Functional Analysis of Corn Husk Photosynthesis[W][OA]
Jasper J.L. Pengelly*, Scott Kwasny, Soumi Bala, John R. Evans, Elena V. Voznesenskaya, Nuria K. Koteyeva,
Gerald E. Edwards, Robert T. Furbank, and Susanne von Caemmerer
Research School of Biology, Australian National University, Canberra, Australian Capital Territory 0200,
Australia (J.J.L.P., S.B., J.R.E., S.v.C.); High-Resolution Plant Phenomics Centre, Commonwealth Scientific and
Industrial Research Organization Plant Industry, Canberra, Australian Capital Territory 2601, Australia (S.K.,
R.T.F.); V.L. Komarov Botanical Institute, St. Petersburg, Russia, 197376 (E.V.V., N.K.K.); and School of
Biological Sciences, Washington State University, Pullman, Washington 99164 (G.E.E.)
The husk surrounding the ear of corn/maize (Zea mays) has widely spaced veins with a number of interveinal mesophyll (M)
cells and has been described as operating a partial C3 photosynthetic pathway, in contrast to its leaves, which use the C4
photosynthetic pathway. Here, we characterized photosynthesis in maize husk and leaf by measuring combined gas exchange
and carbon isotope discrimination, the oxygen dependence of the CO2 compensation point, and photosynthetic enzyme
activity and localization together with anatomy. The CO2 assimilation rate in the husk was less than that in the leaves and did
not saturate at high CO2, indicating CO2 diffusion limitations. However, maximal photosynthetic rates were similar between
the leaf and husk when expressed on a chlorophyll basis. The CO2 compensation points of the husk were high compared with
the leaf but did not vary with oxygen concentration. This and the low carbon isotope discrimination measured concurrently
with gas exchange in the husk and leaf suggested C4-like photosynthesis in the husk. However, both Rubisco activity and the
ratio of phosphoenolpyruvate carboxylase to Rubisco activity were reduced in the husk. Immunolocalization studies showed
that phosphoenolpyruvate carboxylase is specifically localized in the layer of M cells surrounding the bundle sheath cells, while
Rubisco and glycine decarboxylase were enriched in bundle sheath cells but also present in M cells. We conclude that maize
husk operates C4 photosynthesis dispersed around the widely spaced veins (analogous to leaves) in a diffusion-limited manner
due to low M surface area exposed to intercellular air space, with the functional role of Rubisco and glycine decarboxylase in
distant M yet to be explained.
The maize (Zea mays) leaf utilizes CO2 to make
sugars using the C4 photosynthetic pathway, in which
carbon assimilation is essentially split into two distinct
cycles within the leaf. The partitioning of these two
cycles is facilitated by two specialized photosynthetic
cell types within the leaf: the bundle sheath (BS) cells,
which are clustered around vascular bundles (VB) and
are surrounded by mesophyll (M) cells, forming a
wreath-like formation known as Kranz anatomy
(Dengler and Nelson, 1999). Initially in the C4 cycle,
CO2 is fixed by phosphoenolpyruvate carboxylase
(PEPC) in M cells, which leads to the formation of
the C4 dicarboxylic acids malate and Asp. These acids
then diffuse to BS cells via plasmodesmata, where they
are decarboxylated. The released CO2 is subsequently
fixed by Rubisco in the C3 cycle (Edwards and Walker,
1983; Hatch, 1987). This cellular partitioning of the two
cycles enables Rubisco to operate in a CO2-rich envi* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Jasper J.L. Pengelly ([email protected]).
[W]
The online version of this article contains Web-only data.
[OA]
Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.111.176495
ronment, limiting photorespiration and maximizing
photosynthetic CO2 assimilation.
C4 photosynthesis is suggested to have more than
50 independent evolutionary origins (Sage, 2004;
Muhaidat et al., 2007). Biochemical and structural diversity within different C4 pathways include variation
in the primary decarboxylating enzyme, the position
of chloroplasts in BS cells, the degree of M and BS
chloroplast granal development, and the size, number,
and structure of BS mitochondria (Edwards and
Voznesenskaya, 2011). Maize is a well-studied monocot
example of the NADP-malic enzyme (ME) biochemical
C4 subtype, characterized anatomically by the absence
of a mestome sheath, centrifugal position of organelles
in chlorenchymatous BS, grana-deficient chloroplasts
in BS cells, and chloroplasts with well-developed grana in
M cells (Gutierrez et al., 1974).
A feature considered essential in C4 photosynthesis
for maintaining efficient transport of C4 acids from M
to BS cells is high vein density or fewer cells between
VB (Sage, 2004; McKown and Dengler, 2007). Typically,
there are very few M cells between adjacent BS cells in
most C4 grasses, including maize (Hattersley and
Watson, 1975; Dengler et al., 1994). However, in maize,
the photosynthetically active husk that covers the ear
exhibits a very low vein density, similar to C3 plants,
with up to 20 M cells between VB (Langdale et al.,
1988). A study by Langdale et al. (1988) looking at in
Plant PhysiologyÒ, June 2011, Vol. 156, pp. 503–513, www.plantphysiol.org Ó 2011 American Society of Plant Biologists
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
503
Pengelly et al.
situ localization of C3 and C4 enzymes in maize husk
hypothesized that M cells distant from the VB were in
fact performing C3 photosynthesis. They identified
Rubisco mRNA transcripts present throughout husk
M cells and also observed a slight oxygen dependence
on the CO2 assimilation rate, a feature typically absent
in C4 plants. Further evidence for this theory was
reported by Yakir et al. (1991), who measured the
natural abundance of carbon isotopes (13C and 12C) in
maize husk and leaf dry matter, calculating that there
was significant C3 fixation of CO2 in the husk, contributing to the production of husk cellulose (16%).
A later study used real-time PCR analysis of photosynthetic enzyme transcripts, including Rubisco,
PEPC, NADP-ME, and pyruvate phosphate dikinase,
in maize leaves and husk, which again showed
Rubisco transcripts in husk M cells distant from the
BS clusters surrounding the VB (Hahnen et al., 2003).
Results from these studies have contributed to the
theory that vein spacing influences the pattern and
degree of photosynthetic gene expression and that
the accumulation of C4 enzymes is regulated locally
around individual veins.
At present, an international focus on engineering
new C4 crop plants from existing C3 plants to supply
future food demand has led to a considerable interest in
understanding the evolutionary path from C3 to C4
photosynthesis (Sheehy et al., 2007; Westhoff and
Gowik, 2010). This understanding is crucial to deciding
which structural and genetic features of photosynthesis
must be adapted or developed to make a C4 plant and
those that are common to both systems. Using maize as
a model of possible coexistence of C3 and C4 photosynthetic tissues, the aim of this study was to compare the
form of photosynthesis carried out in maize husk with
that in leaves using the standard physiological, anatomical, and biochemical techniques employed to
distinguish between C3 and C4 photosynthesis. Here,
we present to our knowledge the first detailed gasexchange perspective of husk photosynthesis, including measurements of compensation points and carbon
isotope discrimination, and link it to anatomical fea-
tures, photosynthetic enzyme activities, and immunolocalization of Rubisco, PEPC, and Gly decarboxylase
(GDC).
