The Calvin-Benson-Bassham cycle in C4 and Crassulacean acid metabolism species.

The Calvin-Benson-Bassham (CBB) cycle is the ancestral CO2 assimilation pathway and is found in all photosynthetic organisms. Biochemical extensions to the CBB cycle have evolved that allow the resulting pathways to act as CO2 concentrating mechanisms, either spatially in the case of C4 photosynthesis or temporally in the case of Crassulacean acid metabolism (CAM). While the biochemical steps in the C4 and CAM pathways are known, questions remain on their integration and regulation with CBB cycle activity. The application of omic and transgenic technologies is providing a more complete understanding of the biochemistry of C4 and CAM species and will also provide insight into the CBB cycle in these plants. As the global population increases, new solutions are required to increase crop yields and meet demands for food and other bioproducts. Previous work in C3 species has shown that increasing carbon assimilation through genetic manipulation of the CBB cycle can increase biomass and yield. There may also be options to improve photosynthesis in species using C4 photosynthesis and CAM through manipulation of the CBB cycle in these plants. This is an underexplored strategy and requires more basic knowledge of CBB cycle operation in these species to enable approaches for increased productivity.

Producing fast and active Rubisco in tobacco to enhance photosynthesis.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) performs most of the carbon fixation on Earth. However, plant Rubisco is an intrinsically inefficient enzyme given its low carboxylation rate, representing a major limitation to photosynthesis. Replacing endogenous plant Rubisco with a faster Rubisco is anticipated to enhance crop photosynthesis and productivity. However, the requirement of chaperones for Rubisco expression and assembly has obstructed the efficient production of functional foreign Rubisco in chloroplasts. Here, we report the engineering of a Form 1A Rubisco from the proteobacterium Halothiobacillus neapolitanus in Escherichia coli and tobacco (Nicotiana tabacum) chloroplasts without any cognate chaperones. The native tobacco gene encoding Rubisco large subunit was genetically replaced with H. neapolitanus Rubisco (HnRubisco) large and small subunit genes. We show that HnRubisco subunits can form functional L8S8 hexadecamers in tobacco chloroplasts at high efficiency, accounting for ∼40% of the wild-type tobacco Rubisco content. The chloroplast-expressed HnRubisco displayed a ∼2-fold greater carboxylation rate and supported a similar autotrophic growth rate of transgenic plants to that of wild-type in air supplemented with 1% CO2. This study represents a step toward the engineering of a fast and highly active Rubisco in chloroplasts to improve crop photosynthesis and growth.

Kalanchoë PPC1 Is Essential for Crassulacean Acid Metabolism and the Regulation of Core Circadian Clock and Guard Cell Signaling Genes.

Unlike C3 plants, Crassulacean acid metabolism (CAM) plants fix CO2 in the dark using phosphoenolpyruvate carboxylase (PPC; EC 4.1.1.31). PPC combines phosphoenolpyruvate with CO2 (as HCO3 -), forming oxaloacetate. The oxaloacetate is converted to malate, leading to malic acid accumulation in the vacuole, which peaks at dawn. During the light period, malate decarboxylation concentrates CO2 around Rubisco for secondary fixation. CAM mutants lacking PPC have not been described. Here, we employed RNA interference to silence the CAM isogene PPC1 in Kalanchoë laxiflora Line rPPC1-B lacked PPC1 transcripts, PPC activity, dark period CO2 fixation, and nocturnal malate accumulation. Light period stomatal closure was also perturbed, and the plants displayed reduced but detectable dark period stomatal conductance and arrhythmia of the CAM CO2 fixation circadian rhythm under constant light and temperature free-running conditions. By contrast, the rhythm of delayed fluorescence was enhanced in plants lacking PPC1 Furthermore, a subset of gene transcripts within the central circadian oscillator was upregulated and oscillated robustly in this line. The regulation of guard cell genes involved in controlling stomatal movements was also perturbed in rPPC1-B These findings provide direct evidence that the regulatory patterns of key guard cell signaling genes are linked with the characteristic inverse pattern of stomatal opening and closing during CAM.

