Journal of Experimental Botany, Vol. 64, No. 18, pp. 5485–5496, 2013
doi:10.1093/jxb/ert314 Advance Access publication 14 October, 2013
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
Research paper
How succulent leaves of Aizoaceae avoid mesophyll
conductance limitations of photosynthesis and survive
drought
Brad S. Ripley1,*, Trevor Abraham1, Cornelia Klak2 and Michael D. Cramer3
1 Department of Botany, Rhodes University, 6140 Grahamstown, South Africa
Bolus Herbarium, Department of Biological Sciences, University of Cape Town, 7701 Rondebosch, South Africa
3 Department of Biological Sciences, University of Cape Town, 7701 Rondebosch, South Africa
2 * To whom correspondence should be addressed. E-mail: [email protected]
Received 23 May 2013; Revised 30 July 2013; Accepted 22 August 2013
Abstract
In several taxa, increasing leaf succulence has been associated with decreasing mesophyll conductance (gM) and an
increasing dependence on Crassulacean acid metabolism (CAM). However, in succulent Aizoaceae, the photosynthetic tissues are adjacent to the leaf surfaces with an internal achlorophyllous hydrenchyma. It was hypothesized
that this arrangement increases gM, obviating a strong dependence on CAM, while the hydrenchyma stores water and
nutrients, both of which would only be sporadically available in highly episodic environments. These predictions were
tested with species from the Aizoaceae with a 5-fold variation in leaf succulence. It was shown that gM values, derived
from the response of photosynthesis to intercellular CO2 concentration (A:Ci), were independent of succulence, and
that foliar photosynthate δ13C values were typical of C3, but not CAM photosynthesis. Under water stress, the degree
of leaf succulence was positively correlated with an increasing ability to buffer photosynthetic capacity over several
hours and to maintain light reaction integrity over several days. This was associated with decreased rates of water
loss, rather than tolerance of lower leaf water contents. Additionally, the hydrenchyma contained ~26% of the leaf
nitrogen content, possibly providing a nutrient reservoir. Thus the intermittent use of C3 photosynthesis interspersed
with periods of no positive carbon assimilation is an alternative strategy to CAM for succulent taxa (Crassulaceae and
Aizoaceae) which occur sympatrically in the Cape Floristic Region of South Africa.
Key words: Aizoaceae, Crassulaceae, Crassulacean acid metabolism, drought avoidance, leaf succulence, mesophyll
conductance
Introduction
Leaf succulence has evolved in diverse phylogenetic lineages
worldwide (Landrum, 2002; Ogburn and Edwards, 2010) and
is common in plants subject to frequent drought. Succulents
form a major component of the flora of the Succulent Karoo
within the Greater Cape Floristic Region (GCFR; sensu Born
et al., 2007) and leaf succulence (as opposed to stem succulence) is found in some 30 lineages within the Aizoaceae,
Asphodelaceae, and Crassulaceae (Linder et al., 2010). This
region contains some 1200 species of Aizoaceae (Goldblatt
and Manning, 2000), one clade of which has undergone very
Abbreviations: A, net photosynthetic rate; CAM, Crassulacean acid metabolism; Ci, intercellular CO2 concentration; δ13C, stable carbon isotope ratio; Fv/Fm, potential PSII photochemical efficiency; gST, stomatal conductance; gM, mesophyll conductance; Ja, rate of linear electron transport; Jmax, maximum of rate of electron
transport; PPFD, photosynthetic photon flux density; Rd, dark respiration; τ*, photorespiratory compensation point; Vcmax, Rubisco carboxylation; VPD, vapour
pressure deficit; WC, leaf water content.
© The Author 2013. Published by Oxford University Press on behalf of the Society for Experimental Biology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/),
which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
5486 | Ripley et al.
rapid and recent diversification (Klak et al., 2004). The driving force behind this rapid radiation is thought to be the
innovation of several key morphological characters, including succulent, subcylindrical to trigonous shaped leaves, in
response to an increasingly arid environment at the end of the
Miocene (Klak et al., 2004). However, the physiological role
of leaf succulence in the tolerance of drought has not been
investigated in the Aizoaceae.
Succulent leaves have high water contents, and this, combined with elastic cell walls and apoplastic polysaccharides,
allows leaves to avoid low leaf water potentials even after
extensive dehydration (Nobel, 1988; von Willert et al., 1992;
Martin, 1994; Pimienta-Barrios et al., 2002). This serves to
buffer transpirational water loss, prolonging stomatal opening and positive gas exchange, the magnitude of which correlates with the degree of leaf succulence (Nobel, 1976; Acevedo
et al., 1983; Martin and Adams, 1987; Gravatt and Martin,
1992; Martin, 1994). Subsequent to stomatal closure, succulence and the possession of relatively impervious leaf cuticles
prevents cellular dehydration and allows succulent leaves to
endure long periods of drought (Hoffman et al., 2009).
