Journal of Experimental Botany, Vol. 54, No. 389, pp.

1969±1975, August 2003

DOI: 10.1093/jxb/erg197

RESEARCH PAPER

Development of C4 photosynthesis in sorghum leaves

grown under free-air CO2 enrichment (FACE)

A. B. Cousins1, N. R. Adam1,2, G. W. Wall2, B. A. Kimball2, P. J. Pinter Jr2, M. J. Ottman3, S. W. Leavitt4

and A. N. Webber1,*
1
Department of Plant Biology and Center for the Study of Early Events in Photosynthesis,

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Arizona Sate University, PO Box 871601, Tempe, AZ 85287±1601, USA
2
USDA, Agricultural Research Service, US Water Conservation Laboratory, Phoenix, AZ 85040, USA
3
Department of Plant Science, University of Arizona, Tucson, AZ 85721, USA
4
Laboratory of Tree Ring Research, University of Arizona, Tucson, AZ 85721, USA

Received 12 April 2003; Accepted 22 April 2003

Abstract Key words: C4 expression, elevated CO2, leaf tissue.

The developmental pattern of C4 expression has been
well characterized in maize and other C4 plants.
However, few reports have explored the possibility
that the development of this pathway may be sensi-
tive to changes in atmospheric CO2 concentrations.
Therefore, both the structural and biochemical
development of leaf tissue in the ®fth leaf of
Sorghum bicolor plants grown at elevated CO2 have
been characterized. Ribulose-1,5-bisphosphate carb-
oxylase/oxygenase (Rubisco) and phosphoenol-
pyruvate carboxylase (PEPC) activities accumulate
rapidly as the leaf tissue differentiates and emerges
from the surrounding whorl. Rubisco was not
expressed in a cell-speci®c manner in the youngest
tissue at the base of the leaf, but did accumulate
before PEPC was detected. This suggests that the
youngest leaf tissue utilizes a C3-like pathway for car-
bon ®xation. However, this tissue was in a region of
the leaf receiving very low light and so signi®cant
rates of photosynthesis were not likely. Older leaf tis-
sue that had emerged from the surrounding whorl
into full sunlight showed the normal C4 syndrome.
Elevated CO2 had no effect on the cell-speci®c local-
ization of Rubisco or PEPC at any stage of leaf
development, and the relative ratios of Rubisco to
PEPC remained constant during leaf development.
However, in the oldest tissue at the tip of the leaf, the
total activities of Rubisco and PEPC were decreased
under elevated CO2 implying that C4 photosynthetic
tissue may acclimate to growth under elevated CO2.
Sorghum bicolor, structural and biochemical development.
Introduction
The ability of C4 plants to elevate the levels of CO2
around ribulose-1,5-bisphosphate carboxylase/oxygenase
(Rubisco) in the bundle sheath cells (BSC) limits the rate
of photorespiration and its associated loss of energy, as
well as allowing Rubisco to function at nearly its
maximum rate of catalysis (Edwards and Walker, 1983).
Based on this it is generally assumed that C4 plants will not
respond to growth under elevated atmospheric [CO2]
concentrations. However, a number of studies have shown
enhanced photosynthesis and/or growth of C4 plants under
elevated atmospheric [CO2] (Poorter et al., 1996; Wand
et al., 1999; Cousins et al., 2001; Ottman et al., 2001; Wall
et al., 2001). Typically, the increased growth response to
elevated atmospheric [CO2] of C4 plants is attributed to the
indirect CO2 effect of stomatal closure and subsequent
increased water use ef®ciency (WUE) and water potential
(Ziska et al., 1999; Wall et al., 2001). However, it has also
been shown that C4 species respond directly to increased
atmospheric [CO2] independent of any improvement in
leaf water potential (Ghannoum et al., 1997; Maroco et al.,
1999; Ziska et al., 1999; Cousins et al., 2001).
Recent studies indicate that growth of C4 plants under
elevated atmospheric [CO2] may lead to acclimation of
certain photosynthetic enzymes. For example, Rubisco,
but not phosphoenolpyruvate carboxylase (PEPC), activity
was reduced in mature maize plants grown under 3-fold

