Plant Cell Physiol. 42(1): 1–8 (2001)
JSPP © 2001
Why Abaxial Illumination Limits Photosynthetic Carbon Fixation in Spinach
Leaves
Jindong Sun 1, 2 and John N. Nishio 1, 3
1
Department of Botany, University of Wyoming, Laramie, WY 82071-3165, U.S.A.
;
1980, Lloyd et al. 1992, Loreto et al. 1992, Parkhurst 1994,
Thoday 1931)
Bifacial leaves contain palisade mesophyll (PM) near the
adaxial surface and spongy mesophyll (SM) near the abaxial
surface and receive light mainly from the adaxial surface
(Clements 1905). Gas exchange studies show that bifacial
leaves have a different light response curve for photosynthesis,
when they are illuminated either on the adaxial or abaxial surface, and that the gas exchange rate is usually higher when the
leaf is illuminated on the adaxial surface compared to the abaxial surface (Evans et al. 1993, Leverenz and Jarvis 1979, Moss
1964, Ögren and Evans 1993, Oya and Laisk 1976, Syvertsen
and Cunningham 1979, Terashima 1986, Turner and Singh
1984). Information about the effect of light direction on photosynthetic activity at different depths within the leaf is lacking.
Light gradients and the patterns of carbon fixation across
leaves do not correlate well. Maximal 14CO2 fixation per paradermal section occurs midway through the PM (Nishio et al.
1993, Sun et al. 1996b). In contrast, the light microenvironment (Cui et al. 1991, Vogelmann 1993, Vogelmann et al.
1989) and light absorption (Terashima and Saeki 1985) decline
exponentially with leaf depth, and only small amounts of transmitted and reflected light reach the chloroplasts near the abaxial surface.
It is possible that the observed maximum of carbon fixation in the middle of the PM is due to limitations of CO2 diffusion. For example, when gas exchange occurs only on one surface of the leaf as in a hypostomatous leaf, or when only one
side of an amphistomatous leaf is fed CO2, a CO2 gradient may
develop across the leaf, and mesophyll resistance of CO2 diffusion may limit photosynthesis within the leaf. Mesophyll
resistance of CO2 diffusion has been reported to be both small
(Farquhar and von Caemmerer 1982, Mott and O’Leary 1984,
Sharkey et al. 1982) and large (Farquhar et al. 1980, Parkhurst
and Mott 1990, Parkhurst et al. 1988), and it appears to be species dependent. Mesophyll resistance of CO2 diffusion is small
in agricultural crops and is large in woody plants (Lloyd et al.
1992, Parkhurst et al. 1988).
The effect of CO2 diffusion on the rates of carbon fixation
at different depths within the leaf is unknown. The objectives
of the present study were to investigate how CO2 diffusion
across spinach leaves and the distribution of Ribulose-1,5bisphosphate carboxylase/oxygenase (Rubisco) activity and
Limitations of carbon fixation within spinach leaves
due to light and CO2 were investigated. Under equivalent
photon fluxes, carbon fixation was higher when leaves were
irradiated adaxially compared to abaxially. Maximal carbon fixation occurred in the middle of the palisade mesophyll under adaxial illumination, whereas, maximal carbon
fixation occurred in the spongy mesophyll under abaxial
illumination. Total carbon fixation and the pattern of carbon fixation across leaves were similar, when leaves were
irradiated with 800 mmol quanta m–2 s–1 either adaxially
alone or adaxially plus abaxially (1,600 mmol quanta m–2 s–1).
In contrast, when both leaf surfaces were irradiated simultaneously with 200 mmol quanta m–2 s–1, total carbon fixation increased and carbon fixation in the middle of the leaf
was higher compared to leaves irradiated unilaterally with
the low light. Feeding 14CO2 through either the adaxial or
abaxial leaf surface did not change the pattern of carbon
fixation across the leaf. Increasing 14CO2 pulse-feeding
times from 5 s to 60 s allowed more 14CO2 to be fixed but
did not change the pattern of 14CO2 fixation across the leaf.
We concluded that in spinach, the distribution of both light
and Rubisco activity within leaves has significant effects on
the patterns of carbon fixation across leaves; whereas CO2
diffusion does not appear to affect the carbon fixation pattern within spinach leaves.
Key words: Carbon fixation — CO2 diffusion — Photosynthetic limitations — Rubisco — Spinacia oleracea.
