What is the potential for dark CO2 fixation in the facultative crassulacean acid metabolism species Talinum triangulare?

Journal of Plant Physiology 174 (2015) 55–61

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Physiology

What is the potential for dark CO2 fixation in the facultative crassulacean acid metabolism species Talinum triangulare? Ana Herrera a,∗ , Caín Ballestrini a , Enrique Montes b a b

Centro de Botánica Tropical, Instituto de Biología Experimental, Universidad Central de Venezuela, Caracas 1041, Venezuela College of Marine Sciences, University of South Florida, St. Petersburg, FA 33701-5016, USA

a r t i c l e

i n f o

Article history: Received 23 July 2014 Received in revised form 15 October 2014 Accepted 15 October 2014 Available online 23 October 2014 Keywords: Carbon isotopic composition Facultative CAM Water-use efficiency

a b s t r a c t In obligate Crassulacean acid metabolism (CAM) plants, dark CO2 fixation is almost the sole route of CO2 fixation and, under drought, continues for long periods. In contrast, in plants of the facultative CAM species Talinum triangulare under experimental drought, dark CO2 fixation provides a small proportion of the daily assimilation observed in watered plants and occurs only for a few days, after which almost nil CO2 fixation is observed. Under field conditions, with a practically unlimited substrate volume, droughtinduced CAM might operate for a longer period and make a higher contribution to daily CO2 fixation. Greenhouse-grown plants of T. triangulare were subjected to low and nearly constant soil water content; the operation of CAM was assessed through the measurement of nocturnal proton accumulation and dark CO2 fixation. Dark CO2 fixation appeared 19 d after the onset of drought; its contribution during three months of experiment to daily CO2 assimilation ranged from 0.5 to 30.7% with a mean of 13.5%. Twenty days after the beginning of treatment, nocturnal proton accumulation increased six times and remained high for over three months. In spite of low soil water content, leaves did not engage in dark CO2 fixation all the time but dark CO2 fixation was large enough to produce an increase in relative 13 C composition of mature leaves compared to watered plants but not to the value in short-term drought experiments. Leaf anatomical characteristics may guarantee the achievement of higher rates of dark CO2 fixation but results evidence the occurrence of a limit to the expression of CAM that remains to be determined. © 2014 Elsevier GmbH. All rights reserved.

Introduction In obligate CAM plants, such as agaves, the proportion of diel CO2 uptake due to dark CO2 fixation (DF) may be 90% in watered plants and becomes 100% after 30 d of drought (calculated from data in Nobel, 1988a). In contrast, in many facultative CAM plants CO2 uptake in watered plants takes place only during the day, droughtinduced DF occurs during a few days and afterward net CO2 fixation is almost nil; generally, DF constitutes a small proportion (22% on average) of C3 CO2 assimilation of watered plants (Herrera, 2009). Facultative CAM may help extend the time the plant is physiologically active during prolonged drought (Martin and Jackson, 1986). In annual species, such as Mesembryanthemum crystallinum

Abbreviations: A, assimilation rate; ABA, abscisic acid; CAM, crassulacean acid metabolism; Ci /Ca , ratio of intercellular to ambient CO2 concentration; DF, dark fixation; DM, dry mass; FM, fresh mass; IR, internal recycling; IRGA, infrared gas analyzer; %DF, proportion of daily CO2 fixed by night; PPFD, photosynthetic photon flux density; WUE, water-use efficiency. ∗ Corresponding author. Tel.: +58 2127510544. E-mail address: [email protected] (A. Herrera). http://dx.doi.org/10.1016/j.jplph.2014.10.006 0176-1617/© 2014 Elsevier GmbH. All rights reserved.

and Calandrinia polyandra, the induction of CAM at the beginning of the dry season prolongs net carbon gain at low water cost, thereby aiding reproduction (Winter and Holtum, 2014). Plants of M. crystallinum begin exhibiting CAM as the dry summer approaches and remain in this mode until the end of their life cycle. In contrast, in Talinum triangulare, a deciduous perennial which loses its leaves under extreme drought, DF occurs after about 7–9 d under drought and idling, i.e. no net CO2 fixation during 24 h and occurrence of small nocturnal proton accumulation, H+ (definition of idling in Cushman, 2001), sets in after 25–30 d (Herrera et al., 1991; Pieters et al., 2003). Plants of T. triangulare inhabit seasonally-dry as well as semiarid environments; in the latter, they may experience short periods of drought followed by rains in an unpredictable manner. These plants possibly spend a significant length of time in the CAM mode, if soil water content is low enough. Plants of T. triangulare watered to 80% field capacity showed zero H+ , whereas 50 and 30% field capacity increasingly promoted H+ , which was higher at 30 than at 50%, remaining high for four months (Irazábal, 2005). Similarly, low DF was observed after 80 d in plants of the inducible-CAM species Sedum pulchellum maintained at a soil water potential of −1.5 to −4.0 MPa (Smith and Eickmeier, 1983).

