Crassulacean Acid Metabolism Cycle Research Articles

Elevated nocturnal respiratory rates in the mitochondria of CAM plants: current knowledge and unanswered questions.

Crassulacean acid metabolism (CAM) is a metabolic adaptation that has evolved convergently in 38 plant families to aid survival in water-limited niches. Whilst primarily considered a photosynthetic adaptation, CAM also has substantial consequences for nocturnal respiratory metabolism. Here, we outline the history, current state and future of nocturnal respiration research in CAM plants, with a particular focus on the energetics of nocturnal respiratory oxygen consumption. Throughout the 20th century, research interest in nocturnal respiration occurred alongside initial discoveries of CAM, although the energetic and mechanistic implications of nocturnal oxygen consumption and links to the operation of the CAM cycle were not fully understood. Recent flux balance analysis (FBA) models have provided new insights into the role that mitochondria play in the CAM cycle. Several FBA models have predicted that CAM requires elevated nocturnal respiratory rates, compared to C3 species, to power vacuolar malic acid accumulation. We provide physiological data, from the genus Clusia, to corroborate these modelling predictions, thereby reinforcing the importance of elevated nocturnal respiratory rates for CAM. Finally, we outline five unanswered questions pertaining to nocturnal respiration which must be addressed if we are to fully understand and utilize CAM plants in a hotter, drier world.

Low internal air space in plants with crassulacean acid metabolism may be an anatomical spandrel.

Crassulacean acid metabolism (CAM) is a photosynthetic adaptation found in at least 38 plant families. Typically, the anatomy of CAM plants is characterized by large photosynthetic cells and a low percentage of leaf volume consisting of internal air space (% IAS). It has been suggested that reduced mesophyll conductance (gm) arising from low % IAS benefits CAM plants by preventing the movement of CO2 out of cells and ultimately minimizing leakage of CO2 from leaves into the atmosphere during day-time decarboxylation. Here, we propose that low % IAS does not provide any adaptive benefit to CAM plants, because stomatal closure during phase III of CAM will result in internal concentrations of CO2 becoming saturated, meaning low gm will not have any meaningful impact on the flux of gases within leaves. We suggest that low % IAS is more likely an indirect consequence of maximizing the cellular volume within a leaf, to provide space for the overnight storage of malic acid during the CAM cycle.

Morphological, photosynthetic, and CAM physiological responses of the submerged macrophyte Ottelia alismoides to light quality

Light quality strongly affect a great many physiological processes in terrestrial plants. Whatever, few studies have investigated how the submerged macrophytes response to different light quality. The present research aimed to understand how light quality affect morphogenesis, photosynthesis, as well as crassulacean acid metabolism (CAM) operation during the growth of Ottelia alismoides , a freshwater facultative CAM macrophyte. The plants were grown under three light qualities of white (W), red (R) and blue (B) lights with same light intensity of 100 μmol m −2 s −1 . Leaf development, leaf anatomic characteristics, photosynthetic traits, as well as CAM metabolism were determined on the newly produced leaves during light treatment. Monochromatic R stimulated leaf blade and petiole elongation, leaf area and dry weight, but reduced leaf thickness, negatively influenced chlorophyll content and Fv/Fm. On the contrary, B- grown plants were dwarf and with compact size as well as lower leaf dry weight. The content of photosynthetic product starch under B was significantly lower as compared with R, moreover, combined with the analysis of ultrastructural characteristics of epidermal and mesophyll chloroplasts, it showed that B inhibited starch to be translocated out of epidermal chloroplasts. Furthermore, both R- and B- grown plants exhibited obvious diurnal acidity fluctuation comparable to W- grown plants, and had high activity of enzymes including ribulose-bisphosphate carboxylase/oxygenase (Rubisco), phosphoenolpyruvate carboxylase (PEPC) and pyruvate phosphate dikinase (PPDK) that required for CAM cycle, indicating that CAM was present in both R- and B- grown plants and monochromatic R and B did not affect CAM activity. In conclusion, monochromatic red and blue light differentially played their special roles in regulating leaf growth and morphogenesis, as well as photosynthetic metabolism in O. alismoides . • O. alismoides displayed strong morphological plasticity to light quality. • Leaf elongation was promoted by red light but inhibited by blue light. • Red light favoured O. alismoides leaf development and photosynthesis. • Blue light inhibited translocation of starch out of epidermal chloroplasts. • O. alismoides exhibited comparable CAM activity under white, red and blue light.