Abbreviations: PM, palisade mesophyll; Rubisco, Ribulose-1,5bisphosphate carboxylase/oxygenase; SM, spongy mesophyll.
Introduction
The distribution of photosynthetic activity within the leaf
is heterogeneous because of interactions among different tissue
types within a leaf (Bil’ 1993, Laetsch 1974, Nishio and Ting
1987), the light microenvironment (Cui et al. 1991, Martin et
al. 1989, Terashima and Saeki 1985, Vogelmann 1993), protein
and pigment distribution (Nishio et al. 1993, Sun et al. 1996a,
Terashima and Inoue 1985), and possibly a CO2 gradient
caused by CO2 diffusion (Epron et al. 1995, Farquhar et al.
2
3
Present address: Monsanto Gene Mining and Development, Mail Zone N2SB, 800 N Lindbergh, Blvd., St. Louis, MO 63167, U.S.A.
Corresponding author: E-mail, [email protected]; Fax, +1-307-766-2851.
1
Directional light and 14CO2 feeding to leaves
2
light limit photosynthesis within the leaf. To investigate the interactions between light and Rubisco in determining the pattern of carbon fixation within spinach leaves, which are amphistomatous, carbon fixation profiles across leaves were
measured under high light and low light directed to the adaxial
and/or abaxial leaf surfaces. 14CO2 was fed abaxially or adaxially under adaxial and/or abaxial illumination to test whether
the pattern of carbon fixation across leaves is due to CO2 diffusion. The data further corroborate the finding that the CO2 fixation pattern across leaves is dependent on the distribution of
Rubisco within leaves (Nishio et al. 1993).
Materials and Methods
Plant growth conditions
Spinach (Spinacia oleracea cv hybrid 424, Ferry-Morse Seed
Company, Modesto, CA) was cultured hydroponically in 0.5
Hoagland solution (Hoagland and Arnon 1950) in controlled environmental growth chambers as previously described (Nishio et al. 1993).
Plants were grown at a photon flux density of 800 mol quanta (400–
700 nm) m–2 s–1 (other light measurements refer to the same wavelengths). Five- to six-week old plants were used for all experiments.
The daytime temperature was 232C and the nighttime temperature
was 172C. The photoperiod was 12 h light and 12 h dark. The average leaf thickness was 640 m. The PM corresponds to the upper 48%
of the leaf depth, and the remaining portion of each leaf type corresponds to the SM.
14
CO2 fixation
Attached leaves were activated by irradiating them under white
light (800 mol m–2 s–1) on their adaxial surfaces for more than 1 h in
ambient air at room temperature. They were then irradiated with assay
light for 5 min before being clamped in a small leaf chamber (1.27 cm
diameter) for 14CO2 feeding. 14CO2 labeling and measurement were
conducted as previously described (Nishio et al. 1993, Sun et al.
1996b). 700 ppm 14CO2 (specific activity of 5 Ci mol–1) was fed with a
syringe at a constant flow rate of 15 ml min–1 for 10 s, unless stated
otherwise. After CO2 feeding, a flat leaf disc (0.47 cm2) was obtained
by rapid freezing with a paper punch that was precooled in N2 (l). The
flat, frozen leaf disc was then transferred to the stage of a freezing
microtome and cut paradermally into 40-m thick sections. Each section was immediately put into 1 ml 95% ethanol in a liquid scintillation vial. A few drops of 30% acetic acid were added to bleach the Chl
and drive off unfixed 14CO2. 14CO2 incorporation was measured by
liquid scintillation counting (TriCarb 4430; United Technologies
Packard, Downers Grove, IL). The counting efficiency was 855%. In
our experiments, volume (per section) and area are equivalent, because each paradermal section has an equivalent area and volume.
Rubisco activity assay
Attached leaves were activated for more than 1 h by irradiating
their adaxial surface with 800 mol quanta m–2 s–1. Then a leaf disc
(0.47 cm2) was sampled as above. After each section was cut, it was
immediately transferred into a 1.5 ml microcentrifuge tube containing
80 l 150 mM Tricine-KOH (pH 7.95, 4C), containing 20 mM
MgCl2, 1 mM EDTA-Na2, 20 mM DTT, and 0.5% BSA. The sample
was immediately macerated with a microhomogenizer. Then 10 l of
200 mM NaH14CO3 (1 Ci mol–1) was added to the sample, and the
mixture was incubated at 25C for 5 min. The reaction was started by
adding 10 l of 10 mM RuBP-Na4. After incubating at 25C for 30 s
the reaction was stopped by adding 100 l 30% acetic acid. The mix-
ture was dried in an oven at 80C, and then 100 l distilled water was
added to dissolve the sample. Six ml of scintillation cocktail
(Scintiverse E, Fisher, Denver, CO) was then added and 14C-incorporation was measured by liquid scintillation counting (TriCarb 4430;
United Technologies Packard, Downers Grove, IL).