* To whom correspondence should be addressed. Fax: +1 480 965 6899. E-mail: andrew.webber@asu.edu

Journal of Experimental Botany, Vol. 54, No. 389, ã Society for Experimental Biology 2003; all rights reserved
1970 Cousins et al.
ambient levels of atmospheric [CO2] (Maroco et al., 1999).
In another growth chamber experiment, the total amount of
PEPC, but not Rubisco, was reduced in the mature leaves
of sorghum plants grown under double atmospheric [CO2]
(Watling et al., 2000). Although the developmental pattern
of C4 expression has been well characterized in maize and
other C4 leaves, the possibility that the development of the
C4 pathway may be sensitive to changes in growth
atmospheric [CO2] has not been addressed.
In graminaceous plants, leaf cells divide from a basal
meristem, which causes older cells to be displaced by
younger cells below them (Nelson and Langdale, 1989).
This type of developmental pattern creates a positional
gradient of cell ages; with younger, less differentiated cells
near the base and older, more differentiated cells toward
the tip (Martineau and Taylor, 1985). It has been well
documented in maize, a NADP-type C4 plant closely
related to sorghum, that as young leaf tissues differentiate
from the leaf primordia they switch from a C3-type protein
expression to an expression characteristic of C4 photo-
synthesis (Nelson and Dengler, 1992; Langdale et al.,
1988a, b, 1989a, b).
The development and expression of C4 photosynthetic
genes shows a temporal and spatial pattern that mirrors the
developmental and age gradients of the leaf cells (Sheen,
1999). Additionally, light signals enhance C4 gene
expression in the BSC (e.g. NADP-malate dehydrogenase
and Rubisco) as well as suppress expression of Rubisco in
the mesophyll cells. It has also been shown that C4 gene
expression is regulated by signals derived from a diverse
set of abiotic factors. For example, nitrogen starvation
induces the accumulation of transcripts encoding PEPC
and other C4 genes by both transcriptional and post-
transcriptional mechanisms (Sakakibara et al., 1998;
Sugiyama, 1998). Additionally, the amphibious sedge
E. vivipara develops Kranz anatomy and uses C4 photo-
synthesis under terrestrial conditions but switches back to
C3 photosynthesis and anatomy when submerged under
water (Ueno, 1996). Taken together, these observations
imply that the expression and regulation of C4 photo-
synthesis is a dynamic process, which may help C4 plants
cope with changing environmental conditions and carbon
metabolism requirements.
These observations also raise the intriguing possibility
that development of the C4 pathway may be sensitive to
elevated atmospheric [CO2]. It is possible that enhanced
productivity of C4 crops is in part due to the fact that
younger leaves are more C3-like and are thus more
sensitive to elevated CO2. Indeed, it has previously been
observed that C4 photosynthesis in young fully expanded
Sorghum bicolor leaves was more responsive then older
leaves to growth under elevated atmospheric [CO2] in a
free-air CO2 enrichment (FACE) experiment (Cousins
et al., 2001). If elevated atmospheric [CO2] delayed the
development of the C4 pathway, this delay may further
increase the sensitivity of C4 crops to elevated atmospheric
[CO2].
The goals of this research are (1) to characterize the
development of the C4 pathway in sorghum leaves; (2) to
assess whether or not growth under elevated atmospheric
CO2 concentrations would alter the development and cell
speci®c expression of key C3 and C4 enzymes; and (3) to
determine if the photosynthetic enzyme activity is affected
by growth under elevated atmospheric [CO2]. To test these
questions, a Sorghum bicolor crop was grown in the ®eld
under a free-air CO2 enrichment (FACE) experiment at
control (370 ppm) and FACE (570 ppm) atmospheric
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CO2 concentrations. the anatomical development, tissue