In addition to increasing water storage, leaf succulence
is frequently associated with the use of some form of photosynthetic Crassulacean acid metabolism (CAM), including obligate CAM, CAM-idling, CAM-cycling, and flexible
CAM/C3 systems (Veste et al., 2001; Herrera, 2009). Leaf
succulence and CAM may be mechanistically linked, or may
have evolved independently to optimize water use strategies
in arid environments (Ogburn and Edwards, 2010). The proposed dependence of leaf succulence on CAM arises from
two considerations. First, dependence on CAM is thought to
require the additional storage capacity for the C4 acids provided for by enlarged succulent cells (Gibson, 1982; Nelson
et al., 2005) associated with high vacuolar volumes (up to
98% of cell volume; Lüttge, 2004). Secondly, as leaf succulence increases, the mesophyll conductance (gM) for the diffusion of CO2 through hydrated succulent leaf tissue to the
photosynthetic tissue decreases. In most C3 and C4 plants
CO2 transfer across the mesophyll is largely through gaseous
pathways, with gM being determined by the ratio of chloroplast surface to leaf area and cell wall thickness (Tomás et al.,
2013). In contrast, the mesophyll of fully hydrated succulent
leaves at least partially lacks gas spaces, and CAM may function to overcome gM limitations on photosynthesis (Maxwell
et al., 1997; Gillon et al., 1998; Griffiths et al., 2008). CAM
plants can tolerate extremely low gM values, partially due to
the capacity of phosphoenolpyruvate carboxylase (PEPc) to
acquire HCO3– (Maxwell et al., 1997) and because nocturnal
carboxylation is limited by PEP derived from starch metabolism, and not by CO2 supply from the atmosphere (Nelson
and Sage, 2008). Reduced gM is advantageous to CAM plants,
as it limits daytime CO2 efflux from photosynthetic tissues
ensuring high intercellular CO2 concentrations during the refixation of CO2 into the C3 cycle (Nelson et al., 2005, 2008).
The extent to which increasing succulence reduces gM is likely
to be dependent on leaf anatomy and how photosynthetic tissue is distributed within the leaf. In some species, increasing
succulence is via additional layers of chloroplast-containing
mesophyll cells, such that photosynthetic tissue is distributed
throughout the leaf in what has been termed ‘all cell succulence’ (von Willert et al., 1992). This anatomy is likely to
result in a reciprocal relationship between succulence and gM,
and may account for the tight relationship of succulence to
CAM in groups such as the Crassulaceae, Orchidaceae, and
some other families (Teeri et al., 1981; Winter et al., 1983;
Kluge et al., 1991; Nelson et al., 2005; Silvera et al., 2005;
Nelson and Sage, 2008). In contrast, increasing succulence in
taxa such as the Aizoaceae is associated with an inner cortex
of water-storing cells, or epidermal and other peripheral cells
that are achlorophyllous, and has been termed ‘storage succulence’ (Ihlenfeldt, 1989). The chlorenchyma in these species
is often distributed around the periphery of the leaves and is
distinct from the water-storing hydrenchyma (Ernshaw et al.,
1987). Many storage succulents do not show a strong dependence on CAM and assimilate the majority of their carbon
diurnally through the C3 cycle (Rundel et al., 1999; Winter and
Holtum, 2002). These species may not benefit from reduced
gM, which would limit their diurnal CO2 uptake. The anatomy
of storage succulence suggests that they may acquire the benefit of stored water, without becoming gM limited and may
thus be less strongly dependent on CAM. This reasoning suggests no ubiquitous mechanistic link between leaf succulence
and CAM per se, but rather that the co-occurrence of CAM
and succulence should be confined only to leaves that display
all cell succulence (e.g. Silvera et al., 2005; Herrera, 2009).
This does not imply that species that develop leaf succulence via a central achlorophyllous hydrenchyma are precluded from having CAM or being facultatively CAM. For
example, Delosperma tradescantioides can rapidly switch
between C3 and CAM (Herppich et al., 1996). This has been
demonstrated in other storage succulents via intermediate
isotopic values (e.g. Mooney et al., 1977; Rundel et al., 1999)
and by more direct measures of CAM activity (Herppich
et al., 1996; Veste et al., 2001). CAM-idling has been shown
to be important for enduring drought and, subsequent to stomatal closure, the ability to capture and release respiratory
CO2 allows continued light reaction activity that protects the
photosynthetic apparatus from photoinhibition (Ting, 1985;
Adams et al., 1987; Griffiths et al., 1989; Herrera, 2009). Some
succulent species increase photosynthetic efficiency by CAMcycling that can save up to 10% of daily assimilated CO2 that
would otherwise be lost to the atmosphere (Patel and Ting,
1987; Herrera, 2009). These forms of CAM may capture only
a small proportion of assimilated carbon through PEPc, and,
when this is less than a third of total CO2 fixation, do not
have δ13C values distinct from C3 plants (Winter and Holtum,
2002; Herrera, 2009), and hence cannot not be identified in
isotopic surveys for CAM photosynthesis.
A range of Aizoaceae species were used to determine the role
of increasing leaf succulence in prolonging gas exchange and
maintaining photosynthetic integrity during leaf desiccation.
The relationship between increasing succulence, CAM, and
gM was examined and the hypothesis was tested that the anatomy of Aizoaceae succulent leaves avoids low gM and hence
a strong dependence on CAM. This CAM–succulence relationship is contrasted between species from the Aizoaceae and
How succulent Aizoaceae avoid conductance limitations and survive drought | 5487
Crassulaceae. The comparison was thus between plants that
possess storage succulence and those that are all cell succulent.
