Plant Physiol. (1986) 82, 147-153
0032-0889/86/82/01 47/07/$0 1.00/0
Recovery of Turgor by Wilted, Excised Cabbage Leaves in the
Absence of Water Uptake'
A NEW FACTOR IN DROUGHT ACCLIMATION
Received for publication September 3, 1985 and in revised form April 3, 1986
JACOB LEVITrT
Carnegie Institution of Washington, Department of Plant Biology, Stanford, California 94305
30 to 60 d.
It was previously shown (5) that mature, moderately wilted
cabbage leaves absorb water extremely slowly, and remain wilted
even when floated on water in a covered dish for 24 h or more.
However, when a fresh cut is made, water is absorbed rapidly
through this surface by the floating leaf, and full turgor is
recovered within minutes. It is thus obvious that the recovery of
turgor in an intact leaf cannot be followed by a daily removal of
samples (e.g. leaf discs for thermocouple psychrometry). Turgor
recovery was therefore followed by a simple method which
distinguishes between cell walls under tension (turgid leaf) and
cell walls not under tension (wilted leaf). Leaves were excised
from fully turgid plants, weighed, then allowed to lose water until
distinctly limp (usually within 1 h). This was determined by
holding the petiole in a horizontal position. In the turgid state,
the leaf blade remained stiffly horizontal, whether held right-side
up or down. In the wilted state, the blade bent limply downward,
whether held either way. Due to the extremely small amount of
mechanical tissue (sclerenchyma and fibrovascular bundles), any
rigidity of the leaf can only be due to turgor and any loss of
rigidity to loss of turgor. No attempt was made to measure
degrees of turgor or wilting.
The wilted leaves (numbered upward, number 1 being the first
true leaf) were then acclimated to drought by transfer to leaf
chambers with an atmosphere near 100% RH. This was accomplished as follows. Two plastic weighing dishes (12 cm square)
were lined with a double layer of wet paper towel covered by a
It has long been known (6) that slightly wilted leaves of plastic screen. One dish was then inverted and allowed to rest on
droughted plants are capable of recovering their turgor without the other forming a high humidity leaf chamber. Several leaf
an addition of water to their roots. This kind of wilting is called chambers were then placed in the growth chamber, exposed to
'temporary wilting' and recovery of turgor normally occurs at the same light regime as the growing plants but to a lower internal
night. If recovery does not occur unless the drought is ended by illumination due to the paper
towel and the translucent plastic
an additional supply of water to the plant, this is called 'perma- dishes. Each leaf rested on top of the screen. This prevented
nent wilting'. Temporary wilting is usually explained by a more contact between the leaf and
wet paper. The two plates of
rapid loss than uptake of water, and recovery-a more rapid the humidity chamber were the
not
sealed, so as to permit gas
uptake than loss.
This also ensured an atmosphere near, but not quite
exchange.
The following investigation describes another kind of tempo- at, 100% RH. Since the interior of the leaf is also near saturation
rary wilting-wilting from which the leaf can recover its turgor (about 99% when just limp), water exchange
the leaf
without any uptake of water, in fact when accompanied by a and chamber was very slow-usually a loss ofbetween
about 1 to 5%
further slow loss of water. This recovery from wilting may be RWC/d, after a 1st d loss of about 5 to 10%. Since
the lid was
called TA,2 by analogy with OA.
not clamped to the bottom half, water evaporated slowly from
the leaf chamber. Every 24 h when the leaves were weighed, a
MATERIALS AND METHODS
few ml water were added to the wet towel to replace that lost.
Cabbage (Brassica oleracea var capitata. Early Jersey Wake- Rate of dehydration of the leaf was controlled, within limits, by
amount of water added. The rate of water loss could be
field) plants were grown from seed in a growth chamber (4) for the
further reduced by partial clamping of the lid to the bottom with
'Carnegie Institution of Washington Department of Plant Biology paper clips. RWC was calculated in the usual way:
ABSTRACT
Cabbage leaves excised from a fully turgid plant wilt within 20 minutes
to 2 hours (depending on plant age) with a loss of about 10% relative
water content (RWC). If droughted for 2 to 4 days in a high relative
humidity leaf chamber, they may acclimate, recovering their turgor
without the absorption of water, in fact at a loss of 15 to 25% RWC.