The starch-deficient plastidic PHOSPHOGLUCOMUTASE mutant of the constitutive crassulacean acid metabolism (CAM) species Kalanchoë fedtschenkoi impacts diel regulation and timing of stomatal CO2 responsiveness.

BACKGROUND AND AIMS: Crassulacean acid metabolism (CAM) is a specialized type of photosynthesis characterized by a diel pattern of stomatal opening at night and closure during the day, which increases water-use efficiency. Starch degradation is a key regulator of CAM, providing phosphoenolpyruvate as a substrate in the mesophyll for nocturnal assimilation of CO2. Growing recognition of a key role for starch degradation in C3 photosynthesis guard cells for mediating daytime stomatal opening presents the possibility that starch degradation might also impact CAM by regulating the provision of energy and osmolytes to increase guard cell turgor and drive stomatal opening at night. In this study, we tested the hypothesis that the timing of diel starch turnover in CAM guard cells has been reprogrammed during evolution to enable nocturnal stomatal opening and daytime closure. METHODS: Biochemical and genetic characterization of wild-type and starch-deficient RNAi lines of Kalanchoë fedtschenkoi with reduced activity of plastidic phosphoglucomutase (PGM) constituted a preliminary approach for the understanding of starch metabolism and its implications for stomatal regulation in CAM plants. KEY RESULTS: Starch deficiency reduced nocturnal net CO2 uptake but had negligible impact on nocturnal stomatal opening. In contrast, daytime stomatal closure was reduced in magnitude and duration in the starch-deficient rPGM RNAi lines, and their stomata were unable to remain closed in response to elevated concentrations of atmospheric CO2 administered during the day. Curtailed daytime stomatal closure was linked to higher soluble sugar contents in the epidermis and mesophyll. CONCLUSIONS: Nocturnal stomatal opening is not reliant upon starch degradation, but starch biosynthesis is an important sink for carbohydrates, ensuring daytime stomatal closure in this CAM species.

A matter of time: regulatory events behind the synchronization of C4 and crassulacean acid metabolism in Portulaca oleracea.

Portulaca species can switch between C4 and crassulacean acid metabolism (CAM) depending on environmental conditions. However, the regulatory mechanisms behind this rare photosynthetic adaptation remain elusive. Using Portulaca oleracea as a model system, here we investigated the involvement of the circadian clock, plant hormones, and transcription factors in coordinating C4 and CAM gene expression. Free-running experiments in constant conditions suggested that C4 and CAM gene expression are intrinsically connected to the circadian clock. Detailed time-course, drought, and rewatering experiments revealed distinct time frames for CAM induction and reversion (days versus hours, respectively), which were accompanied by changes in abscisic acid (ABA) and cytokinin metabolism and signaling. Exogenous ABA and cytokinins were shown to promote and repress CAM expression in P. oleracea, respectively. Moreover, the drought-induced decline in C4 transcript levels was completely recovered upon cytokinin treatment. The ABA-regulated transcription factor genes HB7, NFYA7, NFYC9, TT8, and ARR12 were identified as likely candidate regulators of CAM induction following this approach, whereas NFYC4 and ARR9 were connected to C4 expression patterns. Therefore, we provide insights into the signaling events controlling C4-CAM transitions in response to water availability and over the day/night cycle, highlighting candidate genes for future functional studies in the context of facultative C4-CAM photosynthesis.

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.

Crassulacean acid metabolism guard cell anion channel activity follows transcript abundance and is suppressed by apoplastic malate.

