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ORIGINAL PAPER
Stress responses in Salix gracilistyla cuttings subjectedto repetitive alternate flooding and drought
Arisa Nakai • Yutaka Yurugi • Hiromitsu Kisanuki
Received: 9 December 2009 / Revised: 13 July 2010 / Accepted: 30 July 2010 / Published online: 14 August 2010
� Springer-Verlag 2010
Abstract To determine the tolerance of Salix gracilistyla
to repetitive alternate flooding and drought, we measured
leaf stomatal conductance, pre-dawn water potential,
osmotic adjustment, and biomass production under green-
house conditions. We used a control and nine crossed
treatments (F1-D1–F3-D3) in which we combined 1-, 2-, or
3-week floodings (F) and droughts (D). Leaf stomatal
conductance was lowest in 3 weeks of flooding or drought
when the preceding event (flood or drought) was also of a
3-week duration. Leaf pre-dawn water potential was
reduced in 3 weeks of drought when preceded by 2 or
3 weeks of flooding. Cuttings had slight osmotic adjust-
ments in repetitions of long floodings and droughts. During
longer durations of drought in crossed experiments, plants
had low root and shoot mass, few hypertrophic lenticels,
and reduced leaf mass; when flooding duration increased in
crossed experiments, root mass was reduced, there were
more hypertrophic lenticels, and the leaf area was reduced.
Cuttings achieved stress tolerance by inhibition of tran-
spiration, osmotic adjustment, reduction of transpiration
area, and development of hypertrophic lenticels. Stress
tolerance was weak when repetitive 2- or 3-week floodings
were combined with 3-week droughts. The duration of
flooding and drought periods under which S. gracilistyla
achieves stress tolerance may be critical in determining
distributions along riverbanks.
Keywords Drought tolerance � Flooding and drought
treatment � Flooding tolerance � Leaf pre-dawn water
potential � Leaf stomatal conductance � Osmotic adjustment
Introduction
Gravel bars along rivers experience variable degrees of
flooding that affect plant distribution and growth. Flood-
plain ground surfaces are frequently submerged through
water level fluctuations induced by rainfall in upper
watershed lands. In general, ground surface flooding is
followed by soil oxygen depletion as plant roots and
microorganisms respire (Ponnamperuma 1972). Replen-
ishment is slow because gas diffusion rates in water are
orders of magnitude lower than in aerated soil (Armstrong
and Drew 2002; Jackson 1985). Ground surface flooding
negatively affects plant growth through reduction in leaf
stomatal conductance and net photosynthetic rate (Li et al.
2004; Pezeshki et al. 1998), cessation of root growth
(Kozlowski 2000), and growth inhibition (Jackson and
Attwood 1996; Pezeshki et al. 1998). When flood-tolerant
plants experience flooding, they show some anatomical
modifications, such as the development of aerenchyma and
hypertrophic lenticels, excess stem hypertrophy at the
water surface, and development of adventitious roots
(Crawford 1982; Pezeshki 2001). These anatomical modi-
fications may contribute to root aeration and removal of
toxic ethanol accumulated by oxygen deficiency (hypoxia)
in roots during flooding (Crawford 1982). Flood-tolerant
plants also have a metabolic adaptation to oxygen defi-
ciency, i.e. the absence of the induction of alcohol dehy-
drogenase, which is a catalyst of ethanol production
through increased concentrations of acetaldehyde during
anaerobiosis (Larcher 1980). Furthermore, when the root
Communicated by E. Beck.
A. Nakai (&) � Y. Yurugi � H. Kisanuki
Graduate School of Bioresources, Mie University,
1577 Kurimamachiya, Tsu 514-8507, Japan
e-mail: arisan@bio.mie-u.ac.jp
123
Trees (2010) 24:1087–1095
DOI 10.1007/s00468-010-0481-2
apex is subjected to hypoxic conditions, the synthesis and
translocation of hormones are altered, causing hormonal
imbalance (Crawford 1982). Increased concentrations of
ethylene and abscisic acid have been reported in many
flood-tolerant species (Crawford 1982; Jackson 1991;
Yamamoto et al. 1995a, b). Ethylene acts as a morpho-
logical controller of anatomical modification (Crawford
1982), and abscisic acid leads to stomatal closure
(Milborrow 1974).
In contrast, soil becomes desiccated during long periods
with little rain, low water levels, and absence of flooding.
Protracted periods without flooding lead to lower substrate
water content (Schaff et al. 2003), which declines with
distance from the river waterline (Nakai and Kisanuki
2007a, b; Williams and Levine 2004). Substrate drought
also decreases leaf stomatal conductance and net photo-
synthetic rate, leading to plant growth inhibition (Pezeshki
et al. 1998). Drought-tolerant plants respond through
efficient water absorption, inhibition of transpiration, and
increased hydraulic conductivity to enhance water use
efficiency (Larcher 1980; Wikbergi and Ogreni 2007).
Effects of flooding and drought on tree growth have
been reported in many previous studies (e.g. Li et al. 2004;
Pezeshki et al. 1998). Plants growing near riverbanks are
repeatedly subjected to periods with and without ground
surface flooding, depending on water level fluctuations
(Nakai and Kisanuki 2007a). Trees on gravel bars along
rivers are subjected to repetitive waterlogging and drought.
