Crassulacean acid metabolism-cycling in Euphorbia milii

Photosynthetic characteristics of Euphorbia milii are reported for the first time. The occurrence of CAM-cycling is shown to serve as a mechanism of water conservation. This is a detailed and novel report of such CAM mode in the genus, which is abundant in constitutive CAM and C₄ species. Anatomical evidence for the possible operation of a CO₂-concentrating mechanism around the vascular bundle, the C₂ route, is provided. Findings are important for our understanding of evolution of CAM in the genus.

Abstract

Crassulacean acid metabolism (CAM) occurs in many Euphorbiaceae, particularly Euphorbia, a genus with C₃ and C₄ species as well. With the aim of contributing to our knowledge of the evolution of CAM in this genus, this study examined the possible occurrence of CAM in Euphorbia milii, a species with leaf succulence and drought tolerance suggestive of this carbon fixation pathway. Leaf anatomy consisted of a palisade parenchyma, a spongy parenchyma and a bundle sheath with chloroplasts, which indicates the possible functioning of C₂ photosynthesis. No evidence of nocturnal CO₂ fixation was found in plants of E. milii either watered or under drought; watered plants had a low nocturnal respiration rate (R). After 12 days without watering, the photosynthetic rate (P_N) decreased 85 % and nocturnal R was nearly zero. Nocturnal H⁺ accumulation (ΔH⁺) in watered plants was 18 ± 2 (corresponding to malate) and 18 ± 4 (citrate) μmol H⁺ (g fresh mass)⁻¹. Respiratory CO₂ recycling through acid synthesis contributed to a night-time water saving of 2 and 86 % in watered plants and plants under drought, respectively. Carbon isotopic composition (δ¹³C) was −25.2 ± 0.7 ‰ in leaves and −24.7 ± 0.1 ‰ in stems. Evidence was found for the operation of weak CAM in E. milii, with statistically significant ΔH⁺, no nocturnal CO₂ uptake and values of δ¹³C intermediate between C₃ and constitutive CAM plants; ΔH⁺ was apparently attributable to both malate and citrate. The results suggest that daily malate accumulation results from recycling of part of the nocturnal respiratory CO₂, which helps explain the occurrence of an intermediate value of leaf δ¹³C. Euphorbia milii can be considered as a CAM-cycling species. The significance of the operation of CAM-cycling in E. milii lies in water conservation, rather than carbon acquisition. The possible occurrence of C₂ photosynthesis merits research.

Introduction

Crassulacean acid metabolism (CAM) is of frequent occurrence among the Euphorbiaceae and has appeared polyphyletically several times in the family, particularly in the genus Euphorbia. In this genus, C₄ species seem to be rare, whereas they are abundant in the genus Chamaesyse (Webster et al. 1975). In Euphorbia, CAM has been reported in 21 species, and values of δ¹³C suggest its presence in 44 species (Table 1). Several of these species belong to three different clades within the genus (cladograms in Zimmermann et al. 2010). Twenty-four species can be considered constitutive CAM on the basis of having values of δ¹³C higher than −17 ‰, a criterion established by Mooney et al. (1977). In the remaining species, values of δ¹³C average −24.7 ‰. A value as low as −28.9 ‰ found in E. aphylla falls into the lower mode of the bimodal frequency distribution of δ¹³C in CAM plants, designated as low-level (weak) CAM (Winter and Holtum 2002; Silvera et al. 2005).

Table 1.

Carbon isotopic composition of species of Euphorbia with high to intermediate values of δ¹³C and CAM mode assigned by authors on the basis of leaf gas exchange, acid accumulation, δ¹³C and enzyme activity.

