Departamento De Ciencias Biológicas Universidad De Los Andes

Departamento de Ciencias Biológicas Universidad de los Andes

Trabajo de Grado Para optar al título de Maestro en Ciencias Biológicas

CAM or not CAM: A study on juveniles of Guzmania lingulata, Guzmania monostachia and Werauhia sanguinolenta (Bromeliaceae)

Juan David Beltrán Parra

Co-Director Santiago Madriñán PhD. Co-Directora Eloisa Lasso PhD. Asesor externo Klaus Winter PhD. Jurado interno Catalina Gonzalez PhD. Jurado externo Katia Silvera PhD.

Abril de 2012

A mis amigos Margarita Gomez y Mateo Matamala

ABSTRACT

CAM photosynthesis is a water conserving strategy that many epiphytes exhibit.

Species belonging to the bromeliad clade Tillandsioideae exhibit C3 or CAM photosynthesis. Several species have tank structures that allow them to hold rainwater when adult. By contrast, juveniles that lack this structure are susceptible to drought. We explored whether there was a distinction between the photosynthetic pathway of juveniles and adults. Juveniles and adults of Werahuia sanguinolenta, Guzmania lingulata and Guzmania monostachia were collected at

Barro Colorado Natural Monument, Panama. For adults of these species, C3

(G. lingulata), weak CAM (W. sanguinolenta) and facultative CAM (G. monostachia) have been reported. Juveniles have not yet been extensively studied. Net CO2 exchange, titratable acidity, and δ13C values were measured in juveniles and adults of all three species. Both juveniles and adults performed C3 photosynthesis under well-watered conditions, and both stages showed features of weakly expressed CAM under conditions of drought stress except for the adult stage of Guzmania lingulata.

Key words: CAM : Crassulacean acid metabolism, Bromeliaceae, C3, Rubisco:

Ribulose-1,5-bisphosphate carboxylase-oxygenase , CO2 Fixation.

INTRODUCTION

CAM photosynthesis is mainly considered as a water-conserving mechanism, characterized by nocturnal CO2 uptake and acid accumulation (Holtum et al. 2007,

Winter et al. 2005). Consequently, CAM plants are commonly found in arid and semiarid environments as deserts, Mediterranean, and epiphytic habitats, where water is intermittently available (Smith & Winter 1996). Even in the wet forest, water is an irregular accessible resource for epiphytes like bromeliads and orchids that exhibit

CAM as a water-conserving strategy (Zotz & Ziegler 1997). In contrast, some C3 epiphytes have other strategies to conserve water like is the case of epiphytic bromeliads that develop water-holding structures (Benzing 2000).

Subfamily Tillandsioideae —family Bromeliaceae— their members are epiphytes that may exhibit C3 or CAM photosynthesis (Griffiths & Smith 1983, Martin 1994,

Crayn et al. 2002). The C3 tank epiphytes are “drought avoider” plants with overlapping broad-leaves forming a tank where rainwater is stored (Zotz & Thomas

1999). On the other hand, CAM epiphytes, known as atmospheric, are “drought tolerant” plants have linear and succulent leaves, with trichomes along the whole blade surface that can absorb rainwater and dew (Tomlinson 1970).

Many C3 tank-epiphytes are heteroblastic; there are abrupt changes between adult and juvenile stages in morphological, anatomical and physiological traits (Benzing 2000,

Zotz et al. 2011). Heteroblasty is probably linked to water availability for the plant

(Zotz et al. 2011). In the juvenile stage the plant looks like an atmospheric bromeliad with no tank, scales along the blade surface and linear leaves as well as drought tolerant atmospheric bromeliads (Benzing 2000, Adams & Martin, 1986a,b).

On the other hand, the adult stage develops a tank structure that allows them to store water making them “drought avoiders” (Tomlinson 1970, Benzing 2000, Zotz et al. 2011).

Little is known about differ from juvenile bromeliads in their photosynthetic pathways. Few studies on C3 heteroblastic bromeliads exist and they had focused on

Tillandsia deppeana (Adams & Martin 1986 a, b) and Werauhia sanguinolenta

(Schmidt & Zotz 2001, Zotz et al. 2004). W. sanguinolenta juvenile and adult stages showed an increase in nocturnal acid production after being exposed to water scarcity.

More prolonged exposure to this condition has shown to stop diurnal CO2 exchange. No apparent signals of CAM metabolism were reported for adults and juveniles, the acid accumulation has not been considered as a CAM signal by the authors (Zotz et al.