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A. Herrera et al. / Journal of Plant Physiology 174 (2015) 55–61

Due to the small effect of DF on daily carbon balance of facultative CAM plants, plant carbon isotopic composition (ı13 C) resembles more that in C3 than in obligate-CAM plants, with the exception of M. crystallinum, in which ı13 C was −26‰ (C3 -like) during spring, reaching −14‰ (obligate CAM-like) at the end of summer (Winter and Troughton, 1978). Extremely low values of ı13 C (−30‰) have been reported in the inducible-CAM species Sedum nuttallianum (Martin and Jackson, 1986) and the orchid Trigonidium egertonianum (−32.3‰), in which a small value of H+ significantly higher than zero suggested the occurrence of weak CAM (Silvera et al., 2005). In plants of Sedum wrightii values of ı13 C as high as −14‰ were found in low-altitude populations, whereas at higher altitudes ı13 C tended towards −23‰ (Kalisz and Teeri, 1986). This suggests that the lowland populations spent more of their life cycle performing DF than the highland ones. Many of the data reported in the literature (reviewed by Herrera, 2009) have been collected in experiments done in pots to which watering was withheld and pots let to dry out. Operation of CAM in the field, with a very large rooting volume and slow drying, plausibly continues for much longer. Results found in M. crystallinum (Winter and Troughton, 1978) and S. wrightii (Kalisz and Teeri, 1986) may be a reflection of the actual situation in the field: C3 photosynthesis during the rainy season and induced CAM at a moderate level in partly dry soil for a considerable length of time. In potted plants of T. triangulare, ı13 C was not significantly different between the beginning of treatment and after 24 d of drought, averaging −26.50‰ (Herrera et al., 1991), nor was ı13 C different between short-day plants (with CAM) and long-day plants (without CAM) plants, averaging −25.89‰ (Herrera, 1999). In a field study where plants were left to receive rainfall water or were watered frequently, ı13 C was similar between treatments, averaging −24.60‰ (Taisma and Herrera, 2003). Dark fixation in T. triangulare under short-term drought amounted to 39% of daily CO2 assimilation in short-term drought (recalculated from data of Herrera et al., 1991). Using data of Winter and Holtum (2002), Pierce et al. (2002) and references in Herrera (2009), we did the regression of %DF (the proportion of daily CO2 fixed by night) against ı13 C in obligate and facultative CAM plants, which gave the following equation: ı13 C (‰) = −26.8753 + 0.2043 × DF − 0.0007 × (%DF)2 (r2 = 0.78). We calculated that in T. triangulare ı13 C could increase to approximately −20‰ if DF remained constant at 39%. The ı13 C may increase in an unwatered plant for two reasons: (1) An increase in water-use efficiency (WUE) due mainly to stomatal closure, as shown in wheat cultivars by Farquhar and Richards (1984), because ı13 C is directly related to the ratio of intercellular to ambient CO2 concentration (Ci /Ca ) as ı13 C = −12.44–26.2 × Ci /Ca , and WUE is inversely related to Ci /Ca (Flanagan and Farquhar, 2014), and (2) An enrichment in 13 C due to the operation of phosphoenolpyruvate carboxylase during CAM, because this enzyme discriminates less than rubisco against 13 C and provides a significant amount of the CO2 used by the Calvin cycle (Cushman and Borland, 2002). In view of the lack of differences in ı13 C of plants of T. triangulare experimentally subjected to short-term drought in pots or naturally in the field during a slowly imposed soil water deficit, the implications in carbon balance and survival of the facultative operation of CAM in this species, as in many others, remain obscure (Herrera, 2009). In order to aid in our understanding of facultative CAM and elucidate the potential of facultative CAM for biomass production, plants should be maintained for a long period of time at a water status that promoted DF. We subjected plants of T. triangulare watered to field capacity to a long-term course of drought during which bulk soil volumetric water content () was maintained low by the calculated addition of small volumes of water. CAM activity was determined

by DF and H+ . The ı13 C was determined near the end of the experiment and compared to that in frequently watered plants. We predicted that under these conditions ı13 C would become higher than values previously reported for T. triangulare.