Chl determination
Chl content in leaf paradermal sections was measured as previously reported (Nishio et al. 1993). Chl was extracted and measured in
95% ethanol (Lichtenthaler 1987).
Results
Effects of light direction on 14CO2 fixation across the leaves
Carbon fixation was 37% higher when the leaves were
illuminated adaxially compared to abaxially under illumination of 800 mol quanta m–2 s–1 or 200 mol quanta m–2 s–1
(Table 1, line numbers 1, 2, 5, and 7). The direction of light impinging on the leaf also significantly affected the pattern of carbon fixation within the leaf. Under adaxial illumination, maximal carbon fixation occurred in the PM, while under abaxial
illumination the peak of carbon fixation shifted to the SM (Fig.
1A, B). When expressed on a Chl basis, the gradients of 14CO2
fixation across the leaves decreased exponentially from the
illuminated surface under adaxial illumination of 800 mol
quanta m–2 s–1 (Fig. 2A). A similar result was observed when
leaves were illuminated with 200 mol quanta m–2 s–1 on the
adaxial leaf surface (Fig. 2C). When leaves were irradiated
with 200 mol quanta m–2 s–1 on the abaxial leaf surface, saturated rates of Rubisco activity were attained in the three bottom slices.
When each leaf surface was illuminated with 200 mol
quanta m–2 s–1 at the same time (total 400 mol quanta m–2 s–1),
carbon fixation increased 38% (Table 1, line numbers 7 and 8),
and more carbon fixation occurred in the central part of the leaf
compared to leaves illuminated only adaxially with 200 mol
quanta m–2 s–1 (Fig. 1B). Carbon fixation, when the leaves were
illuminated on both surfaces with 200 mol quanta m–2 s–1, was
lower than the sum of the carbon fixation under adaxial illumination of 200 mol quanta m–2 s–1 plus the carbon fixation
under abaxial illumination of 200 mol quanta m–2 s–1 (Fig.
1B). The predicted pattern (not shown), however was similar to
the measured pattern. On a Chl basis, illumination of both leaf
surfaces followed the pattern of unilateral illumination on each
leaf surface, and in the middle of the leaf, carbon fixation/Chl
was higher than unilateral illumination (not shown to improve
clarity in Fig. 2C, but see Fig. 1B).
When leaves were irradiated with 800 mol quanta m–2 s–1
on both leaf surfaces, the total carbon fixation was 17% higher
(Table 1, line numbers 3 and 4), but not statistically different
from when the leaves were only illuminated adaxially (Table 1,
line numbers 1 and 2). The patterns of carbon fixation across
the leaves were similar whether the leaf was irradiated adaxially with 800 mol quanta m–2 s–1 alone or adaxially plus abaxially (total 1,600 mol quanta m–2 s–1) (Fig. 1).
Directional light and 14CO2 feeding to leaves
Table 1 Effects of adaxial and/or abaxial illumination and
fixation
14
3
CO2 feeding side on total
14
CO2
No.
Irradiance
(mol m–2 s–1)
Light direction
Side fed 14CO2
(Percent of Treatment 1)
14
1
2
3
4
5
6
7
8
9
800
800
800
800
800
800
200
200
200
Adaxial
Adaxial
Adaxial + Abaxial
Adaxial + Abaxial
Abaxial
Abaxial
Adaxial
Adaxial + Abaxial
Abaxial
Abaxial
Adaxial
Abaxial
Adaxial
Abaxial
Adaxial
Abaxial
Abaxial
Abaxial
10012 (n=6) a
10411 (n=6) a
12124 (n=5) a
11712 (n=2) a
787 (n=4) b
708 (n=3) b
603 (n=3) A
8314 (n=4) B
443 (n=3) C
CO2 fixation
14
CO2 concentration was 700 ppm. All values are relative to Treatment No. 1 (“sun” grown control measured
at growth irradiance). Values are mean±SD. Sample size is given as n. The sum of all paradermal leaf sections was used to calculate the total 14CO2 fixation rate.
a
Significantly different from “b” (P<0.01).