RESULTS
Anatomical Measurements of Leaf and Husk
In the C4 monocot maize, there are two types of
photosynthetic organs, the leaf and the leaf sheath. In
the ear, the husk (which is an expanded sheath) has a
leaf at the terminal end in some genotypes. The leaf
emerging from the husk is analogous to a leaf emerging from the sheath of the stem. Two varieties of corn/
maize, B73 and Sweet Corn, Kelvedon Glory, F1 (SWC),
were used to analyze the anatomy of the husk and leaf.
In both maize varieties, we use “husk” to refer to the
outer layer husk that covers the ear. We use the term
“leaf” to refer to a standard leaf originating at the stem
in B73 and for the leaf protruding from the terminal
end of the husk in SWC.
The classic Kranz-type anatomy, with characteristic
centrifugal position of chloroplasts in BS cells, is
clearly present in all maize leaves (Fig. 1, A, C, and
E), but there is a highly modified version in the husk
(Fig. 1, B, D, and F). In the husk, a clear layer of BS cells
containing a few mostly centrifugally located chloroplasts (Fig. 1, D and F) surround the veins, with large,
rounded adjacent M cells (M1) and more distant M
cells between the veins. In leaf cross-sections, the M:BS
cell ratio was 2:3, with the total adjacent M:BS area
ratio of 1.6 6 0.1. In the husk, the BS cells were similar
in size to those in the leaves but were surrounded by
large rounded M cells (Table I; Fig. 1, B and D). Thus,
from cross-sections of the husk, the M:BS cell ratio
(0.5:1) is lower than in the leaves and the M:BS area
ratio is much higher (5.6 6 0.3). Chloroplasts appear in
both BS and M cells in the husk, with an obviously
lower density than in the leaves. In the husk, there is
also a higher density of chloroplasts toward the abaxial side (the side exposed to the atmosphere) and an
Figure 1. Light microscopy images of B73 leaf (A)
and husk (B) and SWC leaf (C and E) and husk (D
and F). BS cells and the first (M1) and second (M2)
layer of M cells from BS are labeled. Centrifugal
positioning of chloroplasts in BS cells is shown at
higher magnification (arrows in E and F). Bars =
100 mm for A, C, and D, 200 mm for B, and 50 mm
for E and F.
504
Plant Physiol. Vol. 156, 2011
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Functional Analysis of Corn Husk Photosynthesis
Table I. Leaf and husk anatomical parameters of maize
Values represent means 6 SE of five or more replicate observations. Asterisks indicate a significant difference between leaf and husk in each maize
variety (P , 0.05). Sb, BS surface area/leaf area as defined previously (von Caemmerer et al., 2007); Sm, M surface area exposed to intercellular air
space/leaf area.
B73
Parameter
Leaf
Stomata per mm2 adaxial leaf surface or inner
husk surface
Stomata per mm2 abaxial leaf surface or outer
husk surface
Vein density (mm mm22)
Interveinal distance (mm)
BS cell area (mm2)
M cell area (mm2)
VB area (mm2)
Sm (m2 m22)
Sb (m2 m22)
Sm/Sb
SWC
Husk
Leaf
Husk
90 6 7
16 6 1*
88 6 4
27 6 2*
119 6 19
78 6 10
80 6 4
117 6 15
8.6 6 0.3
134 6 5
410 6 24
422 6 36
1,006 6 92
9.8 6 0.6
1.85 6 0.7
5.32 6 0.3
1.9 6 0.3*
606a
417 6 36
4,178 6 565*
6,062 6 530*
3.3 6 0.3*
0.66b
4.98b
6
6
6
6
6
6
6
6
1.6 6 0.2*
606a
413 6 32
3,507 6 242*
7,197 6 770*
5.3 6 0.1*
0.75b
7.14b
7.0
129
403
451
1,553
8.8
2.29
3.86
a
0.2
9
57
75
54
0.8
0.2
0.1
Husk vein density was measured, and interveinal distance was calculated based on the vein density-interveinal distance ratio found in leaf.
from estimated interveinal distances.
apparent higher density of chloroplasts around the
veins than in the more distant M cells (Supplemental
Fig. S1).
Anatomical measurements on the surfaces of the
leaves and husks included measurements of vein
density and stomatal number per area. Stomatal numbers were measured for both the upper (adaxial) and
lower (abaxial) leaf surfaces and the inner (morphologically adaxial) and outer (abaxial) husk surfaces.
The stomatal frequency on the abaxial side of the
husks (mean of 98 mm22 for the two varieties) is
similar to that on the abaxial and adaxial surfaces of
the leaves (mean of 94 mm22; Table I). However, the
husk had four to five times more stomata per mm2 on
the abaxial than on the adaxial epidermis in B73 and
SWC. Vein density in the husk was four to five times
lower than in the leaf in both SWC and B73 (Table I).
Anatomical measurements taken from cross-sectional
light microscope images of B73 included the average
BS and M cell areas and VB area (including BS and
vein tissue), the interveinal distance, and the surface
area of M cells exposed to intercellular air space (Sm)
and the BS surface area (Sb), both expressed per unit of
leaf area as defined previously (Pengelly et al., 2010).
Although no significant difference was observed between the husk and leaf BS cell area (Table I), 10-fold
and 6-fold increases were observed in the husk M cell
area and VB area, respectively, in comparison with the
leaf (Table I). The values of Sm and Sb were considerably less in the husk compared with the leaf (by 65%),
indicating a potential for limitations to CO2 diffusion.
b
Calculated
microscopy (Fig. 2) and transmission electron microscopy (TEM; Fig. 3). Using confocal microscopy, Rubisco was clearly labeled in BS chloroplasts of SWC
leaf (Fig. 2A), while PEPC was found primarily in M
cells (Fig. 2B). Occasional bright labeling was also observed in the epidermis due to the antibody binding
nonspecifically to epidermal cell walls (Voznesenskaya
et al., 2001). In the husk, immunolabeling of Rubisco
In Situ Immunolocalization of Photosynthetic Enzymes
Immunolocalization of Rubisco, PEPC, and GDC in
the leaf and husk was performed using the immunogold technique in combination with both confocal
Figure 2. Reflected/transmitted confocal imaging of in situ immunolocalization of Rubisco and PEPC in SWC leaf (A and B) and husk (C
and D). Immunolabeling appears as yellow dots. M1, M cells adjacent
to BS cells. Bars = 40 mm.
Plant Physiol. Vol. 156, 2011
505
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Pengelly et al.
(Fig. 3, G and H). Statistical analysis of the number of
gold particles per mm2 showed that the amounts of
labeling for Rubisco in M cells (Fig. 3C) and for PEPC
in BS (Fig. 3I) were comparable to background levels.
In the husk, TEM analysis of Rubisco labeling showed
a different level of expression with distance from the
veins (Fig. 3, D and E). Quantification of labeling (Fig.
