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STOMATAL RESPONSES TO LIGHT IN
XANTHIUM STRUMARIUM AND OTHER SPECIES

 

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Thomas David Sharkey

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STOMATAL RESPONSES TO LIGHT IN
XANTHIUM STRUMARIUM AND OTHER SPECIES

By

Thomas David Sharkey

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1979

ABSTRACT

STOMATAL RESPONSES TO LIGHT IN
XANTHIUM STRUMARIUM AND OTHER SPECIES

By
Thomas David Sharkey

The stomatal response to light was investigated with detached leaves
in an attempt to answer the following questions:
l. To what degree do stomata respond to light indirectly via the
effect of photosynthesis on the concentration of C02 inside the
leaf?

2. How do factors other than light influence the stomatal response
to light?

3. what are the photoreceptors involved in the stomatal response to
light?

Stomatal conductance was monitored by measuring the humidification
of air that had passed over a leaf and the water vapor concentration
difference between the inside and outside of the leaf.

It was shown that stomata of leaves of Xanthium strumarium responded
to light when photosynthesis was eliminated by the electron transport
inhibitor'cyanazine (2-chloro-4-(l-cyano-l-methylethyl amino)-6-ethyl
amino-s-triazine). An analysis of slopes of curves relating stomatal
conductance to light intensity and intercellular C02 concentration
indicates that the response to light of stomata of g, strumarium,

Phaseolus vulgaris, Perilla frutescens, and Gossypium hirsutum is for

 

the most part not mediated by changes in the intercellular CO2 concen-
tration. However, in ggg_mays at low irradiance, the light-dependent
depletion of C02 inside the leaf provided more than one-half of the

7

opening stimulus. At high irradiance in 5, mays, the intercellular C02

Thomas David Sharkey

concentration did not vary with changes in irradiance and since it is
known that the stomatal response to 602 is not strong enough to keep
the intercellular C02 concentration constant, it is concluded that
the stomata were responding directly to light.

Although the stomatal response to light generally did not depend on
the stomatal response to C02, the stomata of all of the species studied
here did open when the C02 concentration was lowered. However, in
E, frutescens, E. vulgaris, and 5, strumarium the stomatal response to
CO2 was diminished or absent at high irradiance. In 1, strumarium, it
was demonstrated that the direct stomatal response to light was dimin-
ished at high C02 concentrations. Abscisic acid, humidity, and leaf
temperature affected the stomatal response to light in such a way that
the various curves were coincident when they were plotted as a percentage
‘ of the conductance at the highest irradiance.

Experiments with leaves illuminated on either the adaxial or abaxial
surface indicate that the photoreceptor pigment for the direct stomatal
response to light is in the epidermis, presumably in the guard cells.

An action spectrum of stomatal opening in §: strumarium showed that

 

blue light was very effective, while red light was one-tenth to one-fifth
as effective as blue light, and green light was hardly effective at all.
The responses of stomata and C02 assimilation to red light had similar
action spectra and were both eliminated by cyanazine. This evidence
shows that chlorophyll is the pigment responsible for the red light
response of stomata. The red light response was not, however, mediated
by photosynthesis-dependent changes in the intercellular C02 concen-
tration, since the ambient C02 concentration was manipulated to keep

the internal C02 concentration constant.

Thomas David Sharkey

The blue light response of stomata was only slightly reduced by
cyanazine. This indicates that a photoreceptor pigment other than

chlorophyll is also involved in the stomatal response to light.

ACKNOWLEDGEMENTS

I thank the people of the Plant Research Laboratory and the rest of
the University for the rich and rewarding experiences I have had while
carrying out this research.

Each member of my guidance committee, Klaus Raschke, Norman Good,
Kenneth Poff, and Jan Zeevaart, has given me thoughtful advice and
constructive criticism which has been of great help. I thank my major
professor, Klaus Raschke, for providing an environment that was stimu-
lating and challenging. .

I thank Graham Farquhar for his teaching, advice, and encouragement
while I was first learning about measuring gas exchange of leaves.

For encouragement, editorial assistance, and typing, I thank my wife,
Paulette Bochnig.

This research was supported by the U.S. Department of Energy under
contract EY-76-C-02-1338.

11°

TABLE OF CONTENTS

LIST OF TABLES
LIST OF FIGURES
KEY TO SYMBOLS

INTRODUCTION .

The Stomatal Mechanism .

Stomatal Responses to Light and Other Environmental Factors
Effect of Light Quality on Stomatal Opening .
Possible Effects of Light on Guard Cells .

MATERIALS AND METHODS

Analysis of Slopes

Gas Analysis .

Light . . . .

Plants . . . .

Procedure .
RESULTS

Measurement of Direct and Indirect Stomatal Responses to
White Light . .

Interactions between Stomatal Responses to White Light
and CO .
Effect Of ABA, Humidity, and Leaf Temperature on the
Stomatal Response to White Light . .
Inverted Leaf Experiments .

Effect of Light Quality on Stomatal Conductance and
Assimilation . .

DISCUSSION

Direct Versus Indirect Effects of Light on Stomata . .

Mesophyll- Dependent Versus Mesophyll-Independent Effects
of Light on Stomata .

Interactions between Responses to Light and CO2, ABA, Leaf
Temperature, and Vapor Pressure Deficit. . .
Effects of Light, C02, and ABA on Guard Cells

Effect of Light Quality on Stomatal Opening

iii

Page

vi

viii

(DUINN H

19
29

32
43

43
71
71
73
73
76

Page

Action Spectra . . . . . . . . . . . 77

‘ Inhibitors . . . . . . . . . . . . 79
SUMMARY . . . . . . . . . . . . . . 82
LIST OF REFERENCES . . . . . . . . . . . 84
APPENDIX . . . . . . . . . . . . . . 89
Absorption of Light by Leaves . . . . . . . 89

iv

Table

LIST OF TABLES

Effect of light on stomatal conductance and assimilation
using cyanazine treated leaves.

Effect of light on stomatal conductance and assimilation
of leaves.

Values for partial differentials.

Intercellular C02 concentration, stomatal conductance,
and assimilation rate at four light levels.

Effect of red or blue light on assimilation rate, inter-
cellular C02 concentration, and stomatal conductance in
leaves of Xanthium-strumarium. ‘

Effect of DCMU on stomatal conductance and assimilation
in red and blue light.

Transmissivity, reflectivity, and absorptivity of leaves.

Light absorbed by leaves after reflection from the lower
surface.

Page'

22

° 24
26

28

51

65
9O

95

Figure

1.

10.

11.

12.
13.

14.

LIST OF FIGURES

Effect of cyanazine on stomatal response to 002.

Stomatal response to C02 of leaves of Xanthium strumarium
fed cyanazine at three irradiance levels.

Stomatal response to C0 of Xanthium strumarium (without
cyanazine) at fbur irraéiance levels.

Stomatal response to C02 at various irradiance levels for
Perilla frutescens, Phaseolus vulgaris, Gossypium hirsutum,
and Zea mays.

Stomatal response to light of leaves of Xanthium strumarium
fed cyanazine at three concentrations of C02.

Stomatal response to light of leaves of Xanthium strumarium
fed cyanazine and abscisic acid.

Stomatal response to light of leaves of Xanthium strumarium
at various humidity and leaf temperature combinations.

Stomatal response to light of leaves of Xanthium strumarium
fed cyanazine showing differences between adaxial and
abaxial responses.

Stomatal response to light of leaves of Xanthium strumarium
as light was increased and then decrease .

Stomatal response to light of leaves of Xanthium strumarium
in the normal or inverted orientation.

Stomatal response to light quality of leaves of Xanthium
strumarium.

Action spectrum of stomatal opening.

Stomatal conductance and assimilation rate in various wave-
lengths of red light.

Page
21

31

34

36

38

40

42

45

47

49

53
56

59

Stomatal closure in response to turning off the light (674 nm)

with the intercellular C02 concentration held constant.‘

vi

61

Figure . - Page

15. Stomatal conductance and assimilation of Xanthium
strumarium leaves fed cyanazine and placed in red or

 

bTue light. 63
16. Effect of 602 on the stomatal response to red or blue

light. . 67
17. Stomatal response to monochromatic blue or red light

in the normal or inverted orientation. 7O
l-A. Paths of light through leaves. 92

vii

ABA

cyanazine

DCMU

KEY TO SYMBOLS

assimilation rate (usual units are umol m"2 sec-1)

abscisic acid

plants in which the first identifiable product of photo-
synthesis is 3-phosphoglyceric acid (All plants reported
herein except Zga_mays are C3 plants.)

plants in which the first identifiable product of photo-
synthesis is a four carbon dicarboxylic acid (Of the plants
reported herein, only Zea mays is a C4 plant.)

ambient C02 concentration (usual units are p2 2'1)

CO2 concentration in the intercellular spaces of the leaf
(usual units are um 2'1)

2-chloro-4-(l-cyano-l-methylethyl amino)-6-ethyl amino-s-
triazine

3-(3,4-dichlorophenyl)-1,1 dimethylurea
conductance to water vapor, inverse of the more common but
less appropriate resistance to water vapor (usual units are

cmol m' sec' )

irradiance or quantum flux (usual units are w m'2 for white
light and uE m'2 sec'1 for monochromatic light)

viii

PEP phosphoenolpyruvate

r resistance to water vapor (usual units are 1112 set cmol'l)

RH relative humidity

RuBP ribulose bisphosphate

VPD vapor pressure deficit (usual units are ml water vapor/l air)
A wavelength of light (usual units are nm)

(ax/3y)z the slope of the curve of x versus y with 2 held constant

ix

INTRODUCTION

Stomata regulate gas exchange between leaves and their environment.
More water vapor diffuses through the stomata than any other gas; on
the order of 1000 times more water vapor is lost through the stomata
than is carbon dioxide taken up over the life of the plant. Although
this water loss is usually seen as a deleterious consequence of sto-
matal opening for carbon dioxide uptake, it plays an important role in
dissipating the energy received from the sun. Two other important
gases diffusing through the stomata are carbon dioxide and oxygen.
During photosynthesis, C02 diffuses into the leaf and 02 diffuses out.
The 002 concentration difference between the inside and the outside of
the leaf can never be greater than 320 um 2'1, since this is the 002
concentration in the air. Since oxygen diffuses faster than 002 (1.17
times faster theoretically). the 02 concentration difference between
the inside and outside of the leaf is less than the 002 concentration
difference. This concentration difference is insignificant when com-
pared to the concentration of 02 in the air (2 X 105 us 2'1) and so
stomatal movements do not exert large effects on the 02 concentration.
Since water vapor and 002 are the most important gases diffusing through
the stomata, it is reasonable to expect that stomatal function will be
related to plant water status and photosynthesis in some way.

Two reasons to study gas exchange and stomata are the following:

first, to develop a basic understanding of stomata and how they move,

and second, to understand how stomatal responses to various stimuli
allow plants to be successful in diverse environments. This information
can be used to determine what, if any, limitations stomata impose on
photosynthesis and plant growth. It may also help plant breeders to

fit crop plants to the environment, for example, to determine which
plants would best take advantage of an irrigated field and which plants
would be able to survive a dry land farming situation. There may also
be room for improvement by selecting stomatal behavior that is best
suited to a large economic yield from crop plants, rather than the
stomatal behavior best suited to propagation, which natural selection

would have favored.

The Stomatal Mechanism

Stomata open by filling with salts of potassium which increase the
osmotic pressure inside the guard cells (11,37). The resultant increase
in turgor pressure allows the guard cells to push the surrounding epi-
dermal cells apart, leaving a hole between the two guard cells. Malate
and chloride serve as anions and, in epidermal strips of Vicia faba,
the proportion of potassium ions balanced by malate depends on the
availability of chloride (40,47). The processes initiating the increase
in osmotic pressure that leads to stomatal opening are unknown at this

time.

Stomatal Responses to Light and Other Environmental Factors

Stomata open in response to an increase in irradiance, an increase
(or decrease {27}) in leaf temperature, a decrease in the water vapor
pressure deficit, or a decrease in the intercellular C02 concentration.

Studying stomatal responses to environmental factors is difficult

because changing one factor often causes changes in other factors. For
example, an increase in the irradiance falling on a leaf can also cause
at least three other environmental factors to change. They are:
increased leaf temperature because of the increased heat load, increased
water vapor pressure deficit because of the increased leaf temperature,
and finally decreased intercellular 002 concentration because of
increased 002 assimilation. After the stomata have responded to these
changes in their environment, these factors may again be different.

For example, if the stomata respond to light to a large degree, the
intercellular CO2 concentration could eventually be higher than before
the increase in irradiance.

