Journal of Experimental Botany, Vol. 65, No. 13, pp.

3513–3523, 2014
doi:10.1093/jxb/eru022  Advance Access publication 7 February, 2014

Research paper

Leaf anatomical traits which accommodate the facultative

engagement of crassulacean acid metabolism in tropical
trees of the genus Clusia
V. Andrea Barrera Zambrano1, Tracy Lawson2, Enrique Olmos3, Nieves Fernández-García3 and

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Anne M. Borland1,4,*
1 
School of Biology, Newcastle University, Newcastle upon Tyne NE17RU, UK
2 
School of Biological Sciences, University of Essex, Colchester CO4 3SQ, UK
3 
CEBAS–CSIC Campus Universitario de Espinardo, Department of Abiotic Stress and Plant Pathology, 30100 Murcia, Spain
4 
Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6407, USA

* To whom correspondence should be addressed. E-mail: Anne.Borland@ncl.ac.uk

Received 1 November 2013; Revised 17 December 2013; Accepted 19 December 2013

Abstract
Succulence and leaf thickness are important anatomical traits in CAM plants, resulting from the presence of large
vacuoles to store organic acids accumulated overnight. A higher degree of succulence can result in a reduction in
intercellular air space which constrains internal conductance to CO2. Thus, succulence presents a trade-off between
the optimal anatomy for CAM and the internal structure ideal for direct C3 photosynthesis. This study examined how
plasticity for the reversible engagement of CAM in the genus Clusia could be accommodated by leaf anatomical
traits that could facilitate high nocturnal PEPC activity without compromising the direct day-time uptake of CO2 via
Rubisco. Nine species of Clusia ranging from constitutive C3 through C3/CAM intermediates to constitutive CAM were
compared in terms of leaf gas exchange, succulence, specific leaf area, and a range of leaf anatomical traits (% inter-
cellular air space (IAS), length of mesophyll surface exposed to IAS per unit area, cell size, stomatal density/size).
Relative abundances of PEPC and Rubisco proteins in different leaf tissues of a C3 and a CAM-performing species
of Clusia were determined using immunogold labelling. The results indicate that the relatively well-aerated spongy
mesophyll of Clusia helps to optimize direct C3-mediated CO2 fixation, whilst enlarged palisade cells accommodate
the potential for C4 carboxylation and nocturnal storage of organic acids. The findings provide insight on the optimal
leaf anatomy that could accommodate the bioengineering of inducible CAM into C3 crops as a means of improving
water use efficiency without incurring detrimental consequences for direct C3-mediated photosynthesis.

Key words: CAM, Clusia, leaf anatomy, PEPC, photosynthesis, stomata.

Introduction
The photosynthetic organs of plants with crassulacean acid
metabolism (CAM) share a number of anatomical traits
which reflect functional requirements of this metabolic spe-
cialization. The generally thick and succulent leaves and
stems of CAM plants accommodate large central vacuoles
which serve as storage reservoirs for malic acid produced at
night from dark CO2 uptake mediated via phosphoenolpyru-
vate carboxylase (PEPC). Malic acid is subsequently released
from the vacuole and decarboxylated during the day to satu-
rate Rubisco with CO2 behind closed stomata, thereby con-
serving water. Positive relationships between succulence and
the magnitude of CAM have been demonstrated for diverse
phylogenetic lineages (Winter et  al., 1983; Nelson et  al.,
2005). Leaf and stem succulence can improve water storage
and capacitance (von Willert et al., 1990; Griffiths, 2013) and
it has been suggested that possession of this anatomical trait