3F) shows the highest labeling in BS chloroplasts and
less in both the adjacent M (40% of BS) and distant M
cells, with similar labeling in M2 and M3 cells (60% of
BS). The density of labeling for PEPC was highest in M
cells adjacent to BS, while the labeling in BS and
distant M2 cells was not significantly different from
background levels (Fig. 3, J–L). Quantification of mitochondrial GDC labeling in the leaf (Fig. 3, M–O)
supports selective labeling confined to the BS mitochondria and lower density of labeling in the M cells.
In the husk (Fig. 3, P–S), the highest density occurred
in BS mitochondria, with a lower density in M cell
mitochondria adjacent to BS and an increase in labeling in mitochondria of distant M cells.
Chlorophyll Content and in Vitro Activity of PEPC
and Rubisco
Chlorophyll content and the activity of Rubisco and
PEPC were measured on extracts from the leaf and
husk discs from B73 and SWC on which gas-exchange
measurements had been made. The husk in both B73
and SWC contained much less chlorophyll than the
leaf (Table II). PEPC activity expressed on an area basis
was significantly reduced in the husk, approximately
10-fold and 5-fold less than that in the leaf in B73 and
SWC, respectively (Table II). Rubisco activity (expressed on an area basis) in the husk was only half
that in the leaf. The PEPC-Rubisco ratio was much
lower in the husk compared with the leaf in both SWC
and B73 (Table II).
Gas Exchange and Carbon Isotope Discrimination
Figure 3. Immunogold labeling on the TEM level for Rubisco, PEPC, and
GDC in SWC leaf (A–C, G–I, and M–O) and husk (D–F, J–L, and P–S)
chlorenchyma, and graphs showing the density of labeling for respective
proteins in different tissues and organelles (right panels). Density of
labeling is shown for Rubisco in chloroplasts (A–F), PEPC in the cytosol
(G–L), and GDC in the mitochondria (M–S). Labeling appears as black
dots. In the graphs, the y axis represents the number of gold particles per
mm2; for each cell type, 10 to 15 cell fragments were used for counting.
The white areas near the base of the bars show the level of background.
Ch, Chloroplast; M1, M cells adjacent to BS; M2, second layer of M cells;
Mt, mitochondria. Bars = 10 mm for A and B, 5 mm for G, J, and M to S,
and 2 mm for B, E, H, and K.
(Fig. 2C) was largely confined to the few chloroplasts of
the BS cells, and while some labeling for PEPC was
apparent in the cytoplasm of M cells, there was substantial nonspecific labeling of cell walls (Fig. 2D).
Observation of leaf anatomy using TEM confirmed
strict selective labeling for Rubisco in BS chloroplasts
(Fig. 3, A and B) and for PEPC in the cytosol of M cells
The CO2 assimilation rate (A) at ambient CO2 partial
pressure (pCO2) in the husk was 15% of that in the leaf
on a leaf area basis in B73 and 30% in SWC (Fig. 4, A
and E; Table II). On a chlorophyll basis, rates of CO2
fixation under high irradiance and pCO2 did not differ
greatly (Fig. 4, B, D, and F; Table II). Dark respiration
rates were lower in the husk than in the leaf for both B73
and SWC, whereas stomatal conductance did not differ
significantly between the leaf and husk (Table II). CO2
assimilation rate in response to increasing irradiance
was substantially less in the husk than that of the leaf in
B73 on a leaf area basis and saturated at lower irradiance than CO2 assimilation rate in the leaf (Fig. 4C).
The response of CO2 assimilation rate to increasing
intercellular pCO2 (Ci) in the leaves was that typically
observed for C4 species, exhibiting a low compensation point and saturation of CO2 assimilation rates
below ambient pCO2. In contrast, the CO2 response of
the husk exhibited a high CO2 compensation point (G)
506
Plant Physiol. Vol. 156, 2011
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Functional Analysis of Corn Husk Photosynthesis
Table II. Photosynthetic and biochemical properties of maize leaf and husk
Measurements represent averages 6 SE of three or four replicate observations. Gas-exchange measurements were made at 25°C, an irradiance of
1,500 mmol quanta m22 s21, and ambient CO2 between 360 and 380 mbar. Asterisks indicate a significant difference between leaf and husk in each
maize variety (P , 0.05). A, CO2 assimilation rate.
B73
Property
A (mmol CO2 m22 s21)
A [mmol CO2 (mol chlorophyll)21 s21]
Dark respiration rate (mmol CO2 m22 s21)
Stomatal conductance (mol water m22 s21)
Chlorophyll (mmol m22)
Chlorophyll a/b ratio
PEPC activity (mmol CO2 m22 s21)
Rubisco activity (mmol CO2 m22 s21)
PEPC:Rubisco
Dry matter d13C (‰)
Online D (‰)
Ci/Ca
SWC
Leaf
Husk
27.5 6 0.85
36.7 6 3
2.4 6 0.2
0.43 6 0.03
0.76 6 0.04
4.81 6 0.17
237 6 25
44 6 3
5.9 6 0.7
214.78 6 0.09
–
–
4.1 6 0.6*
33.5 6 4.4
1.3 6 0.1*
0.40 6 0.04
0.12 6 0.01*
5.14 6 0.14
22 6 46*
19 6 2*
1 6 0.1*
215.02 6 0.18
–
–
and nonsaturating kinetics, even well above ambient
pCO2 (Fig. 4, A, B, E, and F). Although the G was
higher in the husk compared with the leaf for both B73
and SWC, it did not increase with increasing oxygen
partial pressure (pO2), as is commonly observed for C3
species (Fig. 5).
Carbon isotope discrimination (D, defined as D = Rair/
Rp 2 1, where Rp is the ratio 13C/12C in the photosynthetic product), measured in real time concurrently
with gas exchange on SWC, was similar between the
leaf and husk (Table II; Fig. 6B). However, the intercellular-to-ambient CO2 ratio (Ci/Ca) in the husk was
double that in the leaf over a range of Ci values (Fig.
6C). When plotted against Ci/Ca, D measurements for
both the leaf and husk formed discrete clusters lying
around a theoretical C4 line estimating the relationship
between D and Ci/Ca, using a leakiness (w) of 0.25 and
Leaf
27.3
53.1
2.4
0.39
0.51
4.79
260
40
6.0
213.13
3.45
0.42
6
6
6
6
6
6
6
6
6
6
6
6
Husk
2.4
4.9
0.1
0.02
0.01
0.24
22
1
0.6
0.09
0.12
0.01
7.7 6
53.7 6
1.9 6
0.34 6
0.14 6
4.37 6
52 6
21 6
2.8 6
215.08 6
4.0 6
0.85 6
0.4*
4.6
0.3
0.03
0.01*
0.13
5*
1*
0.6*
0.19*
0.49
0.01*
assuming saturating amounts of carbonic anhydrase
such that the reversible conversion of CO2 and HCO32 is
at isotopic equilibrium (Cousins et al., 2006; Fig. 7).