Materials and methods Plant material and growth conditions Plants of Talinum triangulare (Jacq.) Willd. (Talinaceae) grow in Venezuela in semi-arid to seasonally-dry habitats at less than 20 m of altitude. Seedlings were collected during the rainy season near the town of Carayaca (10◦ 32 N–67◦ 8 W), where mean microclimatic conditions for 18 years were (min/max) 17.9/25.3 ◦ C air temperature, 0.12/3.11 kPa air water-vapour saturation deficit, w (data from Instituto Nacional de Meteorología e Hidrología, Venezuela), and maximum PPFD 1200–2400 ␮mol m−2 s−1 ; mean annual rainfall was 854 mm. Twenty seedlings, approximately onemonth-old, were grown in the greenhouse under natural light for eight months in 6-L-pots filled with silt-clay loam (Viveros Exotica Raphia, S.R.L., Caracas); plants were fertilized monthly with commercial fertilizer (Pokon Naturado BV, Veenendaal, The Netherlands) dissolved as instructed by the manufacturer. Day length was 12 h (06:00–18:00 h). Ten plants maintained at field capacity by watering on alternate days were then subjected to soil drying until soil water content attained approximately 30% field capacity or  = 0.09 m3 m−3 and water added approximately every three d to maintain this value of . Another ten plants continued to be watered at field capacity and used as controls. Photosynthetic photon flux density (PPFD) was measured with a 190-S quantum sensor connected to a LI-185 meter (LI-COR Inc., Lincoln, NE). Air temperature and relative humidity were measured using two HOBO Pro V2 loggers and data dumped with a HOBO Waterproof Shuttle (Onset Computer Corporation, Pocasset, MA). Microclimatic conditions in the greenhouse were (min/max): air temperature 19.8 ± 0.8/26.0 ± 0.7 ◦ C, w 0.35 ± 0.06/2.17 ± 0.98 kPa, and maximum PPFD 1800 ␮mol m−2 s−1 . Soil water content The  was determined as  = (FM − DM)/pot volume; FM was the fresh mass of soil, DM the soil mass after 72 h at 60 ◦ C, and soil volume 6 L. Mass of pot plus plant was weighed with an electronic scale mod. SP-208C (ACS System, China) with a precision of 5 g.

Leaf succulence and anatomical characteristics Leaf succulence was determined as the ratio of FM to leaf area and specific leaf area as the ratio leaf area to leaf DM (48 h at 60 ◦ C). Leaf thickness was measured with a precision caliper (±0.1 mm). Mesophyll cell number and cross-sectional area were determined in free-hand cross sections of leaves from watered plants photographed under the microscope at 200×; images were processed with the ImageJ software.

Carbon isotopic composition Leaf ı13 C was determined in samples collected from natural populations during the rainy season and from experimental watered plants and after 72 d of drought. Samples were dried and ground and ı13 C determined using a ThermoFinnigan DeltaPlusXL Isotope Ratio Mass Spectrometer (San Jose, CA) with a precision of 0.15‰ and Pee Dee Belemnite as standard.

A. Herrera et al. / Journal of Plant Physiology 174 (2015) 55–61

Nocturnal H+ accumulation

20

Length and width of whole leaves were measured for area calculation using a previously determined allometric relationship and leaves weighed fresh and set to boil in 50 mL distilled water for 5 min in a microwave oven at maximum power; samples were sieved through a plastic colander, leaf segments and colander rinsed and the solution taken up to 100 mL. Solutions were titrated to pH 7.0 for the estimation of H+ corresponding to malate according to Nobel (1988b), and to pH 8.4 for citrate after Franco et al. (1990). The H+ was calculated as the difference between dawn and dusk H+ contents.

