ARTICLE IN PRESS

Journal of Physiology 167 (2010) 526–533

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Journal of Plant Physiology

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Specific leaf areas of the tank bromeliad monostachia perform distinct functions in response to water shortage

Luciano Freschi a, Cassia Ayumi Takahashi a, Camila Aguetoni Cambui a, Thais Ribeiro Semprebom a, Aline Bertinatto Cruz a, Paulo Tamoso Mioto a, Leonardo de Melo Versieux a, Alice Calvente a, Sabrina Ribeiro Latansio-Aidar b, Marcos Pereira Marinho Aidar b, Helenice Mercier a,n a Department of Botany, Institute of Biosciences, University of Sao~ Paulo, CEP 05508-900, Sao~ Paulo, SP, b Institute of Botany, C.P. 4005, 01061-970 Sao~ Paulo, SP, Brazil a r t i c l e i n f o a b s t r a c t

Article history: Leaves comprise most of the vegetative body of tank bromeliads and are usually subjected to strong Received 18 September 2009 longitudinal gradients. For instance, while the leaf base is in contact with the water accumulated in Received in revised form the tank, the more light-exposed middle and upper leaf sections have no direct access to this water 28 October 2009 reservoir. Therefore, the present study attempted to investigate whether different leaf portions of Accepted 29 October 2009 , a tank-forming C3-CAM bromeliad, play distinct physiological roles in response to water shortage, which is a major abiotic constraint in the epiphytic habitat. Internal and external Keywords: morphological features, relative water content, pigment composition and the degree of CAM expression Crassulacean acid metabolism were evaluated in basal, middle and apical leaf portions in order to allow the establishment of CAM-idling correlations between the structure and the functional importance of each leaf region. Results indicated Drought stress that besides marked structural differences, a high level of functional specialization is also present Epiphytes Photosynthesis along the leaves of this bromeliad. When the tank water was depleted, the abundant hydrenchyma of basal leaf portions was the main reservoir for maintaining a stable water status in the photosynthetic tissues of the apical region. In contrast, the CAM pathway was intensified specifically in the upper leaf section, which is in agreement with the presence of features more suitable for the occurrence of

photosynthesis at this portion. Gas exchange data indicated that internal recycling of respiratory CO2 accounted for virtually all nighttime acid accumulation, characterizing a typical CAM-idling pathway in the drought-exposed . Altogether, these data reveal a remarkable physiological complexity along the leaves of G. monostachia, which might be a key adaptation to the intermittent water supply of the epiphytic niche. & 2009 Elsevier GmbH. All rights reserved.

Introduction bromeliads, lack an absorptive root system and completely depend on direct precipitation for their water supply (Benzing, The epiphytic habitat represents a highly dynamic environ- 1990). Therefore, a suite of adaptations must be employed by ment, subject to temporal and spatial variations in irradiation, these plants in order to cope with an intermittent water supply nutrient and water supply. Among these abiotic factors, sporadic (Zotz and Hietz, 2001). or seasonal periods of water shortage are perhaps one of the most Approximately half of all bromeliads are epiphytic, and the common challenges even for epiphytes occurring in humid success of this family in the epiphytic niche is frequently tropics, which are characterized by a high annual rainfall associated with the development of strategies to intercept, absorb (Zotz and Thomas, 1999). Some of these epiphytes, such as the and store rainwater more efficiently (Benzing, 2000). In the tank- forming bromeliads, for example, the rainwater accumulates in external tanks (phytotelma) formed by the overlapping of the leaf

Abbreviations: CAM, Crassulacean acid metabolism; Cars, carotenoids; Chls, bases, allowing the plant to draw upon their water reservoir chlorophylls; DW, dry weight; DH+ , dawn-dusk titratable acidity; MDH, malate during periods of drought (Schmidt and Zotz, 2001). Moreover, dehydrogenase; PEPC, phosphoenolpyruvate carboxylase; RWC, relative water the presence of epidermal trichomes on the leaf surfaces ensures content. an efficient way for water absorption in most of these epiphytic n Corresponding author at: Departamento de Botanica,ˆ Instituto de Biociencias,ˆ bromeliads (Benzing, 2000). Universidade de Sao~ Paulo, Rua do Matao,~ 277 CEP 05508-900 Sao~ Paulo, SP, Brasil. Fax: +55 11 30917547. In addition to these morphological specializations, a large E-mail address: [email protected] (H. Mercier). number of epiphytic bromeliads display Crassulacean Acid

0176-1617/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2009.10.011 ARTICLE IN PRESS