Species	δ13C (‰)	Mode	Reference
angusta	−24.9	NA	Webster et al. (1975)
antiquorum	−14.2	NA	Batanouny et al. (1991)
aphylla	ND	Facultative	Mies et al. (1996)
avasmontana	−15.1	Constitutive	Mooney et al. (1977)
bothae	−14.6	Constitutive	Mooney et al. (1977)
bubalina	−13.2	NA	Webster et al. (1975)
burmannii	−18.3	NA	Mooney et al. (1977)
caducifolia	ND	Constitutive	Webster et al. (1975), Sayed (2001)
caput-medusae	−13.3	Constitutive	Mooney et al. (1977)
cyathophora	−25.8	NA	Mies et al. (1996)
didieroides	−26.6	NA	Webster et al. (1975)
didieroides	−24.3	NA	Winter (1979)
dregeana	ND	Constitutive	Sayed (2001)
drupifera	−14.1	NA	Webster et al. (1975)
gariepina	−14.6	Constitutive	Mooney et al. (1977)
genoudiana	−22.7	NA	Winter (1979)
gorgonis	−12.9	Constitutive	Mooney et al. (1977)
gragaria	−11.6	Constitutive	Mooney et al. (1977)
grandidens	ND	Constitutive	Webster et al. (1975), Sayed (2001)
inermis	−13.4	Constitutive	Mooney et al. (1977)
ingezalahiana	−23.6	NA	Winter (1979)
inocua	−28.1	NA	Webster et al. (1975)
leucodendron	−13.2	NA	Winter 1979
macropodoides	−28.3	NA	Webster et al. (1975)
macropus	−28.9	NA	Webster et al. (1975)
mauritanica	−16.0	Constitutive	Mooney et al. (1977)
milii	ND	Non-CAM	Webster et al. (1975)
milii		CAM	McWilliams (1970)
nesemannii	−11.6	Constitutive	Mooney et al. (1977)
nivulia	−15.7	NA	Webster et al. (1975)
nubica	−14.5	NA	Batanouny et al. (1991)
pentagona	−14.9	Constitutive	Mooney et al. (1977)
peperomioides	−25.6	NA	Webster et al. (1975)
plagiantha	−13.2	NA	Winter (1979)
polygona	−10.7	Constitutive	Mooney et al. (1977)
pulcherrima	−25.5	NA	Mies et al. (1996)
squarrosa	−12.5	Constitutive	Mooney et al. (1977)
stenoclada	−12.6	NA	Winter (1979)
submamillaris	ND	Constitutive	Webster et al. (1975), Sayed (2001)
tetragona	−14.7	Constitutive	Mooney et al. (1977)
thi	−13.2	NA	Batanouny et al. (1991)
tirucalli	−15.3	Constitutive	Mies et al. (1996)
triangularis	−13.6	Constitutive	Mooney et al. (1977)
trigona	−19.4	NA	Webster et al. (1975)
xylophylloides	ND	Constitutive	Webster et al. (1975), Sayed (2001)

NA, no mode assigned; ND, not determined.

Since values of δ¹³C alone are not sufficient to distinguish between C₃ species and plants that obtain up to one-third of their carbon during the night, which include weak CAM plants (Winter and Holtum 2002), measurements of physiological and biochemical variables are necessary. In order to demonstrate the operation of CAM, routine determinations include, among others, ΔH⁺, δ¹³C and nocturnal CO₂ fixation. Griffiths et al. (2007) devised an ingenious method of ascertaining the occurrence of nocturnal CO₂ fixation by examining the response of the night-time CO₂ exchange rate to intercellular CO₂ concentration (C_i).

Intermediate values of δ¹³C can also suggest the occurrence of C₃ metabolism with high water-use efficiency, as the data of Farquhar and Richards (1984) on wheat indicate, or of C₂ photosynthesis, as in the case of Euphorbia acuta. In wheat and maize, C₂ photosynthesis is responsible for an increase of 8–11 % in photosynthetic rate through re-assimilation of photorespired CO₂ (Busch et al. 2013).

Plants of Euphorbia milii subgenus Euphorbia, Section Goniostema, common name crown of thorns, originally from Madagascar, are cultivated worldwide for their ornamental value. Plants are perennial armed shrubs as tall as 1 m, with fleshy stem and branches, and partly succulent leaves. According to observations by Mooney et al. (1977), CAM is present in the weak mode in leafy species of the genus. The medicinal and molluscicidal properties of the latex in E. milii have been extensively investigated (e.g. Mwine and Van Damme 2011); in contrast, literature on the physiology of the species is practically non-existent.

