Molecular Plant
•
Volume 1
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Number 2
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Pages 262–269
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March 2008
RESEARCH ARTICLE
Dual Functions of Phospholipase Da1 in Plant
Response to Drought
Yueyun Hong, Suqin Zheng and Xuemin Wang1
Department of Biology, University of Missouri, St Louis, MO 63121, USA
Donald Danforth Plant Science Center, St Louis, MO 63132, USA
Key words:
drought; phospholipase; phosphatidic acid; lipid oxidation; lipid signaling.
INTRODUCTION
Drought is an ever existing stress that causes the loss of crop
yield (Vij and Tyaqi, 2007). Plants are sessile and can not escape
from adverse environments such as water and temperature
stress. To survive, plants have evolved by adapting to different
stresses via morphological modification and changes in physiological, biochemical, or molecular responses (Vij and Tyaqi,
2007; Sreenivasulu et al., 2007). One such change is the membrane lipid turnover. Membranes are the initial point to perceive stress cues and are a target of degradation in response
to drought (Gigon et al., 2004; Wang, 2004). Phospholipase
D (PLD), which hydrolyzes phospholipids to produce phosphatidic acid (PA) and a head group, is a major family of lipid-hydrolyzing enzymes (Wang, 2001). PLD activity has been shown
to increase under water deficits and hyperosmotic conditions
(Frank et al., 2000; Munnik et al., 2000; Katagiri et al., 2001).
Activation of PLD has been reported to signal plant response
to abscisic acid (ABA) to close stomata and decrease transpirational water loss in response to water deficits (Jacob et al.,
1999; Sang et al., 2001b; Mishra et al., 2006). On the other hand,
it has also been observed that under drought stress, the activity
and expression of PLD increased faster in drought-sensitive
cultivars than in drought-tolerance ones (EI Maarouf et al.,
1999; Guo et al., 2006). These results could mean that a
high level of PLD activity might have a negative effect on plant
tolerance to drought.
These seemingly discrepant observations suggest that PLD
has different effects on plant response to water deficits. The
PLD family in plants consists of multiple enzymes (Wang,
2001). Arabidopsis has 12 identified PLDs that are classified into a(3), b(2), c(3), d, e, f(2) (Qin and Wang, 2002; Wang, 2005).
In addition, PLD can be divided into two subfamilies—C2-PLDs
and PH/PX-PLDs, based on their domain structures. C2-PLDs,
which include PLDas, bs, cs, d, and e, contain a Ca2+-dependent
phospholipid-binding domain and require Ca2+ for activity,
whereas PH/PX-PLDs (PLDfs) possess a phox homology (PX) domain and plecktrin homology (PH) domain (Qin and Wang,
2002). Several Arabidopsis PLDs have been shown to display
distinguishable regulatory and catalytic properties (Wang,
2005). Genetic manipulation of PLDa1, PLDd, and PLDfs have
resulted in growth alterations in response to different stimuli,
including ABA, auxin, reactive oxygen species, freezing, and
phosphorus deficiency (Zhang et al., 2003; Li et al., 2004,
2006, 2007; Cruz-Ramı́rez et al., 2006; Mishra et al., 2006).
1
To whom correspondence should be addressed. E-mail
danforthcenter.org, fax 314–587–1519, tel. 314–587–1419.
swang@
ª The Author 2008. Published by the Molecular Plant Shanghai Editorial
Office in association with Oxford University Press on behalf of CSPP and
IPPE, SIBS, CAS.
doi: 10.1093/mp/ssm025, Advance Access publication 8 February 2008
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ABSTRACT Phospholipase Da1 (PLDa1) has been shown to mediate the abscisic acid regulation of stomatal movements.
Arabidopsis plants deficient in PLDa1 increased, whereas PLDa1-overexpressing tobacco decreased, transpirational water
loss. In the early stage of drought, the decrease in water loss was associated with a rapid stomatal closure caused by a high
level of PLD in PLDa1-overexpressing plants. However, in the late stage of drought, the overexpressing plants displayed
more susceptibility to drought than control plants. PLDa1 activity in the overexpressing plants was much higher than that
of control plants in which drought also induced an increase in PLDa1 activity. The high level of PLDa1 activity was correlated to membrane degradation in late stages of drought, as demonstrated by ionic leakage and lipid peroxidation.