Gene co-expression reveals the modularity and integration of C4 and CAM in Portulaca.

C4 photosynthesis and Crassulacean acid metabolism (CAM) have been considered as largely independent adaptations despite sharing key biochemical modules. Portulaca is a geographically widespread clade of over 100 annual and perennial angiosperm species that primarily use C4 but facultatively exhibit CAM when drought stressed, a photosynthetic system known as C4 + CAM. It has been hypothesized that C4 + CAM is rare because of pleiotropic constraints, but these have not been deeply explored. We generated a chromosome-level genome assembly of Portulaca amilis and sampled mRNA from P. amilis and Portulaca oleracea during CAM induction. Gene co-expression network analyses identified C4 and CAM gene modules shared and unique to both Portulaca species. A conserved CAM module linked phosphoenolpyruvate carboxylase to starch turnover during the day-night transition and was enriched in circadian clock regulatory motifs in the P. amilis genome. Preservation of this co-expression module regardless of water status suggests that Portulaca constitutively operate a weak CAM cycle that is transcriptionally and posttranscriptionally upregulated during drought. C4 and CAM mostly used mutually exclusive genes for primary carbon fixation, and it is likely that nocturnal CAM malate stores are shuttled into diurnal C4 decarboxylation pathways, but we found evidence that metabolite cycling may occur at low levels. C4 likely evolved in Portulaca through co-option of redundant genes and integration of the diurnal portion of CAM. Thus, the ancestral CAM system did not strongly constrain C4 evolution because photosynthetic gene networks are not co-regulated for both daytime and nighttime functions.

Engineering of Crassulacean Acid Metabolism.

Crassulacean acid metabolism (CAM) has evolved from a C3 ground state to increase water use efficiency of photosynthesis. During CAM evolution, selective pressures altered the abundance and expression patterns of C3 genes and their regulators to enable the trait. The circadian pattern of CO2 fixation and the stomatal opening pattern observed in CAM can be explained largely with a regulatory architecture already present in C3 plants. The metabolic CAM cycle relies on enzymes and transporters that exist in C3 plants and requires tight regulatory control to avoid futile cycles between carboxylation and decarboxylation. Ecological observations and modeling point to mesophyll conductance as a major factor during CAM evolution. The present state of knowledge enables suggestions for genes for a minimal CAM cycle for proof-of-concept engineering, assuming altered regulation of starch synthesis and degradation are not critical elements of CAM photosynthesis and sufficient malic acid export from the vacuole is possible.

Phosphorolytic degradation of leaf starch via plastidic α-glucan phosphorylase leads to optimized plant growth and water use efficiency over the diel phases of Crassulacean acid metabolism.

In plants with Crassulacean acid metabolism (CAM), it has been proposed that the requirement for nocturnal provision of phosphoenolpyruvate as a substrate for CO2 uptake has resulted in a re-routing of chloroplastic starch degradation from the amylolytic route to the phosphorolytic route. To test this hypothesis, we generated and characterized four independent RNAi lines of the obligate CAM species Kalanchoë fedtschenkoi with a >10-fold reduction in transcript abundance of plastidic α-glucan phosphorylase (PHS1). The rPHS1 lines showed diminished nocturnal starch degradation, reduced dark CO2 uptake, a reduction in diel water use efficiency (WUE), and an overall reduction in growth. A re-routing of starch degradation via the hydrolytic/amylolytic pathway was indicated by hyperaccumulation of maltose in all rPHS1 lines. Further examination indicated that whilst operation of the core circadian clock was not compromised, plasticity in modulating net dark CO2 uptake in response to changing photoperiods was curtailed. The data show that phosphorolytic starch degradation is critical for efficient operation of the CAM cycle and for optimizing WUE. This finding has clear relevance for ongoing efforts to engineer CAM into non-CAM species as a means of boosting crop WUE for a warmer, drier future.