Materials and methods
Species selection and propagation
For both the Aizoaceae and Crassulaceae, taxa were selected which
showed a range of >5-fold differences in leaf succulence. Species
from the Aizoaceae were selected from three of the four subfamilies in order to cover the phylogenetic and leaf morphological diversity of the group. Two species were selected from the Aizooideae
with small, planar succulent leaves where the photosynthetic tissue is distributed throughout the leaf, termed ‘all cell succulence’
(Galenia africana L. and Tetragonia fruticosa L.); one species from
the Mesembryanthemoideae (Mesembryanthemum cordifolium L.f.)
with planar succulent leaves and ‘all cell succulence’; nine species
from the Ruschieae (Ruschioideae), with subcylindrical to trigonous
shaped succulent, rarely planar leaves, with an inner cortex of waterstoring cells that are achlorophyllous, termed ‘storage succulence’
[Antimima dasyphylla (Schltr.) H.E.K.Hartmann, Carpobrotus edulis
(L.) L. Bolus, Delosperma echinatum Schwantes, D. tradescantioides
L. Bolus, Drosanthemum speciosum Schwantes, Glottiphyllum
depressum N.E.Br., G. longum N.E.Br., Lampranthus aureus N.E.Br.,
and Oscularia cedarbergensis (L. Bolus), H.E.K.Hartmann].
Similarly, from the Crassulaceae, genetic and leaf morphological
diversity within the group were selected, with most species selected
from Crassula, since it is the largest genus of Crassulaceae in South
Africa [Adromischus fallax Toelken, Crassula atropurpurea (Haw.)
Dietr., C. fascicularis Lam., C. ovata (Mill.) Druce, C. arborescens
(Mill.) Willd., C. orbicularis L., C. pellucida L., C. sarmentosa Harv.,
C. multicava Lem., C. cordata Thunb., C. sarcocaulis Eckl.&Zeyh.,
C. dejecta Jacq., C. ericoides Haw., Sedum pachyphyllum Rose,
Tylecodon singularis (R.A.Dyer) Toelken, and T. racemosus (Harv.)
Toelken]. The study included mostly field-collected plants from the
winter-rainfall region of the Cape and a few from the summer-rainfall region of the Eastern Cape, South Africa. In addition, plants
were procured from nurseries, which were established as a collection
of potted plants at the University of Cape Town, Biological Sciences
Department Greenhouse. Plants were grown in native or nursery
soils, were well watered and were supplied with Long Ashton nutrient medium (Hewitt, 1966) modified to contain 2 mm NaNO3 (pH
6.5) during pot establishment.
Dehydration of excised shoots
A subsample of nine species from the family Aizoaceae (A. dasyphylla, M. cordifolium, C. edulis, D. speciosum, G. africana, G. depressum, L. aureus, O. cedarbergensis, and T. fructicosa) were used to
determine the decline in photosynthetic rate (A), stomatal conductance (gST), and potential photochemical efficiency (Fv/Fm) during a
prolonged period of shoot dehydration. Shoots, or individual leaves
in the case of large-leaved species, were excised from plants and
the cut ends were sealed with nail varnish. Shoots or leaves were
enclosed in a conifer chamber of a LI-6400 photosynthesis system (LI-COR Biosciences Inc., Lincoln, NE, USA). The chamber
was illuminated from three sides using dichroic halogen spot lights
(ECO-3000, Eurolux) such that all leaf surfaces received a phosynthetic photon flux density (PPFD) >1500 μmol m–2 s–1, leaf temperatures (estimated from energy balance using instrument software)
controlled at 25 ± 3 °C, and a vapour pressure deficit (VPD) <1.5
kPa. Gas exchange parameters were recorded at intervals during
the next 10–20 h. Measurements were made in triplicate and shoots
or leaves were removed from the chamber between measurements,
weighed, and kept under common (~25 °C, PPFD ~100 μmol m–2
s–1) conditions in the laboratory. The photosynthesis system was
‘matched’ before each data point was recorded and after gST and
photosynthesis were stable at the prevailing conditions.
At the end of the experiment, leaf areas were measured by scanning cylindrical or ovoid leaves from two sides and triangular
leaves from three sides. The accuracy of this method was checked
by applying the same method to cylindrical, ovoid, and triangular shapes of known surface area. Gas exchange parameters were
expressed on the basis of these multisided leaf areas (in contrast
to the norm of expressing only on the area of one side of planar
leaves).
Leaf or shoot turgid weights were recorded prior to the first measurement, immediately after the leaves or shoots were excised from
well-watered plants. Dry weights were determined at the end of the
experiments after drying plant material in an oven at 60 °C to constant weight. Leaf succulence was calculated as (turgid weight–dry
weight)/dry weight, and leaf water content (WC) as (wet weight–dry
weight)/turgid weight.