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Metabolism (CAM), which is a specialized photosynthetic path- levels of DH + when compared to their middle and upper leaf way that minimizes the plant evaporative demand by opening its portions. stomata mainly throughout the night when atmospheric vapor Based on these studies, the present work was designed pressure deficits are lower (Luttge,¨ 2004). In fact, it is well-known to evaluate whether different leaf portions of G. monostachia, a that CAM bromeliads are especially abundant in epiphytic tank-forming C3-CAM bromeliad, play distinct physiological communities of dry forests (Griffiths and Smith, 1983), which roles during the modulation of the photosynthetic pathway in reinforces the ecological importance of the CAM pathway for the response to changes in water availability. The level of CAM survival of vascular epiphytes in water-limited environments. was evaluated in basal, middle and apical leaf portions of this Besides water economy, another key feature of the CAM is the bromeliad by analyzing the changes in DH+ and in the activities remarkable plasticity provided by this mode of photosynthesis, of key enzymes of the CAM pathway. Additionally, internal and especially among the C3-CAM facultative , which can external morphological features, relative water content and perform either C3 or CAM depending on the environmental chlorophylls and carotenoids levels were analyzed in these conditions (Cushman, 2001). Among the bromeliads, the best portions in order to allow the establishment of correlations recognized facultative CAM species is Guzmania monostachia between the structure and the functional importance of each leaf (Medina et al., 1977; Griffiths and Smith, 1983; Maxwell et al., region. Finally, gas exchange analyses were performed in the leaf 1992). This epiphytic tank bromeliad is widespread throughout portions to obtain more information about possible differences in the middle to upper canopy in neotropical forests and shows a the photosynthetic capacity along the leaf blade. high capacity of acclimation in response to variations in the natural environment (Maxwell et al., 1994). Water availability and light intensity seem to be the major environmental factors controlling the degree of CAM expression Material and methods in G. monostachia (Medina et al., 1977; Maxwell et al., 1994). Medina et al. (1977) observed that this bromeliad exhibits Plant growth and treatments atmospheric CO2 uptake only during the day with a small day/ night malate fluctuation when water is not limited. On the other Adult plants of Guzmania monostachia (L.) Rusby ex Mez var. hand, after approximately one week of water shortage, the CO2 monostachia (see Supplementary Table S1 for growth stage uptake occurred predominantly during nighttime, and larger acid characterization) were taken from stocks maintained in a green- fluctuations were observed. Moreover, re-watering drought- house of the Department of Botany at the University of Sao~ Paulo, exposed G. monostachia plants resulted in a reversible shift from Sao~ Paulo, Brazil, and transferred to controlled environment CAM to C3 photosynthesis (Medina et al., 1977). chambers. By analyzing the gas exchange of G. monostachia growing as an Throughout the experiments, and for the preceding three weeks epiphyte, Luttge¨ et al. (1986) found that nocturnal recycling of acclimation, the plants were maintained in a growth chamber at of respired CO is particularly important under field conditions. 2 1 2 a photosynthetic flux density (PFD) of about 250 mmol mÀ sÀ Afterwards, studies carried out by Maxwell et al. (1994) also supplied by fluorescent lamps (Sylvania, Germany), 12 h photo- indicated that the nocturnal acid accumulation in drought- period, day/night air temperature of 25/20 1C, and day/night exposed G. monostachia resulted mainly from re-fixation of relative humidity of 60/70%. PFD inside the growth chamber was respiratory CO2. This nocturnal regeneration of respiratory CO2 monitored with an LI-190 quantum sensor connected to an LI-250 can be considered an important CAM response to severe stress A meter (LI-COR Instruments, USA). All plants were cultivated in conditions since the removal of this internal CO2 source pots containing vermiculite, with one plant per pot. They were significantly increased the susceptibility to photoinhibition as watered with distilled water on a daily basis and received a 10% demonstrated for Pyrrosia piloselloides (Griffiths et al., 1989). (v:v) dilution of Hoagland’s solution once a week (Hoagland and Typical for most bromeliads, G. monostachia exhibits long- Arnon, 1938). lived leaves arranged in rosettes (Griffiths and Smith, 1983). After the acclimation period, plants were separated into three This rosette-habit creates a longitudinal light gradient along the experimental groups, each one submitted to a different condition leaf as the top portion receives more light than the leaf bases of watering: (a) control plants watered daily for a week, (b) water during the entire life span (Popp et al., 2003). Moreover, in stress treatment, in which water was withheld for a week and leaves of tank-forming bromeliads there is the establishment (c) rewatered treatment, which consisted of plants subjected to of additional gradients along the leaf blade. In these plants, only drought for a week and, then, watered daily for another week. the leaf bases (also called leaf sheaths) are in direct contact with the water accumulated in the tank and have the ability to absorb this resource (Schmidt and Zotz, 2001). On the other hand, the presence of water in the tank also limits the capacity of Leaf sampling the leaf bases to exchange gas with the atmosphere (Benzing, 2000). After each treatment, the 2nd to the 8th youngest fully Consequently, we should expect strong functional differences developed leaves from five individual plants were collected 1 h along the leaves of bromeliads, especially among the tank- after the onset of illumination and divided in three portions: (a) forming species. However, only few studies have analyzed the basal, corresponding to the part of the leaf that forms the tank; (b) existence of physiological gradients along the bromeliad leaves. In middle, corresponding to the lower half of the green portion of the CAM species Aechmea aquilega, for instance, the highest values the leaf blade; and (c) apical, corresponding to upper half of + of nocturnal acid accumulation (DH ) and CO2 uptake were found the green part of the leaf blade. All samples corresponding to each at the distal third of the leaf (Luttge¨ et al., 1986). Also, significant leaf portion were fragmented into small pieces of about 5 mm differences in the content of nitrogen and lipids and in the d13C length, weighed, frozen in liquid nitrogen and stored at 20 1C À values were detected between the chlorophyll-containing tissues until use in the biochemical analyses. Additional samples for and the non-chlorophyllous leaf bases of pineapple leaves titratable acidity determination were taken 1 h before the end (Medina et al., 1994). Further, Popp et al. (2003) observed of the light period, frozen in liquid nitrogen and also stored at that the leaf bases of five CAM bromeliads possessed the lowest 20 1C until use. À ARTICLE IN PRESS