Plants utilising crassulacean acid metabolism (CAM) concentrate CO2 around RuBisCO while reducing transpirational water loss associated with photosynthesis. Unlike stomata of C3 and C4 species, CAM stomata open at night for the mesophyll to fix CO2 into malate (Mal) and store it in the vacuole. CAM plants decarboxylate Mal in the light, generating high CO2 concentrations within the leaf behind closed stomata for refixation by RuBisCO. CO2 may contribute to stomatal closure but additional mechanisms, plausibly including Mal activation of anion channels, ensure closure in the light. In the CAM species Kalanchoë fedtschenkoi, we found that guard cell anion channel activity, recorded under voltage clamp, follows KfSLAC1 and KfALMT12 transcript abundance, declining to near zero by the end of the light period. Unexpectedly, however, we found that extracellular Mal inhibited the anion current of Kalanchoë guard cells, both in wild-type and RNAi mutants with impaired Mal metabolism. We conclude that the diurnal cycle of anion channel gene transcription, rather than the physiological signal of Mal release, is a key factor in the inverted CAM stomatal cycle.

C4 and crassulacean acid metabolism within a single leaf: deciphering key components behind a rare photosynthetic adaptation.

Although biochemically related, C4 and crassulacean acid metabolism (CAM) systems are expected to be incompatible. However, Portulaca species, including P. oleracea, operate C4 and CAM within a single leaf, and the mechanisms behind this unique photosynthetic arrangement remain largely unknown. Here, we employed RNA-seq to identify candidate genes involved exclusively or shared by C4 or CAM, and provided an in-depth characterization of their transcript abundance patterns during the drought-induced photosynthetic transitions in P. oleracea. Data revealed fewer candidate CAM-specific genes than those recruited to function in C4 . The putative CAM-specific genes were predominantly involved in night-time primary carboxylation reactions and malate movement across the tonoplast. Analysis of gene transcript-abundance regulation and photosynthetic physiology indicated that C4 and CAM coexist within a single P. oleracea leaf under mild drought conditions. Developmental and environmental cues were shown to regulate CAM expression in stems, whereas the shift from C4 to C4 -CAM hybrid photosynthesis in leaves was strictly under environmental control. Moreover, efficient starch turnover was identified as part of the metabolic adjustments required for CAM operation in both organs. These findings provide insights into C4 /CAM connectivity and compatibility, contributing to a deeper understanding of alternative ways to engineer CAM into C4 crop species.

Phosphorylation of Phosphoenolpyruvate Carboxylase Is Essential for Maximal and Sustained Dark CO2 Fixation and Core Circadian Clock Operation in the Obligate Crassulacean Acid Metabolism Species Kalanchoë fedtschenkoi.

Phosphoenolpyruvate carboxylase (PPC; EC 4.1.1.31) catalyzes primary nocturnal CO2 fixation in Crassulacean acid metabolism (CAM) species. CAM PPC is regulated posttranslationally by a circadian clock-controlled protein kinase called phosphoenolpyruvate carboxylase kinase (PPCK). PPCK phosphorylates PPC during the dark period, reducing its sensitivity to feedback inhibition by malate and thus enhancing nocturnal CO2 fixation to stored malate. Here, we report the generation and characterization of transgenic RNAi lines of the obligate CAM species Kalanchoë fedtschenkoi with reduced levels of KfPPCK1 transcripts. Plants with reduced or no detectable dark phosphorylation of PPC displayed up to a 66% reduction in total dark period CO2 fixation. These perturbations paralleled reduced malate accumulation at dawn and decreased nocturnal starch turnover. Loss of oscillations in the transcript abundance of KfPPCK1 was accompanied by a loss of oscillations in the transcript abundance of many core circadian clock genes, suggesting that perturbing the only known link between CAM and the circadian clock feeds back to perturb the central circadian clock itself. This work shows that clock control of KfPPCK1 prolongs the activity of PPC throughout the dark period in K. fedtschenkoi, optimizing CAM-associated dark CO2 fixation, malate accumulation, CAM productivity, and core circadian clock robustness.

