Rethinking the potential productivity of crassulacean acid metabolism by integrating metabolic dynamics with shoot architecture, using the example of Agave tequilana.

Terrestrial CAM plants typically occur in hot semiarid regions, yet can show high crop productivity under favorable conditions. To achieve a more mechanistic understanding of CAM plant productivity, a biochemical model of diel metabolism was developed and integrated with 3-D shoot morphology to predict the energetics of light interception and photosynthetic carbon assimilation. Using Agave tequilana as an example, this biochemical model faithfully simulated the four diel phases of CO2 and metabolite dynamics during the CAM rhythm. After capturing the 3-D form over an 8-yr production cycle, a ray-tracing method allowed the prediction of the light microclimate across all photosynthetic surfaces. Integration with the biochemical model thereby enabled the simulation of plant and stand carbon uptake over daily and annual courses. The theoretical maximum energy conversion efficiency of Agave spp. is calculated at 0.045-0.049, up to 7% higher than for C3 photosynthesis. Actual light interception, and biochemical and anatomical limitations, reduced this to 0.0069, or 15.6 Mg ha-1 yr-1 dry mass annualized over an 8-yr cropping cycle, consistent with observation. This is comparable to the productivity of many C3 crops, demonstrating the potential of CAM plants in climates where little else may be grown while indicating strategies that could raise their productivity.

Atmospheric CO2 decline and the timing of CAM plant evolution.

BACKGROUND AND AIMS: CAM photosynthesis is hypothesized to have evolved in atmospheres of low CO2 concentration in recent geological time because of its ability to concentrate CO2 around Rubisco and boost water use efficiency relative to C3 photosynthesis. We assess this hypothesis by compiling estimates of when CAM clades arose using phylogenetic chronograms for 73 CAM clades. We further consider evidence of how atmospheric CO2 affects CAM relative to C3 photosynthesis. RESULTS: Where CAM origins can be inferred, strong CAM is estimated to have appeared in the past 30 million years in 46 of 48 examined clades, after atmospheric CO2 had declined from high (near 800 ppm) to lower (<450 ppm) values. In turn, 21 of 25 clades containing CAM species (but where CAM origins are less certain) also arose in the past 30 million years. In these clades, CAM is probably younger than the clade origin. We found evidence for repeated weak CAM evolution during the higher CO2 conditions before 30 million years ago, and possible strong CAM origins in the Crassulaceae during the Cretaceous period prior to atmospheric CO2 decline. Most CAM-specific clades arose in the past 15 million years, in a similar pattern observed for origins of C4 clades. CONCLUSIONS: The evidence indicates strong CAM repeatedly evolved in reduced CO2 conditions of the past 30 million years. Weaker CAM can pre-date low CO2 and, in the Crassulaceae, strong CAM may also have arisen in water-limited microsites under relatively high CO2. Experimental evidence from extant CAM species demonstrates that elevated CO2 reduces the importance of nocturnal CO2 fixation by increasing the contribution of C3 photosynthesis to daily carbon gain. Thus, the advantage of strong CAM would be reduced in high CO2, such that its evolution appears less likely and restricted to more extreme environments than possible in low CO2.

The CAM lineages of planet Earth.

BACKGROUND AND SCOPE: The growth of experimental studies of crassulacean acid metabolism (CAM) in diverse plant clades, coupled with recent advances in molecular systematics, presents an opportunity to re-assess the phylogenetic distribution and diversity of species capable of CAM. It has been more than two decades since the last comprehensive lists of CAM taxa were published, and an updated survey of the occurrence and distribution of CAM taxa is needed to facilitate and guide future CAM research. We aimed to survey the phylogenetic distribution of these taxa, their diverse morphology, physiology and ecology, and the likely number of evolutionary origins of CAM based on currently known lineages. RESULTS AND CONCLUSIONS: We found direct evidence (in the form of experimental or field observations of gas exchange, day-night fluctuations in organic acids, carbon isotope ratios and enzymatic activity) for CAM in 370 genera of vascular plants, representing 38 families. Further assumptions about the frequency of CAM species in CAM clades and the distribution of CAM in the Cactaceae and Crassulaceae bring the currently estimated number of CAM-capable species to nearly 7 % of all vascular plants. The phylogenetic distribution of these taxa suggests a minimum of 66 independent origins of CAM in vascular plants, possibly with dozens more. To achieve further insight into CAM origins, there is a need for more extensive and systematic surveys of previously unstudied lineages, particularly in living material to identify low-level CAM activity, and for denser sampling to increase phylogenetic resolution in CAM-evolving clades. This should allow further progress in understanding the functional significance of this pathway by integration with studies on the evolution and genomics of CAM in its many forms.