16

57

Day 0

12 8 4 0 Day 21 8 6

Leaf gas exchange

A (μmol m-2 s-1)

4

Assimilation rate (A; Aday , daytime and Anight , nighttime), stomatal conductance (gs ), Ci /Ca and WUE were measured in mature leaves in the laboratory with a CIRAS 2 infrared CO2 and H2 O gas analyzer (IRGA) connected to a PLC(B) assimilation chamber (PP Systems, Amesbury, MA) at a chamber temperature tracking ambient (24.7 ± 0.1 ◦ C), which remained relatively constant during 24 h of measurement. Air entering the IRGA was drawn from outside the laboratory and passed through a 500-mL Erlenmeyer flask to buffer abrupt changes in CO2 concentration; ambient CO2 concentration was 395 ± 2 ␮mol mol−1 . The w in the chamber was 1.17 ± 0.02 (day) and 0.92 ± 0.02 kPa (night); PPFD during the daytime was 500 ␮mol m−2 s−1 , a value previously shown to saturate photosynthetic rate. Records were taken automatically every 30 min. Values presented are data points of measurements made on one leaf for 24 h. Daily courses of gas exchange were done about every other day in different plants for 85 d; only courses during which DF was found are shown, as courses where no DF or idling was found were not infrequent. Measurements were stopped after 84 d of drought and plants re-watered. With the daytime values of Ci /Ca and applying the equation ı13 C = −12.44–26.2 × Ci /Ca (Flanagan and Farquhar, 2014), we calculated the expected values of ı13 C. Instantaneous gas exchange was measured in apical leaves, mature leaves and fruits (four detached fruits enclosed in the assimilation chamber).

2 0 Day 27 8 6 4 2 0 Day 36 8 6 4 2 0 18

00

06

12

18

Time of day (h) Internal CO2 recycling (IR) The amount of CO2 fixed during the night was determined integrating the night-time courses of CO2 exchange using the Sigmaplot graphic software and expressed as mmol CO2 m−2 . Internal CO2 recycling (IR) was calculated as IR = 100 × (mmol malate m−2 − mmol CO2 m−2 )/mmol malate m−2 , using H+ content determined at pH 7.0 as a proxy for malate H+ .

Fig. 1. Representative daily courses of CO2 exchange in plants of Talinum triangulare subjected to short-term drought. Days of drought indicated in each panel. The filled bar on the abscissa indicates the length of the night.

Statistics Values are mean ± SE. Statistical significance was assessed where indicated through one- or two-way analysis of variance (P < 0.05), depending on the variables, with the Statistica package.

Water relations

Results

Xylem water potential ( ) was measured at 06:00 h in leafbearing branches using a pressure bomb (PMS, Corvallis, OR). Leaf osmotic potential ( s ) was determined in leaf disks (n = 6) taken at the same time as for the measurement of , frozen and placed without defrosting in C-52 chambers connected to an HR-33T micro-voltmeter (Wescor Inc., Logan, UT, USA) operated in the psychrometric mode. Osmotic adjustment was determined as the difference in saturated s between unwatered and watered plants all re-watered over field capacity the previous afternoon, enclosed in black polythene bags and measured after dawn the following day.

Leaf gas exchange A typical time-course of daily leaf gas exchange in plants subjected to short-term drought is shown in Fig. 1. Watered plants showed no nocturnal CO2 fixation but respiration instead; after 21 d of drought, Aday decreased to nearly half the value in watered plants and dark respiration rate decreased to zero. Nocturnal net CO2 uptake occurred after 27 d, when integrated Aday decreased 74% and integrated Anight amounted to 8% of the 24-h C gain. After 36 d, 24-h net CO2 exchange was very low but Anight was still 10% of the 24-h C gain.

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A. Herrera et al. / Journal of Plant Physiology 174 (2015) 55–61

8

-1 -2

4 2 0 6

A

4

A (μmol m s )

6

5

57 d plant 26

19 d plant 15

20 d plant 19

70 d plant 8

3 2 1 0

4 2

B

-1

gs (mmol m s )

30

-1

6

-2

4

29 d plant 12

77 d plant 25

2

20

10

0 84 d plant 12

35 d plant 17

-1

6

WUE (mmol mol )

A (μmol m s )

-2

0

4 2 0 6

85 d plant 12

49 d plant 17

C 8 6 4 2

4

D

2

1.4

6

52 d plant 26

Δw (kPa)