Published by Oxford University Press on behalf of the Society for Experimental Biology.
3514 | Zambrano et al.
might have predisposed ancestral CAM taxa towards the evo-
lution of this photosynthetic specialization in water-limited
habitats (Sage, 2002).
The degree of succulence also has important consequences
for net CO2 exchange. Within the undifferentiated (i.e. no
distinct layers of palisade or spongy mesophyll) leaves and/
or stems that typify most CAM species, increased succulence
tends to reduce internal air space (IAS) and the length of
the mesophyll surface that is directly exposed to intercellular
air spaces per unit area (Lmes/area). Whilst these anatomical
traits improve the carbon economy of CAM during day-time
decarboxylation because net CO2 efflux from the leaf is mini-
mized, the reduced leaf internal conductance to CO2 curtails
direct uptake of atmospheric CO2, particularly during late
photoperiod when stomata may re-open and direct Rubisco-
mediated CO2 uptake occurs (Griffiths, 1992; Nelson and
Sage, 2008). A perceived incompatibility between the optimal
anatomy for high nocturnal PEPC activity and the internal
structure ideal for C3 photosynthesis may account for the
bimodal distribution of weak and strong CAM plants that
is indicated by δ13C across various families known to contain
both C3 and CAM species (Winter and Holtum, 2002; Crayn
et al., 2004; Silvera et al., 2005).
The concept of a functional anatomical threshold for the
dominance of C3 or C4 carboxylation in all CAM species
is confounded by tropical trees of the genus Clusia, many
of which exhibit a dynamic range of C3 and C4 carboxyla-
tion patterns. It has been proposed that Clusia species are
adapted to drought and high irradiance through the use of
CAM and that this is associated with the degree of leaf suc-
culence (Borland et al., 1998). Clusia can exist in a variety of
habits (i.e. lianas, epiphytes, shrubs, trees) and habitats (i.e.
semi-arid coastal restingas to montane cloud forest) which
might reflect the physiological plasticity in photosynthetic
characteristics that exist across the genus (Luttge, 2006,
2008). The genus is monophyletic, but, within it, CAM has
appeared and possibly been lost several times (Gehrig et al.,
2003; Gustafsson et al., 2007). Some Clusia species are fully
C3 (e.g. C. multiflora), others (e.g. C. minor, C. pratensis) are
capable of reversibly switching between C3 photosynthesis
and strong CAM (Winter et  al., 2008) whilst other species
often considered as strong CAM (e.g. C. rosea, C. quadran-
gula) may exhibit an almost continuous range of CO2 uptake
strategies as indicated by highly variable δ13C within indi-
vidual species (Holtum et al., 2004; Vargas-Soto et al., 2009).
Thus, anatomical traits that can accommodate potentially
strong CAM activity without compromising direct day-time
uptake of CO2 by Rubisco must exist in Clusia species that
show a propensity for facultative and reversible engagement
of CAM.
In contrast to the majority of other CAM species, the
leaves of Clusia possess clearly differentiated palisade and
mesophyll tissues along with an adaxial layer of water stor-
age parenchyma (Borland et  al., 1998). Tissue differentia-
tion presents the potential for variation both in cell size
and intercellular air space distribution through the leaf and
for accommodating differential distribution of PEPC and
Rubisco proteins in palisade mesophyll, spongy mesophyll,
and water storage parenchyma. To determine which leaf
anatomical characteristics might accomodate both noctur-
nal net CO2 uptake and direct day-time uptake of atmos-
pheric CO2 in Clusia, measurements were compared of leaf
succulence, specific leaf area (SLA), % IAS, Lmes/area, and
the cell sizes of spongy mesophyll, palisade mesophyll, and
water storage parenchyma across nine species. The species
of Clusia that were studied ranged from constitutive C3
through C3/CAM intermediates to constitutive CAM The
relative abundance of PEPC and Rubisco proteins in dif-
ferent leaf tissues of a C3-performing and a CAM species
of Clusia was assessed using immunogold labelling. The
density and size of stomata were also measured in the nine
species to determine if the potentially lower internal con-
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ductance within thicker-leaved CAM Clusia species could
be countered by the possession of more and/or larger sto-
mata, thereby creating greater potential maximum stomatal
conductance compared with the C3 species.
Materials and methods
Plant material
The species of Clusia chosen for this study were based on the degree
of leaf thickness, previous reports of CAM activity, and phylogeny
(species were sampled from five different sections within the genus).
Species included were: C. multiflora H. B. K., an obligate C3 species
(Grams et al., 1998) and C. tocuchensis Britton 1921, an obligate C3
species (Lüttge, 2007). Both of these species are found in section
Anandrogyne, the most derived section according to the phylogeny
of Gustafsson et  al. (2007). From section Retinostemon, C.  minor
L. a C3/CAM intermediate (Borland et al., 1992) and C. alata Pl et
Tr. (1860), a CAM species (Lüttge, 1999), were studied. From sec-
tion Phloianthera, C. lanceolata Camb., a C3/CAM intermediate spe-
cies (Roberts et  al., 1996; Lüttge, 1999) and C.  hilariana Schlecht.,
a CAM species, were included. From the Omphalanthera complex,
C. aripoensis Britton 1923, a C3/CAM intermediate species (Borland
et  al., 1998), was studied. From section Chlamydoclusia, C.  rosea
Jacq 1760 a CAM species (Lüttge, 2007) and C.  grandiflora Splitg
1842, a C3 species that has reportedly lost CAM (Gehrig et al., 2003)
were included.
Cuttings of each species, originally taken from mature plants
growing at Moorbank Botanic Garden, Newcastle University,
were maintained in heated glasshouses (day-time minimum 25  °C,
night-time minimum 15  °C) with supplementary lighting provided
in the winter months (Oct–Feb). Rooted cuttings that were between
2–3-years-old and about 80–100 cm in height were grown in 20 cm
diameter pots in a mix of commercial loam-based compost (John
Innes No. 2, Sinclair Horticulture Ltd, Lincoln, UK) and sand
(3:1 v/v) and watered with a commercial nutrient mix (Phostrogen,
Bayer UK, Newbury, UK) every 3 weeks. Experimental plants were
transferred to a controlled environment chamber with a 12 h pho-
toperiod, 65–75% relative humidity, 300 μmol m–2 s–1 PFD at plant
height (provided by fluorescent lamps, F100W/840, Polylux cool
white, Light Emission Technology, New York, USA) and day/night
temperatures of 27/18 °C. The plants were maintained under these
conditions for 4–6 weeks before gas exchange measurements and
anatomical studies commenced.
Leaf gas exchange
CAM expression was assessed by monitoring diel gas exchange
patterns of net CO2 uptake in all nine species under compara-
ble controlled conditions. Gas exchange profiles were obtained
for plants grown under well-watered conditions (water supplied