Carbon isotope discrimination measured from dry matter (d13C, relative to the standard V-Pee Dee Belemnite)
was slightly more negative (depleted in 13C) in the husk
compared with the leaf (Table II).
DISCUSSION
Corn Husk Displays Unusual
Gas-Exchange Characteristics
Two of the photosynthetic organ types found in the
C4 monocot maize, the standard foliar leaf (originating
either from the stem or the terminal end of the husk)
and the husk surrounding the ear, have been previFigure 4. A and B, CO2 assimilation
rate as a function of Ci in maize (B73)
leaf (black circles) and husk (white
circles). Gas-exchange measurements
were made in the glasshouse at 1,500
mmol quanta m22 s21 and a leaf temperature of 28°C. C and D, CO2 assimilation rate as a function of irradiance
in maize (B73) leaf and husk. Gasexchange measurements were made at
ambient CO2 between 360 and 380
mbar and a leaf temperature of 28°C. E
and F, CO2 assimilation rate as a function of Ci in maize (SWC) leaf and
husk. Gas-exchange measurements
were made either in the laboratory or
the glasshouse at 1,500 mmol quanta
m22 s21 and a leaf temperature of
25°C.
Plant Physiol. Vol. 156, 2011
507
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Pengelly et al.
of a plant and has frequently been used to distinguish
between C3, C3-C4 intermediate, and C4 species
(Holaday and Chollet, 1983; Ku et al., 1991; Furbank
et al., 2009). The G is consistently higher in C3 compared with C4 species at ambient pO2 and is linearly
dependent on pO2 due to increasing photorespiratory
CO2 release. In C4 species, there is no discernible
oxygen dependence of G (von Caemmerer, 2000). C3-C4
intermediate species frequently display a nonlinear
dependence of G on pO2, with low G values at low pO2
but marked increases at higher pO2 (Holaday and
Chollet, 1983; Ku et al., 1991; von Caemmerer, 2000).
Compensation points of the husk for both SWC and
B73 were high at ambient pO2 compared with that for
the leaves, and this is most readily explained by the
Figure 5. CO2 compensation point (G) as a function of oxygen partial
pressure in maize B73 leaf (black circles) and husk (white circles),
maize SWC leaf (black squares) and husk (white squares), and rice
(Oryza sativa; stars). Measurements were made in the laboratory at
1,500 mmol quanta m22 s21 and a leaf temperature of 25°C.
ously thought to manage carbon assimilation in different ways, with the suggestion that in the husk, photosynthesis may have C3-like characteristics (Langdale
et al., 1988; Yakir et al., 1991; Hahnen et al., 2003). Maize
leaf is known to be C4 with archetypal Kranz anatomy,
differentiation between cells with the complement of
C3 and C4 enzymes, and a typical C4 photosynthetic rate
at ambient CO2. Our physiological measurements confirm typical CO2 response curves for these leaves (Fig.
4). In contrast, the husk exhibits an unusual response of
CO2 assimilation rate to CO2 that does not saturate in
the measured range of pCO2. This is unlike the response
of the leaves of C4 species or responses commonly
observed for leaves of C3 species, which show marked
change in the CO2 response above ambient pCO2,
where CO2 assimilation rate becomes limited by the
rate of ribulose 1,5-bisphosphate regeneration (von
Caemmerer and Farquhar, 1981). Together with the
saturation of CO2 assimilation rate at low irradiance,
this suggests that CO2 assimilation rate in the husk
could be severely limited by CO2 diffusion from intercellular air space to the M cells. This conclusion is
supported by the substantially lower values of M
surface area exposed to intercellular air space (Sm) in
husk compared with the leaf (see below). Insufficient
carbonic anhydrase in M cytosol to facilitate the conversion of CO2 to HCO32 for PEP carboxylation could
also contribute to the diffusion-limited phenotype
(von Caemmerer et al., 2004).
Corn Husks Have High Compensation Points That Lack
Oxygen Sensitivity
The oxygen dependence of G is usually an excellent
indicator of the nature of the photosynthetic pathway
Figure 6. Concurrent measurements of CO2 assimilation rate (A) and
carbon isotope discrimination (B) in leaf (black symbols) and husk
(white symbols) of SWC as a function of Ci. The ratio of intercellular to
ambient CO2 (Ci/Ca) is also shown (C). Different symbols denote
replicate leaf and husk samples. Measurements were made at 1,500
mmol quanta m22 s21 and a leaf temperature of 28°C.
508
Plant Physiol. Vol. 156, 2011
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Functional Analysis of Corn Husk Photosynthesis
Figure 7. Carbon isotope discrimination (D) in leaf (black symbols) and
husk (white symbols) as a function of the ratio of intercellular to
ambient CO2 (Ci/Ca). The C4 line represents the theoretical relationship
between D and Ci/Ca during C4 photosynthesis with a leakiness value of
w = 0.25 [D = 4.4 + (210.1 + w 28.2)(Ci/Ca)]. The C3 line represents the
theoretical relationship between D and Ci/Ca during C3 photosynthesis
[4.4 + 25.6(Ci/Ca)]. Measurements were made as described in Figure 4.
high ratios of respiration rate to CO2 assimilation rate
(Table II). The fact that, like leaves, the husk did not
display an oxygen dependence of G suggests that the
husk primarily fixes CO2 via the C4 photosynthetic
pathway and that the high level of Rubisco evident
from immunolabeling in distant M cells plays a minor
role in husk photosynthesis. However, if C3 photosynthesis was operational in those cells at a low level (less
than 10%), it would generate only a small rise in G with
pO2, which would be difficult to detect experimentally
(von Caemmerer, 2000).
Carbon Isotope Discrimination of Maize Husk
Photosynthesis Is C4 Like
Photosynthetic carbon isotope discrimination (D) is
determined by fractionation occurring during CO2
diffusion from the atmosphere to the site of CO2
fixation and the discrimination factors associated
with the carboxylation steps. Carbon isotope discrimination is much larger in C3 compared with C4 species
because of Rubisco’s preference for 12CO2. In C4 species, both the lower discrimination of the PEP carboxylation step and the fact that Rubisco’s ability to
discriminate against 13CO2 is reduced by being compartmentalized in the gas-tight BS mean that D is much
less than in C3 species (Farquhar et al., 1989). Carbon
isotope discrimination measurements made on corn
dry matter were consistent with measurements
reported by Yakir et al. (1991) indicating a slight
difference in discrimination between the leaf and
husk. Dry matter carbon isotope discrimination values
are confounded by the fact that the source of the
carbon is not necessarily derived from local photosynthesis, and postphotosynthetic fractionation can also
affect the values (Yakir et al., 1991; Henderson et al.,
1992; Badeck et al., 2005). Therefore, we made D
measurements in real time concurrently with measurements of CO2 assimilation rates. Our measurements showed that both the leaf and husk displayed
C4-like carbon isotope discrimination similar to D
measurements made previously for C4 monocot species,
3‰ to 4‰ (Henderson et al., 1992; von Caemmerer
et al., 2007), and in contrast to typical C3 species measurements of approximately 20‰ (Evans et al., 1986;
von Caemmerer and Evans, 1991).