Metabolic Modeling of the C3-CAM Continuum Revealed the Establishment of a Starch/Sugar-Malate Cycle in CAM Evolution.

The evolution of Crassulacean acid metabolism (CAM) is thought to be along a C3-CAM continuum including multiple variations of CAM such as CAM cycling and CAM idling. Here, we applied large-scale constraint-based modeling to investigate the metabolism and energetics of plants operating in C3, CAM, CAM cycling, and CAM idling. Our modeling results suggested that CAM cycling and CAM idling could be potential evolutionary intermediates in CAM evolution by establishing a starch/sugar-malate cycle. Our model analysis showed that by varying CO2 exchange during the light period, as a proxy of stomatal conductance, there exists a C3-CAM continuum with gradual metabolic changes, supporting the notion that evolution of CAM from C3 could occur solely through incremental changes in metabolic fluxes. Along the C3-CAM continuum, our model predicted changes in metabolic fluxes not only through the starch/sugar-malate cycle that is involved in CAM photosynthetic CO2 fixation but also other metabolic processes including the mitochondrial electron transport chain and the tricarboxylate acid cycle at night. These predictions could guide engineering efforts in introducing CAM into C3 crops for improved water use efficiency.

Modelling nonlinear dynamics of Crassulacean acid metabolism productivity and water use for global predictions.

Crassulacean acid metabolism (CAM) crops are important agricultural commodities in water-limited environments across the globe, yet modelling of CAM productivity lacks the sophistication of widely used C3 and C4 crop models, in part due to the complex responses of the CAM cycle to environmental conditions. This work builds on recent advances in CAM modelling to provide a framework for estimating CAM biomass yield and water use efficiency from basic principles. These advances, which integrate the CAM circadian rhythm with established models of carbon fixation, stomatal conductance and the soil-plant-atmosphere continuum, are coupled to models of light attenuation, plant respiration and biomass partitioning. Resulting biomass yield and transpiration for Opuntia ficus-indica and Agave tequilana are validated against field data and compared with predictions of CAM productivity obtained using the empirically based environmental productivity index. By representing regulation of the circadian state as a nonlinear oscillator, the modelling approach captures the diurnal dynamics of CAM stomatal conductance, allowing the prediction of CAM transpiration and water use efficiency for the first time at the plot scale. This approach may improve estimates of CAM productivity under light-limiting conditions when compared with previous methods.

Alternative Crassulacean Acid Metabolism Modes Provide Environment-Specific Water-Saving Benefits in a Leaf Metabolic Model.

Crassulacean acid metabolism (CAM) evolved in arid environments as a water-saving alternative to C3 photosynthesis. There is great interest in engineering more drought-resistant crops by introducing CAM into C3 plants. However, it is unknown whether full CAM or alternative water-saving modes would be more productive in the environments typically experienced by C3 crops. To study the effect of temperature and relative humidity on plant metabolism in the context of water saving, we coupled a time-resolved diel (based on a 24-h day-night cycle) model of leaf metabolism to an environment-dependent gas-exchange model. This combined model allowed us to study the emergence of CAM as a trade-off between leaf productivity and water saving. We show that vacuolar storage capacity in the leaf is a major determinant of the extent of CAM. Moreover, our model identified an alternative CAM cycle involving mitochondrial isocitrate dehydrogenase as a potential contributor to initial carbon fixation at night. Simulations across a range of environmental conditions show that the water-saving potential of CAM strongly depends on the daytime weather conditions and that the additional water-saving effect of carbon fixation by isocitrate dehydrogenase can reach 11% total water saving for the conditions tested.

Constitutive and facultative crassulacean acid metabolism (CAM) in Cuban oregano, Coleus amboinicus (Lamiaceae).