Moreover, cycles of repetitive flooding and drying may be
interrupted, and these interruptions also affect tree growth
(Pezeshki et al. 1998). Responses to interruptions in
flooding or drought rhythms have been examined in some
salicaceous species. For example, when surface flooding is
interrupted, Salix nigra Marshall does not increase root
porosity, a common plant response to flooding (Pezeshki
et al. 1998). These observations suggest that plant
responses to temporary releases from flooding or drought
are different from responses to protracted waterlogging or
drying out. Therefore, plants may respond to the rhythm of
flooding or drought in addition to responding to each factor
separately.
Ground surfaces along rivers, especially low elevation
sites near the waterline, frequently suffer from repeated
flooding and drought (Nakai and Kisanuki 2007a). How
then do plants, especially those growing near the waterline,
deal with repetitive flooding and drought? Plants experi-
encing drought may increase water absorption efficiency
by allocating more resources to their roots to promote root
elongation deeper into the soil (Marron et al. 2003).
However, when plants whose root growth is inhibited by
flooding (Kozlowski 1997, 2000) are exposed to desiccat-
ing conditions, growth inhibition may be greater than in
plants grown solely in dry soil. Growth inhibition caused
by soil waterlogging may be greater in plants transferred
from desiccating conditions than in those continually
maintained in flooded soil.
Salix gracilistyla Miquel, one of the major components
of riverside vegetation in Japan, is found on coarse gravel
substrates near the waterline more often than other Salix
species (Ishikawa 1982, 1997; Niiyama 1987). Thus,
S. gracilistyla may be expected to have adaptations to
repeated cycles of flooding and drought. The objective of
this study was to determine the mechanisms by which
S. gracilistyla deals with the soil water cycle: (1) Does
S. gracilistyla maintain drought tolerance through incre-
asingly protracted periods of flooding? (2) Does S. gracili-
styla maintain flood tolerance through increasingly
protracted periods of drought?
Materials and methods
Plants
Cuttings of Salix gracilistyla Miquel were collected from
1-year-old shoots of three genets in an established S. gra-
cilistyla population growing on a gravel bar in the Miya
River, Mie Prefecture, central Japan. The 1-year-old shoots
were cut to lengths of 20 cm. The diameters at the centers
of the cuttings ranged from 6.6 to 9.8 mm (mean ±
SD = 8.0 ± 0.8 mm).
Experimental procedures
Cuttings were planted in a greenhouse on 11 March 2007.
Pots (20 cm high and 18 cm in diameter) were filled with a
2:1 sand (particle size less than approximately 2–3 mm):
sandy soil (particle size of 90% of the substrate was
106–850 lm, including a small amount of organic matter)
substrate mixture. A single cutting was planted in each pot,
with 10 cm of each shoot buried below pot soil level. At
planting, no cutting had leaves, shoots, or roots. Natural
light in the greenhouse provided a photon flux density
(PPFD) of 1,000–1,200 lmol m-2 s-1 at the tips of the
cuttings. The average temperature in the greenhouse was
23.7 ± 3.7�C (maximum 30.6�C, minimum 14.8�C) during
the experiment. Cuttings were maintained under well-
watered conditions for 4 weeks to promote establishment.
To determine the response of S. gracilistyla cuttings to
repetitive soil flooding and drought, treatments were
applied in which these factors were crossed. Nakai and
Kisanuki (2007a) showed that the longest period without
flooding and rain during the growing period (214 days) was
approximately 3 weeks (24.3 days) at the highest elevation
on a riverbank where current-year seedlings of S. gracili-
styla survived until autumn; and at this elevation, the
1088 Trees (2010) 24:1087–1095
123
longest continual flooding was \1 week in a low water
level year. Thus, the longest duration for flooding or
drought was set at 3 weeks. There were nine treatments and
a control. The treatment durations were 1, 2, and 3 weeks,
designated F1, F2, and F3, for flooding treatments, and D1,
D2, and D3, respectively, for drought treatments. The nine
treatment combinations were set as follows: F1-D1, F1-D2,
F1-D3, F2-D1, F2-D2, F2-D3, F3-D1, F3-D2, and F3-D3,
corresponding to combinations of flooding and drought
durations. Switching between flooding and drought treat-
ments was repeated during the course of the experiment. In
flooding treatments, pots were filled with water to com-
pletely cover the soil surface, and 400–600 ml of water was
added approximately once a week before the substrate
surface emerged. In drought treatments, water was with-
held. In the control, cuttings were watered with[2,400 ml
daily and allowed to drain freely. The experiment ran for
119 days (17 weeks) from 9 April to 6 August 2007.
There were ten replicate cuttings (comprising four, three,
and three ramets from each of three genets) per treatment,
with a total of 100 cuttings. The average diameter of cut-
tings at the center point and average fresh weight did not
significantly differ among treatments and the control at the
outset [diameter df (9, 90), F = 0.382, P = 0.94; fresh
weight df (9, 90), F = 0.712, P = 0.697, ANOVA, ns].
Pots were arranged randomly regardless of treatment.
Substrate measurements
Substrate redox potential (Eh) was monitored weekly
5–10 cm below the soil surface using an Eh meter
(EHS-120, Fujiwara Factory, Japan) for all ten treatments.
This measurement occurred in the same three fixed pots in
each treatment throughout the experiment. The substrate
volumetric water content (SWC) was monitored 0–12 cm
below the surface every week using a Hydro Sense probe
(CS-620, Campbell Scientific, Inc., Australia) for five
cuttings in drought and control conditions.
Physiological responses of cuttings
Leaf stomatal conductance (gs) was measured using a leaf
porometer (SC-1, Decagon Devices, Inc., USA) every
week at 10:00–13:00 h in all treatments. Three sufficiently
developed leaves of one shoot from each of three cuttings
for each treatment were used for measurements.