Stomatal responses to environmental variables often show interactions.
For example, Hall and Kaufmann (8) found that stomata responded more
strongly to humidity when the intercellular 002 concentration was above
200 pt 2'1 than when it was below 100 at 2‘1. Raschke (36) found that
stomata of Xanthium strumarium in light of 340 w m‘2 did not close in
response to an increase in the 002 concentration unless the leaves had
been fed abscisic acid. Similarly, he found that the stomata did not
close after feeding the leaves abscisic acid unless there was C02 in
the air. Heath and Russell (10) and Gaastra (7) found that stomata
responded less to 002 at high irradiance than in darkness.

The stomatal response to light can be very complex. In 1932, Scarth
(41) proposed that stomata respond indirectly to light. According to
Scarth, an increase in irradiance causes an increase in the rate of €02
assimilation, which lowers the concentration of 002 in the intercellular
spaces. Stomata respond to the lowered intercellular 002 concentration

by opening. There is some evidence that stomata of gga_mays can respond

4

to light in this way (39), and this view has been favored by recent
reviewers (24,35). This mechanism for stomatal response to light is
feasible in C4 plants where the C02 concentration drop across the sto-
mata is large and the stomatal conductance is usually small. However,
it cannot be very important in C3 plants, which often have a 002 con-
centration drop of 30 uz 1‘1 or even less (6) and very large stomatal
conductances.

Heath and Russell (10) suggested that there might be "An indirect
effect not operating by the reduction of the internal carbon dioxide in
the guard cells, transmitted . . . from the mesophyll cells by some
agent (chemical or electrical) as yet unknown" (p.290). Wong (54) also
suggested that some type of messenger travels from the mesophyll to the
epidermis such that the photosynthetic rate controls the stomatal con-
ductance. The evidence in favor of this view is primarily the obser-
vation that stomatal conductance responds to many environmental factors
in the same way as does photosynthesis. The photosynthetic and stomatal
responses are so similar that the intercellular 002 concentration
remains constant when environmental influences such as light are changed
over a wide range.

The simplest explanation for stomatal responses to light, however,
is that light absorbed in the guard cells themselves results in stomatal
opening. The fact that stomata of epidermes stripped from the mesophyll
can respond to light indicates that this mechanism of stomatal response
to light occurs in at least some situations.

Wong gt_al, (53) measured stomatal responses to light and 002 in
Eucalyptus pauciflora. They found that the stomatal conductance and

the assimilation rate responded to changes in irradiance (in the range

of 0.25 to 2 mE m‘z sec'l) in such a way that the intercellular 002
concentration remained constant. In this case, despite the stomatal
sensitivity to 002 that they observed, the indirect stomatal response

to light mediated by 002 played no role in the overall stomatal response

to light.

Effect of Light Quality on Stomatal Opening_

Stomata respond primarily to blue light (23). Voskresenskaya and
Polyakov (50) found that when blue light was added to red light, the
photosynthetic rate gradually went up until it was 2 to 3 times greater
than in red light alone, even though the added blue light had an inten-
sity of 1% that of the red. The enhancement of the 002 assimilation
rate by blue light was correlated with an enhancement of transpiration,
which was interpreted as an indication that the blue light effect was
caused by stomatal opening.

The action spectrum of stomatal opening determined by Liebig (21)
showed a relative effectiveness of blue:green:red of 168:42:100. Since
she had calculated the absorption of a mixture of chlophyll a and b to
be similar, she concluded that chlorophyll was the only pigment involved
in light reception leading to stomatal opening. Heath (9) recalculated
Liebig's data taking into account that a quantum of blue light has more
energy than a quantum of red light and found that the relative quantum
efficiency for stomatal opening was blue:green:red, 234:50:100. This
relationship he felt was indicative of the participation of carotenoids
or xanthophylls as well as chlorophyll.

Karvé'(17) criticized Liebig's work and that of many other investi-

gators because they used the "steady state" method. For this method,

the leaf is put into the light of known intensity and quality and the
stomatal response is monitored until it no longer changes; a reading

is then taken. Karvé's criticism was that this method does not take
into account the power (i.e. dose) received by the leaf. He believed
that the reading of the stomatal response to light should be taken
after a certain time in the light, regardless of whether the stomata
were still opening or not. In this way, the dose received by the leaf
at each wavelength would be the same. However, some of the implicit
assumptions of this view are unfounded. For example, there is no
a_pgiggi_reason to believe that the rate of stomatal opening is related
to or controlled by the dose of light received by the stomata. If
light is a signal for stomatal opening rather than an energy source,
then the rate of stomatal opening need not depend on the light intensity
or its effectiveness in causing stomatal opening. Karvé's action spec-
trum, nonetheless, was very similar to that of Liebig.

Kuiper'(20)determined an action spectrum for the maintenance of
stomatal opening with epidermal strips of Senecio QQQEIE: He found that
blue light was more than twice as effective as red light in maintaining
stomatal opening.

Hsiao gt_al, (12) determined the action spectrum for Rb+ (as a tracer.
for K+) uptake as well as for stomatal opening. Stomatal opening and
Rb+ uptake were well correlated and at low irradiance responded only to
blue light. At a higher irradiance, some activity in the red became
apparent.

Ogawa gt_al, (29) found the action spectrum for malate formation in
sonicated epidermal strips to be similar to the action spectra already

described except that the peak in the blue was about 10 times higher

than the peak in the red. Ogawa gt_al, (29) showed that a small amount
of blue light added to a background of red light was much more effective
than either the red light or the blue light alone. They postulated
that there must be two photoreactions involved in the stomatal response
to light: one mediated by chlorophyll and accounting fOr the red peak
and part of the blue peak, and a separate blue light photoreaction.
They measured the action spectrum of the blue light photoreaction by
giving small amounts of blue light in a background of red light. In
this way, the peak in the blue was not distorted by the fact that the
proposed chlorophyll reaction would absorb some wavelengths of blue
light but not others. The resultant action spectrum had two peaks, one
at 460 nm and one at 380 nm, which they interpreted as evidence that

a flavin is involved in the blue light response of stomata.

BrogSrdh (2) also was able to show that stomatal responses to red
and blue light are fundamentally different. He found that blue light
caused a large, rapid increase in transpiration that quickly fell,

i.e. an overshoot. Red light caused only a slow increase in transpi-
ration rate. Johnsson gt_al, (14) found that this response was limited
to grasses. Skaar and Johnsson (44) determined an action spectrum for
the blue light induced transpiration response in Aygna_and fbund a broad
peak of activity around 446 nm. Pretreatments (2) or a continuous
background (44) of red light enhanced the blue light response.

In summary, most action spectra determined since 1940 have been in
basic agreement that blue light is 2 to 10 times more effective in
causing stomatal opening than red and that green light has very little,
if any, effect. Although there is no direct evidence for it, most

authors assume that the red peak is caused by the absorption of light

by chlorophyll and some (Liebig and Kuiper) believe that photosynthesis
is sufficient to account for the entire action spectrum. Ogawa st 31.
(29) and Brogfirdh (2) showed, by virtue of their observed "synergistic"
action of red and blue light, that the blue and red photoreactions are
fundamentally different. As a result, the overall action spectrum of
stomatal opening probably does not correspond to any single photo-
receptor absorption spectrum. What is needed is the determination of
the action spectra of the separate photoreactions as Ogawa et 31. (29)

did for the blue light reaction.

Possible Effects of Light on Guard Cells

Most investigators assume that red light affects stomata via photo-
synthesis. For example, Brogfirdh (2) proposed that the only effect of
red light was indirect and mediated by changes in the intercellular C02
concentration. Clearly this view is wrong since three separate investi-
gators found that red light was active in stomatal opening using strips
of epidermis removed from the mesophyll (12,20,29). It is interesting
to note, however, that the activity of red light relative to the activity
of blue light was usually higher in intact leaves than in stripped
epidermis. For example, Heath's (9) recalculation of Liebig's (21)
data (intact leaves) gives a ratio of 0.4 to 1 while Ogawa gt_al, (29)
(epidermis only) found a ratio of about 0.1 to 1. Part of the activity
found for red light in intact leaves may very well be a COZ-mediated
effect.

It was been proposed that the effect of red light on guard cells is
via photosynthesis in the guard cells. Guard cells contain chloroplasts
while, in most species, the other epidermal cells do not. Guard cells

of Paphiopedilum spp., however, do not contain chlorophyll (26) and

 

still respond to light. Von Mohl (25) suggested in 1856 that photo-
'synthesis in the guard cells could produce the osmotica necessary for
stomatal opening. Lloyd showed in 1908 (22) that stomata responded to
light in COZ-free air. He also found that the starch content in the
guard cells decreased in the light and increased in the dark in air
containing C02. This pattern is opposite to that which one would expect
for a photosynthetic organ and opposite that which he found in the
underlying mesophyll tissue of the leaves he was studying. From his
experiments, Lloyd concluded that there was a stomatal response to
light, but that photosynthesis occurring in the guard cells was of minor
significance in stomatal function. Shaw and Maclachlan (43) found that
the rate of carbon dioxide fixation was too low to account for the
osmotic pressure increase necessary for stomatal opening. More recently,
it has been shown that guard cells do not fix carbon dioxide by the
reductive pentose pathway; they lack the ability to convert ribose-S-
phosphate to RuBP (38). The C02 fixation that is always found associated °
with guard cells is carboxylation of PEP by PEP carboxylase (30,45,52)
with the ultimate formation of malate and aspartate (31,38,42,51). The
rate of 002 fixation by guard cells is usually (but not always {421)
stimulated by light (38,43,51).

Many attempts to determine whether or not chlorophyll is necessary
for stomatal opening have involved the use of variegeted leaves, but
as Virgin (48) pointed out, "so-called non-chlorophyllous parts of
variegated plants contain small amounts of chlorophyll pigments" (p.184).
Virgin (48) did find an albino mutant of barley that was devoid of
chlorophyll and found no stomatal response to light. He also found (49)

that stomata of etiolated leaves of wheat did not begin to respond to

10

light until they had been exposed to light for 2 to 3 hours, at which
time chlorophyll was beginning to form in some of the treatments.
However, stomatal responsiveness to light was not correlated with the
chlorophyll content as he measured it.

Kuiper (20) found that stomata of epidermal strips floating on a

5M DCMU, a photosynthetic electron transport inhibitor,

solution of 10'
closed. Allaway and Mansfield (1) found that stomata of leaves fed

CMU, which acts in the same way as DCMU, closed slightly but reopened
when COz-free air was passed over the leaf. They criticized Kuiper's
interpretation that photophosphorylation was involved in the stomatal
response to light, since they felt that the mode of action of inhibitors
of photosynthesis was to raise the intercellular CO2 concentration.
However, Kuiper had used epidermal strips and so the closure he observed
could not have been the result of an increased intercellular C02 con-
centration. Perhaps of more importance is the fact that Kuiper used

an incandescent lamp for illumination, which should provide primarily
red light. Humble and Hsiao (13) showed that stomata of Vicia faba
epidermal strips closed only slightly in the presence of DCMU in a
nitrogen atmosphere. However, when Cl-CCP, an uncoupler of phosphory-
lation, and DCMU were both present, the stomata closed. From these
experiments they concluded that "the energy derived from photosynthetic
cyclic electron flow can be sufficient and possibly necessary for K+
uptake and stomatal opening in the light" (p.487). This conclusion,
however, is no longer certain in view of the facts that there are prob-
ably two separate photoreactions responsible for the stomatal response

to light and that Cl-CCP upcouples oxidative phosphorylation as well as
photophosphorylation.

11

The stomatal response to blue light is clearly not mediated by
changes in the C02 concentration in the intercellular spaces (23). Two
effects of blue light that may be operating in the guard cells are the
blue light stimulation of respiration (15,18,19) and the blue light
stimulation of PEP carboxylase activity (15,16,28). Both of these
responses have action spectra (15,18,32) similar to that determined

by Ogawa gt_al, (29) for the blue light response of stomata.

