Plant defence compounds occur in floral nectar, but their ecological role is not well-understood. We provide the first evidence that plant compounds pharmacologically alter pollinator behaviour by enhancing their memory of reward. Honeybees rewarded with caffeine, which occurs naturally in nectar of Coffea and Citrus species, were three times more likely to remember a learned floral scent than those rewarded with sucrose alone. Caffeine potentiated responses of mushroom body neurons involved in olfactory learning and memory by acting as an adenosine receptor antagonist. Caffeine concentrations in nectar never exceeded the bees' bitter taste threshold, implying that pollinators impose selection for nectar that is pharmacologically active but not repellent. By using a drug to enhance memories of reward, plants secure pollinator fidelity and improve reproductive success.Many drugs commonly consumed by humans are produced by plants as a form of toxic defence against herbivores (1, 2). While plant-derived drugs like caffeine or nicotine are lethal in high doses (3-5), they have pharmacological effects at low doses that affect mammalian behaviour. For example, low doses of caffeine are mildly rewarding and enhance cognitive performance and memory retention (6). Interestingly, caffeine has been detected in low doses in the floral nectar and pollen of Citrus (7), but whether it has an ecological function is unknown.Two caffeine-producing plant genera, Citrus and Coffea, have large floral displays with strong scents and produce more fruits and seeds when pollinated by bees (8, 9). If caffeine confers a selective advantage when these plants interact with pollinators, we might expect it to be commonly encountered in nectar. We measured caffeine in the nectar of 3 species of Coffea (C. canephora, C. arabica, and C. liberica) and 4 species of Citrus (C. paradisi, C. maxima, C. sinesis, C. reticulata) using liquid chromatography-mass spectrometry (10, Fig. Europe PMC Funders Group Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts S1A). When caffeine was present, its concentration ranged from 0.003 -0.253 mM. The median caffeine concentration in both genera was not significantly different (Fig. 1A, MannWhitney, Z = −1.09, P = 0.272). Caffeine was more common in the nectar of C. canephora than in C. arabica or C. liberica (Coffea: logistic regression χ 2 2 = 11.1, P = 0.004); it was always present in Citrus nectar. The mean total nectar sugar concentration ranged from 0.338-0.843 M (Fig. 1B, see Fig S1B for individual sugars). Caffeine concentration in nectar did not correlate with total sugar concentration (Pearson's r = 0.063, P = 0.596).We hypothesized that caffeine could affect the learning and memory of foraging pollinators.To test this, we trained individual honeybees to associate floral scent with 0.7 M sucrose and 7 different concentrations of caffeine and tested their olfactory memory. Using a method for classical conditioning of feeding responses (proboscis extension reflex, 11), bees...

Crassulacean acid metabolism (CAM) is a photosynthetic adaptation that facilitates the uptake of CO(2) at night and thereby optimizes the water-use efficiency of carbon assimilation in plants growing in arid habitats. A number of CAM species have been exploited agronomically in marginal habitats, displaying annual above-ground productivities comparable with those of the most water-use efficient C(3) or C(4) crops but with only 20% of the water required for cultivation. Such attributes highlight the potential of CAM plants for carbon sequestration and as feed stocks for bioenergy production on marginal and degraded lands. This review highlights the metabolic and morphological features of CAM that contribute towards high biomass production in water-limited environments. The temporal separation of carboxylation processes that underpins CAM provides flexibility for modulating carbon gain over the day and night, and poses fundamental questions in terms of circadian control of metabolism, growth, and productivity. The advantages conferred by a high water-storage capacitance, which translate into an ability to buffer fluctuations in environmental water availability, must be traded against diffusive (stomatal plus internal) constraints imposed by succulent CAM tissues on CO(2) supply to the cellular sites of carbon assimilation. The practicalities for maximizing CAM biomass and carbon sequestration need to be informed by underlying molecular, physiological, and ecological processes. Recent progress in developing genetic models for CAM are outlined and discussed in light of the need to achieve a systems-level understanding that spans the molecular controls over the pathway through to the agronomic performance of CAM and provision of ecosystem services on marginal lands.

Crassulacean acid metabolism (CAM) is a water-use efficient adaptation of photosynthesis that has evolved independently many times in diverse lineages of flowering plants. We hypothesize that convergent evolution of protein sequence and temporal gene expression underpins the independent emergences of CAM from C3 photosynthesis. To test this hypothesis, we generate a de novo genome assembly and genome-wide transcript expression data for Kalanchoë fedtschenkoi, an obligate CAM species within the core eudicots with a relatively small genome (~260 Mb). Our comparative analyses identify signatures of convergence in protein sequence and re-scheduling of diel transcript expression of genes involved in nocturnal CO2 fixation, stomatal movement, heat tolerance, circadian clock, and carbohydrate metabolism in K. fedtschenkoi and other CAM species in comparison with non-CAM species. These findings provide new insights into molecular convergence and building blocks of CAM and will facilitate CAM-into-C3 photosynthesis engineering to enhance water-use efficiency in crops.

Summary 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.