Plant Physiol.
(1988) 86, 782-785
0032-0889/88/86/0782/04/$01 .00/0
Gas Exchange Analysis of the Relative Importance of Stomatal
and Biochemical Factors in Photosynthetic Induction in
Alocasia macrorrhizal
Received for publication June 30. 1987 and in revised form September 3. 1987
MIKO U. F. KIRSCHBAUM AND ROBERT W. PEARCY*
Department of Botany, University of California, Davis, California 95616
ABSTRACT
When leaves of Alocasia macrorrhiza adapted to 10 micromole quanta
per square meter per second were transferred to 500 micromole quanta
per square meter per second, the rate of photosynthetic CO2 assimilation
increased for over 45 minutes. For the first 10 to 15 minutes, increases
in both stomatal conductance and the leaf's photosynthetic capacity were
responsible for the increase in assimilation rate. Thereafter, continuing
increases in stomatal conductance were almost entirely responsible for
further increases in assimilation rate. When conductances were initially
high, assimilation rates 1 minute after the increase in photon flux density
could be more than six times as high as for similar leaves with initially
low conductance. Further increases in assimilation rate in these leaves
with high conductance were predominantly due to increases in the in-
duction state at the biochemical level and followed an exponential time
course. When stomatal conductances were initially low, then increases in
conductance were predominantly responsible for the increases in assim-
ilation rate, with both following a sigmoidal time course. In these leaves,
it was important to also consider the effect of cuticular water loss on the
calculation of the intracellular partial pressure of C02, and an assessment
of the relative importance of stomatal conductance differed considerably
from one that did not include cuticular water loss.
Plants that grow in the forest understory typically experience
long periods of shade followed by sudden and substantial in-
creases in PFD2 during sunflecks. These increased levels of PFD
may last from fractions of a second to many minutes, and con-
tribute a substantial proportion of total daily photon flux (2, 5,
11).
The plant's ability to use this photon flux for photosynthesis
is strongly influenced by the photosynthetic induction state. As
induction tends to be low in plants in low PFD (6), photosynthetic
carbon gain immediately following an increase in PFD is less
than would be predicted from measurements on fully induced
leaves. To predict carbon gain of plants experiencing strongly
fluctuating PFD levels, photosynthetic induction must therefore
be understood and incorporated into any analysis.
It has been known for a long time that there is a requirement
I
Supported by the United States Department of Agriculture Com-
petitive Research Grants Office under Agreement No. 85-CRCR-1-1620.
2Abbreviations: PFD, photon flux density (of photosynthetically ac-
tive radiation); A, net CO2 assimilation rate; g, leaf conductance (i.e.
the sum of stomatal and cuticular); pi. intercellular partial pressure of
C02; RuBPCase, ribulose-1 ,5-bisphosphate carboxylase/oxygenase (EC
4.1.1.39).
782
for photosynthetic induction (15), but there is still no consensus
about the factors contributing to it. Studies on isolated chloro-
plasts (reviewed by Edwards and Walker [7]), protoplasts (17),
and on leaves in high CO2 (14) show that the induction require-
ment is a feature at the biochemical level and not solely de-
pendent on increases in stomatal conductance. However, in these
systems that are not limited by CO2 diffusion, induction tends
to be completed in a matter of minutes (7), while in intact leaves
at normal atmospheric partial pressure of CO, it requires half
an hour or more, and is paralleled by increases in stomatal con-
ductance (6).
While much work has been done on the biochemical changes
occurring over the first few minutes, it is not yet understood to
what extent the longer term increase in A is caused by stomatal
versus biochemical factors. A particular problem is cuticular
evaporation which has not been taken into account in earlier
work (6, 18). The cuticular path is likely to be much more re-
strictive for CO2 uptake than water loss because CO2 must tra-
verse the epidermal cell layer in addition to the cuticle before
reaching a photosynthetically active mesophyll cell. When sto-
matal conductances are low, failure to take cuticular conductance
into account can lead to an overestimation of the intercellular
partial pressure of CO2.
Because of the importance of understanding the causes of a
low photosynthetic induction state and its relationship to carbon
gain of leaves in understory environments, we investigated with
gas exchange techniques the extent to which stomatal or bio-
chemical factors were responsible for the leaf's induction state.
This was primarily done by comparing induction responses of
leaves with high and low conductance. We used the Australian
tropical understory plant, Alocasia macrorrhiza, which has been
the subject of previous studies on induction (6).
MATERIALS AND METHODS
Plant Material. In all experiments, approximately 1-year-old
plants of Alocasia macrorrhiza (L.) G. Don, grown from seed
collected in a tropical rain forest near Atherton, Queensland,
Australia, were used. Plants were grown in 4-L pots in a 50:50
mixture of pumice rock and potting soil, fertilized with half-
strength Hoagland solution twice a week and watered daily. All
plants were grown under shade cloth in a glasshouse that reduced
natural PFD to a maximum of about 30 ,tmol quanta m-' s
at midday in summer. In winter the natural PFD was supple-
mented by a series of incandescent lights that added about 20
,umol quanta m-2 S-1 to the PFD received by the leaves for
approximately 14 h daily. Experiments were carried out over a
period of approximately 1 year. No difference was apparent be-
tween induction responses in different seasons.
Gas Exchange. Gas exchange measurements were done on
 PHOTOSYNTHET4C INDUCTION IN ALOCASIA 783
young, fully expanded leaves in an open system as described
previously (13). Gas with a desired CO2 concentration was ob- A |
^