Using a pressure chamber (model 600, PMS Instrument
Company, USA), we measured pre-dawn leaf water
potential (Wwpd) at the end of the first or second cycle of
drought treatments in all treatments other than the control
from 1 to 19 May 2007 (corresponding to days 22 and 40 of
the experiment). Wwpd was measured in controls on 1 May
(22 days after onset). Two cuttings from each of three
genets (six cuttings in total) were measured per treatment.
For each cutting, the third largest shoot was selected for
measurement.
The various components of osmotic adjustment, water
potential at turgor loss point (Wwtlp) and osmotic potential
at full turgor (Wosat), were obtained for each treatment
from the P–V curve at the conclusion of the experiment.
Leaf samples were cut in water and placed in a bucket of
water overnight to promote water absorption; thereafter, we
collected data for construction of P–V curves (Maruyama
and Morikawa 1983). Measurements were replicated four
times (one shoot from each of four cuttings) per treatment.
Plant growth
For each cutting, all shoots other than the two largest were
removed 34–40 days after onset of the experiment, termi-
nating with Wwpd measurement. At the conclusion of the
experiment, the numbers of attached and defoliated leaves
were counted for all shoots, and the cuttings were divided
into stem, branches, leaves, hypertrophic lenticels, and root
biomass. Hypertrophic lenticels were sliced off from the
stem with a knife. The dry weights of all biomass com-
ponents were obtained after drying at 80�C for 48 h. Leaf
area was also measured at the conclusion of the experi-
ment. Of the largest mature leaves (those that had devel-
oped sufficiently on the upper half of each shoot), 3–12 on
each of the six longest shoots in each treatment were
selected for measurements. Three leaves were selected per
shoot, but more than three leaves were selected when the
sizes of leaves on a shoot differed greatly.
Data analysis
Growth was determined based on the difference between
initial and final dry weight. The significance of differences
in gs, Wwpd, Wwtlp, and Wosat between treatments and
controls was tested with Dunnett’s t test. To analyze the
effects of flooding (F) and drought (D) duration on gs,
Wwpd, Wwtlp, Wosat, and plant growth, we used two-way
ANOVA (with F and D as factors), followed by Bonferroni
adjustment or Tukey’s HSD when significant or non-
significant F–D interactions were detected, respectively.
The significance level adopted was 5%, and analyses were
conducted with SPSS (Ver. 10.0) for Windows.
Results
Substrate measurements
Substrate redox potential (Eh) in controls was [524 mV
throughout the experiment, indicating aerobic conditions.
Trees (2010) 24:1087–1095 1089
123
In all flooding treatment combinations, except F1-D3, Eh
decreased during the second half of the experiment. Upon
flooding, Eh often decreased to \350 mV in combination
with long flooding (F) and short drought (D) durations, i.e.
F2-D1, F3-D1, and F3-D2, indicating hypoxic conditions.
Substrate volumetric water content (SWC) in controls
averaged 22.4% (range 16.2–25.9%) throughout the
experiment and varied remarkably with F and D treatment
combinations. In the final week of drought treatment, SWC
decreased to 9.2–21.3% in the three D1 repetitions,
6.8–10.0% in D2 repetitions, and 6.0–7.0% in D3 repeti-
tions. The SWC differed significantly among C, D1, D2,
and D3 (Tamhane, P \ 0.01).
Physiological responses of Salix gracilistyla cuttings
Leaf stomatal conductance (gs) of cuttings was lower in
most of the treatment combinations compared to the con-
trol (Dunnett’s t test, P \ 0.05, Fig. 1). In most cuttings, gs
decreased after D treatments and then recovered fully
during the first half of the experiment in all F treatments of
some treatment combinations (Fig. 1a, c–e); however, gs
did not fully recover in any treatment combination during
the second half of the experiment. In particular, gs of
cuttings in the three D3 repetitions decreased markedly to
26.0% (mean) of control values (range 12.2–42.2%) by the
third week of drought treatment. In some combinations, gs
decreased during D treatments (Fig. 1a, b, g, i).
Two-way ANOVA with F and D as treatment factors
demonstrated a significant F 9 D interaction and signifi-
cant main effects of F and D on gs in the final week of the
final D treatment [gs(D)], on Wwpd at the end of D
treatments, and on Wosat (P \ 0.05, Table 1). Two-way
ANOVA results also revealed a significant F 9 D inter-
action and a significant main effect of D (P \ 0.05) on gs
in the final week of the final F treatment [gs(F)] and on
Wwtlp.
The gs(D) in F3-D3 decreased remarkably to 12.2% of
that of the control, while values in other treatment combi-
nations decreased to 34.6–54.0% of control values
(Fig. 2a). In F3 repetitions, gs(D) was lower in D3 than in
D1 or D2 (significant two-way interaction using Bonferroni
adjustment, P \ 0.05). In D3 repetitions, gs(D) was lowest
in F3 (P \ 0.05). In D1 or D2 repetitions, there were no
differences in gs(D) among treatment combinations with
different durations of F (P [ 0.05). gs(F) values in F3-D3
were remarkably low, viz. 28.1% of control values
(Fig. 2b). Under F1 or F2 repetitions, gs(F) was lower in D2
than in D1 (P \ 0.05), while under F3 repetition, gs(F) was
lowest in D3 among the three durations of D (P \ 0.05).
Wwpd was high in controls: -0.018 ± 0.008 MPa.
