Plant, Cell and Environment (2011) 34, 65–75
doi: 10.1111/j.1365-3040.2010.02226.x
Drought limitation of photosynthesis differs between C3
and C4 grass species in a comparative experiment
pce_2226
65..75
S. H. TAYLOR1, B. S. RIPLEY2, F. I. WOODWARD1 & C. P. OSBORNE1
1
Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK and 2Botany Department, Rhodes
University, Grahamstown 6139, Republic of South Africa
ABSTRACT
Phylogenetic analyses show that C4 grasses typically occupy
drier habitats than their C3 relatives, but recent experiments
comparing the physiology of closely related C3 and C4
species have shown that advantages of C4 photosynthesis
can be lost under drought. We tested the generality of
these paradoxical findings in grass species representing the
known evolutionary diversity of C4 NADP-me and C3 photosynthetic types. Our experiment investigated the effects
of drought on leaf photosynthesis, water potential, nitrogen,
chlorophyll content and mortality. C4 grasses in control
treatments were characterized by higher CO2 assimilation
rates and water potential, but lower stomatal conductance
and nitrogen content. Under drought, stomatal conductance declined more dramatically in C3 than C4 species,
and photosynthetic water-use and nitrogen-use efficiency
advantages held by C4 species under control conditions
were each diminished by 40%. Leaf mortality was
slightly higher in C4 than C3 grasses, but leaf condition
under drought otherwise showed no dependence on
photosynthetic-type. This phylogenetically controlled
experiment suggested that a drought-induced reduction
in the photosynthetic performance advantages of C4
NADP-me relative to C3 grasses is a general phenomenon.
Key-words: C3 photosynthesis; C4 photosynthesis; drought;
grasses; metabolic limitation; NADP-me; nitrogen; Poaceae;
stomatal conductance; water potential.
INTRODUCTION
Grasses utilizing the C4 photosynthetic pathway have
evolved repeatedly over the last ~32 Ma (Christin et al.
2007, 2008; Vicentini et al. 2008; Bouchenak-Khelladi et al.
2009). These species play a major ecological role at the
global scale, dominating warm climate grassland ecosystems (Still et al. 2003), and are important as agricultural
crops (e.g. millets, maize, sugarcane), forage (Brown 1999)
and biofuel feedstocks (Heaton, Dohleman & Long 2008).
The potential importance of contrasts between C3 and C4
photosynthesis in determining ecological patterns, at scales
Correspondence: C. P. Osborne. Fax: +44 114 222 0002; e-mail:
[email protected]
© 2010 Blackwell Publishing Ltd
up to and including the continental and global, has long
been recognized and debated (Hatch, Osmond & Slatyer
1971; Osmond, Winter & Ziegler 1982; Pearcy & Ehleringer
1984).
Controlling for phylogeny is crucial when comparing the
ecophysiological traits of C3 and C4 grasses (Edwards, Still
& Donoghue 2007; Edwards & Still 2008; Taylor et al. 2010).
Molecular phylogenies place most commonly studied C3
grasses from temperate climates into a clade known as BEP
(three subfamilies, Bambusoideae, Ehrhartoideae and Pooideae, exclusively C3), whilst C4 photosynthesis has arisen
only in its largely tropical sister clade known as PACMAD
(six subfamilies, Panicoideae, Aristidoideae, Chloridoideae,
Micrairoideae, Arundinoideae and Danthonioideae, including both C3 and C4 photosynthetic types). These two clades
diverged more than 50 Ma ago (Christin et al. 2008; Vicentini et al. 2008; Bouchenak-Khelladi et al. 2009), and recent
work has shown that evolutionary divergences both
between and within these clades may explain ecophysiological differences that were previously attributed to differences between C3 and C4 photosynthetic types and
subtypes (Taub 2000; Edwards et al. 2007; Cabido et al. 2008;
Edwards & Still 2008; Edwards & Smith 2010).
Comparative analyses based on large molecular phylogenies indicate that C4 grasses tend to occupy a drier niche
than their C3 relatives, and that the evolution of C4 photosynthesis facilitated ecological transitions into drier, open
habitats (Edwards & Still 2008; Osborne & Freckleton 2009;
Edwards & Smith 2010). These results are consistent with
the experimental observation that, in mesic high irradiance
conditions, C4 grasses typically achieve higher rates of net
leaf photosynthesis (A) than their closest C3 relatives, whilst
their stomatal conductance to water vapour (gs) is markedly
lower (Taylor et al. 2010), i.e. C4 grasses exhibit higher
intrinsic water-use efficiency (A/gs = iWUE). The ratio of A
to leaf nitrogen content per unit area, photosynthetic
nitrogen-use efficiency (A/Narea = PNUE), also tends to be
greater for C4 grasses. However, recent experiments have
suggested that such physiological advantages of C4 photosynthesis may not persist under drought. In two independent comparisons of C3 and C4 grasses from the subfamily
Panicoideae, drought eliminated differences in A that were
observed between C3 and C4 species under well-watered
control conditions (Ibrahim et al. 2008; Ripley, Frole &
Gilbert 2010). In pot-based studies, under well-watered
65
66 S. H. Taylor et al.
conditions, gs in C3 species was initially higher, but under
drought declined to values similar to or smaller than those
observed in their C4 relatives (Ripley et al. 2007, 2010).
Whilst stomatal limitation explained a large proportion of
the total decline in A in C3 species, metabolic limitation was
proposed to be the dominant effect on A in their C4 relatives. Although these experiments considered only a limited
range of C4 species, their results undermine the hypothesis
that the iWUE advantage of C4 grasses under mesic conditions, which is associated with high A and PNUE (Long
1999), persists under drought.
We tested the generality of these findings with a comparative experimental approach, using a grass phylogeny to
sample species representing multiple comparisons between
independent C4 lineages and C3 sister groups. We concentrated on comparisons between C4 NADP-me and C3
photosynthetic types, which contribute the majority of phylogenetic diversity in photosynthetic types within the
Poaceae (Christin et al. 2009). Our design did not control for
habitat or life history, but relied on random sampling of
species to represent the ecological diversity of the photosynthetic types. We imposed a drought treatment that consisted of a controlled decline in soil water content and
addressed the key question: does drought have differential
effects on A and gs in C3 and C4 species? In addition, to
investigate the extent to which plants differing in photosynthetic type were tolerant of drought, we measured the
response to drought of: (1) photosynthetic resource use
efficiency (iWUE and PNUE); and (2) leaf senescence and
leaf water potential.
