tmpEB87 TMP
tmpEB87 TMP
tmpEB87 TMP
Trehalose is widely distributed in nature, from bacteria to higher organisms, both plants and mammals.
It is a non-reducing disaccharide composed of two
glucose units linked by an o~(1 ~ 1) bond, and is synthesized by the sequential action of trehalose-6-phosphate synthase (TPS; EC 2.4.1.15) and trehalose-6phosphate phosphatase (TPP; EC 3.1.3.12) (Cabib
and Leloir, 1958; E[bein, 1974). Because of several
physical properties, e.g., high hydrophilicity, chemical inertness, and non-hygroscopic glass formation,
trehalose works as a protective molecule under various abiotic stresses, especially desiccation and high
temperatures, in organisms such as bacteria, yeast,
nematodes, fungi, and primitive plants (Thevelein,
1984; Larsen et al., 1987; Crowe et al., 1992, 1998;
Bianchi et al., 1993; Strom and Kaasen, 1993; de Virgi[io et al., 1994; Arg0elles, 2000). In higher plants,
however, its occurrence and role are dubious. Nevertheless, in the presence of validamycin A, an inhibitor of trehalase, small quantities of trehalose have
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Dehydration was induced by 1) withholding irrigation, 2) air-drying, or 3) immersing the roots of whole
plants in a Hoagland solution containing 10% (w/v)
PEG-6000. Polyethylene glycol was chosen for its
chemical inertness (Michel, 1970). Salt stress was
applied by supplementing 250 mM NaCI twice a
week during the irrigation of 4-week-old plants.
RNA Gel Blot Analysis
Leaf water potential was measured psychrometrically with an HR-33T Dewpoint Microvo[tmeter (Wescor, USA) in a C-52 sample chamber at room
temperature (Boyer and Knipling, 1965). Discs (0.7cm diam.) were placed in the chamber and allowed
to equilibrate for 15 min, then cooled for 45 s before
the voltage was recorded. Water potential was calculated from this voltage value and corrected for temperature.
Measurement of Photosynthesis
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have an approximately equal Chl content when measured by a Ch[ meter (SPAD-502; Minolta, Japan), but
was then normalized to the control.
Germination Test
Tobacco seeds were surface-sterilized and imbibed
for 2 d in the refrigerator for stratification. To measure salinity tolerance, 100 seeds each were placed
on plates containing three layers of filter paper soaked
with 10 mL of various concentrations of NaCI solutions. Germination was defined as the emergence of
the radicle from the seed coat.
RESULTS
Generation of Trehalose-Producing Transgenic
Tobacco Plants
Through leaf-disc transformation, we generated
tobacco plants over-expressing the E. coil TP5 gene,
otsA. Transgenic plants were first identified by PCR,
then confirmed by northern hybridization. Levels of
otsA expression varied depending on the line. The
amount of trehalose detected in transgenic leaves,
Figure1. Northern hybridization of otsA expression,and trehaloseamounts in leavesof transgenictobacco plants. Numbers indicatetransgeniclines. Data are mean values -- SEfor
4 measurements.NT, non-transformants;ND, not detected.
To re-evaluate the effectiveness of trehalose production in improving tolerance against water stress,
we placed our non-transformed and transgenic
tobacco plants under various water-deficit conditions. Non-transformant leaves showed apparent signs
of wilting beginning 3 d after irrigation was first withheld, while leaves of plants from Lines 6 and 8
remained turgid during that period. Leaves from Lines
4 and 5 appeared less turgid but without clear wilting. This difference became more prominent after 5
d without watering, with leaves from transgenic Lines
6 and 8 maintaining their turgidity while those of the
non-transformants, and even those of Lines 4 and 5,
showing evident wilting (Fig. 2A).
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Figure 2. Increased tolerance to dehydration in trehalose-producing plants. Photographs of 4-week-old tobacco plants after 5 d
without water supply (A) and after PEG treatment for 3 h (B). Note the maintenance of leaf turgidity in transgenic plant from
Line 6 compared with withered leaves in non4ransformants (NT). Decrease in fresh weights of detached leaves (C) and whole
plants (D, E) after dehydration treatment. Water deficit was induced by air-clrying (C, D) or by immersing roots of whole plants
in 10% (w/v) PEG solution (E). Data are mean values +_ SE (n - 5).
