Caffeine in floral nectar enhances a pollinator's memory of reward.

Plant defense compounds occur in floral nectar, but their ecological role is not well understood. We provide evidence that plant compounds pharmacologically alter pollinator behavior by enhancing their memory of reward. Honeybees rewarded with caffeine, which occurs naturally in nectar of Coffea and Citrus species, were three times as likely to remember a learned floral scent as were honeybees 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 did not exceed 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.

Diel shifts in carboxylation pathway and metabolite dynamics in the CAM bromeliad Aechmea 'Maya' in response to elevated CO2.

BACKGROUND AND AIMS: The deployment of temporally separated carboxylation pathways for net CO(2) uptake in CAM plants provides plasticity and thus uncertainty on how species with this photosynthetic pathway will respond to life in a higher-CO(2) world. The present study examined how long-term exposure to elevated CO(2) influences the relative contributions that C(3) and C(4) carboxylation make to net carbon gain and to establish how this impacts on the availability of carbohydrates for export and growth and on water use efficiency over the day/night cycle. METHODS: Integrated measurements of leaf gas exchange and diel metabolite dynamics (e.g. malate, soluble sugars, starch) were made in leaves of the CAM bromeliad Aechmea 'Maya' after exposure to 700 micromol mol(-1) CO(2) for 5 months. KEY RESULTS: There was a 60 % increase in 24-h carbon gain under elevated CO(2) due to a stimulation of daytime C(3) and C(4) carboxylation in phases II and IV where water use efficiency was comparable with that measured at night. The extra CO(2) taken up under elevated CO(2) was largely accumulated as hexose sugars during phase IV and net daytime export of carbohydrate was abolished. Under elevated CO(2) there was no stimulation of dark carboxylation and nocturnal export and respiration appeared to be the stronger sinks for carbohydrate. CONCLUSIONS: Despite the increased size of the soluble sugar storage pool under elevated CO(2), there was no change in the net allocation of carbohydrates between provision of substrates for CAM and export/respiration in A. 'Maya'. The data imply the existence of discrete pools of carbohydrate that provide substrate for CAM or sugars for export/respiration. The 2-fold increase in water-use efficiency could be a major physiological advantage to growth under elevated CO(2) in this CAM bromeliad.

High degree of conservation of nuclear microsatellite loci in the genus Clusia.

In plants, microsatellites and their flanking DNA are rarely conserved across a whole genus, let alone other genera in the same family. Therefore, the possibility of using microsatellite primers developed for a species across a large number of plant species in the same genus is often limited. Remarkably, dinucleotide nuclear microsatellites developed for Clusia minor and for Clusia nemorosa amplified homologous microsatellites in species across the whole genus Clusia. In this present study, we report on the DNA sequence variation across the genus of 3 microsatellite loci with varying levels of variation. Compared over the species, there was a correlation between the lengths of the microsatellite loci. Interrupts occurred multiple times and did not seem to lead to the death of the microsatellite. These highly conserved markers will be useful for studying the variable reproductive systems in the genus Clusia.

Induction of Crassulacean acid metabolism by water limitation.

Crassulacean acid metabolism (CAM), a key adaptation of photosynthetic carbon fixation to limited water availability, is characterized by nocturnal CO2 fixation and daytime CO2 re-assimilation, which generally results in improved water-use efficiency. However, CAM plants display a remarkable degree of photosynthetic plasticity within a continuum of diel gas exchange patterns. Genotypic, ontogenetic and environmental factors combine to govern the extent to which CAM is expressed. The ecological diversity of CAM is mirrored by plasticity in a range of biochemical and physiological attributes. In C3/CAM-intermediate plants, limited water availability can induce or enhance the expression of CAM. CAM induction is controlled by a combination of transcriptional, post-transcriptional and post-translational regulatory events. Early events in CAM induction point to a requirement for calcium and calcium-dependent protein kinase activities. Gene discovery efforts, improved transformation technologies and genetic models for CAM plants, coupled with detailed physiological investigations, will lead to new insights into the molecular genetic basis of induction processes and the circadian oscillator that governs carbon flux during CAM. Future integration of genomic, biochemical and physiological approaches in selected CAM models promise to provide a detailed view of the complex regulatory dynamics involved in CAM induction and modulation by water deficit. Such information is expected to have broad significance as the ecological and agricultural importance of CAM species increases in the face of global warming trends and the associated expansion of desertification in semi-arid regions around the world.