This turgor recovery in the absence of water uptake occurs only if (a) the
rate of water loss is slow enough (about 1-5% RWC per day after the
first 24 hours drought loss of about 15% RWC), (b) if the leaves are no
longer growing actively. Osmotic adjustment accompanies the turgor
adjustment, but cannot be the cause in the absence of water uptake. The
recovery of turgor by wilted cabbage leaves in the absence of water uptake
cannot be explained by (a) transfer of reserve water from apoplast to
symplast either from the cell walls or from the vessel lumens by cavitation
or (b) metabolic loss of dry matter and gain of water. It can be explained
by a contraction of the cell walls around the partially dehydrated protoplasts, until they regain their elastic extensibility. These proposed cell
wall changes during drought acclimation are therefore the opposite of
those occurring during growth. This hypothesis therefore explains the
long recognized inverse relation between growth and acclimation. Two
predictions of this hypothesis were tested and substantiated.
Publication No. 918.
2
Abbreviations: TA, turgor adjustment; RWC, relative water content;
OA, osmotic adjustment; TR, turgor recovery; WA, wall adjustment.
RWC =
147
fresh wt -dry wt Xl00
saturated wt - dry wt
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148
LEVITT
Plant Physiol. Vol. 82, 1986
(2)-the more slowly dehydrated leaf 3(2) showed TA when
dehydrated to a RWC of 71% within 4 d, leaf 4(2) failed to show
TA when dehydrated more rapidly to 64% RWC within the same
time. When a series of some 20 leaves drying at different rates
were compared, the daily threshold rate of dehydration (shown
by the daily loss of RWC) was found (Table II). This table was
useful for monitoring the daily rate of dehydration, in order to
maintain it at a slow enough level to permit TA.
Relation of TA to Leaf Age and Growth Rate. The abovedescribed turgor recovery in the absence of water uptake (TA)
occurred only in fully grown leaves. Leaves excised from very
young (32-d) plants were not fully grown and failed to recover
their turgor (Table III). In slightly older (40-d) plants, only the
RESULTS
fully grown lowermost leaves showed TA. Leaves 2 to 5 from
Turgor Recovery during Acclimation. When a fully turgid leaf 47-d and leaves 3 to 7 from 50-d plants showed TA.
was excised from an unacclimatd (well watered) cabbage plant
Relation to Osmotic Concentration. A marked increase in
and exposed to the atmosphere in the laboratory, it became limp osmotic concentration of the excised leaf occurred during the
within 0.5 to 2 h (depending on age and environmental factors), turgor recovery of the wilted leaf (Table IV).
when it had lost about 10% of its water (Fig. 1). On transfer to a
leaf chamber, it continued to lose water, though much more
DISCUSSION
slowly. By the 2nd to 4th d, when it had lost a total of about 15
to 25% of its water, acclimation had occurred and it recovered
Drought Resistance of Cabbage Leaves. Three kinds of
its turgor (Fig. 1). It retained this turgor until it had lost between drought resistance have been established in cabbage leaves (5).
26 and 33% of its RWC, when secondary wilting occurred (Fig. (a) Drought avoidance due to an efficient cuticle which markedly
1). Therefore, the acclimated leaf was able to remain turgid until decreases water loss through the leaf surface when stomata are
it had lost about 3 times as much water as was required to induce closed, and which is equally developed in unacclimated and
wilting in the unacclimated state. No injury resulted from the acclimated leaves of the same age and stage of development. (b)
wilting, and water was rapidly reabsorbed (Fig. 1).
Drought tolerance due to dehydration avoidance as a result of
In agreement with the above results, when several leaves were OA. (c) Drought tolerance due to dehydration tolerance-the
wilted to different degrees, all were able to recover their turgor ability to survive cell dehydration. Unlike drought avoidance,
(in the absence of water uptake) after wilting to 74% RWC and both of these kinds of drought tolerance are markedly increased
above, but not after wilting to 71% RWC or below (Table I). by drought acclimation. Now, a fourth kind of drought resistThere are, therefore, two wilting zones: (a) the primary wilting ance, that develops during acclimation, has been establishedzone, from which the leaf can recover its turgor in the absence TR of the wilted leaf in the absence of water uptake.
of water uptake, and (b) the secondary wilting zone from which
At first sight, this TR does not appear to be a new phenomeit cannot recover in the absence of water uptake.