MATERIALS AND METHODS

Analysis of Slopes

In order to separate and measure stomatal responses to light, I used
the method described by Farquhar gt a1. (6), stated briefly here. For
the purposes of this investigation, I say that stomatal conductance (g)
is a function of irradiance (I) and intercellular 002 concentration (c1)
in order to separate responses mediated by C02 from those that are not.
Other factors known to affect stomatal conductance, such as leaf tem»
perature and humidity, were held nearly constant and so are not consid-

ered in this analysis. Therefore the following can be written:

g=g<c,.11 <1)
Differentiating equation 1 gives:
e 29. 3
dg (aci)1dci + (5%)cidl (2)

The partial differentials in equation 2 can be evaluated by determining
the slopes of the curves representing the relationships between stomatal
conductance and intercellular CO2 concentration or light intensity.
Since the intercellular C02 concentration is a function of assimilation
and stomatal conductance, we can write:

dci= (g fli) an + (3%11Aag (3)

Assimilation is a function of irradiance and intercellular CO2 concen-
tration if temperature, etc. is held constant so:

dA=—iIc)d.+ (33-de (4)

12

13

By substituting equation 4 into equation 3 and the result into

equation 2, we derive the following:

direct response indirect response

1331c1{1-133i 1113—3 191} + 1331 113311 11933ci11

1-13—3i13-3111 191 439 Ci1113—31,,1

 

 

‘

(5)

 

01 D.
H
II

Equation 5 shows the response of stomatal conductance to light as the
sum of two terms. The first term describes the effects of light that
are not mediated by C02 (including mesophyll-dependent and mesophyll-
independent effects) and will be referred to as the direct stomatal
response to light. The second term describes the indirect effect of
light on stomata mediated by changes in the intercellular C02 concen-
tration.

In a similar way, the response of assimilation to light can be
derived to show the direct response of assimilation to light and the
effect of the increased availability of C02 caused by the direct sto-

matal response to light:

direct response indirect response

A .
M. 13—36 {1-1331311— 311A} + 13311113311A13—31c1

ET- 1-13—3111133119-31—31111-3311A

 

(6)

 

Most of the partials in equations 5 and 6 are easy to determine,
but the partial (ag/aI)ci is not. Since a change in irradiance affects
assimilation, Ci may vary as irradiance is changed. Two approaches
were used to overcome this problem. First, an inhibitor of photosyn-

thesis that did not affect stomatal function was added to the

14

transpiration stream. When the net exchange of CO2 fell to zero, changes
in irradiance no longer affected the concentration of CO2 in the leaf.
The second approach was to determine the way that stomatal conductance
varied with c1 at various irradiances, then to choose one c1 and deter-
mine what the conductance would have been at each irradiance. A graph
of conductance versus irradiance at constant Ci can then be constructed.

Calculations were done at two or four arbitrarily chosen light levels.
For calculations made when a photosynthesis inhibitor was present, an

1

intercellular C02 concentration of 250 pt 2' was chosen as being repre-

sentative for C3 plants. In other calculations, Ci was determined from
a plot of ci versus ambient C02 concentration, assuming an ambient C02
concentration of 320 um 1'1. 3
It was not possible to use this approach to separate mesophyll-
dependent from mesophyll-independent stomatal responses to light, since

we must begin with the following equation:
. as a 1
d9 (aci)I,Adci ” (3T)c1.,AdI I ('a‘A)I,c1.dA

The first two partial differentials can be evaluated when photosynthesis
is eliminated by feeding an inhibitor, but there is no satisfactory way

to evaluate the third partial differential.

Gas Analysis

The gas analysis system consisted of five URAS II (Hartmann und Braun,
Frankfurt a.M., W. Germany) gas analyzers, five specially constructed
temperature-controlled leaf chambers, a digital voltmeter, and a mini-
computer.

Lab-compressed air was passed through two columns of soda lime to

remove the C02. After the air was humidified, the CO2 concentration was

15

adjusted by allowing 1 or 5% C02 to flow into the air through capillaries
of varying resistance. The air was then passed through a glass condenser.
The temperature of the condenser was maintained by a constant temperature
water bath and was measured with a thermocouple. The air stream was
split and the air stream passing over the leaf was adjusted to 50 l hr'1
over each surface. The leaves were mounted in aluminum chambers through
which water was pumped to control the air temperature. The petioles of
the leaves dipped into beakers containing water or solutions to be fed
to the leaves. The chambers allowed 2.44 cm2 leaf area to be exposed to
the air stream. A small copper-constantan thermocouple was pressed
against the nonilluminated surface of the leaf. Those parts of the
leaves not covered by the chambers were trimmed off to prevent excessive
water loss (34). The molar fluxes of 002 and H20 for both the upper
and lower leaf surfaces were measured with four gas analyzers used as
differential analyzers to increase the sensitivity. For 1. strumarium,
E, frutescens, and Q, hirsutum, the temperature of the leaf chamber was
kept at 23 C. For 2, gays. it was 27 C and for P. vulgaris it was 21 C.
Assimilation and evaporation rates. conductance (stomatal and boundary
layer together), and intercellular C02 concentration were calculated by
computer. The intercellular CO2 concentration (c1) was calculated using

the following equation:

ci=ca-1.6A r

where ca is the CO2 concentration in the air passing over the leaf, A is
the assimilation rate, and r is the resistance to water vapor loss of the
stomata and the boundary layer. The factor 1.6 is the ratio of diffusi-
vities of water vapor and C02 in the air. The units used for gas exchange

parameters are consistent with those of Cowan (3) and Farquhar gt_al, (6).

16

Light

White light was provided by an Osram XBF 6000 w water-cooled xenon
arc lamp shining through a Corning no. 4600 infrared-absorbing glass
filter. The irradiance was reduced with neutral density Plexiglas
filters (no. 800 and 838. Rohm und Haas, Darmstadt, Germany). Irradiance
was monitored with a silicon cell in the same plane as the leaf chambers
that had been calibrated with an Eppley pyranometer. White light mea-
surements are reported in w 111"2 but can be converted to quantum flux by
the conversion factor 4.5 uE sec'1=1 w. This conversion factor can only
be considered approximate since iron oxide from the cooling water accumu-
lated on the lamp over the course of the day and changed the spectral
distribution of the light.

Monochromatic light was produced by air-cooled xenon arc lamps and
fecused through band pass filters to select about a 150 nm band of light.
This light passed through a water-cooled interference filter, then into
a box from which all other light was excluded (33). The interference
filters had a half-band width of 20 nm except for the experiment re—
ported in Figure 13, for which a different set of interference filters
with a half-band width of 12 nm was used. Type DAL (20 nm half-band
width) or DIL (12 nm half-band width) tandem filters made by Schott,
Mainz, W. Germany were used. Two 2.5 kw lamps were set up to shine into
one box. Inside. the leaf chamber was mounted on an optical bench and
could be positioned under either lamp. With this setup. the wavelength
of the light shining on the leaf could be changed in less than three
seconds by sliding the chamber from one lamp to the other. A 6.5 kw lamp
was available when very high intensities were needed. The leaf chamber

used for monochromatic light work was fitted with a beam splitter

17

(a microscope slide fixed at a 45 degree angle to the light beam) which
reflected about 10% of the light to two silicon cells. These silicon
cells were calibrated by the following method: An intermediate pair

of silicon cells was calibrated against an Eppley thermopile (model 0-3,
Eppley Lab., Newport. RI). then put into the leaf chamber. The silicon
cells of the beam splitter were then calibrated against the intermediate
pair of silicon cells so that the signal from the beam splitter silicon
cells could be converted directly into quantum flux inside the leaf

chamber. This calibration was done for each wavelength of light used.

Plants

Xanthium strumarium L. (Chicago strain) and Perilla frutescens (L.)

 

Britt. (red-leaved Perilla) were grown in a gravel-soil mixture in a
greenhouse. The natural photoperiod was extended to 20 hours with
0.3 w 111'2 light from Sylvania Gro-lux fluorescent tubes. Temperature
maxima were between 23 C and 29 C; the relative humidity was between
70 and 80%. For 1. strumarium, the fifth or sixth leaf from the apex
of 6 to 12 week old plants was used. For E, frutescens. the youngest
fully expanded leaf pair from one month old plants was used for analysis.

5, strumarium plants used for the-monochromatic light experiments
were grown in a growth chamber in an attempt to produce unifbrm leaves.
A 20 cm X 2.5 cm wick was placed in a pot so that about 15 cm was in
contact with the soil and 5 cm dipped into a gravel bed containing
distilled water. The temperature of the growth chamber was 24 C/20 C,
day/night, daylength was 20 hours. and the highest of three levels of
irradiance was 230 w m'z. The RH was 75%.

Zea mays L. cv. Michigan 500 was grown in a soil-perlite mixture in a

growth chamber. The temperature was 30 C/20 C. day/night; the RH was

18

about 75%. The day length was 12 hours. The highest of three levels of

irradiance was 230 w 111"2

between 400 and 700 nm. The fifth leaf (by
emergence) of 3 to 4 week old plants was used for analysis. Gossypium
hirsutum L. cv. Acala SJ-l was grown in a soil-perlite mixture in a
growth chamber. The temperature was 32 C/22 C. day/night; the RH was

2 and

about 75%. The highest of three levels of irradiance was 230 w m-
day length was 20 hours. The fifth leaf from the apex of 1 to 2 month
old plants was used for analysis. Phaseolus vulgaris L. cv. Montcalm
was grown in a gravel-vermiculite mixture in a growth chamber. The
temperature was 23 C/20 C, day/night; the RH was about 80%. Irradiance
was 180 w 111'2 between 400 and 700 nm. The primary leaves of plants

10 to 15 days old were used for analysis.

Procedure

For experiments where cyanazine was used as an inhibitor of photosyn-
thesis, the leaves were put into the gas analysis chambers in the morning
and cyanazine was added to the irrigation stream. The CD2 concentration

1 2 until

was held at 250 pt 2' and the light level was about 280 w m'
the stomata were open and photosynthesis was reduced enough that the
intercellular C02 concentration was within 10 pt 2’1 of the CO2 concen-
tration in the air (usually about 11:00 a.m., 2 hours after the light
was turned on). Except where noted. stomatal behavior was fellowed as
the CO2 concentration was increased from O or light was decreased from

about 280 w m'z.

This procedure was followed fbr experiments described
by Figures 1. 2, 4-6. 8, and 10 to eliminate the small effect of hys-

teresis on the results (see Figure 9).

RESULTS

Measurement of Direct and Indirect Stomatal Responses to White Light
The photosynthesis inhibitor cyanazine was added to the irrigation
water of leaves of Xanthium strumarium and Gossypium hirsutum. A con-

centration of 10"5

M cyanazine reduced photosynthesis to the compensation
point so that there was no measurable C02 exchange. Under these con-

. ditions, the intercellular £02 concentration is equal to the C02 con-
centration in the air around the leaf and a change in the irradiance
does not cause a change in the intercellular C02 concentration. Stomata
of 1. strumarium were apparently unaffected by cyanazine, while stomata
of g, hirsutum became more sensitive to C02 (Figure 1). Atrazine and
DCMU were also tested onyx, strumarium leaves and found to be suitable.
(This method of analyzing stomatal responses to light could not be used
on leaves of gee may§_or Commelina communis because a high concentration
of cyanazine was required to inhibit photosynthesis within 2 hours. As
a result. respiration was stimulated by the end of the experiment. so
that the intercellular CO2 concentration was no longer independent of
changes in stomatal conductance.) The total stomatal response to light
for x, strumarium and G, hirsutum was calculated for irradiances of 50
and 300 w 111'2 (Table 1). For these calculations, an intercellular CO2

concentration of 250 pa 2’1

was chosen (an average value for C3 plants).
In 1. strumarium. the direct stomatal response to light accounted for
79% of the total response at low irradiance and 55% at high light. while

in g, hirsutum nearly 90% of the response to light was a direct response

19

20

Figure 1. Effect of cyanazine on stomatal response to C02.
Detached, trimmed leaves were put into l0 ml beakers containing
lO'SM cyanazine. The light intensity was 290 w/mz. Each point is the

sum of the conductance of the adaxial and abaxial leaf surfaces.

111.

50

25

25

21

CONDUCTANCE

 

 

 

Xanrhhm struna'un
o/.’.’.\
E/' °\\\\

\conhol
\wonazlne

l l . Gossprn 1111:5111»
‘7.<.\.

\-

 

\ °\o '
°\o§gzlne
3‘3“
o ‘ 260 400

 

22

Table 1. Effect of light on stomatal conductance and assimilation

using cyanazine treated leaves.

Derivations of dg/dI and dA/dI are given in the Materials and Methods

section. All partial derivatives not involving photosynthesis used in

the calculations were determined with leaves fed cyanazine. The inter-

cellular C02 concentration was 250 pals.

derived in part from Figures 5 and 8.