0 86 d plant 8

4

1.2 1.0 0.8

2 0

0.6 18

00

06

12

18

18

00

06

12

18

04

08

After 20 d,  in unwatered pots abruptly decreased from 0.331 ± 0.013 m3 m−3 to 0.093 ± 0.002 m3 m−3 and was maintained at that value for the duration of the experiment by the weekly addition of a mean of 50 mL water, depending on water loss per pot. The time-course of changes in CO2 exchange of mature leaves of seven different plants measured at random during the experimental long-term drought is shown in Fig. 2. Dark fixation occurred when  was approximately 0.09 m3 m−3 . Plants under similar values of  might not show DF, but respiration instead. Dark fixation was found to take place when leaves were 0.3 mm thick or lower but neither leaf thickness nor succulence proved reliable criteria for predicting when a leaf would show DF. A significant leaf shedding and re-sprouting was observed throughout the experimental period, average leaf duration being two months. Relatively low variation was found when hourly values of A, gs and WUE in these daily courses were averaged (Fig. 3). Values of gs were very low all day long and a strong reduction of 53% relative to earlier values was found at midday; this decrease was not due to higher w, because at midday w was lower than during the early

16

20

00

04

08

Time of day (h)

Time of day (h) Fig. 2. Daily courses of CO2 exchange of plants in pots where bulk volumetric water content was maintained at approximately 0.09 m3 m−3 . The filled bar on the abscissa indicates the length of the night. The duration of the measurement period and plant number are indicated in each panel.

12

Fig. 3. Diel changes in plants of Talinum triangulare under drought in: (A) assimilation rate; (B) stomatal conductance; (C) water-use efficiency and (D) air water-vapor saturation deficit during measurements. Each value is the mean ± SE of 12 daily courses of leaf gas exchange done at random on ten different plants during 83 d of drought. The filled bar on the abscissa indicates the length of the night.

hours of the light period. In contrast, WUE decreased only 35% at midday, mainly due to a relative maintenance of Aday (Fig. 3). Young and mature leaves of watered plants had values of Aday measured between 1100 and 1200 h not statistically different (11.2 ± 1.1 and 13.3 ± 1.1 ␮mol m−2 s−1 ; P = 0.20); fruits showed only respiration. Carbon isotopic composition A significant increase with drought in ı13 C was found (Table 1). Values were significantly higher than in watered plants, the highest value occurring in fruits of unwatered plants. The difference in ı13 C between unwatered and watered greenhouse plants was 3.16 (young leaves), 2.05 (mature leaves) and 4.43‰ (fruits). Nocturnal acid accumulation Nocturnal acid accumulation increased with time under drought, remaining relatively stable until the end of the drought period (Fig. 4). Dawn, but not dusk H+ , content corresponding

A. Herrera et al. / Journal of Plant Physiology 174 (2015) 55–61

to malate increased significantly after 19 d of drought, whereas neither dawn nor dusk content corresponding to citrate showed significant variations. Changes in H+ were apparently not caused by changes in leaf succulence, which remained unchanged at 0.442 ± 0.013 kg m−2 . Leaf anatomical characteristics Specific leaf area, with a value of 192 ± 20 cm2 g−1 , did not change with treatment, whereas leaf thickness decreased from 860 ± 50 to 446 ± 20 ␮m after 22 d of drought. Mesophyll cell cross-sectional area was 3953± 609 ␮m2 and there were 77 ± 12 cells mm−2 mesophyll cross-sectional area.

Added water (% total)

6 4 2 0

-1

B 200 150 100 50 C 20 10 0 -10 %DF IR

80

D

75

60

50

40

25

20 0

Osmotic adjustment

20

A

+

ΔH -2 (μmol cm )

malate citrate

*

15

*

10

*

*

*

5 0

B Leaf succulence -2 (kg m )

0 day night

dusk dawn

0.6

WUE -1 (mmol mol )

12

After 65 d of treatment, ␺ was significantly higher in watered than unwatered plants (−0.23 ± 0.03 v. −0.81 ± 0.11 MPa; P = 0.000), whereas ␺s was not significantly different between watered and unwatered plants (−0.61 ± 0.05 v. −0.86 ± 0.11 MPa; P = 0.06) and turgor potential was not significantly different in watered than unwatered plants (0.38 ± 0.07 v. 0.28 ± 0.17 MPa; P = 0.58). In order to determine the possible mechanism whereby watering so rapidly reduced DF and increased Aday , we examined in an independent experiment the occurrence of osmotic adjustment,

100

IR (%)

Different letters indicate significant differences at P < 0.05 (two-way analysis of variance).