These findings indicate that a high level of PLDa1 expression has different effects on plant response to water deficits.
It promotes stomatal closure at earlier stages, but disrupts membranes in prolonged drought stress. These findings
are discussed in relation to the understanding of PLD functions and potential applications.
Hong et al.
A
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263
80
vector
70
OE 1
OE 2
Closed stomata (%)
60
50
40
30
20
10
PLDa1 and PA Mediate Plant Response to ABA and
Stomatal Movement
0
0 min
B
10 min
20 min
9
8
vector
OE 1
7
Stomatal aperture (µm)
PLDa1 is presented in guard cells in a much higher level than
other PLDs in Arabidopsis (Sang et al., 2001b). PLDa1 is activated by ABA to produce PA (Zhang et al., 2004)—a minor class
of phospholipids that act as important second messengers
(Meijer and Munnik, 2003; Wang et al., 2006). PLDa1suppressed or knockout plants displayed less sensitivity to
ABA-induced stomatal closure and lost more water than
wild-type (WT; Sang et al., 2001b; Zhang et al., 2004). Further
studies have found that PLDa1 and PA regulate stomatal closure via a bifurcating pathway (Mishra et al., 2006). On one
hand, PLDa1-derived PA binds to ABI1—a protein phosphatase
2C that functions as a negative regulator in the ABA signaling
pathway. The PA interaction with ABI1 may tether ABI1 to the
plasma membrane, sequester its negative function on ABA signaling, and enhance ABA promotion of stomatal closure via
reducing its translocation to the nucleus (Zhang et al.,
2004). On the other hand, PLDa1 interacts with the Ga1 subunit of the heterotrimeric G protein to inhibit stomatal opening (Zhao and Wang, 2004; Mishra et al., 2006).
The role of PLDa1 in promoting ABA sensitivity and stomatal
closure was further supported by overexpressing PLDa1 in tobacco plants under the control of the 35S promoter
(35S::PLDa1). PLDa1 activity in OE plants was much higher than
in the control plants that were transformed with an empty vector (Sang et al., 2001b). Under well watered normal growth
conditions, 35S::PLDa1 plants grew and developed indistinguishably from WT or vector control plants. When leaves were
detached, more than 90% of stomata were open, and the percentage of open stomata was similar between OE and vector
control plants at the beginning. However, dehydration of the
detached leaves promoted stomatal closure faster in
35S::PLDa1 plants than vector control plants. The percentage
of closed stomata of 35S::PLDa1 OE1 and OE2 were 58 and
42%, respectively, but was only 13% for vector control plants
10 min after detachment (Figure 1A). In addition, when the
apertures of remaining open stomata were measured,
35S::PLDa1 and control plants displayed a clear difference.
Phospholipase D in Drought
OE 2
6
5
4
3
*
2
*
* *
1
0
0 min
10 min
20 min
Figure 1. Stomatal Closure of PLDa1-OE and Control Leaves in Response to Dehydration.
(A) Percentage of closed stomata in response to dehydration after
detachment. Each treatment in each genotype used 50 stomata. A
binary Ti plasmid with a 2.8-kb cDNA encoding the full-length
PLDa1, under the control of the 35S promoter, was transferred into
tobacco by leaf disc inoculation (Sang et al., 2001b). The empty vector was also transferred into leaf tissue to produce vector control
plants. Leaves were detached and exposed to cool, white light
(125 lmol m 2 s 1) at 23C.
(B) Stomata aperture in response to rapid dehydration. Epidermal
peels were collected from the detached leaves to measure stomatal
aperture using a microscope equipped with a digital camera and
analyzed by IMAGEPRO software. Values are means 6 SD (n = 30).
The stomatal aperture was 1.4 and 1.3 lm for OE1 and OE2
plants, respectively, but was 4.9 lm for vector control plants
10 min after detachment (Figure 2B). The results in tobacco
are consistent with the findings in Arabidopsis that PLDa1
plays an important role in promoting stomatal closure.
