Journal of Experimental Botany, Vol. 53, No. 368, pp. 551–558, March 2002
Drying rate and dehydrin synthesis associated
with abscisic acid-induced dehydration tolerance in
Spathoglottis plicata orchidaceae protocorms
Xing-Jun Wang, Chiang-Shiong Loh, Hock-Hin Yeoh and Wendell Q. Sun1
Department of Biological Sciences, National University of Singapore, 10 Kent Ridge Crescent,
Singapore 119260
Received 19 July 2001; Accepted 5 November 2001
Abstract
Dehydration tolerance of in vitro orchid protocorms
was investigated under controlled drying conditions
and after abscisic acid (ABA) pretreatment. Protocorms were obtained by germinating seeds on
Murashige and Skoog (MS) medium containing 10%
(vuv) coconut water, 2% (wuv) sucrose and 0.8% (wuv)
agar, and were dehydrated in relative humidities (RH)
ranging from 7% to 93% at 25 8C. The critical water
content of dehydration tolerance was determined,
using the electrolyte leakage method. Drying rate
affected the critical water content. Slow drying under
high RH conditions achieved the greatest tolerance
to dehydration. ABA pretreatment decreased the
drying rate of protocorms, and increased dehydration
tolerance. Improved tolerance to dehydration after
ABA treatment was correlated with the effect of ABA
on drying rate of protocorms. When critical water
content of protocorms dried under different RH was
plotted as a function of actual drying rate, no
significant difference in tolerance to dehydration
was observed between ABA-treated and control
protocorms. ABA pretreatment and dehydration of
orchid protocorms induced the synthesis of dehydrin,
especially under the slow drying conditions. ABA
pretreatment also promoted dry matter accumulation
such as carbohydrates and soluble proteins and
increased the concentration of Kq and Naq ions in
protocorms. The ABA-induced decrease in drying
rate was correlated with lower osmotic potential,
the enhanced maturity of protocorms and the
accumulation of dehydrin in protocorms during
pretreatment.
1
Key words: Abscisic acid, critical water content, dehydrin,
desiccation tolerance, drying rate, soluble sugars,
Spathoglottis plicata, water stress.
Introduction
Abscisic acid (ABA) plays a crucial role in higher plants
in their response to various environmental stresses and
serves as a regulatory link between stress factors and
plant responses (Seemann and Sharkey, 1987; Chandler
and Robertson, 1994). Physiological studies and molecular analysis have demonstrated that ABA may regulate
the adaptation of plants to environmental stresses
(Stewart and Voetberg, 1985; Skriver and Mundy, 1990).
The increase in endogenous ABA concentration under
water stress conditions, or the application of exogenous
ABA is often accompanied by the expression of a number
of specific genes in plant tissues (Gomez et al., 1988;
Mundy and Chua, 1988; Parcy et al., 1994), and with
enhanced synthesis of dehydration-related proteins
(Gomez et al., 1988; Blackman et al., 1991, 1992; Bray,
1988; Beardmore and Charest, 1995) and other compatible solutes such as proline, glycine-betaine, cyclitols, and
soluble carbohydrates (Hanson et al., 1985; Stewart and
Voetberg, 1985; Blackman et al., 1992; Sun et al., 1999).
In seed development and maturation, ABA also plays an
important role in the acquisition of desiccation tolerance
(Quatrano, 1987; Blackman et al., 1991, 1992; Meurs
et al., 1992). Exogenously applied ABA was able to
induce desiccation tolerance in somatic embryos (Park
et al., 1988; Bochicchio et al., 1991; Etienne et al., 1993)
and immature zygotic embryos (Wakui et al., 1994;
Present address and to whom correspondence should be sent: LifeCell Corporation, One Millennium Way, Branchburg, NJ 08876, USA.
Fax: q1 908 947 1085. E-mail: [email protected]
ß Society for Experimental Biology 2002
552
Wang et al.
Blackman et al., 1992). It is thought that the synthesis of
dehydration-related proteins and small molecular weight
compatible solutes would help plant tissues to tolerate
severe desiccation, possibly by stabilizing cellular structures and protecting metabolic functions from stress
disruption.