In spite of the succulence of its leaves and the various reports of CAM in the genus, E. milii has been reported as non-CAM (Webster et al. 1975). Nevertheless, recalculation of the data of McWilliams (1970) gives a ΔH⁺ of 100 μmol (g fresh mass)⁻¹ and a dark CO₂ fixation rate of 0.1 μmol m⁻² s⁻¹, suggesting that CAM in E. milii operates in the cycling mode, i.e. nocturnal H⁺ accumulation and daytime but nearly no night-time CO₂ fixation (for the definition of CAM modes, see Cushman 2001).

With the aim of contributing to our knowledge of the evolution of CAM in Euphorbia, this study re-examined the possible occurrence of CAM in E. milii through daily leaf gas exchange, including P_N/C_i and R/C_i curves (where P_N is the photosynthetic rate and R is the respiration rate), measurements of dawn and dusk H⁺ content, and determinations of δ¹³C.

Methods

Plant material and cultivation

Plants of E. milii were propagated from one plant purchased at a nursery by inserting cuttings into the soil of 2-L pots filled with silty clay loam (Viveros Exotica Raphia, S.R.L., Caracas); plants were fertilized monthly with N : P : K 15 : 15 : 15 and grown in the garden for 1 year before the beginning of experiments. Plants, ∼50 cm tall, were maintained in the greenhouse under natural light, fully watered every other day and fertilized weekly. Day length was 12 h (06:00–18:00 h), mean maximum daily photosynthetic photon flux density (PPFD) between 09:00 and 14:30 h 507 ± 22 μmol m⁻² s⁻¹, mean air temperature 32 ± 5/18.4 ± 0.5 °C (day/night) and relative humidity 60 ± 10 %. Water deficit was imposed by withholding watering.

Anatomy

Free-hand cross-sections of stems (average thickness 5 mm) and leaves were observed under the microscope at ×40 (stem) and ×400 (leaf). Leaf sections were stained with toluidine blue.

Succulence

Leaf and stem water content was determined as the difference between the fresh mass (FM) and the mass after drying for 72 h at 60 °C [dry mass (DM)], divided by the area in the case of leaves (FM/A) and by DM in the case of stems. Leaf thickness was measured with precision calipers. Chlorophyll (Chl) content was determined after Bruinsma (1963) in 80 % cold acetone extracts of leaf or stem sections collected at 18:00 h. The mesophyll succulence index was calculated as S_m = g water (mg Chl)⁻¹ after Kluge and Ting (1978).

Stable carbon isotope composition

The δ¹³C was determined with a precision of 0.15 ‰ using a ThermoFinnigan DeltaPlusXL Isotope Ratio Mass Spectrometer (San Jose, CA, USA) and PDB as the standard.

Nocturnal H⁺ accumulation

Whole leaves were weighed fresh and set to boil in 50 mL distilled water for 10 min in a microwave oven at maximum power; samples were sieved through a plastic colander, leaf segments and the colander were rinsed, and the solution was made up to 100 mL. Samples were titrated to pH 7.0 for the estimation of H⁺ corresponding to malate according to Nobel (1988), and to pH 8.4 for citrate. Since Franco et al. (1990) noted that there was a strong linear relationship between concentrations of malate and citrate determined enzymatically and by titration, in the absence of an enzymatic method for the determination, titration is an adequate alternative. Latex was collected from cut stems and leaves, suspended in distilled water and titrated likewise. The ΔH⁺ was calculated as the difference between dawn and dusk H⁺ contents.

Leaf gas exchange

The P_N, R, stomatal conductance (g_s) and transpiration rate (E) were measured in the laboratory with a CIRAS 2 IRGA connected to a PLC(B) assimilation chamber (PP Systems, Amesbury, MA, USA) at an incoming CO₂ concentration (C_a) of 380 μmol mol⁻¹, a chamber temperature tracking ambient (24 ± 1 °C) and an incident PPFD of 200 (the first morning hours) or 1000 μmol m⁻² s⁻¹ (the rest of the daytime). Records were automatically taken every 30 min. Response curves were done in six different leaves of P_N and C_i between 10:00 and 11:00 h, and of nocturnal CO₂ exchange to C_i between 20:00 and 05:00 h.