Data for x, strumarium were

 

direct stomatal

effect of direct

 

SPECIES I d9/dI response to dA/dI stomatal response
light to Tight
111/1112 111ml 11 of dg/dI mnol 11 of dA/dI
w sec w sec

Xanthium 300 67 55 62 3
strumarium 50 610 79 100 14
Gbssypium 300 60 87 18 13
hirsutum 50 250 89 54 13

 

23

at both light levels. (The increased sensitivity of G. hirsutum stomata
to C02 caused by cyanazine results in an overestimation of the indirect
stomatal response to light.) The proportion of the assimilation response
to light caused by the direct stomatal response to light never exceeded
14%, indicating that photosynthesis was not greatly limited by stomatal
restriction of the C02 supply in either species once stomata had opened.
Since it generally required 1 day to evaluate the stomatal response
to C02 at one irradiance. the plant material had to behave consistently
for 4 days in order for the graphic analysis method to be useful. (Ex-
periments with g, communis and Amaranthus powelli failed because the
day to day variations in stomatal responses were too large.) The inter-
cellular CO2 concentrations used for these calculations were determined
from graphs of intercellular versus ambient C0z concentration for each
irradiance. given an ambient C02 concentration of 320 um 2'1. Table 2
shows the stomatal responses to light at four light levels for four
plant species. The results for Perilla frutescens and Phaseolus vulgaris
were similar to those in Table 1 in that most of the stomatal response
to light was caused by the direct response to light and the proportion
of the assimilation response to light caused by the direct stomatal
response to light was small. At high irradiance. the proportion of the
increase in assimilation caused by the stomatal response to light in
E, frutescens and Z, may§_was 100%, but this only occurred because
assimilation was saturated with respect to light and does not mean that
stomata had a large effect on photosynthesis. In g, m§y§_at low irra-
diance the indirect stomatal response to light was stronger than the
direct response. (The calculations could not be done for Z. gays in

darkness since the stomatal conductance was so small that gas exchange

24

Table 2. Effect of light on stomatal conductance and assimilation of
leaves.

Derivations of dg/dI and dA/dI are given in the Materials and Methods
section. The intercellular 002 concentrations corresponded to those
that would occur in an ambient 002 concentration of 320 palm and are

listed in Table 4. The data were derived in part from Figures 3 and 4.

 

direct stomatal effect of direct

 

 

SPECIES I dg/dI response to dA/dI stomatal response
Ilght to light
111/1112 moi 11 of dg/dI moi z of dA/dI
w sec w sec

PbriZZa 285 2.8 100 0.06 100
f?utescen8 155 46.4 94 ‘ 17.4 4
45 127 82 29.0 3
O 163 79 29.5 0
Phaseolus 280 66.4 99 6.2 37
vulgaris 160 100 92 25.7 15
45 133 65 53.2 2
0 112 66 63.6 0
Zea mays 620 2.5 100 0 1 100
265 25 3 81 10 9 13
145 60.2 49 51.9 3
50 489 31 113 O
ththium 265 0.24 0 14.5 0
strumarium 155 53.0 95 34.3 2
45 231 96 6O 4 0
0 2230 98 99 O O
xanthium 285 29.1 73 16.6 4
strumarium 150 90.6 76 43.5 2
+ 10-6M 45 249 78 87.6 1
(2)-ABA o 191 97 56.0 o

 

25

could not be measured accurately.) However, as the irradiance increased
so did the relative importance of the direct effect of light on the
stomata. The 3, strumarium plants used for these experiments were less
sensitive to 002 than were those used for the experiments reported in
Table 1. This was reflected in the large proportion of the light
response caused directly. At the highest irradiance the stomatal
‘ response to light was saturated and so the apparent complete indirect
response to light is meaningless. The addition of a low concentration
of ABA to the irrigation water sensitized the stomata to C02, which in-
creased the relative importance of the indirect response to light
mediated by 002.

The partial differentials used for evaluations presented in Table 2
are given in Table 3. The partial differential (ag/aci)I describes the
stomatal sensitivity to 002. For the C3 plants, increased irradiance
caused decreased stomatal sensitivity to 002 (excluding values obtained
in darkness). For the C4 plant 2, gays, however; the stomatal sensi-
tivity to CO2 was constant over the range of 145 to 620 w m'z. When
3, strumarium was fed a low concentration of ABA. the stomatal sensitiv-
ity to CO2 was greatly increased. Abscisic acid did not cause large
changes in any other partial differential, indicating that the only
effect of ABA on the light response of stomata was via its action on
the CO2 sensitivity of stomata.

The intercellular 002 concentration used in the calculations listed
in Tables 2 and 3 are given in Table 4 along with the assimilation rates
and stomatal conductances. As the irradiance increased. the intercel-
lular 002 concentration calculated for 5, strumarium decreased. This

was not true, however. for the other species. For example. for

26

Table 3. Values for partial differentials.

The first partial differentials were evaluated according to the
following equations: (acilag)A=1.6A/g2 and (aci/aA)g=-1.6/g. All of
the other partial differentials were evaluated by determining the
slopes of the curves representing the relationships described by the
partial differentials. The intercellular C02 concentrations, assimi-

lation rates. and stomatal conductance are listed in Table 4.

 

 

 

Species I (aci/ag)A (ag/aci)I (ag/aI)c1
2
w/m um2 sec mol mmol
fiaT——_— EFF:;;; w sec
Perilla frutescens 285 163 -50 2.8
155 145 -200 45
45 252 -550 118
O -473 -175 118
Phaseolus vulgaris 280 128 -300 68
160 218 -530 102
45 378* -630 106
0 -1094 -105 66
Zea mays 620. 470 -1150 3.8
265 605 -1120 33
145 898 -1150 58
50 -1333 -450 60
ththium strumarium 265 60 -62 O
155 48 -280 51
45 19 -400 224
0 -53 -330 2140
ththium strumarium 285 83 -1§g0 g;
-6 150 81 -1 0
’" 1° " (“"3" 45 149 -550 212
0 -272 -30 183

 

27

Table 3 continued -

 

 

Species I (aci/aA)g (aA/aci)I (aA/aI)c1
w/m2 m2 sec mmol gmol
mol "'2 SEC W SEC
Perilla frutescens 285 -7.4 15 O
. 155 -7.8 11 180
45 -16.8 3 300
0 -61.5 0 300
Phaseolus vulgarie - 280 -4.9 32 40
160 -7.3 22 250
45 -17.4 3 540
0 -50 0 640
Zea mays 620 -6.8 9 O
265 -7.8 13 100
145 -10.8 7 520
.50 -26.7 0 1130
ththium strumarium 265 -2.7 39 160
155 -2.8 25 360
45 -3.6 4 610
0 -14.5 0 990
ththium strumarium 285 -3.2 :3 180
-6 150 -3.7 450
* 1° M ”MBA 45 -10.7 2 890
0 -38.1 0 560

 

23

Table 4. Intercellular CO2 concentration, stomatal conductance, and
assimilation rate at four light levels.
Some values were obtained by interpolation, so that all values

correspond to those that would occur in a constant ambient C02 concen-

tration of 320 uz/z.

 

 

species I ci 9 A
w/m2 ut/t cmol/m2 sec umol/m2 sec

Perilla frutescens 285 285 21.5 4.7

155 293 20.5 3.8

45 293 9.5 1.4

0 326 2.6 -O.2

Phaseolus vngario 280 284 32.4 8.4

160 278 A 22.0 6.6

45 278 9.2 2.0

0 356 3.2 -0.7

Zea mayo 620 215 23.4 16.1

265 198 20.5 15.9

145 198 14.8 12.3

50 236 6.0 3.0

Kanthium strumarium 265 288 58.6 12.8

155 293 57.5 10.0

45 312 44.7 2.4

0 326 11.0 -O.4

xanthium strumarium 285 277‘ 50.5 13.2

-6 150 288 43.2 9.4

* 1° M (*I‘ABA 45 303 15.0 2.1

O 333 4.2 -0.3

 

29

P, frutescens, at 155 w 111'2

2

the intercellular 002 concentration was the
same as at 45 w m' . and in both P, vulgaris and ;, gays the intercellular
C02 concentration was higher at the highest irradiance than at an
intermediate light intensity.

The intercellular 002 concentrations in Table 4 were obtained by
interpolation between data points on a graph of intercellular C02 con-
centration versus ambient CO2 concentration. The intercellular CO2
concentrations calculated directly from the assimilation rates and sto-
matal conductances given in Table 4 differ from those listed by as much
as 9 on 9'1 (P, Vulgaris at 45 w m'z). However, twelve of the values
differ by 2 us 2'1 or less. The intercellular CO2 concentrations listed”
in Table 4 are more conservative than those obtained by calculations
based on the assimilation rates and stomatal conductances; the inter-
cellular C02 concentration at the highest irradiance i526 and 17 pt 1'1
higher than at an intermediate irradiance for E. vulgaris and ;, gays

respectively, rather than 7 and 23 am 2‘1.

Nevertheless. the uncertainty
in the determination of intercellular CO2 concentration indicates that
the increase in intercellular C02 concentration with an increase in

irradiance may not be significant in E, vulgaris. but certainly is in

Z, mays.

Interaction between Stomatal Responses to White Light and C02

The response of stomata of greenhouse-grown E: strumarium to C02
varies, although most of the time they are insensitive to CO2 in the
light. Stomata of leaves used for the experiments with cyanazine (done
in the fall of 1978) were sensitive to C02. Even so, light obviously
reduced the stomatal response to C02, especially when the C02 concen-

tration was between 150 and 250 oz 2’1 (Figure 2). Stomata of leaves

30

Figure 2. Stomatal response to 002 of leaves of Xanthium strumarium
fed cyanazine at three irradiance levels.

Detached, trimmed leaves were put into 10 ml beakers containing IO'SM
cyanazine. Each curve represents the average of the sum of the conduc-

tance of the abaxial and adaxial leaf surfaces for four leaves.

31

CONDUCTANCE

 

anol'r‘
hrs—ac ‘

 

 

 

75 "
Own"
o\° - ‘
501. \. \\
dwknes\ .
25 " o\ \
d o ' 260 '

32

used in later experiments (February 1979, Table 2) were insensitive to
C02 in the light. in agreement with earlier observations (34), but in
darkness or low light intensities. the stomata did respond to CO2 (Figure
3). The pattern of decreasing stomatal response to C02 with increasing
irradiance was also observed in E, frutescens and P, vulgaris (Figure 4).
Stomata of Poa pratensis cv. Merion and Triticum aestivum cv. Genesee
were also found to be insensitive to C02 in strong light (data not
shown).

In Figure 5. the response to light of stomata of 5. strumarium leaves
fed cyanazine is shown at three concentrations of C02. The initial slope
of the curve representing the relationship between conductance and
irradiance was steepest in COZ-free air. Similar results were obtained
with G, hirsutum. These experiments could not be performed with other
species since the inhibitor of photosynthesis could not be used.

Effect of ABA. Humidity. and Leaf Temperature on the Stomatal Response
to White Light_

Stomatal conductance at light saturation was lower in ABA-treated
leaves than in control leaves of 1. strumarium fed cyanazine (inset
Figure 6). but the pattern of the relationship between conductance and
irradiance was not affected. The normalized curves representing control.

7. and 10‘5M (t)-ABA-fed leaves were indistinguishable from each

10'
other (Figure 6).

Changing the leaf temperature or the humidity affected the stomatal
response to light in a similar manner. Lowering the leaf temperature
from 22C to 15.5C or raising the vapor pressure deficit from 8 ml l"1

1

to 17.5 ml l' decreased the conductance at light saturation by about

half, but the normalized curves were virtually indistinguishable from

33

Figure 3. Stomatal response to C02 of Xanthium strumarium (without
cyanazine) at four irradiance levels.

Detached, trinmed leaves were used. Each curve represents the average
of the sum of the conductance of the abaxial and adaxial leaf surfaces

for four leaves.

34

CONDUCTANCE

 

2102'
mzsec 265wm’z
O/.I:=N\: .
.%- 155 1111112 \'
50 "
A—‘""""’ ‘\
5/45 1mm“? ‘\4
\‘
25 - \\orkness
\o
\o

 

 

 

 

35

Figure 4. Stomatal response to CO2 at various irradiance levels for
Perilla frutescens, Phaseolus vulgaris, Gossypium hirsutum, and Zea_mays.
Detached, trimmed leaves were used. Cyanazine was present only with

Gossypium hirsutum. Each curve represents the average of the sum of

 

the conductance of the abaxial and adaxial leaf surfaces for feur leaves.
n=ozo w/mz; O=265-285 111/1112; I=145-160 w/mz; A=45-50 w/mz;

O=darkness .

 

ddddd

 

 

 

 

 

37

Figure 5. Stomatal response to light of leaves of Xanthium strumarium

fed cyanazine at three concentrations of C02.

511

Detached, trimmed leaves were put into 10 ml beakers with 10’
cyanazine. Each curve is the average of the sum of the conductance of

the abaxial and adaxial leaf surfaces for four leaves.

cmol)

C

75

50

38

 

9.111191191111119:

 

 

 

 

 

460

39

Figure 6. Stomatal response to light of leaves of Xanthium strwmarium
fed cyanazine and abscisic acid.