Wwpd of cuttings under D1 and D2 combinations did not
decrease and did not differ among combined various
flooding durations (Fig. 3). Wwpd in F2-D3 and F3-D3 was
-1.28 ± 0.71 MPa and -1.54 ± 0.20 MPa, respectively,
both of which were significantly lower than control values
(Dunnett’s t test, P \ 0.05), indicating that cuttings suf-
fered from water stress. In D3 repetitions, Wwpd was lower
in F2 and F3 than in F1 (two-way interaction after
Bonferroni adjustment, P \ 0.05).
Wwtlp and Wosat values for control cuttings were
-1.27 ± 0.10 and -0.92 ± 0.05 MPa, respectively. These
values were higher than those in all treatment combina-
tions except F1-D1 (Dunnett’s t test, P \ 0.05, Fig. 4a, b),
Fig. 1 Response of leaf
stomatal conductance in Salixgracilistyla cuttings. Response
of leaf stomatal conductance is
represented as a percentage of
the control value. Values are
mean ± standard deviation for
nine leaves taken from three
cuttings (three leaves per
cutting). Closed and opensquares represent flooding and
drought treatments,
respectively. Open circlesrepresent the control. Black and
gray bands at the bottomrepresent the duration of
flooding (F) and drought
(D) treatments, respectively.
Asterisks indicate significantly
different from the control
(Dunnett’s t test, P \ 0.05)
1090 Trees (2010) 24:1087–1095
123
indicating osmotic adjustment of cuttings in these combi-
nations. In F1 repetitions, Wwtlp and Wosat were lower in D2
and D3 than in D1 (two-way interaction after Bonferroni
adjustment, P \ 0.05). There were no significant differ-
ences in either Wwtlp or Wosat among different durations of
D treatment in the F2 or F3 repetitions (P [ 0.05).
Table 1 Two-way ANOVA of the physiological response of Salixgracilistyla cuttings
Source of variation df F P
gs(D)
Duration of flooding 2, 72 5.037 0.009
Duration of drought 2, 72 13.917 \0.001
Flooding 9 drought 4, 72 8.101 \0.001
gs(F)
Duration of flooding 2, 72 0.054 0.947
Duration of drought 2, 72 7.703 0.001
Flooding 9 drought 4, 72 8.436 \0.001
Wwpd
Duration of flooding 2, 50 16.197 \0.001
Duration of drought 2, 50 68.602 \0.001
Flooding 9 drought 4, 50 22.676 \0.001
Wwtlp
Duration of flooding 2, 30 2.154 0.134
Duration of drought 2, 30 5.697 0.008
Flooding 9 drought 4, 30 5.757 0.001
Wosat
Duration of flooding 2, 30 3.812 0.033
Duration of drought 2, 30 4.115 0.026
Flooding 9 drought 4, 30 6.342 0.001
gs(D) leaf stomatal conductance in the final week of the final drought
treatment, gs(F) in the final week of the final flooding treatment, Wwpd
pre-dawn leaf water potential at the end of the first or second cycle of
drought treatments, Wwtlp water potential at turgor loss point, and
Wosat osmotic potential at full turgor
Fig. 2 Leaf stomatal conductance in Salix gracilistyla cuttings. gs(D)
(a) and gs(F) (b). Leaf stomatal conductances are expressed as
percentages of the control values. Values are mean ? standard
deviation of nine leaves from three cuttings (three leaves per cutting).
Different letters represent significant differences among treatment
combinations (two-way interaction using Bonferroni adjustment,
P \ 0.01). ns no significant differences among treatments (P [ 0.05)
Fig. 3 Pre-dawn leaf water potential at the end of the first or second
cycle of drought treatment of Salix gracilistyla cuttings. Values are
mean - standard deviation (n = 6). Different letters represent
significant differences among treatments (two-way interaction using
Bonferroni adjustment, P \ 0.05). ns no significant differences
among treatments (P [ 0.05). Asterisks indicate significantly differ-
ent from control value (Dunnett’s t test, P \ 0.05)
Fig. 4 Leaf water relations for Salix gracilistyla cuttings. Wwtlp
water potential at turgor loss point (a) and Wosat osmotic potential at
full turgor (b). Values are mean - standard deviation (n = 4).
Different letters represent significant differences among treatments
(two-way interaction using Bonferroni adjustment, P \ 0.05). Aster-isks indicate significantly different from control value (Dunnett’s
t test, P \ 0.05)
Trees (2010) 24:1087–1095 1091
123
Growth of Salix gracilistyla cuttings
Two-way ANOVA (F and D as factors) showed significant
F main effects on shoot to root ratio and individual leaf
area (P \ 0.05, Table 2). There were significant F and D
main effects on root and hypertrophic lenticel biomass
(P \ 0.05) and a significant main effect of D on leaf and
shoot biomass, number of attached leaves, and defoliation
rate (P \ 0.05). We found a significant D main effect and
an F–D interaction on total cutting biomass (P \ 0.05).
Growth parameters of cuttings were compared among
control and three F repetitions differing in flooding dura-
tion (Table 3). Shoot and leaf biomass and number of
attached leaves were highest in controls than in F1, F2, and
F3 (Tukey’s HSD, P \ 0.05). Root biomass was highest in
controls and lower in F3 than in F1 (P \ 0.05). Shoot to
root ratio was higher in F2 and F3 than in controls and F1
(P \ 0.05). Hypertrophic lenticel biomass was greater in
F2 and F3 than in controls and F1 (P \ 0.05). Defoliation
rate was lower in controls than in F1, F2, and F3
(P \ 0.05). Individual leaf area was largest in controls and
smaller in F3 than in F1 (P \ 0.05). As duration of
flooding increased, cuttings developed less root mass,
higher shoot to root ratios, more hypertrophic lenticels
mass, and smaller individual leaf areas. Growth parameters
of cuttings were also compared among control and three D
repetitions (Table 3). Shoot and leaf biomass and the
number of attached leaves were highest in controls fol-
lowed by D1, and lowest in D3 (Tukey’s HSD, P \ 0.05).
