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Antisense Reduction of NADP-Malic Enzyme in Flaveria
bidentis Reduces Flow of CO2through the C4Cycle[W][OA]
Jasper J.L. Pengelly*, Jackie Tan, 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.v.C.); National University of Singapore, Singapore 119077 (J.T.); and High Resolution Plant
Phenomics Centre, Commonwealth Scientific and Industrial Research Organization Plant Industry, Canberra
2601, Australia (R.T.F.)
An antisense construct targeting the C4isoform of NADP-malic enzyme (ME), the primary enzyme decarboxylating malate in
bundle sheath cells to supply CO2to Rubisco, was used to transform the dicot Flaveria bidentis. Transgenic plants (a-NADP-ME)
exhibited a 34% to 75% reduction in NADP-ME activity relative to the wild type with no visible growth phenotype. We
characterized the effect of reducing NADP-ME on photosynthesis by measuring in vitro photosynthetic enzyme activity, gas
exchange, and real-time carbon isotope discrimination (D). In a-NADP-ME plants with less than 40% of wild-type NADP-ME
activity, CO2assimilation rates at high intercellular CO2were significantly reduced, whereas the in vitro activities of both
phosphoenolpyruvate carboxylase and Rubisco were increased. Dmeasured concurrently with gas exchange in these plants
showed a lower Dand thus a lower calculated leakiness of CO2(the ratio of CO2leak rate from the bundle sheath to the rate of
CO2supply). Comparative measurements on antisense Rubisco small subunit F. bidentis plants showed the opposite effect of
increased Dand leakiness. We use these measurements to estimate the C4cycle rate, bundle sheath leak rate, and bundle sheath
CO2concentration. The comparison of a-NADP-ME and antisense Rubisco small subunit demonstrates that the coordination of
the C3and C4cycles that exist during environmental perturbations by light and CO2can be disrupted through transgenic
manipulations. Furthermore, our results suggest that the efficiency of the C4pathway could potentially be improved through
a reduction in C4cycle activity or increased C3cycle activity.
In the leaves of a range of plants including maize
(Zea mays), sorghum (Sorghum bicolor), sugarcane
(Saccharum officinarum), and millet (Pennisetum ameri-
canum), a biochemical pathway known as C4photo-
synthesis has evolved to concentrate CO2at the site of
Rubisco such that Rubisco can operate at close to its
maximal activity and photorespiration is reduced, en-
hancing the rate of photosynthesis in air (Hatch, 1987;
Sage, 2004). In most C4plants, CO2is fixed by phos-
phoenolpyruvate carboxylase (PEPC) in the mesophyll
cells into four-carbon acids, which diffuse to an inner
ring of bundle sheath cells, where they are decar-
boxylated and the CO2is refixed by Rubisco. Plants
using the C4photosynthetic mechanism have been sub-
divided into three primary subtypes, the NADP-malic
enzyme (ME), NAD-ME, and phosphoenolpyruvate
carboxykinase types, according to the decarboxylat-
ing enzyme used to generate CO2from C4acids in the
bundle sheath cells (Hatch, 1987). Flaveria bidentis is a
typical NADP-ME dicot in which malate and Asp
contribute equally in the transfer of CO2to bundle
sheath cells (Meister et al., 1996). Presumably, in most
C4plants, the reactions that facilitate the appropria-
tion, transformation, transport, and eventual concen-
tration of CO2in the bundle sheath cell chloroplasts
(C4cycle) are balanced with the reactions that incor-
porate CO2into usable carbon compounds for energy
(C3/Calvin cycle) such that energy is not lost or
wasted as environmental conditions fluctuate. This
process is important in maintaining the efficiency of
the CO2-concentrating mechanism and of C4photo-
synthesis overall. The nature of the controlling mech-
anisms for balance and coordination between the C3
and C4cycles is still unclear, however, and concrete
evidence for the coordinated regulation of primary
carboxylation in the mesophyll and decarboxylation of
C4acids in the bundle sheath has not been forthcom-
ing. A key approach to revealing these mechanisms
has been the use of antisense RNA in the C4dicot F.
bidentis to reduce levels of key photosynthetic enzymes,
including Rubisco (Furbank et al., 1996), NADP-malate
dehydrogenase and pyruvate phosphate dikinase
(Furbank et al., 1997), Rubisco activase (von Caemmerer
et al., 2005), carbonic anhydrase (Cousins et al., 2006),
and PEPC protein kinase (Furumoto et al., 2007). This
has proven to be a valuable method to help gain insight
into enzyme function and regulation during C4photo-
synthesis and to potentially alter the balance between
the C3and C4cycles.
In this study, we targeted the gene encoding the
chloroplastic C4isozyme of NADP-ME in F. bidentis
* Corresponding author; e-mail jasper.pengelly@anu.edu.au.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy de-
scribed in the Instructions for Authors (www.plantphysiol.org) is:
Jasper J.L. Pengelly (jasper.pengelly@anu.edu.au).
[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.112.203240
1070 Plant PhysiologyÒ,October 2012, Vol. 160, pp. 1070–1080, www.plantphysiol.org Ó2012 American Society of Plant Biologists. All Rights Reserved.