Statistics

Values are mean ± SE (n = 6). Statistical significance was assessed where indicated through one- or two-way analysis of variance (ANOVA) (P < 0.05) with the Statistica package.

Results

Leaf cross-sections showed a dorsiventral anatomy, with a compact palisade parenchyma containing many chloroplasts and a spongy parenchyma with large vacuoles and fewer chloroplasts; the spongy parenchyma constituted 40 % of the whole-leaf thickness (Fig. 1). A bundle sheath with large chloroplasts located centrifugally was observed. Cross-sections of the fleshy stems (not shown) have a thick green cortex and colourless pith; the cortex was 54 % of the stem thickness on average.

Figure 1.

Cross-sections of the leaf of E. milii. UE, upper epidermis; PP, palisade parenchyma; VB, vascular bundle; BS, bundle sheath; SP, spongy parenchyma; LE, lower epidermis. Arrowheads point at chloroplasts.

In watered plants, S_m in both leaves and stem green cortex was 2.7 ± 0.2 g water (mg Chl)⁻¹; δ¹³C was −25.2 ± 0.7 ‰ in leaves and −24.7 ± 0.1 ‰ in stems. In the stem green cortex of watered plants, malate- and citrate-H⁺ content was 48 ± 9 and 29 ± 5 μmol H⁺ (g FM)⁻¹, respectively, without daily oscillation. Water suspensions of latex showed no acid content.

Leaves had significant amounts of malate- and citrate-H⁺ at dawn and dusk, contents increasing with time under drought (Fig. 2A and B). A significant accumulation of malate-H⁺ took place in watered plants, which remained constant up to 16 days of drought (P < 0.05). A similar trend in dawn and dusk H⁺ content and ΔH⁺ for citrate-H⁺ was found, except that after 16 days of drought ΔH⁺ became zero. Mean ΔH⁺ was 18 ± 2 (malate) and 18 ± 4 (citrate) μmol (g FM)⁻¹. Changes in either morning and evening H⁺ contents or ΔH⁺ bore no relationship to changes in FM/A, which remained relatively constant for the duration of the experiment, as did Chl content (Fig. 2). The ratio Chl a/b remained unchanged at 3.3 ± 0.2. Stem water content was 11.2 ± 0.5 g water (g DM)⁻¹, twice as high as in leaves, and did not vary with time under drought (P = 0.86).

Figure 2.

Time course of changes with drought in leaves of E. milii in (A) H⁺ content titrated to pH 7.0 (empty circles, dawn; filled circles, dusk); (B) H⁺ content titrated to pH 8.4 (empty circles, dawn; filled circles, dusk); (C) nocturnal H⁺ accumulation (empty triangles, pH 8.4; filled triangles, pH 7.0), and (D) dawn leaf FM per area (circles) and chlorophyll content (triangles). Values are mean ± SE (n = 12). Different letters indicate significant differences at P < 0.05 after a two-way ANOVA (time under drought × hour of day for each pH in A and B) and a one-way ANOVA (time under drought for each pH in C).

As shown in Fig. 3A, P_N of watered plants became saturated at 700 μmol m⁻² s⁻¹ PPFD; apparent quantum yield was 0.047 and light-compensation point 48 μmol m⁻² s⁻¹. The P_N/C_i curves (Fig. 3B) show that P_N did not become saturated by C_i, increasing 47 % with an increase in C_i to 800 μmol mol⁻¹ (C_a = 1240 μmol mol⁻¹). This lack of saturation could have been due to very low g_s, which remained unchanged by C_i. The CO₂ compensation concentration was 32 μmol mol⁻¹.

Figure 3.

Response curve of the leaf photosynthetic rate to (A) photosynthetic photon flux density and (B) leaf intercellular CO₂ concentration in watered plants of E. milii. Values are mean ± SE (n = 6). Filled circles, P_N; empty symbols, g_s. Measurements were made at a CO₂ concentration of 380 μmol mol⁻¹ in (A) and a PPFD of 1000 μmol m⁻² s⁻¹ in (B).

