• The Bromeliaceae encompass predominantly rosette, terrestrial or epiphytic species, including C3 and crassulacean acid metabolism (CAM) photosynthetic types within its three subfamilies. Here, leaf diurnal changes and longitudinal gradients of soluble sugars, organic acids and starch, were quantified to estimate the rates of carbohydrate translocation from mature leaves of C3 and CAM species.
• Leaves of Ananas comosus, Aechmea fendleri, Bromelia humilis, Guzmania mucronata, Tillandsia fendleri, Tillandsia flexuosa and Tillandsia utriculata, were sampled at the base, middle, and upper sections during the day. We measured osmolality in sap from frozen subsamples, sugars and organic acids in hot-water extracts from microwave-dried subsamples, and starch hydrolysed with α-amylase or 1.1% HCl.
• CAM activity was expressed by malate accumulation, citrate was present, but fluctuations were not significant. Nocturnal reductions in sucrose in bromelioid CAM species accounted for most of the acidification requirements. Tillandsioid CAM species used starch for acid synthesis. Both CAM and C3 bromeliads exported significant amounts of hexose during the night, particularly from the leaf base.
• Leaf bases of CAM species showed lowest acid accumulation but similar or more positive δ13C-values to the active CAM sections. Exported carbohydrates probably derive from carbon fixed during the night period.
The rosette life form is widespread in several families with nocturnal CO2-fixation (CAM plants) such as the Agavaceae, Liliaceae and Bromeliaceae. The rosette-habit is unique in that a plant contains a well-defined leaf age-structure. New leaves grow in length from a basal intercalary meristem. Therefore, there is an age gradient from the center to the periphery of the rosette, and from the base to the tip of each leaf (Olivares & Medina, 1990). The leaf arrangement in rosettes creates a highly variable light environment because the angle of inclination of leaves decreases with leaf age and mutual shading increases with number of leaves. In addition, there is a longitudinal light gradient in each leaf, the tip receiving more light than the base during the leaf life span. In the obligate CAM bromeliad Aechmea aquilegaLüttge et al. (1986) detected a strong gradient of nocturnal net CO2-uptake and acid accumulation, as well as osmolality of leaf cell sap at dawn from the leaf tip to the leaf bases. It has been shown that leaf age in rosettes of several cultivars of Ananas comosus is associated with changes in their biochemical composition and δ13C values. These changes result from vari-ations in photosynthesis and nocturnal CO2-fixation activity during the life span of the leaves (Medina et al., 1993).
Carbon budgets, partitioning and translocation in CAM plants have been studied at various different levels. The types of carbohydrates involved (i.e. glucose, fructose, sucrose, starch, fructans, galactomannans, etc.) are species dependent (Christopher & Holtum, 1996, 1998). Oligofructans play a role in some rosette species such as Fourcroya humboldtiana and Agave deserti (Olivares & Medina, 1990; Wang & Nobel, 1998). Translocation of carbohydrates from photosynthesizing organs of CAM plants, as in plants with other modes of photosynthesis, depends on source-sink relationships, such as plant and organ age. Sink removal or girdling in Kalanchoe pinnata reduces CO2 fixation (Mayoral et al., 1991). Wang et al. (1998) studied the sink-to_source transition of cladodes of Opuntia ficus-indica and noted that during development, nocturnal CO2 uptake started early, when cladodes still were sinks.
Another increasingly intriguing question refers to the carbon partitioning between day-time filling of reserves needed as precursors for formation of phosphoenolpyruvate for subsequent nocturnal CO2-fixation and export to sinks for growth, respectively (Borland et al., 2001). It was suggested that the former mainly results from reduction of CO2 coming from the nocturnally stored malate in phase III of CAM (sensuOsmond, 1978) and the latter from direct assimilation of atmospheric CO2 in phase IV (Winter, 1985). Lüttge et al. (1985) attempted to test this, recording stable carbon-isotope signatures in relation to daily nectar secretion rhythms in different CAM plants. However, they did not find δ13C rhythms in the nectar secreted, suggesting internal mixing of carbon pools at least before serving the sink of sugar secretion. Conversely, several studies strongly support the idea that exported carbon in CAM mainly comes from phase IV CO2-fixation, for example in Kalanchoe pinnata (Mayoral & Medina, 1985), Clusia minor (Borland et al., 1994) and Sedum telephium (Borland, 1996).
