Drought stress triggers alterations of adaxial and abaxial stomatal development in basil leaves increasing water-use efficiency.

The physiological control of stomatal opening by which plants adjust for water availability has been extensively researched. However, the impact of water availability on stomatal development has not received as much attention, especially for amphistomatic plants. Therefore, the acclimation of stomatal development in basil (Ocimum basilicum L.) leaves was investigated. Our results show that leaves developed under water-deficit conditions possess higher stomatal densities and decreased stomatal length for both the adaxial and abaxial leaf sides. Although the stomatal developmental reaction to water deficit was similar for the two leaf surfaces, it was proven that adaxial stomata are more sensitive to water stress than abaxial stomata, with more closed adaxial stomata under water-deficit conditions. Furthermore, plants with leaves containing smaller stomata at higher densities possessed a higher water use efficiency. Our findings highlight the importance of stomatal development as a tool for long-term acclimation to limit water loss, with minimal reduction in biomass production. This highlights the central role that stomata play in both the short (opening) and long-term (development) reaction of plants to water availability, making them key tools for efficient resource use and anticipation of future environmental changes.

Far-Red Light Mediated Carbohydrate Concentration Changes in Leaves of Sweet Basil, a Stachyose Translocating Plant.

Photosynthetic active radiation (PAR) refers to photons between 400 and 700 nm. These photons drive photosynthesis, providing carbohydrates for plant metabolism and development. Far-red radiation (FR, 701-750 nm) is excluded in this definition because no FR is absorbed by the plant photosynthetic pigments. However, including FR in the light spectrum provides substantial benefits for biomass production and resource-use efficiency. We investigated the effects of continuous FR addition and end-of-day additional FR to a broad white light spectrum (BW) on carbohydrate concentrations in the top and bottom leaves of sweet basil (Ocimum basilicum L.), a species that produces the raffinose family oligosaccharides raffinose and stachyose and preferentially uses the latter as transport sugar. Glucose, fructose, sucrose, raffinose, and starch concentrations increased significantly in top and bottom leaves with the addition of FR light. The increased carbohydrate pools under FR light treatments are associated with more efficient stachyose production and potentially improved phloem loading through increased sucrose homeostasis in intermediary cells. The combination of a high biomass yield, increased resource-use efficiency, and increased carbohydrate concentration in leaves in response to the addition of FR light offers opportunities for commercial plant production in controlled growth environments.

Soil Moisture Levels Affect the Anatomy and Mechanical Properties of Basil Stems (Ocimum basilicum L.).

As plants would benefit from adjusting and optimizing their architecture to changing environmental stimuli, ensuring a strong and healthy plant, it was hypothesized that different soil moisture levels would affect xylem and collenchyma development in basil (Ocimum basilicum L. cv. Marian) stems. Four different irrigation set-points (20, 30, 40 and 50% VWC), corresponding respectively to pF values of 1.95, 1.65, 1.30 and 1.15, were applied. Basil plants grown near the theoretical wilting point (pF 2) had a higher xylem vessel frequency and lower mean vessel diameter, promoting water transport under drought conditions. Cultivation at low soil moisture also impacted the formation of collenchyma in the apical stem segments, providing mechanical and structural support to these fast-growing stems and vascular tissues. The proportion of collenchyma area was significantly lower for the pF1.15 treatment (9.25 ± 3.24%) compared to the pF1.95 and pF1.30 treatments (16.04 ± 1.83% and 13.28 ± 1.38%, respectively). Higher fractions of collenchyma resulted in a higher mechanical stem strength against bending. Additionally, tracheids acted as the major support tissues in the basal stem segments. These results confirm that the available soil moisture impacts mechanical stem strength and overall plant quality of basil plants by impacting xylem and collenchyma development during cultivation, ensuring sufficient mechanical support to the fast-growing stem and to the protection of the vascular tissues. To our knowledge, this study is the first to compare the mechanical and anatomical characteristics of plant stems cultivated at different soil moisture levels.

The Impact of Hydro-Priming and Osmo-Priming on Seedling Characteristics, Plant Hormone Concentrations, Activity of Selected Hydrolytic Enzymes, and Cell Wall and Phytate Hydrolysis in Sprouted Wheat (Triticum aestivum L.).