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These results provide biochemical, genetic, and physiological
evidence that different PLDs have distinguishable functions.
The unique functions of different PLDs might explain in part
the different effects of PLD in drought responses. PLDa1 and its
derived PA have been reported to promote ABA-mediated stomatal closure and decrease transpirational water loss (Sang
et al., 2001b; Zhang et al., 2004). PLDd promotes PA production
under rapid dehydration and high salinity (Katagiri et al.,
2001). But the role of specific PLDs that are involved in drought
sensitivity have not been established. Another possibility could
be that a same PLD can have different effects on plant
response to drought, depending upon the severity and stages
of the stress. Here, we describe that PLDa1, which is the most
abundant PLD in plant tissues, has dual effects on plant
responses to drought and implications of the findings.
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Relative water content (RWC) in leaves was measured 8, 11, 14, and
17 d after withholding watering. Plants watered regularly were
used as normal condition control. The leaves were detached and
weighed for the initial fresh weight (FW), then were submerged
in water overnight to measure saturated weight (SW), followed
by drying in an oven at 80C for 48 h for dry weight (DW). RWC
was expressed as (FW-DW)/(SW-DW)*100%. Values are means 6 SD
(n = 3) from one of three independent experiments. * indicates
the statistical difference between OE and vector plants at P , 0.05
level.
High PLDa1 Activity Confers Opposite Effects on Plant
Water Status in Early and Late Stages of Drought
Stomatal movements regulate water status in plant response
to drought and more than 95% of water is lost through stomata in terrestrial plants. PLDa1-deficient plants exhibited less
sensitivity to ABA-induced stomatal closure and increased
transpirational water loss than did WT plants (Sang et al.,
2001b; Zhang et al., 2004). When the rates of water loss from
PLDa1-OE and vector control leaves of similar size, age, and
position on tobacco plants were compared, leaves from
35S::PLDa1 lines lost less water than vector control leaves (Sang
et al., 2001b). The results indicate that increased expression of
PLDa1 decreases plant water loss by promoting rapid stomatal
closure in the early stage of response to dehydration.
The effect of increased PLDa1 expression on plant relative
water content (RWC) was determined to test the function of
35S::PLDa1 in plant drought tolerance. Under a well watered
condition, RWC was similar between 35S::PLDa1 and vector
control plants. In the early stage of drought, OE plants lost significantly less water, with RWC of OE and vector control leaves
at approximately 80 and 70%, respectively, on day 8 after withholding watering (Figure 2). However, the RWC was only 60%
in OE, whereas that of vector plants was 67% on day 14 without watering (Figure 2). The greater decrease in RWC in
35S::PLDa1 plants indicates that OE plants fail to maintain wa-
Overexpression of PLDa1 Enhanced Membrane Damage
in Prolonged Drought
The decrease in RWC in OE plants in late stages of drought is
probably due to a higher PLDa1 activity, which degrades membranes, thus accelerating cell damage and water loss. To test
this hypothesis, the accumulation of malonaldehyde (MDA)
and relative membrane conductivity were measured. The level
of MDA is commonly used to indicate lipid peroxidation, and
has been found to increase in response to stress (Weber et al.,
2004; Larkindale et al., 2005). After withholding watering for
14, 15, 16, and 17 d, leaves with similar size, age, and position
on PLDa1-OE and vector control plants were detached to take
measurements. A separate set of plants with the same age under the well watered condition were sampled as reference.
There was no difference in relative conductivity and MDA content between OE and vector control plants when the plants
were well watered (Figure 4A and 4B). However, under
drought stress, the relative conductivity and MDA of PLDa1OE plants were significantly higher than that of the control
plants (Figure 4A and 4B). Both MDA levels and membrane
conductivity increased as drought progressed. The MDA level
in PLDa1-OE leaves was about 60% higher than that in control
plants. At day 15 without watering, PLDa1-OE leaves showed
almost two-fold increase in MDA, but the vector control leaves
had only a slight increase of MDA (Figure 4A).
These results suggest that oxidation damage to the membrane is more severe in PLDa1-OE than in vector control plants.