every day) and under conditions of drought (9 d without water).
Each plant (including soil and pot) was weighed every day follow-
ing the withdrawal of watering to establish the time taken for soil
drying to occur. In all cases, there was no further weight loss from
the plant/pot after 4–6 d without water. Extending the period of
drought beyond 9 d resulted in a gradual decline in nocturnal
and day-time net CO2 uptake in all species (data not shown). Net
CO2 uptake was measured using a compact mini cuvette system,
Central Unit CMS-400 with a BINOS-100 infra-red gas ana-
lyser, working in an open mode (Heinz Walz GmbH, Effeltrich,
Germany). A  fully expanded leaf was clamped in the cuvette,
ensuring it received full light (i.e. 300  µmol m–2 s–1) within the
growth chamber. The temperature of the cuvette was set to track
environmental conditions within the growth room. Data for net
CO2 uptake were collected every 15 min. The gas flow through the
cuvette was maintained between 400 and 500 ml min–1 to avoid
water condensation inside the cuvette. The leaf was maintained
inside the cuvette for at least 48 h to get a complete 24 h gas
exchange profile. Data were analysed using DIAGAS software
based on the area of leaf inside the cuvette. Each gas exchange
curve presented is representative of that obtained from three bio-
logical replicates per species.
Leaf anatomy
In addition to leaf succulence (kg m–2), measurements were made of
specific leaf area (SLA: leaf area (cm2)/dry mass (g), a trait which has
been reported as a good indicator of leaf tissue density and resource
use strategies (Garnier and Laurent, 1994). To study internal leaf
anatomy, transverse sections of 2–3 leaves of each species were
made, following the methodology of Morison et  al. (2005). Small
pieces of leaf (approximately 2–3 × 8–10 mm) were fixed in 5% glu-
taraldehyde via vacuum infiltration and left overnight at 5 °C. The
leaf pieces were then dehydrated using an ethanol series and embed-
ded in resin (LR White acrylic resin, Sigma-Aldrich, Gillingham,
UK). Sections of 1 μm thickness were cut using an ultra-microtome
and stained with toluidine blue. Images of each section were pho-
tographed under 5×, 10×, and 40× magnifications in a light micro-
scope (Leica DM RB, Leica Microsystems CMS GmbH, Wetzlar,
Germany) in order to measure mesophyll thickness, [i.e. depth of
palisade and spongy mesophylls and of water storage parenchyma
(WSP)], cell size (i.e. cell area), % intercellular air space (IAS), and
length of mesophyll surface exposed to IAS per unit area (Lmes/area,
μm–1). All images were analysed using Image J software (National
Institutes of Health, USA).
Immunolabelling for PEPC and Rubisco in leaf sections
To localize and determine the relative abundance of PEPC and
Rubisco in WSP and in palisade and spongy mesophyll cells, trans-
verse sections of leaves of C. aripoensis (weak CAM-inducible but
operating in the C3 mode when sampled) and C. rosea (CAM) were
taken. Previous trials had shown that these two species presented
better tissue preservation after fixation than the other C3 and CAM
species investigated in this study.
Leaf pieces (1 × 1 mm) were fixed in 2.5% paraformaldehyde and
0.5% glutaraldehyde in 0.06 M phosphate buffer (pH 7.2) for 2 h at
4 °C, rinsed in the same buffer and dehydrated in an ethanol series.
Finally, samples were embedded in LR White resin (Fernández-
García et al., 2009).
Ultra-thin sections (60–70 nm) were obtained with a Leica EM
UC6 ultra-microtome (Leica Mikrosysteme, Vienna, Austria) and
collected on formvar-coated nickel grids. The grids were placed in
phosphate-buffered saline (PBS) with 5% bovine serum albumin
(BSA) for 30 min at room temperature and then incubated for 2 h
with the primary antibody, anti-PEPC or Rubisco rabbit polyclonal
immunoglobulin G, diluted 1:250 in PBS containing 5% BSA. The
sections were washed three times in PBS and incubated for 2 h with
the secondary antibody (goat anti- rabbit, IgG, 10 nm, Electron
Functional leaf anatomy of Clusia | 3515
Microscopy Sciences) diluted 1:50 in PBS supplemented with 1%
BSA plus 1% goat serum. The grids were washed in PBS and dis-
tilled water. Samples were viewed with a Philips Tecnai 12 elec-
tron microscope (Philips, Eindhoven, The Netherlands) equipped
with a CCD SIS MegaView III camera (1280 × 1280 pixel, 12 bit).
Morphometrical data were obtained as described by Fernández-
García et  al. (2009). Gold particle densities were counted in the
cytoplasm for PEPC and in the chloroplast for Rubisco, as well in
the mitochondria and vacuole as a background.
Stomatal measurements
The leaves of Clusia are hypostomatous (A Barrera, unpublished
observations) so in order to measure stomatal characteristics, impres-
sions of the abaxial surface of the leaf, were taken using Xantropen
VL plus silicone impression material and hardener (Beyer Dental
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Leverkusen, Germany) with a mixture of approximate ratio 10:1
(Weyers and Johansen, 1985). Stomatal impressions were made for
2–3 leaves of two plants per species. Sections were photographed
under a light microscope (Leica DM RB). At least 25 stomata per
leaf were measured in terms of dimensions and over 50 areas of
1.65 mm2 each were used to estimate stomatal density and stomatal
index. Stomatal dimensions (pore length and width) were measured
under 40× magnification and stomatal density was measured under
10× magnification. All the images were analysed using Image J soft-
ware. The theoretical maximum stomatal conductance (GH2O) was
estimated using the equation described previously by Lawson et al.
(1998) where:
[mean formula mass of air] × [effective diffusion coefficient] × [stomatal density] × [pore area]
GH 2 O =
(pore depth) + (end correction)
The mean formula of air was taken as 40.9 mol m–3 at 25 °C and the
effective diffusion coefficient for water vapour as 2.49 × 10–5 m2 s–1
at 25 °C. Pore area was calculated assuming the pore as an ellipse,
with a constant minor axis (pore length) at different apertures and
maximal aperture as a major axis. The end correction was (pore
area/π)0.5.
All statistical analyses were performed using Minitab 16 (Minitab
Ltd. Coventry, UK).
Results
Leaf gas exchange profiles
Patterns of net CO2 uptake were measured for mature leaves
of all nine Clusia species over an entire 24 h day/night cycle
under well-watered and droughted conditions (Fig. 1). Three
species, C. minor, C. lanceolata, and C. aripoensis showed an
increase in net dark CO2 uptake after drought and so were
categorized as C3/CAM intermediates (Fig.  1d–f; Table  1).
There was no increase in net dark CO2 uptake in C. hilariana,
C. alata or C. rosea after 9 d of drought so these species were
categorized as CAM (Fig. 1a–c). Of the three CAM species,
C.  rosea engaged in the most day-time net CO2 take during
phases 2 and 4 (Fig. 1c). Drought did not elicit any net dark
CO2 uptake or accumulation of acidity (data not shown) in
C. multiflora, C. tocuchensis or C. grandiflora so these species
were categorized as C3 (Table 1). The proportion of net 24 h
CO2 uptake that occurred at night was calculated by integrat-
ing the relevant areas under each of the gas exchange curves
and expressed as a percentage of total 24 h net CO2 uptake
(Table 1).
3516 | Zambrano et al.