The diurnal carbohydrate balance of bromeliads has been documented in several species, but results differ among authors (Borland & Griffiths, 1989; Carnal & Black, 1989; Medina et al., 1993; Christopher & Holtum, 1998). Since the Bromeliaceae contain species of the C3 and CAM photosynthetic types we set out to quantify, under natural conditions, the rates of non-structural carbohydrate translocation out of mature leaves of terrestrial and epiphytic species. We show that differences in the light environment along the leaf determined by the rosette-habit result in the establishment of a physiological gradient reflected in the concentration of organic solutes associated with the carbohydrate metabolism of the leaves. In addition, we present hexose balances of the leaf blade of several bromeliad species to quantify net carbohydrate translocation. To that end, we sampled adult, fully expanded leaves within rosettes of terrestrial and epiphytic bromeliads of the C3 and CAM photosynthetic types, and from environments with contrasting humidity and/or light conditions. We measured the changes in concentration of metabolites (starch, soluble sugars, malate and citrate) at the base, middle and top of the leaves at the end and the beginning of the light period. With these figures we calculated the net nocturnal change in hexose equivalents of adult leaves. This nocturnal balance should be negative and represents the sum of the amount of carbon respired plus the amount of carbon exported from the leaf section analysed.
Materials and Methods
Plant samples were collected in three different sites in northern Venezuela near the end of the dry season (March). Ananas comosus (L.) Merrill cv. Spanish Red was sampled from a plantation in a semiarid area of Lara State (Duaca) (400 m a.s.l). Bromelia humilis Jacq. and Tillandsia flexuosa Swartz were collected from a coastal site close to Chichiriviche (Falcón State, for site description see Medina et al., 1989). Aechmea fendleri André ex Mez, Tillandsia fendleri Grisebach, Tillandsia utriculata L., and Guzmania mucronata (Griseb.) Mez were sampled within the area of the Instituto Venezolano de Investigaciones Científicas, at 1500 m above sea level (a.s.l.) in the Coastal Interior range (Miranda State) close to the city of Caracas, Venezuela.
For A. comosus, leaves in the fifth to seventh position after the first visible leaf were cut at the base every 3 h, with three parallel samples per sampling time. After taking the middle part of the leaves (10 cm above and below the leaf center) the remaining two parts, including either the base or the top, were again divided into halves, giving 5 samples: base, middle basewards, middle, middle topwards and top.
All other plants were sampled at dawn (c. 06.00 h) and dusk (c. 18.00 h). Adult leaves were harvested from the middle section of the plant rosette (position 5–10 after the first visible leaf) and care was taken to have the leaves of the same plants at similar insertion for dawn and dusk samples. After measuring leaf length, these leaves were divided into three parts (base, middle and top). In all cases the number of replicates was three.
In all cases small subsamples were put into plastic syringes and frozen in dry ice, to be used later for leaf sap extraction by centrifugation, and osmolality measurement (Wescor Dew Point Osmometer, Wescor Inc., Utah, USA). After taking the leaf area, the cut leaves were immediately dried in a microwave oven. This procedure was tested in the laboratory and gave the same results for sugars, organic acids and starch as freeze drying at −55°C (Popp et al., 1996). The dried plant material was finely ground and aliquots of the powder were taken for starch determination and hot water extraction. The determination of sugars and organic acids in the hot-water extracts followed Ball et al. (1991). For the analysis of the starch content two different methods were applied, either using 1.1% HCl (Janauer & Kinzel, 1976) or α-amylase (Karkalas, 1985; Rasmussen & Henry, 1990) for the first hydrolysation step. Both procedures yielded the same amount of starch in the samples, showing that 1.1% HCl did not hydrolyse any other macromolecules (cellulose, pectins, etc.) than starch. These procedures were also tested with pure preparations of starch, cellulose and pectin. The δ13C values were determined in dried leaf tissue as in Medina et al. (1994).
Diurnal variations of metabolites in A. comosus cv. Spanish Red.