However, in these earlier studies, actual drying rates of
control tissues and ABA-treated or pre-conditioned
tissues were not quantified. It is known that ABA could
reduce plant water loss under water stress conditions. For
example, ABA could regulate the behaviour of leaf
stomata (Munns and King, 1988; MacRobbie, 1990).
The effect of drying rate on plant desiccation tolerance
has also been well documented. Slow drying significantly
increases desiccation tolerance of somatic embryos and
immature zygotic embryos or seeds (Blackman et al.,
1992; Etienne et al., 1993; Li et al., 1999). In these studies,
slow drying was believed to allow plant tissues to acquire
desiccation tolerance during the period of prolonged
slow dehydration. By contrast, slow drying was found to
be detrimental to desiccation tolerance of many recalcitrant (desiccation-sensitive) seeds and embryos (Farrant
et al., 1985; Pammenter et al., 1998). More recently, it was
reported that there is actually an optimal drying rate for a
plant tissue to achieve its maximum desiccation tolerance
(Liang and Sun, 2000). Therefore, drying rate appears to
modify significantly the ability of plant tissues to withstand dehydration stress. These earlier studies indicate
that ABA-induced dehydration tolerance may be associated with changes in drying rate in plant tissues. As far
as is currently known, there have been no reports about
the effect of ABA treatment on tissue drying rate under
controlled conditions.
The objective of the present study is to test the
hypothesis that ABA-induced dehydration tolerance is
related to the change of drying rate in plant tissues.
The relationship between dehydration tolerance and drying rate, and the effect of ABA pretreatment on drying
rate of orchid protocorms have been investigated.
These data suggest that the improvement of dehydration
tolerance in orchid protocorms by ABA pretreatment
is largely associated with a decrease in drying rate of
the tissue.
Materials and methods
Plant materials
Spathoglottis plicata plants were grown in containers with
garden soil. Orchid flowers were hand-pollinated. Healthy capsules were harvested 30 d after pollination. The capsules were
surface-sterilized in 20% (vuv) commercial bleach (5.75% NaOCl)
for 10 min and rinsed with autoclaved distilled water 3–5 times.
The capsules were aseptically cut open and seeds were germinated in Murashige and Skoog (MS) medium with 2% sucrose,
10% coconut water and 0.8% agar (pH 5.2). Seeds were cultured
at 25 8C with 16 h photoperiod. Protocorms reached an
average diameter of 2 mm before ABA pretreatment and drying
studies.
ABA pretreatment
A stock solution of ABA was made in 95% alcohol, filtersterilized and added to autoclaved MS medium. The final concentration of ABA in the medium was 5 or 10 mg l1. ABA
pretreatment was carried out at 25 8C with 16 h photoperiod.
Changes in protocorm weight, chlorophyll content, carotenoid
content, osmotic potential, and concentrations of Kq, Naq and
Ca2q ions were determined during ABA pretreatment.
Determination of tissue water content and osmotic potential
Cultured protocorms were collected and washed clean with distilled water. Protocorms were then blot-dried with tissue paper.
Water content of samples were determined gravimetrically after
drying for 24 h at 85 8C, and expressed on a dry weight basis
(g water g1 dry mass). Osmotic potential in the protocorm was
determined using a dew point microvoltmeter (Wescor, Logan,
UT, USA). Protocorms were frozen in Eppendorf tubes at
80 8C for 1 d. Samples were thawed at room temperature for
1 h before measurement. The microvoltmeter was calibrated
with standard NaCl solutions, and the osmotic potential of
protocorms calculated according to the standard curve from a
series of NaCl solution. To investigate the possible osmotic
adjustment during drying, the amount of water lost during drying
were recorded, and dried protocorms were rehydrated to the
original water content by adding back the same amount of
lost water. After equilibration, osmotic potential of rehydrated
protocorms was measured.
Determination of chlorophyll, carotenoid, Kq, Naq
and Ca2q content
Contents of chlorophyll and carotenoid in protocorms were
extracted with 80% aqueous acetone solution and determined
spectrophotometrically. The concentrations of Kq, Naq and
Ca2q ions in protocorms were determined using a flame photometer. Weighed samples were first boiled in deionized water for
30 min to extract the soluble Kq, Naq and Ca2q from the
tissues. Extraction solutions were then diluted to the optimal
range for the ion concentration measurement.