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Fig. 1.  Gas exchange curves showing 24 h patterns of net CO2 uptake for nine species of Clusia under well-watered (closed symbols) and droughted
(9 d without water, open symbols) conditions. The shaded areas in each graph represent the dark period and each of the gas exchange curves presented
is representative of three replicate runs for each species.

Table 1.  The percentage of 24 h net CO2 uptake that occurred at night in nine species of Clusia under well-watered conditions and after
9 d without water

The data were calculated from the gas exchange profiles illustrated in Fig. 1, from which species were categorized as CAM, C3/CAM or C3.
Values for leaf succulence and specific leaf area are also shown as the mean of six biological replicates ±standard error of the mean.

Species Net 24 h CO2 uptake Photosynthetic Leaf succulence Specific leaf area
over night (%) category (kg m–2) (cm g–1 dry mass)
C. alata 74 ± 5 CAM 1.641 ± 0.018 91 ± 9
–H2O 89 ± 8
C. hilariana 88 ± 6 CAM 1.018 ± 0.019 112 ± 14
–H2O 92 ± 5
C. rosea 41 ± 10 CAM 0.903 ± 0.017 118 ± 20
–H2O 62 ± 11
C. minor 21 ± 7 C3/CAM 0.693 ± 0.032 132 ± 18
–H2O 77 ± 10
C. lanceolata 14 ± 4 C3/CAM 0.597 ± 0.020 160 ± 15
–H2O 72 ± 10
C. aripoensis 0 C3/CAM 0.569 ± 0.007 190 ± 17
–H2O 31 ± 4
C. grandiflora –8 ± 2 C3 0.512 ± 0.013 160 ± 15
–H2O –10 ± 1
C. tocuchensis –2 ± 1 C3 0.531 ± 0.011 196 ± 20
–H2O –3 ± 1
C. multiflora –5 ± 1 C3 0.554 ± 0.021 212 ± 22
–H2O –7 ± 1
 Functional leaf anatomy of Clusia | 3517