CAM photosynthesis: the acid test.

There is currently considerable interest in the prospects for bioengineering crassulacean acid metabolism (CAM) photosynthesis - or key elements associated with it, such as increased water-use efficiency - into C3 plants. Resolving how CAM photosynthesis evolved from the ancestral C3 pathway could provide valuable insights into the targets for such bioengineering efforts. It has been proposed that the ability to accumulate organic acids at night may be common among C3 plants, and that the transition to CAM might simply require enhancement of pre-existing fluxes, without the need for changes in circadian or diurnal regulation. We show, in a survey encompassing 40 families of vascular plants, that nocturnal acidification is a feature entirely restricted to CAM species. Although many C3 species can synthesize malate during the light period, we argue that the switch to night-time malic acid accumulation requires a fundamental metabolic reprogramming that couples glycolytic breakdown of storage carbohydrate to the process of net dark CO2 fixation. This central element of the CAM pathway, even when expressed at a low level, represents a biochemical capability not seen in C3 plants, and so is better regarded as a discrete evolutionary innovation than as part of a metabolic continuum between C3 and CAM.

Temporal and spatial transcriptomic and microRNA dynamics of CAM photosynthesis in pineapple.

The altered carbon assimilation pathway of crassulacean acid metabolism (CAM) photosynthesis results in an up to 80% higher water-use efficiency than C3 photosynthesis in plants making it a potentially useful pathway for engineering crop plants with improved drought tolerance. Here we surveyed detailed temporal (diel time course) and spatial (across a leaf gradient) gene and microRNA (miRNA) expression patterns in the obligate CAM plant pineapple [Ananas comosus (L.) Merr.]. The high-resolution transcriptome atlas allowed us to distinguish between CAM-related and non-CAM gene copies. A differential gene co-expression network across green and white leaf diel datasets identified genes with circadian oscillation, CAM-related functions, and source-sink relations. Gene co-expression clusters containing CAM pathway genes are enriched with clock-associated cis-elements, suggesting circadian regulation of CAM. About 20% of pineapple microRNAs have diel expression patterns, with several that target key CAM-related genes. Expression and physiology data provide a model for CAM-specific carbohydrate flux and long-distance hexose transport. Together these resources provide a list of candidate genes for targeted engineering of CAM into C3 photosynthesis crop species.

Ammonium intensifies CAM photosynthesis and counteracts drought effects by increasing malate transport and antioxidant capacity in Guzmania monostachia.

Guzmania monostachia (Bromeliaceae) is a tropical epiphyte capable of up-regulating crassulacean acid metabolism (CAM) in its photosynthetic tissues in response to changing nutrient and water availability. Previous studies have shown that under drought there is a gradient of increasing CAM expression from the basal (youngest) to the apical (oldest) portion of the leaves, and additionally that nitrogen deficiency can further increase CAM intensity in the leaf apex of this bromeliad. The present study investigated the inter-relationships between nitrogen source (nitrate and/or ammonium) and water deficit in regulating CAM expression in G. monostachia leaves. The highest CAM activity was observed under ammonium nutrition in combination with water deficit. This was associated with enhanced activity of the key enzyme phosphoenolpyruvate carboxylase, elevated rates of ATP- and PPi-dependent proton transport at the vacuolar membrane in the presence of malate, and increased transcript levels of the vacuolar malate channel-encoding gene, ALMT. Water deficit was consistently associated with higher levels of total soluble sugars, which were maximal under ammonium nutrition, as were the activities of several antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase). Thus, ammonium nutrition, whilst associated with the highest degree of CAM induction in G. monostachia, also mitigates the effects of water deficit by osmotic adjustment and can limit oxidative damage in the leaves of this bromeliad under conditions that may be typical of its epiphytic habitat.

A tonoplast Glu/Asp/GABA exchanger that affects tomato fruit amino acid composition.

