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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: [email protected]

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 drought 2, 72 7.703 0.001


Wwpd

Duration of flooding 2, 50 16.197 \0.001



Wwtlp



Flooding 9 drought 4, 30 5.757 0.001

Wosat




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




Shoot biomass




Root biomass




Hypertrophic lenticel biomass

Duration of flooding 2, 90 10.954 \0.001



Total biomass




Shoot/root




Number of attached leaves




Defoliation rate




Individual leaf area




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)

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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.

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