Plants exhibiting the water-conserving crassulacean acid metabolism (CAM) photosynthetic pathway provide some of the most intriguing examples of photosynthetic diversity and plasticity. Here, a largely unnoticed facet of CAM-plant photosynthesis is highlighted: the co-occurrence of ontogenetically controlled constitutive and environmentally controlled facultative CAM in a species. Both forms of CAM are displayed in leaves of Coleus amboinicus Lour. (Lamiaceae), a semi-succulent perennial plant with oregano-like flavour that is native to southern and eastern Africa and naturalised elsewhere in the tropics. Under well-watered conditions, leaves assimilate CO2 predominantly by the C3 pathway. They also display low levels of CO2 uptake at night accompanied by small nocturnal increases in leaf tissue acidity. This indicates the presence of weakly expressed constitutive CAM. CAM expression is strongly enhanced in response to drought stress. The drought-enhanced component of CAM is reversible upon rewatering and thus considered to be facultative. In contrast to C. amboinicus, the thin-leaved closely related Coleus scutellarioides (L.) Benth. exhibits net CO2 fixation solely in the light via the C3 pathway, both under well-watered and drought conditions. However, low levels of nocturnal acidification detected in leaves and stems indicate that the CAM cycle is present. The highly speciose mint family, which contains few known CAM-exhibiting species and is composed predominantly of C3 species, appears to be an excellent group of plants for studying the evolutionary origins of CAM and for determining the position of facultative CAM along the C3-full CAM trajectory.

Laying the Foundation for Crassulacean Acid Metabolism (CAM) Biodesign: Expression of the C4 Metabolism Cycle Genes of CAM in Arabidopsis.

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that exploits a temporal CO2 pump with nocturnal CO2 uptake and concentration to reduce photorespiration, improve water-use efficiency (WUE), and optimize the adaptability of plants to hotter and drier climates. Introducing the CAM photosynthetic machinery into C3 (or C4) photosynthesis plants (CAM Biodesign) represents a potentially breakthrough strategy for improving WUE while maintaining high productivity. To optimize the success of CAM Biodesign approaches, the functional analysis of individual C4 metabolism cycle genes is necessary to identify the essential genes for robust CAM pathway introduction. Here, we isolated and analyzed the subcellular localizations of 13 enzymes and regulatory proteins of the C4 metabolism cycle of CAM from the common ice plant in stably transformed Arabidopsis thaliana. Six components of the carboxylation module were analyzed including beta-carbonic anhydrase (McBCA2), phosphoenolpyruvate carboxylase (McPEPC1), phosphoenolpyruvate carboxylase kinase (McPPCK1), NAD-dependent malate dehydrogenase (McNAD-MDH1, McNAD-MDH2), and NADP-dependent malate dehydrogenase (McNADP-MDH1). In addition, seven components of the decarboxylation module were analyzed including NAD-dependent malic enzyme (McNAD-ME1, McNAD-ME2), NADP-dependent malic enzyme (McNADP-ME1, NADP-ME2), pyruvate, orthophosphate dikinase (McPPDK), pyruvate, orthophosphate dikinase-regulatory protein (McPPDK-RP), and phosphoenolpyruvate carboxykinase (McPEPCK). Ectopic overexpression of most C4-metabolism cycle components resulted in increased rosette diameter, leaf area, and leaf fresh weight of A. thaliana except for McNADP-MDH1, McPPDK-RP, and McPEPCK. Overexpression of most carboxylation module components resulted in increased stomatal conductance and dawn/dusk titratable acidity (TA) as an indirect measure of organic acid (mainly malate) accumulation in A. thaliana. In contrast, overexpression of the decarboxylating malic enzymes reduced stomatal conductance and TA. This comprehensive study provides fundamental insights into the relative functional contributions of each of the individual components of the core C4-metabolism cycle of CAM and represents a critical first step in laying the foundation for CAM Biodesign.