Vacuolar accumulation of acidic metabolites is an important aspect of tomato fruit flavour and nutritional quality. The amino acids Asp and Glu accumulate to high concentrations during ripening, while γ-aminobutyrate (GABA) shows an approximately stoichiometric decline. Given that GABA can be catabolised to form Glu and subsequently Asp, and the requirement for the fruit to maintain osmotic homeostasis during ripening, we hypothesised the existence of a tonoplast transporter that exports GABA from the vacuole in exchange for import of either Asp or Glu. We show here that the tomato vacuolar membrane possesses such a transport property: transport of Glu across isolated tonoplast vesicle membranes was trans-stimulated in counterexchange mode by GABA, Glu and Asp. We identified SlCAT9 as a candidate protein for this exchanger using quantitative proteomics of a tonoplast-enriched membrane fraction. Transient expression of a SlCAT9-YFP fusion in tobacco confirmed a tonoplast localisation. The function of the protein was examined by overexpression of SlCAT9 in transgenic tomato plants. Tonoplast vesicles isolated from transgenic plants showed higher rates of Glu and GABA transport than wild-type (WT) only when assayed in counterexchange mode with Glu, Asp, or GABA. Moreover, there were substantial increases in the content of all three cognate amino acids in ripe fruit from the transgenic plants. We conclude that SlCAT9 is a tonoplast Glu/Asp/GABA exchanger that strongly influences the accumulation of these amino acids during fruit development.

The regulatory roles of ethylene and reactive oxygen species (ROS) in plant salt stress responses.

Soil salinity is one of the most commonly encountered environmental stresses affecting plant growth and crop productivity. Accordingly, plants have evolved a variety of morphological, physiological and biochemical strategies that enable them to adapt to saline growth conditions. For example, it has long been known that salinity-stress increases both the production of the gaseous stress hormone ethylene and the in planta accumulation of reactive oxygen species (ROS). Recently, there has been significant progress in understanding how the fine-tuning of ethylene biosynthesis and signaling transduction can promote salinity tolerance, and how salinity-induced ROS accumulation also acts as a signal in the mediation of salinity tolerance. Furthermore, recent advances have indicated that ethylene signaling modulates salinity responses largely via regulation of ROS-generating and ROS-scavenging mechanisms. This review focuses on these recent advances in understanding the linked roles of ethylene and ROS in salt tolerance.

ROS-mediated vascular homeostatic control of root-to-shoot soil Na delivery in Arabidopsis.

Sodium (Na) is ubiquitous in soils, and is transported to plant shoots via transpiration through xylem elements in the vascular tissue. However, excess Na is damaging. Accordingly, control of xylem-sap Na concentration is important for maintenance of shoot Na homeostasis, especially under Na stress conditions. Here we report that shoot Na homeostasis of Arabidopsis thaliana plants grown in saline soils is conferred by reactive oxygen species (ROS) regulation of xylem-sap Na concentrations. We show that lack of A. thaliana respiratory burst oxidase protein F (AtrbohF; an NADPH oxidase catalysing ROS production) causes hypersensitivity of shoots to soil salinity. Lack of AtrbohF-dependent salinity-induced vascular ROS accumulation leads to increased Na concentrations in root vasculature cells and in xylem sap, thus causing delivery of damaging amounts of Na to the shoot. We also show that the excess shoot Na delivery caused by lack of AtrbohF is dependent upon transpiration. We conclude that AtrbohF increases ROS levels in wild-type root vasculature in response to raised soil salinity, thereby limiting Na concentrations in xylem sap, and in turn protecting shoot cells from transpiration-dependent delivery of excess Na.

An Arabidopsis soil-salinity-tolerance mutation confers ethylene-mediated enhancement of sodium/potassium homeostasis.

High soil Na concentrations damage plants by increasing cellular Na accumulation and K loss. Excess soil Na stimulates ethylene-induced soil-salinity tolerance, the mechanism of which we here define via characterization of an Arabidopsis thaliana mutant displaying transpiration-dependent soil-salinity tolerance. This phenotype is conferred by a loss-of-function allele of ethylene overproducer1 (ETO1; mutant alleles of which cause increased production of ethylene). We show that lack of ETO1 function confers soil-salinity tolerance through improved shoot Na/K homeostasis, effected via the ethylene resistant1-constitutive triple response1 ethylene signaling pathway. Under transpiring conditions, lack of ETO1 function reduces root Na influx and both stelar and xylem sap Na concentrations, thereby restricting root-to-shoot delivery of Na. These effects are associated with increased accumulation of respiratory burst oxidase homolog F (RBOHF)-dependent reactive oxygen species in the root stele. Additionally, lack of ETO1 function leads to significant enhancement of tissue K status by an RBOHF-independent mechanism associated with elevated high-affinity K(+) TRANSPORTER5 transcript levels. We conclude that ethylene promotes soil-salinity tolerance via improved Na/K homeostasis mediated by RBOHF-dependent regulation of Na accumulation and RBOHF-independent regulation of K accumulation.