Root biomass was largest in controls followed by D1 and
D2, and smallest in D3 (P \ 0.05). Shoot to root ratio was
higher in F1, F2, and F3 than in controls (P \ 0.05).
Hypertrophic lenticel biomass was larger in D1 than in D3
(P \ 0.05). Defoliation rate was lowest in controls, and
higher in D3 than in D1 (P \ 0.05). Individual leaf area
was smaller in F1, F2, and F3 than in controls (P \ 0.05).
As drought duration increased, values for root, hypertro-
phic lenticel, leaf, and shoot biomasses decreased and
numbers of attached leaves decreased and defoliation rate
increased.
Discussion
Water stress on Salix gracilistyla cuttings was not always
influenced by flooding duration between droughts and by
drought duration between floodings of any duration
(Fig. 3), indicating that S. gracilistyla responses to flooding
and drought would be altered by repetitive flooding and
drought (RFD). In addition, S. gracilistyla responded to
flooding and drought through osmotic adjustment, even in
RFD, except with flooding and drought duration as short as
1 week. In general, osmotic adjustment of leaves increases
with increasing severity of drought stress (Dichio et al.
2006). However, under RFD conditions, osmotic adjust-
ment of S. gracilistyla did not increase as duration of
combined flooding increased, even when combined with
protracted periods of drought (Fig. 4). Drought stress in
Cleopatra mandarin (Citrus resnhi), a drought and flood-
tolerant citrus tree, tends to increase levels of soluble
sugars in leaves and the concentrations of Ca, K, Mg, Na,
and Cl in both leaves and roots. Flooding tends to decrease
Table 2 Two-way ANOVA of growth of Salix gracilistyla cuttings
Source of variation df F P
Leaf biomass
Duration of flooding 2, 90 0.047 0.954
Duration of drought 2, 90 3.984 0.022
Flooding 9 drought 4, 90 1.423 0.233
Shoot biomass
Duration of flooding 2, 90 0.114 0.892
Duration of drought 2, 90 5.436 0.006
Flooding 9 drought 4, 90 1.959 0.108
Root biomass
Duration of flooding 2, 90 6.815 0.002
Duration of drought 2, 90 9.876 \0.001
Flooding 9 drought 4, 90 1.410 0.237
Hypertrophic lenticel biomass
Duration of flooding 2, 90 10.954 \0.001
Duration of drought 2, 90 4.553 0.013
Flooding 9 drought 4, 90 1.182 0.324
Total biomass
Duration of flooding 2, 90 0.985 0.377
Duration of drought 2, 90 7.303 0.001
Flooding 9 drought 4, 90 2.574 0.043
Shoot/root
Duration of flooding 2, 90 5.387 0.006
Duration of drought 2, 90 0.391 0.677
Flooding 9 drought 4, 90 1.316 0.270
Number of attached leaves
Duration of flooding 2, 90 0.999 0.372
Duration of drought 2, 90 4.610 0.012
Flooding 9 drought 4, 90 2.452 0.052
Defoliation rate
Duration of flooding 2, 90 0.529 0.591
Duration of drought 2, 90 5.710 0.005
Flooding 9 drought 4, 90 1.613 0.178
Individual leaf area
Duration of flooding 2, 50 3.985 0.025
Duration of drought 2, 50 2.606 0.084
Flooding 9 drought 4, 50 0.606 0.660
The biomass values were determined as the difference between the
dry weight at the end of the experiment and the estimated initial dry
weight of each cutting. Shoots consisted of leaves and branches
1092 Trees (2010) 24:1087–1095
123
concentrations of soluble sugars and all of these ions
except Ca in leaves (Garcia-Sanchez et al. 2007). In this
manner, decreasing concentrations of soluble sugars and
diverse ions in leaves during flooding stress may lead to
decreasing osmotic adjustment of S. gracilistyla when it is
waterlogged. Therefore, if the durations of drought and
flooding are both long, osmotic adjustment to drought
would be inadequate, so that the severity of water stress on
S. gracilistyla increases.
Not only under drought conditions, but also increased
stomatal regulation to improve water balance under
flooding conditions is an adaptive mechanism that prevents
leaf dehydration (Domingo et al. 2002). Salix gracilistyla
responded to flooding and drought through continuous
transpiration inhibition even under RFD conditions
(Fig. 1). Plant growth is affected by stress for a longer
period after stress removal when the stress factor is
flooding rather than drought. For example, when two
hybrid poplar clones are subjected to drought for
8–10 days, photosynthesis recovered fully and quickly
upon removal of stress, but when these same clones are
stressed by root flooding with tap water for 16–20 days,
there is no recovery until 9 days after removal of stress
(Liu and Dickmann 1993). The gs decreased through the
drought period, but increased immediately after drought
stress replacement by flooding (Fig. 1). This temporary
increase in gs might be due to the decrease in severity of
drought stress as water supply increases upon flooding.