In a similar but independent experiment, the reduction photochemical efficiency of photosystem II (PSII; Fv/Fm) over a long
period of dehydration was measured. Excised leaves or shoots were
dark-adapted for 60 min using leaf clips, and chlorophyll fluorescence parameters were recorded using a Walz PAM 2000 fluorometer
(Walz, Effeltrich, Germany). After measurements, leaves of shoots
were stored on a sunlit windowsill in the laboratory and measurements were repeated routinely for a period of 40 d. Measurements
were made in duplicate for each plant species.
For each individual replicate, the decay in A, gST, and WC over
time (t) were fitted with the equation:
A, gST or WC =
a
1 + b exp(−ct ) (1)
The declines in A and gST in response to decreasing leaf WC were
fitted with the same equation, but WC was substituted for t in the
equation above. The decrease in Fv/Fm over time was fitted with the
equation:
Fv
1
=
Fm a + bt( c ) (2)
The parameters a, b, and c were fitted using least squared differences
(using the Solver add-in of Microsoft Excel). Equation 1 was used
to derive the time required to reduce A or gST by 50%, and WC by
10%. Equation 1 was also used to calculate the WC, and the reduction in turgid WC (turgid WC–WC), required to decrease A and gST
by 50%. The latter measure accounts for the positive relationship
between succulence and WC, and shows the effect of water loss from
the initial turgid WC, and not from 100%. Equation 2 was used to
calculate the time required to decrease Fv/Fm by 10%. The choice of
10% or 50% for calculating the decline constants of A, gST, WC, and
Fv/Fm was to ensure that estimates were not extrapolated beyond the
measured data.
A:Ci responses and mesophyll conductance determinations
Individual leaves of shoots were excised from the subsample
of nine Aizoaceae species and cut ends were sealed into waterfilled Eppendorf tubes using multiple wraps of Parafilm (Brand,
Wertheim, Germany). These were then enclosed in a conifer
chamber (Licor 6400-005) of a Licor 6400 photosynthesis system with chamber conditions set as before. The response of A
to intercellular CO2 concentration (Ci) was constructed according to Long and Bernacchi (2003) and individual replicate (n
≥3) curves fitted according to von Caemmerer (2000). Fitted
parameters were used to generate A:Ci responses with points at
2 Pa intervals. gM was then determined according to the constant J method (Warren, 2006), where the rate of linear electron
transport (Ja) was calculated from generated gas exchange data
according to Equation 3.
5488 | Ripley et al.
4  (Ci − A / gM ) + 2τ *
J a = ( A + Rd ) 
(Ci − A / gM ) − τ *
(3)
Where A=photosynthetic rate, Rd=dark respiration, Ci=intercellular
CO2 partial pressure, gM=mesophyll conductance, and τ*=the photorespiratory compensation point. The value of gM was obtained by
iteratively changing the value between the limits of 0.01 μmol m–2 s–1
Pa–1 and 10 μmol m–2 s–1 Pa–1. The gM value was selected that minimized the variance between Ja values determined at each Ci value
(2 Pa intervals, between 90 Pa and 130 Pa) and the average Ja value
calculated over this same range in partial pressures. This method
of calculating gM has acknowledged limitations (Pons et al., 2009;
Tholen et al., 2013), but given that the measurements were made on
a range of species within a single family and that the plants were all
grown under common conditions, the method retains its value for the
interspecific comparison made in this study.
In some species it was possible to align the fibre optic of the fluorometer with leaves enclosed in a modified conifer chamber during
the construction of A:Ci curves. This modification involved replacing the hinged ‘conifer chamber’ lid with a Perspex plate equipped
with a port for the fibre optic probe. In these cases, simultaneous gas
exchange and PSII fluorescence measurements were used to confirm
that ФPSII was constant when Ci ranged from 90 Pa to 130 Pa.
Leaf δ13C isotopic ratios and nitrogen concentration
Whole leaves were removed from well-watered plants for the entire
Crassulaceae and Aizoaceae species collection (n=3) at ~10:00 h.
Leaf areas, and fresh and dry weights were recorded, and leaves were
oven-dried at 60 °C to constant weight. Dried leaves were ground
to a homogeneous powder using a Wiley mill with a 0.5 mm mesh
(Arthur H. Thomas, CA, USA) and weighed into 8 × 5 mm tin capsules (Elemental Microanalysis Ltd, Devon, UK). Additional leaves
from well-watered plants and plants dehydrated for 1 or 2 weeks were
used to make extracts of leaf carbohydrates. Fresh leaves collected at
~10:00 h were rapidly transferred into chilled 80% methanol, homogenized in a mixer-mill (Retsch MM 200, Retsch, Haan, Germany),
and centrifuged in a microfuge. The supernatant was transferred
to ion exchange columns for the separation of the neutral fraction
(mostly carbohydrate) using a modification of the method of Atkins
and Canvin (1971). The column was filled with a mixed bed ionexchange resin (Dowex Marathon MR-3, Sigma-Aldrich, St. Louis,
MO, USA) with deionized water as the mobile phase. The eluted
neutral fraction was collected, air-dried under an N2 stream, and
re-dissolved in 1 ml of water. Aliquots (50 μl) of this sample were
placed into mass spectrometer tin capsules and oven-dried at 65 °C.
The neutral fraction of the methanol extraction contained no N,
demonstrating that the carbohydrates had been effectively separated
from amino compounds.
