REGULAR PAPER

Simulations and observations of patchy stomatal behaviorin leaves of Quercus crispula, a cool-temperate deciduousbroad-leaved tree species

Mai Kamakura • Yoshiko Kosugi • Kanako Muramatsu •

Hiroyuki Muraoka

Received: 20 June 2011 / Accepted: 23 September 2011

� The Botanical Society of Japan and Springer 2011

Abstract We investigated the occurrence of patchy sto-

matal behavior in leaves of saplings and a forest canopy

tree of Quercus crispula Blume. Through a combination of

leaf gas-exchange measurements and numerical simulation,

we detected patterns of stomatal closure (either uniform or

patchy bimodal) coupled with depression of net assimila-

tion rate (A). There was a clear inhibition of A associated

with stomatal closure in leaves of Q. crispula during the

day, but the magnitude of inhibition varied among days and

growing conditions. Comparisons of observed and simu-

lated A values for both saplings and the canopy tree iden-

tified patterns of stomatal behavior that shifted flexibly

between uniform and patchy frequency distributions

depending on environmental conditions. Bimodal stomatal

closure explained severe depression of A in saplings under

conditions of relatively high leaf temperature and vapor

pressure deficit. Model simulations of A depression through

bimodal stomatal closure were corroborated by direct

observations of stomatal aperture distribution using Su-

zuki’s Micro-Printing method; these demonstrated that

there was a real bimodal frequency distribution of stomatal

apertures. Although there was a heterogeneous distribution

of stomatal apertures both within and among patches,

induction of heterogeneity in intercellular CO2 concentra-

tion among patches, and hence severe depression of A,

resulted only from bimodal stomatal closure among pat-

ches (rather than within patches).

Keywords Bimodal stomatal closure � Leaf gas exchange �Midday depression � Leaf temperature �Vapor pressure deficit

Introduction

Gas exchange rates between plants and atmosphere are

determined by adjustment of photosynthetic capacity and

changes in the apertures of stomata on the leaf epidermis.

Photosynthesis and transpiration rates in individual leaves

vary in response to environmental factors such as light,

temperature, water and nutrient supplies. High light, coin-

cident with high leaf temperature or water deficit, often

causes midday depression of net assimilation rate (A),

which is coupled with stomatal closure (Tenhunen et al.

1984; Epron et al. 1995; Valladares and Pearcy 1997; Pathre

et al. 1998; Muraoka et al. 2000). Stomatal closure, induced

by increased leaf-to-air vapor pressure deficit (VPD) and/or

low leaf water potential (Maier-Maercker 1983; Mott and

Parkhurst 1991; Brodribb and Holbrook 2004), decreases

CO2 concentration within intercellular spaces (Ci). The

reduction of A at midday is the result of both an increase in

stomatal limitation and a reduction in the photochemical

process (Ishida et al. 1999; Muraoka et al. 2000).

Patterns of stomatal closure in canopy leaves of tropical

trees may be patchy during severe midday depression

(Ishida et al. 1999). Patchy stomatal behavior occurs in

M. Kamakura (&) � K. Muramatsu

KYOUSEI Science Center for Life and Nature,

Nara Women’s University, Kita-uoya Higashimachi,

Nara 630-8506, Japan

e-mail: mkamakura@cc.nara-wu.ac.jp

Y. Kosugi

Laboratory of Forest Hydrology, Division of Environmental

Science and Technology, Graduate School of Agriculture,

Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku,

Kyoto 606-8502, Japan

H. Muraoka

Institute for Basin Ecosystem Studies, Gifu University,

1-1 Yanagido, Gifu 501-1193, Japan

123

J Plant Res

DOI 10.1007/s10265-011-0460-8

plants with heterobaric leaves. In these leaves, vertical

extensions of bundle sheath cells delimit the mesophyll and

restrict CO2 diffusion (Mott and Buckley 1998, 2000; West

et al. 2005; Kenzo et al. 2007). Patchy stomatal behavior

occurs in response to low humidity (Loreto and Sharkey

1990; Beyschlag et al. 1992; Mott et al. 1993), water stress

(Sharkey and Seemann 1989; Gunasekera and Berkowitz

1992) and increased internal abscisic acid (ABA) concen-

tration (Downton et al. 1988; Terashima et al. 1988).

Because uniform stomatal behavior is assumed in leaf gas-

exchange calculations based on the Farquhar–von Caem-

merer–Berry model (von Caemmerer and Farquhar 1981;

Warren et al. 2003), the effect of patchy stomatal closure

on depression of A may well have been underestimated.