However, when the flooding treatment continued longer
than a week, gs decreased remarkably under combined long
durations of flooding and drought (Fig. 2b). Stomatal
closures following flooding result from an abscisic acid
hormonal signal transmitted from the roots to shoots so
that leaves may avoid water deficit (Dat et al. 2004),
which, nevertheless, develops progressively over time
(Garcia-Sanchez et al. 2007). Therefore, S. gracilistyla
will close more stomata when crossed flooding duration
increases.
Flood-tolerant plants have several survival mechanisms,
such as the maintenance of oxygen supply in flooded roots
and adaptive metabolic responses to anoxia (Crawford
1982). When roots are flooded, the accumulation of toxic
ethanol invariably occurs. To remove ethanol and with-
stand flooding, flood-tolerant trees undergo anatomical
modifications, such as the growth of adventitious roots,
root branching, lenticel proliferation, and hypertrophy
(Crawford 1982). On flooding, ethylene levels increase,
which, in turn, promotes the development of hypertrophic
lenticels (Kludze et al. 1994; Topa and McLeod 1988).
Hypertrophic lenticels, which increase internal gas trans-
port from the stem to the roots upon flooding (Hook et al.
1970), are a morphological adaptation to substrate oxygen
shortage (e.g. Angeles et al. 1986; Kozlowski 1984). The
development of hypertrophic lenticels in S. gracilistyla
cuttings increased with flooding durations of 2 or 3 weeks,
but was more inhibited when longer droughts occurred
between floodings (Table 3). Hypertrophic lenticel devel-
opment was inhibited when oxygen conditions in the
rooting zone were improved temporarily between floo-
dings, suggesting that when long periods of drought occur
between floodings, S. gracilistyla does not respond readily
to low oxygen substrate conditions following flooding.
Table 3 Growth of Salix gracilistyla cuttings under different durations of flooding and drought
Variable Treatment
Control (C) 1-week
flooding (F1)
2-weeks
flooding (F2)
3-weeks
flooding (F3)
1-week
drought (D1)
2-weeks
drought (D2)
3-weeks
drought (D3)
Shoot biomass (g) 2.47 ± 0.57bC 1.65 ± 0.69a 1.59 ± 0.65a 1.65 ± 0.66a 1.87 ± 0.76B 1.68 ± 0.57AB 1.35 ± 0.53A
Root biomass (g) 2.89 ± 0.42cC 1.49 ± 0.54b 1.24 ± 0.37ab 1.13 ± 0.35a 1.47 ± 0.44B 1.34 ± 0.44B 1.04 ± 0.36A
Shoot/root ratio 0.86 ± 0.17aA 1.14 ± 0.36a 1.32 ± 0.44b 1.45 ± 0.32b 1.26 ± 0.39B 1.30 ± 0.41B 1.34 ± 0.40B
Leaf biomass (g) 1.20 ± 0.31bC 0.55 ± 0.22a 0.55 ± 0.23a 0.53 ± 0.24a 0.62 ± 0.28B 0.56 ± 0.17AB 0.45 ± 0.21A
Hypertrophic
lenticel
biomass (g)
0.002 ± 0.002aA 0.014 ± 0.009a 0.024 ± 0.015b 0.030 ± 0.019b 0.029 ± 0.021C 0.022 ± 0.015BC 0.018 ± 0.010B
Number of
attached leaves
48.5 ± 10.5bC 25.1 ± 8.22a 26.9 ± 8.84a 28.1 ± 8.20a 30.4 ± 9.41B 25.2 ± 6.76AB 24.6 ± 7.90A
Defoliation
rate (%)
30.8 ± 11.1aA 54.3 ± 11.6b 51.5 ± 12.9b 51.8 ± 12.7b 47.6 ± 13.3B 52.3 ± 7.80BC 57.7 ± 13.2C
Individual leaf
area (cm2)
3.89 ± 0.73cB 2.95 ± 0.85b 2.48 ± 0.67ab 2.33 ± 0.50a 2.69 ± 0.78A 2.77 ± 0.69A 2.29 ± 0.63A
Values are mean ± standard deviation. Control: n = 10; F1, F2, F3, D1, D2, and D3: n = 30. For leaf area, control: n = 6; F1, F2, F3, D1, D2,
and D3: n = 18. The biomass values were determined as the difference between the dry weight at the end of the experiment and the estimated
initial dry weight of each cutting. Shoots consisted of leaves and branches. Different lowercase letters and capital letters represent significant
differences among the control, F1, F2, and F3, and among the control, D1, D2, and D3, respectively (Tukey’s HSD, P \ 0.05)
Trees (2010) 24:1087–1095 1093
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Deeper roots and a smaller shoot to root biomass ratio
help protect plants from dehydration (van Splunder et al.
1996). However, root biomass of S. gracilistyla declined
under longer drought conditions (Table 3), and shoot to
root ratio was not affected by drought (Table 2). Therefore,
S. gracilistyla would not be able to protect itself from
dehydration following drought by developing roots and
allocating resources to roots. Furthermore, roots have more
sensitive responses than shoots to reductions in substrate
Eh (Kludze and DeLaune 1994), because root growth is an
energy-dependent process requiring oxygen (Bertani and
Brambilla 1982; Drew 1990). Thus, deterioration of root
function will take place rapidly following flooding. This is
because under longer flooding, root biomass and root ratio
are inhibited (Table 3). Root growth of S. gracilistyla
cuttings is also inhibited by each hypoxic flooding or
drought event (Nakai et al. 2009). S. gracilistyla will not
increase drought tolerance, as water absorption ability
increases under conditions of longer flooding between
droughts, and may suffer dehydration during the following
drought. Negative effects of each flooding or drought on
root growth are offset after removal of stresses (Jackson
and Attwood 1996; Li et al. 2004). However, our results
show that RFD does not ameliorate flooding or drought
stress; rather, there is an additive stress effect on root
growth from successive RFD.