(Marshall et al., 1996) with an antisense construct
designed to reduce its activity in vivo. This isoform is
thought to catalyze the decarboxylation of L-malate to
pyruvate and CO2and of NADP to NADPH in bun-
dle sheath chloroplasts during C4photosynthesis
(Ashton, 1997; Drincovich et al., 2001), allowing the
CO2to be fixed into the C3cycle by Rubisco and py-
ruvate to return back to mesophyll cells to be recycled
into PEP. These antisense lines were generated for two
purposes. First, these plants could be used to confirm
the identity of the gene encoding the NADP-ME
isozyme involved in C4photosynthesis. Several other
functioning isoforms of NADP-ME have also been
identified within Flaveria spp.: a chloroplastic but po-
tentially nonphotosynthetic NADP-ME form and a
cytosolic NADP-ME (Marshall et al., 1996; Drincovich
et al., 1998; Lai et al., 2002). The specific role and
regulation of a C4NADP-ME isozyme in F. bidentis is
of interest in relation to the “transfer”or “genera-
tion”of a functioning C4cycle to C3plants (Sheehy
et al., 2007; Furbank et al., 2009). A greater under-
standing of the balance and interactions between this
enzyme and others in the C4and C3cycles will aid in
deciding the expression locations and levels needed
for C3plants to gain a functional CO2-concentrating
mechanism.
The second use of these antisense plants was to in-
vestigate the degree of coordination between the C4/
C3cycles in F. bidentis and the possibility of manipu-
lation to improve photosynthetic efficiency. As men-
tioned above, the mechanisms of regulation (if any) of
the C3pathway enzymes such as Rubisco in response
to the activity and CO2supply rate of the C4cycle are
unknown. It is similarly unclear how much the reac-
tions of the C3cycle affect the rates of the initial CO2-
fixing reactions (carbonic anhydrase and PEPC).
Leakiness (f), defined as the ratio of CO2leak rate
from the bundle sheath to the rate of CO2supply, re-
flects the coordination of the C4and C3cycles by de-
scribing the amount of overcycling of the C4cycle that
has to occur to support a given rate of net CO2as-
similation (Furbank et al., 1990; von Caemmerer and
Furbank, 1999). As a major C4enzyme functioning
within the bundle sheath, a reduction in NADP-ME
should affect both the C4cycle rate and the bundle
sheath CO2concentration (Cs), possibly disrupting the
enzymatic balance and coordination in F. bidentis.
Here, we have designed experiments to simultaneously
look at in vitro photosynthetic enzyme activity, gas
exchange, and real-time carbon isotope discrimination
(D), facilitating estimates of f,C
4cycle rate, and the
possible range of Cswithin transgenic a-NADP-
ME and antisense Rubisco small subunit (a-SSu) F.
bidentis plants (Furbank et al., 1996). These measure-
ments aim to show the impact of our perturbations of
the C3/C4balance, highlighting possible communi-
cation pathways between the cycles and also other
possible targets for future genetic manipulation to
improve the rate and/or efficiency of photosynthesis
in C4plants.
RESULTS
Generation and Characterization of Transformants
Agrobacterium tumefaciens transformation of F. bidentis
with an antisense construct targeting the chloro-
plastic NADP-ME yielded 12 independent primary
transformants. Of these, eight lines were successfully
regenerated through selective tissue culture and replan-
ted in soil: 1a4, 1a5, 1a6, 1a7, 1a8, 2a1, 2a2, and 4a1.
Primary transformants were screened using the in
vitro NADP-ME, Rubisco, and PEPC assays for per-
centage activity relative to a tissue culture-regenerated
control plant and standard wild-type F. bidentis.All
mutants showed NADPH-ME activity from 34% to
75% of control plants, while Rubisco and PEPC activ-
ities were slightly elevated above controls (data not
shown). All lines were selfed and grown to seed, and
from these, seed was germinated from three of the lines,
1a4, 2a1, and 4a1, and the segregating T1 seedlings
again were screened for low NADP-ME activity. Six
plants were selected from each line (18 plants in total),
encompassing a range of NADP-ME activities for use
in experiments. Six plants from a wild-type line with
normal NADP-ME activity were grown in parallel
for experimental comparison. NADP-ME activity levels
in all plants were confirmed by western blotting
(Supplemental Fig. S1). Measurements were also made
on F. bidentis plants with antisense constructs target-
ing the Rubisco small subunit (Furbank et al., 1996)
grown under identical conditions for comparison.
These plants showed wild-type PEPC activity and re-
duced Rubisco activity (20%–35% of wild-type values;
Supplemental Fig. S3).