2004). Adams & Martin (1986b) concluded that the juveniles and adults of this species are C3 plants according to the absence of evidence for a constitutive CAM, however, these authors do not take into account the considerable plasticity of CAM metabolism (Dood et al. 2002), or the C3-CAM continuum (Silvera et al. 2010) even when their net CO2 exchange suggest an expression of weak CAM (see Fig 4 in Adams &

Martin 1986b).

The adult stage of the species included in the present study had been studied before, particularly Guzmania monostachia, the adult stage has been classified as a facultative CAM plant (Medina et al. 1977, Maxwell et al. 1992), but according to Pierce et al. (2002) juveniles and adults can be classified as a weak CAM plant.

W. sanguinolenta adults have been classified as weak CAM plants under well water conditions (Pierce et al. 2002) and Guzmania lingulata adults have been classified as obligated C3 plants (Smith et al. 1985, Smith et al. 1986, Griffiths et al. 1986, Griffiths

& Maxwell 1999, Maxwell 2002). Previous researches have not studied juvenile stages of G. lingulata.

The present study evaluated if tank tillandsioids exhibit CAM photosynthesis during juvenile stages, but change their photosynthetic pathway as they grow and develop impounding structure. To evaluate the photosynthetic pathway we compare anatomical features, the 13C:12C ratio of plant organic matter, leaf extract titratable acidity, and daily fluctuation of the net CO2 exchange between juveniles and adults.

METHODOLOGY

Plant material

Guzmania lingulata, Guzmania monostachia, and Werauhia sanguinolenta were collected at Barro Colorado Natural Monument (Panama) during the end of the wet season and the beginning of the dry season (November- December 2010). During the dry season, rainless periods can extend more than two weeks (Windsor 1990,

Zotz et al. 2004). G. monostachia and W. sanguinolenta are commonly found on

Annona glabra trees and grow in exposed areas. G. lingulata is a shade tolerant epiphyte (Smith et al. 1986), commonly found in the understory of the forest. Species were identified using keys to Bromeliaceae (Smith 1957,Smith & Downs

1977).

Anatomy

Hand-made sections from the middle of the longest leaf were made in 27

G. monostachia, 32 G. lingulata and 37 W. sanguinolenta plants, covering the entire ontogenetic spectrum. The length of the longest leaf was used as age indicator. The dissections under light microscope were photographed, and the thickness of the hydrenchyma— a parenchyma tissue for water storage—and chlorenchyma — the photosynthetic green tissue— were estimated using image J software (Abramoff

2004). The chlorenchyma-hydrenchyma ratio was calculated as the thickness of chlorenchyma divided by the thickness of hydrenchyma (Cl/Hy). This ratio describes the level of succulence and the inner water storage capacity of the plants.

Maintenance conditions

After being collected, the plants were kept in a greenhouse at the main Smithsonian

Tropical Research Institute facility in Panama City, watered twice in a day, without fertilizer, for more than two weeks. The plants were transferred from the greenhouse to an environmental chamber (Environmental Growth Chambers, OH,

USA) at were 12 h of light, —28°C, relative humidity (RH) 66%, PFD 300 mol m-2 s-

1—and 12 h of dark—22°C, RH 90%—, The drought experiment was carried under these conditions. CO2 exchange and Drought experiment

Given the small size of the juveniles several plants were used to measure the net CO2 exchange; for 63 W. sanguinolenta, 98 G. monostachia and 80 G. lingulata whole juvenile plants and for an entire adult plant. The dial CO2 exchange was first determinate in well-watered plants and during several days after being deprived of water. CO2 exchange measures were stop when plants closed the stomata and no further signal was detected. In juveniles this happened after 4-5 days, in adults after

6-7 days. The juveniles were sealed in translucent cuvette of 1.2 l (11 cm x 11 cm x

10 cm) and the adults sealed in translucent cuvette of 6 l (20 cm x 20 cm x 15cm).

Net CO2 exchange was determined using a through-flow system (Walz, Effeltrich,

Germany), the air was pumped through the cuvette at 1.26 L min-1, dehumidified in a cold-trap at 2 C° (Walz KF-18/2) and the CO2 concentration was determined using an infra-red gas analyzer (LI-6252, LICOR, Lincon, NE, USA), as described by Pierce et al. (2002). All the measurements were replicated two or three times. After the experiment the dry mass was measured. The Net CO2 exchange will be useful to know if the plant can uptake CO2 during the night under well water conditions or during the subsequent days without water.