Light quality affects light harvesting and carbon sequestration during the diel cycle of crassulacean acid metabolism in Phalaenopsis.

Crassulacean acid metabolism (CAM) is a specialized photosynthetic pathway present in a variety of genera including many epiphytic orchids. CAM is under circadian control and can be subdivided into four discrete phases during a diel cycle. Inherent to this specific mode of metabolism, carbohydrate availability is a limiting factor for nocturnal CO2 uptake and biomass production. To evaluate the effects of light quality on the photosynthetic performance and diel changes in carbohydrates during the CAM cycle. Phalaenopsis plants were grown under four different light qualities (red, blue, red + blue and full spectrum white light) at a fluence of 100µmol m-2 s-1 and a photoperiod of 12h for 8 weeks. In contrast to monochromatic blue light, plants grown under monochromatic red light showed already a significant decline of the quantum efficiency (ΦPSII) after 5 days and of the maximum quantum yield (Fv/Fm) after 10 days under this treatment. This was also reflected in a compromised chlorophyll and carotenoid content and total diel CO2 uptake under red light in comparison with monochromatic blue and full spectrum white light. In particular, CO2 uptake during nocturnal phase I was affected under red illumination resulting in a reduced amount of vacuolar malate. In addition, red light caused the rate of decarboxylation of malate during the day to be consistently lower and malic acid breakdown persisted until 4h after dusk. Because the intrinsic activity of PEPC was not affected, the restricted availability of storage carbohydrates such as starch was likely to cause these adverse effects under red light. Addition of blue to the red light spectrum restored the diel fluxes of carbohydrates and malate and resulted in a significant enhancement of the daily CO2 uptake, pigment concentration and biomass formation.

Protection by light against heat stress in leaves of tropical crassulacean acid metabolism plants containing high acid levels.

Heat tolerance of plants exhibiting crassulacean acid metabolism (CAM) was determined by exposing leaf sections to a range of temperatures both in the dark and the light, followed by measuring chlorophyll a fluorescence (Fv/Fm and F0) and assessing visible tissue damage. Three CAM species, Clusia rosea Jacq., Clusia pratensis Seem. and Agave angustifolia Haw., were studied. In acidified tissues sampled at the end of the night and exposed to elevated temperatures in the dark, the temperature that caused a 50% decline of Fv/Fm (T50), was remarkably low (40-43°C in leaves of C. rosea). Conversion of chlorophyll to pheophytin indicated irreversible tissue damage caused by malic acid released from the vacuoles. By contrast, when acidified leaves were illuminated during heat treatments, T50 was up to 50-51°C. In de-acidified samples taken at the end of the light period, T50 reached ∼54°C, irrespective of whether temperature treatments were done in the dark or light. Acclimation of A. angustifolia to elevated daytime temperatures resulted in a rise of T50 from ∼54° to ∼57°C. In the field, high tissue temperatures always occur during sun exposure. Measurements of the heat tolerance of CAM plants that use heat treatments of acidified tissue in the dark do not provide relevant information on heat tolerance in an ecological context. However, in the physiological context, such studies may provide important clues on vacuolar properties during the CAM cycle (i.e. on the temperature relationships of malic acid storage and malic acid release).

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.

Nonlinear dynamics of the CAM circadian rhythm in response to environmental forcing.

Crassulacean acid metabolism (CAM) photosynthesis functions as an endogenous circadian rhythm coupled to external environmental forcings of energy and water availability. This paper explores the nonlinear dynamics of a new CAM photosynthesis model (Bartlett et al., 2014) and investigates the responses of CAM plant carbon assimilation to different combinations of environmental conditions. The CAM model (Bartlett et al., 2014) consists of a Calvin cycle typical of C3 plants coupled to an oscillator of the type employed in the Van der Pol and FitzHugh-Nagumo systems. This coupled system is a function of environmental variables including leaf temperature, leaf moisture potential, and irradiance. Here, we explore the qualitative response of the system and the expected carbon assimilation under constant and periodically forced environmental conditions. The model results show how the diurnal evolution of these variables entrains the CAM cycle with prevailing environmental conditions. While constant environmental conditions generate either steady-state or periodically oscillating responses in malic acid uptake and release, forcing the CAM system with periodic daily fluctuations in light exposure and leaf temperature results in quasi-periodicity and possible chaos for certain ranges of these variables. This analysis is a first step in quantifying changes in CAM plant productivity with variables such as the mean temperature, daily temperature range, irradiance, and leaf moisture potential. Results may also be used to inform model parametrization based on the observed fluctuating regime.