Additional leaves harvested from well-watered plants in the
Aizoaceae were, where possible, separated into achlorophyllous
hydrenchyma and chlorophyllous mesophyll tissue using a sterile
scalpel blade. Separated tissues were oven-dried, weighed, milled,
and subject to elemental analysis to determine tissue N concentrations. The stable carbon isotope ratios (δ13C) and tissue N concentrations for the foliar samples and the δ13C values for the carbohydrate
samples were determined using a Thermo Flash EA 1112 series elemental analyser (Thermo Electron Corporation, Milan, Italy) and
Delta Plus XP isotope ratio mass spectrometer (Thermo Electron
Corporation).
Estimates of photosynthetic and achlorophyllous
cross-sectional areas
Fresh leaves taken from a subset of 10 species from the Crassulaceae
and 11 species from the Aizoaceae were hand sectioned with a
razor blade and photographed with a light microscope (Nikon,
SMZ 1500). Digital micrographs were converted to 8-bit black and
white images, and photosynthetic and achlorophyllous areas were
measured using the ‘Analyse Particles’ function in Image-J (Version
1.44p).
Results
In order to retain graphical clarity, the responses of A, gST,
WC, and Fv/Fm to shoot excision and A:Ci responses are presented for select examples of species in the Aizoaceae. Data
for the remaining species are given in the Supplementary
Figures available at JXB online.
Dehydration of excised shoots
A, gST, and WC declined over time after leaf or shoot
excision, were non-linear, and varied between species
(Fig. 1A–C; Supplementary Fig. S1 at JXB online). The
time required to reduce A and gST by 50% (t50%), and WC by
10% (t10%), was calculated from these curves and showed a
linear response to increasing succulence (Fig. 1D–F). The
most succulent species had t50% values for A and gST that
were >10-fold larger than those of the least succulent species. The t50% values for A were higher than those for gST,
indicating that photosynthesis continued despite changes
in gST. This is due to a curvilinear relationship between A
and gST, particularly at high values of gST (data not shown).
Water loss was sustained for longer from more succulent
leaves, which also had the highest WC, and lost the initial
10% of leaf WC 10-fold more slowly than the least succulent species.
The manner in which A and gST declined with WC
was non-linear and varied between species (Fig. 2A, B;
Supplementary Fig. S2 at JXB online). The WC at which
A and gST were decreased by 50% was strongly correlated
to leaf succulence (Fig. 2C, D) and showed that the most
succulent species were the least tolerant of leaf dehydration in comparison with the least succulent species. There
was no correlation between succulence and the decline in
turgid leaf WC required to halve A (P > 0.5) or gST (P >
0.11; Fig. 2E, F). This expression accounts for the fact that
the turgid WC of leaves varied with the degree of succulence. This shows that both A and gST were decreased by
50% when turgid WC was decreased by between 6% and
16% (average of 11%) across all the Aizoaceae species sampled. Only L. aureus tolerated more severe dehydration and
required a reduction in WC of 23% to halve photosynthesis
(Supplementary Fig. S2).
Similarly, the decline in potential PSII photochemical efficiency (Fv/Fm) differed between species (Fig. 3A;
Supplementary Fig. S3 at JXB online). However, a much
longer dehydration period was required to affect Fv/Fm than
either A or gST, and the most succulent species exhibited very
little change in Fv/Fm over a period of up to 20 d. As for A
and gST, however, the derived times required to reduce Fv/Fm
by 10% (t10%) were also positively correlated with increasing
succulence, such that the most succulent species had a t10% of
37 d, nearly 18-fold longer than the least succulent species
(Fig. 3B).
How succulent Aizoaceae avoid conductance limitations and survive drought | 5489
Fig. 1. Decline over time in (A) average photosynthesis (A), (B) stomatal conductance (gST), and (C) water content (WC) for selected
examples of Aizoaceae species following leaf or shoot excision. Average time required to decrease (D) A and (E) gST by 50%, and (F)
WC by 10%, for nine Aizoaceae species (n=3; mean ±SE). Linear regression lines with coefficients of determination and significance of
correlations are shown. Time response data for A. dasyphylla, C. edulis, D. speciosum, L. aureus, O. cedarbergensis, and T. fruticosa
are available in Supplementary Fig. S1 at JXB online.
Leaf δ13C isotopic ratios
The δ13C values for leaves harvested from the 16 wellwatered Crassulaceae species were positively correlated
to leaf succulence (Fig. 4A). The range of values spanned
from those that would be typical of C3 to CAM plants
(Winter and Holtum, 2002). A similar relationship constructed for 12 of the Aizoaceae species showed a 4-fold
weaker relationship (i.e. slope), and all values fell within the
range typical of C3 plants or where CAM contributes only
a small proportion to total carbon assimilation (Fig. 4B).
To test if CAM could be induced by water stress, the δ13C
values of carbohydrates extracted from well-watered leaves
were compared with those from water-stressed plants.
Water stress made δ13C less negative, by on average 1.0‰
and 1.5‰ after 7 d and 14 d, respectively (Fig. 4C). This
small increase in δ13C is more likely to reflect the effect
of decreased gST on gas exchange than a switch to CAM,
which should have a much larger effect on the δ13C of
recently assimilated carbohydrates.