Although, the importance of patchy stomatal closure

effects on leaf gas-exchange rates is recognized, only a few

studies have evaluated the pattern of stomatal behavior

(uniform or patchy bimodal) associated with A depression

in tree leaves under natural conditions (Takanashi et al.

2006; Kosugi et al. 2009; Kamakura et al. 2011).

By combining a numerical analysis that calculated

simulated A at a given stomatal conductance (gs) for both

uniform and patchy stomatal behavior with a pressure-

infiltration method, Takanashi et al. (2006) demonstrated

that patchy stomatal closure with a bimodal pattern

explains midday A depression in top-canopy leaves of a

tropical rainforest. Direct observation of stomatal aperture

distributions using Suzuki’s Universal Micro-Printing

(SUMP) method showed a real patchy bimodal distribution

of stomatal apertures during midday depression in leaves of

mid- and upper-layers in tropical tree vegetation. Further-

more, there was a heterogeneous distribution of stomatal

apertures among adjacent stomata rather than among

compartments delimited by bundle sheath extensions (Ka-

makura et al. 2011). By numerical analysis, we also found

that patchy bimodal stomatal behavior occurs only during

midday depression, suggesting that stomatal aperture dis-

tribution varies flexibly within single days. On the basis of

these procedures, there have been arguments for the

occurrence of patchy stomatal closure that leads to midday

A depression in tropical trees growing in natural environ-

ment. However, there are few reported studies on quanti-

tative effects of such stomatal behavior on gas-exchange

characteristics of heterobaric tree species in other climatic

regions. Moreover, the question of whether or not patchy

stomatal behavior depends on plant species, growth envi-

ronment, or weather conditions.

The objectives of this study were to (1) describe changes

in pattern of stomatal behavior within and among days, (2)

determine factors that induce patchy bimodal stomatal

behavior, and (3) determine the scale of heterogeneity in

frequency distributions of stomatal apertures within leaves

of Quercus crispula Blume, a dominant cool-temperate,

deciduous, broad-leaf tree with heterobaric leaves. Midday

depression of A occurs in leaves of several top-canopy tree

species in cool-temperate forests (Iio et al. 2004). Because

changes in hydraulic architecture through ontogeny affect

photosynthetic capacity (Cavender-Bares and Bazzaz

2000), it is possible that leaf stomatal behavior under high

light differs between saplings and canopy trees. Thus, we

studied both life stages of Q. crispula.

We measured and numerically simulated CO2 exchange

associated with patterns of stomatal behavior under various

photosynthetic photon flux densities (PPFD), leaf temper-

atures (Tleaf), and VPD conditions in a nursery and in a

cool-temperate deciduous broadleaf forest. To predict

A values by numerical simulation, we assumed uniform and

patchy bimodal stomatal behaviors, which are the most

extreme patterns of stomatal aperture frequency distribu-

tion in leaves. Because maximum depression of A is caused

by patchy stomatal closure with a bimodal frequency dis-

tribution (Takanashi et al. 2006), we simulated largest

effects of patchy stomatal behavior on A by assuming

bimodal stomatal closure. Our comparison of observed and

simulated A detected fluctuations in A values caused by

stomatal behavior. Moreover, corroborative data on real

stomatal aperture distribution were provided by SUMP

method observations. We predicted that (1) observed

A would fluctuate between simulated A values calculated

under assumptions of uniform and bimodal stomatal

behaviors within and among days and (2) stomatal aperture

distribution with a bimodal pattern would occur among

adjacent compartments delimited by bundle sheath exten-

sions in leaves under severe environmental conditions that

cause midday depression associated with patchy bimodal

stomatal closure.

Materials and methods

Site and materials

Saplings were studied in a nursery at Kyoto University,

Kyoto, Japan (35�010N, 135�460E). Three-year-old saplings

of Quercus crispula were planted into pots (18 cm diam-

eter, 20 cm deep) filled with soil (Akadama soil:leaf

mold = 7:3, by vol.) on April 7, 2010. The leaves flushed

1–2 weeks after planting. Plants were watered daily and

treated with a Hyponex solution (Hyponex Japan, Osaka,

Japan; N:P:K = 6:10:5 at 1 g l-1) once per week. We

measured in situ leaf gas exchange and observed stomatal

apertures on 1, 7 and 24 June, 2010. Three leaves from

different saplings were used. Mean leaf number was

39 ± 3 (±SD).

We studied a full-sized tree in a cool-temperate decid-

uous broadleaf forest (36�080N, 137�250E, 1,420 m a.s.l.)