A
Significantly different from “B” and “C” (P<0.01).
Total carbon fixation was 40% lower when the abaxial
surface of the leaf was illuminated with 200 mol quanta m–2 s–1
compared to 800 mol quanta m–2 s–1 (Table 1, No. 5, 6, 9).
However, carbon fixation in the three most abaxial layers was a
little higher when the abaxial surface of the leaf was illuminated with an irradiance of 200 mol quanta m–2 s–1 compared
to 800 mol quanta m–2 s–1 (Fig. 1A, B).
Carbon fixation expressed on a Rubisco basis accentuates
the limitation of abaxial light on CO2 fixation (Fig. 3). The actual 14CO2 counts/section (shown in Fig. 1) were divided by the
percent Rubisco content/section, where 100% was represented
by the maximum Rubisco/section measured previously (Nishio
et al. 1993). When leaves were illuminated adaxially with
800 mol m–2 s–1, the fixation per Rubisco was relatively flat
across the leaves (Fig. 3A). Fixation per Rubisco when leaves
were illuminated on both sides with 800 mol m–2 s–1 was
slightly higher than under unilateral adaxial illumination, but
not significantly. As with adaxial illumination alone, the rate
was relatively flat across the leaf under bilateral illumination
with 800 mol m–2 s–1 (Fig. 3A). In contrast, when leaves were
illuminated unilaterally from the abaxial side, CO2 fixation per
Rubisco was significantly lower in the upper half of the leaf
(Fig. 3A).
When leaves were illuminated with 200 mol m–2 s–1 on
the adaxial surface alone, the fixation rate per Rubisco was
lower than when the same leaf surface was illuminated with
800 mol m–2 s–1. As with 800 mol m–2 s–1, the fixation rate
per Rubisco was relatively flat across the leaves when illuminated adaxially with 200 mol m–2 s–1. The general trend was
that the rate of fixation/Rubisco with adaxial light alone was
flat across the top of the leaf with a slight decrease towards the
bottom of the leaves (Fig. 3A, B). When leaves were illumi-
nated on the abaxial leaf surface with 200 mol m–2 s–1, the fixation per Rubisco in the lower half of the leaf was more than
double the rate when illuminated adaxially (Fig. 3B). The
higher rate of fixation under 200 mol m–2 s–1 abaxial light
compared to 800 mol m–2 s–1 abaxial light in the lower part of
the leaf (Fig. 1A, B) is accentuated when expressed on a
Rubisco basis (Fig. 3B).
Effects of 14CO2 diffusion, [14CO2], and 14CO2-feeding times on
14
CO2 fixation profiles across leaves
Feeding 700 ppm 14CO2 to either the abaxial or adaxial
leaf surface did not significantly change the total amount of
carbon fixed (Table 1, No. 1–6) or the pattern of carbon fixation across the leaf (Fig. 1A).
The concentration of 14CO2 used did not affect the pattern
of carbon fixation across the leaves, shown on a Chl basis. At
an irradiance of 200 mol quanta m–2 s–1, the patterns of carbon
fixation across the leaves were similar on a relative basis, when
leaves were fed either 350 ppm 14CO2 or 700 ppm 14CO2 (Fig.
2C), even though the total amount of carbon fixed in leaves fed
350 ppm 14CO2 was 60% of that in the leaves fed 700 ppm
14
CO2.
Increasing the 14CO2 pulse-feeding time from 5 s to 60 s
linearly increased 14CO2 fixation (Fig. 4A), but the pattern of
14
CO2 fixation across the leaf was not changed significantly
(Fig. 4B). Additionally, when leaves were pulsed with 14CO2
for 10 s, and then chased for 5 s to 25 s, the amount of 14CO2
fixed and the pattern of 14CO2 fixation across the leaves were
not statistically different (data not shown).
Rubisco activity across leaves
Rubisco total activity per paradermal section was maxi-
4
Directional light and 14CO2 feeding to leaves
Fig. 1 CO2 Fixation and Rubisco distribution across spinach leaves.