Daily courses of leaf gas exchange done in plants progressively under drought showed a decrease in P_N of 85 % with drought; R became nearly zero after 12 and up to 16 days without watering (Fig. 4). Mean daytime water-use efficiency calculated from these courses of leaf gas exchange was relatively high, decreasing significantly with drought only 18 %, from 4.2 ± 0.1 to 3.5 ± 0.1 mmol mol⁻¹ (P = 0.00).

Figure 4.

Daily course of the leaf photosynthetic rate, stomatal conductance and transpiration rate in plants of E. milii under drought for 0, 7, 12 and 16 days. Measurements were made at a CO₂ concentration of 380 μmol mol⁻¹, leaf temperature of 24.0 ± 1.0 °C and PPFD of 200 (06:00–10:00 h) and 1000 μmol m⁻² s⁻¹ (10:00–18:00 h). The dark bar on the abscissa indicates the length of the dark period.

Stem cross-sections of 1.5 cm² average area from watered plants introduced in the assimilation chamber showed daytime CO₂ assimilation at rates similar to those determined in leaves on a Chl basis (Table 2). Stem sections, as opposed to leaves, showed dark CO₂ uptake.

Table 2.

Photosynthetic and respiration rate of stem cross-sections inserted into the IRGA assimilation chamber.

Organ	PPFD (μmol m−2 s−1)	PN
		(μmol m−2 s−1)	(μmol (g Chl)−1 s−1)
Leaf	1500	9.9 ± 1.2	15.1 ± 0.3
Stem	1500	10.0 ± 0.2	9.1 ± 1.9
Leaf	0	−1.5 ± 0.2	−2.3 ± 0.3
Stem	0	0.5 ± 0.2	4.7 ± 1.3

Values are mean ± SE (n = 6). Incident photosynthetic photon flux density is indicated.

A significant decrease of 65 % in R with C_i was observed without significant changes in g_s (Fig. 5).

Figure 5.

Response curves to leaf intercellular CO₂ concentration of nocturnal respiration rate and stomatal conductance in watered plants of E. milii. Filled symbols, R; empty symbols, g_s. Values are mean ± SE (n = 6).

The regression of E vs. R is shown in Fig. 6. Assuming that the accumulated acids were the products of the recycling of respiratory CO₂, the absolute recycling, i.e. the amount of CO₂ contained in acids, was calculated. Together with the E vs. R regression, it was found that recycling recovered 10 and 37 % of nocturnal CO₂ loss in watered plants and after 12 days of drought, respectively, and helped in saving water during the night by 15 % in watered plants and 2 % in plants under drought. Daytime water saving, calculated as the ratio of the absolute amount of CO₂ in acid equivalents to integrated P_N (after Fetene and Lüttge 1991), was 2 and 86 % in watered plants and plants under drought, respectively.

Figure 6.

Change in nocturnal leaf transpiration rate of plants of E. milii watered and under different degrees of drought as a function of nocturnal respiration rate. Values are data points. The regression line (solid), 95 % confidence intervals (broken lines) and determination coefficient (P < 0.05) are shown.

Discussion

Evidence was found for the operation of weak CAM in E. milii, with statistically significant ΔH⁺ in watered plants and plants under drought, low δ¹³C and no nocturnal CO₂ uptake; ΔH⁺ was apparently attributable to both malate and citrate. Results suggest that daily malate and citrate accumulation results from recycling of part (watered plants) or all (plants under drought) of the nocturnal respiratory CO₂. Recycling of CO₂ through malate synthesis, together with the absence of nocturnal CO₂ uptake, helps explain the occurrence of values of leaf δ¹³C intermediate between C₃ and constitutive CAM plants.

Values of ΔH⁺ determined at pH 7.0 were low compared with CAM species such as Kalanchoe tubiflora and Clusia minor, comparable with the cycling species T. parviflorum and T. mengessi and not as low as in Talinum teretifolium or Zamioculcas zamiifolia (Table 3).

Table 3.

Values of nocturnal acid accumulation and carbon isotopic composition reported for CAM species.