Discrimination Processes and Shifts in Carboxylation during the Phases of Crassulacean Acid Metabolism.

The magnitude and extent of Crassulacean acid metabolism (CAM) activity in two Clusia species was manipulated to investigate the regulation of the distinct CAM phases. First, in response to leaf-air vapor pressure deficit at night, changes in leaf conductance altered on-line carbon-isotope discrimination throughout the theoretical range for dark CO2 uptake during CAM. These ranged from the limit set by phosphoenolpyruvate carboxylase (PEPc) (-6[per mille (thousand) sign], [delta]13C equivalent of -2[per mille (thousand) sign]) to that imposed by diffusion limitation (+4[per mille (thousand) sign], [delta]13C equivalent of -12[per mille (thousand) sign]), but the lowest carbon-isotope discrimination occurred when P[square root]pa was only 0.7. Second, when the availability of external or internal sources of CO2 was reduced for both field- and greenhouse-grown plants, CO2 uptake by day via PEPc during phase II largely compensated. Third, by reducing the dark period, plants accumulated low levels of acidity, and CO2 uptake occurred throughout the subsequent light period. Discrimination switched from being dominated by PEPc (phase II) to ribulose 1,5-bisphosphate carboxylase/oxygenase (phase III), with both enzymes active during phase IV. Under natural conditions, photochemical stability is maintained by extended PEPc activity in phase II, which enhances acid accumulation and delays decarboxylation until temperature and light stress are maximal at midday.

On the Mechanism of Reinitiation of Endogenous Crassulacean Acid Metabolism Rhythm by Temperature Changes.

Under continuous light the endogenous Crassulacean acid metabolism (CAM) rhythm of Kalanchoe daigremontiana Hamet et Perrier de la Bathie disappears at high (>29.0[deg]C) or low (<8.0[deg]C) temperatures. We investigated the reinitiation of rhythmicity when temperature was reduced from above the upper and increased from below the lower threshold level via measurements of (a) short-term changes in carbon-isotope discrimination to illustrate shifts between C3 and C4 carboxylation in vivo, and (b) the malate sensitivity of phosphoenolpyruvate carboxylase (PEPC) in vitro. When the net CO2-exchange rhythm disappears at both temperatures, the instantaneous discrimination indicates low PEPC activity. Leaf malate concentration and osmolarity attain high and low values at low and high temperatures, respectively. After small temperature increases or reductions from the low and high temperatures, respectively, the rhythm is reinitiated, with phases shifted by 180[deg] relative to each other. This can be related to the contrasting low and high leaf malate concentrations due to direct inhibition of PEPC and possibly also of the phosphorylation of PEPC by malate. The experimental results were satisfactorily simulated by a mathematical CAM-cycle model, with temperature acting only on the passive efflux of malate from the vacuole. We stress the important role of the tonoplast in malate compartmentation and of malate itself for the reinitiation and generation of endogenous CAM rhythmicity.

A comparative study on the regulation of C(3) and C (4) carboxylation processes in the constitutive crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana and the C(3)-CAM intermediate Clusia minor.