non. It has long been known that plants undergo temporary
Relation of TA to Rate of Dehydration. The above-described wilting in the daytime, due to a more rapid loss to the atmosphere
turgor recovery in the absence of water uptake occurred only if than uptake from the soil, and a recovery at night, due to the
the dehydration rate was slow enough. Thus, when two leaves reverse relation. Furthermore, as in the above two kinds of
from the same plant (4) were compared, leaf 6(4) was dehydrated drought tolerance, it has been shown that the TR after temporary
slowly to a RWC of 85% in 3 d, and showed TA, leaf 2(4) was wilting may improve due to acclimation. Thus, droughted soydehydrated more rapidly to a RWC of 71% in the same time bean leaves at first wilt during the day and recover at night, but
and failed to develop TA (Fig. 2). At rates intermediate between after a few days' drought remain turgid even during the day. This
these two, the same results were obtained with leaves from plant kind of acclimation, however, is due to an increased deposit of
Since the pots with the growing plants stood in trays of nutrient
solutions, the leaves were assumed to be saturated (100% RWC).
Tests with leaves floating on distilled H20 (cut petiole surface
submerged) supported this assumption, although the leaves continued a very slow uptake beyond the 100% point due, presumably, to continued slow expansion of fully turgid leaves.
Osmotic potential was measured by the method of incipient
plasmolysis (5). This classical method has the advantage of
measuring osmotic concentration of the cell sap at the point of
zero turgor, regardless of the water status (wilted or turgid) of the
leaf.
0
120
0
3::
20
I
40
60
80
120
100
140
160
180
120
II
110
110
100 _
100
90 _
90
wilting zone
secondary
wilting zone
80 F
80
70 F
60
FIG. 1. Recovery of turgor in the absence of water
uptake by excised, initially turgid, cabbage leaf after
3 d in the wilted state. The leaf wilted rapidly in the
laboratory for 2.5 h after excision, then slowly in a
leaf chamber. Recovered turgor was retained during
3 d of slow water loss. Secondary wilting occurred
after 6 d. Broken line, wilted; solid line, turgid.
70
---
0
20
40
60
80
100
Time (h)
120
140
I160
60
180
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Copyright © 1986 American Society of Plant Biologists. All rights reserved.
RECOVERY OF TURGOR IN THE ABSENCE OF WATER UPTAKE
Table I. Turgor Recovery (TR) in the Absence of Water Uptake after
Wilting to Different R WCs
Time to attain corresponding RWC. Recovery at RWC of 74% and
above, failure to recover turgor at 71 % RWC and below. Leaves numbered from 1 for the first true leaf, followed by plant number in parentheses.
RWC at RWC without
RWC at
Time after
Leaf No.
TR
TR
Excision 1st Wilting
%
h
91
0.5
4(4)
85
72
92
5 (4)
0.5
82
72
92
0.5
6 (4)
76
74
93
7 (4)
0.5
77
74
94
0.5
96
0.5
96
96
1.0
96
0.5
96
8 (4)
9 (4)
8 (5)
9 (5)
10 (5)
74
87
71
61
81
77
87
69
lipids on the leaf surface, or an increased drought avoidance,
leading to a decreased water loss (1). Therefore, this kind of TR
is again due to a net uptake of water.
In contrast to this kind of TR, when fully turgid, excised
cabbage leaves were wilted rapidly (0.5-2 h) to a RWC of 88 to
93%, and acclimated as the RWC gradually decreased over 2 to
4 d, they recovered their turgor without absorbing water, in fact
even though slowly continuing to lose water. This is a new and
unexpected factor that may explain the acclimation of plants to
mild or moderate drought (2). Presumably, the recovery of turgor
leads to photosynthesis, growth, and development, perhaps by
inducing stomatal opening at a dehydration causing wilting and
consequent stomatal closure in unacclimated leaves.
The inability of the leaves to acclimate when dehydrated
rapidly agrees with previous results (5). Similarly, the inability of
rapidly growing leaves to acclimate and develop TA is in agree30
0
0
100
-
90
[
80
[
70
[
60
90
120
A
IIl,
I'
149
ment with the well-known inverse relation between growth and
hardiness or acclimation (3).
The recovery of turgor by the wilted leaves is accompanied by
an osmotic adjustment-i.e. a decrease in osmotic potential due
to an increase in osmotic concentration of about 50% (Table
IV). At first sight, the turgor recovery may seem explainable by
this concurrent osmotic adjustment during the acclimation.
From the fundamental equation for water potential, any decrease
in osmotic potential should be accompanied by an uptake of
water and a consequent increase in turgor.