Equations predicting carbon isotope discrimination
during C3 or C4 photosynthesis (Farquhar, 1983;
Cousins et al., 2006; Tazoe et al., 2009) can be used to
infer various parameters from the average D and Ci/Ca
measured at ambient CO2 (Table II). In C3 plants, D
increases with an increase in the Ci/Ca ratio, while in
C4 plants, the response depends on other factors. This
includes the degree of leakiness (w, the ratio of the rate
of CO2 leakage from the BS to the rate of CO2 supply to
this compartment). For example, with increasing Ci/
Ca, the modeled response for C4 plants shows that D
values decrease with increasing Ci/Ca at the low w
values that are typical for C4 plants. In this case, the
husk (at high Ci/Ca) could have a similar D value to the
leaf (at the lower Ci/Ca) by having greater leakage in
the C4 system, by an increase in the ratio of PEPC
carboxylation to CO2 hydrations due to a lack of
carbonic anhydrase, or by the occurrence of 5% to
10% of C3 photosynthesis depending on assumptions
made for CO2 diffusion conductance in the M. The
results with isotope discrimination do not support a
significant contribution by C3 photosynthesis in the
husk, although it is not possible to come up with an
estimate of any C3 contribution due to the above
uncertainties. However, we made measurements at
several ambient CO2 concentrations that we hypothesized might bring about different ratios of C3 to C4
photosynthesis. Since we observed no discernible difference in measured D values (Fig. 6), this is consistent
with little contribution from C3 photosynthesis in the
M cells compared with C4 photosynthesis in the husk.
Maize Husk Displays Kranz-Like Anatomy at Low
Vein Density
Kranz anatomical features were present in all maize
leaves (distinctive and classical NADP-ME), while in
the husk, the anatomy was altered, having a weak
Kranz-like structure around the veins and many interveinal M cells. Maize leaves (Fig. 1, A and C) exhibited
large BS cells and adjacent, more or less radially
arranged, palisade-shaped M cells. M cells adjacent to
BS cells (M1) in the husk do not differ in shape from the
distant M cells but were smaller. The M-BS ratio in the
husk, which is 3.5 times higher than in the leaves, lies
between the numbers characteristic of C4 NADP-ME
and C3 species (Hattersley, 1984). Light microscopy of
the husk showed that chloroplasts are more concentrated in chlorenchyma cells around the VB than in the
Plant Physiol. Vol. 156, 2011
509
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Pengelly et al.
distant M cells. Moreover, the density of chloroplasts in
the layer of BS and adjacent M cells in the husk is only
about 30% to 40% of that in the leaf, indicating less
development of Kranz-type anatomy around the veins
of the husk (Supplemental Fig. S1).
Most notably, the husk has a significantly lower
vein density than the leaves, observed previously by
Langdale et al. (1988) and others, which was quantified in this study as 4.5-fold higher than the husk. This
distance was bridged by M cells roughly 10-fold
bigger than those found between VB in leaves. In C4
species having Kranz anatomy around individual
veins, a small physical distance between veins is often
thought of as a necessity for C4 function, due to the
need for C4 acids to move from M to BS cells. Analysis
of 119 grasses (Hattersley and Watson, 1975) suggested
that in C4 monocot species, no M cell is separated from
the nearest BS cell by more than one other M cell (the
maximum cell distant count). Yet, recent work has
shown that in the C4 dicot Flaveria bidentis, this variable
is surprisingly plastic (Araus et al., 1991; Sage and
McKown, 2006; Pengelly et al., 2010). The M conductance to CO2 diffusion from the intercellular air space
to the M cytosol has been suggested in C4 plants to
correlate with the M surface area exposed to intercellular air space (Sm). The fact that Sm is significantly less
in the husk compared with the leaf suggests a low
conductance to CO2 diffusion from intercellular air
space to M cells. Since the measurements of Sm included distal M cells that lack PEPC (Table II), the
actual conductance is likely to be even less, supporting
the conclusions reached from gas-exchange measurements that in the husk, photosynthesis is diffusion
limited. In C4 plants, the BS conductance to CO2
diffusion can be estimated in part by measurement
of the BS surface area per unit of leaf area (Sb; von
Caemmerer and Furbank, 2003; Pengelly et al., 2010).
Our 3-fold lower estimates of Sb in the husk compared
with the leaves suggest lower BS conductance in the
husk compared with the leaf.
Corn Husk Displays Unusual Expression Patterns of
Rubisco, PEPC, and GDC
The in vitro activity of Rubisco and PEPC on a leaf
area basis was substantially less in the husk compared
with the leaf, in accordance with its low chlorophyll
content and low photosynthetic capacity. However, the
ratio of PEPC to Rubisco activity was also substantially
less, demonstrating that the difference between the
husk and leaf is not just one of scale. Immunolocalization showed in the leaf and husk PEPC selectively
expressed in the cytosol of M cells around the VB but
not in BS or distal M cells, similar to the gene expression pattern of PEPC observed by Hahnen et al. (2003).
The labeling of Rubisco in the leaf was typical of a
standard C4 expression pattern shown previously
(Langdale et al., 1987), with Rubisco selectively expressed in BS chloroplasts. In the husk, there was not
only significant labeling for Rubisco in BS chloroplasts
but also in the M cells adjacent to the BS cells and the
second M cell layer (Table II; Figs. 2 and 3). Detection
of the protein here in distant M cells confirms previous
reports based on the localization of Rubisco transcripts
(Langdale et al., 1988; Hahnen et al., 2003). The presence of Rubisco in M cells was accompanied by the
presence of GDC, suggesting that these M cells could
perhaps operate C3 photosynthesis. However, the lack
of an oxygen dependence of G and the low carbon
isotope discrimination argue against substantial CO2
fixation occurring via the C3 photosynthetic pathway
in these cells. The expression of Rubisco and the lack of
PEPC expression in these distant husk M cells are
puzzling, but there are examples of similar phenotypes
in C4-like species such as Flaveria brownii (Bauwe, 1984;
Reed and Chollet, 1985; Cheng et al., 1988). F. brownii
had originally been considered a typical C4 species
based on anatomical, physiological, and biochemical
criteria. It exhibits low G values that lack oxygen
sensitivity and has C4-like carbon isotope values
(Holaday et al., 1984; Monson et al., 1987, 1988). However, immunolocalization studies have shown the presence of Rubisco in M as well as BS cells (Bauwe, 1984;
Reed and Chollet, 1985). Furthermore, the activity of
Rubisco and of a number of other Calvin cycle enzymes
was detected in isolated M protoplasts (Cheng et al.,
1988). It is not clear whether a complete complement of
Calvin cycle enzymes is expressed in distal M cells in
maize husk, and this deserves further investigation.