A

8

-2

Long-term drought

(mmol m d )

−28.14 ± 0.69a −28.02 ± 0.42a −27.85 ± 0.82a −24.98 ± 0.43bc −25.97 ± 0.38ab −23.42 ± 0.81c

%DF

Young leaves Mature leaves Fruits Young leaves Mature leaves Fruits

Integrated Aday

Watered

-1

ı13 C (‰)

-2

Organ

(mmol m d )

Treatment

10

Integrated Anight

Table 1 Carbon isotopic composition of different organs in plants of Talinum triangulare frequently watered or maintained for 72 d at low soil water content in the greenhouse.

59

E

10 8 night day

6 4 2 0

20

40

60

80

Time (d) Fig. 5. Time-course of changes in: (A) proportion relative to total volume of water added to maintain bulk volumetric soil water content at 0.09 m3 m−3 ; (B) daytime diel CO2 assimilation; (C) night-time diel CO2 assimilation; (D) proportion of daily assimilation due to dark fixation (closed symbols) and internal CO2 recycling (open symbols), and (E) daytime (open symbols) and night-time (closed symbols) mean water-use efficiency. Values in (A) and (E) are mean ± SE (n = 12); values in (B) (C) and (D) are data points.

which could help rapid rehydration and stomatal re-opening. When  reached 0.09 ± 0.04 m3 m−3 after 27 d of drought, was not different between treatments, while s was significantly lower in unwatered than watered plants (P = 0.008), which resulted in an osmotic adjustment of 0.49 ± 0.10 MPa and turgor potentials of 0.21 ± 0.13 and 0.37 ± 0.04 MPa for watered and unwatered plants, respectively (P = 0.20). Carbon balance and ı13 C

0.4

0.2 0

20

40

60

80

Time (d) Fig. 4. Time-course of changes in plants of Talinum triangulare subjected to drought in: (A) nocturnal proton accumulation corresponding to malate (circles) and citrate (triangles), and (B) dawn (open symbols) and dusk (closed symbols) leaf succulence. Each value is the mean ± SE of samples taken at random from six different plants. An asterisk indicates significant differences due to treatment in dawn and dusk H+ contents.

The integrated daily courses of Aday and Anight , IR, WUE and ı13 C calculated from Ci /Ca were calculated from data in Figs. 2 and 4. The time-course of changes in these variables is shown in Fig. 5. The daily courses of integrated Aday showed a strong decrease after a few days of drought; in contrast, integrated Anight increased from respiration to DF. A small volume of water added to maintain  constant, as in day 76, was enough to reduce DF even down to zero and cause significant increases in Aday . The %DF ranged from 0.5 to 30.7% and averaged 13.5%. Values of IR increased markedly with drought, attaining 77% at the end of treatment. Mean WUE was higher during the day than during the night after 36 d of drought, remaining so until the end of the experiment.

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Calculated ı13 C increased during the dry period from −31.14 to −28.13‰, similar to the value corresponding to young and mature leaves of plants receiving sufficient water both in the greenhouse and in the field, but lower by an average of 2.63‰ than the value measured in unwatered plants at the end of treatment. Diurnal water saving by unwatered relative to watered plants was 87 ± 28%, while the nocturnal value oscillated between 14 and 82%, with an average of 45%. Twenty-four hours after plants were re-watered at the end of the experiment on day 84, nocturnal CO2 exchange changed from DF to respiration, and after only 24 h Aday (188.7 mmol m−2 d−1 ) increased and H+ (2.2 ± 0.8 ␮mol cm−2 ) decreased to values similar to those on day 0.