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Anatomical analyses direction in a 2 mL reaction medium containing 50 mM Tris–HCl

(pH 8.0), 2 mM OAA, 5 mM MgCl2 and 200 mM NADH (Cuevas and Freehand transverse sections were taken from fresh Podesta´, 2000). For both enzymatic determination, the reaction samples of basal, middle and apical leaf portions of four indivi- was started by adding an aliquot of enzyme extract and change in dual well-watered plants and analyzed by optical microscopy absorbance was continuously measured at 340 nm. All reported (Zeiss Standard 25 ICS, Carl Zeiss, Germany) in order to evaluate rates are from linear portions of absorbance vs. time curves mesophyll, hydrenchyma and chlorenchyma thickness as well as (usually between 0 and 10 min). The enzymes were assayed the hydrenchyma cell diameter. For each parameter, about 80 at 30 1C. measurements per leaf portion were carried out. Furthermore, stomatal and trichomes densities were assessed using epidermal Leaf gas exchange and incident light measurements silicon imprints prepared from the abaxial surfaces of each leaf portion (10 fields of 1 mm2 per leaf portion). All anatomical measurements were examined using KS100 3.0 software (Carl Gas-exchange measurements were made continuously on the Zeiss, Germany). middle and apical portions of the same leaf over 24 h by using an infra-red gas exchange system (LI-6400, Li-Cor, USA). Every leaf portion was enclosed in a chamber, which tracked the environ- Measurements of tissue water content mental conditions inside the growth cabinet. A CO2 cylinder was 2 used to keep CO2 concentration constant under 380 mmol mÀ The tissue water content of the leaf portions was determined 1 sÀ . Three plants were analyzed for each treatment and all according to Martin and Schmitt (1989). After determining the measurements were carried out in the second youngest fully fresh weight (FW), the samples were dried to a constant weight developed leaf of each plant. Incident photosynthetic flux density at 65 C and allowed to cool down for 3 h before determining 1 (PFD) on the adaxial surfaces of basal, middle and apical portions the dry weight (DW). Tissue water content was calculated using of these leaves was also determined by using an LI-190 quantum the formula ((FW DW)/DW) 100. Measurements were made in À Â sensor connected to an LI-250 A meter (LI-COR Instruments, USA). triplicates.

Chlorophylls and carotenoids determination Statistical analysis

Chlorophylls (Chls) and carotenoids (Cars) were extracted by All data are presented as mean 7SD. One-way ANOVA was homogenizing 1 g of fresh leaf tissue in 7 mL of cold 80% (v/v) used to analyze the results and the means were compared by the aqueous acetone. The homogenate was filtered, and the total Tukey test at 5% probability level. volume of filtrate was completed to 20 mL with cold 80% acetone. Chls a and b and total Cars were determined spectrophotome- trically according to Lichtenthaler (1987). Measurements were Results 1 made in triplicates and the results were expressed as mg gÀ DW. The relative levels of Chl a and Chl b (Chl a/b) as well as the ratio Longitudinal differences in the leaf structure and of total Chls to total Cars (Chls/Cars) were also calculated for each pigment composition leaf portion. In order to investigate the existence of morphological Titratable acidity gradients along the leaf blade of G. monostachia, anatomical analyses were carried out on the basal, middle and apical leaf + To determine the nocturnal accumulation of acidity (DH ), leaf portions of this bromeliad. These analyses revealed an inverse samples collected before dawn and dusk were ground in liquid relationship between the densities of trichomes and stomata nitrogen and then homogenized with 20 volumes (v/w) boiled along the leaf blade (Table 1). Trichome density was twofold distilled water. The crude extracts were boiled for 20 min, filtered higher in basal than in middle and apical leaf portions. In contrast, and then allowed to cool down to room temperature. Titration the middle and apical leaf regions showed the highest values of the water extracts was performed with 20 mM NaOH to an end of stomatal density, while in the basal portion there were almost point of pH 11. Titration up to pH 11 covered the ionization range no stomata (Table 1). Moreover, internal anatomical analyses of malic, citric and isocitric acids. Measurements were made in demonstrated that the mesophyll and hydrenchyma thickness + 1 triplicates and the results were expressed as mmol H gÀ DW. increased significantly from the apical to the basal region of the leaves (Table 1). The chlorenchyma thickness, on the other hand, PEPC and MDH extraction and assay was relatively uniform along the three leaf portions. In addition, while the apical and middle portions exhibited hydrenchyma cells To measure phosphoenolpyruvate carboxylase (PEPC, EC of approximately the same diameter, these cells were significantly 4.1.1.31) and malate dehydrogenase (MDH, EC 1.1.1.37) activities, larger in the leaf bases (Table 1). leaf samples stored in liquid nitrogen were ground to a fine Besides these anatomical differences, the content of Chls powder and extracted in five volumes (v/w) of buffer containing and Cars also varied markedly along the leaf blade. As expected, 200 mM Tris–HCl (pH 8.0), 1 mM EDTA, 5 mM dithiothreitol the lowest content of total Chls and Cars were found in the