speci®c compartmentalization and activity of obligatory
enzymes involved in C4 photosynthesis grown under
FACE and control conditions have been characterized.
Materials and methods
FACE methodology
Free-air CO2 enrichment (FACE) experiments were conducted at the
University of Arizona, Maricopa Agricultural Center (MAC),
Maricopa, AZ, USA in 1998 to determine the interactive effects of
elevated CO2 and drought on Sorghum bicolor (L.) Moench (see
Ottman et al., 2001, for a comprehensive description of the sorghum
FACE experiment).
CO2 treatments
The free-air CO2 enrichment (FACE) technique was used to enrich
the air in circular plots within a sorghum ®eld in a similar way to
earlier experiments (Hendrey et al., 1993; Wechsung et al., 1995;
Hunsaker et al., 1996; Kimball et al., 1999). Brie¯y, four replicate
25 m diameter toroidal plenum rings constructed from 0.305 m
diameter pipe were placed in the ®eld shortly after planting. The
mean daytime values were 566 ml l±1 and 373 ml l±1 and the mean
night-time values were 607 ml l±1 and 433 ml l±1 for FACE and
control, respectively.
Crop culture
Certi®ed grain sorghum seed (Dekalb DK54), which had been
treated with fungicide (Captan, Chloropyrifos-methyl, Fluxofenium,
and Metalaxyl), was planted into relatively dry soil in north±south
rows spaced 0.76 m (30 inches) apart at a rate of 318 000 seeds ha±1.
Immediately after planting, erection of the FACE and control
apparatus commenced and was completed when the ®rst irrigation
was applied to all plots.
Leaf sampling
Two weeks after the initial irrigation, leaf tissue was sampled prior
to the initiation of the ligule on the ®fth leaf to emerge after the
coeloptile on 8 August 1998. Six randomly chosen plants from each
CO2 treatment were harvested. The lengths of the ®fth and sixth
leaves were measured from the base of the plant to the tip of each
leaf respectively. The coeloptile and all prior sheaths surrounding
the ®fth leaf were removed down to the seed. The ®fth leaf was
subsequently sectioned into ®ve portions as illustrated in Fig. 1 and
the area of leaf tissue for each section was determined with a CI-202
leaf area meter (CID, Inc. Vancouver, WA USA). Leaf tissue
sections from three of the six leaves were immediately stored in
liquid nitrogen in prelabelled vials for future biochemical analysis as
previously described. The remaining leaf sections were ®xed in a
 Development of C4 photosynthesis under elevated CO2 1971
Fig. 1. Leaf sectioning and harvesting. The coleoptile and all prior
sheaths surrounding the ®fth leaf were removed down to the seed. The
®fth leaf was subsequently sectioned into ®ve portions (1±5) prior to
the differentiation of the ligule.
FAA ®xative (2% formaldehyde, 50% ethanol and 5% glacial
acetic acid) overnight at room temperature (Robertson and Leech,
1995).
Immunolocalization
Leaf sections were dehydrated in water to ethanol dilution series
followed by an ethanol to tert-butanol (2-methyl-2-propanol)
dilution series. Sections were then in®ltrated in a tert-butanol to
paraplast-plus dilution series before being washed and embedded in
100% paraplast-plus (Oxford Labware, St Louis MO). Transverse
sections (10 mm) were cut using a Spencer No. 820 rotary microtome
(American Optical Company, Buffalo, NY, USA) and adhered to a
poly-lysine coated slide. Sections were then dewaxed in 100%
xylenes and rehydrated in an ethanol to water series. Subsequently,
the slides were placed in a phosphate buffer PBS (0.16 M NaCl,
8.0 mM Na2HPO4, 2.7 mM KCl, and 1.5 mM KH2PO4) for 15 min
then incubated at 4 °C overnight with the polyclonal primary rabbit
anti-Rubisco and rabbit anti-PEPC (1:2000) in 0.5% BSA in PBS.
Sections were then washed and incubated with ¯uorescein isothio-
cyanate (FITC) conjugated secondary IGg antibodies (1:3000) for 1 h
(Jackson-immuno, West Grove, PA USA). Protein compartmental-
ization was then visualized on a Leica DM RBE microscope