MATERIALS AND METHODS
Species selection
To provide a phylogenetically controlled sample of the
diversity of C4 NADP-me types and their C3 relatives within
the grasses, species were selected from genera placed on
two recently published phylogenies (Christin et al. 2008;
Vicentini et al. 2008). Genera known to be polyphyletic or
paraphyletic (e.g. Setaria), were avoided. A species list was
drawn up based on existing collections of plants and seeds,
and availability from several suppliers. We categorized the
list according to C4 origins and C3 sister clades (Christin
et al. 2008), and chose species at random from within each
category. Inferred evolutionary relationships at genus level
for the species used are shown in Fig. 1.
Plant material and growth conditions
Species and sources for plant material are listed in Table 1.
Most species were grown from seed; however, Cortaderia
selloana and Stipa gigantea were obtained as adult plants
and Phragmites australis was propagated from rhizome segments. Seeds were surface-sterilized before germination on
unlit shelves in a growth room (MTPS 120, Conviron, Winnipeg, Manitoba, Canada; conditions 30 °C, 0800–1500 h,
declining to 25 °C 1800–0500 h, relative humidity 80%).
Stipa
Aristida
Cortaderia
Phragmites
Eriachne
Chasmanthium
Digitaria
Cenchrus
Sacciolepis
Echinochloa
Oplismenus
10 Ma
0.5
Ischaemum
Paspalum
Figure 1. Inferred evolutionary relationships, at genus level, for
species included in the experiment, after Christin et al. (2008).
Species with C4 photosynthesis are indicated by black labels. We
assume that the genera are monophyletic. Branch lengths are
proportional to time. All genera except Stipa belong to the
PACMAD clade.
Germinated seedlings were transferred into a 3:1 mix of
John Innes no. 3 compost : perlite, under moderate illumination [mean ⫾ SEM photosynthetic photon flux density
(PPFD), 761 ⫾ 25 mmol mol-1 0600–1700 h, ramping from
darkness, 1800–0500 h] but otherwise similar conditions.
Rhizome segments were treated similarly. When large
enough, seedlings were transferred to a separate walk-in
growth room (BDW 160, Conviron), and transplanted into
4.5 L pots with an 185 mm top diameter (LBS Group,
Colne, Lancashire, UK), filled with a 1:1 mix of John Innes
no. 3 compost : washed silica sand (Chelford 52, WBB Minerals, Sandbach, Cheshire, UK).
Maximum PPFD at mid-canopy height was
1014 ⫾ 17 mmol m-2 s-1 (mean ⫾ SEM). Each day, lighting
was ramped from darkness 1800–0500 h, to maximum light
0700–1600 h. Temperature was 25 °C in darkness, ramping
to 30 °C between 0800 and 1500 h. Relative humidity was
80%. The two species (C. selloana and S. gigantea) obtained
as adult plants were transferred directly into the growing
media and final growth conditions 2 weeks before measurements began. Plants were arranged into five blocks, each on
a 1.5 ¥ 0.75 m tray, including a pair of plants for each
species. To minimize shading, species within each block
were separated according to stature, but within the two
groups (tall/short), species order was randomized.
The plants were fertilized four times in the course of the
experiment; after week one, during weeks four and five, and
after week six (except C. latifolium, which received an additional feed during week two). A commercially available
plant food (Bayer Lawnfood and Tonic, N : P : K 38:5:5) was
diluted into the appropriate volume of water being added
to each treatment.
Soil-drying treatment
We created a controlled decline in gravimetric soil water
content (w, g H2O g dry matter-1). The saturated water
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 65–75
Drought limitation in C3 and C4 grass species 67
Table 1. Species and sources
Species
Photosynthetic type
Life-history
Sourcea
Accession ID
Stipa gigantea
Aristida adoensis
Cortaderia selloana
Phragmites australis
Eriachne aristidea
Chasmanthium latifolium
Digitaria ciliaris
Cenchrus ciliaris
Sacciolepis vilvoides
Echinochloa frumentacea
Oplismenus hirtellus
Ischaemum afrum
Paspalum malacophyllum
C3
C4
C3
C3
C4
C3
C4
C4
C3
C4
C3
C4
C4
Perennial
Perennial
Perennial
Perennial
Annual/perennial
Perennial
Annual
Perennial
Annual
Annual
Perennial
Perennial
Perennial
A
B
A
C
D
E
D
B
B
E
F
B
D
–
PI 385318
–
29212
AusTRCF 322433
40528
KP 5148
PI 147685 701SD
PI 338609 01SD
436583
–
PI 364924 02SD
CPI 27690
a
Sources: A) Ferndale Garden Centre, Dronfield, Derbyshire, UK; B) USDA National Plant Germplasm System (NPGS), BARC-West
Beltsville, MD, USA; C) Millenium Seed Bank Project, Royal Botanical Gardens, Kew, UK; D) Australian Plant Genetic Resource Information Service (AusPGRIS), Queensland, Australia; E) B & T World Seeds, Paguignan, France; F) Seed from a field collection made near
Grahamstown, South Africa.
content of our compost mix was first determined. Compost
mix in five 4.5 L pots was watered to drip point and left to
stand in a tray of water overnight before being allowed to
drain freely for 24 h. Samples were collected using a
15-mm-diameter soil core and w determined based on measurements of fresh mass and dry mass (after drying to constant weight at 100 °C). The mean saturated water content
was used to calculate an expected dry mass for the contents
of each pot in the experiment based on their saturated
weights (plant included) at the start of the experiment,
assuming that plants would contribute only a small fraction
of the total mass. Expected dry masses were used to predict
target masses for each pot during the drying phase of the
experiment (weeks two to four), to obtain a decrease in w of
approximately 2.4 g g-1 d-1 (comparable with rates of soil
drying previously observed in natural mixed C3/C4 grassland in South Africa; Ripley et al. 2010).