Determination of dehydration tolerance
The dehydration tolerance of cultured protocorms was examined at 25 8C in a wide range of drying rates. To maintain a
constant drying rate, protocorms (;180 mg) were dried under
specific relative humidity (RH) in sealed containers (Sun and
Gouk, 1999). Saturated solutions of the following salts were
used to control various RH conditions (in brackets): KNO3
(93%), MgSO4, (89%), K2CrO4 (87%), KCl (85%), NaCl
(75%), (NH4)NO3 (63%), MnCl2 (56%), Ca(NO3)2 (52%),
K2CO3 (43%), MgCl2 (32%), K acetate (23%), and NaOH
(7%). Glycerol solutions at concentrations of 32% (wuw), 36%,
52%, and 64% were also used to generate relative humidities
of 91%, 90%, 80%, and 70%. For each drying experiment,
;15–20 independent samples were prepared for repeated
sampling to eliminate RH fluctuation that was caused by opening containers. Dehydration tolerance was expressed by the
critical water content that was determined using the electrolyte
Drying rate and ABA-induced dehydration tolerance
leakage method. After dehydration to different water contents,
the samples were incubated and gently shaken in 20 ml
deionized water for 1 h. The amount of electrolytes leaked
from protocorms was then measured with a conductivity meter.
The total amount of electrolytes in the tissues was determined
after the sample was boiled for 20 min in caped vials and cooled
to room temperature. The percentage electrolyte leakage was
used as an indicator for dehydration damage. The water content
below which the percentage electrolyte leakage increased sharply
was calculated (according to Sun and Leopold, 1993), and taken
as the critical water content. The critical water content determined by the electrolyte leakage method was further confirmed,
using the protocorm survival test after dehydration. To test the
survival of protocorms that were dried to different water
contents, protocorms were rehydrated and cultured on 0.8%
agaruMS medium containing 2% sucrose and 10% coconut
water. The percentage of protocorm survival was recorded after
14 d of culture.
Determination of carbohydrate content
Soluble carbohydrates were extracted with 50% ethanol containing 100 mg PGS (phenyl-a-D-glucoside) as an internal standard at
70 8C in a water bath for 60 min. Extraction was then repeated
three times with 50% ethanol (without PGS). Extracts were
pooled and dried under vacuum, redissolved in 1 ml distilled
water, and passed through a column containing Dowex 50W,
IRA-94 and PVPP (polyvinylpolypyrrolidone). Filtrate was
collected and freeze-dried. Carbohydrates were derivatized with
1 ml of trimethylsilylimidazole (TMSI) dissolved in pyridine
(1 : 4 vuv) at 90 8C for 1 h. Soluble carbohydrates were separated
with a 15 m fused capillary HP-1 column (0.32 mm ID, 0.25 mm
film thickness) (Hewlett-Packard, USA).
553
Results
Effects of drying rate on dehydration tolerance
of protocorms
Figure 1 shows representative drying curves of orchid
protocorms at a constant RH of 7%, 80% and 93%.
When the water content of protocorms was expressed on
dry weight basis (g water g1 dry matter), the loss of
water from the tissue followed the first-order-kinetics at
all RH levels ranging from 7% to 93%, until the protocorms almost achieved equilibrium with the salt solutions.
Therefore, these drying curves could be described by a
simple exponential function, WC ¼ a exp(–bt), where a
was the initial water content, b was the rate constant of
water loss, and t was time of drying. In the present study,
the rate constant of water loss was used to express the
drying rate quantitatively.
Figure 2A shows the change of electrolyte leakage in
protocorms after drying to different water contents at
7%, 70% and 89% RH. The electrolyte leakage of
protocorms did not increase much during the early stages.