Stable carbon isotopes ratios - δ13C

Ratios of 13C/12C were calculated for 28 individuals of G. monostachia, 37 G. lingulata and 38 W. sanguinolenta plants with all the ontogenetic stages represented. Leaf dry matter of ~ 2 mg were sampled, combusted in an elemental analyzer (CE

Instruments, Milan, Italy) and swept by a helium carrier gas via a constant flow interface into a mass spectrometer (Delta V, Thermo Fischer Scientific, Waltham,

MA). Stable carbon isotope ratios were measured as described Cernusak et al.

(2009). 13C/12C Ratio was calculated relative to the Pee Dee belemnite standard

(Belemni- tella americana) using the relationship:

δ13C(‰) = [(13 C /12 C sample)/ (13 C/ 12 C standard) − 1] × 1000

The typical CAM have δ13C values between -13‰ to -20‰ meanwhile C3 plants have δ13C values between -20‰ to -30‰.

Leaf tissue titratable acidity.

The plants were placed in the growing chamber in the conditions described above..

During two days the plants had well water conditions, then during the subsequent four days, these plants were not watered again. Leaf samples were collected before and after the drought experiment at the end of the light period (17:00 h) and at the end of the dark period (5:00 h). Analysis was done, for the adults, a small portion of the middle of a healthy mature leaf, and for juveniles the entire plant. 20 juveniles and 5 adults of each species were used. The tissues were frozen in liquid nitrogen and then boiled per 50 min in ethanol 20%. Extracts were made up to 25 ml with distilled water and titrated against 10 mM KOH until pH 6.5 as indicated by pH meter as describe by Pierce et al. (2002).

Statistical analysis All the statistical analyses were done using “R” statistics package (R Development

Core Team 2011). The dry mass data was log-transformed before analysis for the normality and their linearity. The relationship between the Cl/Hy ratio and δ13C values with plant size was evaluate with a Pearce-correlation test. T-student test was conduced to evaluate if there are differences between day and night acid content, before and after drought.

RESULTS

The Cl/Hy ratio increased with plant age for all species (Fig. 1). The percentage of water storage tissue was greater than the chlorenchyma for all juveniles. Besides, the green tissue of the adults was represented approximately 60% of the thickness of the leaf, compared with 16% for juveniles; chlorenchyma is the principal tissue of the adult’s leaf (Fig. 1a,b). The air spaces in the leaves are more conspicuous in adult stages (data not shown).

The 13C isotopic composition decreased with the age of the plants for all species

(Fig. 2). For G. monostachia, δ13C values were in the range of -26.1 to -29.6‰,

W. sanguinolenta -25.2 to –30.8‰ and G. lingulata -30.1 to –34.5‰. G. lingulata exhibit δ13C values more negative than G. monostachia and W. sanguinolenta. All plant showed values more negative than -20‰.

On the first day, all plants showed a CO2 gas exchange pattern typical of a C3 plant—

CO2 uptake during the light period, constant respiration during the dark period—, and showed a decrease in the photosynthetic activity as the drought experiment continued (Figs. 3, 4, & 5). All species showed a relief of respiratory losses of carbon in the dark period during the drought experiment. G. monostachia and

W. sanguinolenta adults showed negative net CO2 exchange during the light period in subsequent days without water (Figs 3, 4, & 5). None of the juvenile plants exhibited negative CO2 net balance during the light period.

W. sanguinolenta and G. monostachia, displayed a weak midday depression—phase

III of CAM photosynthesis described by Osmond 1976—during the drought experiment (Fig. 3 & 4). G. monostachia juveniles exhibited during the second night positive CO2 net balance in contrast to the adults that exhibited that trait during the fifth night. G. lingulata adult show the typical pattern of stressed C3 plant (Fig. 5B)

The titration results show that G. monostachia —juveniles and adults— exhibit a significant acidification during the day, before and after stress (Table 1).

W. sanguinolenta adult exhibit acidification only after drought stress, and

G. lingulata (juveniles and adults) did not exhibit acidification before and after drought stress,

DISCUSSION

The possible function of the hydrenchyma is providing water to the green tissue during rainless periods as has been documented in epiphytic cacti (Nobel 1988), which suggest that for juveniles water stored in the hydrenchyma is critical for survival. We suggest that the adult’s hydrenchyma might function as a connecting tissue that link the water hold in the tank with the green tissue in the leaf. It is uncommon to find empty bromeliad tanks during the dry seasons in wild populations (Zotz & Thomas 1999).

Our results show that well-watered adults and juveniles use the same photosynthetic pathway they are all C3. δ13C values more negative than -20‰

(Fig. 1) are evidence of not constitutive CAM (Winter et al. 2005). The relief in respiratory losses during the night is a signal of the expression of weak CAM

(Holtum & Winter 1999), all juveniles we studied showed weak CAM pattern under drought. Doubtless, this mechanism allows the survival of the bromeliads during the rainless periods, especially during their juvenile stage.