(DTT), 10 mM MgCl2, 10% (v/v) glycerol, 0.5% (w/v) bovine serum leaf bases (Table 2). Moreover, the leaf bases exhibited the albumine (BSA). The homogenate was centrifuged for 5 min at lowest values of Chl a/b ratio (Chl a/b) and the highest 15.000g, and the supernatant was immediately used for the weight ratio of total Chls to total Cars (Chls/Cars), as is usually enzymatic assays. The PEPC activity was assayed in a 2 mL found in leaves adapted to low light conditions (Lichtenthaler standard reaction medium containing 50 mM Tris–HCl (pH 8.0), et al., 2007). On the other hand, much higher values of Chl a/b 1 mM DTT, 10 mM MgCl2, 10 mM NaHCO3, 200 mM NADH, 3 mM ratio, total Chls and Cars contents were observed in the apical leaf phosphoenolpyruvate (PEP) and 10 units MDH (Nievola et al., portion, suggesting higher photosynthetic capacity in this region 2005). The MDH activity was assayed in the OAA-reducing (Table 2). Incident PAR was also significantly higher in the leaf top ARTICLE IN PRESS

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Table 1 Anatomical characteristics of different leaf portions of well-watered plants of G. monostachia.

Leaf Stomatal density Trichome density Mesophyll thickness Hydrenchyma thickness Hydrenchyma cell Chlorenchyma 2 2 portions (mmÀ ) (mmÀ ) (lm) (lm) diameter (lm) thickness (lm)

Top 22(74.9) a 23(74.6) b 303.5(720.8) c 190.7(725.2) c 59.1(711.2) b 112.8(722.1) c Middle 11(74.0) b 26(74.2) b 357.9(724.3) b 225.9(722.5) b 60.5(79.3) b 132.3(718.2) a Base 1(70.5) c 53(711.9) a 429.8(725.2) a 304.1(728.5) a 66.6(79.3) a 123.8(726.8) b

Data for stomatal and trichomes densities were derived from light microscopy of 10 replicates per leaf portion. Internal anatomical data were determined from transverse leaf sections mounted in water and examined by light microscopy (n=80 per leaf portion). Data are expressed as the mean 7SD. Different letters indicate a significant difference between the leaf portions (Po0.05%; Tukey test).

Table 2 Total chlorophylls, total carotenoids, chlorophyll a/b ratio (Chl a/b) and chlorophylls/carotenoids ratio (Chls/Cars) in different leaf portions of G. monostachia plants maintained under well-watered conditions or exposed to 7 d of drought.

1 1 Treatment Leaf portion Total chlorophylls (lg gÀ DW) Total carotenoids (lg gÀ DW) Chl a/b ratio Chls/cars ratio

Well-watered Top 1602.5(7194.9) a 564.0(793.9) a 2.7(70.1) a 2.8(70.2) b Middle 1211.7(7123.6) b 420.8(759.8) a 2.7(70.1) a 2.8(70.1) b Base 357.3(7101.1) c 89.9(736.1) b 1.8(70.1) b 3.8(70.8) a

Drought-exposed Top 2162.5(7 186.3) a 762.7(7 79.2) a 2.5(70.1) a 2.8(70.1) b Middle 1184.8(781.3) b 407.2(734.4) b 2.6(70.1) a 2.9(70.1) b Base 199.8(759.9) c 54.1(711.5) c 1.9(70.2) b 3.7(70.4) a

Data are expressed as the mean (7SD) of three replicate samples. Different letters indicate a significant difference between the leaf portions (Po0.05%; Tukey test).

1000 hand, basal and middle portions of drought-exposed plants showed base a reduction in their RWC of about 34% and 25%, respectively. Consequently, these results indicated that the RWC in the apical Aa middle Aa 800 portion was maintained unchanged during the water stress top Ba treatment, probably at the expense of water translocation from Aa Bb Ca the basal and middle leaf sections. In these more basal leaf regions, 600 Ca ABb drought caused a reduction in leaf thickness due to shrinkage of the Bc hydrenchyma but not of the chlorenchyma (data not shown). As also presented in Fig. 1, the drought-induced reduction in the RWC of the 400 basal and middle leaf portions was completely reverted after one week of re-watering.

Relative Water Content 200

CAM expression 0 well-watered drought-exposed rewatered Since reductions in the RWC are believed to influence Treatments the degree of CAM expression in C3-CAM facultative species (Cushman, 2001), we further investigated whether the differential Fig. 1. Relative water content of different leaf portions of well-watered, drought- exposed (7 d of drought) and rewatered (7 d of drought followed by 7 d of changes in the water content along the leaf blade of recovery) plants of G. monostachia. Means followed by the same letter are not G. monostachia were correlated with modifications in the photo- significantly different. Small letters represent the comparison among the synthetic pathway (C3 or CAM) performed by each leaf region. As treatments and capital letters the comparison among the leaf portions shown in Fig. 2, the analyses revealed a marked increase in the (Po0.05%; Tukey test). activities of PEPC and MDH in the drought-exposed plants, which