During the drying phase, pots were weighed each
morning and watered with an appropriate volume of water
to obtain the target mass. This was continued until substantial wilting and discolouration of leaves was observed in the
most susceptible species, at the end of week four. During
week five, w was maintained at the level reached at the end
of week four, and in week six, the pots were rewatered to
drip point for a 2 week recovery period. Pots in the control
treatment were saturated by watering to drip point every
day, as were pots in the drought treatment during weeks one
(prior to drying), six and seven (recovery from drying).
To estimate soil water potential (Ysoil) from w, a moisture
characteristic curve was produced for the compost mix. The
dry mass of subsamples of soil was determined after drying
at 100 °C. Samples were placed into pre-weighed polystyrene vials (60 mL, 125AP, Sterilin, Caerphilly, UK), and
deionized water added to obtain a range of values
for w, augmented by repeated measurements as samples
dried or were re-wetted. To determine Ysoil, soil psychrometers (PST-55-15 thermocouple psychrometer/hygrometer,
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 65–75
Psypro, Wescor Inc., Logan, UT, USA) sealed into the vials
and calibrated against standard solutions of 0.1 to 1 molal
NaCl, were used with a datalogger (Psypro, Wescor Inc.).
The non-linear relationship between Ysoil and w was estimated as Ysoil = aw-b (Campbell & Norman 1998) where
a = -0.399, b = -0.642.
Leaf physiology
Leaf gas exchange was measured for every plant in each
week during the experiment, using a portable open system
(LI-6400; LI-COR, Inc., Lincoln, NE, USA), equipped with
a CO2 mixer, 30 mm ¥ 20 mm chamber and red–blue lightemitting diode light source (LI-6400-02B). Spot measurements were made between 1000 and 1530 h, aiming to
determine the maximum A at the operating gs of youngest
mature leaves on randomly selected tillers. Within each
block, a full set of species were measured each morning,
followed by the second plant for each species pair in the
afternoon (28 plants in total per day). Plants from both
drought and control treatments were measured in the
morning and afternoon sessions, and for each species, the
first treatment to be measured (drought or control) was
rotated between each set of measurements.
For fine-leaved species, to increase the leaf area and size
of fluxes measured, gas exchange was measured on a pair of
leaves from adjacent tillers. A PPFD of 2500 mmol m-2 s-1
was used. Preliminary measurements established that this
PPFD was required to saturate photosynthesis in the C4
species and caused no decline in A in shade tolerant species.
Plants were acclimated to high light conditions by raising
them towards the growth chamber lights for 10–15 min
prior to measurement. Leaf chamber conditions were
matched to those of the growth environment; temperature
was controlled at 30 °C and chamber humidity was maintained between 60 and 85%, to allow minimal adjustment
when switching rapidly between leaves with different rates
68 S. H. Taylor et al.
of water efflux (different leaf areas and drought/control
plants). This compromise was demanded by the large
numbers of samples required to satisfy the experimental
design and resulted in values for mean chamber vapour
pressure deficit (VPD) ranging between 0.79 and 1.55 kPa.
Leaf temperature was estimated using an energy balance
calculation.
Additional measurements were taken alongside measurements of gas exchange in week five, when w was at its lowest.
Chlorophyll content was estimated using a SPAD meter
(Konica Minolta Sensing Inc., Osaka, Japan). Meter readings
were calibrated against total chlorophyll content in the
leaves of eight grass species following the method of Porra,
Thompson & Kriedemann (1989), and were related to SPAD
a
readings using the conversion μ mol Chl a + b m −2 = 10 x
(Markwell, Osterman & Mitchell 1995); where x = SPAD
reading and a is a fitted coefficient.
A 30 mm segment from the centre of the leaf was collected following gas exchange. The area of segments was
calculated based on their width at either end and masses
were determined after drying at 80 °C for at least 48 h,
allowing the calculation of specific leaf area (SLA, cm2 g-1).
Dried leaf segments were stored in an airtight container
over silica gel prior to analysis for nitrogen concentration.
Leaves were ground using a ball mill (TissueLyser,
Quiagen, Crawley, West Sussex, UK), and nitrogen concentrations were determined using a stable isotope ratio mass
spectrometer (PDZ Europa 20-20, PDZ Europa Ltd,
Cheshire, UK).
During the same period in which gas exchange measurements were made, operating leaf water potential (Yop) was
measured for a youngest fully emerged leaf from each
plant, using a Scholander pressure-chamber (model 1000,
PMS Instrument Company, Albany, OR, USA) and following the methods described by Turner (1981).
Statistics
Where summaries are presented for C3 and C4 groups,
values are weighted means (Gelman & Hill 2007), i.e. the
average of species means, weighted by the proportion of the
total number of individuals used to estimate them. Standard
errors for weighted means are the square root of the
summed variances of the species means, weighted by
sample size.
Statistical analyses were carried out using the R Language and Environment (R Development Core Team
2005). A generalized least squares approach (gls function,
‘nlme’ package in R (Pinheiro et al. 2009), was used to estimate the responses of species to treatments within each
week of the experiment, allowing models to be fitted to
untransformed data whilst accounting for differences in
variance between treatment groupings. Count data for the
number of leaves on a stem were loge transformed for
analysis.
Models estimated the effects of species, soil drying, and
species ¥ soil drying interactions and were simplified by
step-wise deletion according to the Aikake’s information
criterion (AIC) criterion. Linear contrasts (Crawley 2007)
were formulated using tools in the R package ‘gmodels’
(Warnes et al. 2009) and used to address the a priori hypothesis that the C3 and C4 groups would differ in their average
response to treatments. To address differences between C3
and C4 species in the control treatment, when the overall
model indicated a significant effect of species, contrasts
were calculated between predicted species means. To
address the effect of photosynthetic type on species
responses to drought, in models for which the interaction
term (species ¥ soil drying) was statistically significant, contrasts were calculated comparing the average size of interaction coefficients between photosynthetic types.
Scaling of leaf nitrogen with SLA was estimated by using
loge transformed data and the package ‘smatr’ in R (Warton
& Ormerod 2007), to apply a standardized major axis
regression with adjustment for measurement error. Differences in slopes were tested using a likelihood ratio test and
differences in elevation using a Wald statistic.