J Plant Res

123

on the north-western slope of Mt. Norikura, Japan. At the

nearby (500 m from the study site) ‘‘Takayama (TKY)

AsiaFlux site’’, annual mean air temperature and precipi-

tation from 1994 to 2008 were 7.2�C and 2,075 mm,

respectively; mean air temperature in the forest we studied

was 6.6�C. The forest canopy was dominated by Q.

crispula, Betula platyphylla Sukatchev var. japonica Hara

and B. ermanii Cham. Betula ermanii is a pioneer species

with a thin crown in the multispecies deciduous forest.

Quercus crispula is a late-successional species that forms a

multi-layered crown that often occupies space down to the

forest shrub layer. Leaf flush in all of these deciduous tree

species occurs after snow-melt. Measurements were made

on leaves collected in top layer of a Q. crispula canopy tree

using a canopy access tower (10 9 10 m on the base and

18 m tall) on 30, 31 July and 2 August, 2010. Three leaves

were collected from a single adult tree.

Measurement of leaf gas exchange

Diurnal changes in net assimilation rate (A, lmol m-2 s-1)

and stomatal conductance for water vapor (gs,

mol m-2 s-1) in intact leaves were measured under natural

photosynthetic photon flux density (PPFD, lmol pho-

tons m-2 s-1) using a LI-6400 gas-exchange measurement

system (Li-Cor Inc., Lincoln, NE, USA) with a 2 9 3 cm

clear-top chamber. Incident PPFD beside the leaf chamber

was measured with a photon sensor (LI-190SA, Li-Cor

Inc.). CO2 concentration, temperature, and relative

humidity of the air in the chamber were adjusted to

ambient levels. Ambient air temperature and relative

humidity were measured with thermo-hygrometers (HOBO

ProV2, Onset Computer corp., Bourne, MA, USA). Leaf-

to-air VPD was calculated from air temperature and rela-

tive humidity in the chamber. Air entered the chamber at a

flow rate of 500 lmol s-1. Gas exchange measurements

were made at about 15-min intervals.

Model description

To obtain normalized maximum rates of carboxylation at

25�C (Vcmax25, lmol m-2 s-1), we used the one-point

method (Wilson et al. 2000; Kosugi et al. 2003; Grassi

et al. 2005; Kosugi and Matsuo 2006); this is an inverse

method based on the Farquhar–von Caemmerer–Berry

model, and it can be used to determine actual responses of

leaves in the field. With the one-point method, apparent

partial pressure of CO2 within the intercellular space

(p(Ci)*) is estimated from variables measured with gas-

exchange methodology, and the ‘apparent’ normalized

maximum carboxylation rate at 25�C (Vcmax25*) is calcu-

lated from values of A, p(Ci)*, and Tleaf. We calculated

Vcmax25* assuming that the infinite internal conductance

(gi = ?), and thus the CO2 concentration in the chloro-

plast (p(Cc)), was identical to p(Ci)*. Hence, Vcmax25* was

calculated as follows:

Vc max 25� ¼ Aþ Rdð ÞpðCcÞ þ Kc 1þ pðOÞ

Ko

� �

pðCcÞ � pðOÞ2s

�1þ exp

DSðVc maxÞðTleafþ273Þ�DHdðVc maxÞRðTleafþ273Þ

h i

expDHaðVc maxÞðTleaf�25Þ

298RðTleafþ273Þ

h i ð1Þ

where Rd is the non-photorespiratory respiration rate

(lmol m-2 s-1); s is the specificity factor of Rubisco;

p(O) (21,000 Pa) is the partial pressure of O2 at the sites of

oxygenation; Kc and Ko are the Rubisco Michaelis–Menten

constants for CO2 and O2, respectively; Tleaf is leaf tem-

perature (�C); R is the gas constant (8.31 J K-1 mol-1);

DHa (Vcmax) is the activation energy for Vcmax; DHd (Vcmax)

is the deactivation energy for Vcmax; and DS (Vcmax) is an

entropy term. Values of Kc and Ko, s and their activation

energies used to calculate temperature dependence were

obtained from Harley et al. (1992). The Arrhenius function

was used to estimate temperature dependence of parame-

ters Kc, Ko, s, Vcmax and Rd (detailed in Kosugi et al. 2003;

Kosugi and Matsuo 2006; Takanashi et al. 2006). Vcmax25

was estimated from Vcmax25* at maximum gs (gsmax)

because Vcmax25 is the intrinsic value that represents car-

boxylation ability of leaves and is not always identical to

the apparent value (Vcmax25*), which has significant diurnal

fluctuations coupled with patchy stomatal closure (Takan-

ashi et al. 2006).