Discussion A significant increase in ı13 C was found in plants of T. triangulare maintained under controlled long-term drought relative to watered plants. Values of ı13 C in mature leaves were higher than average ı13 C calculated using Ci /Ca values; the latter value would be theoretically due only to high WUE without taking into account DF. Despite the observed increase with prolonged drought in ı13 C of mature leaves, values were not higher than those previously reported for short-term pot experiments or measurements in the field. Values of ı13 C in young and mature leaves were significantly lower than, and values in fruits were not different from, previously published values of plants growing in the field (data in Taisma and Herrera, 2003). Sustained water deficit did not allow achieving a higher leaf ı13 C, as expected, but it resulted in stomatal closure producing significant water saving relative to watered plants. We interpret the absence of a larger increase in ı13 C as indicating that there is a limit to the expression of CAM that prevents a higher acquisition of CO2 via DF. The limit to DF may be related to various factors. First, because DF needs carbohydrates synthesized during the day, this supply becomes limiting when photosynthetic rate decreases with drought. Additionally, the size of the photosynthetic machinery may condition the amount of carbohydrate synthesized. The number of chloroplasts per cell in T. triangulare was similar to that in the obligate CAM species, Ananas comosus (Kondo et al., 1998), although a more detailed anatomical study is needed in the former species. Second, the size of the mesophyll in general and more specifically the vacuole impose limitations to the amount of acid that a cell may accumulate (Griffiths et al., 2008). Leaf thickness, succulence and specific leaf area in T. triangulare were similar to values for C3 /CAM species of Clusia after correcting variables in the latter species for hydrenchyma thickness (Zambrano et al., 2014). Comparing our results with those of Nelson et al. (2005), we found that in watered plants leaf thickness was within the range of CAM plants. Additionally, cell cross-sectional area fell at the high end of the range for CAM plants. In view of anatomical variables, plants of T. triangulare should be able to perform DF at a rate closer to obligate CAM species. Third, as in any CAM plant, CAM capacity in T. triangulare must be genetically determined. The induction and expression of CAM in response to environmental factors is highly variable among species that possess the same machinery for this route of CO2 fixation (Taybi et al., 2002) and may not, by manipulating environmental variables, be pushed any further than the characteristics of the species allow. Plant of S. wrightii from populations growing at different altitudes and cultivated in a common environment showed the same range of ı13 C values as in their native environment, indicating that capacity was genetically rather than environmentally determined (Gurevitvh et al., 1986; Kalisz and Teeri, 1986).

Differences in air ı13 C occurring in the different growth conditions where plant ı13 C in T. triangulare has been measured could help explain differences between the present values of plant ı13 C and higher values obtained in the field. Air ı13 C may vary with, among others, time of year and surrounding vegetation (Farquhar et al., 1989). For example, air ı13 C at floor level in an Amazonian forest was −13.00‰ (van Der Merwe and Medina, 1989), while the value for open air during 1991–2008 in Mauna Loa (19◦ 32 N) was −8.04‰ (data from Keeling et al., 2010). Values of plant ı13 C reported by Herrera et al. (1991), Herrera (1999) and here were determined in plants growing in greenhouses or growth chambers, whereas values given by Taisma and Herrera (2003) were collected in dense populations in the field, where the presence of many neighbors may have affected canopy air ı13 C differently. The operation of nocturnal CO2 fixation was evidenced by ı13 C attaining a final value significantly higher than that in watered plants and that due merely to increased daytime WUE. The reason why measured ı13 C was lower than the initially expected value of −20‰ may lie in the fact that there were pronounced oscillations in DF due to the rapid reversal by watering from a high to a low rate, implying that DF was not sustained in time. A similarly rapid reversal of CAM upon water supply has been reported in Clusia minor (Herrera et al., 2008) and T. triangulare (Winter and Holtum, 2014). Additionally, ı13 C in T. triangulare may not become higher than in previous and the current reports because, until they enter idling, plants always show net CO2 exchange and this may be sustained by internal, discriminated CO2 generated by de-carboxylation as well as ambient, non-discriminated CO2 . Continuous records of whole-plant gas exchange in plants maintained under controlled substratum water potential by hydroponic cultivation or automatically controlled soil water content could help elucidate this issue. The direct causes for CAM induction by water deficit and reversal by watering in T. triangulare are not clear. In plants of M. crystallinum subjected to increasing substrate salinity, DF was directly related to the increase in turgor caused by the osmotic adjustment effected by the absorption of salt (Winter and Gademann, 1991). In the present experiment, although osmotic adjustment may have aided in the rapid water absorption by unwatered plants, turgor potential was not different in unwatered than watered plants; therefore, increased DF was apparently not caused by a change in turgor. The prompt reversal to a C3 gas exchange pattern produced in T. triangulare by a very small amount of water, together with the lack of relationship between leaf thickness, succulence and DF, suggests that cell water status is not the necessary signal for CAM induction. In plants of the inducible CAM species Guzmania monostachia, only the apical leaf region performed CAM under water deficit, yet showed no reduction in water content relative to the same region in watered plants performing only C3 photosynthesis (Freschi et al., 2010). Abscisic acid (ABA) may be among the secondary regulators of CAM in T. triangulare. Exogenously applied ABA has been shown to induce acid accumulation and DF in CAM plants (Taybi and Cushman, 1999). In plants of the CAM species Aptenia cordifolia, endogenous ABA concentration after 15 d of water deficit was 64% higher in unwatered than watered plants (Cela et al., 2009). Given the prompt response of DF to a very little change in soil water content, it is plausible that ABA production by roots is promptly inhibited by watering or that leaf ABA content decreases with a small increase in leaf water content. Such a decrease in ABA would prevent the expression of Ppc 1, a gene encoding the CAM-specific isoform of phosphoenolpyruvate carboxylase which is promoted by dehydration, salinity and exogenous ABA (Taybi and Cushman, 1999). The highest ı13 C values found in this experiment corresponded to young leaves and fruits of unwatered plants. This could suggest