RESULTS
Progress of the soil-drying treatment
During the drying phase of the drought treatment, which
began in week two, the average daily decline in mass prior
to watering was 0.02 ⫾ 0.05 kg (mean ⫾ SEM) for C4
species, and 0.03 ⫾ 0.04 kg for C3 species (i.e. less than 1%
of average saturated pot mass at the start of the experiment). Between-species variation in the rate of soil drying
at the diurnal scale was therefore high, and the difference
contrast between C3 and C4 groups was not significant.
Overall, the drying phase resulted in a decrease in w from a
mean value of greater than 0.6 g g-1 in week one, to
0.19 g g-1 in week five (Fig. 2a). Rewatering from the start of
week six returned w to values above 0.6 g g-1. Estimated
Ysoil ranged between -0.42 and -1.77 MPa, with a mean
value in week five of -1.22 MPa (Fig. 2b).
Response of leaf gas exchange to drought
In the control treatment, C4 grasses showed a 41% decline
in A over the course of the experiment (Fig. 3a). A 32%
decrease in A was also observed for the C3 grasses. Despite
the greater decline in A for C4 grasses compared with C3
grasses, a significant difference between the two groups was
supported at every time point and A remained higher for
the C4 group throughout (Fig. 3a). The response of A to dry
soil, modelled as the difference between control and
drought treatments, differed significantly between species
in week five, but the species responses did not differ
between the photosynthetic types (Fig. 3a).
In the control treatment, and under high soil water availability in the dry-down treatment, the gs of C3 grasses was
three times that for C4 species (Fig. 3b). There was a significant difference in control values between the two groups at
every time point (Fig. 3b). During weeks four, five and six,
the response of gs to the drought treatment differed
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 65–75
Drought limitation in C3 and C4 grass species 69
w (g g−1)
0.8
(a)
0.6
0.4
0.2
0.0
Ysoil (MPa)
(b)
−0.5
−1.0
−1.5
1
2
3
4
5
6
increases in iWUE in C3 species that resulted in a significant
interaction between photosynthetic type and drought
(t102 = 2.9, P = 0.004) and a 40% reduction in the C4 iWUE
advantage. Both C3 and C4 species tended to show reduced
PNUE under drought (Fig. 4). The extent of this response
differed between species (drought ¥ species F12,87 = 5.1,
P < 0.001) and the average size of reductions in PNUE was
greater for C4 species, resulting in a 41% decline in the C4
PNUE advantage (t87 = 2.0, P = 0.045).
Rewatering produced a general recovery of gas exchange
(Figs 3 & 4), with no significant difference in A or ci
between plants in the drought and control treatments by
week seven (Fig. 3a,c). The effect of the drying treatment remained significant for gs (Fig. 3b) in week seven;
however, the size of this effect was small, mean gs being
7
week
Figure 2. Response of soil water availability to experimental
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 65–75
A (mmol m−2s−1)
(a)
40
30
20
10
NS
0
gs (mol m−2 s−1)
between species, and the average size of the responses differed significantly between photosynthetic types (Fig. 3b).
In week five, when the drought was at its most extreme, the
decline in gs relative to controls was 76% for C3 species, but
only 37% for C4 species. Although the mean for C4 species
remained 20% lower than that for C3 species in the drought
treatment, the difference was 69% in the control treatment.
The lack of a difference in the response of A, combined
with the evidence for a larger response of gs to drought in
the C3 species, suggested that the effect of drought on the
relationship between A and CO2 supply differed between
C3 and C4 species. This was supported by the response of ci
(Fig. 3c), which, if the relationship between photosynthesis
and CO2 supply was unaffected under drought, should have
declined as well. Overall, a decline in ci was observed for
the C3 species, but not for the C4 species under drought
(Fig. 3c). For each week in which the responses of gs to
drought differed between species, this was also the case for
estimates of ci (Fig. 3c). However, the average response of ci
to the drought treatment only showed a significant difference between photosynthetic types during the drying phase
of the experiment. Differences in the response of ci between
photosynthetic types were lost immediately upon rewatering (Fig. 3c), although the size of responses to drought continued to differ between species.
An examination of the response of both iWUE and
PNUE to drought during week five (Fig. 4) indicated
that the extent of variation in iWUE both within- and
between-species increased under drought, and that there
was greater overlap in values of iWUE between C3 and C4
species (Fig. 4; drought ¥ species F12,102 = 7.4, P < 0.001).
This increased overlap occurred against the background of
constant atmospheric VPD and was driven primarily by
50
(b)
1.0
0.8
0.6
0.4
0.2
*** *** *
0.0
c i (mmol mol−1)
drought treatment. Mean ⫾ SEM for 65 pots (SEM < 5% of
mean in all cases). (a) Gravimetric soil water content (w, g H2O g
dry matter-1). (b) Soil water potential (Ysoil, MPa) calculated
using a psychrometer soil suction curve. Control 䊊; soil drying
treatment .
(c)
300
200
100
* **
0
1
2
3
4 5
week
NS
6
7
Figure 3. Response of leaf gas exchange to declining soil water
availability (weeks one to five), and rewatering (weeks six and
seven), summarized according to photosynthetic type. Weighted
mean ⫾ SEM, n = 3 to 5, for seven C4 and six C3 species; C4
control 䊊, C4 drought , C3 control 䊉, C3 drought 䉱. (a) net
CO2 assimilation (A, mmol m-2 s-1). (b) stomatal conductance (gs,
mol m-2 s-1). (c) leaf internal CO2 concentration (ci, mmol mol-1).
In the control treatments, between-species effects were significant
(P < 0.001) for all comparisons, as were a priori contrasts
between photosynthetic types (P < 0.001, except for A in week
five, P = 0.041). Comparisons for which the species ¥ drought
interaction was significant (P < 0.01) are indicated according to
the statistical significance of between-photosynthetic type
contrasts: ***P < 0.001, *0.01 < P < 0.05, NSP > 0.05. When there
was no significant species ¥ drought interaction, the soil-drying
treatment had significant effects (P < 0.05) on; A, weeks
three–six; gs, weeks three–six; ci, weeks two–six.