A comparison of carbon metabolism in the constitutive crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana Hamet et Perr. and the C(3)-CAM intermediate Clusia minor L. was undertaken under controlled environmental conditions where plants experience gradual changes in light intensity, temperature and humidity at the start and end of the photoperiod. The magnitude of CAM activity was manipulated by maintaining plants in ambient air or by enclosing leaves overnight in an atmosphere of N(2) to suppress C(4) carboxylation. Measurements of diel changes in carbonisotope discrimination and organic acid content were used to quantify the activities of C(3) and C(4) carboxylases in vivo and to indicate the extent to which the activities of phosphoenolpyruvate carboxylase (PEPCase), ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) and decarboxylation processes overlap at the start and end of the photoperiod. These measurements in vivo were compared with measurements in vitro of changes in the diel sensitivity of PEPCase to malate inhibition. The results demonstrate fundamental differences in the down-regulation of PEPCase during the day in the two species. While PEPCase is inactivated within the first 30 min of the photoperiod in K. daigremontiana, the enzyme is active for 4 h at the start and 3 h at the end of the photoperiod in C. minor. Enclosing leaves in N(2) overnight resulted in a two-to threefold increase in PEPCase-mediated CO(2) uptake during Phase II of CAM in both species. However, futile cycling of CO(2) between malate synthesis and decarboxylation does not occur during Phase II in either species. In terms of overall carbon balance, C(4) carboxylation accounted for approximately 20% of net daytime assimilation in both species under control conditions, increasing to 30-34% after a night in N(2). Although N(2)-treated leaves of K. daigremontiana took up 25% more CO(2) than control leaves during the day this was insufficient to compensate for the loss of CO(2) taken up by CAM the previous night. In contrast, in N(2)-treated leaves of C. minor, the twofold increase in daytime PEPCase activity and the increase in net CO(2) uptake by Rubisco during Phase III compensated for the inhibition of C(4) carboxylation at night in terms of diel carbon balance.

Carbon-Isotope Composition of Biochemical Fractions and the Regulation of Carbon Balance in Leaves of the C3-Crassulacean Acid Metabolism Intermediate Clusia minor L. Growing in Trinidad.

Carbon-isotope ratios ([delta]13Cs) were measured for various bio-chemical fractions quantitatively extracted from naturally exposed and shaded leaves of the C3-Crassulacean acid metabolism (CAM) intermediate Clusia minor, sampled at dawn and dusk on days during the wet and dry seasons in Trinidad. As the activity of CAM increased in response to decreased availability of water and higher photon flux density, organic acids and soluble sugars were enriched in 13C by approximately 3.5 to 4%[per mille (thousand) sign] compared to plants sampled during the wet season. The induction of CAM was accompanied by a doubling in size of the reserve carbohydrate pools. Moreover, stoichiometric measurements indicated that degradation of both chloroplastic reserves and soluble sugars were necessary to supply phosphoenolpyruvate for the synthesis of organic acids at night. Results also suggest that two pools of soluble sugars exist in leaves of C. minor that perform CAM, one a vacuolar pool enriched in 13C and the second a transport pool depleted in 13C. Estimates of carbon-isotope discrimination expressed during CAM, derived from the trafficking among inorganic carbon, organic acids, and carbohydrate pools overnight, ranged from 0.9 to 3.1%[per mille (thousand) sign]. The [delta]13C of structural material did not change significantly between wet and dry seasons, indicating that most of the carbon used in growth was derived from C3 carboxylation.

Short-term changes in carbon-isotope discrimination in the C3-CAM intermediate Clusia minor L. growing in Trinidad.

On-line instantaneous carbon isotope discrimination was measured in conjunction with net uptake of CO2 in leaves of exposed and shaded plants of the C3-CAM intermediate Clusia minor growing under natural conditions in Trinidad. At the end of the rainy season (late January-early February, 1992) C3 photosynthesis predominated although exposed leaves recaptured a small proportion of respiratory CO2 at night for the synthesis of malic acid. Citric acid was the major organic acid accumulated by exposed leaves at this time with a citric: malic acid ratio of 11:1. Values of instantaneous discrimination (Δ) in exposed leaves during the wet season rose from 17.1‰ shortly after dawn to 22.7‰ around mid-day just before stomata closed, suggesting that most CO2 was fixed by Rubisco at this time. During the late afternoon, instantaneous Δ declined from 22.2‰ to 17‰, probably reflecting the limited contribution from PEPc activity and an increase in diffusional resistance to CO2 in exposed leaves. Shaded leaves showed no CAM activity and CO2 uptake proceeded throughout the day in the wet season. The decrease in instantaneous Δ from 27‰ in the morning to 19.2‰ in the late afternoon was therefore entirely due to diffusional limitation. Leaves sampled in the dry season (mid-March, 1992) had by now induced full CAM activity with both malic and citric acids accumulated overnight and stomata closed for 4-5 h over the middle of the day. Values of instantaneous Δ measured over the first 3 h after dawn (6.4-9.1‰) indicated that C4 carboxylation dominated CO2 uptake for most of the morning when rates of photosynthesis were maximal, implying that under natural conditions, the down regulation of PEPc in phase II occurs much more slowly than laboratory-based studies have suggested. The contribution from C3 carboxylation to CO2 uptake during phase II was most marked in leaves which accumulated lower quantities of organic acids overnight. In exposed leaves, measurements of instantaneous Δ during the late afternoon illustrated the transition from C3 to C4 carboxylation with stomata remaining open during the transition from dusk into the dark period. Uptake of CO2 by shaded leaves during the late afternoon however appeared to be predominantly limited by decreased stomatal conductance. The short-term measurements of instantaneous Δ were subsequently integrated over 24 h in order to predict the leaf carbon isotope ratios (δp) and to compare this with the δp measured for leaf organic material. Whilst there was close agreement between predicted and measured δp for plants sampled in the wet season, during the dry season the predicted carbon isotope ratios were 5-9‰ higher than the measured isotope ratios. During the annual cycle of leaf growth most carbon was fixed via the C3 pathway although CAM clearly plays an important role in maintaining photochemical integrity in the dry season.