Crassulacean acid metabolism: a continuous or discrete trait?

The key components of crassulacean acid metabolism (CAM) - nocturnal fixation of atmospheric CO2 and its processing via Rubisco in the subsequent light period - are now reasonably well understood in terms of the biochemical reactions defining this water-saving mode of carbon assimilation. Phenotypically, however, the degree to which plants engage in the CAM cycle relative to regular C3 photosynthesis is highly variable. Depending upon species, ontogeny and environment, the contribution of nocturnal CO2 fixation to 24-h carbon gain can range continuously from close to 0% to 100%. Nevertheless, not all possible combinations of light and dark CO2 fixation appear equally common. Large-scale surveys of carbon-isotope ratios typically show a strongly bimodal frequency distribution, with relatively few intermediate values. Recent research has revealed that many species capable of low-level CAM activity are nested within the peak of C3 -type isotope signatures. While questions remain concerning the adaptive significance of dark CO2 fixation in such species, plants with low-level CAM should prove valuable models for investigating the discrete changes in genetic architecture and gene expression that have enabled the evolutionary transition from C3 to CAM.

A macro-ecological perspective on crassulacean acid metabolism (CAM) photosynthesis evolution in Afro-Madagascan drylands: Eulophiinae orchids as a case study.

Crassulacean acid metabolism (CAM) photosynthesis is an adaptation to water and atmospheric CO2 deficits that has been linked to diversification in dry-adapted plants. We investigated whether CAM evolution can be associated with the availability of new or alternative niches, using Eulophiinae orchids as a case study. Carbon isotope ratios, geographical and climate data, fossil records and DNA sequences were used to: assess the prevalence of CAM in Eulophiinae orchids; characterize the ecological niche of extant taxa; infer divergence times; and estimate whether CAM is associated with niche shifts. CAM evolved in four terrestrial lineages during the late Miocene/Pliocene, which have uneven diversification patterns. These lineages originated in humid habitats and colonized dry/seasonally dry environments in Africa and Madagascar. Additional key features (variegation, heterophylly) evolved in the most species-rich CAM lineages. Dry habitats were also colonized by a lineage that includes putative mycoheterotrophic taxa. These findings indicate that the switch to CAM is associated with environmental change. With its suite of adaptive traits, this group of orchids represents a unique opportunity to study the adaptations to dry environments, especially in the face of projected global aridification.

A roadmap for research on crassulacean acid metabolism (CAM) to enhance sustainable food and bioenergy production in a hotter, drier world.

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that features nocturnal CO2 uptake, facilitates increased water-use efficiency (WUE), and enables CAM plants to inhabit water-limited environments such as semi-arid deserts or seasonally dry forests. Human population growth and global climate change now present challenges for agricultural production systems to increase food, feed, forage, fiber, and fuel production. One approach to meet these challenges is to increase reliance on CAM crops, such as Agave and Opuntia, for biomass production on semi-arid, abandoned, marginal, or degraded agricultural lands. Major research efforts are now underway to assess the productivity of CAM crop species and to harness the WUE of CAM by engineering this pathway into existing food, feed, and bioenergy crops. An improved understanding of CAM has potential for high returns on research investment. To exploit the potential of CAM crops and CAM bioengineering, it will be necessary to elucidate the evolution, genomic features, and regulatory mechanisms of CAM. Field trials and predictive models will be required to assess the productivity of CAM crops, while new synthetic biology approaches need to be developed for CAM engineering. Infrastructure will be needed for CAM model systems, field trials, mutant collections, and data management.

Adaptive radiation, correlated and contingent evolution, and net species diversification in Bromeliaceae.