The response was similar with our PEG treatment.
When dehydration was induced by 10% (w/v) PEG,
the trehalose-producing tobacco plants maintained
leaf turgidity better than the non-transformants. After
3 h of treatment, leaves from Lines 6 and 8 remained
firm while those of the non-transformants were
severely withered (Fig. 2B). Leaves from Lines 4 and 5
showed an intermediate response despite containing
at least as much trehalose as that measured from
Lines 6 and 8. Finally, when detached leaves were airdried, those of the non-transformants wilted faster
than the transgenic tissues. A similar trend was
observed with the whole plants (data not shown).
Interestingly, the degree of tolerance against dehydration was more related to the level of otsA expression than to trehalose content. This suggests that T6P,
not trehalose, acts as the protective molecule. Furthermore, when whole plantg were re-hydrated after
12 h of air-drying or 24 h of PEG treatment, all the
non-transformants failed to recover and eventually
died whereas most of the transgenic plants revived
and fully regained their turgidity (data not shown).
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Jun et al.
Table l. Changes in Pmax (retool m-2 s 1) of 02 evolution and maximal photochemical efficiency of PSII (Fv/Fm) after
dehydration, which was induced by PEG treatment for 2 h.
Treatment
Pmax
NT
#4
Fv/Fm
#6
NT
#4
#6
Control
8.41 + 1.51
7.55 _+0.68
10.07 _+1.01
0.81
0.80
0.81
Dehydration
3.25 _+0.29
2.55 + 0.34
4.12 + 0.57
0.81
0.81
0.82
*Data presented are mean values_+SDfor 3 to 5 measurements. Standard deviations for Fv/Fm are not shown, but are lessthan
2%.
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Figure 4. Increased tolerance to salinity in transgenic plants. A) Photographs are of non-transformant (NT) and transgenic
tobacco plants grown with or without the supplementation of 250 mM NaCI. Top row shows tobacco plants grown without
salinity stress; bottom row, those under salinity stressfor 20 d. Note the smaller size of plants after sustainedgrowth under salinity, although extensive chlorosis and necrosisoccurred only in leavesof non-transformants. B) Changes in Chl content in leaves
after sustained growth under 250mM NaCI. Chl content was measured with Minolta Chl meter. Data are mean values _+ SE (n
= 3).
tial, thereby proving that neither of them functions as
an osmo[yte.
NaCI treatment, we monitored changes in Chl contents in leaves with no apparent damage or necrosis.
Chlorophyll content in the leaves of non-transformants was maintained for 5 d after the salt treatment, but sharply declined by about 40% on Day 15
despite there being no apparent damage or chlorosis
(Fig. 4B). In contrast, no significant reduction in Chl
content was observed in the transgenic plants of Lines
6 and 8 (Fig. 4B). However, those that expressed low
TPS levels (Lines 4 and 5) were less efficient in maintaining their leaf Chl content (data not shown). Thus,
trehalose or T6P appears to protect the thylakoid
membranes by delaying the chlorosis normally
induced by high salinity.
As additional parameters for thylakoidal integrity,
we monitored initial Chl fluorescence (Fo) and the
maximal photochemical efficiency of PSII (Fv/Fm),
both of which are often used to assess PSII function
(Renger and Schreiber, 1986). After salt treatment for
up to 20 d, the Fv/Fm and Fo values did not change
significantly for either non-transformants or transgenic plants, indicating that NaCI causes no substantial decline in PSII function (data not shown).
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Jun et al.
DISCUSSION
Morphological Features of Trehalose-Producing
Tobacco Plants
The identification of two functional homologs of
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ACKNOWLEDGEMENTS
We are grateful to Prof. Chong Wook Park at Seoul
National University for the use of HPLC equipment.
This work was supported by a grant from the Korea
Research Foundation (KRF2001-015-DP0481 ) and, in
part, by research funding from the Catholic University
of Korea, Pucheon (2004). We also appreciate the
financial support from the BK21 Program.
Received October 7, 2005; accepted November 21, 2005.
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