511

Detached, trinmed leaves were put into 10 ml beakers with 10"
cyanazine and the indicated concentration of abscisic acid (ABA). Each
point is the sum of the conductance of the adaxial and abaxial leaf
surfaces for one leaf. The data are presented as percentages of the
conductance measured at the highest light intensity; the absolute data

are shown in the inset. The C02 concentration in the ambient air and

inside the leaf was 100 uz/z.

4O

CONDUCTANCE

 

 

 

 

 

 

 

 

 

 

%
'00 ’- l I ‘-
I
‘ I
. I
. 1cm_o_l_ Control
"m sec[ /‘ ‘-
- 5° / /l(; 71111141-ABA
so - , pf
25 [ Mamas-ABA
. f
o zoo
0 o L 260 400

I,wrn"2

41

Figure 7. Stomatal response to light of leaves of Xanthium strumarium

 

at various humidity and leaf temperature combinations.

Detached, trimmed leaves were used. The ambient C02 concentration
was 320 ut/z (cyanazine was not present). Data are plotted as a per-
centage of the conductance at the highest light intensity. Each point
is the average of the sum of the conductance of the abaxial and adaxial
leaf surfaces for four leaves. The units are: leaf temperature-degrees C,

vapor pressure deficit (VPD)-ml HZO/l air, conductance-cmol/m2 sec.

42

CONDUCTANCE

 

 

 

9“ .
'00 ' ' ‘
o . ‘
o"
‘ Loaf 2.0%
50- I ,, 1 temp. vro (406%)
A ' o 22 8 60.6
. ‘ '55 8 338
. ' 22 17.5 $2
.
. I
.
o l 4* L i 4h]
0 200 400

 

I ,wm"

43

each other (Figure 7).

Inverted Leaf Experiments

Stomatal conductance of g. strumarium leaves fed cyanazine decreased
as the irradiance decreased (Figure 8). The relationship between sto-
matal conductance and light was similar when the light was increased
or decreased and stomata were opening or closing (Figure 9). Surpris-
ingly, the stomata of the upper (adaxial) epidermis required a higher
irradiance for saturation than did those of the lower (abaxial) epi-
dermis (Figure 8), despite the fact that the stomata on the lower

surface were shaded by the mesophyll. When leaves of 5, strumarium were

 

put into the chambers upside down, the stomata on the abaxial surface
(now directly illuminated) began to open at a much lower irradiance
than when the leaf was in the normal orientation (Figure 10). The
(adaxial stomata required much more light to open when the leaf was in-
verted than when it was in the normal orientation. Identical results
were obtained with leaves not fed cyanazine. Based on the saturation
light intensity. it can be calculated that the abaxial stomata were
about 20 times more sensitive to light than were the adaxial stomata.
Absorptivity of light by the leaf did show a slight dependence on
orientation, but it was considered insignificant in this context (see

Appendix).

Effect of Light Quality on Stomatal Conductance and Assimilation
Stomatal conductance and assimilation changed when a leaf was irra-

diated first with blue light and when with red light of equal quantum

flux density. Stomata were substantially more open in blue light than

in red light, deSpite a lower assimilation rate and a much higher

44

Figure 8. Stomatal response to light of leaves of Xanthium strumarium
fed cyanazine showing differences between adaxial and abaxial responses.
Detached, trimmed leaves were put into a 10 ml beaker with IO'SM
cyanazine. The C02 concentration in the air (and inside the leaf) was

270 ut/z. Each curve represents the stomatal behavior of one leaf.

 

 

 

 

 

 

 

)3;
o I l l “W" «Lemme
25 . / “3:: :5.
o . 1 . ‘

 

25

 

 

 

46

Figure 9. Stomatal response to light of leaves of Xanthium strumarium

 

as light was increased and then decreased.

Detached, trinmed leaves were used. The C02 concentration inside the
leaf was held constant at 290 us/z (along/2) by adjustments of the C02
concentration in the ambient air. Each point is the average of the sum
of the conductance of the abaxial and adaxial leaf surfaces for four

leaves.

47

CONDU CTANCE
r______

 

 

 

cmol o
m’zsec 1 s/
50 '
25 r //
o _.I 1 fi___L_1
0 ISO

 

48

Figure 10. Stomatal response to light of leaves of Xanthium strumarium

in the normal or inverted orientation.

5

Detached. trimmed leaves were put into 10 ml beakers containing 10’ M

cyanazine. The leaf blade was positioned so that the light was shining
on either the adaxial surface (normal) or abaxial surface (inverted).

The C02 concentration in the air and inside the leaf was 255 us/z.

CONDUCT ANCE

49

 

 

 

 

 

 

 

£le adaxial epidermis
mzsec 4W'
25 - / 7
Af// 010W
/
O Q I I Said opklfl'mll a
/,.."2m'
O/e/‘immd
25 ' I.
1“! -
. I . . 10,,
75 ' "3""9'
/./
501‘ /0 /Tnvertod
o o/o
.5. 17"
, l I l l
G o

50

intercellular 002 concentration (Table 5). This result is consistent
with the results presented above, which indicate that the stomatal
response to light is for the most part a direct response to light and
is not mediated by changes in C02 concentration or by hypothetical ,
changes in the supply of assimilates to the guard cells. 2

Since it had been determined that stomata on the abaxial (lower)
surface of leaves of 3, strumarium were much more sensitive to light
than were the stomata on the adaxial surface (Figure 10), all subse-
quent work with monochromatic light was perfOrmed with the leaf inverted
so that the abaxial surface was directly illuminated. Conductance
values reported are for the directly illuminated abaxial surface only.
while the assimilation rates reported are the sums from both sides of
the leaves. 7

An action spectrum for stomatal opening was constructed using the
"steady state" method (i.e. a leaf was exposed to one quantum flux at
a particular wavelength until the conductance no longer changed. In
general, the stomatal response to a new quantum flux took 30 minutes.)
The stomatal response to increasing quantum flux is shown for 14 wave-
lengths of light in Figure 11. There were three responses that could
be distinguished (Figures 16, 17). At low quantum flux, the stomata
responded weakly, if at all, to light. At intermediate quantum fluxes.
the stomata responded linearly to the logarithm of the quantum flux.
At very high quantum fluxes, the stomata were once again insensitive to
changes in the quantum flux. A linear regression was performed for the
three or four points that fell in the linear response range fOr each
wavelength (except at 711 nm for which only two points could be used).

No correlation coefficient was less than 0.98 and six correlation

51

Table 5. Effect of red or blue light on assimilation rate, intercellular
C02 concentration, and stomatal conductanee in leaves of Xanthium

strumarium.

 

Incident quantum flux was 740 uE/m2 sec.for both red and blue light.

The CD2 concentration was 305 ut/z.

 

Light 1 Assimilation Intercellular C02 Stomatal
rate concentration conductance
nm umol/m2 sec us/t cmol/m2 sec

 

red 681 11.6 255 35.9
blue 436 9.9 273 52.9

 

52

Figure 11. Stomatal response to light quality of leaves of Xanthium
strumarium.

Detached, trimmed leaves were positioned in the leaf chamber so that
the abaxial leaf surface was directly illuminated. The lines are linear
regressions of the three or four points that fell on the linear response
portion of the curve. The extent of the line indicates the range over
which the stomata responded linearly to log changes in irradiance. Each
line was derived from data for one leaf. There was no response up to
1000 uE/m2 sec at 749 and 731 nm and up to 100 uE/m2 sec (limit of the
light source) at 386 and 372 nm.

53

CONDUCTANCE

mf—

mzsec

 

I

479
455448436
467 68!
25 ' 592
4|6 55'
523

20 49

l5

'0 —

I IIIIIII L I IIIIIII I

6l2
7"

4L

 

IOO
I, 115/ mzsec

IOOO

 

54

coefficients were equal to or greater than 0.998. The length of the
line in Figure 11 indicates the range over which the stomatal response
was linear with logarithmic changes in quantum flux. The slopes of
the lines in blue light were greater than the slopes in red light.

The average slope of all lines between 400 and 500 nm was 19.4:3.7 cmol

111'2 sec'1 per decade change in quantum flux. while the average slope

of all the lines between 600 and 700 nm was 12.8:0.8 cmol m"2 sec"1 per
decade. Using data from Figure 11, an action spectrum was constructed
by plotting the inverse of the quantum flux required to produce a con-
ductance of 15 cmol 111'2 sec'1 versus wavelength (Figure 12). This
method of construction for the action spectrum deemphasizes the peak of
activity in red light, since the slopes of the fluence response curves
are less in the red light than in the blue. Another action spectrum was
constructed (not shown) by extrapolating the regression lines to the
conductance value obtained in the dark fer the individual leaf and
plotting the inverse of the quantum flux thus obtained versus wavelength.
In that case, the red peak was 20% of the blue peak. rather than 10%

as in Figure 12.

Many investigators have hypothesized that chlorophyll is involved in
the stomatal response to light. While chlorophyll cannot be the only
pigment involved, it could be the pigment responsible fer the activity
of red light in causing stomatal opening. To test this. a leaf was put
into the gas analysis chamber and the steady state conductance in a

'1 at each of 5 wavelengths of red light

quantum flux of 300 uE m-2 sec
was determined at the long wavelength end of the chlorophyll absorption
spectrum where interference by accessory pigments should be minimal.

For two leaves. the wavelength was increased from the shortest to the

55

Figure 12. Action spectrum of stomatal opening.
The data were derived from Figure 11 by reading the quantum flux
required to give a stomatal conductance of 15 cmol in.2 sec'1 and

plotting the inverse of that quantum flux versus wavelength.

56

0?.

E: . 505.963
08 000 w

 

 

 

 

57

longest and for two additional leaves the wavelength was decreased from
the longest to the shortest. For each leaf, the shape of the curve
relating conductance to wavelength was the same, although the conduc-
tance was variable from leaf to leaf. In each of the feur leaves, the
conductance at 691 nm oscillated with a period of about 25 minutes and
decreasing amplitude. The results, averaged from the four leaves, are
shown in Figure 13. The intercellular C02 concentration was controlled

1

at about 250 oz 9' and the measured average concentration was 253:8

pt 2'1 for all wavelengths except 711 nm. for which the intercellular

1, providing evidence that the stomatal

C02 concentration was 282 pt 2'
response to red light shown in Figure 13 was not the result of changes
in the intercellular CO2 concentration. The stomatal response to a

change in wavelength of red light was nearly identical to the response-
of assimilation. Figure 14 shows additional evidence that the stomatal

response to red light does not depend on changes in the intercellular
CO2 concentration. Stomata of a leaf in 674 nm light (300 uE m'2 sec'l)
closed when the light was-turned off, even though the ambient C02 con-
centration was lowered enough to maintain the intercellular C02 concen-
tration constant. (The intercellular C02 concentration determined after
a large change in the ambient C02 concentration is unreliable so there
are no lines connecting the value obtained at 13:43.)

The stomatal response to red light was sensitive to DCMU and cyanazine.
Figure 15 shows the results of one of three similar experiments. De-
tached leaves were put into 10 ml beakers containing 5 X 10'5M cyanazine
(as in Figure 15) or DCMU (as in Table 6). When these leaves were put

2

into 455 nm light (650 uE m' sec'l). both conductance and assimilation

increased rapidly until the photosynthesis inhibitor began to reduce

58

Figure 13. Stomatal conductance and assimilation rate in various wave-
lengths of red light.

Detached. trimmed leaves were positioned in the gas analysis chamber
such that the abaxial surface was directly illuminated. The data are
the average of four leaves. Stomatal conductance data are for the
directly illuminated abaxial surface only, while the assimilation data
are for both leaf surfaces. The CD2 concentration varied to keep the
intercellular C02 concentration constant at 250 palm and the quantum
flux was 300 uE/m2 sec at each wavelength. Assimilation (squares) is
plotted against the left scale and stomatal conductance is plotted
against the right scale. The average stomatal conductance in darkness
was 4.6 cmol/m2 sec. The interference filters used had a half-band

width of 12 nm and the one-tenth band width was 20 nm.

E... . 385.325
0:. 00h 0mm 08 Ohm

 

59

(D

o
E

 

1

u u u - u o
8.6.52

I/ 1V

./.\. We.

. 85:5
Bu 0 .25

 

 

60

Figure 14. Stomatal closure in response to turning off the light (674 nm)
with the intercellular 002 concentration held constant.
The data are for one of the leaves used in the experiment described

in Figure 13.