We present an integrative model predicting associations among epiphytism, the tank habit, entangling seeds, C3 vs. CAM photosynthesis, avian pollinators, life in fertile, moist montane habitats, and net rates of species diversification in the monocot family Bromeliaceae. We test these predictions by relating evolutionary shifts in form, physiology, and ecology to time and ancestral distributions, quantifying patterns of correlated and contingent evolution among pairs of traits and analyzing the apparent impact of individual traits on rates of net species diversification and geographic expansion beyond the ancestral Guayana Shield. All predicted patterns of correlated evolution were significant, and the temporal and spatial associations of phenotypic shifts with orogenies generally accorded with predictions. Net rates of species diversification were most closely coupled to life in fertile, moist, geographically extensive cordilleras, with additional significant ties to epiphytism, avian pollination, and the tank habit. The highest rates of net diversification were seen in the bromelioid tank-epiphytic clade (D(crown) = 1.05 My⁻¹), associated primarily with the Serra do Mar and nearby ranges of coastal Brazil, and in the core tillandsioids (D(crown) = 0.67 My⁻¹), associated primarily with the Andes and Central America. Six large-scale adaptive radiations and accompanying pulses of speciation account for 86% of total species richness in the family. This study is among the first to test a priori hypotheses about the relationships among phylogeny, phenotypic evolution, geographic spread, and net species diversification, and to argue for causality to flow from functional diversity to spatial expansion to species diversity.

Uncoupling of reactive oxygen species accumulation and defence signalling in the metal hyperaccumulator plant Noccaea caerulescens.

The metal hyperaccumulator plant Noccaea caerulescens is protected from disease by the accumulation of high concentrations of metals in its aerial tissues, which are toxic to many pathogens. As these metals can lead to the production of damaging reactive oxygen species (ROS), metal hyperaccumulator plants have developed highly effective ROS tolerance mechanisms, which might quench ROS-based signals. We therefore investigated whether metal accumulation alters defence signalling via ROS in this plant. We studied the effect of zinc (Zn) accumulation by N. caerulescens on pathogen-induced ROS production, salicylic acid accumulation and downstream defence responses, such as callose deposition and pathogenesis-related (PR) gene expression, to the bacterial pathogen Pseudomonas syringae pv. maculicola. The accumulation of Zn caused increased superoxide production in N. caerulescens, but inoculation with P. syringae did not elicit the defensive oxidative burst typical of most plants. Defences dependent on signalling through ROS (callose and PR gene expression) were also modified or absent in N. caerulescens, whereas salicylic acid production in response to infection was retained. These observations suggest that metal hyperaccumulation is incompatible with defence signalling through ROS and that, as metal hyperaccumulation became effective as a form of elemental defence, normal defence responses became progressively uncoupled from ROS signalling in N. caerulescens.

Metal hyperaccumulation armors plants against disease.

Metal hyperaccumulation, in which plants store exceptional concentrations of metals in their shoots, is an unusual trait whose evolutionary and ecological significance has prompted extensive debate. Hyperaccumulator plants are usually found on metalliferous soils, and it has been proposed that hyperaccumulation provides a defense against herbivores and pathogens, an idea termed the 'elemental defense' hypothesis. We have investigated this hypothesis using the crucifer Thlaspi caerulescens, a hyperaccumulator of zinc, nickel, and cadmium, and the bacterial pathogen Pseudomonas syringae pv. maculicola (Psm). Using leaf inoculation assays, we have shown that hyperaccumulation of any of the three metals inhibits growth of Psm in planta. Metal concentrations in the bulk leaf and in the apoplast, through which the pathogen invades the leaf, were shown to be sufficient to account for the defensive effect by comparison with in vitro dose-response curves. Further, mutants of Psm with increased and decreased zinc tolerance created by transposon insertion had either enhanced or reduced ability, respectively, to grow in high-zinc plants, indicating that the metal affects the pathogen directly. Finally, we have shown that bacteria naturally colonizing T. caerulescens leaves at the site of a former lead-zinc mine have high zinc tolerance compared with bacteria isolated from non-accumulating plants, suggesting local adaptation to high metal. These results demonstrate that the disease resistance observed in metal-exposed T. caerulescens can be attributed to a direct effect of metal hyperaccumulation, which may thus be functionally analogous to the resistance conferred by antimicrobial metabolites in non-accumulating plants.