The PLDa1-OE plants displayed more wilting than vector control plants (Figure 5A). There was no difference in leaf senescence between the two genotypes under well watered
conditions. However, the PLDa1-OE plants had more yellow
leaves than the control plants after prolonged drought treatments (Figure 5B). These results indicate that PLDa1-OE leaves
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Figure 2. Leaf Water Status during Prolonged Drought.
ter status and become more susceptible to water deficits than
vector control plants in the late stages of drought.
The expression of RAB18, which is widely used as a molecular
marker in plant response to dehydration and drought treatments, was assessed to determine whether the increased expression of PLDa1 affected the expression of this gene. PLD
has also been reported to mediate ABA-induced expression
of RAB18 (Hallouin et al., 2002). Under normal conditions,
RAB18 in both PLDa1-OE and vector control plants was undetectable. However, the level of RAB transcript was induced
greatly under drought stress (Figure 3A). RAB18 expression
level in OE plants was slightly higher than that of vector control plants, but the difference was small (Figure 3A).
When the change in PLDa1 activity was measured after
withholding watering for 11, 14, 17, 20, and 23 d, the PLDa1
activity in 35S::PLDa1 plants was much higher and remained
stable throughout the drought period. In contrast, the PLDa1
activity in vector control plants showed three-fold increases
from the 11-d to 23-d drought period (Figure 3B). At 23 d,
the stimulated activity of PLDa1 in vector control plants was
still three-fold lower than the activity in OE plants.
Hong et al.
MDA content (nmol/g FW)
A
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Phospholipase D in Drought
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4
vector
OE1
OE2
3
Drought
2
Watered
1
0
B
14
16
17
18
35
vector
OE1
OE2
30
Relative conductivity (%)
15
Drought
25
20
15
Watered
10
5
0
0
Figure 3. RAB18 Expression and of PLDa1 Levels in Response to
Drought Stress.
(A) Expression of RAB18 in response to drought stress. Total RNA
was extracted from leaves of plants watered regularly or withholding watering for 7, 10, 13, 16, 19, and 22 d. RNA (10 mg/lane) was
subjected to denaturing 1% formaldehyde agarose gel electrophoresis and transferred onto a nylon membrane. rRNA was used
as loading control. The fragment of Arabidopsis RAB18 cDNA
was labeled with (a-32P) dCTP by random priming. Hybridization
was performed in the same solution at 60C overnight. W, well
watered.
(B) PLDa activity in response to drought stress. Five week old plants
were withheld watering for 11, 14, 17, 20, and 23 d and proteins
were extracted by grinding fully expanded leaves in liquid N2 in
a buffer A (50 mM Tri-HCl (pH 8.0), 10 mM KCl, 1 mM EDTA,
0.5 mM PMSF, 2 mM DTT) followed by centrifugation at 6000 xg
for 10 min at 4C. PLDa1 activity was assayed in a reaction mixture
containing 100 mM MES, pH 6.0, 25 mM CaCl2, 0.5 mM SDS, 1%
ethanol, 2 mM phosphatidylcholine (egg yolk) mixed with dipalmmitoyl-3-phosphate-(methyl-3H)-choline, and 15 lg leaf protein in
a final volume of 200 ll at 30C for 30 min. The release of 3H-choline into the aqueous phase was quantified by scintillation counting. Values are means 6 SD (n = 5).
14
15
16
17
18
Days after withholding water
Figure 4. Membrane Ion Leakage and Oxidation during Drought.
(A) MDA contents in leaves were measured from PLDa1-OE and vector control plants 15, 16, and 17 d after withholding water. MDA
was measured by thiobarbituric acid-reactive substances (TBARS)
assay with 0.5 g of leaves from different treatments. Samples were
ground in liquid N2 and then were added to 2 ml of 0.5% thiobarbituric acid in 20% trichloroacteic acid (TCA) and 2 ml of buffer containing 175 mM NaCl and 50 mM Tri-HCl (pH 8.0). The
homogenates were heated to 95C for 25 min and centrifuged
at 3000 xg for 20 min. The absorbance for the supernatant was
measured at both 532 and 600 nm. The non-specific absorbance
at 600 nm was subtracted from the absorbance of 532 nm, and
the difference was used to calculate the amount of MDA using
an extinction coefficient of 155 mM 1 cm 1(Larkindale et al.,
2005). Values are the means 6 SD (n = 3) from one of three independent experiments.