In each case, dehydration damage, as measured by electrolyte leakage, increased significantly after the protocorms
Western blot of dehydrin-related proteins
One gram of fresh protocorms was homogenized with 5 ml of
extraction buffer containing 50 mM TRIS-HCl, pH 7.5, 50 mM
KCl and 1.0 mM phenylmethylsulphonyl fluoride (PMSF) at
4 8C. Homogenate was centrifuged at 15 000 g for 20 min. The
supernatant was desalted using a 10-DG column (Bio-Rad
Laboratories, Hercules, CA, USA). Protein concentration was
determined by the Bio-Rad protein assay using bovine serum
albumin as a standard. Proteins (30 mg) were separated by 15%
SDS gel, and then transferred to a nitrocellulose membrane
using the mini trans-blot electrophoretic transfer cell. Nonspecific binding sites were blocked with 3% gelatin in PBS
(phosphate buffer with NaCl and KCl, pH 7.4) buffer for 3 h at
room temperature. The nitrocellulose membrane was incubated
for 2 h at room temperature with primary antibody (rabbit
anti-dehydrin), which was raised from a peptide with the
sequence CTGEKKGIMDKIKEKLPGQH (Close et al., 1993).
The membrane was washed with PBS before it was incubated
with goat anti-rabbit IgG alkaline phosphatase conjugate
(5000 g dilution with 2% gelatin in PBS) for 2 h. The second
antibody was detected using nitroblue-tetrazolium chloride
(NBT) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP).
The relative quantity of dehydrin in the images of Western
blots was determined, using the Bio-Rad image analysis
software ‘Quantity One’. The images were digitized, and the
density of dehydrin bands was integrated. The relative changes
in dehydrin content after dehydration and ABA treatment were
expressed over dehydrin content in fresh control protocorms
(i.e. using the dehydrin content of fresh control protocorms
as 100%).
Fig. 1. (A) Representative drying curves of Spathoglottis plicata
protocorms. Samples were dried under constant relative humidities.
(B) The first-order-plot of protocorm water loss during drying. The slope
of the first-order-plots was used to express drying rate (rate constant).
554
Wang et al.
Fig. 2. (A) Percentage electrolyte leakage of Spathoglottis plicata
protocorms that were dried at 7%, 70% and 89% RH to different
water contents. Water content below which the percentage leakage
increased sharply was determined by fitting the data with two separate
linear regression lines, and the x-axis intercept of the best statistical fit
was taken as the critical water content (indicated by arrows). (B) A
comparison between electrolyte leakage and survival of protocorms that
were dried at 43% RH. The rate of survival was recorded after 14 d of
culture on MS medium.
were dried to below a threshold water content. This
critical water content was used to express the degree of
dehydration tolerance in protocorms. The survival test
was also used to confirm the dehydration tolerance of
protocorms. Figure 2B showed the good correlation
between electrolyte leakage and survival of protocorms
dried at 43% RH. Because of its simplicity, the electrolyte leakage method was used as a routine method for
dehydration tolerance measurements in the present study.
Figure 3 shows the effects of RH on drying rate and
dehydration tolerance of orchid protocorms. Drying rate
changed greatly under different RH conditions, with the
b-value ranging from 0.20 to 0.01 h1. The critical water
content of protocorms was also affected by RH conditions. RH between 7% and 60% did not significantly
affect dehydration tolerance. However, slow drying at
higher RH enhanced tolerance to water loss of protocorms. Critical water content of protocorms decreased
from 3.0 g g1 DW to 1.5 g g1 DW as RH increased
from 60% to 93%.
Fig. 3. (A) Drying rate and (B) critical water content of Spathoglottis
plicata protocorms that were dried at different relative humidities.
Effects of ABA pretreatment on growth and
development of protocorms
Figure 4 shows the relative increases in fresh weight and
dry weight of protocorms during ABA pretreatment.
During the pretreatment of 2 weeks, fresh weight and dry
weight of control protocorms increased by 200% and
280%, respectively. The addition of ABA at concentrations of 5 mg l1 and 10 mg l1 inhibited protocorm
growth. The fresh weight of ABA-treated protocorms
increased only by 20–30%; however, dry weight increased
by 80–100%. ABA treatment appeared to enhance the
maturation of protocorms, as indicated by the change of
colour. The content of chlorophyll a and carotenoid
decreased by more than 40%, relative to control
protocorms, after 7 d pretreatment with 10 mg l1 ABA
(Table 1). As a result of the change in the growth and
development pattern, control and ABA-treated protocorms were in a different physiological status. ABA
treatment for 7 d at 10 mg l1 resulted in (a) a decrease
in osmotic potential from 0.81"0.03 MPa to
1.01"0.04 MPa, (b) an increase in Kq and Naq
concentrations in the tissue by 12–18%, (c) an increase
of soluble carbohydrate concentration by more than
80%, of which 90% was sucrose, and (d) an increase in
soluble protein content by 39% (Table 1). All of these
physiological parameters showed significant differences
statistically.