5490 | Ripley et al.
Fig. 2. (A and B) Decline in average photosynthesis (A) and stomatal conductance (gST) in response to water content (WC) for selected
examples of Aizoaceae species following leaf or shoot excision. (C and D) Average WC at which A and gST were reduced by 50% for
nine Aizoaceae species. (E and F) Average reduction in turgid WC required to decrease A and gST by 50% for nine Aizoaceae species
(n=3; mean ±SE). Linear regression lines with coefficients of determination and significance of correlations are shown. Water content
response data for A. dasyphylla, C. edulis, D. speciosum, L. aureus, O. cedarbergensis, and T. fruticosa are available in Supplementary
Fig. S2 at JXB online.
Photosynthetic cross-sectional area and leaf N content
Leaf N concentrations of neither the achlorophyllous hydrenchyma nor the photosynthetic mesophyll (Table 1) were correlated to succulence (all R2 < 0.05, data not shown). The
N contents of the achlorophyllous hydrenchyma were high,
being on average (across species) 26 ± 6% of the total leaf N
content, suggesting that this could be an important N store in
these leaves. The cross-sectional area of photosynthetic tissue
was linearly correlated to succulence in both the taxonomic
groups; however, the slope of the relationship was 3-fold
higher in the Crassulaceae than in the Aizoaceae (Fig. 5). The
difference in the slopes results from the fact that the more
succulent species of the Aizoaceae possess achlorophyllous
hydrenchyma while the less succulent Aizoaceae species and
the Crassulaceae exhibit chlorophyllous ‘all cell succulence’.
A:Ci responses and mesophyll conductance
A:Ci responses for the Aizoaceae (Fig. 6A; Supplementary
Fig. S4 at JXB online) produced a wide range of Vcmax
(16–70 μmol m–2 s–1) and Jmax values (41–148 μmol m–2 s–1;
Supplementary Table S1 at JXB online) that were significantly
different between the species (Vcmax, F8,22=11.3, P < 0.0001;
Jmax, F8,22=8.0, P < 0.0001). These parameters were not correlated to leaf succulence (all R2 >0.1; data not shown).
How succulent Aizoaceae avoid conductance limitations and survive drought | 5491
Fig. 3. (A) Decline in efficiency of PSII (Fv/Fm) for individual
replicates of selected examples of Aizoaceae species following
leaf or shoot excision. (B) Time required to decrease Fv/Fm by
10% for nine Aizoaceae species (n=2). For each individual,
replicate data are shown. Linear regression lines with coefficients
of determination and significance of correlations are shown. Fv/
Fm response data for A. dasyphylla, C. edulis, D. speciosum,
L. aureus, O. cedarbergensis, and T. fruticosa are available in
Supplementary Fig. S3 at JXB online.
The gM values derived from the CO2-saturated portions of
the A:Ci responses were significantly different between species
(F8,22=4.1, P < 0.004) and ranged from 0.59 μmol m–2 s–1 Pa–1
to 1.73 μmol m–2 s–1 Pa–1, excluding the apparent outlier value
of 5.9 μmol m–2 s–1 Pa–1 for C. edulis (Fig. 6B). The gM values
were not correlated to leaf succulence (P > 0.11).
Discussion
Fig. 4. δ13C (‰) values for well-watered leaves for species
from (A) Crassulaceae (n=16) and (B) Aizoaceae (n=12). Linear
regression lines with coefficients of determination and significance
of correlations are shown. (C) Response of δ13C values for select
species from the Aizoaceae that were well watered or subject to
water stress for 7 d or 14 d.
As hypothesized, increasing succulence in the Aizoaceae was
not accompanied by a decrease in mesophyll conductance
(gM). The range of gM values of the Aizoaceae that were measured were generally higher than those reported for the CAM
succulent Kalanchoe daigremontiana (~0.5 μmol m–2 s–1 Pa–1;
Maxwell et al., 1997; Griffiths et al., 2007). The present data
are, however, expressed on a total leaf area basis since several
5492 | Ripley et al.
Table 1. Leaf succulence and N concentrations (%, w/w) measured separately for achlorophyllous hydrenchyma and photosynthetic
tissue (where possible) for nine species of Aizoaceae
Species
Succulence (g H2O g–1 dry weight)
Achlorophyllous hydrenchyma N (%)
Chlorophyllous mesophyll N (%)
Antimima dasyphylla
Mesembryanthemum cordifolium
Carpobrotus edulis
Drosanthemum speciosum
Galenia Africana
Glottiphyllum depressum
Lampranthus aureus
Oscularia cedarbergensis
Tetragonia fruticosa
6.0 ± 0.9
17.1 ± 1.6
20.0 ± 2.3
14.8 ± 0.6
4.5 ± 0.5
28.0 ± 3.4
18.1 ± 0.6
25.5 ± 3.8
11.6 ± 2.5
–
–
3.83 ± 0.77
0.73 ± 0.06
–
0.59 ± 0.12
2.51 ± 0.15
1.79 ± 0.48
–
1.97 ± 0.38
1.78 ± 0.12
2.91 ± 0.04
1.15 ± 0.12
2.98 ± 0.23
1.73 ± 0.14
3.95 ± 0.30
2.45 ± 0.09
1.23 ± 0.06
Values are means ±SE (n=3).