Trees experiencing soil moisture shortage decrease
whole plant transpiration rate (Braatne et al. 1992) by
reducing transpiration surface area (e.g. leaf mass and leaf
size) (e.g. Metcalfe et al. 1990) to reduce the risk of xylem
embolism. Leaf dehydration also occurs when roots are
submerged, despite the abundant moisture in the rhizo-
sphere (Pezeshki 2001), because water uptake is slower in
waterlogged soil than in aerated soil (Everard and Drew
1989; Kramer and Jackson 1954) and root permeability
decreases in flooded soil (Hiron and Wright 1973; Kramer
1940). Drought and flood of long duration negatively
affected leaf mass and individual leaf size (Table 3) indi-
cating that, under RFD conditions, S. gracilistyla could
cope with water shortage due to flooding or drought by
reducing transpiration area. However, lack of F–D inter-
action on leaf mass and leaf size (Table 2) reveals that
RFD neither promote nor inhibit S. gracilistyla response to
flooding and drought through reducing transpiration area
compared with sole flooding or drought.
Under RFD conditions, S. gracilistyla increases drought
tolerance by inhibiting transpiration under drought condi-
tions, but decreases osmotic adjustment so as to decrease
drought tolerance, as flooding durations between droughts
increase. Furthermore, the drought tolerance of S. gracili-
styla becomes stronger by inhibiting leaf mass, as drought
duration increases regardless of flooding duration. In con-
trast, under RFD conditions, long droughts inhibit
transpiration of S. gracilistyla, which enhances flood tol-
erance, while long droughts also decrease the mass of
hypertrophic lenticels, which decreases flooding tolerance
as drought duration between floodings increases. In addi-
tion, S. gracilistyla increases flood tolerance by decreasing
leaf size as flooding duration increases, regardless of
drought duration. Repeated cycles of flooding and drought
alter the response to flooding and drought in S. gracilistyla.
At sites along rivers, the duration of flooding, which
depends on elevation, is expected to strongly affect the
population dynamics of S. gracilistyla, because adult trees
of this species are primarily distributed at specific eleva-
tions (Nakai and Kisanuki 2007b). Furthermore, most
S. gracilistyla current-year seedlings at low elevations with
long flooding duration and at high elevations with short
flooding duration cannot survive until autumn (Nakai and
Kisanuki 2007a; Nakai unpublished data). Thus, for trees
such as S. gracilistyla, which grow in habitats where
waterlogging and drought repeatedly occur, the flooding
and drought combinations that can be tolerated by the trees
may be key to understanding their distribution.
Acknowledgments The authors thank their colleagues for their
assistance with the survey.
References
Angeles G, Evert RF, Kozlowski TT (1986) Development of lenticels
and adventitious roots in flooded Ulmus americana seedlings.
Can J For Res 16:585–590
Armstrong W, Drew MC (2002) Root growth and metabolism under
oxygen deficiency. In: Waisel Y, Eshel A, Kafkafi U (eds) Plant
roots: the hidden half. Marchel Dekker, USA, pp 729–761
Bertani A, Brambilla I (1982) Effect of decreasing oxygen concen-
tration on wheat roots: growth and induction of anaerobic
metabolism. Z Pflanzenphysiol 108:283–288
Braatne JH, Hinckley TM, Stettler RF (1992) Influence of soil-water
on the physiological and morphological components of plant
water balance in Populus trichocarpa, Populus deltoides and
their F1 hybrids. Tree Physiol 11:325–339
Crawford RMM (1982) Physiological responses to flooding. In:
Lange OL, Nobel PS, Osmond CB, Ziegler H (eds) Physiological
plant ecology II water relations and carbon assimilation.
Springer, Berlin, pp 453–477
Dat JF, Capelli N, Folzer H, Bourgeade P, Badot PM (2004) Sensing
and signalling during plant flooding. Plant Physiol Biochem
42:273–282
Dichio B, Xiloyannis C, Sofo A, Montanaro G (2006) Osmotic
regulation in leaves and roots of olive trees during a water deficit
and rewatering. Tree Physiol 26:179–185
Domingo R, Perez-Pastor A, Ruiz-Sanchez C (2002) Physiological
responses of apricot plants grafted on two different rootstocks to
flooding conditions. J Plant Physiol 159:725–732
Drew MC (1990) Sensing soil oxygen. Plant Cell Environ 13:681–696
Everard JD, Drew MC (1989) Mechanisms controlling changes in
water movement through the roots of Helianthus annuus L.
during continuous exposure to oxygen deficiency. J Exp Bot
40:95–104
1094 Trees (2010) 24:1087–1095
123
Garcia-Sanchez F, Syvertsen JP, Gimeno V, Botia P, Perez-Perez JG
(2007) Responses to flooding and drought stress by two citrus
rootstock seedlings with different water-use efficiency. Physiol
Plantarum 130:532–542
Hiron RWP, Wright STC (1973) The role of endogenous abscisic acid
in the response of plants to stress. J Exp Bot 24:769–781
Hook DD, Brown CL, Kormanik PP (1970) Lenticels and water root
development of swamp tupelo under various flooding condition.