To determine the fluctuations in observed A values

between simulated A values calculated under assumptions

of uniform and patchy bimodal stomatal behaviors within

and among days, we performed simulations to predict net

assimilation rates at a given stomatal conductance for both

uniform and non-uniform stomatal behaviors using photo-

synthetic parameters obtained by the one-point method.

Uniform stomatal behavior indicates that whole-leaf con-

ductance reflects uniform change in stomatal conductance.

For non-uniform stomatal behavior, we assumed a patchy

bimodal frequency distribution of stomata, which indicates

that whole-leaf conductance reflects either open or closed

stomatal conductance. In heterobaric leaves, the bundle

sheath extensions separate mesophyll into many compart-

ments, and we termed each compartment a ‘patch’. For

patchy bimodal distribution, stomatal conductances for

open and closed patches were maximum and minimum gs

(gsmax and gsmin), and the open/closed patch ratio (roc) was

determined from the observed gs for a whole leaf. Net

assimilation rate and intercellular CO2 concentration for

each patch were estimated with the Farquhar–von Caem-

merer–Berry model used for determining patch stomatal

conductance values. We assumed uniform photosynthetic

J Plant Res

123

parameters for each patch, and average net assimilation

rate for the whole leaf was calculated by integrating net

assimilation rates of individual patches. In model simula-

tions, the net assimilation rate of each patch was calculated

following Kosugi et al. (2003), Kosugi and Matsuo (2006),

and Takanashi et al. (2006). Briefly, A values of open (Aop)

and closed (Acl) patches were determined from the mini-

mum difference between the RuBP-saturated rate or car-

boxylation-limited net assimilation rate (Ac) and the

electron transport- or RuBP regeneration-limited net

assimilation rate (Aj) calculated as follows:

Ac ¼ Vc max 25

expDHaðVc maxÞðT1�25Þ

298RðT1þ273Þ

h i

1þ expDSðVc maxÞðT1þ273Þ�DHdðVc maxÞ

RðT1þ273Þ

h i

�pðCcÞ � pðOÞ

2s

pðCcÞ þ Kc 1þ pðOÞKo

� �� Rd ð2Þ

Aj ¼J

4

pðCcÞ � pðOÞ2s

pðCcÞ þ pðOÞs

� Rd: ð3Þ

The partial pressure of CO2 in the chloroplasts of open

patches (p(Cc)op) was determined from Aop and gsmax, and

that for closed patches (p(Cc)cl) was determined from Acl

and gsmin. A J value was required to estimate the influence

of depression in the electron transport rate on net

assimilation rate. To estimate J, we used optimal Jmax

values estimated from the relationship between Vcmax and

Jmax as follows:

Jmax ¼ kjVc max: ð4Þ

Net assimilation rate (Aleaf) and CO2 concentration in

the chloroplasts (p(Cc)leaf) of the whole leaf were

calculated by multiplying the open and closed patch ratio

in a leaf by A or p(Cc) values for open and closed patches.

The equations were as follows:

Aleaf ¼ rocAop þ ð1� rocÞAcl ð5Þ

pðCcÞleaf ¼ rocpðCcÞop þ ð1� rocÞpðCcÞcl: ð6Þ

The estimated maximum rate of carboxylation at 25�C

(Vcmax25*) was determined by substituting values of Aleaf

and p(Cc)leaf into Eq. 1.

For uniform photosynthetic parameters, we used the

observed optimal values of Vcmax25 (lmol m-2 s-1) and

gsmax (mol m-2 s-1), i.e., 41.1 and 0.19 in saplings and

40.3 and 0.19 in the canopy tree, respectively. We also

used values of 0.005 mol m-2 s-1 for gsmin and 2.2 for kj

following Takanashi et al. (2006). Based on the average

line or median value listed by Kosugi and Matsuo (2006),

we used the following parameterizations: 55,200 J mol-1

for DHa (Vcmax), 220,000 J mol-1 for DHd (Vcmax),

650 J mol-1 for DS (Vcmax), and 41,500 lmol m-2 s-1 for

DHa (Rd). Other parameters were taken from Table 2 of

Takanashi et al. (2006).