2 1 2 1 occurred specifically in the apical portion of the leaves (Fig. 2). (23574 mmol mÀ sÀ ) than in the middle (16876 mmol mÀ sÀ ) 2 1 Increase in the activity of these key enzymes of the CAM cycle was and basal (68 5 mol mÀ sÀ ) leaf regions. 7 m positively correlated with a significant rise in the levels of nocturnal accumulation of titratable acidity (DH+ ) also in the Water relations along the leaf blade upper leaf region (Fig. 2). In these water stressed plants, total Chls and Cars also increased particularly in the leaf top, even though Leaves of G. monostachia also showed a marked longitudinal both Chl a/b and Chls/Cars ratios remained virtually unchanged gradient in terms of relative water content (RWC). Under well- (Table 2). When compared with the leaf top, basal and middle leaf watered condition, basal and middle leaf portions exhibited RWC portions of drought-exposed plants showed no or much less values significantly higher than those detected in the apical region marked changes in pigment composition, DH+ and PEPC and MDH (Fig. 1). On the other hand, under drought conditions, these activities. After re-watering, the values of both enzymatic activity differences practically disappeared, indicating that the reduction in and DH+ returned to levels similar to or even lower than those RWC caused by water shortage did not occur equally along the leaf observed for well-watered plants (Fig. 2). However, it is blade. In fact, the values of RWC observed in the leaf top portion interesting to mention that a significant level of nocturnal after the drought treatment were statistically similar to those acidification was detected in middle and apical leaf portions of detected in the same region of well-watered plants. On the other both well-watered and rewatered individuals of G. monostachia, ARTICLE IN PRESS

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considerable levels of nocturnal acidity accumulation observed 12 base Aa in the apical and middle regions of drought-exposed plants middle (Fig. 2C) occurred in almost complete absence of atmospheric CO2 top uptake and, therefore, could only be explained by re-fixation of 9 DW) the respiratory CO2 produced during the dark period. -1 g -1 6 Ba Discussion PEPC activity mol min µ

( Ab Ab Leaves compose most of the vegetative body of epiphytic tank 3 Bb Ca Bb Bc bromeliads and are responsible not only for photosynthesis but Bc also for water absorption and storage (Benzing, 2000; Popp et al., 0 2003). Therefore, besides anatomical modifications, functional well-watered drought-exposed rewatered differences are also expected to occur along the leaf blade of these 500 bromeliads (Benzing, 2000; Takahashi et al., 2007). In agreement, Aa the data obtained here demonstrated that different leaf regions of G. monostachia can perform distinct roles during the physiological 400 responses triggered by changes in one of the most significant

DW) variables in the epiphytic niche, the water availability (Zotz and -1 300 Hietz, 2001). g

-1 During sporadic or seasonal periods of water deprivation, Ba epiphytic tank bromeliads can rely basically on only two sources 200 Ab MDH activity of water storage, the external tank and the leaf water-storing Ab mol min Bb tissues (e.g. hydrenchyma). As pointed out by Zotz and Thomas µ Ca ( Cb Bb 100 Cc (1999), the presence of relatively large tanks does not imply that bromeliads such as G. monostachia are ‘continuously supplied’ with water since without resupply, the water accumulated in this 0 well-watered drought-exposed rewatered structure will dry out after little more than one week under natural conditions. Therefore, given the intermittence in the Aa water availability typical of the epiphytic niche, the water-storing tissues of the leaf can be considered a key adaptation for the 600 survival of these plants during medium to long-term periods of water shortage (Benzing, 2000). As demonstrated in the present DW)

-1 study, these internal water reservoirs are not uniformly distrib- + 400 H uted along the leaf blade of a tank bromeliad, being especially

∆ Ab Ab Aa Ba abundant in basal leaf regions (Table 1). In fact, the gradual Ba increase in the mesophyll thickness observed from the top to the mol H+ g

(µ 200 leaf bases can be explained almost exclusively by a progressive increment in the abundance of the hydrenchyma tissues. There- Bb Ca Cb fore, the predominance of water-storing tissues closer to the leaf bases correlates well with the high capacity of water absorption at 0 well-watered drought-exposed rewatered this region, as indicated by the elevated density of trichomes Treatments (Table 1) and the direct contact with the water accumulated in the tank (Benzing, 2000). Fig. 2. Activities of PEPC (A), MDH (B), and titratable acidity (C) of different leaf Moreover, the drought-induced changes in the relative water portions of well-watered, drought-exposed (7 d of drought) and rewatered (7 d of content also occurred unevenly along the leaf blade once drought followed by 7 d of recovery) plants of G. monostachia. Means followed by the same letter are not significantly different. Small letters represent the significant reductions in the RWC occurred in the basal comparison among the treatments and capital letters the comparison among the and middle portions, while no considerable variations were leaf portions (Po0.05%; Tukey test). found in the water content of the leaf top (Fig. 1). Given the fact that the drought treatment was imposed by removing the water supply from both roots and tanks, the maintenance of a stable water status in the leaf apex during drought was suggesting the occurrence of some degree of CAM even under probably due to water translocation from tissues closer to these conditions (Fig. 2). the leaf bases. Therefore, when water is no longer available