Our data suggest that W. sanguinolenta is a C3 plant under well water conditions and resort to a weak CAM under drought, this been evident by gas exchange during drought and titration (Fig. 3, Table. 1). Contrary to general believe that tillandsioids are not plastic in their CAM expression (Pierce et al. 2002) we found that

W. sanguinolenta, G. lingulata (juveniles), and G. monostachia shows a plastic weak

CAM expression under drought, weakly express phase III and relief of respiratory losses at the night during the experiment.

W. sanguinolenta adults and juveniles showed a positive net gas exchange during the light period through the experiment (Fig. 3), indicating that the juveniles and adults did not cease their gas exchange after one day or three days of drought as found by Zotz et al. (2004). The stress conditions imposed by Zotz et al. (2004) was different than the stress conditions in the present work as the light intensity. Our CO2 exchange data support that G. monostachia adult is a CAM facultative plant (Medina et al. 1977). Our data (Fig. 4A) is different to the gas exchange of G. monostachia “seedling” found by Pierce et al. (2002) where the plants never uptake CO2 during the dark period, the differences could be related with the size of the plant, probably because the plant that we used was smaller.

G. lingulata juvenile stages exhibited weak CAM only evident by the net CO2 exchange but undetectable and not supported by titration analyses. We suggest that the juvenile stage of G. lingulata has the same enzymatic machinery of a CAM plant, but only weakly expressed under drought stress, to uptake and/or recycle the CO2 during the dark period. Plausibly, the oldest parts of the leaves only exhibit CAM as occurs with G. monostachia leaves (Freschi et al. 2010) and we use the whole juvenile plant for titration and the middle of the leaf for the adults. G. lingulata shown a midday depression during the experiment (Fig. 5) as found by Maxell

(2002), however the environmental conditions used by Maxwell were different.

According to Maxwell the midday depression is related to high light intensity, nevertheless the experimental conditions in the present study light were held constant through the day. Therefore, the midday depression in G. lingulata not only has a light component but a CO2 component related to the weak expression of CAM.

Differences on δ13C values among species are likely to be related to habitat preferences. G. monostachia and W. sanguinolenta growth in the canopy, an exposed area. The biotic process enriched the understory atmosphere with 12C isotopes

(Smith et al. 1985, Broadmeadow et al. 1999); the atmosphere in the canopy is less enriched with 12C isotopes than the understory atmosphere. This might explains why G. lingulata exhibit more negative values that the other plants.

Differences on δ13C values during the ontogeny are probably explained by changes in the anatomy or changes in the stressful conditions. We found that Juveniles has less air spaces than adults, so the juveniles leaves had less porosity. The intercellular partial pressure may be lower in leaves with low porosity and high cell density (Parkhurst 1988). The partial pressure of CO2 became less in the juvenile’s intercellular spaces that in the adult’s intercellular spaces and in the atmosphere, as a consequence the δ13C values became less negative in juveniles (Farquhar et al.

1989).

Another scenario is that juveniles are more water stressed than adults (Benzing

2000), therefore during drought stress the plant closed the stomata, the partial pressure of CO2 decreased in the intercellular spaces that in the atmosphere

(Farquhar et al. 1989); as a consequence the δ13C values became less negative in juveniles than adults. In this scenario where more water stress implies that the juveniles resort to weak CAM according to our data (Fig. 3, 4 & 5). Using the same enzymatic machinery of a CAM plant, but weakly expressed, the δ13C values became less negative but not as much as δ13C values of a constitutive CAM plant (Pierce et al.

2002). The three different processes may be occurring at the same time. Our data do not allow us to discriminate that process is occurring and those not give us much information on its prevalence. In conclusion, there is no evidence of constitutive CAM in well-watered juveniles, however our data suggest a weak CAM induction under drought in juveniles of

G. lingulta, a typical shade tolerant C3 plant. Our data confirm the CAM facultative of

G. monostachia in adults and show that the juveniles are CAM facultative, too.

W. sanguinolenta is a C3 plant under well water conditions, different to the conclusion of Pierce et al. (2002). These results open questions about the nature of

CAM plants in the genus Guzmania because the only known member that exhibit

CAM was G. monostachia. The evolution of CAM photosynthesis in the subfamily

Tillandsioideae must consider the weak expression, not only under well water conditions, more over under water stress.