The effects of the direction of illumination and direction of 14CO2
feeding on the pattern of carbon fixation across spinach leaves are
shown in A and B. The adaxial leaf surface is zero on the abscissa. The
curve fits in A and B represent adaxial illumination (solid lines), abaxial illumination (dotted lines) and both surface illumination (dashed
lines). Data points represent means. The SD is similar to that illustrated in Fig. 3B, but slightly larger, because the data here is not normalized. A. 14CO2 (700 ppm) incorporation was conducted under
800 mol quanta m–2 s–1. Filled triangles are adaxial surface illumination, adaxial 14CO2 feeding (abbreviated as “light direction/CO2 direction” or adaxial/adaxial), n=6; unfilled triangles, adaxial/abaxial, n=6;
filled circles, abaxial/adaxial 14CO2 feeding, n=3; unfilled circles,
abaxial/abaxial 14CO2 feeding, n=4; filled squares, both leaf surfaces/
adaxial, n=2; unfilled squares, both leaf surfaces/abaxial, n=4. B. Effects of “low” light direction on the pattern of carbon fixation across
spinach leaves. 200 mol quanta m–2 s–1 was directed either to the
adaxial (unfilled triangles, n=3), abaxial (unfilled circles, n=3), or both
(unfilled squares, n=4) leaf surfaces in the presence of 700 ppm 14CO2
fed to the abaxial surface. C. Total Rubisco activity across spinach
leaves preilluminated on the adaxial leaf surface with 800 mol quanta
m–2 s–1. Relative Rubisco activity expressed on equal tissue volume
basis.
Fig. 2 14CO2 fixation and Rubisco activity distribution across leaves
expressed on a per unit Chl basis. Figure 1 shows the data on an equal
volume (equivalent to area) basis. The uppermost point containing
epidermis was not included in the logarithmic curve fits, when leaves
were illuminated adaxially. A. 14CO2 fixation. filled triangles, adaxial/
adaxial (see Fig. 1 for nomenclature), n=6; unfilled triangles, adaxial/
abaxial, n=6; filled circles, abaxial/adaxial, n=3; unfilled circles, abaxial/abaxial, n=4; filled squares, both leaf surfaces/adaxial, n=2; unfilled squares, both leaf surfaces/abaxial, n=4. The curve fits are
adaxial illumination (solid lines), abaxial illumination (dotted lines)
and both surface illumination (dashed lines). B. Rubisco activity. C.
Two concentrations of CO2 did not affect the relative distribution of
carbon fixation across the leaves. Leaves were irradiated with
200 mol quanta m–1 s–1 either adaxially or abaxially in the presence
of 350 ppm or 700 ppm CO2. Unfilled circles, Adaxial illumination,
350 ppm CO2; filled circles, Adaxial illumination, 700 ppm; unfilled
triangles, Abaxial illumination, 350 ppm; filled triangles; Abaxial illumination, 700 ppm CO2. Sample size was three for each case (typical
SD is shown in 4B).
Directional light and 14CO2 feeding to leaves
Fig. 3 Effect of light direction on carbon fixation per Rubisco across
spinach leaves. A. 14CO2 (700 ppm) incorporation was conducted under 800 mol quanta m–2 s–1. As in Fig. 1, unfilled circles, adaxial/
abaxial, n=6; filled triangles, abaxial/abaxial 14CO2 feeding, n=4;
unfilled squares, both leaf surfaces/abaxial, n=4. B. 200 mol quanta
m–2 s–1 was directed either to the adaxial (unfilled circles, n=3), abaxial
(filled triangles, n=3), or both (unfilled squares, n=4) leaf surfaces in
the presence of 700 ppm 14CO2 fed to the abaxial surface.
mal midway through the PM at a depth of about 150–200 mm
(Fig. 1C). When expressed on a Chl basis, the gradients of
Rubisco activity declined exponentially with leaf depth (Fig.
2B).
Discussion
Light direction had a profound effect on the patterns of
carbon fixation across spinach leaves. Our previously published profiles of carbon fixation across leaves were all done
under adaxial light and showed that maximum fixation occurred in the middle of the PM (Nishio et al. 1993, Sun et al.
