Plant Cell Physiol. 42(2): 245–249 (2001)
JSPP © 2001
Improved Salt Tolerance of Transgenic Tobacco Expressing Apoplastic YeastDerived Invertase
Eiichi Fukushima 1, Yuuto Arata 1, Tsuyoshi Endo 1, 3, Uwe Sonnewald 2 and Fumihiko Sato 1, 3, 4
1
2
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502 Japan
Institute for Plant Genetics and Crop Plant Research, Corrensstrasse 3, D-06466 Gatersleben, Germany
;
improve the stress tolerance of plants (Tarczynski et al. 1993,
Hayashi et al. 1997, Sheveleva et al. 1998).
Very few reports, however, have dealt with the in vivo
function of sucrose metabolism and its effects on photosynthesis under water stress, whereas stress causes an increase in the
sucrose concentration in water-stressed plants, and sucrose synthase and sucrose phosphatase genes are up-regulated (Ingram
and Bartels 1996). Furthermore, recent QTL investigation suggested that some invertase activities are closely related to water
stress responses (Pelleschi et al. 1999). We therefore investigated the salt tolerance of transgenic tobacco plants that
express yeast invertase in the apoplast (Apo-Inv) or vacuole
(Vac-Inv) and that accumulate more sucrose and hexoses in the
leaves in light than does wild-type tobacco (von Schaewen et
al. 1990, Sonnewald et al. 1991, Heineke et al. 1994). In these
transformants, sugar transportation to the sink organ has been
inhibited by ectopically expressed yeast invertase in the apoplastic space or vacuole, resulting in accumulation of sucrose
in the cytoplasm of the source organ. The sucrose remained in
cytoplasm of mesophyll cells would be in part converted to glucose and fructose. The sucrose contents in Apo-Inv and Vac-Inv
tobacco are reported to be respectively 15 and 5 times that in
wild-type tobacco, whereas glucose contents are 50 and 90
times greater, and fructose contents 61 and 25 times greater
(Sonnewald et al. 1991, Buessis et al. 1997). It is expected that
high sugar contents in these transformants may protect photosynthetic apparatus under salt stress.
We investigated the salt tolerance of transgenic
tobacco, in which yeast invertase is expressed in the apoplastic (Apo-Inv) spaces. Whereas photosynthetic activities
in wild-type tobacco in light were inhibited under salt
stress, transgenic Apo-Inv tobacco maintained constant
photosynthetic activities. The physical appearance of plants
under salt stress also indicates that yeast invertase expression in the apoplastic space is beneficial for inducing salt
tolerance. Apo-Inv tobacco had a much higher osmotic
pressure increase in the cell sap than did wild-type tobacco
under this type of stress. The physiological importance of
sucrose metabolism under salt stress is discussed.
Key words: Invertase — Nicotiana tabacum — Photosynthesis
— Salt stress — Sucrose.
Abbreviations: Apo-Inv, apoplastic invertase; Apo-Inv tobacco,
transgenic tobacco expressing yeast invertase in apoplastic space; Fm,
maximum yield of Chl fluorescence; Fo, minimum yield of Chl fluorescence; Fv, variable fluorescence Fm-Fo.
To clarify stress tolerance in plants and to improve plant
productivity, various investigations have been made of gene
expression in plants under water stresses (e.g. drought, salt,
freezing), the most severe limiting factors of plant growth and
yields. Under stress conditions, plants accumulate a set of proteins, such as LEAs, and low molecular weight compounds, socalled ‘compatible solutes’ (see review, Bohnert et al. 1995,
Ingram and Bartels 1996). Compatible solutes are compounds
that accumulate in stress-tolerant plants under water stresses.