Thus, when water uptake is allowed, the OA can account for
the 17% excess RWC and, therefore, expansion of the acclimated
leaf (Fig. 1). The equation, however, is for equilibrium conditions. Under the above near but not quite equilibrium conditions,
the OA (decrease in osmotic potential) of excised, wilted cabbage
leaves is accompanied by a water loss, in spite of the recovery of
turgor. Therefore, the OA due to an increase in cell sap concentration in the excised leaves cannot induce the TR that accompanies it without an uptake of water.
There must, therefore, be a second kind of cell adjustment
during acclimation, which parallels OA, and leads to TR in the
absence of water uptake. It is proposed that this adjustment
occurs in the cell wall, as opposed to OA which results in
accumulation of solutes in the vacuole and protoplasm.
To distinguish it from TR due to water uptake, TR in the
absence of water uptake will be called TA. This terminology is
in agreement with the use of the term OA, for both TA and OA
occur in the absence of a transfer of materials (water or solutes,
respectively) from outside to inside the leaf.
POSSIBLE MECHANISM OF TA
a. Transfer of Reserve Water from Apoplast to Symplast. As
already pointed out, although OA parallels the TA during acclimation, it cannot explain the TA, since there is no net water
uptake from the environment. Another mechanism is, however,
conceivable. The OA of the leaves occurs in the protoplasts of
the cells (and therefore the symplasm of the leaf). It may,
therefore, be proposed that the TA is due to movement of reserve
water from the apoplast (the part of the leaf outside the protoplasts) to the symplast, as a result of the water potential gradient
induced by the OA. There are two components of apoplast water:
(a) the cell wall water, and (b) the water contained in the vessels.
Cell Wall Component ofApoplast Water. When the leaf is first
excised, it wilts rapidly (0.5-3 h) due to the large water potential
150
100
"
90
6(4)
80
3:
3(2)
OAd
-0 t
2(4)
60
30
60
90
70
O
4(2)
.-
0
FIG. 2. Relation of turgor adjustment to rate of dehydration, as shown by rate of decrease of RWC. Broken
line, wilted; solid line, turgid.
120
1
60
150
Time (hours)
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150
LEVITT
Table II. Limiting Rate of Dehydration (as Measured by Decrease in
R WC) Permitting TA (Turgor Recovery in the Absence of Water
Uptake)
No TA if dehydration rate is more rapid. First column is time to attain
corresponding RWC.
Time
RWC
Turgor
h
%
0
100
Turgid
1-2
90
Limp
24
79
Limp
48
75
Limp
72
73
Turgid
96
71
Turgid
120
69
Turgid
Table III. Relation of Plant Age to Recovery of Turgor by Wilted
Excised Leaves during Continued Slow Loss of Water (TA)
Oldest leaves (I of 47-d and leaves 1 and 2 of 54-d plant) were
senescent (chlorotic) and therefore not used.
% RWC
Leaves
Leaves Showing
Age
At lh
At TA
Used
TA
wilting
(3-6 d)
d
32
1-6
86-93
64-69
None
40
1-6
89-95
70-88
1, 2
47
2-7
84-89
75-77
2, 3, 4, 5
54
3-8
91-96
75-80
3, 4,5,6,7
between the leaf and its environment. Since both the apoplast
and the symplast are initially at water saturation, both must lose
water during this wilting, and water must move from symplast
to apoplast to environment. On transfer to the leaf chamber, the
gradient between leaf and environment decreases markedly and
the water loss from the leaf drops to a very low value. With
acclimation, OA begins to occur in the protoplasts, reversing the
water potential gradient (and therefore the water flow) between
apoplast and symplast. At TA, therefore, it may be proposed that
there is no net loss of water from the symplast, and the total net
loss from the leaf would be from the apoplast. This means that,
since the loss of water from the TA leaf is as much as 25% of
the RWC in the originally turgid leaf, the apoplast would have
to account for all of this loss of 25% of the total symplast plus
apoplast. At the cell level, the apoplast is the hydrated cell wall
and the symplast is the protoplast. Therefore, either the hydrated
cell wall of the living cell, or the vessel water would have to
account for this 25% of the turgid cell. Simple visual observation
of the living, turgid cells, especially after vital staining of the
vacuole with neutral red, leads to the conclusion that the actual
value for cell wall water is less than 10%, and that therefore, this
component of the water reserve in the apoplast is far too small
to account for the turgor recovery. This conclusion is supported
Time
h
0
3
48
96
Plant Physiol. Vol. 82, 1986
by the fact that the outer cell walls at the two leaf surfaces are
cutinized and therefore, hydrophobic and nonhydrated. Those
walls that are hydrated must, therefore, carry the greater part of
the burden.