1996b, Sun et al. 1998). In the present paper, we showed that
abaxial illumination caused carbon fixation to occur mainly in
the SM (Figs. 1 and 3). Furthermore, the total carbon fixation
was decreased when leaves were illuminated with the same
5
Fig. 4 Effect of 14CO2 pulse-feeding time on total 14CO2 fixation
across spinach leaves. 700 ppm 14CO2 was fed for various times at a
constant flow rate of 15 ml min–1. A. Total carbon fixation. Sample
size is n=4 for all feeding times except 60 s, where n=3. MeanSD is
shown; where not shown, it is smaller than the symbol. B. 14CO2 fixation profile across spinach leaves. Unfilled circles, 5 s pulse, n=4; unfilled squares, 10 s pulse, n=4; unfilled triangles, 30 s pulse, n=4;
diamonds, 60 s pulse, n=3.
photon flux abaxially compared to adaxially (Table 1, no. 1, 2,
5, 6, 7, 9). A decrease in absorption cannot account for the difference. It is likely that light penetration into the palisade tissue was limited, when leaves were irradiated abaxially, because light is highly scattered and absorbed in the SM
(Terashima and Saeki 1983, Terashima and Saeki 1985). Thus,
one reason why the gas exchange rate is usually higher, when a
bifacial leaf is illuminated on the adaxial surface compared to
the abaxial surface (Evans et al. 1993, Leverenz and Jarvis
1979, Moss 1964, Ögren and Evans 1993, Oya and Laisk 1976,
Syvertsen and Cunningham 1979, Terashima 1986, Turner and
Singh 1984), is that when a leaf is illuminated abaxially, CO2
fixation is mainly limited to the SM, due to light scattering in
the SM.
The exponential decline in carbon fixation per Chl with
6
Directional light and 14CO2 feeding to leaves
leaf depth under adaxial illumination (Fig. 2) supports the
notion that a significant amount of light absorption within
spinach leaves obeys the Beer-Lambert Law. However, light
scattering and reflection also need to be considered, because
the leaf structure, cell anatomy, and pigment distribution are
not homogeneous (Richter and Fukshansky 1996). Multiple
scattering in the SM leads to more absorption of the light not
strongly absorbed, such as green light (Richter and Fukshansky
1996, Sun et al. 1998, Terashima and Saeki 1983, Terashima
and Saeki 1985). Our data showed that the red and blue component of the white light must also have been strongly absorbed
in the SM, but that is more likely due to the high extinction of
blue and red light by Chl.
Since adaxial illumination allowed significant fixation in
the SM, our data also support the notion that the structure of
the PM allows light to penetrate deeply into the leaf (Thoday
1931, Vogelmann and Martin 1993). Light utilization across
the leaf is more efficient under adaxial illumination, because
PM cells have a low apparent extinction coefficient per Chl
(Terashima and Saeki 1983) and less light scattering (Knapp et
al. 1988). However, in spinach the average light absorption
profiles can be attributed mainly to Chl alone (Nishio 2000).
When light was not limiting, the pattern of photosynthetic
(carbon fixation) capacity across the leaf closely correlated
with the Rubisco activity profile across the leaf (Fig. 1). The
data corroborate our earlier finding, that CO2 fixation across
leaves correlates well with the Rubisco distribution across
leaves (Nishio et al. 1993), and support the model of Farquhar
et al. (1980), that predicts Rubisco limits carbon fixation under
saturating light.
14
CO2 fixation and Rubisco content (Nishio et al. 1993)
exhibit profiles similar to Rubisco activity across leaves (Fig.
2B) on both a per section (Fig. 1C) and Chl basis (Fig. 2B). In
a bifacial leaf, the SM is less compact than the PM, which
partly explains why Rubisco activity was higher in the PM than
in the SM, but it does not account for the lower activity at the
top of the leaf compared to midway through the PM. The very
low fixation per Chl and per section in the uppermost paradermal section is due to the epidermis, which has little Rubisco.
The elevated fixation/Chl was due to elevated Rubisco activity
(Fig. 2) and content per Chl (Nishio et al. 1993) at the top of
spinach leaves. The relatively fixed rate of 14CO2 fixation per
Rubisco across leaves (Fig. 3) further illustrates that the
Rubisco/Chl ratio must be elevated at the top of leaves. Deeper
within the leaf, the Chl content does not decline as rapidly from
the adaxial surface as Rubisco protein does, since more light
harvesting capacity is associated with the reaction centers in
the lower part of the leaf (Nishio et al. 1993).
In Vicia faba, PM and SM cells exhibit comparable
Rubisco activity on a Chl basis (Outlaw et al. 1976), and in
Zinnia elegans Rubisco activity in the SM and PM were equivalent on a protein basis (Seeni et al. 1983). Because the units of
Rubisco activity are expressed differently, direct comparisons
of the data are difficult. The growth conditions, leaf orienta-
tion and structure, and whether the assays are done in vivo or in
vitro may also affect the results. It is likely that in spinach,
Rubisco activity expressed on a protein basis would be similar
in PM and SM cells, because the gradient of Rubisco protein,
which constitutes about 50% of the total protein (Nishio et al.