(B) Membrane ion leakage in response to drought. Fully expanded
leaves were sampled in the late stage of drought, 14, 15, 16, and
17 d after withholding watering and washed briefly with deionized
water. The same number of leaf discs from different genotypes was
immersed in 10 ml water with degassing for 30 min, followed by
gentle agitation for 2 h. The initial conductivity of the bathing solution was measured. The leaf discs in bathing solution were boiled
for 15 min to determine the total conductivity. The membrane ion
leakage was expressed as the percentage of initial conductivity versus total conductivity. Values are means 6 SD (n = 3) from one of
three independent experiments.
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(A) Leaf wilting of representative PLDa1-OE and control plants after
15 d without watering. At the onset of drought treatment, the initial soil water content was adjusted to the same and plant growth
stages were similar between the vector control and OE plants. For
drought treatment, 5 week old plants grown in a greenhouse, under light intensity of 280–300 lmol m 2 s 1at 28/21C, were withheld watering. Plants watered regularly were used as normal
growth conditions.
(B) Number of yellow, senescent leaves at the bottom of 5 week old
plants withholding watering for 15 d. Values are means 6 SD
(n = 25).
senesce earlier and are less tolerant to prolonged drought
stress than control plants.
Dual Functions of One PLD and Implications
The studies show that increased PLDa1 expression has different
effects on plant response to water deficiency. In early stages of
drought, the increase in PLDa1 in tobacco plays a positive role
in maintaining leaf water status, as indicated by faster stomatal closure and a higher RWC than control plants. PLDa1 and its
derived PA interact with ABI1 and a G-protein to mediate ABApromoted stomatal closure through a bifurcating pathway
(Zhang et al., 2004; Mishra et al., 2006). Transcriptional profil-
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Figure 5. Growth Phenotype of PLDa1-OE and Vector Control
Plants in a Later Stage of Drought Treatment.
ing reveals that WT and PLDa1-deficient plants share only
about 15% of the genes that are up- or down-regulated by
drought (Mane et al., 2007). Results from the molecular interaction and expression profiling suggest that PLDa1 mediates
early events in signaling plant response to ABA and water deficits (Figure 6).
In prolonged, severe drought stress, however, increased
PLDa1 activity has a negative impact on drought tolerance,
as indicated by decreased RWC and increased membrane oxidation and leakage in PLDa1-OE plants. As a major family of
membrane lipid-hydrolyzing enzymes, PLD activity has been
implicated in membrane degradation. Cell membranes are
the barrier separating the cell from its surrounding, and membrane integrity is vital for cells to respond to stresses. Decreases
in lipid contents are associated with the stimulation of lipolytic
activities (Navari-Izzo, et al., 1993) and with the induction of
genes involved in lipid degradation (Gigon et al., 2004). The activity and expression of PLD increased faster in drought-sensitive
cultivars than in drought-tolerance ones of cowpea and peanut
under drought stress (EI Maarouf et al., 1999; Guo et al., 2006).
A high level of PLD activity can have detrimental effects on
membrane stability and function under drought stress.