Leaf anatomy
The CAM species of Clusia had higher values for leaf succu-
lence (kg m–2) than the C3/CAM intermediates which, in turn,
were higher than those of C3 species (Table 1). Specific leaf
area showed an inverse relationship with succulence and with
the proportion of CO2 taken up at night under well-watered
conditions across the nine Clusia species (P<0.01; Fig.  2a).
Thus, leaves of CAM Clusias were thicker than those of the
C3/CAM species which were thicker than those of the C3
species.
There was a weak (but not significant, P>0.1) negative rela-
tionship between the percentage of internal air space (% IAS)
in the leaf mesophyll and the propensity for dark CO2 uptake

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(Fig. 2b). The length of the mesophyll surface exposed to IAS
per unit area (Lmes/area) was found to show a strong negative
relationship (P<0.01) with the magnitude of CAM in the nine
species of Clusia (Fig.  2c). Those species which engaged in
more dark CO2 uptake had lower Lmes/area compared with the
species which engaged in more day-time net CO2 uptake. The
values of Lmes/area calculated for the nine Clusia species were
compared with the mean Lmes/areas calculated by Nelson et al.
(2005) for a phylogentically diverse assemblage of 18 CAM
species and six C3 species (Fig. 2c). The Lmes/areas of all the
Clusia species were higher than the reported CAM average
(Fig.  2c). Values for Lmes/area in most of the Clusia species
examined in the present study (including all the C3/CAM spe-
cies as well as the CAM-performing C. rosea) were in the range
of Lmes/areas reported for C3 species (Nelson et al., 2005).
The leaves of Clusia possess clearly differentiated layers of
water storage parenchyma, palisade mesophyll, and spongy
mesophyll and the proportions of these tissues vary between
photosynthetic types as illustrated in Fig. 3. The relative con-
tributions from the different tissue types to leaf thickness was
quantified by measuring the depth of the layers of water stor-
age parenchyma (WSP), palisade mesophyll, and spongy mes-
ophyll and expressing this as a percentage of leaf thickness
(Fig. 4a, c, e). The size of the cells in each of the three tissue
types was also measured (Fig.  4b, d, f). Each of these ana-
tomical characteristics was plotted against the propensity for
CAM (measured as the proportion of CO2 taken up at night).
In those species that engaged in a greater proportion of dark
CO2 uptake, the palisade mesophyll represented a greater
percentage of leaf thickness (40–45%) compared with the C3
species of Clusia where palisade mesophyll constituted ~25–
Fig. 2.  Relationships between the proportion of net CO2 uptake
30% of leaf thickness (Fig. 4a, P=0.02). Cell size of palisade conducted at night in nine species of Clusia and (a) specific leaf area,
mesophyll also showed a positive relationship with the pro- (b) intercellular air space, and (c) Lmes/area (mean±SE, n=3). Average Lmes/
pensity for CAM (Fig. 4b, P<0.01). The cells of the palisade areas for C3 and CAM species were taken from Nelson et al. (2005) and
mesophyll in the four species that showed the highest rates of are shown in relation to Lmes/areas of the nine Clusia species (c).
net dark CO2 uptake (i.e. C. alata, C. hilariana, C. rosea, and
droughted C. minor; Fig. 1) were >4× larger than those of the
C3 species and the weak C3/CAM intermediate, C. aripoensis CAM species as reported by Nelson et al. (2005). The spongy
(Fig. 4b). The size of palisade cells in the C3 Clusias and the mesophyll constituted ~40–60% of leaf thickness across all
weak C3/CAM intermediate C. aripoensis fell within the range the different photosynthetic types and there was a trend (not
of average cell size for C3 species as reported by Nelson et al. statistically significant) for increased cell size of the spongy
(2005). The size of palisade cells of the CAM Clusias and mesophyll in those species which engaged in more dark CO2