Priming improves the seed germination rate and grain yield. Before this work was executed, little, if any, research has been reported on priming wheat for improving its nutritional properties. The impact of hydro-priming and osmo-priming using solutions with different water potentials on selected hydrolytic enzyme activities and their breakdown of starch, cell wall materials, and phytates during subsequent sprouting was studied here. A higher germination rate in the early growth stage of seedlings was found for hydro-primed or osmo-primed (-0.3, -0.6 MPa) grains. Hydro-primed sprouted grains had the longest radicles and coleoptiles and the highest hydrolytic enzyme activities. The latter lead to a 90% increase in reducing sugar, a 20% increase in water-extractable arabinoxylan, and an 8% decrease in phytate contents after 5 days of sprouting. This study thus offers opportunities for optimizing agricultural practice. The presence of different plant hormones and their concentrations are generally not affected by priming. However, the plant hormone concentrations in grains primed at -1.2 MPa and subsequently sprouted were lower than those in all other samples under study. The induction of too high osmotic stresses in these grains leads to disruption of the sprouting processes. Finally, it was for the first time found, based on the known biosynthesis pathways of wheat, that gibberellic acid (GA)20-oxidase in (primed) sprouted wheat is more active than GA3-oxidase and much more active than GA13-oxidase.

The Role of Auxin-Ethylene Crosstalk in Orchestrating Primary Root Elongation in Sugar Beet.

It is well-established in Arabidopsis and other species that ethylene inhibits root elongation through the action of auxin. In sugar beet (Beta vulgaris L.) ethylene promotes root elongation in a concentration dependent manner. However, the crosstalk between ethylene and auxin remains unknown during sugar beet seedling development. Our experiments have shown that exogenously applied auxin (indole-3-acetic acid; IAA) also stimulates root elongation. We also show that auxin promotes ethylene biosynthesis leading to longer roots. We have further demonstrated that the auxin treatment stimulates ethylene production by redirecting the pool of available 1-aminocyclopropane-1-carboxylic acid (ACC) toward ethylene instead of malonyl-ACC (MACC) resulting in a prolonged period of high rates of ethylene production and subsequently a longer root. On the other hand we have also shown that endogenous IAA levels were not affected by an ACC treatment during germination. All together our findings suggest that the general model for auxin-ethylene crosstalk during early root development, where ethylene controls auxin biosynthesis and transport, does not occur in sugar beet. On the contrary, we have shown that the opposite, where auxin stimulates ethylene biosynthesis, is true for sugar beet root development.

Ethylene is differentially regulated during sugar beet germination and affects early root growth in a dose-dependent manner.

MAIN CONCLUSION: By integrating molecular, biochemical, and physiological data, ethylene biosynthesis in sugar beet was shown to be differentially regulated, affecting root elongation in a concentration-dependent manner. There is a close relation between ethylene production and seedling growth of sugar beet (Beta vulgaris L.), yet the exact function of ethylene during this early developmental stage is still unclear. While ethylene is mostly considered to be a root growth inhibitor, we found that external 1-aminocyclopropane-1-carboxylic acid (ACC) regulates root growth in sugar beet in a concentration-dependent manner: low concentrations stimulate root growth while high concentrations inhibit root growth. These results reveal that ethylene action during root elongation is strongly concentration dependent. Furthermore our detailed study of ethylene biosynthesis kinetics revealed a very strict gene regulation pattern of ACC synthase (ACS) and ACC oxidase (ACO), in which ACS is the rate liming step during sugar beet seedling development.

Light quality modulates metabolic synchronization over the diel phases of crassulacean acid metabolism.

Temporal compartmentation of carboxylation processes is a defining feature of crassulacean acid metabolism and involves circadian control of key metabolic and transport steps that regulate the supply and demand for carbon over a 24h cycle. Recent insights on the molecular workings of the circadian clock and its connection with environmental inputs raise new questions on the importance of light quality and, by analogy, certain photoreceptors for synchronizing the metabolic components of CAM. The present work tested the hypothesis that optimal coupling of stomatal conductance, net CO2 uptake, and the reciprocal turnover of carbohydrates and organic acids over the diel CAM cycle requires both blue and red light input signals. Contrasting monochromatic wavelengths of blue, green, and red light (i.e. 475, 530, 630nm) with low fluence rates (10 µmol m(-2) s(-1)) were administered for 16 hours each diel cycle for a total treatment time of 48 hours to the obligate CAM bromeliad, Aechmea 'Maya'. Of the light treatments imposed, low-fluence blue light was a key determinant in regulating stomatal responses, organic acid mobilization from the vacuole, and daytime decarboxylation. However, the reciprocal relationship between starch and organic acid turnover that is typical for CAM was uncoupled under low-fluence blue light. Under low-fluence red or green light, the diel turnover of storage carbohydrates was orchestrated in line with the requirements of CAM, but a consistent delay in acid consumption at dawn compared with plants under white or low-fluence blue light was noted. Consistent with the acknowledged influences of both red and blue light as input signals for the circadian clock, the data stress the importance of both red and blue-light signalling pathways for synchronizing the metabolic and physiological components of CAM over the day/night cycle.