Longitudinal variations in water content are strongly developed in rosette-plant leaves. In A. comosus, absolute water content decreased from the base to the top of leaves, and it appeared to have a diurnal course, with higher values at midnight (Fig. 1a). Osmolality showed the reverse pattern, being lower in the leaf base and increasing by up to 400 mmol kg−1 at the top of the leaf (Fig. 1d). No consistent diurnal variations were observed in osmolality, possibly because of simultaneous changes in levels of different metabolites in opposite directions. Concentration of sucrose showed a very clear diurnal pattern, with the highest values recorded at the end of the day, and minimum values recorded at the end of the night (Fig. 1b). The magnitude of the diurnal change in sucrose content increased from the base to the top of the leaf. Daily course of starch was more variable, but the concentrations at dusk were higher than those at dawn, except at the leaf base. The middle leaf section showed a diurnal pattern similar to that of sucrose (Fig. 1e). Titratable acidity (not shown) and concentrations of malate also showed strong diurnal patterns, but in opposite phase to sucrose (Fig. 1c). Again, the most pronounced changes were recorded at the leaf top. Citrate concentrations changed little during the day, and increased markedly from the base to the top of the leaf (Fig. 1f).
The balance of metabolites measured in different leaf sections of A. comosus in the field shows that during the night, titratable acidity and malate consistently increased, while soluble sugars and starch decreased (Table 1). The proportions of glucose, fructose and starch consumed during the night were not the same in all leaf sections. Acidity changes were almost quantitatively explained by the changes in malate concentrations.
Table 1. Dawn–dusk values and their differences of titratable acidity, malate, citrate, glucose, fructose, sucrose and starch in different leaf parts of Ananas comosus cv. Spanish Red
[H+] (mEq m−2 leaf)
Malate (mmol m−2 leaf)
Citrate (mmol m−2 leaf)
Glucose (mmol m−2 leaf)
Fructose (mmol m−2 leaf area)
Sucrose (mmol m−2 leaf)
Starch (mmol hexose units m−2 leaf)
Mean values with SE in parenthesis, n = 3. Missing SEs indicate sample loss. Standard error of the dawn–dusk difference = square root ((standard errordawn)2+ (standard errordusk)2). Hexose balance = (Δmalate/2) + Δcitrate + Δglucose + Δfructose + (Δsucrose × 2) + Δstarch. aΔvalues larger than twice the standard error of the difference are considered different from zero.
The balance of carbon in the diurnal changes of metabolites was calculated as follows by assuming that in nocturnal organic-acid synthesis of CAM, a hexose molecule will produce two moles of malate and one mole of citrate:
In the leaf base, the nocturnal reduction in sucrose levels was equivalent to 2.6 times the amount required for malic acid synthesis. Conversely, in the light-exposed parts of the leaf blades sucrose reduction was sufficient for only about 80–90% of the acid accumulation.
Hexose balance was negative in all cases but was particularly so in the leaf base. These results indicate that the leaves measured are actively translocating carbohydrates during the night period towards other parts of the plant, stem and meristems.
Dusk–dawn variations of metabolites in C3 and CAM bromeliads
Acidity increased consistently during the night in all CAM bromeliads, the changes being less pronounced in the leaf bases (Table 2). In C3 bromeliads, acidity did not change during the night and their proton concentration was still lower than that measured in CAM plants in the deacidified state (dusk). Malate concentration increased markedly during the night in the CAM bromeliads, as expected, while citrate increased slightly or not at all, particularly in the leaf bases (Table 2). In the species studied, those belonging to the subfamily Bromelioideae (Bromelia humilis, Aechmea fendleri, and A. comosus) had consistently higher citrate and sucrose concentrations in leaf sap than the species of the genus Tillandsia (data not shown; the complete data set is available upon request). In addition, sucrose decreased during the night consistently in the bromelioids, the lowest values recorded for shade plants (B. humilis and A. fendleri). In all the tillandsioid species measured, sucrose reduction was rather small or absent (Table 2). Sucrose reduction was enough to explain nocturnal acidification in B. humilis, and nearly so in A. comosus, but it explained only between 15% and 47% of the acidification in A. fendleri.
Table 2. Diurnal net changes in osmolality, titratable acidity and carbon balance of bromeliads
Δ Osmolality (mmol kg−1)
Δ (mmol m−2) Acids
Figures are the differences recorded between collections at 18.00 h and collections at 06.00 h (Δ = a.m. – p.m.); the standard error (in parenthesis) of the difference = square root ((standard errordawn)2+ (standard errordusk)2). CAM, crassulacean acid metabolism. Hexose balance = (Δmalate/2) + Δcitrate + Δglucose + Δfructose + (Δsucrose × 2) + Δstarch. Δvalues larger than twice the standard error of the difference are considered different from zero.