Relationship of CO2Assimilation Rate, Rubisco and PEPC
Activity, and Leaf Nitrogen Content to NADP-ME Activity
The in vitro activity of NADP-ME, Rubisco, and
PEPC was measured in all 24 plants spectrophoto-
metrically (Fig. 1). NADP-ME activity in wild-type
plants (n= 6) varied from 57 to 80 mmol m22s21(av-
erage of 72 63), whereas variation within the mutants
(n= 18) was from 25 to 75 mmol m22s21. Gas exchange
of all greenhouse-grown plants was conducted in situ
over a period of 3 d, during which the CO2assimila-
tion rate of each plant was measured three times. The
CO2assimilation rate was significantly reduced in
mutant plants with less than 30 mmol m22s21NADP-
ME activity (Fig. 1A), yet above this level, little impact
on CO2assimilation rate was observed. Activity of
PEPC and Rubisco in relation to NADP-ME activity
showed a slight negative correlation for both enzymes
(Fig. 1, B and C), while no change in the PEPC/
Rubisco was observed (Supplemental Fig. S2A). A
slight negative correlation was also observed between
leaf nitrogen and NADP-ME activity, indicating an
increase in nitrogen per leaf area in plants with less
NADP-ME activity (Fig. 1E). The average total leaf
nitrogen for wild-type plants (n= 6) was 112.3 65.3
Plant Physiol. Vol. 160, 2012 1071
Reduction of NADP-Malic Enzyme in Flaveria bidentis
mmol m22. This differed from the average total leaf
nitrogen for NADP-ME plants (with less than 30 mmol
m22s21NADP-ME activity; n= 4) at 146.6 62.6 mmol
m22(P#0.001). No significant differences were ob-
served in either chlorophyll content or the chlorophyll a/b
ratio between mutants and wild-type plants (Supplemental
Fig. S2, B and C).
CO2Response of CO2Assimilation Rate
Figure 2 shows the response of the CO2assimilation
rate to increasing intercellular CO2partial pressure
(Ci) in four wild-type and eight a-NADP-ME mutant
plants. The mutant population included four plants
that exhibited the most significant reduction in the
initial NADP-ME activity screening relative to the
wild-type (activities between 34% and 40% of wild-
type values). The steep initial rise in CO2assimilation
rate from 10 to 60 mbar Cicharacteristic of C4plants
did not differ between the wild type and mutants
(Fig. 2B), yet assimilation rate at high Ciwas signifi-
cantly reduced in the a-NADP-ME plants, with
NADP-ME activity below 40% of the wild-type level
(Fig. 2A).
Concurrent Gas Exchange and D
Carbon isotope composition measured on dried leaf
discs (d13C, relative to the standard V-Pee Dee Belemnite)
taken from glasshouse-grown wild-type and a-NADP-
ME plants showed no significant differences (Fig. 1D).
Online measurements of Dmeasured concurrently
with gas exchange were performed on three individual
wild-type and three a-NADP-ME plants with low
(under 30 mmol CO2m22s21)NADP-MEactivityover
a range of both increasing Ci(Fig. 3) and irradiance
(Fig. 4). Stomatal conductance remained similar be-
tween the wild type and mutants (Figs. 3B and 4B), yet
as assimilation rates were lower in the a-NADP-ME
plants (Figs. 3A and 4A), the ratio of intercellular to
ambient CO2(Ci/Ca) was greater in mutants than in
wild-type plants (Figs. 3C and 4C). Online measure-
ments indicated that Dwas significantly lower in
a-NADP-ME plants (Figs. 3D and 4D), and fwas also
calculated to be less than in wild-type plants (Figs. 3E
and 4E).
When plotted against Ci/Ca(Fig. 5A), Δmeasure-
ments for wild-type and a-NADP-ME plants spread
discretely along theoretical lines estimating the rela-
tionship between Dand Ci/Causing the C4model by
Farquhar (1983): fvalues of 0.25 and 0.184 were used,
respectively, and assuming saturating amounts of
carbonic anhydrase such that the reversible conversion
of CO2and HCO3
2is at isotopic equilibrium (Cousins
et al., 2006). Identical measurements made for com-
parison on three transgenic a-SSu F. bidentis plants
(Supplemental Fig. S3) showed increased fcompared
with wild-type plants (fof approximately 0.34) and
also plotted alongside the equivalent C4model line
Figure 1. CO2assimilation rate (A), PEPC activity (B), Rubisco activity
(C), dry matter carbon isotope composition (D), and nitrogen (E) as a
function of NADP-ME activity in wild-type (open circles) and
a-NADP-ME (closed circles) F. bidentis plants. Error bars show three
technical replicates of individual plants. Linear correlations were fitted
to B, C, D, and E, yielding r2values of 0.17, 0.33, less than 0.01, and
0.46 respectively.
1072 Plant Physiol. Vol. 160, 2012
Pengelly et al.
(Fig. 5A). Figure 5B shows the different fcalculations
in wild-type, a-NADP-ME, and a-SSu plants yet com-
paratively negligible changes to each as a function of
increasing Ci.
Anatomical Measurement of Smand Sb
The surface area of mesophyll cells exposed to in-
tercellular airspace (Sm) and the bundle sheath cell sur-
face area (Sb) were measured in embedded leaf sections
of wild-type and a-NADP-ME plants (Supplemental
Fig. S4). No significant difference was found in Sb
(2.1 60.1 and 2.0 60.1 m2m22, respectively), and a
small change was found in Smfor a-NADP-ME plants
(15.3 60.6 m2m22compared with the wild type at
17.5 60.7 m2m22).
Figure 3. Concurrent measurement of CO2assimilation rate (A), sto-
matal conductance (B), Ci/Ca(C), D(D), and f(E) as a function of Ci.
Lines and error bars represent averages and SE of measurements on
three individual wild-type (open circles) and a-NADP-ME (closed
circles) F. bidentis plants, respectively. Measurements were made at
1,500 mmol quanta m22s21and a leaf temperature of 25˚C.