Tissue specific analysis reveals a differential organization and regulation of both ethylene biosynthesis and E8 during climacteric ripening of tomato.

BACKGROUND: Solanum lycopersicum or tomato is extensively studied with respect to the ethylene metabolism during climacteric ripening, focusing almost exclusively on fruit pericarp. In this work the ethylene biosynthesis pathway was examined in all major tomato fruit tissues: pericarp, septa, columella, placenta, locular gel and seeds. The tissue specific ethylene production rate was measured throughout fruit development, climacteric ripening and postharvest storage. All ethylene intermediate metabolites (1-aminocyclopropane-1-carboxylic acid (ACC), malonyl-ACC (MACC) and S-adenosyl-L-methionine (SAM)) and enzyme activities (ACC-oxidase (ACO) and ACC-synthase (ACS)) were assessed. RESULTS: All tissues showed a similar climacteric pattern in ethylene productions, but with a different amplitude. Profound differences were found between tissue types at the metabolic and enzymatic level. The pericarp tissue produced the highest amount of ethylene, but showed only a low ACC content and limited ACS activity, while the locular gel accumulated a lot of ACC, MACC and SAM and showed only limited ACO and ACS activity. Central tissues (septa, columella and placenta) showed a strong accumulation of ACC and MACC. These differences indicate that the ethylene biosynthesis pathway is organized and regulated in a tissue specific way. The possible role of inter- and intra-tissue transport is discussed to explain these discrepancies. Furthermore, the antagonistic relation between ACO and E8, an ethylene biosynthesis inhibiting protein, was shown to be tissue specific and developmentally regulated. In addition, ethylene inhibition by E8 is not achieved by a direct interaction between ACO and E8, as previously suggested in literature. CONCLUSIONS: The Ethylene biosynthesis pathway and E8 show a tissue specific and developmental differentiation throughout tomato fruit development and ripening.

Dynamic changes of the ethylene biosynthesis in 'Jonagold' apple.

In this study, the short-term and dynamic changes of the ethylene biosynthesis of Jonagold apple during and after application of controlled atmosphere (CA) storage conditions were quantified using a systems biology approach. Rapid responses to imposed temperature and atmospheric conditions were captured by continuous online photoacoustic ethylene measurements. Discrete destructive sampling was done to understand observed changes of ethylene biosynthesis at the transcriptional, translational and metabolic level. Application of the ethylene inhibitor 1-methylcyclopropene (1-MCP) allowed for the discrimination between ethylene-mediated changes and ethylene-independent changes related to the imposed conditions. Online ethylene measurements showed fast and slower responses during and after application of CA conditions. The changes in 1-aminocyclopropane-1-carboxylate synthase (ACS) activity were most correlated with changes in ACS1 expression and regulated the cold-induced increase in ethylene production during the early chilling phase. Transcription of ACS3 was found ethylene independent and was triggered upon warming of CA-stored apples. Increased expression of ACO1 during shelf life led to a strong increase in 1-aminocyclopropane-1-carboxylate oxidase (ACO) activity, required for the exponential production of ethylene during system 2. Expression of ACO2 and ACO3 was upregulated in 1-MCP-treated fruit showing a negative correlation with ethylene production. ACO activity never became rate limiting.

Sedoheptulose accumulation under CO2 enrichment in leaves of Kalanchoë pinnata: a novel mechanism to enhance C and P homeostasis?

