100), and AtTPPG are induced 6-28 fold, while AtTPS3 is
9
repressed 24 fold after 12 h, and AtTPPD is repressed 48 fold after 24 h.
Osmotic stress, transiently induced TPS3 5-fold after 6 h, while it repressed
AtTPPA 14-fold after 12 h. AtTPS3 is also regulated during salt stress in
tandem with AtTPPD, early on (1-3 h) induced 8-fold, then repressed 16-32
fold after 12-24 h. AtTPPI is weakly induced after 12 h while AtTPS11 is
weakly repressed after 24 h. AtTPPD, AtTPS8, AtTPS11 and AtTPPI are all
weakly induced within 3 h of drought treatment. AtTPS4 is weakly repressed
following 6 h of oxidative treatment. AtTPPD is induced up to 15-fold, whereas
AtTPS3, AtTPPB and AtTPPI are repressed during UV-B treatment. Following
wounding, AtTPS3 is induced in tandem with AtTPPI and AtTPPG. During
heat stress, AtTPP8 is induced, whereas AtTPS3, AtTPPD and AtTPPI are
repressed.
Most of the Arabidopsis trehalose biosynthetic genes showed changes in
transcript accumulation following plant exposure to the wide range of abiotic
stresses. However, most significant changes were observed with cold,
osmotic and salt stresses. For the majority of them, enzymatic activity is not
proven yet. AtTPS3 appear to be a major player in Arabidopsis responses to
abiotic stresses, both in root and shoot in Arabidopsis. Its tandem regulation
in shoot following wounding together with AtTPPI and AtTPPG and during salt
stress together with AtTPPD indicates a putative role for trehalose and/or
trehalose-6-phosphate in response to abiotic stresses. Furthermore, AtTPPD
is induced over 160-fold in response to infiltration with Pseudomonas syringae
(data not shown), indicating its involvement in response to biotic stress as well.
Concluding Remarks
The recent discovery of trehalose biosynthesis in higher plants had a high
impact on plant metabolism, physiology and development researches. Thus
far, most of the studies focused on trehalose-6-phosphate and its function on
plant development. However, transgenic researches with microbial trehalose
biosynthesis genes indicated that trehalose accumulation leads to stress
tolerance. It is becoming clearer that plant trehalose biosynthesis pathway is
tightly regulated by multiple stress cues. Our in silico study showed that many
10
of trehalose biosynthesis genes are differentially regulated by abiotic stresses.
This further suggests the importance of trehalose in stress tolerance.
Although the involvement of trehalose metabolism in stress tolerance is
indubitable, our understanding of how exactly interacts and acts upon stress
pathways is far from complete. Studies of individual trehalose biosynthesis
genes will help us to precisely assess their specific roles in the abiotic stress
context, and may enable us to develop new strategies to enhance abiotic
stress tolerance of crop plants.
References
Albini FM, Murelli C, Patritti G, Rovati M, Zienna P, Vita Finzi P (1994).
Low-molecular weight substances from the resurrection plant Sporobolus
stapfianus. Phytochemistry 37, 137-142.
Avonce N, Leyman B, Mascorro-Gallardo JO, Van Dijck P, Thevelein JM,
Iturriaga G (2004). The Arabidopsis trehalose-6-P synthase AtTPS1 gene
is a regulator of glucose, abscisic acid, and stress signaling. Plant Physiol.
136, 3649-3659.
Bae HH, Herman E, Bailey B, Bae HJ, Sicher R (2005). Exogenous
trehalose alters Arabidopsis transcripts involved in cell wall modification,
abiotic stress, nitrogen metabolism, and plant defense. Physiol. Plant. 125,
114-126.
Bell W, Sun W, Hohmann S, Wera S, Reinders A, De Virgilio C, Wiemken
A, Thevelein JM (1998). Composition and functional analysis of the
Saccharomyces cerevisiae trehalose synthase complex. J. Biol. Chem.
272, 33311-33319.