Figure 2. Gas exchange of wild-type (n= 4) and a-NADP-ME (n=8)F.
bidentis plants. CO2assimilation rates are given over a complete (A)
and low (B) range of Ci. Lines depicted are four wild-type plants (open
circles), four a-NADP-ME plants with NADP-ME activity from 55% to
95% of the wild type (closed triangles), and four a-NADP-ME plants
with NADP-ME activity below 40% of the wild type (closed circles).
Error bars show three technical replicates of individual plants. Mea-
surements were made in the glasshouse at 1,500 mmol quanta m22s21and
a leaf temperature of 25˚C.
Plant Physiol. Vol. 160, 2012 1073
Reduction of NADP-Malic Enzyme in Flaveria bidentis
Estimation of f,C
4Cycle Rate, Bundle Sheath Leak Rate,
and Bundle Sheath CO2
We used the concurrent measurements of fand CO2
assimilation rates to estimate the C4cycle and bundle
sheath leak rate (Fig. 6), as outlined in “Materials and
Methods.”In a-NADP-ME plants with the lowest CO2
assimilation rates, both the rate of the C4cycle and the
bundle sheath leak rate have been reduced (Fig. 6, B, C,
E, and F), whereas in the a-SSu plants, the bundle
sheath leak rate was similar to the wild-type rate ac-
companied by a reduction in the C4cycle rate.
The anatomical measurements above showed no dif-
ferences in Sb; therefore, we assumed the same bundle
sheath resistance of 333 m2s21mol21bar21to estimate
Cs.Lowa-NADP-ME plants exhibited a reduced Cs
estimation (compared with the wild-type) in response to
increasing Ci(Fig. 7A) and irradiance (Fig. 7B), whereas
Csin a-SSu plants was predicted to be more similar to
the wild-type value. Estimation of Csis linearly depen-
dent on the assumed bundle sheath resistances, and this
Figure 4. Concurrent measurement of CO2assimilation rate (A), sto-
matal conductance (B), Ci/Ca(C), D(D), and f(E) as a function of
irradiance. Lines and error bars represent averages and SE of mea-
surements on three individual wild-type (open circles) and a-NADP-ME
(closed circles) F. bidentis plants, respectively. Measurements were
made at a CO2concentration of 380 mmol mol21and a leaf temperature
of 25˚C. Figure 5. A, Dof wild-type (open circles, open squares), a-NADP-ME
(closed circles), and a-SSu (closed squares) F. bidentis plants as a
function of Ci/Ca. Lines represent the theoretical relationship between
Dand Ci/Caduring C4photosynthesis (Eq. 1) at infinite gmwith fof
0.184, 0.25, and 0.34 [(D= 4.64 + (25.9 24.64 + 29.2 3f)3(Ci/
Ca)]. Measurements were made as described in Figure 3. B, fof wild-
type (open circles, open squares), a-NADP-ME (closed circles), and
a-SSu (closed squares) plants as a function of Ci. Measurements made
as described in Figure 3.
1074 Plant Physiol. Vol. 160, 2012
Pengelly et al.
highlights the uncertainty in the absolute values of Cs,
but relative differences remain the same (Fig. 7C).
DISCUSSION
An Antisense Construct Targets the Photosynthetic C4
NADP-ME Isoform in F. bidentis, Reducing CO2
Assimilation Rate
An antisense construct targeted to the central coding
region of ChlMe1 reduced total NADP-ME leaf activity
in transformed F. bidentis plants considerably. In F.
bidentis leaves, ChlMe1 has been shown to be the
dominant expressing isoform at both the mRNA and
protein levels (Marshall et al., 1996; Drincovich et al.,
1998), so it is likely that the reduction seen in the in
vitro NADP-ME activity from whole leaf extracts is
mostly due to the reduction in ChlMe1 transcripts
rather than other isoforms. Despite this, enough se-
quence similarity exists between ChlMe1 and ChlMe2
that ChlMe2 transcripts may also have been reduced
in the antisense plants. The probable function of the
Figure 6. CO2assimilation rate (A and D), C4cycle rate (B and E), and
bundle sheath leak rate (C and F) as a function of Ci(A–C) and irradiance
(D–F) in wild-type (open circles, open squares), a-NADP-ME (closed
circles), and a-SSu (closed squares) F. bidentis plants. The C4cycle and
bundle sheath leak rate were calculated from Equations 6 and 7.
Figure 7. Estimated Csas a function of Ci(A), irradiance (B), and
bundle sheath resistance (C). Symbols represent estimated bundle
sheath CO2levels at the bundle sheath resistance assumed in this study
for fmeasurements (approximately 333 m2s21bar21mol21).
Plant Physiol. Vol. 160, 2012 1075
Reduction of NADP-Malic Enzyme in Flaveria bidentis
NADP-ME isoform encoded by ChlMe1 is in C4pho-
tosynthesis, as the major enzyme responsible for de-
carboxylation of the C4acids malate and Asp, releasing
CO2in close proximity to Rubisco for CO2assimilation
in the Calvin cycle. This role is based on studies linking
the light-regulated, leaf-specific expression of ChlMe1
in C4Flaveria species with immunolocalization of the
enzyme at high concentrations in bundle sheath chlo-
roplasts (Marshall et al., 1996, 1997; Drincovich et al.,
1998). The data shown here support this role, linking
the specific decline of NADP-ME activity (less than
30 mmol CO2m22s21)inF. bidentis leaves to a mea-
surable decrease in photosynthetic CO2assimilation
rate at saturating Ci.