It is possible to test this conclusion semiquantitatively. Since
the transfer of water from apoplast to symplast would be passive,
it should be fully and quickly reversible. Therefore, acclimated
leaves showing TA after 2 to 4 d wilting, should be able to regain
their original 100% RWC as rapidly as briefly wilted leaves that
have not been permitted to acclimate and recover their turgor.
Figure 4 shows that recovery of 100% RWC does occur in the
TA leaves when submerged and their petioles excised in water,
but much more slowly than in the control, briefly wilted leaves
(Fig. 3). In fact, due to the OA, which is retained for at least 24
h after regaining full turgor (3), the turgor of the acclimated
leaves should exceed the original at excision and so should the
RWC. In agreement with the above conclusion this does not
happen.
Vessel Water and Cavitation. Another component of the apoplast water which may conceivably contribute to the flaccid
symplast is the vascular water, which is said to be as much as 10
to 20% of the water content of some plants. Wilting may conceivably induce sufficient tension to cause cavitation of this vessel
water, releasing it to the symplast. The question is whether this
vascular reserve water is sufficient to account for the above TA
in cabbage leaves.
Cabbage plants produce a rosette of leaves close to the root
system. They, therefore, develop far less conducting tissue for
the transfer of water and solutes than in the case of trees or tall
herbaceous plants. Furthermore, in the above experiments the
plants were grown under minimal water stress conditions (low
light intensity relative to sunlight, and pots standing in nutrient
solution). Since droughting is known to increase the proportion
of vascular tissue (3), the nondroughted leaves may be expected
to develop minimal vascular reserves of water. Also, since excised
leaves were used, air must have entered the vessels at the cut
surface of the petioles as soon as wilting occurred (within minutes), leaving even less of a vascular water reserve for later TA.
Simultaneously, the vascular water rose from the petiole and
entered the living flaccid cells of the blade. In view of these
considerations, the vascular water of the above cabbage leaves
must not be significant relative to the 25% loss of water preceding
TA.
In agreement with the above conclusions, simple observation
of the cabbage leaf with the naked eye reveals a small fraction of
colorless vein tissue (only part of which is vessels) relative to the
green, nonvascular islands between the veins. A more quantitative estimate can be obtained with the aid of dye solutions. When
the petiole of a wilted cabbage leaf is submerged in dye solution
of high concentration (1%) and excised, all the veins become
stained within minutes if it is a basic dye (pyronin-B), and
therefore before an appreciable recovery from wilting. If an acidic
dye (acid fuchsin) is used, all the leaf except the veins becomes
visibly stained, also within minutes and before recovery from
Table IV. Osmotic Adjustment during Acclimation, Accompanying
Leaf No.
2
3
4
RWC Turgor O.P. RWC Turgor O.P. RWC Turgor
%
MPa
%
MPa
%
100
Turgid 1.10
100 Turgid 1.16
100 Turgid
91
88 Limp
Limp
92 Limp
86
86 Turgid 1.29
Turgid 1.23
92 Turgid
72
83 Turgid 1.60
Turgid 1.66
91
Turgid
Turgor Recovery in the Absence of Water Uptake
5
O.P.
MPa
1.16
1.35
1.72
RWC
%
100
92
90
84
Turgor
Turgid
Limp
Turgid
Turgid
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6
O.P.
MPa
1.16
1.35
1.72
RWC
%
100
93
90
85
Turgor
Turgid
Limp
Turgid
Turgid
O.P.
MPa
1.10
1.35
1.77
151
RECOVERY OF TURGOR IN THE ABSENCE OF WATER UPTAKE
0
24
0
40
80
120
the whole petiole is therefore:
160
I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
I I
3(4)
100
100
11(4)
x
(102)2
0.16 x
10-2
(2.5)2
These results clearly show that the total water of all the vessels
in the cabbage petiole is less than 1% of the total petiole water.
Observation of the leaf blade reveals a similar relation.
90
All of the above results point to the same conclusion. Although
trees and certain kinds of herbaceous plants (e.g. some xerophytes) may have a considerable amount of their water content
in the vessels, this is not true of the leaves of cabbage seedlings.
8
0
801
Their vascular water content is a negligible fraction of the total,
and cavitation can play no role in the TA of cabbage leaves.