In contrast to the well-documented roles of its mono- and bisphosphate esters, the occurrence of free sedoheptulose in plant tissues remains a matter of conjecture. The present work sought to determine the origin of sedoheptulose formation in planta, as well as its physiological importance. Elevated CO2 and sucrose induction experiments were used to study sedoheptulose metabolism in the Crassulacean acid metabolism (CAM) plants Kalanchoë pinnata and Sedum spectabile. Experimental evidence suggested that sedoheptulose is produced from the oxidative pentose phosphate pathway intermediate sedoheptulose-7-phosphate, by a sedoheptulose-7-phosphate phosphatase. Carbon flux through this pathway was stimulated by increased triose-phosphate levels (elevated CO2, compromised sink availability, and sucrose incubation of source leaves) and attenuated by ADP and inorganic phosphate (Pi). The accumulation of free sedoheptulose is proposed to act as a mechanism contributing to both C and P homeostasis by serving as an alternative carbon store under elevated CO2 or a compromised sink capacity to avoid sucrose accumulation, depletion of inorganic phosphate, and suppression of photosynthesis. It remains to be established whether this acclimation-avoiding mechanism is confined to CAM plants, which might be especially vulnerable to Pi imbalances, or whether some C3 and C4 plants also dispose of the genetic capacity to induce and accelerate sedoheptulose synthesis upon CO2 elevation.

CIM(®) monolithic anion-exchange chromatography as a useful alternative to CsCl gradient purification of bacteriophage particles.

The use of anion-exchange chromatography was investigated as an alternative method to concentrate and purify bacterial viruses, and parameters for different bacteriophages were compared. Chromatography was performed with Convective Interactive Media(®) monoliths, with three different volumes and two matrix chemistries. Eleven morphologically distinct phages were tested, infecting five different bacterial species. For each of the phages tested, a protocol was optimized, including the choice of column chemistry, loading, buffer and elution conditions. The capacity and recovery of the phages on the columns varied considerably between phages. We conclude that anion-exchange chromatography with monoliths is a valid alternative to the more traditional CsCl purification, has upscaling advantages, but it requires more extensive optimization.

Targeted systems biology profiling of tomato fruit reveals coordination of the Yang cycle and a distinct regulation of ethylene biosynthesis during postclimacteric ripening.

The concept of system 1 and system 2 ethylene biosynthesis during climacteric fruit ripening was initially described four decades ago. Although much is known about fruit development and climacteric ripening, little information is available about how ethylene biosynthesis is regulated during the postclimacteric phase. A targeted systems biology approach revealed a novel regulatory mechanism of ethylene biosynthesis of tomato (Solanum lycopersicum) when fruit have reached their maximal ethylene production level and which is characterized by a decline in ethylene biosynthesis. Ethylene production is shut down at the level of 1-aminocyclopropane-1-carboxylic acid oxidase. At the same time, 1-aminocyclopropane-1-carboxylic acid synthase activity increases. Analysis of the Yang cycle showed that the Yang cycle genes are regulated in a coordinated way and are highly expressed during postclimacteric ripening. Postclimacteric red tomatoes on the plant showed only a moderate regulation of 1-aminocyclopropane-1-carboxylic acid synthase and Yang cycle genes compared with the regulation in detached fruit. Treatment of red fruit with 1-methylcyclopropane and ethephon revealed that the shut-down mechanism in ethylene biosynthesis is developmentally programmed and only moderately ethylene sensitive. We propose that the termination of autocatalytic ethylene biosynthesis of system 2 in ripe fruit delays senescence and preserves the fruit until seed dispersal.

T4-related bacteriophage LIMEstone isolates for the control of soft rot on potato caused by 'Dickeya solani'.

The bacterium 'Dickeya solani', an aggressive biovar 3 variant of Dickeya dianthicola, causes rotting and blackleg in potato. To control this pathogen using bacteriophage therapy, we isolated and characterized two closely related and specific bacteriophages, vB_DsoM_LIMEstone1 and vB_DsoM_LIMEstone2. The LIMEstone phages have a T4-related genome organization and share DNA similarity with Salmonella phage ViI. Microbiological and molecular characterization of the phages deemed them suitable and promising for use in phage therapy. The phages reduced disease incidence and severity on potato tubers in laboratory assays. In addition, in a field trial of potato tubers, when infected with 'Dickeya solani', the experimental phage treatment resulted in a higher yield. These results form the basis for the development of a bacteriophage-based biocontrol of potato plants and tubers as an alternative for the use of antibiotics.

Protocol: An updated integrated methodology for analysis of metabolites and enzyme activities of ethylene biosynthesis.