Bianchi G, Gamba A, Limiroli R, Pozzi N, Elster R, Salamini F, Bartels D
(1993). The unusual sugar composition in leaves of the resurrection plant
Myrothamnus flabellifolia. Physiol.Plant.87, 223–226.
Blazquez MA, Santos E, Flores CL, Martinez-Zapater JM, Salinas J,
Gancedo C (1998). Isolation and molecular characterization of the
Arabidopsis TPS1 gene, encoding trehalose-6-phosphate synthase. Plant
J. 13, 685-689.
11
Chary SN, Hicks GR, Choi YG, Carter D, Raikhel NV (2008). Trehalose-6phosphate
synthase/phosphatase
regulates
cell
shape
and
plant
architecture in Arabidopsis. Plant. Physiol. 146, 97-107.
Cortina C, Culiáñez-Macià FA (2005). Tomato abiotic stress enhanced
tolerance by trehalose biosynthesis. Plant Science 169, 75-82.
Crowe JH, Crowe LM, Chapman D (1984). Preservation of membranes in
anhydrobiotic organisms: the role of trehalose. Science. 223, 701–703.
Drennan PM, Smith MT, Goldsworthy D, Van Staden (1993). The
occurrence of trehalose in the leaves of the desiccation-tolerant
angiosperm Myrothamnus flabellifolius Welw. Plant Physiol. 142, 493–496.
Eastmond PJ, van Dijken AJ, Spielman M, Kerr A, Tissier AF, Dickinson
HG, Jones JD, Smeekens SC, Graham IA (2002). Trehalose-6phosphate synthase 1, which catalyses the first step in trehalose synthesis,
is essential for Arabidopsis embryo maturation. Plant J. 29, 225-235.
Elbein AD, Pan YT, Pastuszak I, Carroll D (2003). New insights on
trehalose: a multifunctional molecule. Glycobiology 13, 17R-27R.
Fougere F, Le Rudulier D, Streeter JG (1991). Effects of salt stress on
amino acid, organic acid, and
carbohydrate composition of roots,
bacteroids, and cytosol of alfalfa (Medicago sativa L.). Plant Physiol. 96,
1228-1236.
Garcia B, de Almeida Engler J, Iyer S, Gerats T, Van Montagu M, Caplan
AB (1997). Effects of osmoprotectants upon NaCl stress in rice. Plant
Physiol. 115, 159-169.
Garg AK, Kim JK, Owens TG, Ranwala AP, Choi YD, Kochian LV, Wu RJ
(2002). Trehalose accumulation in rice plants confers high tolerance levels
to different abiotic stresses. Proc. Natl. Acad. Sci. U.S.A. 99, 15898-15903.
Goddijn OJ, Verwoerd TC, Voogd E, Krutwagen RW, de Graaf PT, van
Dun K, Poels J, Ponstein AS, Damm B, Pen J (1997). Inhibition of
trehalase activity enhances trehalose accumulation in transgenic plants.
Plant Physiol. 113, 181-190.
Han SE, Park SR, Kwon HB, Yi BY, Lee GB, Byun MO (2005). Genetic
engineering of drought-resistant tobacco plants by introducing the
trehalose phosphorylase (TP) gene from Pleurotus sajor-caju. Plant Cell,
Tissue and Organ Culture 82, 151–158.
12
Holmström K-O, Mäntylä E, Welin B, Mandal A, Palva ET, Tunnela OE,
Londesborough J (1996). Drought tolerance in tobacco. Nature 379,
683–684.
Hottiger T, Boller T, Wiemken A (1987). Rapid changes of heat and
desiccation tolerance correlated with changes of trehalose content in
Saccharomyces cerevisiae cells subjected to temperature shifts. FEBS
LETTERS 220, 113-115.
Hounsa CG, Brandt EV, Thevelein J, Hohmann S, Prior BA (1998). Role of
trehalose in survival of Saccharomyces cerevisiae under osmotic stress.
Microbiology 144, 671-680.