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Zotz G & Ziegler H. (1997) The occurrence of crassulacean acid metabolism among vascular epiphytes from Central Panama. New Phytologist 137, 223–229. Table 1. Differences on titratable acidity between juveniles and adults of Guzmania lingulata, Guzmania monostachia, and Werahuia sanguinolenta. Titrated until pH 6.5. Titratable acidity represents the mean± 1 SE, five samples per treatment. Significative differences between means at dusk and dawn were determined by Student’s t test.

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C Werauhia sanguinolenta

50 150 250 350 Length of Longest Leaf (mm)

Fig. 1. (A) Relationship between the length of the longest leaf and percentage of hydrenchyma and epidermis for Werauhia. sanguinolenta r2= 0.7789, p- value<0.001, n=36; Guzmania monostachia r2= 0.1313, p-value<0.001, n=25; Guzmania lingulata r2= 0.4523, p-value<0.001, n=31. The epidermis thickness mean (mm) ± SE of W. sanguinolenta 0.028 ± 0.0003, G. monostachia 0.027 ± 0.0006, G. lingulata 0.027 ± 0.0003. (B) Relationship between the length of the longest leaf and percentage of chlorenchyma for W. sanguinolenta r2= 0.7413, p- value<0.001, n=36; G. monostachia r2= 0.4785, p-value<0.001, n=25; G. lingulata r2= 0.5421, p-value<0.001, n=31. (C) Relationship between the length of the longest leaf and Cl/Hy ratio for W. sanguinolenta r2= 0.8691, p-value<0.001, n=36; G. monostachia r2= 0.617, p-value<0.001, n=25; G. lingulata r2= 0.6144, p- value<0.001, n=31. For all species the thickness of the leaf was not significative correlated with the length of the leaf. Leaf thickness (mean±SE) of W. sanguinolenta 0.44±0.01, G. monostachia 0.28±0.01, G. lingulata 0.21±0.007. -26

-28

)

) -30

‰

(

3 1

1 -32

δ !

-34 Guzmania lingulata Guzmania monostachia -36 Werauhia sanguinolenta

1.5 x 10-3 1.5 x 10-2 1.5 x 10-1 1.5 x 10-0 1.5 x 101 Log Dry Mass (g)

Fig. 2. Relationship between log dry mass and δ13C values. The youngest plants had less negative δ13C values than adults. For all species the correlation between dry mass and δ13C values was significant and negative for Werauhia sanguinolenta correlation factor -0.329, r2= 0.11, p-value<0.05, n=38; Guzmania monostachia correlation factor -0.4419, r2= 0.195, p-value<0.05, n=30; Guzmania lingulata correlation factor -0.5929, r2= 0.352, p-value<0.001, n=37.

Werauhia sanguinolenta 8 A 6

) 1

- 2

D 0

l -2

n (

18 6 18 6 18 6 18 6 18 6 18

e g

n 12

h c

x 9 B

2 O

C 6

t e

N 3

-3

18 6 18 6 18 6 18 6 18 6 18 6 18 6

Time of Day (h)

Fig. 3. Net CO2 exchange rates exhibited by juvenile stage, 61 plants, DM mean: 0.01 g per plant (A) and adult stage, 1 plant DM 2.16 g (B) of Werauhia sanguinolenta under drought. The solid bars represents the dark periods (23°C), the open bars represent the light periods (300 μmol photons m–2 s–1, 28°C). The dashed lines indicate zero net CO2 exchange.

Guzmania monostachia

3 A

) 1

- s

1 -1

o -2

n (

18 6 18 6 18 6

e g

n 5

h c

x 4 B

O 3

e 2 N 1

-1 18 6 18 6 18 6 18 6 18 6 18 6

Time of Day (h)

Fig. 4. Net CO2 exchange rates exhibited by juvenile stage, 98 plants, DM mean: 0.007 g per plant (A) and adult stage, 1 plant DM 3.26 g (B) of Guzmania monostachia under drought. The solid bars represents the dark periods (23°C), the open bars represent the light periods (300 μmol photons m–2 s–1, 28°C). The dashed lines indicate zero net CO2 exchange.

Guzmania lingulata

20 A 15

)

- s

1 0

o -5

n (

18 6 18 6 18 6 18 6

e g

n 12

h c x B

E 9

2 O

C 6

t e

N 3

-2

18 6 18 6 18 6 18 6 18 6 18 6 18 6

Time of Day (h)

Fig. 5. Net CO2 exchange rates exhibited by juvenile stage, 80 plants, DM mean: 0.02 g per plant (A) and adult stage, 1 plant DM 2.22 g (B) Guzmania lingulata under drought. The solid bars represents the dark periods (23°C), the open bars represent the light periods (300 μmol photons m–2 s–1, 28°C). The dashed lines indicate zero net CO2 exchange.