CAM mode	Species	ΔH+	δ13C	Reference
		(μmol (g FM)−1)	(‰)
Facultative	Clusia minor	1400	−24.6	Borland et al. (1992, 1994)
Constitutive	Kalanchoe daigremontiana	152	−16.7	Holtum et al. (1983)
Cycling	Talinum calycinum	39	−25.2	Martin and Zee (1983)
Cycling	Sedum nuttalianum	37	−27.2	Gravatt and Martin (1992)
Cycling	Talinum calcaricum	29	−26.0	Harris and Martin (1991)
Cycling	Sedum telephioides	23	−26.2	Gravatt and Martin (1992)
Cycling	Euphorbia milii	18	−25.2	This study
Cycling	Talinum teretifolium	14	−25.4	Harris and Martin (1991)
Cycling	Talinum parviflorum	11	−25.8	Harris and Martin (1991)
Cycling	Talinum mengessi	6	−24.3	Harris and Martin (1991)
Facultative	Zamioculcas zamiifolia	5	ND	Holtum et al. (2007)

Some values were re-calculated from the data in references. ND, not determined.

A value of S_m higher than 1 g water (mg Chl)⁻¹ was also suggestive of CAM, as proposed by Kluge and Ting (1978). The succulent nature of leaves was corroborated by the microscopic observation of cross-sections, in which cells with a large volume and few chloroplasts are present, as in many leaf-succulent CAM plants (Kluge and Ting 1978). In facultative CAM species, values of S_m are intermediate (Table 4) but, given that low as well as high values of S_m can be found in constitutive CAM species (Table 4), it becomes apparent that for a leaf to perform full CAM a large proportion of vacuole volume to chloroplasts is not required. The lack of significant differences in FM/A between five strong CAM and three weak CAM species (Nelson and Sage 2008) lends support to this hypothesis.

Table 4.

Values of mesophyll succulence index reported for constitutive, facultative- and cycling-CAM species.

Species	Sm g water (mg Chl)−1	CAM mode	Reference
Kalanchoe daigremontiana	1.3	Constitutive	Kluge and Ting (1978)
Euphorbia milii	2.7	Cycling	This study
Talinum paniculatum	3.4	Facultative	Güerere et al. (1996)
Talinum triangulare	6.0	Facultative	Herrera et al. (1991)
Puya floccosa	6.2	Facultative	Herrera et al. (2010)
Sedum morganianum	13.0	Constitutive	Kluge and Ting (1978)

The presence in E. milii of bundle sheath cells with chloroplasts is indicative of the possible operation of C₂ photosynthesis, as reported in E. acuta. This species shares with E. milii low, C₃-like values of δ¹³C (−25.5 ‰ according to Webster et al. 1975, and −28.5 ‰ according to Sage et al. 2011) and an intermediate value of CO₂ compensation concentration (32 mmol mol⁻¹; Sage et al. 2011). The occurrence of CAM, Kranz anatomy and C₄ photosynthesis in the same leaf has been reported in Portulacaceae (Guralnick and Jackson 2001), but to date no report on CAM together with C₂ photosynthesis has been published. Given that the C₂ route of carbon fixation has been proposed as an intermediate evolutionary step from C₃ to C₄ (Sage et al. 2011), investigating the functioning of C₂ photosynthesis in E. milii would bring interesting viewpoints on the evolution of CAM and C₄ plants, particularly in Euphorbia.

A significant oscillation in H⁺ content corresponding to citrate was found, equivalent to 12 μmol citrate (g FM)⁻¹ after 12 days of drought, comparable with the lower end, ∼22 μmol (g FM)⁻¹, of the range in species of Clusia, a genus abundant in CAM species performing different modes (Lüttge 2007). The role of citrate accumulation in carbon or water balance during CAM remains unclear (Lüttge 2007); citrate does not provide net CO₂ gain, as does malate, but should prove more effective than malate in increasing C_i during the day because its breakdown produces three molecules of CO₂ as opposed to one in the case of malate (Lüttge 2007). Increased C_i during the day is a photoprotective mechanism well recognized in CAM plants, as shown in plants of the facultative CAM species Talinum triangulare under drought (Pieters et al. 2003).