Transgenic perturbation of the decarboxylation phase of Crassulacean acid metabolism alters physiology and metabolism but has only a small effect on growth.

Mitochondrial NAD-malic enzyme (ME) and/or cytosolic/plastidic NADP-ME combined with the cytosolic/plastidic pyruvate orthophosphate dikinase (PPDK) catalyze two key steps during light-period malate decarboxylation that underpin secondary CO(2) fixation in some Crassulacean acid metabolism (CAM) species. We report the generation and phenotypic characterization of transgenic RNA interference lines of the obligate CAM species Kalanchoë fedtschenkoi with reduced activities of NAD-ME or PPDK. Transgenic line rNAD-ME1 had 8%, and rPPDK1 had 5% of the wild-type level of activity, and showed dramatic changes in the light/dark cycle of CAM CO(2) fixation. In well-watered conditions, these lines fixed all of their CO(2) in the light; they thus performed C(3) photosynthesis. The alternative malate decarboxylase, NADP-ME, did not appear to compensate for the reduction in NAD-ME, suggesting that NAD-ME was the key decarboxylase for CAM. The activity of other CAM enzymes was reduced as a consequence of knocking out either NAD-ME or PPDK activity, particularly phosphoenolpyruvate carboxylase (PPC) and PPDK in rNAD-ME1. Furthermore, the circadian clock-controlled phosphorylation of PPC in the dark was reduced in both lines, especially in rNAD-ME1. This had the consequence that circadian rhythms of PPC phosphorylation, PPC kinase transcript levels and activity, and the classic circadian rhythm of CAM CO(2) fixation were lost, or dampened toward arrhythmia, under constant light and temperature conditions. Surprisingly, oscillations in the transcript abundance of core circadian clock genes also became arrhythmic in the rNAD-ME1 line, suggesting that perturbing CAM in K. fedtschenkoi feeds back to perturb the central circadian clock.

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

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

Climate-resilient agroforestry: physiological responses to climate change and engineering of crassulacean acid metabolism (CAM) as a mitigation strategy.

Global climate change threatens the sustainability of agriculture and agroforestry worldwide through increased heat, drought, surface evaporation and associated soil drying. Exposure of crops and forests to warmer and drier environments will increase leaf:air water vapour-pressure deficits (VPD), and will result in increased drought susceptibility and reduced productivity, not only in arid regions but also in tropical regions with seasonal dry periods. Fast-growing, short-rotation forestry (SRF) bioenergy crops such as poplar (Populus spp.) and willow (Salix spp.) are particularly susceptible to hydraulic failure following drought stress due to their isohydric nature and relatively high stomatal conductance. One approach to sustaining plant productivity is to improve water-use efficiency (WUE) by engineering crassulacean acid metabolism (CAM) into C3 crops. CAM improves WUE by shifting stomatal opening and primary CO2 uptake and fixation to the night-time when leaf:air VPD is low. CAM members of the tree genus Clusia exemplify the compatibility of CAM performance within tree species and highlight CAM as a mechanism to conserve water and maintain carbon uptake during drought conditions. The introduction of bioengineered CAM into SRF bioenergy trees is a potentially viable path to sustaining agroforestry production systems in the face of a globally changing climate.

Down-Regulation of Phosphoenolpyruvate Carboxylase Kinase in Grapevine Cell Cultures and Leaves Is Linked to Enhanced Resveratrol Biosynthesis.