100
50
0
0
200
400
600
800
PNUE (mmol CO2 mol N
−1
s−1)
Figure 4. Response of iWUE and PNUE to drought in 13
grasses differing in photosynthetic type. Measurements taken
during week five of drought treatment. Mean ⫾ SEM, n = 3 to 5,
except C3 S. vilvoides in the drought treatment, n = 2 for PNUE;
C4 control 䊊, C4 drought , C3 control 䊉, C3 drought . In
response to the drought treatment, iWUE showed a significant
(P < 0.05) decline in one of six C3 species, and increased
significantly in four of six C3 and one of seven C4 species; PNUE
showed a significant decline in five of six C3 and four of seven C4
species.
0.03 mol m-2 s-1 greater in the drought treatment than in the
control treatment (F1,112 = 5.36, P = 0.022).
Response of leaf water potential to drought
The difference in mean Yop between drought and control
was similar to or greater than the change in Ysoil for eleven
of the 13 species (Fig. 5a). Two C4 species (Digitaria ciliaris
and Echinochloa frumentacea) were excluded from the
analysis of Y, because values for Yop in the drought treatment were substantially less negative than estimated Ysoil.
The ‘false’ end points detected for these two species may be
explained as a result of water contained in non-vascular
mesophyll and parenchyma cells being squeezed out of the
leaf by the chamber gasket as pressure was applied. There
was a significant difference in Yop between drought and
control (P < 0.02) for all species except Sacciolepis vilvoides
(t84 = -1.53, P = 0.131). There was also a significant difference in Yop between C3 and C4 species in the control treatment (t84 = -4.40, P < 0.001), the mean for C3 species being
23% more negative than for the C4 species. However,
although the size of the significant responses to drought
differed between species (F10,84 = 6.4 P < 0.001), they did not
differ significantly between the photosynthetic types
(t84 = 1.94, P = 0.055). Two species showed an unusually
large response of Yop to drought (>1.5 MPa, Fig. 5a),
which was greater in Ischaemum afrum (C4) than in S.
gigantea (C3).
The average size of gradients in water potential between
leaf and soil, DY (Yop - Ysoil), showed a significant difference between C3 and C4 species in the control treatment
(t84 = -3.878, P < 0.001), the average gradient for C3 species
being 0.28 MPa larger than for C4 species. The effect of the
drought treatment on DY was strongly dependent upon
Leaf and whole plant condition under drought
Comparisons between species during week five, at the most
extreme phase of the drying treatment, showed that foliar
nitrogen content (Narea, Table 2) differed between species in
the control treatment (F12,91 = 32.0, P < 0.001) and that
mean values for C4 species were significantly lower than for
C3 species (t91 = 29.37, P < 0.001). Although pooled means
for Narea increased under drought, and the effect of drought
was significant (F1,91 = 21.945, P < 0.001), it was underlain
by differences in the size of response between species
(drought ¥ species F12,91 = 3.25, P < 0.001) that were independent of photosynthetic type (t91 = 1.10, P = 0.273).
The pattern in leaf chlorophyll content (Table 2) matched
that seen for Narea. In the control treatment, differences
between species (F12,102 = 61.7, P < 0.001) were consistent
0
(a)
−1
−2
−3
−4
(b)
0
−1
−2
−3
C. ciliaris
P. malaco.
E. aristidea
I. afrum
A. adoensis
O. hirtellus
C. latifolium
S. vilvoides
C. richardii
S. gigantea
P. australis
150
species (F10,84 = 5.89 P < 0.001), including a significant effect
of photosynthetic type (t84 = 2.08, P = 0.041), that eliminated the difference in average DY between C3 and C4
species. The importance of this effect is unclear, because a
significant difference in DY between treatments (P < 0.009,
Fig. 5b) was detected for only 3 of the 11 species, I. afrum
(C4), S. gigantea (C3) and Chasmanthium latifolium (C3).
Thus, over the range of Ysoil generated, most species maintained a relatively constant DY in both control and drought
treatments; however, a small number of highly responsive
species drove an increase in between-species differences in
DY under drought (Fig. 5b).
Yop (MPa)
200
ΔY (MPa)
iWUE (mmol CO2 mol H2O−1)
70 S. H. Taylor et al.
Figure 5. Response of leaf water potential to drought in eleven
grasses differing in photosynthetic type. Measurements taken
during week five of drought treatment; (a) Operating leaf water
potential (Yop); (b) Leaf-soil gradient in water potential (DY).
Mean ⫾ SEM, n = 4 to 5; C4 control 䊊, C4 drought , C3 control
䊉, C3 drought 䉱. Reference lines in (a) are mean Ysoil, for
control (dashed line) and drought (dotted line). Within each
photosynthetic type, species order follows mean Yop under
control conditions.