the C3/CAM intermediate C.  minor cells fell just below the uptake (Fig. 4c, d). A weak inverse correlation (P=0.15) was
range of average cell size reported for a diverse range of other found between the magnitude of CAM and the percentage
3518 | Zambrano et al.
Fig. 3.  Transverse leaf sections from three species of Clusia with different modes of photosynthetic metabolism and indicating the major tissue types
within the leaf that include water storage parenchyma, palisade mesophyll, and spongy mesophyll. (This figure is available in colour at JXB online.)
of total mesophyll occupied by WSP (Fig.  4c). The highest
percentage of water storage parenchyma was found in the C3
species C. multiflora, C. tocuchensis, and the weak C3/CAM
species C. aripoensis. However, the C3 species C. grandiflora
(a species that is believed to have lost CAM) had a signifi-
cantly reduced percentage of WSP compared with the other
C3 species. There was no relationship between the propensity
for CAM and cell size of the WSP (Fig. 4f).
Immunolocalization of PEPC and Rubisco
The abundance of Rubisco protein was 1.5 times higher in
cells of the palisade mesophyll compared with cells of the
spongy mesophyll in both C.  aripoensis operating in the C3
mode (t=4.436, P<0.0001) (Fig.  5a) and in the CAM spe-
cies C.  rosea (t= 3.619, P=0.001) (Fig.  5b). The abundance
of PEPC was slightly higher in spongy mesophyll cells in
C. aripoensis compared with palisade cells but was three times
higher in palisade cells compared with spongy mesophyll cells
in C. rosea (t=6.770, P<0.0001). As expected, the abundance
of PEPC protein was higher in C. rosea (Fig. 5a) compared
with C.  aripoensis (Fig.  5b). Rubisco and PEPC proteins
were not detectable in cells of the WSP using immunogold
labelling.
Stomatal characteristics
Stomatal density showed a significant (P<0.01) negative cor-
relation with the propensity for dark CO2 uptake across the
nine species of Clusia (Fig. 6a) but there was no clear rela-
tionship between photosynthetic type and stomatal index
(Fig.  6b). There was a trend for increased stomatal pore
size in those species that engaged in more dark CO2 uptake
(Fig. 6c) but the C3 species C. grandiflora (reported to have
lost CAM) was an outlier on this trend (Fig. 6c).
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The maximum measured stomatal conductance (gH2O) of
all nine Clusia species (measured at the start of the photo-
period) was substantially lower than the maximum stomatal
conductance that was predicted (GH2O) based on stomatal
size and density (Fig. 7a, b). There was a negative relationship
between measured maximum stomatal conductance and the
propensity to engage in dark CO2 uptake (Fig. 7a). However,
there was no clear trend across the photosynthetic types in
terms of the potential for water loss based solely on stomatal
dimensions and density on the leaf (Fig. 7b).
Discussion
Succulence and leaf thickness are important anatomical
traits in CAM plants, resulting from the presence of large
vacuoles to store organic acids accumulated during the night.
A  higher degree of succulence means less intercellular air
space (IAS) between mesophyll cells and a reduction in the
length of mesophyll exposed to intercellular air spaces (Lmes/
area), traits that reduce internal conductance to CO2. Thus,
succulence presents a ‘trade-off’ between the optimal leaf
anatomy for CAM and the internal structure ideal for C3
photosynthesis. The aim of the present study was to investi-
gate how plasticity for the reversible engagement of CAM in
the genus Clusia could be accommodated by leaf anatomical
traits that, in principle, could facilitate high nocturnal PEPC
activity without compromising the direct day-time uptake of
CO2 via Rubisco.
Specific leaf area and implications for internal
conductance to CO2 in Clusia
As predicted, Clusia species with more succulent and
thicker, denser leaves (lower specific leaf area, SLA) engaged
in more dark CO2 uptake than the thinner-leaved species.
 Functional leaf anatomy of Clusia | 3519