equipped with a Leica TCS NT confocal scanning head equipped
with the manufacturer's ®lters set-up for FITC dyes and an argon
laser (488 nm) (Leica, Heidelberg, Germany). Images were
composed and analysed using Adobe PhotoShop 5.0.
Biochemical assays
Leaf tissue was removed from liquid nitrogen and ground in an ice-
cold glass homogenizer containing 100 mM Tricine (pH 8), 10 mM
MgCl2, 1 mM EDTA, 14 mM DTT, 2% PVP, 20% glycerol, 1 mM
PMSF, and 1 mM NaFl at a ratio of 1 cm2 leaf tissue to 1 ml buffer.
Aliquots were assayed for maximum activity of Rubisco using a
100 mM Tricine (pH 8) buffer containing 10 mM MgCl2, 2 mM
DTT, 10 mM 14C-labelled sodium bicarbonate, and 0.4 mM RuBP.
For Rubisco maximum activity, the leaf homogenate was allowed to
incubate with sodium bicarbonate for 10 min before the assay.
Additional aliquots were assayed for PEPCase activity using 50 mM
Hepes-KOH, 5 mM MgCl2, 10 mM 14C-labelled sodium bicarbon-
ate, 10 U ml±1 MDH, and 0.2 mM NADH under optimal conditions
(pH 8 and 5 mM PEP). Each reaction was timed for 30 s and then
terminated with HCl/HCOOH (1 N/4 N). After centrifugation, total
soluble protein was measured using Coomassie Plus reagent (Pierce)
according to manufactures methodology.
Statistical analysis
The enzyme activity data were analysed using PROC MIXED for the
analysis of variance in SAS (SAS Institute, Cary, NC, USA). Leaf
section was considered a repeated factor for the enzyme activity data
and post hoc pairwise comparisons were made using Tukey's
probability.
Results
Leaf growth
There was no difference in the length of the ®fth or sixth
leaves due to growth under elevated atmospheric [CO2]
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(Fig. 2a, b). In addition, the total leaf area of the ®fth leaf
did not differ between treatments (Fig. 2c). The individual
sections (1±5) of leaf 5 differed in total area only slightly in
section 1 which is the youngest leaf tissue just above the
apical meristem (Fig. 2d).
Regardless of CO2 treatment, the total extractable
protein in the young developing leaves was highest in
section 1 compared to the remaining sections (Fig. 3a). In
the older leaf sections the protein levels remained
relatively constant (1 g protein m±2 of leaf tissue).
Growth CO2 conditions had no effect on the total protein
content in any of the leaf sections. Total chlorophyll per
leaf area (chl m±2) increased steadily from sections 1 to 5
as the ®fth leaf emerged from the surrounding whorl
(Fig. 3b). There was, however, no treatment effect on the
total amounts of chlorophyll in any section of leaf.
Enzyme activities
Enzyme activities expressed on a total extractable protein
basis (mmol CO2 mg±1 protein) or on a total chlorophyll
content (mmol CO2 mg±1 chlorophyll) increased steadily as
the leaf tissue differentiated and emerged from the
surrounding whorl (Fig. 4a, b). CO2 treatment, however,
had no effect on either Rubisco or PEPC activities when
assayed on a per protein or a per chlorophyll basis (not
shown). However, on a leaf area basis, Rubisco and PEPC
activity (mmol CO2 m±2 s±1) was higher depending on the
leaf section in the control plots as compared to the FACE
plots (Fig. 4c, d; F=4.11; P <0.05 and F=5.51; P <0.01,
respectively). Rubisco and PEPC activities were signi®-
cantly higher in control plants only in sections 4 and 5 as
determined by Tukey's pairwise comparison (P <0.05;
P <0.01 and P <0.01; P <0.01, respectively). The ratio of
PEPC to Rubisco total activity remained relatively
constant during leaf development and was not affected
by growth under elevated atmospheric [CO2].
Development of Kranz anatomy and enzyme
localization
Under both CO2 growth conditions the Kranz anatomy
appeared fully developed and the chloroplasts within the
bundle sheath cells were large and centrifugally arranged
in the third section prior to the emergence of the leaf tissue
1972 Cousins et al.