The above results indicate that transfer of reserve water from
apoplast to symplast is not the mechanism of TA.
b. Metabolic Loss of Dry Matter and Gain of Water. Loss of
70
Dry Matter. It may be suggested that the loss of fresh weight is
due to loss, not of water, but of dry matter. Since TA is possible
during a loss of as much as 25% of the RWC, and therefore of
its fresh weight, and since the dry matter (less than half of which
60 I
is probably respirable) accounts for only 10% of the fresh weight,
24
0
0
40
80
120 160
the maximum respiratory loss of dry weight is an order of
Time (h)
Time (mn)
magnitude too small to account for the loss in fresh weight.
Accumulation of Metabolic Water. It may be suggested that
FIG. 3. Rapid recovery of full turgor (100% RWC) by wilted unacclimated leaves after submerging in water and excising submerged petiole. the evaporative loss of water is counterbalanced by an accumulation of water produced in the respiratory process. From the
wilting. In both cases, this means that the total water content of relation:
the vessel lumena has moved ahead of the dye solution, into the
C6H1206 + 602 6 CO2 + 6 H20
symplast. Yet this total vascular water was insufficient to induce
recovery of leaf turgor, or even an observable decrease in the for every 72 g dry matter lost (02 gained - CO2 lost), 108 g
degree of wilting. It was only after some hours in the dye solution metabolic water are formed, or 1.5 g H20 per g dry matter lost.
(or water) that the leaf recovered its turgor.
Since TA occurs during a loss of as much as 25% RWC,
The reason for these perhaps unexpected results is to be found replacement of this loss by metabolic water would require a
in the simple anatomy of the cabbage leaf. Cross-sections of respiratory loss of dry matter equal to 17% (0.7 x 25%) of the
petioles 2.5 mm in diameter revealed 3 to 5 vascular bundles fresh weight of the leaf, or (since the total dry matter is actually
with a total of about 100 vessels whose lumens averaged 10 gm about 10% the fresh weight) 1.7 x the total dry matter. Furtherin diameter. Since both the petioles and the vessels are cylindrical more, some of the leafs water must be utilized as a result of OA,
in shape, their volumes and therefore their water content in each since this involves hydrolysis of starch to sugar (3). Therefore,
case is proportional to the cross-sectional area and therefore:
the maximum amount of metabolic H20 is far too small to
account for TA.
c
c
V
d2
W,.
These theoretically based conclusions can be tested experimenwhere W, = water content, V = volume, d = diameter of vessel. tally. If respiratory loss of dry matter, and reproduction of water
The total water content of the 100 vessels, relative to that of play a role, then there should be a decrease in percent dry matter
-.
0
24
48
72
96
0
40
80
120 160 200
9(4) 100
4(4)
100
T
90
a
.
6(4)
90
FIG. 4. Slow
l-,
tcx
80 -A
80
excising petiole.
..
-
--
70 _
60
70
I
0
=24
48
Time (h)
72
96
0
40
80
120 160 200
recovery
of full turgor by wilted,
acclimated leaves after submerging in water and
60
Time (min)
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152
Plant Physiol. Vol. 82, 1986
LEVITT
during acclimation. When the percent dry matter (usually 812%) was determined for a series of leaves on the same plant, it
was found to increase with leaf number (or decrease with age).
When some of the leaves are allowed to wilt and undergo TA for
several days, their dry matter contents showed the same relation
when compared to leaves whose dry matter was determined
immediately on excision from the plant (Table V). Therefore,
from both theoretical considerations and experimental tests it
must be concluded that metabolic water is not the explanation
of TA.
In all the above evaluations of the possible role of metabolism
in TA, conditions for maximum respiratory effect are assumed,
specifically the absence of photosynthesis although the leaves
were illuminated.
From all the above evidence, it must be concluded that TA
cannot be explained on the basis of metabolic loss of dry matter
or gain of water.
Wall Adjustment. The turgor pressure of the leaf cells can be
increased in two ways: (a) by uptake of water, (b) in the absence
of water uptake, by decreasing the volume enclosed by the cell
wall.
It has long been known that growth by cell enlargement is the
result of a plastic (nonelastic and, therefore, irreversible) wall
expansion. This involves a rupture of some of the wall's intermolecular bonds accompanied by the insertion of other molecules (intussusception). It is, therefore, conceivable that the
drought acclimating cell can reverse this process and induce a
wall contraction by either removing particles or producing folds
in the wall, and in either case, forming new intermolecular bonds,
and increasing the elastic modulus. This would result in an elastic
tightening of the wall around the partially dehydrated and contracted protoplasts of the wilted leaf, and therefore a TR in the
absence of water uptake. This concept may be called the WA
hypothesis of TR by a wilted plant or its leaves during acclimation, in the absence of water uptake.