 

so. now. 9mm. Ran. o
03 .
com .o \./.1.....I.\. ,l.
S: . . 0.
8:20:38
to so:
oommE
.25

 

 

 

62

Figure 15. Stomatal conductance and assimilation of Xanthium strgmarigm
leaves fed cyanazine and placed in red or blue light.

Detached, trinmed leaves were positioned in the leaf chamber so that
the abaxial surface was directly illuminated. The ends of the petioles

5M cyanazine. Assimilation

were in 10 ml beakers containing 5 X 10'
rates (open symbols, left scale) and stomatal conductance (solid symbols,
right scale) rose initially in response to the light (650 uE/m2 sec at
.both wavelengths). The C02 concentration in the air was 320 pill. The
stomatal conductance data are for the directly illumihated abaxial sur-

face only, while the assimilation rate data are for both surfaces.

63

 

cmol 3 final
mesoc "12ch

455nm ' '0

 

 

 

 

20..
‘5
'0' .
- \‘Xmfllnm
o 9 a a a 4550'": o
l327 14127 1527 1627 I727

Time of day

64

the photosynthetic rate. The stomatal conductance fell slightly; this
may have been a C02 effect. since the intercellular C02 concentration

1 as the photosynthetic rate fell. In light of 681 nm

rose by 30 u2 2'
(same quantum flux), the conductance and assimilation rate both fell as
the inhibitor of photosynthesis began to act. Table 6 contains data
from a similar experiment, this time with DCMU as the photosynthesis
inhibitor. Since ten times more quanta of red light than of blue were

2 sec'1 (Figure 12), the

required to produce a conductance of 15 cmol m'
experiment included one treatment in which the quantum flux of blue
light was one-tenth that of the red light. After 6 hours in the light.
the two leaves in blue light had large stomatal conductances. while the
leaf in red light had a very low stomatal conductance. Because the
stomata closed in red light but remained open in blue light, the amount
of DCMU taken up during the course of the experiments (as judged by the
_ water lost by the leaves) was 50% higher for the blue light treatments
than for the red light treatment. After the stomata were closed by
DCMU in red light, they still responded to blue light. though not as
strongly as during the first opening movement.

Some investigators. notably Brogsrdh (2), believe that the stomatal
response to red light depends on changes in the concentration of C02,
while the blue light response is independent of C02. To test the effect
of C02 on the stomatal response to red or blue light. a fluence response
curve was determined in high or low C02 in red or blue light. The
results. shown in Figure 16. indicate that CO2 affected the stomatal
response to blue light in a similar manner as it did the response to

red light. For both red and blue light, leaves in the high concentration

of C02 required more light for the same stomatal conductance and the

65

Table 6. Effect of DCMU on stomatal conductance and assimilation in
red and blue light.

The dose was calculated by integrating the stripchart record of
water loss for the 6 hour duration of the experiment and multiplying

the result by the concentration of DCMU in the irrigation water (5 x 10'5M).

 

 

Light 1 Quantum Stomatal Assimilation Dose
flux conductance rate
nm uE/m2 sec cmol/m2 sec nmol/m2 sec nmol
blue 455 650 33.2 0.21 36.5
blue 455 65 33.6 0.33 36.3
red 684 650 7.8 1.99 23.0
*blue 455 650 16.8 1.97 --

 

*Same leaf as used for 684 nm. This line shows that when stomata in

red light are closed by DCMU, they can still respond to blue light.

66

Figure 16. Effect of CO2 on the stomatal response to red or blue light.

Detached. trimmed leaves were positioned in the gas analysis chamber
so that the abaxial surface was directly illuminated. The conductance
reported was for the directly illuminated abaxial surface. The open
symbols are at low C02 concentration (110 u2/2 for blue, 130 u2/2 for
red) and the filled symbols are for high C02 concentration (510 u2/2
for blue. 580 u2/2 for red).

67

 

 

 

 

 

 

55% /°/4;3M
30'
0/ /°":/"'
43611111
20_ a/ ’0 68mm /
/ - --——.
[o- .//. 68mm
.//'
7“""
91 ' 1'0 ' oa'

I , pE/ mzsec

 

68

maximum conductance attained was lower.

To test whether the stomatal response to red light was mesophyll-
dependent or mesophyll-independent. the inverted leaf experiment shown
in Figure 10 was repeated with monochromatic blue or red light. Figure
17 shows that when the abaxial surface was illuminated directly it re-
quired much less light than when it was shaded by the mesophyll in both
red and blue light. '

69

Figure l7. Stomatal response to monochromatic blue or red light in the
normal or inverted orientation.

Detached, trimmed leaves were put into l0 ml beakers containing water.
The leaf blade was positioned so that light was shining on the adaxial
surface (abaxial shaded by the mesophyll) or on the abaxial (adaxial
shaded). The filters had half bandwidths of 20 nm. The average trans-
mittance in blue light was 1.7% and in red light it was 3.9% for the

Xanthium strumarium leaves used in this experiment.

7O

 

cououcrche
.9312! 436 nm

'0.

- --—-——-:,. ,

68| nm

a adaxial surface
' 0110be surface shaded
oabaxlal surface
303 Uabaxlal surface shaded

 

 

 

 

 

 

20-
lo-

L _ M‘
o 1 W.

 

 

DISCUSSION

Direct Versus Indirect Effects of Light on Stomata

Stomatal opening in response to light was almost entirely a direct
response (not mediated by C02) in all species.studied here. The species
studied were C3 plants and one C4 plant. Nobel and Hartsock (27) found
that stomata of the CAM plant Agave deserti responded to light but not
to C02 when the plant was well watered and operating as a C3 plant.

Wong et;al, (53)measured the stomatal response to light in Eucalyptus
pauciflora and found that 70% of the stomatal response to light was a
direct response. Based on this evidence, we conclude that stomata of
most well watered plants respond primarily directly to light (i.e. the
response is not mediated by changes in the intercellular C02 concentra-
tion). The only exception to this is Zea may; at low irradiance (Table
2 and reference 39).

The intercellular C02 concentration is determined by the flux of C02
through the stomata and the resistance to.that flux (assuming the external
C02 concentration remains constant). As long as the stomatal response
to light matches the response of assimilation to light. there is no
change in the intercellular CO2 concentration.. In these cases. the sto—
matal sensitivity to C02 plays no role in stomatal responses to light.

For example, for Perilla frutescens, at 155 w m'2

2

the intercellular CO2
concentration was the same as at 45 w m' , even though the stomatal
conductance was more than two times greater (Table 4). Since it is known

that the stomatal response to CO2 is not strong enough to keep the

71

72

intercellular C02 concentration constant, the increase in conductance
from 9.5 to 20.5 cmol m'2 sec'1 is a direct response to lightl From
Table 2 it would be expected that between 82 and 94% of the stomatal
response to light would be a direct response, but instead it is ob-
served that all of the response is accounted for by the direct response
to light. This discrepancy comes about because the values in Table 2

are differentials and are valid only over an infinitesimal light range
and cannot be used to predict what the integrated response will be. In
fact. the direct stomatal response to light is often strong enough to
eliminate the reduction of the intercellular C02 concentration. which

is the signal for the indirect stomatal response to light. In gea_m§y§.
and Phaseolus vulgaris, the stomatal conductance increased more than the
assimilation rate at high irradiance (Table 4), causing a higher inter-
cellular C02 concentration at the highest irradiance than at intermediate
light intensities. In these cases, stomatal sensitivity to C02 should
have caused stomatal closure, but the direct stomatal response to light
completely overcame this closing stimulus. This behavior had been pos-
tulated by Cowan and Farquhar (4) if stomata function to minimize the
amount of water lost for a given amount of carbon dioxide taken up. In

1

.P. vulgaris, the increase was only 6 u2 2' and the stomata were insen-

sitive to C02, so this rise in intercellular CO2 concentration was

probably unimportant. However, in ;, mays the increase was 17 u2 2'1

and the stomata were sensitive to C02. At constant irradiance, such an
increase in intercellular C02 concentration would reduce stomatal con-

ductance by 2 to 3 cmol m'2 sec'l.

73

gesophyll-Dependent Versus Mesophyll-Independent Effects of Light on
tomata

Although it is not possible to quantitatively separate mesophyll-
dependent from mesophyll-independent effects of light on stomata, three
-pieces of evidence lead to the conclusion that. at least in g, strumarium,
the direct stomatal response to light is primarily mesophyll-independent.
First, in leaves in which photosynthetic electron transport was blocked
by cyanazine. the stomata still responded to C02. Second. a higher
irradiance was required for stomatal opening when the surface being
measured was shaded by the mesophyll than when illuminated directly
(Figures 10.17). Similar results were obtained by Turner (46) with
Nicotiana and Sorghum. If the rate of photosynthesis in the underlying
mesophyll plays a large role in stomatal regulation. one would expect
little difference in the stomatal response to light when the leaf was
illuminated from above or below. Third, blue light was more effective in
causing stomatal opening than red light, while the photosynthetic rate
was greater in the red (Table 5). The large number of studies using
epidermal strips to study stomatal opening in response to light also
speaks in favor of a mesophyll-independent light response.

Interactions between Stomatal Responses to Light and C09, ABAg Leaf
Temperature. and Vapor Pressure Deficit '

High irradiance caused the stomata to become insensitive to C02 in
1, strumarium, P, vulgaris, and E, frutescens. In G. hirsutum. the
response to CO2 was smallest at high irradiance. Gaastra (7) found
several years ago that stomata of turnip responded less to C02 in high
light than in low, and Heath and Russell (10) faund that stomata of wheat
became insensitive to C02 at high light intensities. Thus, in all C3

plants studied. light lessens the stomatal response to CO2 and I know

74

_of no counter examples. In a similar way. the response to light of
stomata of 5. strumarium and g, hirsutum depended inversely on the C02
concentration. ;, mgyg, on the other hand, did not fit this pattern.
The slope of the curve representing the relationship between stomatal
conductance and CO2 was independent of the irradiance, although the
curve was displaced along the C02 concentration scale by changing the
irradiance (Figure 4).

If water is not limiting, a high stomatal sensitivity to C02 will be
a liability for a C3 plant. in which the photosynthetic rate does not
saturate with respect to C02 until well above the ambient C02 concentra-
tion of 320 to 340 u2 2'1 (in other words. any increase in stomatal
conductance would increase the photosynthetic rate by increasing the CO2
supply). Consequently, for a well watered C3 plant, the stomata should
open very widely and allow the intercellular C02 concentration to be as
close as possible to the ambient CO2 concentration. In 5, strumarium,
P, vulgaris, and E, frutescens the stomata were insensitive to C02
(Figures 3 and 4) and the intercellular CO2 concentration was close to
ambient (Table 4). Photosynthesis of';, gays, however. saturates between
100 and 200 an 2'1 intercellular co2 (6. and data from this investigation
not presented). In this case, a high sensitivity to C02 is not a lia-
bility as long as the intercellular CO2 concentration does not fall
below the saturation point far photosynthesis. Again, this is the
behavior observed (Figure 4 and Table 4).

If. however. water is in limited supply. a large stomatal conductance
will be a liability. Since stomatal closure will limit transpiration
more than photosynthesis in C3 plants at least at high conductances (37).

the stomata of water-stressed plants should close. A high stomatal

75

sensitivity to C02 assures that stomatal conductance does not greatly
exceed that needed for photosynthesis: This allows a judicious use of
water and only a small reduction in the rate of photosynthesis. It has
already been shown that ABA causes stomata to become more sensitive to
C02 (5.36). It was therefore not surprising to find that the addition
of ABA to the transpiration stream increased the proportion of the sto-
ematal response to light mediated by C02 (Table 2). If the photosynthetic
apparatus becomes impaired (as can occur during water stress) the inter-
cellular C02 concentration will rise. causing stomatal closure and a
decrease in stomatal sensitivity to light. On the other hand, if the
photosynthetic apparatus is not inhibited, if the stomata are restricting
the supply of C02 to a great degree. then the intercellular C02 concen-
tration will fall, which in turn will cause stomatal opening and an
increase in the stomatal sensitivity to light. If leaf temperature is
reduced, the need for CO2 will be diminished by the falling photosyn-
thetic rate and so stomatal closure will not limit photosynthesis. If
the vapor pressure deficit is high. stomata will be less open than when
it is low. so that water loss will not be excessive. Apparently these
complex interactions between environmental and physiological factors
allow stomata to control gas exchange in a way that is appropriate for
the particular state of the plant (e.g. during periods of optimal or
suboptimal water supply. and during periods with conditions favorable to

or not favorable to photosynthesis).