They are water soluble and do not disturb plant cell metabolism. Several compatible solutes are known: sugars, polyols,
amino acids, and amino acid derivatives. These are involved in
osmoregulation, removal of free radicals (Smirnoff and
Cumbes 1989, Shen et al. 1997), and stabilization of the
hydrated structure of proteins to maintain membrane integrity
and protein stability (Inchroensakdi et al. 1986, Papageorgiou
et al. 1991, Murata et al. 1992). Introduction of genes for biosynthesis of compatible solutes has been used successfully to
3
4
Plant culture conditions and salt stress treatment
Transgenic tobacco (Nicotiana tabacum L. cv. Samsun
NN) plants, which express yeast invertase in apoplastic and
vacuolar spaces, as well as wild-type tobacco seedlings were
maintained aseptically on agar media containing a half concentration of Linsmaier-Skoog (LS) inorganic medium (Linsmaier
and Skoog 1965) and cultured for 2 weeks in hydroponic
medium with a quarter concentration of LS at 25C under continuous light (100 mol quanta m–2 s–1) with aeration. Salt tolerance was evaluated under moderate light intensity (200 mol
quanta m–2 s–1) in hydroponic medium with a quarter concentration of LS with or without 300 mM NaCl. The plants were
Present address: Division of Integrated Life Sciences, Graduate School of Biostudies, Kyoto University, Kyoto, 606-8502 Japan.
Corresponding author: E-mail, [email protected]; Fax, +81-75-753-6398.
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Apoplastic invertase and salt tolerance
Fig. 1 Effects of salt stress on various Chl fluorescence parameters in Apo-Inv (circle and triangle) and wild-type (square and diamond) tobacco
plants. Plants were treated with (square, circle) or without (diamond, triangle) 300 mM NaCl in light (200 mol quanta m–2 s–1). Vertical bars are
standard errors for three replicants. (A) quantum yield of PSII (II, F/Fm’), (B) photoinhibition of PSII (Fv/Fm).
pre-adapted to the continuos light illumination at 200 mol
quanta m–2 s–1 for at least 1 week before salt stress treatment.
Photosynthetic activities were measured with a PAM2000 Chl
fluorometer (Heinz Walz, Effeltrich, Germany). The estimated
activities were the quantum yield of photosystem II (II = F /
Fm’ = (Fm’ – F) / Fm’) under white actinic light of 200 mol
quanta m–2 s–1 (Genty et al. 1989); potential activity of photosystem II (Fv/Fm, Fv = Fm – Fo) after 15 min of dark adaptation.
Photosynthetic activities in Apo-Inv tobacco plants under salt
stress
Photosynthetic activity in transgenic tobacco plants that
expressed yeast invertase was evaluated under salt stress and
compared with that in wild-type plants (Fig. 1A). In the latter,
the quantum yield of PSII (II), indicated by the Chl fluorescence parameter F/Fm’, was markedly decreased by salt
stress, whereas the II of Apo-Inv tobacco remained as high as
that of the non-stressed control. In contrast, under salt stress the
II of Vac-Inv tobacco decreased more sharply than that of
wild-type tobacco (data not shown). The reason for the different photosynthetic activity responses of Apo-Inv and Vac-Inv
tobacco is not clear, but this result suggests that not only
sucrose accumulation, but the mechanism of accumulation
(localization of invertase) is important for the growth performance of plants as indicated by the more severe defect in Vac-Inv
tobacco than Apo-Inv tobacco even under normal growth conditions. In the experiments described below, we used Apo-Inv
tobacco to further characterize the function of sucrose metabolism in vivo.
The degree of photoinhibition under salt stress
Apo-Inv tobacco showed tolerance to salt stress and had a
higher quantum yield of PSII than wild-type tobacco under the
same stress (Fig. 1A). To confirm the tolerance under salt
stress, we measured the degree of photoinhibition in wild-type
and Apo-Inv plants under salt stress. The degree of photoinhibition (degradation of PSII core protein D1) can be estimated
from the Chl fluorescence parameter Fv/Fm (Schreiber and
Bilger 1993). Salt stress clearly produced different degrees of
photoinhibition in the wild-type and Apo-Inv plants (Fig. 1B).
Fig. 2 Physical appearance of Apo-Inv (left) and wild-type (right)
tobacco plants under salt stress. After their lower leaves were
removed, the plants were treated with 300 mM NaCl in light
(200 mol quanta m–2 s–1) for 122 h.
Apoplastic invertase and salt tolerance
247
absorption of water from medium (0.3 M NaCl; ca. 600 mOsm
kg–1).