This effect on CO2assimilation rate is similar to that
observed in F. bidentis plants with reduced Rubisco small
subunit levels (Furbank et al., 1996; von Caemmerer
et al., 1997). As predicted in the C4model, the CO2-
saturated portion of an A/Ci curve (response of CO2
assimilation rate to intercellular CO2)inC
4plants at
high irradiance is naturally limited by either PEP or
ribulose 1,5-bisphosphate regeneration or by maxi-
mum Rubisco activity (Berry and Farquhar, 1978; von
Caemmerer and Furbank, 1999). As Rubisco activity in
vitro was not reduced in a-NADP-ME plants (Fig. 1C),
the reduction in CO2assimilation rate at high CO2in
the a-NADP-ME plants is most likely due to reduced
C4cycle regeneration rate rather than a Rubisco limi-
tation. The similarity of maximum CO2assimilation
rates in wild-type and antisense plants exhibiting
greater than 30 mmol CO2m22s21NADP-ME activity
suggests that in wild-type leaves the C4NADP-ME is
either present in excess or is regulated to limit its ac-
tivation, as observed with other C4enzymes. In a-SSu
lines, both Rubisco content and maximal activity were
shown to correlate linearly with CO2assimilation rates
under saturating illumination (Furbank et al., 1996;
von Caemmerer et al., 1997), indicating that Rubisco
was a major limitation for maximal photosynthetic flux,
with a control coefficient of 0.5 to 0.7. In contrast, ex-
tractable activities of NADP-malate dehydrogenase
(Furbank et al., 1997) have been shown to be 10 times
that required to support CO2assimilation rates (i.e. an
effective flux control coefficient of zero). Similarly, it
seems that in F. bidentis, NADP-ME can be reduced to
approximately one-half the wild-type content (based
on in vitro activity assays) without affecting photo-
synthetic rates or growth, again indicating a control
coefficient of effectively zero. It seems unlikely that in
F. bidentis, regulation of NADP-ME is limiting photo-
synthetic flux in any major way or playing a direct
regulatory role in coordinating relative fluxes through
the C3and C4cycles in wild-type plants.
Increase in Rubisco and PEPC Activity and Leaf Nitrogen
in Low-NADP-ME Antisense Plants
The increased activity of PEPC and Rubisco mea-
sured in plants with NADPH-ME activity (and reduced
photosynthetic performance at high Ci) is intriguing
with respect to C3-C4cycle coordination. Strong posi-
tive correlations are commonly observed between both
PEPC and Rubisco activity and maximum photosyn-
thetic rates (Sage et al., 1987; von Caemmerer et al.,
1997), yet here we observed the opposite in antisense
NADP-ME plants. One possibility for these observa-
tions is that the decline in the amount of NADP-ME
has altered the amount of nitrogen available for en-
zyme production, causing general increases in Rubisco
and PEPC protein expression. However, the increase in
PEPC and Rubisco activity was matched by an increase
in leaf nitrogen in this study (Fig. 1E), with plants
exhibiting the most significant reductions in NADP-
ME activity (less than 40% of wild-type values; n=4)
showing on average 30% more leaf nitrogen than the
wild type (n= 6). In C4plants, Rubisco has been esti-
mated to account for between 5% and 15% of total leaf
nitrogen, PEPC for between 2% and 6%, and NADP-
ME for approximately 1% to 1.5% (Sage et al., 1987;
Evans and von Caemmerer, 2000; Makino et al., 2003;
Ghannoum et al., 2005). From the in vitro activity (Fig.
1B) of Rubisco (assuming that Rubisco is 16% nitrogen
by mass and has a molecular weight of 550,000 g mol21;
Ghannoum et al., 2005) and the total nitrogen per leaf
area (Fig. 1E), we estimate that 15.7% 60.9% and
14.8% 60.8% of the total leaf nitrogen present in these
plants can be attributed to Rubisco in wild-type plants
(n= 6) and low-NADP-ME mutants (less than 40% of
the wild type; n= 4), respectively. These estimates in-
dicate that the increase in total leaf nitrogen has been
mirrored by a proportional increase in Rubisco. The
substantially lower proportion of nitrogen estimated for
NADP-ME (Evans and von Caemmerer, 2000) would
indicate that its reduction alone in antisense plants
would not account directly for the increase in leaf
nitrogen observed. It is possible, though, that its re-
duction may have an indirect effect, possibly altering
common transcription factors, metabolites, or other
regulators of activity/expression for PEPC and Rubisco.
A more detailed study of photosynthetic metabolite
levels would be required to examine changes in pho-
tosynthetic flux, but even this approach would be
hampered by the complexities of the anatomical sep-
aration of photosynthetic processes within C4leaves.
Antisense Reduction of NADP-ME Alters C3-C4
Cycle Coordination
While the mechanisms and extent of the communi-
cation between the C3and C4photosynthetic cycles are
not well understood, there are logical reasons (reduc-
ing energy loss, maximizing energy gain) and experi-
mental evidence for both coordination and balance
between the two sets of reactions. Some early evidence
for C3-C4cycle interaction includes metabolite mea-
surements made in maize (Leegood and Furbank,
1984a, 1984b) and Amaranthus edulis (Leegood and von
Caemmerer, 1988) showing concurrent changes of C3
1076 Plant Physiol. Vol. 160, 2012
Pengelly et al.
and C4metabolite levels in response to differing CO2
concentrations and irradiances. Additional evidence is
found in the regulation of the C4enzyme PEPC activity
in maize by the in vivo concentrations of its substrate
PEP and the C4acid malate (Doncaster and Leegood,
1987), suggestive of a mechanism by which PEPC ac-
tivity might be balanced with that of other C3and C4
enzymes.