The carbon isotope ratio of plant organic material reflects temporal and spatial variations in CO2 within tropical forest formations in Trinidad.

A method of monitoring and collecting CO2 samples in the field has been developed which has been used to study both temporal and spatial variations in canopy CO2 isotopic signatures in two contrasting tropical forest formations in Trinidad. These have been related to vertical gradients in the carbon isotope ratio (δ13C) of organic material in conjunction with measurements of other environmental parameters. The δ13C of leaf material from two canopies showed a gradient with respect to height, more negative values being found low in the understorey. The deciduous secondary forest, (Simla) showed a difference of 4.6‰ and the semi-evergreen seasonal canopy (Aripo), 2.8‰. The range of δ13C values at Simla was 4‰ less negative than those at Aripo. In order to relate these measurements to the interaction between diffusion or carboxylation limitation, and source CO2 effects, variations in environmental parameters through the canopy have been compared with changes in CO2 partial pressure (P a) and isotopic composition δ13C throughout the day during the dry season. Values of P a20 m above the ground at Aripo varied from 380 vpm at dawn to 340 vpm at midday, at which time the partial pressure 15 cm above the ground was 375 vpm. The CO2 partial pressure did not stabilise during the course of the day, and there was good correlation (r 2=0.82) between δa and P a, with more negative values of δa occuring in the understorey. Diuraal changes of 2‰ were evident at all canopy positions. In the more open canopy at Simla, these gradients were similar, but less marked. Leaf-air vapour pressure deficit (VPD) showed no relationship with height, possibly as a result of minimal water flux from both the soil and the canopy due to low soil water content; VPD was 1.5 kPa higher at midday than dawn. A 3° C temperature gradient between the understorey and upper canopy was observed at Aripo but not in the more open Simla canopy. CO2 partial pressure stabilised for only 4 h in the middle of the day, while other parameters showed no stable period. The proportion of floor respired CO2 reassimilated at Aripo has been calculated as 26%, 19%, and 8% for the periods 0600-1000, 1000-1400, and 1400-1800 hours. In order to quantify source CO2 effects, measurements of the environmental parameters and assimilation rate must be made at all canopy positions and throughout the day.

On the ecophysiology of the Clusiaceae in Trinidad: expression of CAM in Clusia minor L. during the transition from wet to dry season and characterization of three endemic species.