Observation of stomatal aperture frequency distribution

To determine real stomatal behavior distributions, we cal-

culated the frequency distribution of stomatal apertures for

single leaves from saplings and the canopy tree using the

SUMP method (Kamakura et al. 2011). Immediately fol-

lowing leaf gas exchange measurements, we made

impressions of the surface of one of the three experimental

leaves by fastening thin celluloid plate (2 cm in diameter,

SUMP Laboratory, Tokyo, Japan) dissolved in amyl ace-

tate to the abaxial leaf surface (the site at which gas

exchange rate was measured). Individual stomatal aper-

tures distributed within a patch were observed in a field of

vision under a digital microscope (Model VH-Z450; Key-

ence, Osaka, Japan). Stomatal numbers per patch were

28 ± 5 in saplings and 25 ± 5 in the canopy tree,

respectively (mean ± SD). We examined 40 microscope

fields per leaf for both saplings and the canopy tree. The

widths of individual stomatal pores were determined from

static images using NIH image software (National Insti-

tutes of Health, Bethesda, MD, USA). Because the leaves

used for making impressions were destroyed, we used

adjacent leaves with similar photosynthetic rates for sub-

sequent leaf gas exchange measurements.

Results

Diurnal patterns of leaf gas exchange and stomatal

distribution in Q. crispula saplings

Observed and simulated plots of diurnal changes in gas

exchange rates of Q. crispula saplings are depicted in Fig. 1.

The ratios of simulated A values calculated under assump-

tions of bimodal versus uniform stomatal behaviors (ratios of

gray-dash lines versus gray-solid lines of net assimilation

rate graphs in Fig. 1) were 0.67 ± 0.26, 0.46 ± 0.11, and

0.69 ± 0.08 on 1, 7, and 24 June, respectively

(mean ± SD). These values indicate the maximum effect of

patchy stomatal closure on A. Plots of observed A values fell

between plots of simulated A values calculated under

assumptions of uniform and bimodal stomatal behaviors.

The ratios of observed to simulated A under the assumption

of uniform stomatal behavior were 1.01 ± 0.17,

0.82 ± 0.02, and 0.61 ± 0.15 (mean ± SD). These values

indicate the actual effect of patchy stomatal closure on A,

with maximum depression occurring on 24 June. On 1 and 24

June, observed A and gs reached their maxima in the morn-

ing, and then decreased during the day as PPFD

([1,500 lmol m-2 s-1), Tleaf, and VPD increased to highest

J Plant Res

123

values. Observed A and gs values were rather constant during

7 June when PPFD, Tleaf, and VPD were more moderate. Net

assimilation and transpiration rates (TR) during midday

hours of 24 June were more depressed than those on 1 June.

A plot of observed A values through the day on 1 June fell on

the simulated A line calculated under assumption of uniform

stomatal behavior. On 24 June, however, the plot of observed

A values shifted to the simulated A line calculated under the

assumption of bimodal stomatal behavior (with PPFD, Tleaf,

and VPD increasing to maximum values), suggesting

changes in patterns of stomatal behavior.

Relationships between observed and simulated A values

for three leaves measured on each day demonstrate that

uniform stomatal behavior largely explained observed

trends in A through the day on 1 June (Fig. 2). This was not

the case for 7 June, when neither nor bimodal stomatal

behaviors explained observed trends in A. On 24 June,

uniform stomatal behavior explained only higher values of

observed A in the morning, but patchy stomatal behavior

with a bimodal distribution explained depressed values of

A during midday hours. Thus, there was probably a shift in

stomatal behavior pattern (from uniform to patchy bimo-

dal) during the day on 24 June. A comparison of data for 1

and 24 June also suggests that uniform and bimodal sto-

matal behavior largely explained the magnitude of the

severe depression of A on 24 June and the moderate

depression on 1 June.

Figure 3 depicts images (captured by SUMP method)

and plots of stomatal aperture frequency distributions

within a patch (a, d, g), and pooled frequency distribution

data for both stomatal apertures (b, e, h) and mean stomatal

apertures of patches (c, f, i) within single leaves of Q.

crispula saplings. On 1 June, most of stomata were wide

open at 0900 hours (Fig. 3a, b, c), following which, there

was a depression of stomatal openings at 1100 hours

(Fig. 3d, e, f). On 24 June, however, we observed open

(most stomata were wide open, Fig. 3g-1) and closed pat-

ches (most stomata were closed, Fig. 3g-2) on a single leaf

at 1200 hours (Fig. 3h). We detected heterogeneity of

individual stomatal pores within a patch at each observa-

tion time (Fig. 3a, d, g). However, two peaks in the fre-

quency distribution plots of mean stomatal apertures of

patches within a leaf observed at 1200 hours on 24 June

(Fig. 3i) indicate that there was a natural patchy bimodal

distribution of stomatal apertures among patches rather

than within patches during midday depression. The actual

scale of patchiness coincided with the theoretical scale of

patchy bimodal stomatal closure assuming a bimodal fre-

quency distribution of ‘closed’ and ‘open’ patches.