CONCLUSION
Our observations and measurements have shown
that in maize, the outer husk surrounding the ear
operates a C4-like photosynthetic pathway. Both carbon isotope analysis and the lack of oxygen sensitivity
in the compensation point show that the majority of
Rubisco actively participating in CO2 assimilation is
that compartmentalized within the chloroplasts of BS
cells, which is supported by the C4 cycle via PEPC in
the adjacent M. In C4 species having Kranz anatomy
around individual veins, high vein density is seen as a
requirement for active C4 photosynthesis. Maize husk
operates C4 photosynthesis, but as it is dispersed
around widely spaced veins, photosynthesis is diffusion limited in part due to low M surface area exposed
to intercellular air space. The role of Rubisco in distant
M cells in husk photosynthesis remains unresolved.
We suggest that transcriptome analysis in distal M
cells may help establish whether all enzymes required
for C3 photosynthesis are expressed in this cell type.
Understanding husk photosynthesis in maize, a species where the genome has been sequenced, may
provide another route to understanding the path
from C3 to C4 photosynthesis. Our study has also
highlighted the need for a multidisciplinary approach
in correctly labeling the photosynthetic pathway of a
plant, as neither physiological, biochemical, nor anatomical measurements alone are enough.
510
Plant Physiol. Vol. 156, 2011
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Functional Analysis of Corn Husk Photosynthesis
MATERIALS AND METHODS
Growth Conditions and Sampling of Maize
Photosynthetic Organs
Two varieties of corn/maize (Zea mays), B73 and Sweet Corn, Kelvedon
Glory, F1 (SWC), were grown during the summer months in a glasshouse
under natural light conditions (28°C day and 18°C night temperatures). Plants
were grown in 30-L pots in a garden soil mix with fertilizer (Osmocote; Scotts
Australia) and watered daily. Experiments and sampling were done on both
the husk and leaf tissues. Here, “husk” refers to the outer layer of thick ribbed
sheath surrounding the ear and “leaf” refers to a standard leaf originating at
the stem in B73 and a leaf protruding from the husk in SWC.
Gas-Exchange Measurements
2
Gas-exchange measurements were made with a 6-cm leaf chamber of the
LI-6400 with a red-blue light-emitting diode light source (Li-Cor) either in the
glasshouse or the laboratory.
In the glasshouse, leaves were first equilibrated for 30 min at an ambient
CO2 of 400 mmol mol21, 1,500 mmol quanta m22 s21 irradiance, and a leaf
temperature of 28°C before measurements were taken. To measure the CO2
response, CO2 concentrations were changed every 2 min, with values of 0, 30,
50, 75, 100, 150, 200, 300, 400, 500, 600, 800, and 1,200 mmol mol21 in the
reference cell of the LI-6400. Following this, leaves were acclimated at 400
mmol mol21 for 30 min, and then irradiance was reduced in steps at 2-min
intervals.
In the laboratory, measurements of CO2 response were made as described
above but at 25°C. This was followed by measurements of CO2 compensation
point at different oxygen levels. For this, air entering the LI-6400 was prepared
by using two mass-flow controllers (MKS Instruments) to mix different
concentrations of N2 and oxygen. Compensation points were calculated from
measurement of CO2 response curves at low CO2 concentrations. The average
atmospheric pressure in Canberra, Australia, is 950 mbar.
Measurements of Carbon Isotope Discrimination and
Gas Exchange
Dry matter carbon isotope discrimination measurements were made as
reported previously (Pengelly et al., 2010). For online measurements, two LI6400 systems were coupled to a tunable diode laser (model TGA100; Campbell
Scientific) for concurrent measurements of carbon isotope discrimination and
gas exchange (Bowling et al., 2003; Griffis et al., 2004; Tazoe et al., 2011). Input
gases (N2 and oxygen) were mixed using mass-flow controllers (Omega
Engineering), and measurements were first made at 2% oxygen and at
reference CO2 of 400 mmol mol21, 1,500 mmol quanta m22 s21, and leaf
temperature of 25°C for 30 min. Then, the reference CO2 was changed
stepwise to 1,000, 700, 500, 400, 300, 200, and 100 mmol mol21 and measurements were made for 30 min at each CO2 concentration.
Following gas-exchange measurements, 0.5-cm2 discs were removed from
tested leaves and husks, snap frozen in liquid nitrogen, and stored at 280°C
for later measurement of photosynthetic enzyme activities and chlorophyll
content.
Measurement of Rubisco and PEPC Activity and
Chlorophyll Content
Activities of the photosynthetic enzymes Rubisco and PEPC were measured as described previously (Cousins et al., 2007; Pengelly et al., 2010).
Chlorophyll was extracted from frozen leaf or husk discs in a Tissuelyser II
ball mill (Retsch) with 80% acetone. Chlorophyll a and b contents were
spectrophotometrically quantified (Porra et al., 1989).
Determination of Stomatal Numbers
Stomatal numbers were measured from the same or similar leaf and husk
as used for gas-exchange measurements from silicone rubber impressions
taken from both sides of the leaves or husks (von Caemmerer et al., 2004).
Stomata and epidermal cells were counted from positives made from the
impressions with nail polish, in 10 different fields of view per leaf, with a
compound microscope using a magnification of 200-fold. Three leaves per
plant were measured from three individual plants. Digital photographs of
each field were taken and cells counted with the publicly available ImageJ
software (http://rsb.info.nih.gov/ij/).
Leaf Vein Density Analysis
Vein density was measured on leaf and husk sections taken from three
individual plants. Leaves were initially cleared by immersion in a 95%
ethanol, 5% NaOH solution for 4 d and rehydrated in water for 1 h. Sections
were cut from areas used for gas-exchange measurements avoiding major
veins. Digital images were taken at 503 magnification. Vein density was
determined from each image by measuring total length of veins within a 2-cm2
quadrant using ImageJ quantification software.
Embedding of Leaf and Husk Sections for Light and
Electron Microscopy
Leaf sections measuring approximately 2 mm 3 5 mm were taken from
fully developed husk and leaves of three B73 and three SWC maize plants.
Fresh sections were fixed in buffer containing 1.25% glutaraldehyde and 2%
formaldehyde in 50 mM PIPES buffer, pH 7.2, under a vacuum for 4 h. Fixed
sections were dehydrated in an alcohol dilution series and then embedded in
either Araldite resin (Electron Microscopy Sciences) for light microscopy or
London Resin White acrylic resin (Electron Microscopy Sciences) for use in
immunolocalization.
Anatomical Measurements by Light Microscopy
Semithin sections of 0.5 mm thickness were cut from embedded husk and
leaf pieces using glass knives on a Reichert ultramicrotome (Reichert Technologies), stained with toluidine blue, and heat fixed to glass slides. Slides
were viewed using a Zeiss Axioskop light microscope (Carl Zeiss) at 4003
magnification. Images from each cross-section were analyzed using ImageJ
software for a variety of anatomical measurements, including BS cell area, M
cell area, VB area, interveinal distance, the M surface area exposed to
intercellular air space/leaf area (Sm), and the BS surface area/leaf area (Sb),
as described previously (Pengelly et al., 2010). Both leaf and husk Sm were
calculated using an approximate curvature correction factor of 1.43 (Evans
et al., 1994; von Caemmerer et al., 2007).