Jang IC, Oh SJ, Seo JS, Choi WB, Song SI, Kim CH, Kim YS, Seo HS,
Choi YD, Nahm BH, Kim JK (2003). Expression of a bifunctional fusion of
the Escherichia coli genes for trehalose-6-phosphate synthase and
trehalose-6-phosphate phosphatase in transgenic rice plants increases
trehalose accumulation and abiotic stress tolerance without stunting
growth. Plant Physiol. 131. 516-524.
Karim S, Aronsson H, Ericson H, Pirhonen M, Leyman B, Welin B,
Mäntylä E, Palva E, Dijck P, Holmström K-O (2007). Improved drought
tolerance without undesired side effects in transgenic plants producing
trehalose. Plant Mol. Biol. 64, 371-386.
Lee SB, Kwon HB, Kwon SJ, Park SC, Jeong MJ, Han SE, Byun MO,
Daniell H (2003). Accumulation of trehalose within transgenic chloroplasts
confers drought tolerance. Molecular Breeding 11, 1-13.
Lunn Je, Feil R, Hendriks JHM, Gibon Y, Morcuende R, Osuna D,
Scheible W-R, Carillo P, Hajirezaei M-R, Stitt M (2006). Sugar-induced
increases in trehalose 6-phosphate are correlated with redoxactivation of
ADPglucose pyrophosphorylase and higher rates of starchsynthesis in
Arabidopsis thaliana. Biochem J. 397, 139-148.
Miranda JA, Avonce N, Suárez R, Thevelein JM, Van Dijck P, Iturriaga G
(2007). A bifunctional TPS–TPP enzyme from yeast confers toleranceto
multiple and extreme abiotic-stress conditions in transgenic Arabidopsis.
Planta 226,1411–1421.
Müller J, Boller T, Wiemken A (1995). Trehalose and trehalase in plants:
recent developments. Plant Science 112, 1-9.
13
Müller J, Aeschbacher RA, Sprenger N, Boller T, Wiemken A (2000).
Disaccharide-mediated regulation of sucrose:fructan-6-fructosyltransferase,
a key enzyme of fructan synthesis in barley leaves. Plant Physiol. 123,
265-274.
Pellny TK, Ghannoum O, Conroy JP, Schluepmann H, Smeekens S,
Andralojc J, Krause K-P, Goddijn O, Paul MJ (2004). Genetic
modification of photosynthesis with E. coli genes for trehalose synthesis.
Plant Biotechnol. J. 2, 71-82.
Pilon-Smits EAH, Terry N, Sears T, Kim H, Zayed A, Hwang S, van Dun K,
Voogd E, Verwoerd TC, Krutwagen RWHH, Goddijn OJM (1998).
Trehalose-producing transgenic tobacco plants show improved growth
performance under drought stress. J. of Plant Physiol. 152, 525–532.
Pramanik MHR, Imai R (2005). Functional identification of a trehalose-6phosphatase gene that is involved in transient induction of trehalose
biosynthesis during chilling stress in rice. Plant Mol. Biol. 58, 751-762.
Romero C, Belles JM, Vaya JL, Serrano R, Culianez-Macia FA (1997).
Expression of the yeast trehalose-6-phosphate synthase gene in
transgenic tobacco plants: Pleiotropic phenotypes include drought
tolerance. Planta 201, 293-297.
Satoh-Nagasawa N, Nagasawa N, Malcomber S, Sakai H, Jackson D
(2006). A trehalose metabolic enzyme controls inflorescence architecture
in maize, Nature 441, 227–230.
Schluepmann H, Pellny T, van Dijken A, Smeekens S, Paul M (2003).
Trehalose 6-phosphate is indispensable for carbohydrate utilization and
growth in Arabidopsis thaliana. PNAS 100, 6849–6854.
Schluepmann H, van Dijken A, Aghdasi M, Wobbes B, Paul M, Smeekens
S (2004). Trehalose mediated growth inhibition of arabidopsis seedlings is
due to trehalose-6-phosphate accumulation. Plant Physiol. 135, 879–890.