A study was made of photosynthesis and expression of crassulacean acid metabolism (CAM) in naturally exposed and shaded populations of Clusia minor L. during the transition from wet to dry season in Trinidad (mid-February to mid-April, 1990). At the start of the dry season, plants from exposed and shaded habitats showed a capacity for CAM either through the fixation of external or internal (respiratory) CO2 . Exposed plants showed continuous uptake of CO2 over 24 h although dark fixation accounted for only a small proportion of CO2 fixed over the day. The expression of CAM was considerably enhanced as the dry season progressed with substantial increases in the overnight accumulation of titratable acidity, particularly in leaves of exposed plants. This was accompanied by a reduction in day-time photosynthesis and an increase in dark fixation, with shaded plants showing only night-time fixation of CO2 . The magnitude of CAM in C. minor was substantial with a maximum ΔH+ of 1410 mol m-3 measured in leaves from exposed branches. Both malic and citric acids were accumulated overnight. The highest citric:malic acid ratios were found in young leaves from exposed plants with 250 mol m-3 malic and 125 mol m-3 citric acid accumulated near the time of maximum CAM activity. Photosynthetic efficiency, measured as light responses of O2 evolution, also varied on a daily basis dependent on the incident photosynthetic photon flux density (PPFD). Apparent quantum yield and photosynthetic capacity showed marked reductions depending on the degree of exposure, suggesting that photoinhibitory responses are important under natural conditions. An analysis of three members of the Clusiaceae endemic to Trinidad showed that each had the capacity to induce CAM activity, despite being found in a narrow range of habitats which have higher rainfall than those of C. minor. However, despite the variable expression of CAM activity, carbon isotope composition suggested that when integrated throughout the year, carbon accumulation is predominantly mediated via the C3 pathway in all the species studied.

Short-term changes in carbon-isotope discrimination identify transitions between C3 and C 4 carboxylation during Crassulacean acid metabolism.

Short-term measurements of instantaneous carbon-isotope discrimination have been determined from mass-spectrometric analyses of CO2 collected online during gas exchange for the epiphytic bromeliad Tillandsia utriculata L. Using this technique, the isotopic signature of CO2 exchange for each phase of Crassulacean acid metabolism (CAM) has been characterised. During night-time fixation of CO2 (Phase I), discrimination (Δ) ranged from 4.4 to 6.6‰, equivalent to an effective carbon-isotope ratio (δ(13)C) of -12.3 to -14.5‰ versus Pee Dee Belemnite (PDB). These values reflected the gross photosynthetic balance between net CO2 uptake and refixation of respiratory CO2, characteristic of CAM in the Bromeliaceae. When Δ for the relative proportion of external (p a ) and internal (p i) CO2 is taken into account, calculated p i/p a decreased during the later part of the dark period from 0.68 to 0.48. Measurements of Δ during Phase II, early in the light period, showed the transition between C4 and C3 pathways, with carboxylation being increasingly dominated by ribulose bisphosphate carboxylase (Rubisco) as Δ increased from 10.5 to 21.2‰ During decarboxylation in the light period (Phase III), CO2 leaked out of the leaf and the inherent discrimination of Rubisco was expressed. The value of Δ calculated from on-line measurements (64.4‰) showed that the CO2 lost was considerably enriched in (13)C, and this was confirmed by direct analysis of the CO2 diffusing out into a CO2-free atmosphere (δ (13)C = + 51.6‰ versus PDB). Instantaneous discrimination was characteristic of the C3 pathway during Phase IV (late in the light period), but a reduction in Δ showed an increasing contribution from phosphoenolpyruvate carboxylase. The results from this non-invasive technique confirm the observations that "double carboxylation" involving both phosphoenolpyruvate carboxylase and Rubisco occurs during the transient phases of CAM (II and IV) in the light period.

The role of arginine decarboxylase in modulating the sensitivity of barley to ozone.

Polyamines (PA) are known to be involved in the areas of plant physiology and biochemistry which are related to the response of a plant to air pollution. This study examines the role of arginine decarboxylase (ADC), an important rate-limiting enzyme in polyamine synthesis, in barley plants exposed to ozone (O(3)). The activity of ADC increased significantly in O(3)-treated leaves when visible injury was hardly apparent. The increase in ADC activity may be a mechanism to increase the PA levels in O(3)-treated leaves and so minimize the damaging effects of O(3). Supporting this, foliar applications of DL-alpha-difluoromethylarginine (DFMA), a specific inhibitor of ADC, prevented the rise in ADC activity and visible injury was considerable on exposure to O(3). This damage was not due to the foliar sprays, as little visible injury was seen in leaves in the O(3)-free controls. The results are discussed in terms of the roles of PA in conferring O(3) resistance in plants.