Drying rate and ABA-induced dehydration tolerance
555
Effects of ABA pretreatment on drying rate and
dehydration tolerance of protocorms
ABA pretreatment altered the response of protocorms to
drying. Figure 5 shows the effects of ABA pretreatment
on drying rate and critical water content of protocorms.
After ABA pretreatment, the drying rate of protocorms
was reduced to approximately 50% as compared to
control protocorms. The dehydration tolerance of
protocorms, as indicated by the critical water content,
increased after ABA pretreatment. Interestingly, when
critical water content of protocorms dried at different RH
was plotted as a function of actual drying rate, there was
no significant difference between control and ABAtreated protocorms in their responses to drying (Fig. 6).
This result suggests that ABA pretreatment might
enhance the tolerance of protocorms to water stress
through the reduction in the rate of water loss.
Effects of drying rate and ABA pretreatment on the
synthesis of dehydrin
Fig. 4. Relative increases in fresh weight and dry weight of Spathoglottis
plicata protocorms during ABA pretreatment. Control protocorms
were cultured on MS medium. Percentage weight increases were
normalized by the initial weights (Wt ¼ 0) for different treatments:
(Wt–Wt ¼ 0)uWt ¼ 0. Data were mean"SE of three measurements, and
bars smaller than symbols were not shown. Note the scale change of
vertical axis.
ABA pretreatment induced the synthesis of several new
dehydrins. Fresh protocorms that were cultured in control MS medium showed only one visible protein band
that was immunologically related to dehydrin (;30 kDa)
in the Western blot, whereas ABA-pretreated protocorms
contained six additional bands at molecular weights
Table 1. Contents of chlorophylls, carotenoids and water, osmotic
potential, concentrations of Kq, Naq and Ca 2q ions, soluble
protein and sugar contents in control and ABA-pretreated
Spathoglottis plicata protocorms
Data were mean"SE of at least three measurements.
Biochemical and physiological
parameter
Chlorophyll a content
Chlorophyll b content
Carotenoid content
Water content (g g1 DW)
Osmotic potential (MPa)
Fresh protocorms
Fast-dried, rehydratedb
Slowly dried, rehydratedb
Ion concentration
Kq (mmol kg1 tissue water)
Naq (mmol kg1 tissue water)
Ca2q (mmol kg1 tissue water)
Soluble sugar content
Sucrose (mmol kg1 tissue water)
Total sugar (mmol kg1 tissue water)
Soluble protein content
(g kg1 tissue water)
a
Control
ABA
pretreatmenta
3.15"0.19
1.89"0.07
0.72"0.11
10.3"0.4
1.86"0.13
1.93"0.22
0.42"0.04
8.6"0.1
0.81"0.03
0.80"0.04
0.86"0.02
1.01"0.04
1.04"0.02
0.95"0.01
96.6"4.8
129.0"5.9
3.9"0.3
107.7"0.7
152.2"2.9
4.1"0.2
23.3"1.5
26.5"1.8
15.5"1.2
44.2"9.8
48.2"9.1
21.5"1.5
Protocorms were cultured for 7 d at 25 8C with a 16 h photoperiod
on the MS medium alone or the MS medium containing 10 mg l1 ABA.
b
Osmotic potential of dried protocorms was measured after rehydration to the original water content before desiccation treatment.
Fig. 5. (A) Drying rate and (B) critical water content of Spathoglottis
plicata protocorms that were pretreated with 10 mg l1 abscisic acid for
7 d, and then dried at different relative humidities. Drying rate and
critical water content of control protocorms were presented for a
comparison (dashed lines).
556
Wang et al.