tained by injecting 4% CO2 in air into a gas stream of C0,-free

air. Flow rates of both gases were controlled with mass flow 4
controllers (Tylan model FC-260). Humidity was controlled by (n
humidifying the air and then removing excess water vapor with 3-
a temperature-controlled condenser. A single, attached leaf was E
enclosed in a whole-leaf chamber. Enrichment of water by the -a
leaf was measured with a relative humidity sensor (Vaisala HM E
2-
1iP), and depletion of CO, was measured with a differential CO,
analyzer (Horiba model VIA 500R). Overflow bubblers ensured
that the pressure inside these instruments was the same for gas
coming from the leaf chamber and that bypassing the chamber.
Water vapor effects on the CO2 analyzer (4) were mathematically 0 I
taken into account. Leaf temperature was measured with a cop-
per-constantan thermocouple (Omega SCPSS-032E-6). A data N
.. .. .. . .. .
acquisition (Hewlett Packard model HP 3497) and computer .S .S
(IBM PC/AT) system allowed automatic setting of flow rates,
and frequent and accurately timed sampling of gas exchange 6 50 _ ;00
wo~~~~~~~oo
rates. All calculations of gas exchange parameters were based 04
on the equations of von Caemmerer and Farquhar (19). Stomatal E
conductance was calculated as the difference between measured
leaf conductance and assumed cuticular conductance for water. E
3
Cuticular conductance for CO, was assumed to be zero. Photon
flux density was measured with a LI-COR quantum flux sensor
(LI-COR LI-190 SR). Low PFD refers to be a background PFD 30 45 60
of 10,umol quanta M-2 s
supplied by an incandescent light -1 0
',

bulb. High PFD refers to a PFD of approximately 500 ,umol -1

I

0
1T 5 30 45 60
quanta m-2s supplied by a 2.5 kW water-cooled Xenon-arc
lamp. This was more than saturating for photosynthesis in these
low-light grown plants, and appeared to cause no damage even Time (minutes)
if leaves were subject to it for many hours. The timing of PFD FIG. 1. Timecourse of CO2 assimilation rate (A) and leaf conduct-
changes was determined by an electronic shutter that was con- ance (B) following step
a increase in PFD from 10 to 500 Amol quanta
trolled either manually by a switch or directly by the computer. 2 s The arrows in the
m- -'. figure
denote the time when PFD was in-
creased. The leaf was preconditioned
in low CO2 (75 to obtain ,bar)
high leaf conductance. The CO2 concentration was changed to normal
RESULTS ,ubar)
atmospheric (350 about 5 min before the increase in PFD.
When PFD was ,umolm '2s
first increased from 10 to 500 1,