Here, Dhas been used to estimate fto gain a mea-
sure of coordination and balance between C3and C4
cycles. From the C4carbon isotope model, it is clear
that Dis strongly influenced by both Ci/Caand f
(Farquhar, 1983), mostly due to the compartmentali-
zation of Rubisco in the bundle sheath, reducing its
opportunity to discriminate. We measured both the
stored dry matter (d13C) and the real-time Din leaves
for festimations. Dry matter measurement of dis-
crimination did not show a difference between wild-
type and mutant populations. As discussed previously
(Pengelly et al., 2011), this likely reflects the integrated
nature of this measurement, which includes post-
photosynthetic fractionation rather than just photo-
synthetic discrimination.
Using real-time Dmeasurements, we clearly show
that plants with NADPH-ME (less than 40% of the
wild type) showed less 13C discrimination and infer
reduced frelative to wild-type plants over increasing
irradiance and Ci(Figs. 3 and 4). We used the simpli-
fied equations presented in “Materials and Methods,”
which make the assumption that Cs..Ci(for dis-
cussion, see Ubierna et al., 2011). If this were not the
case, we would have overestimated fslightly in the
a-NADP-ME plants relative to the wild type.
Measurements of fin C4plants have been shown to
be remarkably constant over a range of irradiances,
temperatures, and intercellular CO2concentrations
(Henderson et al., 1992). This has been taken as evi-
dence that some level of regulation of the C3and C4
cycles occurs. Our estimations of f(Figs. 3 and 4;
Supplemental Fig. S3) support these earlier results
showing that fwithin the plant is generally main-
tained at a relatively constant level. The exception to
this, as observed previously (Pengelly et al., 2010), is
the apparent increased fat low irradiance, although
this has recently been attributed to an overestimation
due to the incorrect assumption that a large difference
in CO2concentration is maintained between the me-
sophyll and bundle sheath at low irradiances (Ubierna
et al., 2011).
Our measurements of Din a-NADP-ME plants
contrasts with Dmeasurements (repeated in this
study) made on antisense F. bidentis plants with re-
duced levels of the Rubisco small subunit (a-SSu;
Furbank et al., 1996; von Caemmerer et al., 1997; Fig.
5A; Supplemental Fig. S3). Reduction in Rubisco
resulted in an increased frelative to the wild type. We
have used finferred from Dmeasurements from both
a-NADP-ME and a-SSu plants together with the con-
current measurements of CO2assimilation rates to
calculate estimates of the rate of the C4cycle and the
bundle sheath leak rate (Fig. 6). In the a-NADP-ME
plants with the lowest CO2assimilation rates, both the
rate of the C4cycle and the bundle sheath leak rate have
been reduced, whereas in the a-SSu plants, although f
was increased, the bundle sheath leak rate was similar
to the wild-type value, accompanied by a reduction
in the C4cycle rate despite very similar in vitro PEPC
activities (see legend to Supplemental Fig. S3). This
suggests some feedback from the C3to the C4cycle.
The comparison of the performance of a-NADP-ME
and a-SSu plants examined here suggests that the
coordination of the C3and C4cycles that is apparent
during environmental perturbations by light and CO2
can be disrupted through transgenic manipulations.
In effect, the reduction of the decarboxylating action
of NADP-ME within the bundle sheath chloroplasts of
the antisense plants created here has reduced the C4
cycle rate, and the bundle sheath leak rate has also
decreased (Fig. 6). To estimate Cs, an estimate of
bundle sheath resistance is needed. Although attempts
have been made to estimate bundle sheath resistance
by varies techniques, great uncertainty exists about the
actual magnitude of this resistance. We assumed a
value of 333 m2s21mol21bar21(which is within the
range of estimates made by Yin et al. [2011] for maize)
to estimate Cs(Fig. 7). These calculations suggest re-
duced Csfor a-NADP-ME plants, whereas a-SSu
plants had values similar to the wild type. To put these
values into context, the apparent Michaelis-Menten
constant for Rubisco carboxylation in F. bidentis at
ambient oxygen partial pressures is approximately
1,000 mbar and greater if oxygen is elevated in the
bundle sheath (Whitney et al., 2011), which suggests
that Csis not saturating for Rubisco in a-NADP-ME
plants. For wild-type and a-SSu plants, Csit is close to
saturation at four times the Michaelis-Menten constant
at high light and above ambient CO2. Figure 7C illus-
trates how the estimates of Csdepend on the bundle
sheath resistance used in the calculations.