In grapevine, trans-Resveratrol (tR) is produced as a defence mechanism against stress or infection. tR is also considered to be important for human health, which increases its interest to the scientific community. Transcriptomic analysis in grapevine cell cultures treated with the defence response elicitor methyl-ß-cyclodextrin (CD) revealed that both copies of PHOSPHOENOLPYRUVATE CARBOXYLASE KINASE (PPCK) were down-regulated significantly. A role for PPCK in the defence response pathway has not been proposed previously. We therefore analysed the control of PPCK transcript levels in grapevine cell cultures and leaves elicited with CD. Moreover, phosphoenolpyruvate carboxylase (PPC), stilbene synthase (STS), and the transcription factors MYB14 and WRKY24, which are involved in the activation of STS transcription, were also analysed by RT-qPCR. The results revealed that under CD elicitation conditions PPCK down-regulation, increased stilbene production and loss of PPC activity occurs in both tissues. Moreover, STS transcripts were co-induced with MYB14 and WRKY24 in cell cultures and leaves. These genes have not previously been reported to respond to CD in grape leaves. Our findings thus support the hypothesis that PPCK is involved in diverting metabolism towards stilbene biosynthesis, both for in vitro cell culture and whole leaves. We thus provide new evidence for PEP being redirected between primary and secondary metabolism to support tR production and the stress response.

Stomatal Responses to Light, CO2, and Mesophyll Tissue in Vicia faba and Kalanchoë fedtschenkoi.

The responses of stomatal aperture to light intensity and CO2 concentration were studied in both Vicia faba (C3) and Kalanchoë fedtschenkoi (Crassulacean acid metabolism; CAM), in material sampled from both light and dark periods. Direct comparison was made between intact leaf segments, epidermises grafted onto exposed mesophyll, and isolated epidermal peels, including transplantations between species and between diel periods. We reported the stomatal opening in response to darkness in isolated CAM peels from the light period, but not from the dark. Furthermore, we showed that C3 mesophyll has stimulated CAM stomata in transplanted peels to behave as C3 in response to light and CO2. By using peels and mesophyll from plants sampled in the dark and the light period, we provided clear evidence that CAM stomata behaved differently from C3. This might be linked to stored metabolites/ions and signalling pathway components within the guard cells, and/or a mesophyll-derived signal. Overall, our results provided evidence for both the involvement of guard cell metabolism and mesophyll signals in stomatal responses in both C3 and CAM species.

Developing Portulaca oleracea as a model system for functional genomics analysis of C4/CAM photosynthesis.

Previously regarded as an intriguing photosynthetic curiosity, the occurrence of C4 and Crassulacean acid metabolism (CAM) photosynthesis within a single organism has recently emerged as a source of information for future biotechnological use. Among C4/CAM facultative species, Portulaca oleracea L. has been used as a model for biochemical and gene expression analysis of C4/CAM under field and laboratory conditions. In the present work, we focussed on developing molecular tools to facilitate functional genomics studies in this species, from the optimisation of RNA isolation protocols to a method for stable genetic transformation. Eleven variations of RNA extraction procedures were tested and compared for RNA quantity and quality. Also, 7 sample sets comprising total RNA from hormonal and abiotic stress treatments, distinct plant organs, leaf developmental stages, and subspecies were used to select, among 12 reference genes, the most stable reference genes for RT-qPCR analysis of each experimental condition. Furthermore, different explant sources, Agrobacterium tumefaciens strains, and regeneration and antibiotic selection media were tested in various combinations to optimise a protocol for stable genetic transformation of P. oleracea. Altogether, we provide essential tools for functional gene analysis in the context of C4/CAM photosynthesis, including an efficient RNA isolation method, preferred reference genes for RT-qPCR normalisation for a range of experimental conditions, and a protocol to produce P. oleracea stable transformants using A. tumefaciens.

Delayed fluorescence as a universal tool for the measurement of circadian rhythms in higher plants.