The WA hypothesis clearly explains the above found inverse
relation between TA and growth. Since growth-associated intus-
susception and wall contraction cannot occur simultaneously,
the growth process and TA due to drought acclimation must also
be mutually exclusive. There are two predictions of this hypothesis that may be readily tested experimentally.
(a) The wall adjusted cells in the acclimated leaves are turgid,
and therefore have elastically extended cell walls, though at a
RWC well below the original 100% before wilting. When allowed
to reabsorb water, the increased modulus of elasticity of the walls
of the WA cells (see above) must reduce the expansion and,
therefore, prevent the recovery of the leafs original 100% RWC.
If, however, the turgor pressure developed is sufficient to gradually break the new bonds formed during acclimation, the increased modulus of elasticity will be temporary and will only
retard recovery.
(b) The leaf that has recovered its turgor without the uptake
of water, must have a smaller volume than when turgid before
excision.
The first of these predictions can be tested by submerging both
the acclimated and unacclimated leaves in water and excising
their petioles. This technique has been shown to permit very
rapid rehydration of wilted cabbage leaves to maximum RWC
(5). If the fully turgid, acclimated leaf failed to recover its full
original 100% RWC or recovered it very slowly, this would be
in agreement with the hypothesis. In the case of unacclimated
leaves wilted quickly (in 4 h) to a RWC of 78%, full turgor and
100% RWC were recoverd within 40 min (Fig. 3). In the case of
acclimated leaves, which showed TA in 3 d after wilting to 85%
or 76% RWC, recovery of nearly 100% RWC did occur (Fig. 4),
but only after 170 min. After longer periods of TA, full recovery
of 100% RWC did not occur even after reabsorption of water
for 275 min. This slow recovery of the original RWC may
indicate that the WA by shrinking and tightening does indeed
occur, but that it is slowly reversible when subjected to a sufficiently high TA.
Control leaves show a slow increase in RWC even beyond
100% (Table V), when floated on water with the petiole surface
submerged. This increase is presumably due to continued, slow
Table V. Dry Matter Content and R WC of Leaves Immediately on Excision from the Saturated Plant and
after 5 to 7 Days in Leaf Chambers Leading to TR
Dry Matter
RWC
Leaf No.
Immediate on
excision
After
5-7 d and TA
At
wilting
At TA
At full
turgor due to
water uptake
A. 53-d plant
2
3
4
5
6
7
8
2
3
4
5
6
7
8
9
10
11
8.3
9.2
10.6
11.1
90
90
90
80
80
75
11.5
11.9
B. 60-d plant
90
90
80
81
9.1
10.1
9.9
86
82
85
81
79
81
10.3
10.3
89
93
91
75
80
12.0
8.0
9.6
9.9
11.1
10.2
77
100 (on excision)
109 (floated 24 h)
110 (floated 24 h)
ll0(floated24h)
100 (on excision)
99(floated2 h)
104 (floated 2 h)
100 (floated 4.5 h)
100(floated20h)
118(floated20h)
113 (floated 20 h)
102 (floated 4.5 h)
102 (floated 4.5)
115 (floated 20 h)
119 (floated 20 h)
117(floated20h)
101 (floated 4.5 h)
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RECOVERY OF TURGOR IN THE ABSENCE OF WATER UPTAKE
Table VI. Decreases in R WC and Leaf Thickness
Excised, fully turgid leaves were allowed to wilt followed by TA (turgor
recovery in the absence of water uptake).
Decrease in RWC and Leaf Thickness
Leaf No.
49-d plant
56-d Plant
62-d Plant
Leaf
Leaf RWC
Leaf
RWC thickness
thickness RWC thickness
2
3
4
5
6
7
8
9
37
28
28
21
19
21
14
11
15
17
11
21
35
24
24
22
22
20
18
7
11
11
13
12
35
27
27
25
28
31
23
10
19
19
26
26
cell enlargement when subjected to full turgor pressure. In view
of the OA, the walls of the turgid acclimated cells are now
subjected to a higher turgor pressure than in the unacclimated
cells and, therefore, may be expected to stretch more than the
unacclimated cells, if the elastic modulus is unchanged. In point
of fact, the acclimated stretch less (Figs. 3 and 4), in agreement
with the elastic change postulated by the WA hypothesis.