Striking differences in the diurnal pattern of CO2 assimilation in the external tank, the water stored in the hydrenchyma of were also observed between well-watered and drought-treated these basal leaf regions may represent the main reservoir for plants (Fig. 3). In well-watered plants, CO2 assimilation occurred maintaining a favorable water status in the photosynthetic primarily during early morning and late afternoon in the middle tissues. In agreement with these results, several studies have and apical leaf portions, both showing a midday depression. In demonstrated that water movement between distinct leaves or these plants, the apical leaf portion exhibited diurnal levels of gas from tissue to tissue within a leaf are commonly observed in exchanges slightly higher than those found in the middle region, succulent plants exposed to drought (Schulte and Nobel, 1989). In revealing a positive correlation with the highest stomatal density most cases, a preferential movement of water from chlorophyll- also detected in the leaf top (Table 1). In contrast to the well- less hydrenchyma to the chlorenchyma is maintained by an watered plants, those submitted to water stress exhibited osmotic gradient in which the hydrenchyma, with lower solute virtually no CO2 exchange in the middle and apical leaf portions concentration, provides water to the chlorenchyma tissue during the entire day and night (Fig. 3). Therefore, the (Schulte and Nobel, 1989). For instance, Nowak and Martin ARTICLE IN PRESS

L. Freschi et al. / Journal of Plant Physiology 167 (2010) 526–533 531

3 top middle 2 ) -1 s -2 1 mol m µ (

0 Net photosynthetic rate

-1 18:00 20:00 22:00 0:00 2:00 4:00 6:00 8:00 10:00 12:00 14:00 16:00 18:00 3

2 ) -1 s -2 1 mol m µ (

0 Net photosynthetic rate

-1 18:00 20:00 22:00 0:00 2:00 4:00 6:00 8:00 10:00 12:00 14:00 16:00 18:00 Time of the day

Fig. 3. Diurnal pattern of CO2 assimilation in the middle and top leaf portions of G. monostachia plants maintained under well-watered conditions (A) or exposed to 7 d of drought (B). The shaded areas indicate the dark period. (n=3).

(1997) demonstrated that water movement from the hydrench- the presence of several leaf morphological adaptations to drought yma to the chlorenchyma was responsible, at least in part, for the stress, such as hypostomaty, presence of sunken stomata and a maintenance of high physiological activity in the atmospheric mesophyll dominated by a tightly packed hydrenchyma. Although CAM bromeliad Tillandsia ionantha even after 20 d of extreme crucial for the survival in the water-limited epiphytic niche, these drought conditions. leaf morphological adaptations may limit CO2 diffusion from the Taking into account the direct contact of the base leaf portion atmosphere to inside the leaf and, therefore, restrict carbon gain with the water retained in the tank, even if intermittently, and the and growth (Maxwell, 2002). considerably lower light intensities received by this region, the Another key adaptation of G. monostachia to withstand the leaf bases of tank bromeliads are assumed to carry out reduced water shortage periods is the capacity to perform CAM photo- photosynthetic rates (Benzing, 2000). Although the partial super- synthesis under adverse conditions (Medina et al., 1977; Griffiths position of the leaves impeded direct measurements of gas and Smith, 1983; Maxwell et al., 1992). Studies carried out by exchanges in intact leaf bases of G. monostachia, the almost Maxwell et al. (1995) demonstrated that CAM is rapidly induced complete absence of stomata and the low levels of Chls and Cars in this bromeliad at the start of the dry season and is completely in fact suggested little photosynthetic activity at this leaf portion. reverted to C3 photosynthesis as the conditions become more On the other hand, the leaf regions localized outside the tank area favorable. Afterwards, by analyzing the CAM activity in and, consequently, more exposed to light and with less restriction G. monostachia over an entire year, Zotz and Andrade (1998) to carry out gas exchange exhibited a set of characteristics revealed that considerable nocturnal acidification in the leaf indicative of a higher photosynthetic capacity, such as abundant tissues of this bromeliad can occur even during the rainy periods stomatal densities, increased contribution of the chlorenchyma to with just a small, but significant, increase in the dry season. the mesophyll thickness (Table 1) and higher total Chls and Cars In agreement with these field data, our results also indicated contents (Table 2). In general, similar diurnal patterns of gas significant levels of nocturnal H + accumulation in middle and exchange were observed in the middle and apical leaf portions of apical leaf portions of well-watered plants of G. monostachia well-watered plants, with maximal photosynthesis restricted to (Fig. 2), suggesting that some level of CAM can occur in this the beginning and end of the photoperiod (Fig. 3). In agreement bromeliad even when water supply is abundant and fairly with previous laboratory and field studies (Luttge¨ et al., 1986; constant. However, the data obtained also reinforce the occur- Maxwell et al., 1994, 1995; Maxwell, 2002), G. monostachia under rence of a high photosynthetic plasticity in G. monostachia and well-watered conditions exhibited low values of diurnal atmo- provide evidence demonstrating that the intensification of the spheric CO2 (Fig. 3), which may be attributed, at least in part, to CAM in response to drought does not occur homogenously along ARTICLE IN PRESS