In Situ Immunolocalization
Antibodies used (all raised in rabbit) were anti-Spinacea oleracea Rubisco
(large subunit specific) IgG (courtesy of Dr. Bruce McFadden), commercially
available anti-maize PEPC IgG (Chemicon), and anti-Pisum sativum mitochondrial GDC IgG (courtesy of Dr. David Oliver). Preimmune serum was
used in all cases as a control.
Leaf cross-sections (0.8–1 mm thick) were dried from a drop of water onto
gelatin-coated slides and blocked for 1 h with Tris-buffered saline plus Tween
20 + bovine serum albumin (TBST + BSA; 10 mM Tris-HCl, 150 mM NaCl, 0.1%
[v/v] Tween 20, and 1% [w/v] BSA, pH 7.2). They were then incubated for 3 h
with preimmune serum diluted in TBST + BSA (1:100 dilution), anti-Rubisco
(1:50), or anti-PEPC (1:50). The slides were washed with TBST + BSA and then
treated for 1 h with 5-nm Protein A-gold (diluted 1:100 with TBST + BSA).
After washing, the sections were exposed to a silver enhancement reagent for
20 min according to the manufacturer’s directions (Amersham), stained with
0.5% (w/v) Safranin O, and imaged in a reflected/transmitted mode using a
Zeiss Confocal LSM 510 Meta Laser Scanning Microscope (Carl Zeiss). The
background labeling with preimmune serum was very low, although occasional labeling occurred in areas where the sections were wrinkled due to
trapping of antibodies/label (data not shown).
For TEM immunolabeling, thin sections (approximately 70–90 nm) on
Formvar-coated nickel grids were incubated for 1 h in TBST + BSA to block
nonspecific protein binding on the sections. They were then incubated for 3 h
with the preimmune serum diluted in TBST + BSA, anti-Rubisco (1:50), antiPEPC (1:50), or anti-GDC (1:10) antibodies. After washing with TBST + BSA,
the sections were incubated for 1 h with Protein A-gold (5 and 15 nm) diluted
1:100 with TBST + BSA. The sections were washed sequentially with TBST +
BSA, TBST, and distilled water. The sections with 5-nm gold labeling were
treated with the gold enhancement kit GoldEnchance (Nanoprobes) to obtain
higher density of labels and better contrast for observation under low
Plant Physiol. Vol. 156, 2011
511
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Pengelly et al.
magnification and then poststained with a 1:3 dilution of 0.5% (w/v)
potassium permanganate and 2% (w/v) uranyl acetate. The sections with
15-nm gold labeling were poststained with the same solution and used for
statistical analysis of label density. Images were collected using a Philips
CM200 UT transmission electron microscope (FEI Co.). The density of labeling
was determined by counting the gold particles on electron micrographs and
calculating the number of particles per unit of area (mm2) in mitochondria
(GDC), chloroplasts (Rubisco), or cytosol (PEPC) and nonspecific background
labeling. For each cell type, replicate measurements were made on parts of cell
sections (n = 10–12). Cell area was measured using ImageJ software.
Statistical Analysis
The relationship between mean values of photosynthetic, anatomical, and
biochemical data obtained throughout this study was tested using Student’s t
test (P , 0.05). Significant differences are marked with asterisks.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Volume fraction of chloroplasts per cell in husk
versus leaves in maize (SWC).
Received March 15, 2011; accepted April 20, 2011; published April 21, 2011.
LITERATURE CITED
Araus JL, Brown HR, Byrd GT, Serret MD (1991) Comparative effects of
growth irradiance on photosynthesis and leaf anatomy of Flaveria
brownii (C4-like), Flaveria linearis (C3-C4), and their F1 hybrid. Planta
183: 497–504
Badeck F-W, Tcherkez G, Nogués S, Piel C, Ghashghaie J (2005) Postphotosynthetic fractionation of stable carbon isotopes between plant
organs: a widespread phenomenon. Rapid Commun Mass Spectrom 19:
1381–1391
Bauwe H (1984) Photosynthetic enzyme activities and immunofluorescence
studies on the localization of ribulose-1,5-bisphosphate carboxylase/
oxygenase in leaves of C3, C4 and C3-C4 intermediate species of Flaveria
(Asteraceae). Biochem Physiol Pflanz 179: 253–268
Bowling DA, Sargent SD, Tanner BD, Ehleringer JR (2003) Tunable diode
laser absorption spectroscopy for stable isotope studies of ecosystem
atmosphere CO2 exchange. Agric For Meteorol 118: 1–19
Cheng SH, Moore BD, Edwards GE, Ku MSB (1988) Photosynthesis in
Flaveria brownii, a C4-like species: leaf anatomy, characteristics of CO2
exchange, compartmentation of photosynthetic enzymes, and metabolism of 14CO2. Plant Physiol 87: 867–873
Cousins AB, Badger MR, von Caemmerer S (2006) Carbonic anhydrase
and its influence on carbon isotope discrimination during C4 photosynthesis: insights from antisense RNA in Flaveria bidentis. Plant Physiol
141: 232–242
Cousins AB, Baroli I, Badger MR, Ivakov A, Lea PJ, Leegood RC, von
Caemmerer S (2007) The role of phosphoenolpyruvate carboxylase
during C4 photosynthetic isotope exchange and stomatal conductance.
Plant Physiol 145: 1006–1017
Dengler NG, Dengler RE, Donnelly PM, Hattersley PW (1994) Quantitative leaf anatomy of C3 and C4 grasses (Poaceae): bundle sheath and
mesophyll surface area relationship. Ann Bot (Lond) 73: 241–255
Dengler NG, Nelson T (1999) Leaf structure and development in C4 plants.
In RF Sage, RK Monson, eds, C4 Plant Biology. Academic Press, San
Diego, pp 133–172
Edwards GE, Voznesenskaya EV (2011) C4 photosynthesis: Kranz forms
and single-cell C4 in terrestrial plants. In A Raghavendra, RA Sage, eds,
C4 Photosynthesis and Related CO2 Concentrating Mechanisms, Vol 32.