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Fig. 2. Leaf length and area from plants from each CO2 treatment. The length (cm) of the ®fth (a) and sixth (b) leaves measured from the base of
the plant to the tip of each leaf respectively. The total leaf area (c) and the average area for each section (d) for the ®fth leaf (cm2).

from the surrounding whorls (Fig. 5). In the younger

tissues (leaf sections 1 and 2) a de®nitive Kranz anatomy
was not apparent and the chloroplast in both the mesophyll
cells and the BSC were small and arranged primarily
adjacent to all faces of the cell walls (Fig. 5a±b, f±g).
PEPC was undetectable via immunolabelling in the earliest
leaf tissue (sections 1 and 2). However, in older leaf tissue
(sections 3±5) PEPC occurred only in the cytosol of the
mesophyll cells under both growth treatments (Fig. 5f±j).
Rubisco localized exclusively to the chloroplast of the
bundle sheath cells in sections 3±5 and appeared to be BSC
localized even in the second leaf section (Fig. 5b±e). In the
very young leaf tissue (section 1) Rubisco localized in the
chloroplast of both the mesophyll cells and BSC
(Fig. 5a). The differential tissue expression of Rubisco
was unaffected by either growth CO2 condition.

Discussion
In order to determine the effect of elevated atmospheric
[CO2] on the growth and development of the C4 pathway,
both the structural and biochemical development of leaf
tissue in the ®fth leaf of Sorghum bicolor have been
characterized. the appearance, accumulation and cell
speci®c expression of key C4 pathway enzymes during
the development of this particular leaf were measured
directly. Extremely large changes in leaf tissue anatomy
and biochemistry occurred in a very short period of
development. In the youngest leaf tissue the chloroplasts
were small and randomly arranged against the cell wall.
The total enzyme activities of Rubisco and PEPC were low
and virtually undetectable by immunolocalization (Figs 4,
Fig. 3. Protein and chlorophyll development. The total soluble protein
(g protein m±2) and total chlorophyll (mmol Chl m±2) determined on a
leaf area basis for each of the ®ve leaf sections on the ®fth leaf.
5). Similar to what would be expected for a C3 plant,
Rubisco was located in both the mesophyll and BSC
chloroplast in the youngest leaf tissue (Fig. 5a). By
contrast with mature C4 leaf tissue, PEPC immunolocal-
ization was undetectable in these same young leaf tissues
(Fig. 5f). These observations are consistent with previous
work on developing maize leaves where Rubisco appears
signi®cantly before PEPC accumulates (Nelson et al.,
1984). The BSC chloroplasts in the second leaf section
 Development of C4 photosynthesis under elevated CO2 1973
Fig. 4. Enzyme development. Maximum activity of Rubisco and the optimal rates of PEPC for each section of the ®fth leaf for control and
FACE-grown plants: (a, b) on a protein basis (mmol CO2 mg±1 protein s±1); (c, d) on a leaf area basis (mmol CO2 m±2 s±1).
were not enlarged nor centrifugally arranged, as typically
seen in mature leaf tissue. However, Rubisco immuno-
localized exclusively to the BSC chloroplast (Fig. 5b).
These observations are again consistent with earlier work
in developing maize leaves, which showed that mRNA for
the large and small subunits of Rubisco accumulate
exclusively in the BSC chloroplast before the cells are
fully differentiated (Martineau and Taylor, 1985). PEPC
expression was still undetectable in the second leaf section
(Fig. 5g). By the third leaf section the BSC chloroplasts
were enlarged, centrifugally arranged and contained large
amounts of Rubisco (Fig. 5c), and PEPC occurred only in
the cytosol of the mesophyll cells (Fig. 5h). Both Rubisco
and PEPC activity accumulated very rapidly as the leaf
tissue differentiated further and emerged from the sur-
rounding whorl (Fig. 4). The total amount of leaf protein
per unit area remained relatively constant after the ®rst leaf
section. However, total chloroplast and total enzyme
activities of Rubisco and PEPC continued to increase as
the leaf developed and emerged from the whorl.
From these observations it appears that Rubisco is not
expressed in a cell-speci®c manner in very young leaf
tissue. In addition, Rubisco accumulates before signi®cant
amounts of PEPC are detectable. Thus, the cells at very
early stages of leaf development must utilize a C3-like
pathway for carbon ®xation. Although this young leaf