Shima S, Matsui H, Tahara S, Imai R (2007). Biochemical characterization of
rice trehalose-6-phosphate phosphatases supports distinctive functions of
these plant enzymes. FEBS J. 274, 1192-1201.
Styrvold OB, Strom AR (1991). Synthesis, accumulation, and excretion of
trehalose in osmotically stressed Escherichia coli K-12 strains: influence of
amber suppressors and function of the periplasmic trehalase. Journal of
14
Bacteriology 173, 1187-1192.
Suzuki N, Bajad S, Shuman J, Shulaev V, Mittler R (2008). The
transcriptional co-activator mbf1c is a key regulator of thermotolerance in
Arabidopsis thaliana. J. Biol. Chem. DOI: 10.1074/jbc.M709187200.
Toufighi K, Brady SM, Austin R, Ly E, Provart NJ (2005). The Botany Array
Resource: e-Northerns, Expression Angling, and promoter analyses. Plant
J. 43, 153–163.
van Dijken AJH, Schluepmann H, Smeekens SCM (2004). Arabidopsis
trehalose-6-phosphate synthase 1 is essential for normal vegetative
growth and transition to flowering. Plant Physiol. 135, 969–977.
Vogel G, Aeschbacher RA, ller JM, Boller T, Wiemken A (1998).
Trehalose-6-phosphate
phosphatases
from
Arabidopsis
thaliana:
identification by functional complementation of the yeast tps2 mutant.
Plant J. 13, 673–683.
Vogel G, Fiehn O, Jean-Richard-Dit-Bressel L, Boller T, Wiemken A,
Aeschbacher
RA,
Wingler
A (2001). Trehalose metabolism
in
Arabidopsis: occurrence of trehalose and molecular cloning and
characterization of trehalose-6-phosphate synthase homologues. J. Exp.
Bot. 52,1817-1826.
Wagner W, Wiemken A, Matile P (1986). Regulation of fructan metabolism
in leaves of barley (Hordeum vulgare L. cv Gerbel). Plant Physiol. 81, 444447.
Wingler A, Fritzius T, Wiemken A, Boller T, Aeschbacher RA (2000).
Trehalose induces the ADP-glucose pyrophosphorylase gene, ApL3, and
starch synthesis in Arabidopsis. Plant Physiol. 124, 105-114.
15
Figure Legends
Fig. 1. Trehalose biosynthesis pathway
Fig. 2. Stress responsiveness of trehalose biosynthesis genes under
different abiotic stresses in root (A) and shoot (B). For cold, osmotic, salt,
genotoxic and oxidative stress treatment tissue was harvested at 0.5, 1, 3, 6,
12 and 24 h. For drought, UV-B and wounding treatment tissue was harvested
at 0.25, 0.5, 1, 3, 6, 12 and 24 h. For heat treatment tissue was harvested at
0.25, 0.5, 1, 3, 4, 6, 12 and 24 h.
16
Species
transformed
Tobacco
Tobacco
Table 1. Stress tolerant transgenic plants expressing microbial trehalose biosynthetic genes.
Promoter
Microbial genes
Tolerance
Changes in phenotype
Rubisco
35S
TPS1
360-440 μg g FW (NS)
-1
6.3 μmol g FW (NS)**
-1
~8 μg g FW (NS, DS)
TPS1-TPS2
TPS1
No changes
No changes
~16 μg g FW (NS, DS)
-1
1-2 μg g FW (NS, DS)
TPS1-TPS2
TPS1
No changes
No changes
~4 μg g FW (NS, DS)
b
N.A.
No changes
~48 μg g FW (NS)
-1
~508 μg g FW (DS)
-1
~55 μg g FW (DS)
-1
~163 μg g FW (NS)
-1
310-1036 μg g FW (NS)
otsA
Drought
b
35S
Drought
16SrRNA
35S
Rubisco
TPS1
TP
TPS1
Drought
Drought
Drought
Tobacco
Tobacco
Tobacco
Tobacco
AtRAB18 (drought
inducible)
Arabidopsis
Rubisco*
Rice
ABRC1-rice actin1
(ABA-inducible)
rice rbcS*
otsA-otsB
Rice
Ubiquitin
otsA-otsB
Tomato
35S
TPS1
Arabidopsis
rd29A (stress
inducible)
TPS1-TPS2
N.A.