Fig. 6. The relationship between drying rate and dehydration tolerance
(critical water content) of Spathoglottis plicata protocorms. Note that
there was no significant difference in desiccation tolerance between
control and ABA-pretreated protocorm, when critical water content of
protocorms dried at different RH was plotted as a function of actual
drying rate.
dried at RH of 67% and 7.2%, respectively, that achieved
the same drying rate of 0.09 h1; whereas under slow
drying conditions protocorms were dried at RH of
87% and 75% which achieved the same drying rate
of 0.028 h1. Drying resulted in the synthesis of new
dehydrins in control protocorms, and increased the
content of existing dehydrins (Fig. 7A). Drying rate
affected dehydrin synthesis greatly during dehydration,
and slow drying promoted dehydrin synthesis, particularly in control protocorms (Fig. 7A). Figure 7B shows
the relative increase of total dehydrin content in protocorms after ABA pretreatment and drying under
fast and slow drying conditions. Fast and slowly-dried
protocorms led to 300% and 700% increase in dehydrin
content relative to fresh control tissues. ABA-pretreated
protocorms were less responsive to dehydration. Fast
and slow drying resulted in, respectively, only 100% and
180% increase in dehydrin content over ABA-pretreated
fresh protocorms.
Discussion
Fig. 7. (A) Effect of ABA pretreatment and drying rate on dehydrin
synthesis in Spathoglottis plicata protocorms (Western blot). Arrows
indicate two molecular weight protein markers in the correspondent
SDS–PAGE. (B) Quantification of total dehydrin synthesis in control
and ABA-pretreated protocorms. The relative increase was calculated
with the dehydrin content of fresh control protocorm as 100%.
around 27, 22, 19, 16, 13.5, and 12 kDa (Fig. 7A). To
investigate dehydrin synthesis of protocorms in response
to the rate of drying, control and ABA-pretreated
protocorms were dried under both fast and slow drying
conditions. Since ABA pretreatment affected the drying
rate of protocorms (Fig. 5), control and ABA-pretreated
protocorms were dried at different RH conditions that
achieved the same drying rate. Under the fast drying
conditions, control and ABA-pretreated protocorms were
The response of in vitro orchid protocorms to dehydration was investigated under a wide range of drying
conditions (drying rates) in the present study. The critical
water content of dehydration tolerance was a function of
drying rates for both control and ABA-pretreated
protocorms (Figs 3, 5, 6). One hypothesis that proposes
the beneficial effect of slow drying for embryonic tissues is
that slow drying could permit the tissue to synthesize
certain protective substances in response to dehydration.
The data of the present study supports this hypothesis.
Protocorms were damaged at high critical water content
during rapid drying at low RH between 7% and 60%
(Fig. 3). In these conditions, protocorms would lose 50%
of their tissue water in less than 5 h and, therefore, there
is probably insufficient time for the tissues to adapt to
the severe dehydration stress. In order to examine the
relationship between the acquisition of dehydration
tolerance and slow drying rate further, the time needed
for protocorms to lose 50% of their tissue water under
different drying conditions was calculated (Fig. 8). The
plot of critical water content against the time of 50%
water loss indicates clearly the presence of a period for
the adaptation or acquisition of dehydration tolerance
in protocorms. ABA pretreatment has been known to
enhance dehydration tolerance of embryonic tissues (Park
et al., 1988; Etienne et al., 1993; Wakui et al., 1994;
Li et al., 1999). Therefore, it was not surprising that
ABA pretreatment decreased the critical water content
of protocorms (Fig. 5). What was surprising was the
observation that there was no significant difference
between the control and ABA pretreatment, when
dehydration tolerance of protocorms was compared at
Drying rate and ABA-induced dehydration tolerance
Fig. 8. The relationship between the time of 50% water loss (T50) and
dehydration tolerance (critical water content) of Spathoglottis plicata
protocorms. Note that T50 ¼ 0.693ub according to the first-order-kinetics
of drying curves (refer to Fig. 1).
the same drying rate (Figs 6, 8). The increase in dehydration tolerance after ABA pretreatment appeared to be due
mainly to the reduction in water loss from protocorms,
allowing the tissues more time for the acquisition of
dehydration tolerance.