Fig. 1 Plots of observed and simulated (through numerical analysis

based on the Farquhar–von Caemmerer–Berry model) diurnal

changes in gas exchange rates in three Q. crispula sapling leaves.

Measurements were made on 1, 7 and 24 June, 2010. Net assimilation

rates (A), stomatal conductances (gs), transpiration rates (TR),

photosynthetically active photon flux densities (PPFD), leaf temper-

atures (Tleaf), and leaf to air vapor pressure deficits (VPD) are plotted

against time of day. Comparisons were made between observed

A values and simulations assuming uniform and patchy bimodal

patterns of stomatal behavior. Solid vertical lines indicate times when

impressions were made from abaxial leaf surfaces using the SUMP

method

c

J Plant Res

123

Diurnal patterns of leaf gas exchange and stomatal

distribution in a Q. crispula canopy tree at the TKY site

The ratios of simulated A values of the canopy tree of Q.

crispula calculated under assumptions of bimodal versus

uniform stomatal behaviors were 0.47 ± 0.03, 0.55 ±

0.04, and 0.51 ± 0.03 (mean ± SD) on 30 July, 31 July,

and 2 August, respectively (Fig. 4). These values indicate

the maximum effect of patchy stomatal closure on A. The

ratios of observed to simulated A under the assumption of

Fig. 2 Plots of observed and simulated net assimilation rates

(A) calculated by numerical analysis based on the Farquhar–von

Caemmerer–Berry model for three Q. crispula sapling leaves.

Simulated A was calculated assuming uniform stomatal closure

(a) or patchy bimodal stomatal closure (b). Close fits of observed data

to the line calculated under the assumption of uniform stomatal

behavior explained A. A close fit of observed data to the line

calculated under the assumption of patchy bimodal stomatal closure

indicates that patchy stomatal behavior with a bimodal distribution

explained A

Fig. 3 Images (captured by the SUMP method) and frequency

distribution plots of stomatal apertures within a patch (a, d, g), and

pooled frequency distribution plots for stomatal apertures (b, e, h) and

mean stomatal apertures for patches (c, f, i) within single Q. crispulasapling. Data were captured at 0900 and 1100 hours on 1 June, and at

1200 hours on 24 June

J Plant Res

123

uniform stomatal behavior were 1.02 ± 0.06, 1.04 ± 0.02,

and 0.81 ± 0.02 (mean ± SD). Thus, the effect of patchy

stomatal closure on A in canopy tree leaves was smaller

than in sapling leaves. On cloudy days (30 and 31 July),

A and gs were not depressed during midday hours, and

plotted observed A values fell on the simulated A line

calculated under the assumption of uniform stomatal

behavior through the day. On 2 August, however, gs

decreased during midday hours under higher PPFD

([1,200 lmol m-2 s-1), Tleaf, and VPD, indicating mod-

erate depression of A. On this day, observed A moved

between simulated A lines calculated under assumptions of

(1) uniform or (2) bimodal stomatal behaviors.

The relationships between observed and simulated

A values for three tree leaves on each day indicate that

uniform stomatal behavior explained optimum A through

the days of 30 and 31 July (Fig. 5). On the other hand, it

was difficult to determine whether either uniform or

bimodal stomatal behavior explained A through the day on

2 August.

Figure 6 depicts the distribution of leaf stomatal aper-

tures in the Q. crispula canopy tree on 2 August. Most

stomata were wide open at 0930 hours (Fig. 6a, b, c); there

was a depression of stomatal opening was shown at

1200 hours (Fig. 6d, e, f). At 1530 hours, however, open

and closed patches were observed within the leaf (Fig. 6g,

h). Two peaks in the frequency distributions of mean sto-

matal apertures of patches within the leaf (Fig. 6i) indicate

that there was a natural bimodal stomatal closure in late

afternoon hours.

Discussion

Changes in pattern of stomatal behavior by day

and growing condition

Patchy stomatal closure occurs with midday depression of

A in tree species with heterobaric leaves (Beyschlag and

Pfanz 1990; Beyschlag et al. 1992; Takanashi et al. 2006;