Effects of Light! C0,, and ABA on Guard Cells
Light causes stomata to open, whereas CO2 causes stomata to close. If
the irradiance is high enough, the stomata require a greater closing

stimulus than is provided by the 320 to 340 u2 2'1 CO2 normally found in

76

the atmosphere so that the stomata appear to be insensitive to C02.
Stomatal insensitivity to C02 can occur at an intensity one-half that
of full sunlight (Figure 3). Abscisic acid alters the relationship
between light and C02 so that stomata respond to C02 in the light.

Since the saturation irradiance is the same with or without ABA (Figure
6) and stomatal conductance in the dark is reduced by ABA (Table 3), the
effect of ABA must be to add to the closing stimulus of C02 either by
enhancing the effectiveness of C02 or by providing a separate closing

stimulus.

Effect of LightAQuality on Stomatal Opening_

Stomatal conductance is greater in blue light than in red (Table 5).
Previously, it has been shown that gas exchange is enhanced by blue
light (2.23.50) and that uptake of Rb+ into guard cells. production of
malate by guard cells. and swelling of guard cell protoplasts are stim-
ulated by blue light (12.29.55). Of special interest in Table 5 are
the findings that the rate of assimilation fell slightly. and the inter-

1, two signals believed

cellular C02 concentration rose by almost 20 u2 2'
to cause stomatal closure. Thus. the results with monochromatic light
corroborate the results with white light that indicated that light has

a large direct effect on stomatal conductance.

What are the photoreceptor pigments involved in the stomatal response
to light? The results of Brogaroh (2) and Ogawa 91.9.1.- (29) indicate
that there are at least two pigments involved, one that absorbs blue
light (Ogawa et_gl, {29} suggested a flavin) and one that absorbs red
light, which several investigators have hypothesized is chlorophyll (2.

12.20.21). Two methods were employed to determine if chlorophyll is

77

indeed responsible for the red light response: action spectra of
stomatal opening were determined and inhibitors of photosynthetic

electron transport were given to leaves.

Action spectra

One method of determining the photoreceptor involved in response to
light is to measure the relative effectiveness of light of various wave-
lengths in producing the action of interest and comparing the action
spectrum obtained with the absorption spectrum of the presumed photo-
receptor pigment. Usually. an action spectrum is produced by irradiating
with light of various wavelengths but equal quantum flux and determining
the action at each wavelength. If the quantum flux is too law, then
some wavelengths of light will appear to have no effect, either because
the response is too small to be measured or because some threshold quantum
flux was not exceeded. If the quantum flux is too high, the response to
light may be saturated over some portion of the spectrum; peaks will be
broad and the action spectrum may be distorted. To overcome these
problems, Hsiao et 31, (12) used two different quantum fluxes (78 uE
m'2 sec'1 and 380 uE m'2 sec'l). At the low quantum flux, they saw no
activity of red light in causing stomatal opening or Rb+ transport. At
the higher quantum flux they observed that red light was quite effective
in causing stomatal opening and Rb+ transport. though not as effective
as blue light. They interpreted their results as indicating two dif-
ferent photoreactions, one effective at low quantum flux and one
effective at high qUantum flux. There is. however, no need to invoke
two photoreactions based on their evidence alone. The low quantum flux

may have been below the threshold for light activity in the red but not

in the blue. Action spectra similar to those of Hsiao et_gl, (12) can

78

be constructed from the data in Figure 11 of this thesis by reading

2 sec'l. At the low

the stomatal conductance at either 78 or 380 uE m'
irradiance, only blue light was active. i.e. the stomata responded very
little to red light. At the high irradiance, the stomata responded
strongly to red light and the blue light response was saturated. re—
sulting in an overestimation of the red peak of activity relative to

the blue one.

A preferable method for constructing an action spectrum is to deter-
mine fluence response curves at each wavelength of interest and then
determine the quantum flux required to produce a standard response. The
disadvantage of this method is that it requires a different leaf for
each wavelength of light investigated and so the accuracy of the action
spectrum will be limited by variability of the plant material.

A preliminary action spectrum covering the range from 399 to 711 nm
was determined by selecting a conductance of 15 cmol m'2 sec'1 as the
standard response (Figure 12). However. it was observed that the
standard response selected to determine the action spectrum from the
fluence response curves can affect the shape of the action spectrum.

An alternative to the response used to determine the action spectrum

in Figure 12 is to extrapolate the linear regression to the initial con-
ductance for that leaf in the dark. The action spectrum based on these
"threshold" quantum fluxes (not shown) was different from that shown in
Figure 12 in that the shoulder at 478 nm was almost nonexistent and the
height of the red peak relative to the blue peak was 0.2 instead of 0.1.
These differences are the result of the fact that the slopes of the

fluence curves were variable and generally greater in the blue than in

the red. This observation is consistent with the hypothesis that the

79

stomatal response to light is mediated by chlorophyll (shown in this
thesis) and a pigment absorbing blue light, possibly a flavin (29).

A more detailed action spectrum with filters of 12 nm half-band
width (compared to 20 nm half-band width filters used far Figure 12)
was determined between 674 and 711 nm where only chlorophyll should be

2 sec'l,

absorbing light. Five wavelengths of light. all at 300 uE m'
were used and the results of faur leaves were averaged. Since the least
effective wavelength (711 nm) produced a measurable increase in stomatal
conductance over the dark value. and the highest conductance attained
was lower than the maximum attainable stomatal conductance (Figure 11),
this action spectrum avoids the potential errors pointed out above far
this type of action spectrum. The effect of C02 on the stomatal response
to light was eliminated by maintaining the intercellular C02 concentra-
tion constant. The action spectrum for stomatal opening in the red was

very similar to the action spectrum for 602 assimilation. indicating

that the same photoreceptor probably accounts for both processes.

Inhibitors
Both DCMU and cyanazine had little or no effect on stomatal conduc-

tance of g. strumarium leaves in white light or blue light. In red

 

light. however, both inhibitors caused stomatal closure. This result
may explain why Kuiper (20) found that stomata of epidermal strips
floated on solutions of DCMU closed, since he used an incandescent lamp.
which produces primarily red light. The experiment of Allaway and
Mansfield (1) does not show that the effect of DCMU is only the result
of a change in the concentration of C02 inside the leaf, because they
did not keep the concentration of C02 inside the leaf constant, but

rather used COZ-free air. which independently causes stomatal opening.

80

The stomatal opening that occurs in response to COZ-free air has no
significance in stomatal function, since plants are never exposed to
COZ-free air. It is my opinion that the closure of stomata in red
light caused by the inhibitors of photosynthetic electron transport and
the strong similarity of the action spectra for photosynthesis and
stomatal opening in red light constitute conclusive proof that chloro-
phyll is one of two or more photoreceptors involved in the stomatal
response to light.

Wong (54) has suggested that photosynthesis in the underlying meso-
phyll tissue can affect the stomatal response to light. The inverted
leaf experiment (Figure 17) shows. however, that the absorption of light
that causes stomatal opening occurs near the surface of the leaf. In
blue light, it can be seen that it requires about 10 times more light
to cause stomatal opening on the abaxial epidermis when it is shaded
than when it is direct illuminated. Since the average transmittance of
the leaves in blue light is 0.02, stomatal opening will require 50 times
more light when the leaf is inverted than when it is in the normal orien-
tation. One possible reason for this discrepancy is that the average
transmittance is quite different from the transmittance of the leaf above
some of the guard cells (39). Additional evidence that the pigment
absorbing red light is located in the guard cells comes from the work of
Hsiao et 31. (12), Kuiper (20). and Ogawa ethgl, (29), all of whom faund
that stomatal activities were stimulated by red light in isolated
epidermes. I

The photosynthetic electron transport system may provide NADPH for
the reduction of oxaloacetate to malate or ATP for pumping K+ ions, but

I speculate that the most important function is related to information.

81

(The stomata open very nicely in total darkness in COz-free air. so
stomata do not need the light for energy.) Environmental influences
which affect photosynthetic electron transport in the mesophyll will
also affect the photosynthetic electron transport in the guard cells.
and as a result the stomatal response to light may depend to some degree
on the same environmental influences as photosynthesis in the mesophyll.
It is my opinion that this is the function of the chlorophyll-mediated
stomatal response to light and that this is why stomata and photosyn-

thesis often respond in a similar way to various environmental influences.

SUMMARY

Stomatal responses to light were investigated and found to be com-
posed of three separate responses. They are: a stomatal response to
changes in the intercellular CO2 concentration. a direct stomatal
response to light mediated by some blue light photoreceptor, and a
direct stomatal response to light mediated by chlorophyll.

The COZ-mediated indirect stomatal response to light can_be important
at low irradiances, since the stomata respond to changes in the inter-
cellular CO2 concentratibn and the intercellular C02 concentration
changes with variations in irradiance at low irradiance. However, at
high irradiance the intercellular C02 concentration does not change
greatly with variations in irradiance and in the C3 species studied. the
stomata are not sensitive to C02. This interaction between responses to
light and C02 is similar to that reported for ABA and CO2 (35). Leaf
temperature, water vapor pressure deficit. and ABA did not interact
with the stomatal response to light.

Stomata were most sensitive to blue light. Quantum fluxes of 1 uE
m'2 sec'1 or less caused measurable increases in stomatal conductance.
Inhibitors of photosynthetic electron transport caused only a slight
stomatal closure in leaves in blue light.

The stomatal response to red light had the same action spectrum as
did photosynthesis. The effect of red light was eliminated by the
addition of the inhibitors of photosynthetic electron flow, DCMU or

cyanazine, to the transpiration stream. These two pieces of evidence

82

83

lead to the conclusion that chlorophyll is involved in the direct sto-
matal response to light. Since chlorophyll also absorbs blue light.
some of the activity of blue light in causing stomatal opening is
probably the result of the chlorophyll-mediated response. It is specu-
lated that the role of the chlorophyll-mediated response may be to keep
the stomatal response to light matched to the response of assimilation
to light. since environmental influences an electron transport in the
mesophyll would also affect the chlorophyll-mediated stomatal response

to light.

LIST OF REFERENCES

10.

LIST OF REFERENCES

Allaway, W.G. and T.A. Mansfield. 1967. Stomatal responses to
changes in carbon dioxide concentration in leaves treated with 3-
(4-chlorophenyl)-1,1-dimethylurea;

New Phytol. 66:57-63.

Broggrdh. T. 1975. Regulation of transpiration in Avena. Responses
to red and blue light steps.
Physiol. Plant. 35:303-309.

Cowan, I.R. 1977. Stomatal behaviour and environment.
Adv. Bot. Res. 4:117-228.

Cowan. I.R. and 6.0. Farquhar. 1977. Stomatal function in relation
to leaf metabolism and environment.
Symp. Soc. Exp. Biol. 31:471-505.

Dubbe. 0.R., G.D. Farquhar and K. Raschke. 1978. Effect of abscisic
acid on the gain of the feedback loop involving carbon dioxide and

stomata. ~
Plant Physiol. 62:413-417.

Farquhar, G.D., 0.R. Dubbe and K. Raschke. 1978. Gain of the feed-
back loop involving carbon dioxide and stomata. Theory and measurement.
Plant Physiol. 62:406-412.

Gaastra. P. 1959. Photosynthesis of crop plants as influenced by
light, carbon dioxide, temperature. and stomatal diffusion resistance. ,_
Meded. Landb. Hogesch. Wageningen. 59(13):1-68.

Hall, A.E. and M.R. Kaufmann. 1975. Stomatal response to environ-
ment with Sesamum indicum L.
Plant Physiol. 55:455-459.

Heath. 0.V.S. 1959. Light and carbon dioxide in stomatal movements.
In W. Ruhland. ed., Encycl. Plant Physiol., Vol. XVII/1.
Springer-Verlag, Berlin. pp. 415-464.

Heath. 0.V.S. and J. Russell. 1954. Studies in stomatal behaviour VI.
An investigation of the light responses of wheat stomata with the
attempted elimination of control by the mesophyll. Part 2. Inter-
actions with external carbon dioxide, and general discussion.

J. Exp. Bot. 5:269-292.

84

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

85

Hsiao, T.C. 1976. Stomatal ion transport.
In U. Luttge. M.G. Pitman. ed., Encycl. Plant Physiol., New
Series Vol. 2, Part 8. Springer-Verlag, Berlin. pp. 195-221.

Hsiao, T. C. W. G. Allaway and L. T. Evans. 1973. Action spectra
for guard cell Rb+ uptake and stomatal opening in Vicia faba.
Plant Physiol. 51. 82- 88.

Humble, 6.0. and T.C. Hsiao. 1970. Light-dependent influx and
efflux of potassium of guard cells during stomatal opening and
c 051ng.