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 65–75
52 ⫾ 3
79 ⫾ 11
62 ⫾ 6
52 ⫾ 17
68 ⫾ 9
69 ⫾ 9
39 ⫾ 3
61 ⫾ 9
58 ⫾ 6
45 ⫾ 6
56 ⫾ 8
79 ⫾ 10
43 ⫾ 9
47 ⫾ 12
37 ⫾ 7
52 ⫾ 8
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 65–75
201 ⫾ 8
461 ⫾ 50
599 ⫾ 54
673 ⫾ 112
399 ⫾ 80
218 ⫾ 17
341 ⫾ 46
415 ⫾ 62
759 ⫾ 61
585 ⫾ 60
287 ⫾ 79
733 ⫾ 51
664 ⫾ 78
377 ⫾ 115
564 ⫾ 77
206 ⫾ 20
791 ⫾ 78
855 ⫾ 56
339 ⫾ 90
471 ⫾ 122
368 ⫾ 102
438 ⫾ 144
496 ⫾ 95
490 ⫾ 112
646 ⫾ 48
452 ⫾ 59
997 ⫾ 77
750 ⫾ 50
286 ⫾ 78
604 ⫾ 74
320 ⫾ 125
476 ⫾ 107
509 ⫾ 86
672 ⫾ 86
540 ⫾ 162
389 ⫾ 159
358 ⫾ 95
468 ⫾ 123
505 ⫾ 87
558 ⫾ 44
383 ⫾ 38
472 ⫾ 117
404 ⫾ 99
433 ⫾ 102
461 ⫾ 86
Control
Mean ⫾ SE
420 ⫾ 114
537 ⫾ 95
553 ⫾ 91
620 ⫾ 62
609 ⫾ 97
296 ⫾ 83
592 ⫾ 142
513 ⫾ 100
532 ⫾ 85
633 ⫾ 62
515 ⫾ 41
515 ⫾ 125
529 ⫾ 82
413 ⫾ 126
523 ⫾ 92
Drought
Mean ⫾ SE
Stem (Chl a + b)
(mmol Chl m-2)a
3.7 ⫾ 2.8/5.0
5.4 ⫾ 3.3/8.9
5.1 ⫾ 4.7/5.6
2.2 ⫾ 1.4/3.6
4.2 ⫾ 2.7/6.5
3.9 ⫾ 2.5/6.0
9.2 ⫾ 7.6/11.2
4.5 ⫾ 3.2/6.3
6.7 ⫾ 5.2/8.6
6.7 ⫾ 6.1/7.3
3.4 ⫾ 2.3/5.1
6.3 ⫾ 3.8/10.3
5.4 ⫾ 4.9/6.0
3.4 ⫾ 2.1/5.5
5.1 ⫾ 3.7/6.9
Control
Mean ⫾ SE
3.5 ⫾ 2.9/4.3
7.6 ⫾ 5.1/11.2
4.2 ⫾ 3.3/5.3
2.1 ⫾ 1.4/3.3
4.5 ⫾ 3.3/6.1
5.1 ⫾ 3.7/6.9
7.8 ⫾ 6.0/10.0
4.6 ⫾ 3.4/6.2
7.6 ⫾ 6.5/8.9
5.2 ⫾ 4.3/6.3
4.4 ⫾ 3.3/5.9
6.1 ⫾ 3.8/9.7
5.2 ⫾ 4.6/5.9
3.7 ⫾ 2.5/5.6
5.2 ⫾ 4.0/6.9
Drought
Mean ⫾ SE
Number of leaves
on stemb
3.6 ⫾ 2.8/4.7
4.9 ⫾ 3.1/7.6
4.3 ⫾ 3.9/4.8
2.0 ⫾ 1.3/2.9
3.8 ⫾ 2.6/5.7
3.1 ⫾ 2.2/4.4
7.8 ⫾ 7.0/8.8
3.9 ⫾ 2.9/5.3
4.4 ⫾ 3.2/6.2
5.9 ⫾ 5.5/6.4
2.2 ⫾ 1.5/3.1
4.7 ⫾ 3.0/7.4
4.4 ⫾ 3.8/5.1
2.6 ⫾ 1.8/3.7
3.8 ⫾ 2.8/5.1
Control
Mean ⫾ SE
3.2 ⫾ 2.8/3.8
5.1 ⫾ 3.5/7.5
2.9 ⫾ 2.1/3.9
2.1 ⫾ 1.4/3.3
3.1 ⫾ 2.7/3.5
2.9 ⫾ 2.2/3.9
3.5 ⫾ 2.2/5.5
3.2 ⫾ 2.4/4.3
6.1 ⫾ 5.2/7.1
4.6 ⫾ 4.0/5.3
2.8 ⫾ 2.4/3.3
4.2 ⫾ 3.1/5.8
4.0 ⫾ 3.5/4.6
1.9 ⫾ 1.4/2.8
3.7 ⫾ 3.0/4.6
Drought
Mean ⫾ SE
Number of leaves
livingb
Sample size per species in each treatment was four to five, except where shown in parentheses. Pooled sample sizes for weighted averages were 27 to 30 for C3, and 32 to 35 for C4 NADP-me
species.
a
Mean chlorophyll content calculated across all leaves on a stem, with number of living leaves included in model as a covariate.
b
Back-transformed from loge(x).
A. adoensis
C. ciliaris
D. ciliaris
E. frumentacea
E. aristidea
I. afrum
P. malacophyllum
Weighted average
C4 NADP-me
77 ⫾ 9
144 ⫾ 13
66 ⫾ 4 (3)
196 ⫾ 20
79 ⫾ 22 (2)
119 ⫾ 9
122 ⫾ 14
106 ⫾ 10
97 ⫾ 8
46 ⫾ 2 (3)
166 ⫾ 14
77 ⫾ 10
86 ⫾ 4
100 ⫾ 9
C. latifolium
C. richardii
O. hirtellus
P. australis
S. vilvoides
S. gigantea
Weighted average
C3
Drought
Mean ⫾ SE
Control
Mean ⫾ SE
Drought
Mean ⫾ SE
Control
Mean ⫾ SE
Species
Subtype
Leaf (Chl a + b)
(mmol Chl m-2)
Narea (mmol N m-2)
Table 2. Response of leaf condition to drought treatment, for grass species of C3 and C4 (NADP-me) photosynthetic types
Drought limitation in C3 and C4 grass species 71
72 S. H. Taylor et al.
(Table 2) and the total number of leaves per stem (logetransformed) was tested as a covariate with species and
drought effects. The latter were both dropped from the
minimal model, and mean chlorophyll content in the living
leaves showed a significant positive relationship with the
number of leaves on a stem (F1,122 = 5.3, P = 0.023).
Scaling of leaf N with SLA
The scaling of Nmass against SLA (Fig. 6) showed significant
differences between the C3 and C4 types, but drought did
not affect the relationship in either photosynthetic type.The
scaling slope was >1 for the C4 species (slope, +/-95% c.i.
control 1.54, 1.15/2.06; drought 1.56, 1.15/2.12), but was not
significantly different from 1 for the C3 species (control
1.02, 0.77/1.35; drought 0.95, 0.68/1.32). Differences in Nmass
for the C3 species were, therefore, in proportion with differences in SLA, but for C4 species, differences in Nmass were
proportionally greater than inter-specific differences in
SLA.
Although mean SLA values overlapped between C3 and
C4 species in both drought and control treatments, they
were always greater than 150 cm2 g-1 for C4, whereas some
C3 species showed values between 70 and 100 cm2 g-1. In
contrast, the range of Nmass values showed strong overlap
(Fig. 6). The range of values for Nmass was therefore conserved, whilst for SLA, the range differed between the two
photosynthetic types. If the scaling relationship for C4 Nmass/
SLA were to be projected over the entire 6-fold range of
species means for SLA (i.e. across both C3 and C4 species),
it would result in a 14-fold range of Nmass C4 species, rather
than the observed four-fold difference and mean Nmass
N mass (mmol g−1)
with significantly lower chlorophyll contents in the leaves of
C4 species than in C3 species (t102 = 3.94, P < 0.001, Table 2).