Fig. 5. Variation in cross-sectional area of chlorophyllous tissue
of Crassulaceae and the Aizoaceae species (n=11 species for
both) with succulence. Linear regression lines with coefficients of
determination and significance of correlations are shown.
leaves were cylindrical or triangular in cross-section, rather
than as usual for single surfaces of planar leaves, and thus
equivalent values for K. daigremontiana would be ~0.25 μmol
m–2 s–1 Pa–1. The present values mostly fall withinthe range of
gM values reported for C3 species (with correction to consider
both leaf surfaces: ~0.38–2.18 μmol m–2 s–1 Pa–1; Flexas et al.,
2007). This high gM in the Aizoaceae is remarkable considering how succulent some of these leaves are, and that these
succulent leaves largely lack mesophyll airspaces, which contribute to high gM in many C3 species. The relatively high gM
of the Aizoaceae is, however, readily explained by the fact that
anatomically the photosynthetic tissue is in a thin band adjacent to the leaf surface. Avoiding the limitations of low gM
via anatomical modifications is not unique to the Aizoaceae,
and stomatal crypts play a similar role in Banksia leaves with
high dry mass per area (Hassiotou et al., 2010). Additionally,
limiting the water-demanding photosynthetic tissue to a thin
Fig. 6. (A) A:Ci responses of selected examples of Aizoaceae
species. Fitted equations for each replicate were used to
interpolate data to a common series of Ci values for the calculation
of means and standard errors. (B) Derived mesophyll conductance
values (gM) for nine Aizoaceae species. For each species n ≥3, and
vertical bars are standard errors. A:Ci responses for A. dasyphylla,
D. speciosum, L. aureus, O. cedarbergensis, and T. fruticosa are
available in Supplementary Fig. S6 at JXB online).
How succulent Aizoaceae avoid conductance limitations and survive drought | 5493
layer and associating this with three-dimensional venation
(Ogburn and Edwards, 2013) would maintain favourable
hydraulic path lengths, whilst allowing the development of
succulent leaves. This hydraulic supply would be important
for the uptake and recharge of photosynthetic water storage
tissue and to service photosynthetic gM and the water loss
from photosynthetic surfaces (Griffiths, 2013).
The Aizoaceae lack a strong dependence on CAM, as was
evident from the δ13C isotope values, which were all more
negative than –21‰, which is the cut-off value determined for
CAM species (Winter and Holtum, 2002). The average δ13C
value was –29.6‰, which indicates that >60% of carbon gain
would have occurred diurnally via the C3 cycle (Winter and
Holtum, 2002). The fact that the δ13C values only increased
by 1.5‰ after 14 d of water stress indicates that the high δ13C
values relative to those of typical CAM plants were not due
to the well-watered conditions under which the plants were
generally maintained, and that CAM is not induced in these
species by water stress. This small shift in δ13C in response to
water stress could be accounted for by changes in the Ci/Ca
induced by stomatal closure, as in C3 species (Farquhar et al.,
1989).
The succulent Aizoaceae leaves utilized the water stored
in the achlorophyllous hydrenchyma to prolong positive
gas exchange and allowed leaves to withstand long periods
of drought. This photosynthetic buffering and the ability to
endure drought have been demonstrated in numerous succulent species and not just those with achlorophyllous hydrenchyma (e.g. Nobel, 1976; Hoffman et al., 2009). Interestingly,
in the Aizoaceae, the more succulent species were least physiologically tolerant of lowered leaf tissue water content. The
positive correlation between succulence and the WC required
to halve A and gST meant that the gas exchange of the most
succulent species responded at the highest water contents.
When the differences in initial turgid WC were accounted for,
it was apparent that when the initial WC was decreased by
6–16%, the photosynthetic and stomatal response were evident irrespective of the degree of leaf succulence. Hence, the
mechanism for sustained gas exchange was not due to tolerance of dehydration, but the most succulent species had a
large volume of water to buffer dehydration. Maintenance of
photosynthetic tissue water relations at the expense of water
loss from the hydrenchyma has been demonstrated for various species (Schmidt and Kaiser, 1987; Herrera et al., 2000;
Nobel, 2006), and interspecific differences in this might
account for the range in WC loss required to produce the
observed gas exchange responses.
Once positive gas exchange had ceased, the capacity for
photosynthesis was maintained for long periods of drought
and was directly correlated to the degree of leaf succulence.
Most succulent species reached t50% for A within 7 h, but t10%
for Fv/Fm only occurred after 38 d, indicating that the energy
provision from the photosynthetic lights reactions continued
even in the absence of net CO2 acquisition. Maintenance of
photochemistry is likely to be important for energy provision
to offset maintenance costs. Furthermore, a functioning photochemical system might enable rapid resumption of carbon
assimilation after rainfall events. Such rapid gas exchange
responses to water inputs have been demonstrated in various
leaf succulents including Ruschia caroli (Aizoaceae; Midgley
and van der Heyden, 1999), Mesembryanthemum pellitum
(Aizoaceae), and Othonna optima (Asteraceae; Eller et al.,
1993). The maintenance of photochemical integrity may
be aided by CAM-idling, or drought-induced low-level
CAM, which has been demonstrated in D. tradescantioides
(Aizoaceae; Herppich et al., 1996). CAM-idling does not
contribute to overall carbon assimilation and hence does not
result in discernible CAM δ13C signal (Winter and Holtum,
2002). Consequently, the importance of these mechanisms
may have been overlooked in many succulent species that
have C3-like isotopic values and would not have been discernible with the present measurements.