CONCLUSION
From our observations in this study, we confirm the
function of NADP-ME (encoded by ChlMe1) in the
reduction of malate for CO2supply to Rubisco as part
of the C4cycle in F. bidentis. We have shown that under
standard light and temperature conditions in F.
bidentis, NADP-ME activity exceeds what is required
for maximum photosynthetic flux at high Ciand that
this enzyme is unlikely to be rate limiting in the C4
photosynthetic pathway. The comparison of the pho-
tosynthetic performance of a-NADP-ME and a-SSu
plants demonstrates that the coordination of the C3
and C4cycles apparent during environmental pertur-
bations by light and CO2can be disrupted through
transgenic manipulation. Furthermore, our results sug-
gest that the efficiency of the C4pathway could poten-
tiallybeimprovedthroughareductioninC
4cycle
activity or increased C3cycle activity.
Plant Physiol. Vol. 160, 2012 1077
Reduction of NADP-Malic Enzyme in Flaveria bidentis
MATERIALS AND METHODS
Plasmid Construction, Transformation, and
Mutant Regeneration
Total RNA was isolated and purified from Flaveria bidentis (von Caemmerer
et al., 1997) leaves using the TRIzol reagent (Invitrogen). Complementary
DNA was synthesized using the SuperScript III First-Strand Synthesis kit
(Invitrogen) from 1 mg of total RNA using the reverse specific primer
NADPME-R2 (59-ATGGGTGGATCCGTACTTTTCAAG-39). NADPME-R2
and NADPME-F2 (59-TTGGAGCTCTTGGTGGTGGTGTTG-39) primers were
designed based on the chloroplastic C4isoform of the NADP-ME open reading
frame ME1 from Flaveria trinervia and F. bidentis (GenBank accession nos.
X57142 and AY863144). These primers were used to amplify, via PCR, an 845-
bp fragment from the F. bidentis complementary DNA library and introduce
BamHI and SacI restriction enzyme sites at the ends of the section. This PCR
fragment was ligated into the pGEM T-Easy vector (Invitrogen), sequenced to
confirm identity, and designated ME1. The fragment was then digested out
and inserted in the antisense orientation into the BamHI/SacI-digested pBI121
binary vector under the control of a 35S cauliflower mosaic virus promoter
and named pANME2. pANME2 was subsequently transformed into Agro-
bacterium tumefaciens strain AgL1 and maintained in selective culture. F.
bidentis was transformed with pANME2 using the A. tumefaciens method
(Chitty et al., 1994) in two separate transformation experiments involving 100
to 400 explants each. Transformants were grown in three rounds of selective
tissue culture and then transferred to soil after roots had grown.
Plant Growth
Wild-type and antisense NADP-ME F. bidentis plants were grown to seed in
growth cabinets under a 12/12-h day/night cycle at 28°C/25°C, 70% hu-
midity, and an irradiance of 400 mmol quanta m22s21. Subsequently, seed was
germinated and plants grown for experiments during the summer months in a
glasshouse under natural light conditions (28°C day and 18°C night temper-
atures). Plants were grown in 30-L pots in a garden soil mix with fertilizer
(Osmocote; Scotts Australia) and watered daily. Experimental measurements
were conducted on fully expanded leaves from plants approximately 30 to
40 d after germination. F. bidentis plants containing an antisense construct to
the small subunit of Rubisco (Furbank et al., 1996) were grown under identical
conditions for the purpose of comparative measurements.
Measurements of NADP-ME, Rubisco, and PEPC Activity
in Vitro
The activity of photosynthetic enzymes was measured in vitro on leaf ex-
tracts from frozen leaf discs sampled directly after gas exchange. Rubisco and
PEPC were measured as described previously (Cousins et al., 2007; Pengelly
et al., 2010). The activity of NADP-ME was measured indirectly, as described
previously by Hatch and Mau (1977) with minor changes outlined below, by
following the formation of NADPH as malate is decarboxylated to pyruvate.
As the chloroplastic type of NADP-ME is the dominant isoform in F. bidentis
(Marshall et al., 1996; Drincovich et al., 1998), measurements on a whole leaf
extract should predominantly represent the activity of this isoform. A 0.7-cm2
frozen leaf disc was processed in ice-cold glass homogenizers with 500 mLof
extraction buffer (50 mMTricine-KOH, pH 7.8, 1 mMEDTA, 0.1% Triton-X, 10
mMdithiothreitol, and 1% polyvinylpolypyrrolidone) and 10 mL of protease
inhibitor cocktail (Sigma). Homogenate was briefly centrifuged, and the su-
pernatant was used for NADP-ME assays. For each assay, 20 mL of leaf extract
was added to 980 mL of assay buffer (50 mMTricine-KOH, pH 8.3, 5 mM
malate, 0.1 mMEDTA, and 0.5 mMNADP), and the reaction was initiated by
the addition of 10 mL of 200 mMMgCl2. Activity of NADP-ME was calculated
by monitoring the increase of NADPH A340 with a diode array spectropho-
tometer (Hewlett-Packard) after initiation of the reaction. Chlorophyll was
extracted from frozen leaf discs in a Tissuelyser II ball mill (Retsch) with 80%
acetone. Chlorophyll aand bcontents were spectrophotometrically quantified
according to Porra et al. (1989).
Gas Exchange and D
Gas-exchange measurements on T1 plants were done in situ on the youngest
fully expanded leaves using a LI- 6400 (Li-Cor). The CO2assimilation rate was first
measured at ambient CO2using a leaf temperature of 25°C at a blue-red irradiance
of 1,500 mmol quanta m22s21. Following this, CO2assimilation rate was measured
over a range of CO2partial pressures from 50 to 600 mbar. Online Dmeasurements
concurrent with gas exchange were later made on a subset of plants in a constant-
temperature growth cabinet using two LI-6400 systems coupled to a tunable diode
laser (model TGA100; Campbell Scientific) as described by Tazoe et al. (2009).