Bromelia humilis (CAM, terrestrial, semiarid areas, sun plants)
The C3 bromeliads had organic acid concentrations comparable in concentration to those measured in CAM bromeliads at dusk (see Fig. 3), and their soluble sugar and starch contents tended to decrease during the night. Osmolality was comparatively low in all species; the lowest values were recorded in the C3 bromeliad T. fendleri.
Osmolality increased during the night in all CAM bromeliads, and the magnitude of the change increased towards the top of the leaves (Table 2). The C3 bromeliads always showed small but significant decreases in osmolality during the night. The hexose balance of the upper half of leaves was mostly negative or nil, as expected if the processes affecting the hexose content of the leaves are hexose translocation and respiration in both CAM and C3 bromeliads. In CAM bromeliads the increase in organic acid content during the night is capable of increasing leaf sap osmolality despite the net reduction in soluble sugars content. The hexose balance at the leaf base was mostly negative, with the exception of A. fendleri and the shade plants of B. humilis (Table 2).
Malate–citrate relationships in CAM bromeliads
Diurnal oscillations in titratable acidity were essentially determined by the nocturnal accumulation of malate, with a negligible contribution of citrate (Fig. 2).
δ13C-values of leaf tissues, as affected by position
The measurements of δ13C values along the leaves showed only small differences between top and base (Table 3). In the CAM bromeliads, the leaf base showed consistently less negative values, the base–top difference ranging from 0.2‰ in T. flexuosa, to 0.4–0.5‰ in A. comosus, B. humilis and T. utriculata and up to 1‰ in A. fendleri. In the C3 bromeliads, the opposite tendency was observed: more negative values were measured in the leaf bases (base–top, –0.3 to –0.9‰). In addition, within the same forest, the G. mucronata, restricted to shady conditions had more negative δ13C values than T. fendleri, restricted to sunny epiphytic habitats. Similar differences are observed within the sun and shade plants of B. humilis.
Table 3. δ 13C values of leaf tissue of different position
δ 13C (‰) (± SD) Base
CAM, crassulacean acid metabolism.
Ananas comosus (CAM)
Bromelia humilis (CAM)
−12.6 (< 0.1)
−12.6 (< 0.1)
−12.8 (< 0.1)
−13.3 (< 0.1)
−13.7 (< 0.1)
Aechmea fendleri (CAM)
−11.5 (< 0.1)
−11.8 (< 0.1)
Tillandsia flexuosa (CAM)
Tillandsia utriculata (CAM)
−13.8 (< 0.1)
Tillandsia fendleri (C3)
−23.8 (< 0.1)
Guzmania mucronata (C3)
The results show that there are longitudinal gradients in metabolite concentrations in leaves of rosette bromeliads with CAM and C3 photosynthetic pathways. In CAM bromeliads the nocturnal increases in titratable acidity, malate and citrate concentrations, and osmolality, and reductions in sucrose concentration are more pronounced in the most light-exposed middle and upper parts of the leaves. Osmolality always increased during the night in CAM bromeliads, despite the net reduction in soluble sugar content. Results for hexose balance indicate that, in the species belonging to the subfamily Bromelioideae (B. humilis, A. fendleri, and A. comosus) nocturnal reductions in sucrose levels covered a large fraction of the amount of hexose units required for acidification. This fact was previously reported for A. comosus (Sideris et al., 1948; Carnal & Black, 1989; Medina et al., 1993) and B. humilis (Medina et al., 1986). The suggestion that it may be a general characteristic for the bromelioid subfamily raises interesting questions for the evolution of CAM within the family. In the tillandsioid CAM species (T. flexuosa and T. utriculata) starch is apparently the only source of hexose for acid synthesis. These results support previous findings of Christopher & Holtum (1998) who showed accumulation of starch in a CAM tillandsoid species (T. tricolor), and starch and sugars in three bromelioid species (A. comosus, Ortophytum vagans and Nidularium billbergioides). The only CAM bromeliad belonging to the pitcairnioid subfamily studied by these authors, Dickya sp., also accumulated sugars. The pattern of sugar-accumulating pitcairnioid, starch-accumulating tillandsioid, and both sugar- and starch-accumulating bromelioids, constitute a biochemical support for the evolutionary differentiation of the family, and it should be confirmed by analysing a larger set of species. Molecular phylogeny based on intron sequences of the chloroplast genome showed the monophyletic character of tillandosids and bromelioids, and the heterogeneous character of the pitcairnioid, as currently circumscribed (Horres et al., 2000). The published phylogenetic trees appear to separate known CAM from C3 genera within each subfamily.