Photosynthesis and water use by two Sedum species in green roof substrate

Different succulent plants have been shown to exhibit varying degrees of crassulacean acid metabolism (CAM). This variation has not been consistently shown to relate to water use efficiency (WUE) by these species. This raises fundamental questions about the ecophysiological implications of switching between C3 photosynthesis and CAM metabolism. More information is also needed for Sedum species that utilize CAM to varying degrees on green roofs so that plant water use can be included in water balance equations that predict green roof performance. We monitored carbon dioxide exchange and water use by two common green roof species, Sedum album and Sedum kamtschaticum, for three weeks without water under strictly controlled environmental conditions in order to (1) compare daily carbon gain, rates of evapotranspiration (ET), and WUE for these two species and (2) assess the effect of CAM cycling on WUE with increasing drought for entire plant stands. Under similar environmental conditions, S. album and S. kamtschaticum canopies exhibited CAM metabolism to different extents with different ecophysiological consequences. S. kamtschaticum exhibited C3 photosynthesis for the first 14 days without water and outperformed S. album in terms of carbon gain and WUE above a volumetric water content (VWC) of 0.08m3m−3. S. album was the more drought tolerant species under the conditions of this experiment, gaining at least as much carbon and using water more efficiently at VWC below 0.08m3m−3. By switching from C3 to CAM at lower VWC, S. kamtschaticum had an overall greater WUE even though this species used as much if not more water overall compared to S. album. These data further support suggestions that a major adaptive function of CAM is to prolong plant-available water while maintaining a consistent, albeit low level of carbon gain. These findings also demonstrate why Sedum species should be treated independently of each other when considered in green roof designs.

A Diel Flux Balance Model Captures Interactions between Light and Dark Metabolism during Day-Night Cycles in C3 and Crassulacean Acid Metabolism Leaves.

Although leaves have to accommodate markedly different metabolic flux patterns in the light and the dark, models of leaf metabolism based on flux-balance analysis (FBA) have so far been confined to consideration of the network under continuous light. An FBA framework is presented that solves the two phases of the diel cycle as a single optimization problem and, thus, provides a more representative model of leaf metabolism. The requirement to support continued export of sugar and amino acids from the leaf during the night and to meet overnight cellular maintenance costs forces the model to set aside stores of both carbon and nitrogen during the day. With only minimal constraints, the model successfully captures many of the known features of C3 leaf metabolism, including the recently discovered role of citrate synthesis and accumulation in the night as a precursor for the provision of carbon skeletons for amino acid synthesis during the day. The diel FBA model can be applied to other temporal separations, such as that which occurs in Crassulacean acid metabolism (CAM) photosynthesis, allowing a system-level analysis of the energetics of CAM. The diel model predicts that there is no overall energetic advantage to CAM, despite the potential for suppression of photorespiration through CO2 concentration. Moreover, any savings in enzyme machinery costs through suppression of photorespiration are likely to be offset by the higher flux demand of the CAM cycle. It is concluded that energetic or nitrogen use considerations are unlikely to be evolutionary drivers for CAM photosynthesis.