532 L. Freschi et al. / Journal of Plant Physiology 167 (2010) 526–533 the leaf blade of this bromeliad. In fact, the drought treatment perform distinct functional roles in response to water shortage, induced significant increases in DH + exclusively in the upper leaf which may represent a significant adaptation to the survival portion where most of the activities of CAM enzymes were also of this species in the water-limited epiphytic niche. The main detected (Fig. 2). On the other hand, the levels of DH+ and anatomical, biochemical and physiological differences observed activities of CAM enzymes in the middle and basal leaf portions along the leaves of this bromeliad are summarized in the exhibited little or no changes in response to water stress. Supplementary Figure S1, which also presents the internal Therefore, the intensification of CAM in response to drought structure of each leaf region. According to the results, when was detected exclusively in the leaf top, which is the most light- water is no longer available in the external tank, the leaf base exposed leaf portion (Table 2) and also the only region that seems to represent the main reservoir of this resource for retained almost unchanged values of RWC (Fig. 1). Since the maintaining the photosynthetic activity in more apical leaf presence of large vacuoles in the mesophyll cells are considered to regions. On the other hand, another critical drought adaptation, be critical for overnight acid storage in CAM plants (Cushman, the CAM pathway, was intensified specifically in the upper leaf 2001), it seems plausible to assume that, by keeping a relatively section, which was the only region showing an almost constant stable water content, the capacity of C4 acid accumulation at the water status during water deprivation. Finally, gas exchange data leaf top remained unaffected during the drought, which may indicated that under drought conditions practically all nighttime explain the preference for the operation of the CAM cycle at this accumulation of organic acids resulted from the re-fixation of leaf portion. In addition, the higher nocturnal carboxylation respiratory CO2, characterizing, therefore, the establishment of a capacity of the leaf apex might also be associated with a possibly typical CAM-idling pathway. Altogether, these results reinforce higher availability of carbohydrates at this region since it is the remarkable physiological complexity of tank bromeliad leaves considered a limiting resource for nocturnal CO2 fixation in CAM and highlight the importance of further studies regarding the plants (Borland and Dodd, 2002). Moreover, the occurrence of existence of functional gradients along the leaf blade of other drought-induced increases in total Chls and Cars exclusively in highly specialized plants. the leaf top of G. monostachia (Table 2) seems to indicate that the intensification of the CAM at this leaf region was also associated with a general increment in the photosynthetic capacity of the Appendix A. Supplementary material tissue. Irrespective of the reasons for the preferential establish- ment of the CAM cycle in the leaf top, these data are in agreement Supplementary data associated with this article can be found with previous observations in obligate CAM bromeliads, which in the online version at doi:10.1016/j.jplph.2009.10.011. usually show higher values of DH+ from the leaf bases to the tip (Luttge¨ et al., 1986; Popp et al., 2003).

Virtually no exogenous CO2 uptake was observed during the entire diurnal cycle in both middle and upper leaf portions of References drought-exposed plants (Fig. 3) therefore, we can conclude that all nighttime accumulation of organic acids detected in these Benzing DH. In: Vascular Epiphytes: General Biology and Related Biota. tissues (Fig. 2) was originated by nocturnal re-fixation of Cambridge: Cambridge University Press; 1990. Benzing DH. In: : Profile of an Adaptive Radiation. Cambridge: respiratory CO2. This physiological state, known as CAM-idling, Cambridge University Press; 2000. is considered a strong mode of CAM which is characterized by a Borland AM, Dodd AN. Carbohydrate partitioning in Crassulacean acid metabolism substantial reduction in water loss by maintaining the stomata plants: reconciling potential conflicts of interest. Funct Plant Biol 2002;29: 707–16. ¨ closed during day and night (Rayder and Ting, 1983; Luttge, Ceusters J, Borland AM, Londers E, Verdoodt V, Godts C, De Proft MP. Differential 2004). Although plants in CAM-idling mode do not show net usage of storage carbohydrates in the CAM bromeliad Aechmea ‘Maya’ carbon gain or growth, they are able to maintain a basal metabolic during acclimation to drought and recovery from dehydration. Physiol Plant activity even during severe drought conditions and can rapidly 2009;135:174–84. Cuevas IC, Podesta´ FE. Purification and physical and kinetic characterization of an recover the photosynthetic capacity when favorable water NAD + -dependent malate dehydrogenase from leaves of pineapple (Ananas conditions are re-established (Rayder and Ting, 1983). Addition- comosus). Physiol Plant 2000;108:240–8. ally, in these plants, the internal CO recycling may alleviate Cushman JC. Crassulacean acid metabolism. A plastic photosynthetic adaptation to 2 arid environments. Plant Physiol 2001;127:1439–48. photoinhibition by providing a sink for electron transport. There- Griffiths H, Smith JAC. Photosynthetic pathways in the Bromeliaceae of Trinidad: fore, although a number of studies under field and laboratory relations between life-forms, habitat preference and the occurrence of CAM. conditions have already reported that re-fixation of respiratory Oecologia 1983;60:176–84. Griffiths H, Ong BL, Avadhani PN, Goh CJ. Recycling of respiratory CO2 during CO2 forms a significant proportion of the nocturnal acid Crassulacean acid metabolism: alleviation of photoinhibition in Pyrrosia accumulation in G. monostachia (Luttge¨ et al., 1986; Maxwell piloselloides. Planta 1989;179:115–22. et al., 1994; Maxwell, 2002), the data obtained here demonstrate Hoagland DR, Arnon DI. The water culture method for growing plants without soil. Calif Exp Stn Circ 1938;347:1–39. that a typical CAM-idling cycle can also be induced in this Lichtenthaler HK. Chlorophylls and carotenoids: pigments of photosynthetic bromeliad in response to water deprivation. In addition, the biomembranes. Method Enzymol 1987;148:350–82. drought-induced establishment of CAM-idling in G. monostachia Lichtenthaler HK, Ac A, Marek MV, Kalina J, Urban O. Differences in pigment composition, photosynthetic rates and chlorophyll fluorescence images of sun occurred extremely rapid when compared to other bromeliads. and shade leaves of four tree species. Plant Physiol Biochem 2007;45:577–88. For example, in the bromeliad Aechmea ‘Maya’, a typical CAM- Luttge¨ U. Ecophysiology of crassulacean acid metabolism (CAM). Ann Bot idling state occurred only after 150 d of water stress (Ceusters 2004;93:629–52. et al., 2009), while in G. monostachia practically no CO uptake Luttge¨ U, Stimmel KH, Smith JAC, Griffiths H. Comparative ecophysiology of CAM 2 and C3 Bromeliads. 2. Field-measurements of gas-exchange of CAM brome- was detected after one week of water shortage (Fig. 3). Therefore, liads in the humid tropics. Plant Cell Environ 1986;9:377–83. this rapid acclimation of the photosynthetic metabolism to abrupt Martin CE, Schmitt AK. Unusual water relations in the CAM atmospheric epiphyte changes in the water supply may help to explain the widespread Tillandsia usneoides L (Bromeliaceae). Bot Gaz 1989;150:1–8. Maxwell K. Resistance is useful: diurnal patterns of photosynthesis in C3 distribution of this bromeliad across a wide range of exposed and and Crassulacean acid metabolism epiphytic bromeliads. Funct Plant Biol shaded habitats in the epiphytic niche (Medina et al., 1977; 2002;29:679–87. Griffiths and Smith, 1983). Maxwell C, Griffiths H, Borland AM, Broadmeadow MSJ, McDavid CR. Photo- inhibitory responses of the epiphytic bromeliad Guzmania monostachia during In summary, the present study demonstrated that different the dry season in Trinidad maintain photochemical integrity under adverse leaf sections of the tank C3-CAM bromeliad G. monostachia can conditions. Plant Cell Environ 1992;15:37–47. ARTICLE IN PRESS