Drought
Drought, Salt,
Cold
No changes
35S
a
800-3200 μg g DW (NS)
No changes
Aberrant root growth, lanced shaped leaves, stunted
plants
Same as above, but not as pronounced, bleached
interveinal tissue
No changes
b
N.A.
Loss of apical dominance, stunted growth, lancet shaped
leaves, partial sterility
No changes
No changes
No changes
otsA
otsA-otsB
TPS1
Patatin
-1
Drought
otsA-otsB
Potato
Trehalose levels
Drought, Salt,
Cold
Drought, Salt
Drought, Cold,
Salt, Heat
No changes
Thick shoots with short internodes, rigid dark green
leaves, aberrant root development
No changes
Smaller dark green leaves, partial sterility, glucose
insensitive
b
Trehalose-6phosphate
a
N.D.
-1
20-110 μg g FW (NS)
N.D.
a
-1
30-60 μg g FW (NS)
-1
3-20 μg g FW (NS)
a
N.A.
-1
<170 μg g FW (NS)
-1
a
N.D.
a
N.D.
b
N.A.
a
N.D.
a
N.D.
b
N.A.
Reference
Holmström et al.,
1996
Goddijn et al., 1997
Pilon-Smits et al.,
1998
Goddijn et al., 1997
Romero et al., 1997
Lee et al., 2003
Han et al., 2005
Karim et al., 2007
-1
-1
-1
-1
150 μg g FW (NS)
-1
8.2-16.7 μg g FW (NS)
b
Garg et al., 2002
b
Jang et al., 2003
b
Cortina et al., 2005
b
Miranda et al., 2007
N.A.
N.A.
N.A.
N.A.
-1
8.5-38.4 μg g FW (NS)
N.D. Not determined; N.A. Not available; *with added transit peptide for chloroplast targeting; NS not stressed; DS drought stressed; TPS1: Saccharomyces cerevisiae TPS; TPS2:
Saccharomyces cerevisiae TPP; OtsA: Escherichia coli TPS; OtsB: Escherichia coli TPP; TP: Pleurotus sajor-caju trehalose phosphorylase.
Cold
Control
A.
B.
Osmotic
Treatment Control
Treatment
Salt
Control
Treatment
Drought
Control
Treatment
Genotoxic
Control
Oxidative
Treatment Control
Treatment
UV-B
Control
Treatment
Wounding
Control
Treatment
Heat
Control
Treatment
At1g16980 ATTPS2
At1g17000 ATTPS3
At1g22210 ATTPPC
At1g60140 ATTPS10
At1g23870 ATTPS9
At1g70290 ATTPS8
At2g18700 ATTPS11
At4g27550 ATTPS4
At1g68020 ATTPS6
At1g35910 ATTPPD
At5g65140 ATTPPJ
At1g06410 ATTPS7
At4g22590 ATTPPG
At5g51460 ATTPPA
At2g22190 ATTPPE
At4g12430 ATTPPF
At5g10100 ATTPPI
At1g78580 ATTPS1
At1g78090 ATTPPB
At4g39770 ATTPPH
At4g17770 ATTPS5
At1g17000 ATTPS3
At1g22210 ATTPPC
At4g17770 ATTPS5
At1g35910 ATTPPD
At1g16980 ATTPS2
At4g12430 ATTPPF
At5g65140 ATTPPJ
At1g23870 ATTPS9
At1g70290 ATTPS8
At2g18700 ATTPS11
At4g27550 ATTPS4
At1g06410 ATTPS7
At2g22190 ATTPPE
At1g60140 ATTPS10
At5g10100 ATTPPI
At4g22590 ATTPPG
At1g78090 ATTPPB
At5g51460 ATTPPA
At1g68020 ATTPS6
At1g78580 ATTPS1
At4g39770 ATTPPH