Plant Physiol. 46:483-487.

Johnsson, M., s. Issaias, 1. BrogSrdh and A. Johnsson. 1976. Rapid,
blue-light-induced transpiration response restricted to plants with
grass-like.stomata.

Physiol. Plant. 36:229-232.

Kamiya, A. and S. Miyachi. 1974. Effects of blue light on respi-
ration and carbon dioxide fixation in colorless Chlorella mutant
cells.

Plant & Cell Physiol. 15:927-937.

Kamiya, A. and s. Miyachi. 1975. Blue light-induced formation of
phosphoenolpyruvate carboxylase in colorless Chlorella mutant cells.
Plant & Cell Physiol. 16:729-736.

Karvé, A. 1960. Die Wirkung verschiedener Lightqualitaten auf die
Offnungsbewegung der Stomata.
Z. Bot. 49. 47- 72.

Kowallik. W. 1967. Action spectrum fer an enhancement of endogenous
respiration by light in Chlorella.
Plant Physiol. 42:672-676.

Kowallik. W. and H. Gaffron. 1966. Respiration induced by blue
light.
Planta 69:92-95.

Kuiper. P.J.C. 1964. Dependence upon wavelength of stomatal move-
ment in epidermal tissue of Senecio odoris.
Plant Physiol. 39:952-955}

Liebig. M. 1942. Untersuchungen Uber die Abhhngigkeit der Spaltweite
der Stomata von IntensitSt und Qualitet der Strahlung.
Planta 33: 206- 257.

Lloyd. F.E. 1908. The physiology of stomata.
Publ. Carneg. Instn. 82:1-142.

Mansfield. T.A. and H. Meidner. 1966. Stomatal opening in light of
different wavelengths: Effects of blue light independent of carbon

dioxide concentration.
J. Exp. Bot. 17:510-521.

24.

25.

26.

27.

28.

29.

31.

32.

33.

34.

35.

86

Meidner. H. and T.A. Mansfield. 1965. Stomatal responses to
illumination.
Biol. Rev. 40:483-509.

Mohl, H. van. 1856. Nelche Ursache bewirken Erweiterung und
Verengung der Spalt3ffnungen.
Bot. Ztg. 14:697-704 and 713-720.

Nelson, 8.0. and J.M. Mayo. 1975. The occurrence of functional
non-chlorophyllous guard cells in Paphiopedilum spp.
Can. J. Bot. 53:1-7.

Nobel, P.S. and T.L. Hartsock. 1979. Environmental influences on
open stomates of a crassulacean acid metabolism plant, Agave
deserti.

Plant Physiol. 63:63-66.

Ogasawara. N. and S. Miyachi. 1970. Regulation of Goa-fixation in
Chlorella by light of varied wavelengths and intensities.
Plant 8 Cell Physiol. 11:1-14.

Ogawa, T., H. Ishikawa, K. Shimada and K. Shibata. 1978. Syner-
gistic action of red and blue light and action spectra for malate
formation in guard cells of Vicia faba L.

Planta 142:61-65.

 

Outlaw, H.H. and J. Kennedy. 1978. Enzymic and substrate basis for
the anaplerotic step in guard cells.
Plant Physiol. 62:648-652.

Outlaw, w.H. and 0.H. Lowry. 1977. Organic acid and potassium
accumulation in guard cells during stomatal opening.
Proc. Nat. Acad. Sci. USA 74:4434-4438.

Pickett, J.M. and C.S. French. 1967. The action spectrum fOr blue-

light stimulated oxygen uptake in Chlorella.
Proc. Nat. Acad. Sci. USA 57:1587-1593.

Raschke. K. 1967. Eine Anlage zur monochromatischen Bestrahlung
biologischer Objekte. ausgerustet mit Interferenzfiltern und einer
elektronisch geregelten 2,5kw-Xenonlampe.

Planta 75:55-72.

Raschke. K. 1974. Abscisic acid sensitizes stomata to C02 in leaves
of Xanthium strumarium L.
‘Proc. 8th Internat. Conf. on Plant Growth Substances, Tokyo. 1973.
Hirokawa Publishing Co., Tokyo, pp. 1151-1158.

Raschke. K. 1975a. Stomatal action.
Ann. Rev. Plant Physiol. 26:309-340.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

47.

87

Raséhke, K. 1975b. Simultaneous requirement of carbon dioxide and
abscisic acid for stomatal closing in Xanthium strumarium L.
Planta 125:243-259.

Raschke. K. 1979. Movements of stomata.
In H. Haupt, M.E. Feinleib, ed., Encycl. Plant Physiol., New
Series Vol. 7. Springer-Verlag, Berlin, pp. 383-441.

Raschke, K. and P. Dittrich. 1977. {14C} carbon-dioxide fixation
by isolated leaf epidermes with stomata closed or open.
Planta 134:69-75.

Raschke, K., N.F. Hanebuth and 6.0. Farquhar. 1978. Relationship

between stomatal conductance and light intensity in leaves of

ggg_mays L., derived from experiments using the mesophyll as shade.
Planta 139:73-77.

Raschke, K. and H. Schnabl. 1978. Availability of chloride affects
the balance between potassium chloride and potassium malate in

guard cells of Vicia faba L.
Plant Physiol. 62:84-87.

Scarth, G.N. 1932.. Mechanism of the action of light and other

factors on stomatal movement..
Plant Physiol. 7:481-504.

Schnabl. H. 1977. Isolation and identification of soluble poly-
saccarides in epidermal tissue of Allium cepa.
Planta 135:307-311.

Shaw, M. and C.A. Maclachlan. 1954. The physiology of stomata I.
Carbon dioxide fixation in guard cells.
Can. J. Bot. 32:784-794.

Skaar, H. and A. Johnsson. 1978. Rapid, blue-light induced tran-
spriation in Avena.
Physiol. Plant. 43:390-396.

Thorpe, N., C.J. Brady and F.L. Milthorpe. 1978. Stomatal metab-
olism: primary carboxylation and enzyme activities.
Aust. J. Plant Physiol. 5:485-493.

Turner, N.C. 1970. Response of adaxial and abaxial stomata to
light.
New Phytol. 69:647-653.

Van Kirk, C.A. and K. Raschke. 1978. Presence of chloride reduces
malate production in epidermis during stomatal opening.
Plant Physiol. 61:361-364.

Virgin, H.I. 1956a. Stomatal transpiration of some variegated
plants and of chlorophyll-deficient mutants of barley.
Physiol. Plant. 10:170-186.

49.

50.

51.

52.

53.

54.

55.

88

Virgin, H.I. 1956b. Light-induced stomatal transpiration of
etiolated wheat leaves as related to chlorophyll a content.
Physiol. Plant. 9:482-493.

Voskresenskaya, N.P. and M.A. Polyakov. 1975. Enhancement of C02-
gas exchange in the leaves of Convallaria majalis L., its action

. spectrum and light saturation.

Plant Sci. Lett. 5:333-338.

Hillmer, C.M. and P. Dittrich. 1974. Carbon dioxide fixation by
epidermal and mesophyll tissues of Tulipa and Commelina.
Planta 117:123-132.

Hillmer, C.M., J.E. Pallas and C.C. Black. 1973. Carbon dioxide
metabolism in leaf epidermal tissue.
Plant Physiol. 52:448-452.

Wong, S.C., I.R. Cowan and 6.0. Farquhar. 1978. Leaf conductance

in relation to assimilation in Eucalyptus pauciflora Sieb. ex spreng.

Influences of irradiance and partial pressure of cEFbon dioxide.
Plant Physiol. 62:670-674.

Wong, S.C. 1979. Stomatal behaViour in relation to photosynthesis.
Ph.D. thesis. Australian National University, Canberra.

Zeiger, E. and P.K. Hepler. 1977. Light and stomatal function:
blue light stimulates swelling of guard cell protoplasts.
Science 196:887-889.

APPENDIX

APPENDIX

Absorption of Light by Leaves

The lower surface of Xanthium strumarium leaves has a higher reflec-
tance than does the upper surface. As a consequence, the amount of
light absorbed depends on the orientation of the leaf with respect to
the light source. Surprisingly, the transmittance was found to be the
same when the leaf was illuminated from the top or the bottom (Table I-A).
This suggests that the lower epidermis reflects light back up through the
leaf when the leaf is illuminated from above. A model of light absorp-
tion was developed that predicts equal transmittance when the leaf is
illuminated from above and below; this can be used to evaluate the effect
of a highly reflective lower leaf surface. Figure I-A shows 5 paths of
light that can be accounted for by this model. Path 4 is usually very
small and is lumped with path 3 in the mathematical treatment.

The transmittance of light through the leaf (T) is equal to the inci-
dent light (I) minus the reflected light (R) and the absorbed light (A):

T=I-R-A (1)

The amount of light absorbed by the leaf (light paths 2 and 3 of Figure
1) is given by:

A=(I-Iru)a+(I-ru-(I-Iru)a)r1 (2)
where ru and r.l are the reflectances of the upper and lower surfaces
respectively and a is the absorptivity of the mesophyll. Setting I equal
to 1 and substituting equation 2 into equation 1 we obtain:

T=1-a+aru-r.l+rur]+ar1-arur]-ru (3)

From equation 3 it can be seen that the transmission of light is inde-

pendent of the orientation of the light source, since ru and r1 could be

89

90

Table I-A. Transmissivity, reflectivity, and absorptivity of leaves.
Transmissivity, reflectivity, and absorptivity of light of’a leaf of
Xanthium strumarium measured at two wavelengths of light. Measurements
were made with the light shining on the upper surface (normal) or the
lower surface (inverted). All measurements were made with a Zeiss PMQ
II spectrophotometer with an integrating sphere. Absorptivity was cal-

culated using equation 1 in the text.

 

 

436 nm 681 nm
normal inverted normal inverted
Transmissivity (%) 2.6 2.7 5.5 5.6
Reflectivity (%) . 5.2 7.5 6.0 9.9

Absorptivity (%) 92.2 A 89.9 88.5 84.5

 

91

 

Figure l-A. Paths of light through leaves.

The model of light absorption developed in the text can account
for these five paths of light through leaves. Since light path 4 was
determined to be very small, it is lumped with light path 3 in the

analysis.

 

92

 

 

 

 

 

 

 

93

interchanged without changing the equation.

As the reflectance of the lower surface of the leaf increases, light
path 3 of Figure 1 also increases. To evaluate this effect on the total
absorption of light, it is necessary to solve for a (T, ru, and r1 can be
measured directly).

a=(1-r]-ru+r

url-T)/(I-ru-r1+rur1) (4)

Light path 3 (LP3) is given by:

LP3=(I-Iru-(I-Iru)a)r1 ' (5)
By arbitrarily setting the reflectivity of the lower surface equal to
that of the upper surface and subtracting that result from the result
of equation 5, we can determine the extra absorbance caused by the higher
reflectivity of the lower surface. These calculations were done with
data from Xanthium strumarium and Populus 3193, a tree whose leaves have
a hirsute lower surface with a high reflectivity. The refflectivity of
the upper and lower surface and the transmissivity of each leaf was
measured with a Ziess PMQ II spectrophotometer with an integrating
sphere. Measurements were recorded every l0 nanometers from 400 to 700
nanometers. The averaged results are presented in Table 2-A. The effect
of the reflective lower surface was small in the blue and the red because
nearly all of the light was absorbed the first time it passed through the
leaf. However, a fair amount of green light was reflected from the lower
surface of the leaf. At 540 nm, 7% of the incident light was reflected
from the lower surface of leaves of Populus alga, As a result, the rel-
ative intensities of light at various wavelengths is important. The
average was calculated for two light distributions. First, it was as-
.sumed that there was an equal number of quanta at each wavelength and

second, to simulate the light distribution in the understory, it was

94'

assumed that the light had the same distribution as the transmission
spectrum of the leaf being studied. This was done by multiplying each
value by the transmissivity at that wavelength and dividing by the
average transmissivity. The data in Table 2-A indicate that the effect
of the reflective lower surface of Xanthium strumarium leaves is insigni-'
ficant in terms of light absorption, but for'Populus albg_the effect

can be significant, especially for leaves in the understory.

95

Table 2-A. Light absorbed by leaves after reflection from the lower
surface.

Calculations were made assuming an equal quantum flux at all wave-
lengths (labeled "normal light distribution") and assuming that the
incident light had first passed through an identical leaf (labeled
"understory light distribution"). See text fbr more detail on how

these calculations were made.

 

Absorption (%)

Light distribution
Normal Understory

 

PopuZus aZba . 2.1 4. 9

Xanthium strwnamlum 0 . 3 0. 6

 

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