L. Freschi et al. / Journal of Plant Physiology 167 (2010) 526–533 533

Maxwell C, Griffiths H, Young AJ. Photosynthetic acclimation to light regime and Popp M, Janett HP, Luttge¨ U, Medina E. Metabolite gradients and carbohydrate

water stress by the C3-CAM epiphyte Guzmania monostachia: gas-exchange translocation in rosette leaves of CAM and C3 bromeliads. New Phytol characteristics, photochemical efficiency and the xanthophyll cycle. Funct Ecol 2003;157:649–56. 1994;8:746–54. Rayder L, Ting IP. CAM-idling in Hoya carnosa (Asclepiadaceae). Photosynth Res Maxwell C, Griffiths H, Borland AM, Young AJ, Broadmeadow MSJ, Fordham MC. 1983;4:203–11.

Short-term photosynthetic responses of the C3-CAM epiphyte Guzmania Schmidt G, Zotz G. Ecophysiological consequences of differences in plant size: monostachia var monostachia to tropical seasonal transitions under field in situ carbon gain and water relations of the epiphytic bromeliad, conditions. Aust J Plant Physiol 1995;22:771–81. sanguinolenta. Plant Cell Environ 2001;24:101–11. Medina E, Delgado M, Troughton JH, Medina JD. Physiological ecology of CO2 Schulte PJ, Nobel PS. Responses of a CAM plant to drought and rainfall: capacitance fixation in Bromeliaceae. Flora 1977;166:137–52. and osmotic pressure influences on water movement. J Exp Bot 1989;40: Medina E, Ziegler H, Luttge¨ U, Trimborn P, Francisco M. Light conditions 61–70. during growth as revealed by d13C values of leaves of primitive culti- Takahashi CA, Ceccantini GCT, Mercier H. Differential capacity of nitrogen vars of Ananas comosus, an obligate CAM species. Funct Ecol 1994;8: assimilation between apical and basal leaf portions of a tank epiphytic 298–305. bromeliad. Braz J Plant Physiol 2007;19:119–26. Nievola CC, Kraus JE, Freschi L, Souza BM, Mercier H. Temperature determines the Zotz G, Andrade J-L. Water relations of two co-occurring epiphytic bromeliads. occurrence of CAM or C3 photosynthesis in pineapple plantlets grown in vitro. J Plant Physiol 1998;152:545–54. In Vitro Cell Dev Biol Plant 2005;41:832–7. Zotz G, Hietz P. The physiological ecology of vascular epiphytes: current Nowak EJ, Martin CE. Physiological and anatomical responses to water deficits in knowledge, open questions. J Exp Bot 2001;52:2067–78. the CAM epiphyte Tillandsia ionantha (Bromeliaceae). Int J Plant Sci 1997;158: Zotz G, Thomas V. How much water is in the tank? Model calculations for two 818–26. epiphytic bromeliads. Ann Bot 1999;83:183–92.