Kamakura et al. 2011). We showed a clear inhibition of

A coupled with stomatal closures in leaves of both saplings

and a canopy tree of Q. crispula during the daytime hours

on clear days (1 and 24 June in the nursery, and 2 August at

the TKY site), but the magnitude of inhibition varied by

day and growing conditions (Figs. 1, 3). By comparing

observed and simulated A values at a given gs for both

uniform and bimodal stomatal behaviors and observing

stomatal distribution directly (Figs. 2, 3), we demonstrated

Fig. 4 Plots of observed and simulated diurnal changes in gas

exchange rates in three leaves from a Q. crispula canopy tree at the

TKY site. Measurements were made on 30, 31 July, and 2 August,

2010. Net assimilation rates (A), stomatal conductances (gs), transpi-

ration rates (TR), photosynthetically active photon flux densities

(PPFD), leaf temperatures (Tleaf), and leaf to air vapor pressure

deficits (VPD) are plotted against time of day. Comparisons were

made between observed A values and simulations assuming uniform

and patchy bimodal patterns of stomatal behavior. Solid vertical linesindicate times when impressions were made from abaxial leaf

surfaces using the SUMP method

c

J Plant Res

123

that patchy bimodal stomatal closure explained severe

depression of A during midday hours of 24 June; moderate

midday depression of A was coupled with uniform stomatal

closure in Q. crispula saplings on 1 June. Thus, midday

depression of A did not always occur with bimodal sto-

matal closure. Numerical analysis indicated that uniform

stomatal behavior in leaves of the canopy tree best

explained A through cloudy days at the TKY site (30 and

31 July, Fig. 5). However, neither uniform nor bimodal

stomatal behaviors explained A in leaves of either saplings

Fig. 5 Comparisons between observed and simulated net assimila-

tion rates (A) calculated by numerical analysis based on the Farquhar–

von Caemmerer–Berry model for three Q. crispula canopy tree leaves

collected at the TKY site. Simulated A was calculated assuming

uniform stomatal closure (a) or patchy bimodal stomatal closure (b).

Close fits of observed data to the line calculated under the assumption

of uniform stomatal behavior explained A. A close fit of observed data

to the line calculated under the assumption of patchy bimodal

stomatal closure indicates that patchy stomatal behavior with a

bimodal distribution explained A

Fig. 6 Images (captured by the SUMP method) and frequency

distribution plots of stomatal apertures within a patch (a, d, g), and

pooled frequency distributions of stomatal apertures (b, e, h) and

mean stomatal aperture of patches (c, f, i) within a Q. crispula canopy

tree leaf at the TKY site. Data were captured at 0930, 1200, and

1530 hours on 2 August

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123

or the canopy tree through the days of 7 June and 2 August

(Figs. 2, 5). Thus, on sunny days with high temperature and

VPD, the pattern of stomatal closure shifts to a bimodal

frequency distribution and induces severe midday

A depression. In contrast, the patterns of stomatal opening

and closing show a uniform frequency distribution (in some

cases, neither uniform nor bimodal frequency distribution)

on cool, cloudy days. These results suggest that patterns of

stomatal behavior shifted in a flexible manner between

uniform and bimodal frequency distributions depending on

micrometeorological conditions. Different patterns of sto-

matal behavior between saplings and the canopy tree

indicate that growing conditions would also affect stomatal

opening and closing pattern.

Effects of biotic factors on leaf gas exchange

Our numerical simulation predicted that gi was infinite;

thus p(Cc) and p(Ci)* were identical. However, diurnal

change in gi might have been caused by aquaporin, which

affects photosynthetic CO2 diffusion (Terashima et al.

2006). Figure 7 depicts the sensitivity of simulated A under

assumption of uniform stomatal closure (a) and patchy

bimodal stomatal closure (b) to changes in gi. In each

graph, observed A varied much more than the gi range.

Thus, changes in stomatal behavior were more susceptible

to leaf gas exchange than gi.

The SUMP-based analysis demonstrated that ‘‘closed

patches’’ assumed in a numerical simulation might in reality

be rather open (Figs. 3, 6). Hence, the real gsmin might have

been higher than the value we used (0.005). Figure 8 depicts

the sensitivity of simulated A to changes in gsmin. Simulated

A values under the assumption of bimodal stomatal closure

decreased with gsmin. If bimodal stomatal closure with a

higher value of gsmin ([0.005) is assumed, simulated

A values under the assumption of bimodal stomatal closure

would be higher than those of this study (Figs. 1, 4). If

bimodal stomatal behavior with a higher value of gsmin were

to occur in nature, bimodal stomatal behavior might have

explained A on 7 June and/or 2 August since A on those two

dates were not explained neither by uniform nor bimodal

stomatal behavior (Figs. 2, 5).

Which factors induce bimodal stomatal behavior?