Considerable early studies indicated that increases in PLD activity could lead to membrane lipid degradation in plant senescence and under other stresses (Borochov et al., 1982; List et al.,
1992; Salama and Pearce, 1993). A decrease in phospholipid
content and an increase in the ratio of sterol to phospholipids
decrease the fluidity of rose petal membranes with age
(Borochov et al., 1982; ltzhaki et al., 1990). PLDa1-deficient
plants display delayed ABA-promoted leaf senescence (Fan
et al., 1997) and decreased lipid hydrolysis after freezing damage (Welti et al., 2002). PLDa1 hydrolyzes structural phospholipids, such as phosphatidylcholine (PC) to PA, and this activity
is responsible for about 50% of PA formation under freezing
(Welti et al., 2002). Increased levels of PA favor the formation
of lipid hexagonal phases due to its smaller head group to
form a conical shape. Thus, an increase in PA and decrease
in PC increase the propensity of changing membranes from
stable bilayer to unstable nonbilayer structures. In addition,
PLDa1 and PA also activate NADPH oxidase to generate reactive oxygen species (Sang et al., 2001a). The increases in lipid
hydrolysis and oxidization may underlie a basis for the
increases in membrane peroxidation and loss of membrane integrity under severe drought conditions. These results are consistent with the role of PLDa1 in lipid degradation in freezing
and seed-aging stress. It is of interest to note that seed viability
and quality of PLDa1-knockdown plants are better than those
of PLDa1-knockout, suggesting that a lower level but not complete voidance of PLDa1 is beneficial for seed storage (Devaiah
et al., 2007). This indicates that a delicate balance between signaling and catabolic function of PLDa1 is important for plants
to deal with stress response and adaptation. In addition, PLD
has been implicated in many other cellular processes, such as
vesicle trafficking, cytoskeletal reorganization, secretion, and
membrane remodeling (Wang et al., 2006).
Hong et al.
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Drought progression
Early
mild drought
PLDα
PLDα
Prolonged
severe drought
PLDα hydrolysis
of membrane lipids
Membrane
degradation
oxidation
Stomatal
closure
Loss of
membrane
integrity
Water
loss
Figure 6. A Model Depicting the Dual Function of PLDa1 at Different Stages of Water Deficits.
Increased PLD a1 activity promotes stomatal closure via signal transduction and decreases water loss at early phases of water deficits. However, under severe, prolonged drought, a high PLDa activity increases lipid hydrolysis and membrane degradation. This promotes membrane
oxidation and loss of membrane integrity, rendering plants susceptible to drought damage.
Based on the present and previous results, we conclude that
PLDa1 plays important, different roles in plant response to water deficits, depending upon the stages and severity of the
stress (Figure 6). In early stages of drought, the effect on intracellular signaling is predominant, and increased PLDa1 expression is helpful for plants to decrease water transpiration due to
a rapid stomatal closure by the action of PLDa1 in ABA signaling. The signaling effect of PLD may involve selective lipid hydrolysis to produce regulatory PA and also direct PLD
interaction with other proteins, such as G proteins. Under prolonged drought, however, a high PLDa1 activity promotes
membrane degradation and oxidation, resulting in decreased
tolerance to severe drought stress (Figure 6). The positive role
of increased PLDa1 expression in maintaining leaf water status
in early phases of drought stress may be further explored for
improving plant water-use efficiency. The detrimental effect
of the constitutive PLDa1-OE, as driven by the 35S promoter,
may be alleviated by placing the gene under the control of
a guard cell-specific promoter that is inducible in early phases
of drought stress.
METHODS
Plant Materials and Growth Conditions
A binary Ti plasmid with a 2.8 kb cDNA encoding the fulllength castor bean PLDa1, under the control of the 35S promoter, was transferred into agrobacterial strain EHA 105,
and then was transformed into tobacco by leaf disc inoculation
(Sang et al., 2001a). The empty vector was also transferred into
leaf tissue to produce vector control plants. The overexpression of PLDa1 in transgenic tobacco plants was confirmed
by immunoblotting using PLDa1 antibody. For drought treatment, five-week-old plants grown in a greenhouse, under light
intensity of 280–300 lmol m-2s-1 at 28/21C, were withheld
watering. Plants watered regularly were used as normal
growth conditions.