BACKGROUND: The foundations for ethylene research were laid many years ago by researchers such as Lizada, Yang and Hoffman. Nowadays, most of the methods developed by them are still being used. Technological developments since then have led to small but significant improvements, contributing to a more efficient workflow. Despite this, many of these improvements have never been properly documented. RESULTS: This article provides an updated, integrated set of protocols suitable for the assembly of a complete picture of ethylene biosynthesis, including the measurement of ethylene itself. The original protocols for the metabolites 1-aminocyclopropane-1-carboxylic acid and 1-(malonylamino)cyclopropane-1-carboxylic acid have been updated and downscaled, while protocols to determine in vitro activities of the key enzymes 1-aminocyclopropane-1-carboxylate synthase and 1-aminocyclopropane-1-carboxylate oxidase have been optimised for efficiency, repeatability and accuracy. All the protocols described were optimised for apple fruit, but have been proven to be suitable for the analysis of tomato fruit as well. CONCLUSIONS: This work collates an integrated set of detailed protocols for the measurement of components of the ethylene biosynthetic pathway, starting from well-established methods. These protocols have been optimised for smaller sample volumes, increased efficiency, repeatability and accuracy. The detailed protocol allows other scientists to rapidly implement these methods in their own laboratories in a consistent and efficient way.

Bacteriophages LIMElight and LIMEzero of Pantoea agglomerans, belonging to the "phiKMV-like viruses".

Pantoea agglomerans is a common soil bacterium used in the biocontrol of fungi and bacteria but is also an opportunistic human pathogen. It has been described extensively in this context, but knowledge of bacteriophages infecting this species is limited. Bacteriophages LIMEzero and LIMElight of P. agglomerans are lytic phages, isolated from soil samples, belonging to the Podoviridae and are the first Pantoea phages of this family to be described. The double-stranded DNA (dsDNA) genomes (43,032 bp and 44,546 bp, respectively) encode 57 and 55 open reading frames (ORFs). Based on the presence of an RNA polymerase in their genomes and their overall genome architecture, these phages should be classified in the subfamily of the Autographivirinae, within the genus of the "phiKMV-like viruses." Phylogenetic analysis of all the sequenced members of the Autographivirinae supports the classification of phages LIMElight and LIMEzero as members of the "phiKMV-like viruses" and corroborates the subdivision into the different genera. These data expand the knowledge of Pantoea phages and illustrate the wide host diversity of phages within the "phiKMV-like viruses."

Crassulacean acid metabolism under severe light limitation: a matter of plasticity in the shadows?

Despite the increased energetic costs of CAM compared with C(3) photosynthesis, it is hypothesized that the inherent photosynthetic plasticity of CAM allows successful acclimation to light-limiting conditions. The present work sought to determine if CAM presented any constraints to short and longer term acclimation to light limitation and to establish if and how metabolic and photosynthetic plasticity in the deployment of the four phases of CAM might facilitate acclimation to conditions of deep shade. Measurements of leaf gas exchange, organic acids, starch and soluble sugar (glucose, fructose, and sucrose) contents were made in the leaves of the constitutive CAM bromeliad Aechmea 'Maya' over a three month period under severe light limitation. A. 'Maya' was not particularly tolerant of severe light limitation in the short term. A complete absence of net CO(2) uptake and fluctuations in key metabolites (i.e. malate, starch or soluble sugars) indicated a dampened metabolism whilst cell death in the most photosynthetically active leaves was attributed to an over-acidification of the cytoplasm. However, in the longer term, plasticity in the use of the different phases of gas exchange and different storage carbohydrate pools, i.e. a switch from starch to sucrose as the major carbohydrate source, ensured a positive carbon balance for this CAM species under extremely low levels of irradiance. As such, co-ordinated plasticity in the use of C(3) and C(4) carboxylases and different carbohydrate pools together with an increase in the abundance of light-harvesting complexes, appear to underpin the adaptive radiation of the energetically costly CAM pathway within light-limiting environments such as wet cloud forests and shaded understoreys of tropical forests.

Determination of S-adenosyl-l-methionine in fruits by capillary electrophoresis.

INTRODUCTION: S-adenosyl-l-methionine (SAM) plays an important role in many biochemical reactions in plants. It is mainly used as a methyl donor for methylation reactions, but it also participates in, for example, the biosynthesis of polyamines and the plant hormone ethylene. OBJECTIVE: To develop a fast capillary electrophoresis technique to separate SAM in fruits and fruit juices without any pre-purification steps. METHODOLOGY: Four different extraction solutions and two extraction times were tested, of which 5% trichloroacetic acid (TCA) for 10 min was found most suited. A glycine : phosphate buffer (200 : 50 mm, pH 2.5) was found optimal to analyse SAM in TCA extracts. Analyses were preformed on different climacteric and non-climacteric fruits and fruit juices. The calibration curve was created in degraded tomato extract. The CE-method was compared with a more conventional HPLC method described in literature. RESULTS: The CE technique made it possible to completely separate the S,S- and R,S-diastereoisomeric forms of SAM. The CE method proved to be very fast (20 min total running time instead of 42 min) and more sensitive (limit of detection of 0.5 µm instead of 1 µm) compared with the conventional HPLC method. CONCLUSION: Fast measurements of SAM in fruits and juices are favoured by capillary electrophoresis in a 200 : 50 mm glycine : phosphate (pH 2.5) buffer system.