Because the saplings used in this study were well watered

before measurements, midday stomatal closure on 1 and 24

June might have been caused by increased transpirational

demand (Maier-Maercker 1983; Mott and Parkhurst 1991)

rather than decreased leaf water potential. Comparisons of

gas exchange data for 1 and 24 June indicate that magni-

tude of midday depression in Q. crispula saplings was

correlated with Tleaf and VPD, which are strongly related to

transpirational demand (Fig. 1), as reported previously

(Lange 1988; Muraoka et al. 2000). Indeed, Tleaf and VPD

increased rapidly by midday on 24 June, and depression of

A began earlier than on 1 June. Moreover, TR decreased

with severe depression of gs during midday hours of 24

June (Fig. 1). Hence, the pattern of stomatal behavior,

which varied flexibly with Tleaf and VPD, appeared to

determine the magnitude of midday depression. In other

words, bimodal stomatal closure may occur when Tleaf and

VPD exceed certain threshold values even though tree

species with heterobaric leaves would not show bimodal

stomatal behavior at sub-threshold values of these two

parameters.

The effect of patchy stomatal closure on A in the canopy

tree leaves was smaller than in saplings (Figs. 1, 4). Sap-

lings generally have smaller and shallower root systems

Fig. 7 Sensitivity of simulated A under assumption of uniform

stomatal closure (a) and patchy bimodal stomatal closure (b) to

changes in internal conductance (gi). Data from 24 June (leaf 3)

Fig. 8 Sensitivity of simulated A under the assumption of patchy

bimodal stomatal closure to changes in minimum stomatal conduc-

tance (gsmin). Data from 24 June (leaf 3)

J Plant Res

123

than canopy trees, which are able to develop root systems

into deeper soil layers where water may be more abundant

(Lyford 1980). Canopy trees also have great biomass

allocated to storage tissues that may hold reserve water

(Holbrook and Putz 1996). Thus, different age classes of

trees use different strategies when responding to water

stress under high light; saplings resist water stress by

closing stomata, whereas canopy trees avoid water deficit

by accessing deeper water reserves (Cavender-Bares and

Bazzaz 2000). Although Tleaf and VPD values of the can-

opy tree during 2 August were lower than those in saplings

during 24 June (Figs. 1, 3), it is also possible that a dif-

ference in stomatal sensitivity between saplings and can-

opy trees under high light may affect pattern of stomatal

behavior during midday hours.

On 2 August, Tleaf and VPD of the canopy tree reached

maxima during midday hours, and then decreased with

light reduction (Fig. 4). Nevertheless, we assumed

depression of A coupled with bimodal stomatal closure in

Q. crispula leaves during late afternoon hours. Kosugi et al.

(2009) reported that depression of A coupled with bimodal

stomatal closure occurs in tropical rainforest top-canopy

leaves during late afternoon even under relatively moderate

PPFD, Tleaf and VPD. Thus, bimodal stomatal closure may

be cued by circadian rhythms as well as by transpirational

demand (Doughty et al. 2006; Kosugi et al. 2009).

Scale differences in the heterogeneity of stomatal

aperture distribution

Our direct observation of stomatal aperture frequency

distribution in Q. crispula leaves using the SUMP method

agreed with simulated depression of A caused by bimodal

stomatal closure, indicating real occurrence of patchy

bimodal distribution in stomatal apertures (Figs. 3g, 6g).

We detected bimodal distributions of stomatal apertures

among patches by the presence of two peaks in the fre-

quency distributions of mean stomatal apertures of patches

within single leaves (Figs. 3i, 6i); we also observed het-

erogeneity of individual stomatal pores within a patch.

Hence, bimodal stomatal closure occurred among patches

rather than within patches in Q. crispula leaves. Theoret-

ically, the bimodal distribution of stomatal apertures

among patches rather than within patches would lead to Ci

heterogeneity among patches and, hence, to severe

depression of A (Terashima et al. 1988). Our results for Q.

crispula were concordant with this theoretical manner of

‘patchy bimodal stomatal closure’. We reported previously

that a bimodal distribution of stomatal aperture occurs

within patches in top-canopy leaves of several tropical

rainforest species, and we observed that this scale of

patchiness is coincident with severe depression of A (Ka-

makura et al. 2011). In theory, CO2 absorbed from open

stomata diffuses uniformly within a patch and the occur-

rence of patchy bimodal stomatal behavior within patches

rather than between them would not cause heterogeneity of

Ci. However, the effects of real distributions of stomatal

apertures within and between patches on the distribution of

Ci, and hence A, may be more complicated. Further anal-

yses are necessary to determine which scales of hetero-

geneity in the frequency distribution of stomatal apertures

regulate leaf gas exchange capacity.

Acknowledgments We thank Dr. Hibiki Noda of the University of

Tsukuba for her support of field investigations at the Takayama site.

We also thank Mr. Kenji Kurumado and Yasunori Miyamoto at the

Takayama field station, Gifu University, for their arrangement of field

investigations.

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