Bot Gaz 131:217–224
Ishikawa S (1982) Ecological studies of the floodplain willow forests
in the Tohoku district (in Japanese with English abstract). Res
Rep Kochi Univ Natur Sci 31:95–104
Ishikawa S (1997) Distribution behavior of riparian plants and species
diversity of the vegetation on rocky river banks in the Yoshino
River in Shikoku, Japan. Mem Fac Sci Kochi Univ Ser D (Biol)
18:1–7
Jackson MB (1985) Ethylene and the responses of plants to soil
waterlogging and submergence. Annu Rev Plant Physiol
36:145–174
Jackson MB (1991) Regulation of water relationships in flooded
plants by ABA from leave, roots and xylem sap. In: Davies WJ,
Jones HG (eds) Abscisic acid: physiology and biochemistry.
Bios Scientific Publishers, Oxford, pp 217–226
Jackson MB, Attwood PA (1996) Roots of willow (Salix viminalis L.)
show marked tolerance to oxygen shortage in flooded soils and in
solution culture. Plant Soil 187:37–45
Kludze HK, De Laune RD (1994) Methane emission and growth of
Spartina patens in response to soil redox intensity. Soil Sci Soc
Am J 58:1838–1845
Kludze H, Pezeshki SR, De Laune RD (1994) Evaluation of root
oxygenation and growth in baldcypress in response to short-term
soil hypoxia. Can J For Res 24:804–809
Kozlowski TT (1984) Flooding and Plant Growth. Academic Press,
Orlando
Kozlowski TT (1997) Responses of woody plants to flooding and
salinity. Tree Physiol Monogr 1:1–29
Kozlowski TT (2000) Responses of woody plants to human-induced
environmental stresses: issues, problems and strategies for
alleviating stress. Crit Rev Plant Sci 19:91–170
Kramer PJ (1940) Causes of decreased absorption of water by plants
in poorly aerated media. Am J Bot 27:216–220
Kramer PJ, Jackson WJ (1954) Causes of injury to flooded tobacco
plants. Plant Physiol 29:241–249
Larcher W (1980) Physiological plant ecology. Springer, Berlin
Li S, Pezeshki SR, Goodwin S, Shields FD (2004) Physiological
responses of black willow (Salix nigra) cuttings to a range of soil
moisture regimes. Photosynthetica 42:585–590
Liu ZJ, Dickmann DI (1993) Responses of 2 hybrid Populus clones to
flooding, drought, and nitrogen availability. II. Gas-exchange
and water relations. Can J Bot 71:927–938
Marron N, Dreyer E, Boudouresque E, Delay D, Petit JM, Delmotte
FM, Brignolas F (2003) Impact of successive drought and
re-watering cycles on growth and specific leaf area of two Pop-ulus x canadensis (Moench) clones, ‘Dorskamp’ and
‘Luisa_Avanzo’. Tree Physiol 23:1225–1235
Maruyama Y, Morikawa Y (1983) Measurement of leaf water
relations using the pressure–volume technique. J Jpn For Soc
65:23–28
Metcalfe JC, Davies WJ, Pereira JS (1990) Leaf growth of Eucalyptusglobulus seedlings under water deficit. Tree Physiol 6:221–227
Milborrow BV (1974) The chemistry and physiology of abscisic acid.
Annu Rev Plant Physiol 25:259–307
Nakai A, Kisanuki H (2007a) Effect of elevation above the waterline
on the growth of current-year Salix gracilistyla seedlings on a
gravel bar (in Japanese with English abstract). J Jpn For Soc
89:1–6
Nakai A, Kisanuki H (2007b) Effect of inundation duration on Salixgracilistyla density and size on a gravel bar. J For Res
12:365–370
Nakai A, Yurugi Y, Kisanuki H (2009) Growth responses of Salixgracilistyla cuttings to a range of substrate moisture and oxygen
availability. Ecol Res 24:1057–1065
Niiyama K (1987) Distribution of salicaceous species and soil texture
of habitats along the Ishikari River (in Japanese with English
abstract). Jpn J Ecol 37:163–174
Pezeshki SR (2001) Wetland plant responses to soil flooding. Environ
Exp Bot 46:299–312
Pezeshki SR, Anderson PH, Shields FDJ (1998) Effects of soil
moisture regimes on growth and survival of black willow (Salixnigra) posts (cuttings). Wetlands 18:460–470
Ponnamperuma FN (1972) The chemistry of submerged soil. Adv
Agron 24:29–96
Schaff SD, Pezeshki SR, Shields FDJ (2003) Effects of soil conditions
on survival and growth of black willow cuttings. Environ Manag
31:748–763
Topa MA, McLeod KW (1988) Promotion of aerenchyma formation
in Pinus serotina seedlings by ethylene. Can J For Res
18:276–280
van Splunder I, Voesenek LACJ, Coops H, de Vries XJA, Blom
CWPM (1996) Morphological responses of seedlings of four
species of Salicaceae to drought. Can J Bot 74:1988–1995
Wikbergi J, Ogreni E (2007) Variation in drought resistance, drought
acclimation and water conservation in four willow cultivars used
for biomass production. Tree Physiol 27:1339–1346
Williams JL, Levine JM (2004) Small-scale variation in growing
season length affects size structure of scarlet monkeyflower.
Oikos 106:131–137
Yamamoto F, Sakata T, Terazawa K (1995a) Growth, morphology,
stem anatomy and ethylene production in flooded Alnus japonicaseedlings. IAWA J 16:47–59
Yamamoto F, Sakata T, Terazawa K (1995b) Physiological, anatom-
ical and morphological responses of Fraxinus mandshuricaseedlings to flooding. Tree Physiol 15:713–719
Trees (2010) 24:1087–1095 1095
123
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