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Fig. 4.  Relationships between the proportion of net CO2 uptake conducted at night in nine species of Clusia and the percentage of leaf thickness
attributed to (a) palisade mesophyll, (c) spongy mesophyll, and (e) water storage parenchyma. Also shown are cell sizes for (b) palisade mesophyll, (d)
spongy mesophyll, and (f) water storage parenchyma (mean ±SE, n=3). Average cell sizes for C3 and CAM species were taken from Nelson et al. (2005)
and are shown in relation to palisade cell size of the nine Clusia species (b). The dotted circle in (b) highlights the four Clusia species with the higest rates
of net dark CO2 uptake.

SLA serves as a useful indicator of the way in which plants
invest carbon and nutrients (dry biomass) in a given area of
light-intercepting foliage (Vendramini et al., 2002). Species
with lower SLA may be considered to incur a higher leaf-
level cost for light interception (Poorter, 2009), a strategy
that is common in species that inhabit environments where
drought and/or nutrient limitation hamper growth. Thus,
possession of a low SLA may be a trait that predisposed
Clusia species towards the evolution of CAM in water and
nutrient-limited habitats.
The trend of thicker leaves in CAM Clusias was accompa-
nied by a reduced length of mesophyll surface exposed to IAS
per unit area (Lmes/area). This observation is consistent with
the hypothesis that, by constraining leaf internal conductance
to CO2, a reduced Lmes/area will enhance photosynthetic effi-
ciency during decarboxylation in phase III of CAM because
net CO2 efflux from the leaf is minimized (Maxwell et  al.,
1997). Thus, the propensity for CAM in Clusia showed a clear
relationship with Lmes/area. Previous measurements gathered
for a phylogentically diverse group of CAM species indicated
3520 | Zambrano et al.
Fig. 5.  The relative amounts of PEPC and Rubisco in terms of the density of gold particles in cells of the palisade mesophyll and spongy mesophyll for
C. aripoensis operating in the C3 mode (a) and the CAM species C. rosea (b). Gold particle densities were also counted in the mitochondria and vacuole
as background.
that photosynthetic divergence between weak and strong
CAM is mediated by % IAS and Lmes/area which presents a
functional threshold for predominantly Rubisco or predomi-
nantly PEPC-mediated net CO2 uptake (Nelson et al., 2005).
However, the CAM Clusia species investigated in the present
work showed substantially higher % IAS (mean of 30%) com-
pared with the diverse CAM lineages investigated by Nelson
et al. (2005) where average % IAS was ~15%. Similarly, the
measured values for Lmes/area in all of the nine Clusia species
were higher than those of the CAM species studied by Nelson
et  al. (2005). In C.  alata and C.  hilariana which conducted
70–90% of 24 h uptake at night, the average value of Lmes/area
was 50% higher than the mean value for CAM species studied
by Nelson et al. (2005). The Lmes/area for the remaining seven
Clusia species (including the CAM species C. rosea and the
C3/CAM intermediate C.  minor which is capable of strong
CAM activity under drought) fell within the range measured
for C3 species (Nelson et  al., 2005). The ‘C3-like’ Lmes/area
in Clusia leaves indicates a relatively well-aerated mesophyll
which may be a critical anatomical component underpinning
the notable phenotypic plasticity of C3/CAM engagement
within the genus.
Development of the palisade mesophyll is linked to
propensity for CAM in Clusia
Whilst the relatively high (compared with other CAM species)
Lmes/area of Clusia could help to optimize direct day-time
C3-mediated CO2 uptake, adequate vacuolar storage capac-
ity is required to facilitate C4 carboxylation in those species
that engage in constitutive or facultative CAM. The thicker
leaves (lower SLA) of the Clusia species that engaged in more
nocturnal net CO2 uptake showed enhanced development of
the palisade mesophyll, both in terms of cell size and num-
ber of cell layers. The cells of the palisade mesophyll were,
on average, ~4× larger in the four Clusia species that showed
the highest rates of nocturnal net CO2 uptake compared
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with the other C3/CAM and C3 Clusia species. Moreover,
immunolocalization studies showed that the enhanced PEPC
abundance in C.  rosea compared with the weak C3/CAM
intermediate C. aripoensis was predominantly associated with
the palisade mesophyll tissue which contained 3× more PEPC
protein compared with the spongy mesophyll. Enhanced
development of palisade mesophyll tissue is commonly found
in thicker-leaved C3 species and is hypothesized to improve
the harvesting of light, thereby helping to offset the increased
investment in biomass in thicker leaves (Smith and Hughes,
2009). In addition, enhanced development of the palisade
mesophyll could potentially accommodate the increased ener-
getic requirements of CAM. In CAM Clusias, the strategy of
investment in palisade mesophyll cells, alongside a relatively
high Lmes/area could maintain a capacity for direct C3 car-
boxylation whilst the enlarged palisade mesophyll cells would
also offer potential for significant C4 carboxylation by serving
to accommodate more PEPC protein and large vacuoles to
store organic acids overnight. In principle, such anatomical
features could accommodate the dynamic range of photosyn-
thetic manifestations that distinguish Clusia from many other
CAM lineages.
Water storage parenchyma and propensity for CAM in
Clusia
In contrast to the close positive relationship that was estab-
lished between leaf thickness and the depth of palisade
mesophyll tissues within Clusia, there was no such positive
relationship with water storage parenchyma (WSP). In fact,
the presence of a thick layer of WSP was particularly evi-
dent in the C3 species C. tocuchensis and C. multiflora, both
of which belong to the most derived section of the genus in
which CAM does not appear to have evolved (Gustaffson
et  al., 2007). Comparisons of WSP in the genus Peperomia
have shown that the thickness of this tissue shows a nega-
tive relationship with CAM expression (Sipes and Ting,