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that fruits were built at the expense of carbohydrates produced by young leaves performing CAM; young leaves had values of Aday similar to those in mature leaves. Although fruits of T. triangulare are green for an important period of their development, being therefore partly self-sufficient in terms of carbohydrates, they did not show net assimilation. An alternative explanation for a higher fruit ı13 C is that fruits are enriched relative to mature leaves simply because they are heterotrophic. It has been repeatedly reported that heterotrophic organs tend to be enriched in 13 C relative to autotrophic organs (Cernusak et al., 2009). In contrast, in two CAM species of Clusia, fruits had lower ı13 C than leaves, which suggests that fruits were built at the expense of carbon fixed via C3 photosynthesis (Borland and Dodd, 2002). Internal recycling of respiratory CO2 may also have contributed to fruit biomass. In the present investigation, IR continually increased despite changes in %DF. Although theoretically IR is considered not to contribute to net CO2 gain, an enhancement of CAM-cycling in plants of S. telephium subjected to water deficit under high but not low PPFD (low photosynthesis) allowed continued export of carbon to sinks and possibly continued growth (Borland, 1996). The determination of the CO2 sources, whether external or internal, used in biomass construction, as done by Borland (1996) could help explain the increased fruit ı13 C in T. triangulare. Conclusions Long-term controlled drought did not increase leaf ı13 C relative to the higher value predicted for sustained high DF, values in plants under short-term experimental drought or values in plants subjected to the natural rainfall. Possibly, the sporadic short rains of low intensity occurring during the dry season in the regions where plants of T. triangulare grow are sufficient to rapidly shift the pattern of CO2 exchange from CAM to C3 , as our results under experimental conditions have shown. Among the limits imposed to DF may be a leaf anatomy characterized by low thickness, succulence and specific leaf area more typical of C3 species that, while allowing for significant photosynthetic rate under drought, precludes a higher malic acid accumulation. Acknowledgements This research was funded by grant PG 03.7381.2011-1 (CDCHUCV, Venezuela). Field material was kindly collected by W. Tezara. Amalia Brito did the leaf cross-sections. We appreciate a thorough review by E. Medina and help with image processing by A. Mondragón. References Borland AM. A model for the partitioning of photosynthetically fixed carbon during the C3 -CAM transition in Sedum telephium. New Phytol 1996;134:433–44. Borland AM, Dodd AN. Carbohydrate partitioning in crassulacean acid metabolism plants: reconciling potential conflicts of interest. Funct Plant Biol 2002;29:707–16. Cela J, Arrom L, Munné-Bosch S. Diurnal changes in photosystem II photochemistry, photoprotective compounds and stress-related phytohormones in the CAM plant, Aptenia cordifolia. Plant Sci 2009;177:404–10. Cernusak LA, Tcherkez G, Keitel C, Cornwell WK, Santiago LS, Knohl A, et al. Why are non-photosynthetic tissues generally 13 C enriched compared with leaves in C3 plants? Review and synthesis of current hypotheses. Funct Plant Biol 2009;36:199–213. Cushman JC. Crassulacean acid metabolism. A plastic photosynthetic adaptation to arid environments. Plant Physiol 2001;127(4):1439–48. Cushman JC, Borland AM. Induction of CAM by water limitation. Plant Cell Environ 2002;25:295–310. Farquhar GD, Richards RA. Isotopic composition of plant carbon correlates with water-use efficiency of wheat genotypes. Aust J Plant Physiol 1984;11:539–52.

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