Measurements were made over a range of CO2partial pressures and irradiances.
Following gas exchange, 0.5-cm2discs were removed from tested leaves, snap
frozen in liquid nitrogen, and stored at 280°C for subsequent measurements of
enzyme activities, chlorophyll, nitrogen content, and dry matter D.
Calculation of ffrom online Dmeasurements were similar to the previous
description by Pengelly et al. (2010) except that the tertiary formulation sug-
gested by Farquhar and Cernusak (2012) was used, such that
D¼1
ð12tÞa9þ1þt
12tðai2a9ÞA
gmCaþ1þt
12tðb4
9þðb3
92sÞf2a9ÞCi2A
gm
Ca
ð1Þ
where t¼ð1þa9ÞE
2gt
ac
.Edenotes the transpiration rate and gt
ac the total conduc-
tance to CO2diffusion including boundary layer and stomatal conductance
(von Caemmerer and Farquhar, 1981). The symbol a9denotes the combined
fractionation factor through the leaf boundary layer and through stomata
a9¼abðCa2ClsÞþaðCls 2CiÞ
ðCa2CiÞð2Þ
where Cls is the CO2partial pressure at the leaf surface, ab(2.9‰)isthe
fractionation occurring through diffusion in the boundary layer, and a(4.4‰)
is the fractionation due to diffusion in air (Evans et al., 1986). The fractionation
factor associated with the dissolution of CO2and diffusion through water is
given by ai(1.8‰). Here, we assume that s=ai.
b3
9¼b32eRd
ðAþRdÞ20:5Rd
ðAþ0:5RdÞð3Þ
and
b4
9¼b42e0:5Rd
ðAþ0:5RdÞð4Þ
where b3is the fractionation by Rubisco (30‰)andb4is the combined fractionation
of the conversion of CO2to HCO3
2and PEP carboxylation (25.74‰at 25°C). The
fractionation factor eassociated with respiration was calculated from the difference
between d13CintheCO
2cylinder (22.5 to 24.5‰) used during experiments and
that in the atmosphere under growth conditions (28‰; Tazoe et al., 2009). Aand
Rddenote the CO2assimilation rate and day respiration, respectively; Rdwas as-
sumed to equal dark respiration. Equation 1 can be rearranged to calculate f:
f¼
12t
1þtD2a9
1þt2ai2b4
9A
gmCa
2b4
92a9Ci
Ca
b3
92sCi
Ca
2A
gmCað5Þ
In Equation 1, the assumption is made that Cs..Cm. If that is not the case,
this may slightly overestimate f. More complex equations are given by
Ubierna et al. (2011) and Farquhar and Cernusak (2012). We assumed a me-
sophyll conductance (gm) = 1 mol m22s21bar21for these calculations.
Calculations of C4Cycle Rates, Bundle Sheath Leak Rate,
and Bundle Sheath CO2Partial Pressure
We used the values of fcalculated from Dmeasurements together with the
measurements of CO2assimilation rate to calculate the C4cycle rate (Vp)and
the bundles sheath leak rate from the equation:
Vp ¼ ðAþRdÞ=ð1 2fÞð6Þ
in which Adenotes the CO2assimilation rate and Rdthe rate of mitochondrial
day respiration (von Caemmerer and Furbank, 1999). Bundle sheath leak rate
(L) was calculated from
L ¼ f Vpð7Þ
We used Lto estimate the Csfrom:
1078 Plant Physiol. Vol. 160, 2012
Pengelly et al.
Cs ¼ Cm þ L=gsð8Þ
assuming a bundle sheath conductance (gs) of 0.003 mol m22s21bar21for the
wild type and mutants, as there was no difference in the Sb(von Caemmerer
and Furbank, 1999). Mesophyll CO2concentration (Cm) was calculated from A
and Cias
Cm ¼ Ci 2A=gmð9Þ
with gm= 1 mol m22s21bar21.
Anatomical Measurements
Leaf sections measuring approximately 2 mm 35 mm from mature plants
were fixed and embedded in London Resin White (Electron Microscopy Sci-
ences) acrylic resin. Leaf cross-sections were cut and visualized, and the Sm
and Sbwere measured as described previously (Pengelly et al., 2010). Mea-
surements were averaged from data from 20 sections from four different wild-
type and a-NADP-ME plants.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Western blot of NADP-ME content in wild-type
and a-NADP-ME F. bidentis lines.
Supplemental Figure S2. Ratio of PEPC to Rubisco activity, chlorophyll
a/bratio, and chlorophyll a+bcontent as a function of NADP-ME activity
in F. bidentis wild-type and a-NADP-ME plants.
Supplemental Figure S3. Concurrent measurement of CO2assimilation
rate, stomatal conductance, Ci/Ca,D,andfas a function of intercellular
CO2and irradiance in three individual wild-type and a-SSu F. bidentis
plants.
Supplemental Figure S4. Representative images of the leaf cross-sections
in a-NADP-ME and wild-type plants used to measure Smand Sb.
Received July 5, 2012; accepted July 24, 2012; published July 30, 2012.
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