tissue may in fact have C3-like characteristics, the cells are
under very low light conditions inside the whorls and
signi®cant rates of photosynthesis are unlikely. By the time
the leaf tissue emerges from the surrounding whorl and
into full sunlight, the C4 apparatus appears to be fully
expressed.
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By contrast to C3 plants, elevated CO2-induced photo-
synthetic acclimation is not commonly observed in C4
plants. In C3 plants acclimation to long-term exposure to
elevated CO2 usually causes a decrease in the photosyn-
thetic capacity associated with reduced levels of Rubisco
and other C3 cycle enzymes (Stitt, 1991; Sage, 1994;
Webber et al., 1994; Nie et al., 1995; Ghannoum et al.,
2000). In C4 plants elevated CO2 may allow alterations in
the content or activity of some C3 and C4 cycle enzymes
without losses in the rates of CO2 assimilation (Maroco
et al., 1999). In this experiment the relative ratios of
Rubisco to PEPC remained constant during leaf develop-
ment. However, the total activities of Rubisco and PEPC
decreased under elevated CO2 implying that young C4
photosynthetic plant tissue may acclimate to growth under
elevated CO2. These data are consistent with the results of
previously published work, which showed that photosyn-
thesis in the upper most fully expanded ®fth leaf was
consistently lower in response to changes in intercellular
CO2 in FACE-grown plants as compared to control plants
(Cousins et al., 2001; see Fig. 2e, f). Although these gas
exchange data were collected during the second year of the
FACE sorghum experiment, it further substantiates enzym-
atic data indicating that C4 photosynthesis may acclimate
to growth under elevated CO2.
Conclusions
Although young sorghum leaf tissues express C3-like
photosynthetic characteristics, it seems unlikely that rates
of photosynthesis are signi®cant in these cells. By the time
cells emerge from the surrounding whorl and into full
1974 Cousins et al.
Fig. 5. Immunolocalization of Rubisco (a±e) and PEPC (f±j) in
transverse sections of the ®fth leaf from FACE-grown plants. The leaf
was sectioned into ®ve portions from the base to the tip of the leaf
prior to the differentiation of the ligule.
sunlight, the mesophyll and BSC are nearly fully differ-
entiated. In the older leaf tissue, PEPC and Rubisco
activities have increased signi®cantly and appear to be
localized in a cell-speci®c manner. In this same leaf tissue
the total enzyme activities of PEPC and Rubisco are lower
under elevated CO2 compared with control plants. This
apparent acclimation response is possibly attributable
to the increased capacity for carbon ®xation and the
ef®ciency with which the C4-pump is able to concentrate
CO2 within the BSC under CO2 enrichment
Acknowledgements
Asaph Cousins acknowledges support from a NSF Graduate
Research Training Grant (DGE-9553456). The research was
supported by Interagency Agreement No DE-AI03-97ER62461
between the Department of Energy, Of®ce of Biological and
Environmental Research, Environmental Sciences Division and the
USDA, Agricultural Research Service BAK); by Grant No. 97-
35109-5065 from the USDA, Competitive Grants Program to the
University of Arizona (SWL); and by the USDA, Agricultural
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Research Service as part of the DOE/NSF/NASA/USDA/EPA Joint
Program on Terrestrial Ecology and Global Change (TECO III). The
research was also supported by Interagency Agreement No. IBN-
9652614 between the National Science Foundation and the USDA,
Agricultural Research Service (Gerard W Wall, PI) as part of the
NSF/DOE/NASA/USDA Joint Program on Terrestrial Ecology and
Global Change (TECO II); and, by the USDA, Agricultural Research
Service. This work contributes to the Global Change Terrestrial
Ecosystem (GCTE) Core Research Programme, which is part of the
International Geosphere±Biosphere Programme (IGBP). Confocal
imaging was conducted in the WM Keck BioImaging Laboratory at
Arizona State University. Antisera were provided by Dr M Salvucci
(PEPC) and Dr W Frasch (Rubisco). We also acknowledge the
helpful co-operation of Dr Robert Roth and his staff at the Maricopa
Agricultural Center. Portions of the FACE apparatus were furnished
by Brookhaven National Laboratory, and we are grateful to Mr Keith
Lewin, Dr John Nagy, and Dr George Hendrey for assisting in its
installation and consulting about its use.
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