Springer, Dordrecht, The Netherlands, pp 29–61
Edwards GE, Walker DA (1983) C3, C4: Mechanisms, and Cellular and
Environmental Regulation of Photosynthesis. Blackwell Scientific,
Oxford
Evans JR, Sharkey TD, Berry JA, Farquhar G (1986) Carbon isotope
discrimination measured concurrently with gas-exchange to investigate
CO2 diffusion in leaves of higher plants. Aust J Plant Physiol 13: 281–292
Evans JR, Von Caemmerer S, Setchell BA, Hudson GS (1994) The relationship between CO2 transfer conductance and leaf anatomy in transgenic tobacco with a reduced content of Rubisco. Aust J Plant Physiol 21:
475–495
Farquhar G (1983) On the nature of carbon isotope discrimination in C4
species. Aust J Plant Physiol 10: 205–226
Farquhar GD, Ehleringer JR, Hubick KT (1989) Carbon isotope discrimination and photosynthesis. Annu Rev Plant Physiol Plant Mol Biol 40:
503–537
Furbank RT, von Caemmerer S, Sheehy J, Edwards G (2009) C4 rice: a
challenge for plant phenomics. Funct Plant Biol 36: 845–856
Griffis TJ, Baker JM, Sargent SD, Tanner BD, Zhang J (2004) Measuring fieldscale isotopic CO2 fluxes with tunable diode laser absorption spectroscopy
and micrometeorological techniques. Agric For Meteorol 123: 15–29
Gutierrez M, Gracen VE, Edwards GE (1974) Biochemical and cytological
relationships in C4 plants. Planta 119: 279–300
Hahnen S, Joeris T, Kreuzaler F, Peterhänsel C (2003) Quantification of
photosynthetic gene expression in maize C3 and C4 tissues by real-time
PCR. Photosynth Res 75: 183–192
Hatch MD (1987) C4 photosynthesis: a unique blend of modified biochemistry, anatomy and ultrastructure. Biochim Biophys Acta 895: 81–106
Hattersley PW (1984) Characterization of C4 type leaf anatomy in grasses
(Poaceae), mesophyll -bundle sheath area ratios. Ann Bot (Lond) 53: 163–179
Hattersley PW, Watson L (1975) Anatomical parameters for predicting
photosynthetic pathways of grass leaves: ‘the maximum lateral count’
and ‘the maximum cells distant count’. Phytomorphology 25: 325–333
Henderson SA, Von Caemmerer S, Farquhar GD (1992) Short-term measurements of carbon isotope discrimination in several C4 species. Aust J
Plant Physiol 19: 263–285
Holaday AS, Chollet R (1983) Photosynthetic/photorespiratory carbon
metabolism in the C3-C4 intermediate species, Moricandia arvensis and
Panicum milioides. Plant Physiol 73: 740–745
Holaday AS, Lee KW, Chollet R (1984) C3-C4 intermediate species in the
genus Flaveria: leaf anatomy, ultrastructure, and the effect of O2 on the
CO2 compensation concentration. Planta 160: 25–32
Ku MS, Wu J, Dai Z, Scott RA, Chu C, Edwards GE (1991) Photosynthetic
and photorespiratory characteristics of Flaveria species. Plant Physiol 96:
518–528
Langdale JA, Metzler MC, Nelson T (1987) The argentia mutation delays
normal development of photosynthetic cell-types in Zea mays. Dev Biol
122: 243–255
Langdale JA, Zelitch I, Miller E, Nelson T (1988) Cell position and light
influence C4 versus C3 patterns of photosynthetic gene expression in
maize. EMBO J 7: 3643–3651
McKown AD, Dengler NG (2007) Key innovations in the evolution of
Kranz anatomy and C4 vein pattern in Flaveria (Asteraceae). Am J Bot 94:
382–399
Monson RK, Schuster WS, Ku MSB (1987) Photosynthesis in Flaveria
brownii A.M. Powell: a C4-like C3-C4 intermediate. Plant Physiol 85:
1063–1067
Monson RK, Teeri JA, Ku MSB, Gurevitch J, Mets LJ, Dudley S (1988)
Carbon-isotope discrimination by leaves of Flaveria species exhibiting
different amounts of C3- and C4-cycle co-function. Planta 174: 145–151
Muhaidat RM, Sage RA, Dengler NG (2007) Diversity of Kranz anatomy
and biochemistry in C4 eudicots. Am J Bot 94: 362–381
Pengelly JJL, Sirault XR, Tazoe Y, Evans JR, Furbank RT, von Caemmerer
S (2010) Growth of the C4 dicot Flaveria bidentis: photosynthetic acclimation to low light through shifts in leaf anatomy and biochemistry. J
Exp Bot 61: 4109–4122
Porra RJ, Thompson WA, Kriedmann PE (1989) Determination of accurate
extinction coefficients and simultaneous equations for assaying chlorophyll a and b extracted with four different solvents: verification of the
concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim Biophys Acta 975: 384–394
Reed JE, Chollet R (1985) Immunofluorescent localization of phosphoenolpyruvate carboxylase and ribulose 1,5-bisphosphate carboxylase/
oxygenase proteins in leaves of C3 , C4 and C 3 -C 4 intermediate Flaveria
species. Planta 165: 439–445
Sage RA (2004) The evolution of C4 photosynthesis. New Phytol 161: 341–370
Sage RF, McKown AD (2006) Is C4 photosynthesis less phenotypically
plastic than C3 photosynthesis? J Exp Bot 57: 303–317
Sheehy JE, Mitchell PL, Hardy B, editors (2007) Charting New Pathways
to C4 Rice. International Rice Research Institute, Los Banos, Philippines
512
Plant Physiol. Vol. 156, 2011
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Functional Analysis of Corn Husk Photosynthesis
Tazoe Y, von Caemmerer S, Badger MR, Evans JR (2009) Light and CO2 do
not affect the mesophyll conductance to CO2 diffusion in wheat leaves. J
Exp Bot 60: 2291–2301
Tazoe Y, von Caemmerer S, Estavillo GM, Evans JR (2011) Using tunable
diode laser spectroscopy to measure carbon isotope discrimination and
mesophyll conductance to CO2 diffusion dynamically at different CO2
concentrations. Plant Cell Environ 34: 580–591
von Caemmerer S (2000) Biochemical Models of Leaf Photosynthesis.
CSIRO Publishing, Canberra, Australia
von Caemmerer S, Evans JR (1991) Determination of the average partial
pressure of CO2 in chloroplast from leaves of several C3 plants. Aust J
Plant Physiol 18: 287–305
von Caemmerer S, Evans JR, Cousins AB, Badger MR, Furbank RT (2007)
C4 photosynthesis and CO2 diffusion. In JE Sheehy, PL Mitchell, B
Hardy, eds, Charting New Pathways to C4 Rice. International Rice
Research Institute, Los Banos, Philippines, pp 95–115
von Caemmerer S, Farquhar GD (1981) Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. Planta 153: 376–387
von Caemmerer S, Furbank RT (2003) The C4 pathway: an efficient CO2
pump. Photosynth Res 77: 191–207
von Caemmerer S, Lawson T, Oxborough K, Baker NR, Andrews TJ,
Raines CA (2004) Stomatal conductance does not correlate with photosynthetic capacity in transgenic tobacco with reduced amounts of
Rubisco. J Exp Bot 55: 1157–1166
Voznesenskaya EV, Franceschi VR, Kiirats O, Freitag H, Edwards GE
(2001) Kranz anatomy is not essential for terrestrial C4 plant photosynthesis. Nature 414: 543–546
Westhoff P, Gowik U (2010) Evolution of C4 photosynthesis: looking for the
master switch. Plant Physiol 154: 598–601
Yakir D, Osmond B, Giles L (1991) Autotrophy in maize husk leaves:
evaluation using natural abundance of stable isotopes. Plant Physiol 97:
1196–1198
Plant Physiol. Vol. 156, 2011
513
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.