As for Narea, although the drought treatment resulted in a
significant increase in average values for chlorophyll
content (F1,102 = 6.2, P = 0.015), this was underlain by
changes in chlorophyll content that depended upon species
(drought ¥ species F12,102 = 3.58, P < 0.001), but were independent of photosynthetic type (t102 = -0.75, P = 0.449).
The average total number of leaves (live and dead) on a
tiller differed between species within the control treatment
(loge-transformed data, F12,111 = 3.3, P < 0.001). Contrasts
indicated no significant difference in mean values between
the C3 and C4 groups (t111 = 0.14, P = 0.256) and, based on
the AIC criterion, the effect of drought was dropped
from the model. Thus, at the most acute phase of the
drought treatment, there was no indication that drought
had induced a reduction in the rate of leaf accumulation,
and tillers possessed similar numbers of leaves regardless of
treatment.
The number of living leaves per tiller showed a significant
relationship with the total number of leaves (both axes
loge-transformed, F1,109 = 1497.9, P < 0.001) that was consistent across species and treatments (interaction effects were
dropped from the minimal model). On average, a doubling
of the total number of leaves on a stem resulted in only a
77% increase in the number of living leaves, showing that as
stem length increased, further stem extension was associated with leaf mortality and suggesting that for species with
more leaves per stem, leaf turnover was higher. The interaction between drought and species was dropped from the
minimal model, but between species differences in intercepts suggested that, on average, C3 species (t109 = -0.11,
P = 0.035) had 10% fewer living leaves per stem than C4
species. Drought also produced a small but significant
reduction in the number of living leaves per stem (average
7% decline; F1,72 = 6.1, P = 0.016), i.e. leaf mortality
increased. On the original scale, these differences in logscaled relationships corresponded to an increasing difference in the number of living leaves with stem length, both
between C3 and C4, and between drought and control plants
within each photosynthetic type. This effect was, therefore,
consistent with a greater decline in the number of living
leaves per stem in C4 species, which had a greater number of
living leaves in the control condition, than in C3 species: at
the average stem length of ~6 leaves, the difference in the
number of living leaves between C3 and C4 species was
reduced by ~7% in the drought treatment relative to the
control treatment. However, the size of these effects was
small when compared with between species differences
(C3/C4 difference ~26% of between-species range in control
treatment, 3.0 to 4.6, for predicted number of living leaves
for a stem with six leaves) and they were only meaningful,
in terms of predicted whole-leaf counts, for plants whose
stems had a large total number of leaves.
To determine whether differences in the leaf chlorophyll
content in response to drought were influenced by changes
in whole stem properties, mean chlorophyll content was
calculated over all of the living leaves on each stem
3.5
3.0
2.5
2.0
1.5
1.0
0.5
50
100
200
500
SLA (cm2 g−1)
Figure 6. Scaling relationship between foliar nitrogen
concentration (Nmass) and specific leaf area (SLA) in week five,
loge-scaled axes. Mean ⫾ SEM by species, n = 2 to 5; C4 control
, C4 drought
, C3 control
, C3 drought
.
Best-fit lines are standardized major axis relationships,
accounting for average measurement error. The slope between C3
species differed from that between C4 species (L3 = 8.89,
P = 0.031). There was no significant difference in slope between
treatments within each photosynthetic type (C3, L1 = 0.11,
P = 0.742; C4, L1 = 0.01, P = 0.934). There was a significant
difference in the intercept between treatments for the C4 species
(w1 = 4.14, P = 0.042), but not the C3 species (w1 = 2.24, P = 0.135).
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 65–75
Drought limitation in C3 and C4 grass species 73
values for species at the bottom of this range would be
approximately 0.25 mmol g-1, i.e. 0.35%.
DISCUSSION
We found that the photosynthetic advantage of C4 over C3
species under mesic conditions was reduced under drought.
High iWUE in C4 species under mesic conditions resulted
from higher A and lower gs than in C3 grasses, consistent
with the well-known capacity of C4 photosynthesis to draw
down ci to a greater extent than C3 photosynthesis (Long
1999). Under most conditions, the exchange of CO2 and
H2O between atmosphere and leaf occurs predominantly
via the stomatal pathway, and the capacity to draw down ci
through photosynthesis is equivalent to iWUE. Drought
forced a decrease in A in both C3 and C4 grasses, but its most
striking effect on gas exchange was the near elimination of
differences in gs between the C3 and C4 species. The resultant increase in iWUE observed for several C3 species closed
the gap between them and their C4 counterparts under
drought conditions.
Under adequate water supply, the initial slopes of A-ci
relationships for C4 species are steeper than those of C3
species (Pearcy & Ehleringer 1984). Consequently, with no
change in the shape of this relationship, reduced CO2 supply
due to decreased stomatal aperture would force a greater
decline in A for C4 than C3 species (Björkman 1971). The
mismatch observed between the size of the decreases in gs
(smaller in C4) and in A (equivalent in C3 and C4) is consistent with this theoretical expectation. However, declining gs
under drought should also drive down ci, and we found that
overall, this was not the case for C4 species, an effect that
implies a change in the shapes of A–ci relationships, i.e.
declines in A relative to CO2 supply. Our findings are, therefore, consistent with those of previous studies restricted to
Panicoideae species (Ripley et al. 2007, 2010; Ibrahim et al.
2008) and support the hypothesis that drought-limitation of
photosynthetic capacity is greater in C4 NADP-me than C3
grasses. Here, we find that this effect can be generalized to
C4 NADP-me species across the PACMAD clade.
However, certain factors must be considered when interpreting trends in ci values as indicators of photosynthetic
performance under drought (Lawlor & Cornic 2002).