Fig. 3 Effects of salt stress on osmotic pressure in the leaf. Plants
were treated with 300 mM NaCl in light (200 mmol quanta m–2 s–1) for
24 h. Closed bars are the wild-type and hatched ones Apo-Inv tobacco
leaves. Horizontal bars standard errors for three replicants.
Wild-type tobacco showed marked photoinhibition due to salt
stress, whereas no apparent photoinhibition occurred in ApoInv tobacco. This difference is attributable in part to the difference in the reduction level of QA (data not shown), because
acceptor-side photoinhibition is induced by the reduction of QA
(Barber 1994). Alternatively, the reaction center protein may be
protected from photo-degradation in chloroplasts of Apo-Inv
tobacco.
Figure 2 shows the physical appearance of Apo-Inv and
wild-type tobacco plants stressed in the presence of 300 mM
NaCl for 122 h. The Apo-Inv plants appears to be much healthier than the wild-type one because there is less photodamage.
The more turgid appearance of the Apo-Inv plant may be due
to higher osmotic pressure in the cells (see below; Fig. 3).
Osmotic pressure
The osmotic pressure in the cell varies with the plant species, developmental stage, and environment. The generation of
osmotic pressure is essential for maintaining turgor pressure for
the absorption of water and for keeping the cell shape under
salt/water stress. Because Apo-Inv tobacco plants had a better
physical appearance under salt stress, the osmotic pressure in
leaf cells with and without salt treatment was determined.
Fresh leaves were frozen in liquid nitrogen and then thawed in
warm water. After centrifugation of the mixture at 1,000g for
5 min, the leaf pellets obtained were squeezed with a teaspoon,
and the cell sap was collected. The osmotic pressure of the sap
was measured with an osmometer (Shimadzu, model OSM-1,
Kyoto, Japan).
Osmotic pressures in both the wild-type and Apo-Inv
tobacco were similar under the non-stress condition, but salt
stress at 0.3 M NaCl for 24 h clearly increased the osmotic
pressure in Apo-Inv tobacco, from ca. 400 to ca. 1,050 mOsm
kg–1, whereas in wild-type tobacco the increase was only from
ca. 300 to ca. 600 mOsm kg–1 (Fig. 3). This suggests that ApoInv tobacco maintained much higher turgor pressure for the
Concentration of sucrose and hexoses in chloroplasts
Figures 1 and 2 patently show that the photosynthetic
apparatuses in Apo-Inv tobacco were well protected under salt
stress. What then is the agent that protects photosynthetic activity? It is generally believed that sucrose does not appear inside
the plastids (Avigad and Dey 1997), but there are reports of the
presence of sucrose inside chloroplasts (Heineke et al. 1994,
Lohaus et al. 1999) and that an increase in the sucrose concentration in the leaves of transgenic tobacco with yeast-derived
invertase increases the sucrose concentration in the chloroplasts (Heineke et al. 1994). However, there have been no
reports on the sucrose concentration in chloroplasts under salt
stress. We therefore measured the sucrose concentration in
chloroplasts of both the wild-type and Apo-Inv plants under
salt stress.
To prepare intact chloroplasts, leaves were homogenized
at 4C in buffer A (a concentration of sorbitol isotonic to the
osmotic pressure in the leaves with 50 mM of MES, pH 6.1,
and a teaspoon of ascorbate) in a Waring blender for 3 s at
maximum speed. The homogenate was filtered through four
layers of gauze, and chloroplasts obtained were quickly centrifuged at 2,000g for 30 s and then suspended in buffer B (isotonic concentration of sorbitol and 50 mM of HEPES, pH 7.6).
Intact chloroplasts were separated on 40% and 75% Percoll
buffer layers by centrifugation at 2,000g for 5 min and recovered between the 40% and 75% Percoll surface. To evaluate
chloroplast intactness, oxygen evolution (10–20 (mg Chl) ml–1)
in buffer B supplemented with 1 M nigericin and 0.5 mM
potassium ferricyanide was measured with a Clark type oxygen electrode (Hansatech, U.K.) under 2,500 mol quanta m–2
s–1 light. Oxygen evolution in the broken chloroplasts was
measured as described above, and intactness calculated from
the difference in activities. Chloroplasts were suspended in
80% acetone at 4C to measure Chl concentration. Arnon’s
equation (Arnon 1949) was used to calculate the Chl concentration from the absorbances of the extracts.