the rate of photosynthetic CO, assimilation exhibited an initial
fast increase over the first min, followed by a slower increase
over the next 45 to 60 min (Figs. IA, and 2A). This slower
increase was paralleled by increased leaf conductance (Figs. lB
and 2B). When the conductance in 10 ,umol quantam ' was 2 the activation state of the biochemical components and their rate
varied by exposing the leaf to either 980 or 75,ubar CO, (but
returning it to 350,tbar about 5 min before the increase in PFD),
the time course of the increase in assimilation rate varied. The
increase was more strongly sigmoidal for the leaf with a low
initial conductance (Fig. 2) than for the same leaf with high initial
conductance (Fig. 1). It was generally observed in analyses of
over 40 induction responses that time courses were more sig-
moidal if initial leaf conductance was low.
For an assessment of the relative importance of stomatal con-
ductance in determining the induction state, it was important to
consider the effect of cuticular evaporation on the calculation of
gas exchange parameters. This is illustrated in Table I where
values of the intercellular partial pressure of CO, and the initial
slope of the A(pj) relationship were calculated with different
assumptions about cuticular conductance for water. Cuticular
conductance for CO, was assumed to be 0. In the leaf with high
conductance, assumptions about cuticular evaporation had little
effect on pi and the estimated biochemical induction state (as
measured by dAldpj. When conductance was low, however, pi
and consequently the inferred biochemical induction state was
strongly affected by the assumed value of cuticular conductance.
A value of cuticular conductance can be found with which the
same induction state at the biochemical level was obtained for
both leaves. This value was 3.2 MMOI M 2
s Iin this particular
example (TableI). The same analysis was done for 7 leaves. The
mean estimate of cuticular conductance was 2.7 -+- 0.3mmol m-2
s (mean + SE). Implicit in this method of estimating cuticular
conductance is the assumption that in 10,mol quanta m s-
of activation in response to the increase of PFD are independent
of the initial stomatal conductance.
In order to separate the relative contribution of leaf conduct-
ance and biochemical factors in determining the low assimilation
rates early in induction, assimilation rate was plotted as a func-
tion of the intercellular partial pressure of CO2 (pi) during in-
duction. The intercellular partial pressure of CO2 was calculated
using estimate of cuticular conductance of 3.2 mmol m- s-'.
When initial conductances were high, the points initially started
below the steady state A(p1) relationship, shown by the solid
curve, but gradually approached it over the first 10 to 15 min of
the induction response (Fig. 3A). This indicates that an activation
of the biochemical components of the photosynthetic apparatus
was occurring over the first 10 to 15 min. The relatively small
change in p1 between 2 and 10 min was a result of increasing
stomatal conductance in combination with the increasing slope
of the A(p,) relationship. After about 10 min, all points fell on,
or very close to, the steady state A(p,) curve, indicating that
increases in stomatal conductance, resulting in higher pi values,
were responsible for further increases in assimilation rate.
When initial conductances were low (Fig. 3B), there was con-
siderably more variation in the pi values becausevalueseven small
variations in conductance had a large effect. The pi within
the first 5 min after the increase in PFD were as low as 80 to
784 KIRSCHBAUM AND PEARCY Plant Physiol. Vol. 86, 1988
5 after the increase in PFD against leaf conductance (Fig. 4). As-
similation rates were strongly dependent on conductance, with
the leaves with highest conductance having values approximately
- six times as high as those of leaves with lowest conductance (Fig. 4).
En 4 S