High-resolution elemental localization in vacuolate plant cells by nanoscale secondary ion mass spectrometry.

By combining the capabilities of advanced sample preparation methodologies with the latest generation of secondary ion mass spectrometry instrumentation, we show that chemical information on the distribution of even dilute species in biological samples can be obtained with spatial resolutions of better than 100 nm. Here, we show the distribution of nickel and other elements in leaf tissue of the nickel hyperaccumulator plant Alyssum lesbiacum prepared by high-pressure freezing and freeze substitution.

Karatophyllum bromelioides L.D. Gomez revisited: a probable fossil CAM bromeliad.

PREMISE OF THE STUDY: The fossil leaf Karatophyllum bromelioides L. D. Gómez found in Costa Rica was proposed by Gómez (1972) to belong to the Bromeliaceae and to date from the middle Tertiary. If the age and affinity of this specimen were proven to be correct, it would constitute the oldest record of this large and ecologically diverse monocotyledonous family. KEY RESULTS: Morphological features of the fossil (leaf dimensions, marginal spines, cuticular traces) indicate a close affinity with the extant bromeliad Aechmea magdalenae (André) André ex Baker. Leaf thickness (1.6 mm at maximum) suggests that K. bromelioides L. D. Gómez performed CAM photosynthesis. The geological information does not corroborate the estimated age and location of the specimen; the fossil is suggested to be of more recent origin. CONCLUSIONS: The affinity of this fossil to Bromeliaceae was confirmed, but the uncertainties surrounding its age and collection locality mitigate against its use in inferences concerning the evolutionary history of the family.

Phylogeny, adaptive radiation, and historical biogeography in Bromeliaceae: insights from an eight-locus plastid phylogeny.

PREMISE: Bromeliaceae form a large, ecologically diverse family of angiosperms native to the New World. We use a bromeliad phylogeny based on eight plastid regions to analyze relationships within the family, test a new, eight-subfamily classification, infer the chronology of bromeliad evolution and invasion of different regions, and provide the basis for future analyses of trait evolution and rates of diversification. METHODS: We employed maximum-parsimony, maximum-likelihood, and Bayesian approaches to analyze 9341 aligned bases for four outgroups and 90 bromeliad species representing 46 of 58 described genera. We calibrate the resulting phylogeny against time using penalized likelihood applied to a monocot-wide tree based on plastid ndhF sequences and use it to analyze patterns of geographic spread using parsimony, Bayesian inference, and the program S-DIVA. RESULTS: Bromeliad subfamilies are related to each other as follows: (Brocchinioideae, (Lindmanioideae, (Tillandsioideae, (Hechtioideae, (Navioideae, (Pitcairnioideae, (Puyoideae, Bromelioideae))))))). Bromeliads arose in the Guayana Shield ca. 100 million years ago (Ma), spread centrifugally in the New World beginning ca. 16-13 Ma, and dispersed to West Africa ca. 9.3 Ma. Modern lineages began to diverge from each other roughly 19 Ma. CONCLUSIONS: Nearly two-thirds of extant bromeliads belong to two large radiations: the core tillandsioids, originating in the Andes ca. 14.2 Ma, and the Brazilian Shield bromelioids, originating in the Serro do Mar and adjacent regions ca. 9.1 Ma.

Low-level CAM photosynthesis in a succulent-leaved member of the Urticaceae, Pilea peperomioides.

Pilea peperomioides Diels (Urticaceae) is a semi-succulent herbaceous species native to south-western China that has become popular in cultivation as an ornamental plant. To investigate whether this species possesses the capacity for CAM photosynthesis, measurements were made of CO2 gas exchange and titratable acidity in plants under both well-watered and water-deficit conditions. Plants were found to assimilate CO2 almost exclusively in the light via C3 photosynthesis. However, distinct transient reductions in the rate of net nocturnal CO2 release were consistently observed during the course of the dark period, and under water-deficit conditions one plant exhibited a brief period of net nocturnal CO2 uptake, providing unequivocal evidence of CAM activity. Furthermore, nocturnal increases in titratable acidity in both leaf laminas and petioles were observed in all plants exposed to wet-dry-wet cycles. This is the first report of CAM in the family Urticaceae. The results are discussed in relation to the phylogenetic position of Pilea and the partially shaded montane habitats in which this species is typically found. An updated list of all plant families currently known to contain species with CAM is presented.