Influence of germination date on Dioon edule (Zamiaceae) seedling tolerance to water stress

Dioon edule seedling mortality is mostly attributed to dehydration by prolonged drought, even when they present xeromorphic characteristics like the adult plants. The effect of germination date (GD) and soil water deficit on seedling tolerance to water stress was assessed. The seedlings germinated and grown from mature seeds every month from December to April GD were selected to evaluate the leaf area, photosynthetic pigment content, crassulacean acid metabolism (CAM) activity, stomatal conductance (gs) and leaflet anatomy at soil water potential (Ψs) of 0.0MPa (day 1), -0.1MPa (day 40), -1.0MPa (day 90), -1.5MPa (day 130), and a control (0.0MPa at day 130) to recognize differences due to leaf development. The seedlings shifted from C3 to CAM cycling when exposed to water stress at Ψs of -1.0MPa, like adult plants. The March-April GD seedlings with undeveloped sclerified hypodermis and stomata, presented reduced leaf area, lower Chlorophyll a/b ratio, higher CAM activity and midday partial stomatal closure when reached Ψs of -1.0MPa. These have higher probability of dehydration during severe drought (February-April) than those of the December-February GD with similar Ψs. Plants used for restoration purposes must have full leaf development to increase the survival.

Day/night regulation of aquaporins during the CAM cycle in Mesembryanthemum crystallinum

Mesembryanthemum crystallinum exhibits induction of Crassulacean acid metabolism (CAM) after a threshold stage of development, by exposure to long days with high light intensities or by water and salt stress. During the CAM cycle, fluctuations in carbon partitioning within the cell lead to transient drops in osmotic potential, which are likely stabilized/balanced by passive movement of water via aquaporins (AQPs). Protoplast swelling assays were used to detect changes in water permeability during the day/night cycle of CAM. To assess the role of AQPs during the same period, we followed transcript accumulation and protein abundance of four plasma membrane intrinsic proteins (PIPs) and one tonoplast intrinsic protein (TIP). CAM plants showed a persistent rhythm of specific AQP protein abundance changes throughout the day/night cycle, including changes in amount of McPIP2;1, McTIP1;2, McPIP1;4 and McPIP1;5, while the abundance of McPIP1;2 was unchanged. These protein changes did not appear to be coordinated with transcript levels for any of the AQPs analysed; however, they did occur in parrallel to alterations in water permeability, as well as variations in cell osmolarity, pinitol, glucose, fructose and phosphoenolpyruvate carboxylase (PEPc) levels measured throughout the day/night CAM cycle. Results suggest a role for AQPs in maintaining water balance during CAM and highlight the complexity of protein expression during the CAM cycle.

Correlation between citric acid and nitrate metabolisms during CAM cycle in the atmospheric bromeliad Tillandsia pohliana

Crassulacean acid metabolism (CAM) confers crucial adaptations for plants living under frequent environmental stresses. A wide metabolic plasticity can be found among CAM species regarding the type of storage carbohydrate, organic acid accumulated at night and decarboxylating system. Consequently, many aspects of the CAM pathway control are still elusive while the impact of this photosynthetic adaptation on nitrogen metabolism has remained largely unexplored. In this study, we investigated a possible link between the CAM cycle and the nitrogen assimilation in the atmospheric bromeliad Tillandsia pohliana by simultaneously characterizing the diel changes in key enzyme activities and metabolite levels of both organic acid and nitrate metabolisms. The results revealed that T. pohliana performed a typical CAM cycle in which phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase phosphorylation seemed to play a crucial role to avoid futile cycles of carboxylation and decarboxylation. Unlike all other bromeliads previously investigated, almost equimolar concentrations of malate and citrate were accumulated at night. Moreover, a marked nocturnal depletion in the starch reservoirs and an atypical pattern of nitrate reduction restricted to the nighttime were also observed. Since reduction and assimilation of nitrate requires a massive supply of reducing power and energy and considering that T. pohliana lives overexposed to the sunlight, we hypothesize that citrate decarboxylation might be an accessory mechanism to increase internal CO 2 concentration during the day while its biosynthesis could provide NADH and ATP for nocturnal assimilation of nitrate. Therefore, besides delivering photoprotection during the day, citrate might represent a key component connecting both CAM pathway and nitrogen metabolism in T. pohliana; a scenario that certainly deserves further study not only in this species but also in other CAM plants that nocturnally accumulate citrate.