I
3 1*.
E DISCUSSION
2 The results show how both stomatal and biochemical factors
were jointly responsible for the photosynthetic induction re-
1 _ / sponse, with their relative importance depending on stomatal
N conductance at the time of the increase in PFD. When con-
ductance was low, then increases in assimilation rate early in
0- induction were predominantly determined by increases in con-
ductance. Increases in the biochemical induction state alone would
E have led to only minor increases in assimilation rate. When initial
-a conductance was high, biochemical induction assumed a greater
n
60
B S.SSS..SS.S.*.S..... importance over the first 10 to 15 min of induction. The parallel
A**SS
increase in conductance was important, however, in maintaining
I pi values nearly constant. Further increases in assimilation rate
after 10 to 15 min were caused by increases in pi due to continuing
E stomatal opening.
v6 30 _ In leaves with low stomatal conductance it is inherently difficult
to assess the importance of stomatal conductance because of
uncertainties about the cuticular path for water loss. Cuticular
conductance for water is generally believed to be low (16), but
0 it is likely to be even lower for CO2 because CO2 must also
-15 0 15 30 45 60 diffuse through an epidermal cell before reaching the photosyn-
thetically active mesophyll. When stomatal conductance is also
low, a failure to partition transpiration between stomatal and
Time (minutes) cuticular paths can result in a considerable overestimation of pi
(Table I) and an underestimation of the significance of stomatal
FIG. 2. Time course of CO, assimilation rate (A) and leaf conduct- conductance. The problem is less acute when stomatal conduct-
ance (B) following a step increase in PFD from 10 to 500 gmol quanta ance is high, but the cuticular path still should be considered in
m- 2 s- . The arrows in the figure denote the time when PFD was in-
any analysis of induction.
creased. The leaf is the same as shown in Figure 1, but in this case it
was preconditioned in high CO, (980 ,ubar) to obtain low leaf conduct- Unfortunately, there appear to be no ways to directly measure
ance. The CO, concentration was changed to normal atmospheric (350
the magnitude of cuticular evaporation that are independent of
,ubar) about 5 min before the increase in PFD. uncertainties concerning evaporation through the stomatal pore.
The conductance of the adaxial leaf surface of A. macrorrhiza,
which lacks stomata, was measured to be 0.31 + 0.09 mmol m- 2
100 Abar, and caused assimilation rates to be low. Here, the s ' (mean ± SE; n = 10), but this value would not be expected
changes in pi due to stomatal opening were clearly more impor- to be valid for the abaxial surface. Peristomatal evaporation (1,
tant in causing increases in assimilation rate during induction 10) may be important so that the value for a surface with closed
than when initial conductances were high. Nevertheless, for about stomata may be many times higher than for a surface lacking
the first 10 min points largely fell below the steady state A(pi) stomata. In the absence of techniques for directly measuring
relationship. Increases in the biochemical induction state in the cuticular conductance, indirect methods such as the one em-
absence of concomitant stomatal opening, however, would have ployed in this study must be used. If it was assumed that the
led to only minor changes in assimilation rate. biochemical induction state was independent of stomatal con-
There was considerable leaf-to-leaf variation in the value of ductance, then cuticular conductance in average leaves must have
leaf conductance in 10 ,umol quanta m -2 S- ' (9). It also declined been about 2 to 3 mmol m-2 s-'. If, on the other hand, it was
diurnally (9) and responded to humidity (data not shown). It assumed that cuticular conductance was less than 2 to 3 mmol
was therefore possible to obtain induction responses for leaves m- 2 s-1, then leaves with a low initial conductance must have
with a wide range of conductances in low PFD. With these data, also had a lower biochemical induction state than leaves with
the role of leaf conductance in the early phases of induction was higher initial conductance.
further investigated by plotting assimilation rates measured 1 min In understory microenvironments, the induction state of the

Table I. Effect of Including a Cuticular Conductance, g, on the Calculation of the Intercellular Partial
Pressure of CO2 and the Initial Slope of the A(p,) Relationship
The data used for these calculations are the 1-min points in Figures 1 to 3. Calculations assumed a C02-
compensation point of 44 ,bar.
gl = 37.4mmolm-2 S-I g1= 8.6mmol M-2S-I
9c
Pi dAldp, pi dAldp,
mmol m-2S-2 ,ubar mol m-2 s-I bar-' ,ubar mol m s- bar-'
o 216.6 0.0153 183.1 0.0061
2 210.5 0.0159 137.7 0.0091
3.2 206.7 0.0163 96.3 0.0163
4 203.7 0.0166 54.3 0.0830
 PHOTOSYNTHETIC INDUCTION IN ALOCASIA 785
6 photosynthetic apparatus is likely to be an important determinant
of the capacity to utilize sunflecks. The analysis in this paper
shows that both stomatal and biochemical factors can be impor-
tant in the induction response, with the relative importance of
4 each during the first 10 min being a function of the initial con-
ductance in low PFD. Measurements on A. macrorrhiza in a
C-1 shaded tropical forest understory show that leaf conductances
in are quite high (>40 mmol m-) s- 1) even after long periods when
C4
the PFD does not exceed 5 to 10 ,umol quanta m-2 s '(3). With
E 2 initial conductances of this magnitude, the induction response
over the first 10 min would be dominated by biochemical limi-
E tations. However, water stress and even wilting of some plants
-6 in the understory does occur during the dry season in Australian
0 0 tropical forests where A. macrorrhiza is abundant. Under these
L-
conditions a greater role for stomata in limiting the induction
:._
C response might be expected.
0 The high conductances in low PFD would appear to be wasteful
in terms of water. However, it should be remembered that tran-
(O.4
4 spiration rates in these understory microenvironments would still
0 be low because of low leaf-air vapor pressure gradients. More-
0EIn over, the high stomatal conductance may be advantageous in
allowing a faster induction and higher carbon gain during sun-
2 flecks and in maintaining a higher quantum yield because of the
greater intercellular partial pressures of CO2 (12).

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