1993), and the gradient of Rubisco activity (Fig. 1) are similar.
Therefore, our data is most in agreement with the data of Seeni
et al. (1983), which expresses Rubisco activity on a protein
basis. There may, of course, be species differences.
Under abaxial illumination, 14C-fixation per Rubisco was
extremely limited in the PM (Fig. 3). The in vivo results further
illustrated that light is scattered (trapped) in the SM. Carbon
fixation per Rubisco under both non-saturating and saturating
adaxial illumination was reasonably flat across the leaves (Fig.
3). It is possible that there was a minor light limitation or
different Rubisco efficiency in the lower part of the leaves,
because there was a trend toward lower fixation per Rubisco in
the SM, when leaves were irradiated adaxially. Such a result
can also be calculated from our previously published data
(Nishio et al. 1993), where fixation per Rubisco in sun leaves is
remarkably constant across the PM, then slightly decreases in
the SM (not shown). Fixation in the SM under 200 mmol m–2 s–1
directed to the abaxial leaf surface was slightly higher than under 800 mmol m–2 s–1. As in Fig. 1, the difference may be due to
leaf differences, but see discussion about photoinhibition below. Nonetheless, the results illustrate that C-fixation was enzyme limited in the SM under low irradiation.
When the leaves were illuminated on both surfaces with
200 mmol quanta m–2 s–1, the profile of carbon fixation was as
expected based on summing the individual carbon fixation
rates under unilateral adaxial or abaxial 200 mmol quanta m–2 s–1
(Fig. 1B). However, the rate was 20% less than expected. The
result could be physiological, as the quantum yield is not linear
under higher light (McCree 1972). When irradiance increases
from 200 mmol quanta m–2 s–1 to 400 mmol quanta m–2 s–1, the
quantum yield should generally decrease. Additionally, the limitation by Rubisco activity is negligible under low irradiance
but could be larger under higher irradiance. If either or both
possibilities were true, the greatest increase in fixation under
bidirectional illumination, compared to under unilateral illumination, would occur in the middle of the leaf, which was the
case (Fig. 1B). It is also possible that the leaves used for the
bidirectional experiments had more Rubisco, but such is not
likely since the fixation rates near the irradiated surfaces were
similar under either bidirectional or unidirectional illumination.
Carbon fixation in the four most abaxial layers was
slightly higher, when the abaxial surface of the leaf was illuminated with an irradiance of 200 mmol quanta m–2 s–1 compared
to 800 mmol quanta m–2 s–1. However, the total carbon fixation
was 40% lower, when the abaxial surface of the leaf was illuminated with an irradiance of 200 mmol quanta m–2 s–1 compared to 800 mmol m–2 s–1 (Table 1). The results suggest that
photoinhibition and/or low light saturation occurred in the most
abaxial layers of the leaf when the abaxial surface of the leaf
Directional light and 14CO2 feeding to leaves
was illuminated with 800 mmol quanta m–2 s–1. Photoinhibition
is a likely explanation, since the abaxial surface of leaves similar to those used in the present experiment is more susceptible
to high light damage than the adaxial surface of the leaf (Sun et
al. 1996b).
We hypothesized that pulse chase experiments would
demonstrate movement of 14CO2 from the PM to the vascular
tissue or to the SM; however we did not detect such transport.
Since increasing the time of illumination also caused no change
in the pattern of carbon fixation across the leaves (Fig. 3B), we
presume transport equilibration had already been reached, the
leaves were making starch, or there was little export of carbon
during the time course of the experiment from the leaves used
(Sun et al. 1999). A lack of transport may be a reflection of leaf
age and/or physiological status of the plant. Further studies, including chase experiments, are required to distinguish the possibilities.
We reported earlier that CO2 diffusion across the leaf did
not affect the pattern of CO2 fixation (Nishio et al. 1993). In the
present study we further tested the possibility of a limitation of
carbon fixation by CO2 diffusion across the spinach leaf by
feeding 14CO2 (700 ppm) to either the adaxial or abaxial leaf
surface. The direction of CO2 feeding did not significantly
change the total amount of carbon fixed (Table 1) or the pattern of carbon fixation within spinach leaves (Fig. 1). In addition, the patterns of carbon fixation within the leaf were similar on a relative basis when leaves were fed either 350 ppm
14
CO2 or 700 ppm 14CO2 at an irradiance of 200 mmol quanta
m–2 s–1 (Fig. 2C; see also Nishio et al. 1993).