Seasonal influences on carbohydrate metabolism in the CAM bromeliad Aechmea 'Maya': consequences for carbohydrate partitioning and growth.

BACKGROUND AND AIMS: Photosynthetic plasticity in response to a range of environmental factors that include [CO(2)], water availability, light intensity and temperature, is ubiquitous among plants with crassulacean acid metabolism (CAM). The present study examined how seasonal changes in light availability, as experienced by greenhouse CAM crops in northern latitude regions, influence diel carboxylation patterns and impact on carbon gain and seasonal accumulation of biomass. METHODS: In the CAM bromeliad Aechmea 'Maya' integrated measurements of leaf gas exchange, diel metabolite dynamics (e.g. malate, soluble sugars and starch) and biomass accumulation were made four times a year, i.e. in winter, spring, summer and autumn. KEY RESULTS: During the brighter seasons (spring and summer) daytime Phases II and IV were dominated by C(4) carboxylation, whilst the higher diurnal uptake in the autumn and winter was characterized by equal contributions of both Rubisco and PEPC. As a consequence, net CO(2) uptake showed a significant depression at the end of the day in the darker months when supplementary illumination was turned off. Remarkable seasonal consistency was found in the amount of storage reserves available for nocturnal carboxylation, a consequence of predominantly daytime export of carbohydrate in spring and summer whilst nocturnal export was the major sink for carbohydrate in autumn and winter. CONCLUSIONS: Throughout the different seasons Aechmea 'Maya' showed considerable plasticity in the timing and magnitude of C(3) and C(4) carboxylation processes over the diel cycle. Under low PPFD (i.e. winter and autumn) it appears that there was a constraint on the amount of carbohydrate exported during the day in order to maintain a consistent pool of transient carbohydrate reserves. This gave remarkable seasonal consistency in the amount of storage reserves available at night, thereby optimizing biomass gain throughout the year. The data have important practical consequences for horticultural productivity of CAM plants and suggest a scenario for reconciling carbohydrate partitioning between competing sinks of nocturnal acidification and export for growth.

Differential usage of storage carbohydrates in the CAM bromeliad Aechmea 'Maya' during acclimation to drought and recovery from dehydration.

CAM requires a substantial investment of resources into storage carbohydrates to account for nocturnal CO(2) uptake, thereby restricting carbohydrate partitioning to other metabolic activities, including dark respiration, growth and acclimation to abiotic stress. Flexible modulation of carbon flow to the different competing sinks under changing environmental conditions is considered a key determinant for the growth, productivity and ecological success of the CAM pathway. The aim of the present study was to examine how shifts in carbohydrate partitioning could assure maintenance of photosynthetic integrity and a positive carbon balance under conditions of increasing water deprivation in CAM species. Measurements of gas exchange, leaf water relations, malate, starch and soluble sugar (glucose, fructose and sucrose) contents were made in leaves of the CAM bromeliad Aechmea 'Maya' over a 6-month period of drought and subsequently over a 2-month period of recovery from drought. Results indicated that short-term influences of water stress were minimized by elevating the level of respiratory recycling, and carbohydrate pools were maintained at the expense of export for growth while providing a comparable nocturnal carbon gain to that in well-watered control plants. Longer term drought resulted in a disproportionate depletion of key carbohydrate reserves. Sucrose, which was of minor importance for providing substrate for the dark reactions under well-watered conditions, became the major source of carbohydrate for nocturnal carboxylation as drought progressed. Flexibility in terms of the major carbohydrate source used to sustain dark CO(2) uptake is therefore considered a crucial factor in meeting the carbon and energy demands under limiting environmental conditions. Recovery from CAM-idling was found to be dependent on the restoration of the starch pool, which was used predominantly for provision of substrate for nocturnal carboxylation, while net carbon export was limited. The conservation of starch for the nocturnal reactions might be adaptive with regard to responding efficiently to a return of water stress.