Photosynthetic characteristics in E. milii were consistent with those of a sun plant: high apparent quantum yield, saturating PPFD and Chl a/b ratio (Pearcy and Franceschi 1986).

The absence of net dark CO₂ fixation was consistent with the proportion of dark CO₂ uptake calculated using the regression equations of the proportion of CO₂ fixed during the night against δ¹³C found by Winter and Holtum (2002) and Pierce et al. (2002). In many facultative CAM species, δ¹³C tends towards low values. In a review of 23 facultative CAM species (Herrera 2009), the mean, maximum and minimum δ¹³C were −23.9, −14.0 and −30.0 ‰, respectively, indicating that the variability in δ¹³C values may lead researchers to classify a species as a C₃, facultative or constitutive CAM plant. In Euphorbia aphylla, δ¹³C ranged from −27.1 ‰ for the youngest cladode in the dry season during summer to −24.5 ‰ for the oldest cladode in the rainy season during winter (Mies et al. 1996), reflecting the effects on δ¹³C of day/night temperatures, water availability and developmental stage.

Values of δ¹³C higher in stems than in leaves suggest the occurrence of nocturnal CO₂ fixation, although this could not be demonstrated in intact plants. The occurrence of an assimilation rate in the dark amounting to a third of PPFD-saturated leaf P_N strongly suggested that stems are capable of nocturnal CO₂ fixation. Stem internal CO₂ re-fixation in young twigs and branches possessing a green cortex may compensate for 60–90 % of the potential respiratory carbon loss (Pfanz et al. 2002). If stem recycling in E. milii occurred through phosphoenolpyruvate carboxylase (PEPC) activity, that would explain the apparent ¹³C enrichment. There are two alternative explanations for higher δ¹³C in the stems of E. milii. One explanation is that this variable was determined in sections comprising all tissues, green as well as non-autotrophic; non-autotrophic organs of C₃ plants, such as stems, have been found to be enriched in δ¹³C by ∼1–3 ‰ relative to leaves (Cernusak et al. 2009). Another, simpler, explanation is that barriers to CO₂ diffusion into the stem are larger than into leaves, hindering entrance of the heavier ¹³CO₂. Actual nocturnal CO₂ fixation by stems of E. milii remains to be determined accurately by methods such as carbon labelling, involving all stem tissues.

The response of leaf dark respiration to C_i suggests the operation of a carboxylation system, most likely PEPC, which makes recycling of respiratory CO₂ possible. In the constitutive CAM plant Kalanchoe daigremontiana, a P_N/C_i curve during phase I of the CAM cycle showed a pronounced increase at low C_i and saturation at a C_i of ∼250 μmol mol⁻¹ (Griffiths et al. 2007). Our results show that the response of dark respiration to C_i was not an artefact caused by changes in g_s, because g_s remained constant in spite of increasing C_i.

Water saving through respiratory CO₂ recycling was significant, as in the case of T. paniculatum, a facultative CAM species, in which the amount of water saved was 5–12 times that lost by transpiration (Güerere et al. 1996). Similarly, in T. calycinum, 5–44 % of water was potentially conserved by CAM-cycling (Martin et al. 1988).

Leaf water balance in E. milii seems to rest on both recycling of respiratory CO₂ and strict stomatal control, rather than on water supply from the succulent stem, as leaf FM/A remained unchanged after 16 days of drought and stem water content did not vary significantly during this time.

Conclusions

In view of the observations presented here, E. milii can be considered as a CAM-cycling species that in watered plants shows diurnal, but not nocturnal, CO₂ uptake and low ΔH⁺; plants under drought have very low P_N, equally low ΔH⁺ and no net dark CO₂ exchange. The significance of the operation of such a low CAM in E. milii resides in water conservation, rather than carbon acquisition. The occurrence of C₂ photosynthesis remains to be demonstrated.

Sources of Funding

Experiments were done with equipment acquired through grant PG-03.00.6524.2006 and technical assistance provided by grant PG 03.7381.2011-1 (CDCH-UCV).

Conflicts of Interest Statement

None declared.