Similar to our results, sunflower gas exchange rates across
both surfaces and across one surface are identical (Mott and
O’Leary 1984). In contrast, the gas exchange rate, when CO2 is
fed to both surfaces, is similar to that when CO2 is fed to the
adaxial surface only and higher than when CO2 is fed only to
the abaxial surface (Parkhurst et al. 1988). The disparity in
findings is likely due to species differences. The gas exchange
rates occurring across both surfaces and across one surface are
similar in Spinacia, Brassica, and Phaseolus, whereas the gas
exchange rate is lower, when CO2 is fed to the abaxial surface
in Eucalyptus (Parkhurst et al. 1988). Crop leaves are usually
more porous and the intercellular air space generally accounts
for 30% of the leaf volume (Nobel 1991). At low [CO2], the
CO2 exchange rate in helox ranges up to 7% higher than in air
in amphistomatous leaves (Parkhurst and Mott 1990), which
indicates that intercellular diffusion limits CO2 uptake. The
effect, however, is negligible at ambient or higher CO2 concentration. At 290 ppm CO2 the average increase in gas exchange
rate in helox is only 1.9% in seven amphistomatous leaves
(Parkhurst and Mott 1990). Such a small change would not be
detectable using our system, since such differences can occur
in different parts of the same leaf.
The components of mesophyll resistance, including
gaseous intercellular, liquid-phase intracellular resistance, and
biochemical resistance, are difficult to separate. Diffusion in
7
the free air space is a fast process even though the pathway for
the movement of gas in the intercellular air space is tortuous.
Nobel (1991) estimates that an upper limit for the gaseous
diffusion pathway in the intercellular air spaces of a leaf might
be 1,000 mm, and that the time needed for CO2 to diffuse
1,000 mm in air is 16 ms. CO2 diffuses 10,000 times more
slowly through water than through air, so diffusion of CO2
through the cell to the sites of carboxylation is a potentially
important limitation to photosynthesis. Using the equation
given by Nobel (1991), the time needed for CO2 to diffuse
20 mm (a typical radius of a mesophyll cell) in water is 60 ms.
Chloroplasts are generally closer to the plasma membrane
(Haberlandt 1914), so the diffusion time to the chloroplasts
would be considerably less than 60 ms. In addition, cytoplasmic streaming, carbonic anhydrase, and active transport may
also impact CO2 diffusion. Based on our data and the above
analysis, we concluded that the transport of CO2 across spinach leaves is not likely to be limiting to carbon fixation.
We have presented the first investigations on how CO2
diffusion and the direction of illumination on leaves affects the
distribution of CO2 fixation within leaves. The direction by
which a bifacial, spinach leaf is illuminated contributes significantly to the pattern of carbon fixation within the leaf, which
was defined by the distribution of Rubisco within the leaf. On
the other hand, CO2 diffusion had little effect on the pattern of
carbon fixation across spinach leaves, as feeding CO2 either
adaxially or abaxially did not change the pattern of carbon fixation across the leaves. Doubling the concentration of CO2 also
did not change the relative pattern of fixation across spinach
leaves (see also Nishio et al. 1993).
Based on our experiments, we concluded that for spinach
leaves (1) light is more limiting for carbon fixation when
leaves are irradiated abaxially compared to adaxially, because
the SM acts like a light trap, (2) the gradient of carbon fixation
across the leaf, when light is not limiting, correlated with Rubisco activity across the leaf, and (3) CO2 diffusion does not
significantly affect the pattern of carbon fixation under our experimental conditions. The possibility that CO2 gradients alter
gene expression of Rubisco during leaf development is another
question. The molecular mechanism controlling the distribution of enzymes across the leaf remains to be elucidated.
Acknowledgements
Many thanks to Dr. Karl Y. Bil’ and Dr. Thomas Vogelmann for
helpful discussions. Supported in part by grants from Competitive Research Grants Office, U.S. Department of Agriculture (Nos. 91–
37100–6672, 93–37100–8855, and 96–35100–3167).
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(Received February 9, 2000; Accepted October 12, 2000)