Fig. 6.  Relationships between the proportion of net CO2 uptake
conducted at night in nine species of Clusia (mean ±SE, n=3) and (a)
stomatal density, (b) stomatal index, and (c) stomatal pore area (mean
±SE, n=50).
1985). Thus, leaves of the C3-CAM intermediate P.  obtusi-
folia showed a thicker WSP (i.e. 63% of total mesophyll)
compared with P. macrostachya, a constitutive CAM species
(i.e. WSP=19% of total mesophyll; Fondom et al., 2009). It
is tempting to speculate that, by acting as a means of buffer-
ing against water shortage, a thick WSP might obviate the
need for CAM. An exception to the trend for a thicker WSP
Functional leaf anatomy of Clusia | 3521
in C3 species was, however, noted in C. grandiflora, a C3 spe-
cies believed to have lost CAM and belonging to section
Chlamydoclusia (Gustaffson et al., 2007). The depth of WSP
in C. grandiflora was comparable with that in the CAM spe-
cies C. rosea which also belongs to section Chlamydoclusia.
A  more detailed examination of the depth of WSP across
more species and sections of the Clusia genus will be neces-
sary to establish potential relationships between the thickness
of the WSP, phylogeny, and photosynthetic mode.
Stomatal traits and propensity for CAM in Clusia
It was hypothesized that the potentially lower internal con-
ductance to CO2 within the thicker leaves of CAM-performing
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species of Clusia could be countered by the possession of more
and/or bigger stomata compared with the thinner-leaved C3
species. Previous studies have reported that, in general, more
succulent species show lower stomatal densities than less suc-
culent species (Sayed, 1998; Luttge, 2008). The present study
was in agreement with this finding but whilst stomata were
present in lower densities in the thicker-leaved CAM species
of Clusia, the stomatal pore areas tended to be larger in the
CAM Clusias compared with the C3 species. A negative rela-
tionship between stomatal density and stomatal size has been
reported for a diverse range of C3 species and is attributed
to spatial limits in the placing of stomata on the leaf surface
which ultimately constrains the maximum size and density of
stomata (Beaulieu et al., 2008; Franks et al., 2009). Since it has
been suggested elsewhere that smaller stomata are better at
improving WUE due to their more rapid response to changes
in environmental conditions such as humidity (Hetherington
and Woodward, 2003), smaller stomata might have been
predicted for CAM-performing Clusias compared with C3
Clusias. However, the production of more, smaller stomata
for a given leaf area may incur additional metabolic costs and
higher rates of guard cell respiration (Srivastava et al., 1995;
Franks et al., 2009). These extra costs could be compensated
for with high CO2 assimilation rates but only if environmen-
tal resources such as water and light are not limiting (Franks
et al., 2009). Given that the CAM-performing species of Clusia
commonly inhabit water-limited environments and, given the
extra energetic costs associated with CAM, the possession of
larger stomata in lower densities might be the most appropri-
ate strategy in terms of resource use.
The CAM species of Clusia studied here possessed both
larger stomata and palisade mesophyll cells which may be a
consequence of common genetic control of cell sizes, perhaps
via genome size (Beaulieu et al., 2008). It has been proposed
for C3 plants that size correlations between different cell types
(e.g. guard cells, epidermal cells, palisade, and xylem) provide
a highly efficient match between potential maximum water
loss (determined by stomatal conductance) and the leaf vas-
cular system’s capacity to replace that water (determined by
vein density; Brodribb et al., 2013). Calculating the theoreti-
cal maximum stomatal conductance to water in the different
Clusia species showed no clear trend between cell size, pho-
tosynthetic mode or the potential for water loss. However,
actual measurements of maximal stomatal conductance
3522 | Zambrano et al.
Fig. 7.  The relationship between the proportion of net CO2 uptake conducted at night in nine species of Clusia (mean ±SE, n=3) and (a) measured
maximal stomatal conductance (gH2O) taken at the start of the photoperiod and (b) theoretical maximal stomatal conductance (GH2O) based on the
density and dimensions of stomata and using the equation described by Lawson et al. (1998).
showed a strong negative relationship with the propensity for
CAM. Since the difference between theoretical maximum sto-
matal conductance to water and measured stomatal conduct-
ance was greatest in those species which showed the greatest
level of CAM expression, endogenous control over stoma-
tal conductance appears to be more important than stoma-
tal patterning in determining the potential for water loss in
CAM as compared with C3 species. The lower Lmes/area in the
CAM-performing species might also curtail the diffusion of
water vapour from leaves (Kaiser, 2009)
Conclusions
A study of functional leaf anatomy within the genus Clusia,
indicates that a relatively well-aerated spongy mesophyll
helps to optimize direct C3-mediated CO2 fixation, whilst
enlarged palisade cells accommodate the potential for C4
carboxylation and nocturnal storage of organic acids. The
findings provide insight into the optimal leaf anatomy that
could accommodate the bioengineering of inducible CAM
into C3 crops as a means of improving water use efficiency
without incurring detrimental consequences for direct C3-
mediated photosynthesis. Selecting genotypes of C3 target
crops with increased ploidy levels, which is correlated expe-
diently with increased cell size and biomass productivity
(De Veylder et  al., 2011) and with succulence (De Rocher
et  al., 1990), along with a well-developed palisade meso-
phyll, could, in principle, enhance the efficacy of engineered
CAM.
Acknowledgements
VABZ was funded by Colfuturo, and Newcastle University. This material
is based upon work supported by the Department of Energy, Office of
Science, Genomic Science Program under Award Number DE-SC0008834.
Oak Ridge National Laboratory is managed by UT-Battelle, LLC for the
US Department of Energy under Contract Number DE–AC05–00OR22725.
Downloaded from https://academic.oup.com/jxb/article-abstract/65/13/3513/2877477 by guest on 23 March 2019
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