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PLD / PA in ABA
signaling in guard cells
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Leaf Water Loss and Stomatal Behavior
Immunoblotting and PLDa1 Activity Assay
Total proteins were extracted by grinding fully expanded
leaves in liquid N2. Chilled buffer A (50 mM Tri-HCl (pH 8.0),
10 mM KCl, 1 mM EDTA, 0.5 mM PMSF, 2 mM DTT) was added
to homogenate and was then centrifuged at 6,000 xg for 10
min at 4C. Proteins (27 lg/lane) were separated by 8% SDSpolyacrylamide gel electrophoresis and transferred onto a
polyvinylidene difluoride membrane. The membrane was incubated with an antibody raised against the castor bean
PLDa1, followed by incubation with a second antibody. PLDa1
was made visible by staining alkaline phosphatase activity with
a Bio-Rad immunoblotting kit. To assay PLDa activity, 15 lg leaf
protein was incubated in a reaction mixture containing 100
mM MES, pH 6.0, 25 mM CaCl2, 0.5 mM SDS, 1% ethanol,
and 2 mM phosphatidylcholine (egg yolk) mixed with dipalmmitoyl-3-phosphate-(methyl-3H)-choline in a final volume of
200 ll at 30C for 30 min (Wang et al., 2000). The reaction
was stopped by adding chloroform : methanol (2:1, v/v). The
release of 3H-choline into the aqueous phase was quantified
by scintillation counting.
RNA Isolation and Blotting
Total RNA was isolated from fully expanded leaves by the cetytrimethylammonium bromide extraction method described
previously (Wang et al., 1994). RNA (12 lg/lane) was subjected
to denaturing 1% formaldehyde agarose gel electrophoresis
and transferred onto a nylon membrane. RNA was fixed on
the membrane by cross-linking with ultraviolet irradiation.
The membrane was prehybridized in a solution of 1 mM EDTA,
0.5 M Na2HPO4 (pH 7.2), and 7% SDS at 60C for 30 min. The
fragment of Arabidopsis RAB18 cDNA was labeled with (a-32P)
dCTP by random priming. Hybridization was performed in the
same solution at 60C overnight. The blot was washed first
with a solution of 5% SDS, 1 mM EDTA, and 40 mM Na2HPO4
Membrane Permeability and Malonaldehyde
Measurements
During drought treatments, fully expanded leaves were detached from plants and washed briefly with deionized water.
The same number of leaf discs from different genotypes was
immersed in 10 ml water with degassing for 30 min, followed
by gentle agitation for 2 hours. The initial conductivity of the
bathing solution was measured. The leaf discs in bathing solution were boiled for 15 min to determine the total conductivity. The membrane permeability is expressed as percentage of
the initial conductivity versus the total conductivity. MDA was
measured by thiobarbituric acid reactive substances (TBARS)
assay by sampling 0.5 g of leaves from different treatments.
Samples were ground in liquid N2 and then were added to
2 ml of 0.5% thiobarbituric acid in 20% trichloroacteic acid
(TCA) and 2 ml of buffer containing 175 mM NaCl and 50
mM Tri-HCl (pH 8.0). The homogenates were heated to 95C
for 25 min and centrifuged at 3,000 xg for 20 min. The absorbance for the supernatant was measured at both 532 and 600
nm. The non-specific absorbance at 600 nm was subtracted
from the absorbance of 532 nm, and the difference was used
to calculate the amount of MDA using an extinction coefficient of 155 mM-1 cm-1 (Larkindale et al., 2002).
ACKNOWLEDGMENTS
This work was supported by grants from the National Science Foundation NSF (IOS 0454866) and the US Department of Agriculture
(2007–35318–18393). No conflict of interest declared.
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Water loss was measured in detached leaves of similar size,
age, and position on tobacco plants. Leaves were detached
and left at ambient conditions in a greenhouse at various time
intervals after detachment. Decreases in fresh weight were
recorded. The percentage of decrease in the fresh weight
was expressed as percentage of water loss. To examine stomatal movement in response to dehydration, the detached leaves
were exposed to cool white light (125 lmol m-2s-1) at 23C at
various time intervals. Epidermal peels were collected from the
detached leaves to measure stomatal aperture using a microscope equipped with a digital camera and analyzed by IMAGEPRO software. Relative water content was measured in the
early, middle, and late stages of drought. The leaves were detached and weighed for the initial fresh weight (FW), then
were saturated in water overnight to measure saturated
weight (SW), followed by drying in an oven at 80C for 48
hours to measure dry weight (DW). RWC was expressed as
(FW-DW)/(SW-DW)*100%.
(pH 7.2) and then with 1% SDS, 1 mM EDTA, and 40 mM
Na2HPO4 (pH 7.2) two times. The filter was exposed to X-ray
film.
Hong et al.
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