To measure the sugar content, intact chloroplasts were frozen at –20C then thawed and thoroughly broken by sonication
(max power, 5 s). Proteins were removed from the chloroplast
suspension by the Carrez reagent. The chloroplast suspension
(600 ml) was mixed vigorously with 50 ml of Carrez I (ferrocyanide, final concentration, 4.25 mM) and 50 ml of Carrez II
(zinc sulfate, final concentration, 12.5 mM). After centrifugation of the mixture at 20,000g for 15 min, the supernatant was
filtered through a MILLIPORE GV membrane and desalted for
1 h with Amberlite IRN-150L resin (Pharmacia, Plusone). The
concentrations of sucrose and hexoses in the supernatant were
measured with an enzymatic assay kit (Roche Diagnostics
K.K., TC Sucrose/D-Glucose/D-Fructose) according to the manufacturer’s protocol.
Chloroplast volume in wild-type tobacco (osmotic pres-
248
Apoplastic invertase and salt tolerance
Fig. 4 Estimated sugar concentrations in chloroplasts before and after 24 h of salt stress (300 mM NaCl, 200 mmol quanta m–2 s–1) treatment.
suc, sucrose; fru, fructose; glu, glucose; total = suc + fru + glu.
sure ca. 300 mOsm kg–1) was assumed to be 95 ml (mg Chl)–1
on the basis of reported values (Winter et al. 1993, Winter et al.
1994) to calculate sugar concentration. The chloroplast volume in both tobacco types was estimated from the inverse logarithmic relationship of the volume of intact spinach chloroplasts and the sorbitol concentration in the medium considered
to be the osmotic pressure in the cytosol (Heldt and Sauer
1971). The sugar concentration in chloroplasts was calculated
based on the intactness of the chloroplasts, sugar content (mmol
sugar (mg Chl)–1), and chloroplast volume (ml (mg Chl)–1).
Figure 4 shows that sucrose was present in both the wildtype and Apo-Inv tobacco chloroplasts. Estimated sucrose contents in the chloroplasts of both types without salt stress were
similar to those reported previously (Heineke et al. 1994,
Lohaus et al. 1999). After 24 h of salt stress, the estimated
sucrose content in wild-type tobacco had increased slightly
whereas in Apo-Inv tobacco with salt stress it was similar to
the value without salt stress. In contrast, the hexose content of
Apo-Inv tobacco showed a marked increase after salt stress,
whereas without salt stress they were much lower than the
sucrose content. The hexose content of wild-type tobacco were
not altered by salt stress. The chloroplasts of salt-stressed ApoInv tobacco therefore had a much higher total of sucrose, glucose, and fructose than did those of the wild type with or without salt treatment and those of Apo-Inv without salt treatment.
The estimated contribution of the sugars (180 mOsm kg–1) to
osmotic pressure was relatively low in chloroplasts of saltstressed Apo-Inv tobacco because the osmotic pressure of the
leaf was about 1,050 mOsm kg–1. This marked increase in the
sugar concentration in chloroplasts of Apo-Inv tobacco during
salt stress is, however, important for the adjustment of osmotic
balance in the chloroplasts and providing compatible solutes
for the protection of photosynthesis.
Concluding remarks
In Apo-Inv plants, a large accumulation of sugars, mostly
in the form of glucose and fructose, in chloroplasts under salt
stress was observed. Inhibition of sugar transportation to the
sink organ in Apo-Inv plants resulted in accumulation of
sucrose in source organ, and a part of remained sucrose seemed
be converted to glucose and fructose. It is reasonable to conclude that these changes in sucrose metabolism in Apo-Inv
transformants protect photosynthetic apparatus under salt
stress. Molecular mechanisms of the protection, however,
remain to be clarified.
Acknowledgments
This work was supported in part by a Grant-in-Aid of Scientific
Research on Priority Areas (No. 04273103) from the Ministry of Education, Science, Culture and Sports, Japan (F.S); Grant JSPSRFTF9616001 (F.S.); and a NEDO International Joint Research Program grant (F. S.).
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(Received August 21, 2000; Accepted December 6, 2000)