Citrate concentration increased slightly during the night in all CAM bromeliads, the changes were more pronounced in the sun-exposed upper leaf sections. Citrate fluctuations were also larger in sun compared with shade plants. Thus, possibly the stronger citrate fluctuations in the upper leaf sections may be consequence of higher irradiation, as indicated in previous studies on B. humilis (Lee et al., 1989). An interesting distinction is that bromelioid species had consistently higher citrate concentrations in the leaf sap than the species of the genus Tillandsia (Fig. 3). In all cases, the major contribution to total diurnal Δ acidity is from malic acid and the role of citric acid is small (e.g. in comparison with CAM-performing species of Clusia; Popp et al., 1988); these differences are intriguing and deserve further study along the lines discussed by Lüttge (1988).
In both CAM and C3 bromeliads there is a pronounced reduction of leaf starch content during the night. In C3 plants these reductions can be attributed only to the translocation of sucrose, and the amount of hexoses consumed in respiration. In CAM plants, utilization of phosphoenolpyruvate (PEP) from glycolytic hexose breakdown as CO2 acceptor for malate synthesis and accumulation is an additional cause. In growing C3 bromeliads the nocturnal carbon balance must be always negative, while in CAM plants the operation of the malate-synthesizing pathway may result in carbon balances that are either nil (no sugar translocation plus re-fixation of respiratory CO2) or negative (net carbon export by sugar translocation). The actual contribution of mitochondrial respiration to carbohydrate consumption during the night should be small. Rates of dark respiration of CAM plants are difficult to measure directly, owing to the operation of PEP-carboxylase during the night. However, dark respiration rates of C3 bromeliads are seldom above 0.5 µmol m−2 s−1 (Lüttge et al., 1986). At this rate we should expect a maximum respiratory consumption of 3.2 mmol hexose m−2 s−1. Our results show that leaves of actively growing CAM bromeliads under natural conditions export significant amounts of hexoses during the night. Resolution of the analyses, with dawn and dusk samples only, does not allow one to distinguish pools of carbon assimilated during phases III and IV, respectively, and if exported carbon was mainly due to phase IV photosynthesis (see the Introduction). However, the results show complex interactions along the leaves, suggesting that at least some mixing of pools may occur. The contribution of each leaf section is not identical. In A. comosus, the most active CAM plant among the group studied here, the net export ranged from 31 to 90 hexose units m−2 leaf area in the upper leaf section to 126 hexose units m−2 leaf area in the leaf base. These numbers might change according to the plant age and sink strength. In the other CAM bromeliads described here, not all leaf sections had a negative balance, indicating either that the non-structural carbohydrate pool was being used mainly for malate synthesis, or that the sink strength was low. In the C3 bromeliads the balance was either strongly negative or nearly zero in the upper leaf parts, while the bases mostly showed a negative balance.
Differences in δ13C along the leaves of the rosette plants studied here are small. Although not statistically different in all cases, there is a consistent trend of less negative δ13C-values in the leaf bases than in the leaf tips in all CAM bromeliads studied here. This, in fact, may be explained by a possibly higher lipid content in the greener sun-exposed parts of the leaves with their strong development of chloroplast membranes, since lipids always show more negative δ13C concentrations because of the 13C-discrimination of pyruvate dehydrogenase providing the acetyl residues required for fatty acid synthesis (Deniro & Epstein, 1977). However, in the C3 bromeliads described here, differences of δ13C-values along the leaves are even smaller and clearly not significant (if there is a trend, it may even be in the opposite direction). Both CAM and C3 plants under shady conditions had more negative δ13C values. Medina et al. (1994) has already shown that the non-photosynthetically active sections of the leaf bases of several cultivars of A. comosus were significantly less negative than the leaf tips. Apart from the chlorophyllous and nonchlorophyllous leaf blades and bases from sun and shade plants, they also studied stem tissue. They gave a detailed evaluation of the various δ13C signatures and concluded that differences are only partly associated with differences in lipid content. Relative contributions of direct CO2-fixation by phosphoenolpyruvate carboxylase with a low 13CO2-discrimination (phase I of CAM) and ribulose-bis-phosphate carboxylase/oxygenase with a high 13CO2-discrimination (phase IV of CAM) as also isotope effects during translocation of photosynthetic products, may be involved.
Thanks to Sabas Pérez (IVIC) for his help in the field. Dr Ana Herrera (UCV, Caracas) provided useful comments to a previous version of this paper.