Direct measurements of cell volume, or even of leaf volume,
are not easily made without damaging the leaf or changing its
degree of hydration. Leaf volume, however, is the product of leaf
area and leaf thickness. Due to the rather heavy rigid cuticle on
mature cabbage leaves, area changes are likely to be small. This
was corroborated by preliminary measurements. Changes in leaf
volume must, therefore, involve significant changes in leaf thickness. This is easily measured by micrometer calipers.
Table VI shows that TA at RWCs of approximately 65 to 80%
(or a dehydration of 20-35%) was accompanied in all cases by a
decrease in leaf thickness within a range of about 10 to 30%. As
expected, the dehydration was greater than the decrease in leaf
thickness, since there must have been some decrease in leaf area,
especially in the younger plant with less cuticle and therefore
presumably more elastically extensible leaves. Since the measurements were made on leaf area free of veins, these results show
that there was no uptake of water by the mesophyll and other
living cells, in opposition to the cavitation hypothesis but in
agreement with the WA hypothesis.
Both the above tests, therefore support the WA hypothesis of
TA in the absence of water uptake. TA may conceivably be an
important adaptation to drought-induced water stress only when
this is moderate and relatively short-lived. The TA developed
during acclimation could, for instance, permit the plant to continue net photosynthesis, growth, and development by maintaining turgor and presumably stomatal opening at dehydration that
would not permit these processes in the unacclimated plant due
to wilting. Even if dehydrated severely enough to induce wilting
153
in acclimated plants, a light shower may raise their RWC to a
level sufficient for TR in the acclimated but not in the nonacclimated plants.
The above results fully establish the occurrence of TA, and
point to WA as the mechanism of TR in the absence of water
uptake. Just how this WA would be induced can only be guessed
at in the absence of more direct evidence. The postulated wall
contraction, for instance, may not necessarily be by means of
the reverse intussusception suggested above. The WA could
conceivably be achieved by intermolecular bonding between
small, pinched off folds in the wall of the flaccid cell, converting
it to the elastically stretched and, therefore, the turgid state. One
possible mechanism is by the secretion oflectins at the protoplast
surface. Due to the well-known ability of lectins to agglutinate
carbohydrate groups, they may conceivably pinch together parts
of the flaccid wall, thus tightening the wall surrounding the
protoplast. It is interesting to note that Siminovitch (7) observed
such a folding in the plasma membrane of freeze-acclimated
cells.
CONCLUSIONS
On the basis of the above analysis, it must be concluded that:
(a) The reserve water in the apoplast cannot account for TA.
(b) Respiratory loss of dry matter and accumulation of water
cannot account for TA.
(c) WA can account for TA and appears to be the only
plausible explanation.
There are, therefore, two methods of TR available to the wilted
leaf:
(a) By absorbing enough water osmotically to stretch the
flaccid cell wall elastically. This is the only method available to
the nonacclimated plant.
(b) In the absence of water uptake (and even when accompanied by a slow loss of water) by shrinking and tightening its
cell walls around the partially dehydrated and therefore contracted protoplasts. This may be called WA and it is proposed to
occur during drought acclimation.
LITERATURE CITED
1. CLARK JA, J LEVITT 1956 The basis of drought resistance in the soybean plant.
Physiol Plant 9: 598-606
2. HSIAo TC Plant responses to water stress. Annu Rev Plant Physiol 24: 519570
3. LEvTrr J 1980 Responses of Plants to Environmental Stresses. Academic Press,
New York
4. LEvlrr J 1983 Plasmolysis shape in relation to freeze-hardening of cabbage
plants and to the effect of penetrating solutes. Plant Cell Environ 6: 465470
5. LEvrrT J 1985 Relation of dehydration rate to drought avoidance, dehydration
tolerance, and dehydration avoidance of cabbage leaves, and to their acclimation during drought induced water stress. Plant Cell Environ 8: 287-295
6. MAXIMov NA 1929 The Plant in Relation to Water In RH Yapp, ed, Allen
and Unwin, London, p 62
7. SIMINOVITCH D, B RHLAUME, K POMEROY, M LEPAGE 1968 Phospholipid,
protein, and nucleic acid increases in protoplasm and membrane structures
associated with development of extreme freezing resistance in black locust
tree cells. Cryobiology 5: 202-225
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