Firstly, calculation of ci requires an accurate description of
gs, which, due to the need to obtain accurate estimates of
boundary layer conductance (gbl), may be difficult to
achieve for narrow leaved species. Secondly, when estimating leaf conductance to CO2, it is normally assumed that
cuticular conductance represents a negligible component of
leaf gas exchange, an assumption which may be violated
under drought (Boyer, Wong & Farquhar 1997). Finally, the
value of ci is a spatially averaged mean, weighted by leaf
conductance, that behaves as a strong index of photosynthetic performance when the spatial distributions of gs and
A are homogeneous across the entire leaf, but may perform
poorly in response to heterogeneity (Downton, Loveys &
Grant 1988; Terashima et al. 1988; Farquhar 1989). Heterogeneity may result from barriers to lateral (e.g. bundle
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 65–75
sheath extensions, Metcalfe 1960) and dorso-ventral diffusion of CO2 (Long et al. 1989) that are known to occur in the
leaves of grasses, and heterogeneous gas exchange has been
reported for maize (Terashima 1992). Differences in the
occurrence of these effects between C3 and C4 species could
plausibly explain our observation of a differential effect of
drought on ci.
Alternatively, effects on ci may result from drought limitation of photosynthetic capacity. Under mild to severe
water shortage, photosynthetic capacity of C3 species is
maintained and reductions in gs force reversible declines
in ci (Lauer & Boyer 1992; Brodribb 1996). However,
decreases in leaf water status (measured as relative water
content or leaf water potential) will eventually cause metabolic limitations of photosynthetic capacity (Lawlor &
Cornic 2002), and the mechanisms underlying effects of leaf
water status on photosynthetic metabolism are expected to
be qualitatively different between C3 and C4 plants (Ghannoum 2009). We found no strong evidence for a larger
response of leaf water potential to drought in C4 plants
when compared with C3 species, and the latter had more
negative leaf water potentials in our control treatment. Any
proposed metabolic effects on photosynthesis in C4 species
would, therefore, have occurred despite similar or less negative leaf water potentials than were experienced by C3
species.
It has been suggested that lower gs and E associated with
C4 photosynthesis may result in adaptation of plant hydraulics (Kocacinar & Sage 2003). Our observation of less negative Yop and smaller DY in C4 than C3 species in control
treatments is consistent with other recent work implying a
higher capacity for hydraulic supply relative to demand in
the C4 species (Taylor et al. 2010). Few species showed large
changes in DY in response to our drought treatment, suggesting anisohydric-isohydrodynamic behaviour (Franks,
Drake & Froend 2007). Assuming that large changes in DY
were representative of impaired hydraulic function, our
results show that this was no more common in C3 grasses
than in their C4 relatives. The observation that DY was
frequently maintained under drought is interesting from
two perspectives. Firstly, it suggests a link between Ysoil and
Yop that, during the progressive development of drought in
natural soils, might result in a progressive onset of metabolic effects of declining Yop. Secondly, it occurred in the
context of much larger decreases in gs for C3 than for C4
species. Assuming a strong relationship between gs and E,
in addition to differences in whole-plant conductance
(kplant = E/DY) observed between C3 and C4 species under
mesic conditions, the response of kplant to drought will be
greater in C3 grasses than in their C4 relatives.
We found no strong evidence that our drought treatment
reduced the chlorophyll and nitrogen content of leaves
measured for gas exchange. This suggests that senescence
did not drive observed decreases in A or PNUE in response
to drought. However, at the whole plant scale, droughtinduced leaf mortality was detected, and was most important in those species with longer-stems or C4 photosynthesis,
with the result that in the drought treatment, differences
74 S. H. Taylor et al.
between C3 and C4 species in the number of living leaves
per stem were reduced. Thus, changes in leaf area available
for photosynthesis may also contribute to effects of drought
on whole-plant photosynthesis and transpiration in C4
species compared with C3 relatives under drought.
We have previously found evidence that the range
of values for Nmass largely overlapped when comparing
PACMAD C3 and C4 grasses, and differences in Narea
between photosynthetic types were influenced by SLA
(Taylor et al. 2010). The results of the present experiment
further support that result. Conservation of the range of
Nmass values across species, paired with greater conservatism in SLA between C4 species, provided a strong contribution to differences in Narea between photosynthetic
types. Should conservatism in SLA between C4 species be
a general trend, the extent of its relationship with C4
Kranz anatomy will be of interest. For example, for a comparison made between Paniceae species, C3 grasses had
thinner leaves at a given vein density and reduced vein
density in wider leaves, whilst their C4 relatives showed no
relationship between vein density and leaf width (Oguro,
Hinata & Tsunoda 1985).
By concentrating on the NADP-me biochemical
subtype of C4 photosynthesis, we excluded several subtypes of C4 photosynthesis from our comparisons. Drought
tolerance has been shown to differ between C4 subtypes
(Ghannoum, von Caemmerer & Conroy 2002), an important effect to be considered in developing truly global conclusions on the drought tolerance of C4 photosynthesis.
Nonetheless, we provide evidence for a differential
response of photosynthetic types to drought. A difference
in Yop when water supply is abundant is coupled with
lower gs, and smaller changes in gs in response to changing
soil-water conditions in NADP-me grasses than in their C3
relatives. Drought reduced both the PNUE and iWUE
advantages commonly observed in C4 species, and produced a slight increase in leaf mortality in C4 species. Our
results therefore indicated that despite conservative regulation of gs, C4 NADP-me grass species are no more robust
to low water availability than their C3 relatives. In fact,
drought-induced limitations of photosynthesis may
become evident at less negative leaf water potentials in C4
NADP-me species than in C3 species. These results
provide experimental evidence that complements comparative studies of ecological adaptation in C4 grasses
(Osborne & Freckleton 2009; Edwards & Smith 2010).
Our finding that C4 species are no more tolerant of a
‘drought’ treatment imposed under controlled growing
conditions, and the impacts that this has upon their photosynthetic advantages over their C3 relatives, emphasize
the need for studies addressing physiological contrasts
between related C3 and C4 species in native situations.
ACKNOWLEDGMENTS
The authors thank Peter Franks, Andrew Leakey and
Matthew Gilbert for discussions of gas exchange results;
Mark Rees for advice on statistical analyses; Pascal-Antoine
Christin for his phylogenetic tree; Irene Johnson for advice
on plant rearing, and Hui Liu, Stephen Hulme and Sarah
Wilkinson for technical assistance. Research was funded
by the NERC grant NE/DO13062/1 awarded to CPO and
FIW, and a Royal Society University Research Fellowship
awarded to CPO.
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Received 20 June 2010; received in revised form 6 August 2010;
accepted for publication 12 August 2010