The species distribution of the Aizoaceae in the GCFR
overlaps that of the Crassulaceae (von Willert et al., 1990;
Jürgens, 1997; Goldblatt and Manning, 2002), many of
which are constitutive CAM plants (e.g. Mooney, 1977). Of
the ~659 species of the Aizoaceae, ~524 (80%) are endemic
to the winter rainfall GCFR. In contrast, only 35 (26%)
of 134 species of Crassulaceae are endemic to this region
(Goldblatt and Manning, 2002). Where the Crassulaceae are
found in the GCFR, their centre of diversity is in a region
of higher mean annual precipitation (200–290 mm) and less
severe summer drought in the Cedarberg mountains and
surrounding valleys and the Little Karoo (Jürgens, 1997;
Mucina et al., 2006; Rebelo et al., 2006). The centre of diversity for the succulent Aizoaceae (subfamilies Ruschioideae
and Mesembranthemoideae) occurs at lower (90–164 mm)
strongly winter rainfall, in the Richtersveld and Knersvlakte
(Hartmann, 1991). It is speculated that the peripheral photosynthetic tissue and achlorophyllous hydrenchyma of the succulent Aizoaceae may be especially suited to the wet winters
and dry summers found in this region. The greater dependence on CAM of the Crassulaceae may be associated with the
fact that these plants receive moisture and grow during the
hot summers.
The onset of the growing season in the Succulent Karoo
is early May, reaching the mid-growing season in August,
with growth ceasing by early January (Wessels et al., 2011).
The combination of cold and rain eliminates water stress for
the winter and some of the spring period, during which time
CAM would not be beneficial. Under this climate scenario,
the role of succulence in the Aizoaceae may be to extend the
growing season into the drier and warmer period of early summer, after which the plants may become dormant during the
hot dry period of late summer. Thus, the engagement of C3
photosynthesis interspersed with periods of dormancy in net
CO2 acquisition, with or without weak CAM or CAM-idling,
is an alternative to constitutive CAM in these seasonally arid
environments. A similar habitat differentiation based on rainfall has been observed for CAM plants in Madagascar. Here,
CAM plants occur in dry climatic zones or in dry micro-habitats, whereas facultative CAM physiotypes occur in less stressful environments (Kluge et al., 2001). Because CAM is a more
energetically demanding photosynthetic system than C3 (von
Willert et al., 1992; Winter and Smith, 1996; Lüttge 2004), it
might not be ubiquitously suitable for Mediterranean climates.
5494 | Ripley et al.
CAM is particularly beneficial in hot conditions where it enables positive C acquisition, unlike C3 photosynthesis which
becomes increasingly compromised by photorespiratory CO2
losses at higher temperatures (Nobel, 1991). Thus, although
both the Aizoaceae and Crassulaceae tolerate drought, the
seasonality of rainfall and the growing seasons may result in
different photosynthetic strategies being beneficial.
While succulence is commonly associated with retaining tissue hydration, an additional limitation of seasonally arid environments is the availability of soil nutrients, which require water
to become soluble and accessible to plants (Cui and Caldwell,
1997). This limitation is particularly pertinent in shallow soils or
where roots are confined to rock crevices and small soil pools,
as is the case for many succulents in the CFR (Midgley and van
der Heyden, 1999). The extension of the period over which gas
exchange can occur during water stress can only benefit growth
if there is a simultaneous availability of nutrients for new tissue expansion. It is likely that succulent plants increase nutrient uptake during periods of water availability and store those
nutrients. This storage of nutrients is an additional role for the
achlorophyllous hydrenchyma in the succulent Aizoaceae and
explains the high N contents measured in these tissues.
Supplementary data
Supplementary data are available at JXB online.
Figure S1. Decline over time in average photosynthesis (A),
stomatal conductance (gST), and water content (WC) for the
indicated Aizoaceae species following leaf or shoot excision
(n=3; mean ±SE).
Figure S2. Decline in average photosynthesis (A) and stomatal conductance (gST) in response to water content (WC)
for the indicated Aizoaceae species following leaf or shoot
excision (n=3; mean ±SE).
Figure S3. Decline in initial Fv/Fm over time for the indicated Aizoaceae species following leaf or shoot excision. For
each species n=2 and hence individual replicate and not average data are presented.
Figure S4. A:Ci responses for the indicated Aizoaceae species. For each species n ≥3, and vertical bars are standard
errors.
Acknowledgements
Peter Bruyns (Bolus Herbarium, University of Cape Town)
is thanked for supplying leaf samples of the Crassulaceae.
Ian Newton (Department Archeometry, University of Cape
Town) is thanked for conducting the mass spectrometer analyses. Funding for this research was from Rhodes University
(BSR) and the University of Cape Town (MDC).
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