The reduction in water loss from protocorms was one
of the most notable changes after ABA pretreatment
(Fig. 5A). A number of developmental, biochemical
and physiological changes might be associated with the
decrease in drying rate. Developmental changes included
the maturity, size and shape of protocorms. The maturity
of protocorms was accelerated during ABA pretreatment
(Table 1), and the increased thickness of the cuticle might
well contribute to the reduction of drying rate. The shape
of protocorms did not change much during ABA
pretreatment. The change in protocorm size was significant (Fig. 4), but would probably not decrease the
drying rate. ABA-pretreated protocorms were smaller
than control protocorms (Fig. 4), and therefore actually
had a higher surface-to-volume ratio, which should
facilitate the drying of protocorms. Biochemical and
physiological factors included the increase in ion concentration, solute synthesis and osmotic regulation
during ABA pretreatment. Significant osmotic adjustment occurred in protocorms during ABA pretreatment.
The osmotic potential of ABA-treated protocorms
decreased from 0.81 MPa to 1.01 MPa in 7 d
(Table 1). A major part of this adjustment was attributed
to the accumulation of soluble sugars, especially sucrose,
and the increased concentration of Kq and Naq ions
in the tissue. The increases in Kq, Naq and sucrose
concentrations could contribute to this osmotic adjustment as much as 12%, 28% and 31%, respectively.
Altogether they contributed 71% of osmotic adjustment.
Additional contributions might come from the ABAinduced synthesis of other solutes and certain proteins.
The content of soluble proteins in ABA-treated
protocorms increased by 39% (Table 1). The relative
557
contribution of soluble proteins to the osmotic adjustment could not be estimated, because soluble proteins do
not behave like simple solutes osmotically. However, the
extent of osmotic adjustment (0.20 MPa) was unlikely
to be able to influence the drying rate of protocorms
significantly and so any possible additional osmotic
adjustment during drying was also investigated. Dried
protocorms were rehydrated to the original water content
for osmotic potential measurement. There was insignificant osmotic adjustment during drying in either control
or ABA-pretreated protocorms (Table 1). Besides
osmotic adjustment, compatible solutes may increase
dehydration tolerance through other mechanisms. ABA
pretreatment significantly increased the content of soluble
sugars in the protocorms, especially sucrose. These
carbohydrates were found to be a good protectant against
dehydration stress in desiccation-tolerant plant tissues
(Blackman et al., 1992; Sun et al., 1994, 1999; Pelah et al.,
1997; Sun and Leopold, 1997). In an ongoing study, the
preculture of protocorms in medium with a high sucrose
content (10%, wuv) greatly increased the dehydration
tolerance of protocorms. However, sucrose treatment did
not affect the drying rate of protocorms (data not shown).
The effect of ABA pretreatment on protein synthesis
in protocorms was investigated. The increase of soluble
protein content in ABA-treated protocorms by 39%
(Table 1) led to the hypothesis that certain proteins
may modulate water loss in tissues during dehydration.
Recently, Walters et al. reported that desiccation-related
LEA proteins had strong water-binding capacity (Walters
et al., 1997). ABA-pretreatment has been known to
induce the synthesis of a variety of similar proteins (Ried
and Walker-Simmons, 1990; Skriver and Mundy, 1990;
Blackman et al., 1991, 1992; Chandler and Robertson,
1994). In the present study, it was observed that ABA
pretreatment induced the synthesis of six new dehydrins,
and resulted in a 130% increase in total dehydrin content
over control protocorms (Fig. 7). It remains unclear how
ABA-induced dehydrin reduces water loss in protocorms.
Drying rate affected both dehydration tolerance and
dehydrin synthesis (Fig. 7). The drying rate experiment
on dehydrin synthesis in control and ABA-pretreated
protocorms showed a significant correlation (r ¼ 0.89,
n ¼ 6) between critical water content and dehydrin content in dried protocorms. The good correlation, however,
did not necessarily suggest a cause–effect relationship
between the accumulation of dehydrin and dehydration
tolerance.
In conclusion, the present study showed that dehydration tolerance of orchid protocorms was a function of
drying rate. Slow drying and ABA pretreatment increased
the dehydration tolerance of protocorms and the synthesis of dehydrin. The improvement of dehydration
tolerance after ABA pretreatment was associated with the
decrease in drying rate of protocorms.
558
Wang et al.
Acknowledgements
The research was supported by a grant from the National
University of Singapore to WQS (RP-960366). We thank
Dr Andreas Richter (University of Vienna) for his assistance in
GCuMS identification of two carbohydrates, and Professor
Timothy J Close (University of California at Riverside) for his
generous gift of dehydrin antibody.
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