The plant circadian clock plays an important role in enhancing performance and increasing vegetative yield. Much of our current understanding of the mechanism and function of the plant clock has come from the development of Arabidopsis thaliana as a model circadian organism. Key to this rapid progress has been the development of robust circadian markers, specifically circadian-regulated luciferase reporter genes. Studies of the clock in crop species and non-model organisms are currently hindered by the absence of a simple high-throughput universal assay for clock function, accuracy and robustness. Delayed fluorescence (DF) is a fundamental process occurring in all photosynthetic organisms. It is luminescence-produced post-illumination due to charge recombination in photosystem II (PSII) leading to excitation of P680 and the subsequent emission of a photon. Here we report that the amount of DF oscillates with an approximately 24-h period and is under the control of the circadian clock in a diverse selection of plants. Thus, DF provides a simple clock output that may allow the clock to be assayed in vivo in any photosynthetic organism. Furthermore, our data provide direct evidence that the nucleus-encoded, three-loop circadian oscillator underlies rhythms of PSII activity in the chloroplast. This simple, high-throughput and non-transgenic assay could be integrated into crop breeding programmes, the assay allows the selection of plants that have robust and accurate clocks, and possibly enhanced performance and vegetative yield. This assay could also be used to characterize rapidly the role and function of any novel Arabidopsis circadian mutant.

Silencing the cytokine storm: the use of intravenous anakinra in haemophagocytic lymphohistiocytosis or macrophage activation syndrome.

The term cytokine storm syndromes describes conditions characterised by a life-threatening, fulminant hypercytokinaemia with high mortality. Cytokine storm syndromes can be genetic or a secondary complication of autoimmune or autoinflammatory disorders, infections, and haematological malignancies. These syndromes represent a key area of interface between rheumatology and general medicine. Rheumatologists often lead in management, in view of their experience using intensive immunosuppressive regimens and managing cytokine storm syndromes in the context of rheumatic disorders or infection (known as secondary haemophagocytic lymphohistiocytosis or macrophage activation syndrome [sHLH/MAS]). Interleukin (IL)-1 is pivotal in hyperinflammation. Anakinra, a recombinant humanised IL-1 receptor antagonist, is licenced at a dose of 100 mg once daily by subcutaneous injection for rheumatoid arthritis, systemic juvenile idiopathic arthritis, adult-onset Still's disease, and cryopyrin-associated periodic syndromes. In cytokine storm syndromes, the subcutaneous route is often problematic, as absorption can be unreliable in patients with critical illness, and multiple injections are needed to achieve the high doses required. As a result, intravenous anakinra is used in clinical practice for sHLH/MAS, despite this being an off-licence indication and route of administration. Among 46 patients admitted to our three international, tertiary centres for sHLH/MAS and treated with anakinra over 12 months, the intravenous route of delivery was used in 18 (39%) patients. In this Viewpoint, we describe current challenges in the management of cytokine storm syndromes and review the pharmacokinetic and safety profile of intravenous anakinra. There is accumulating evidence to support the rationale for, and safety of, intravenous anakinra as a first-line treatment in patients with sHLH/MAS. Intravenous anakinra has important clinical relevance when high doses of drug are required or if patients have subcutaneous oedema, severe thrombocytopenia, or neurological involvement. Cross-speciality management and collaboration, with the generation of international, multi-centre registries and biobanks, are needed to better understand the aetiopathogenesis and improve the poor prognosis of cytokine storm syndromes.

Exploring C4-CAM plasticity within the Portulaca oleracea complex.

Portulaca oleracea is a C4 herb capable of performing CAM under drought stress. It is distributed worldwide and is either considered a polymorphic species or a complex of subspecies, due to its numerous morphological variations. We evaluated CAM plasticity within P. oleracea genotypes since the complexity surrounding this species may be reflected in intraspecific variations in photosynthetic behavior. Eleven subspecies of P. oleracea from distant geographical locations and one cultivar were morphologically and physiologically characterized. C4 and CAM photosynthesis were monitored in plants exposed to well-watered, droughted and rewatered treatments, and data obtained were compared among individual genotypes. All subspecies expressed CAM in a fully-reversible manner. Transcript abundance of C4-CAM signature genes was shown to be a useful indicator of the C4-CAM-C4 switches in all genotypes. C4-related genes were down-regulated and subsequently fully expressed upon drought and rewatering, respectively. CAM-marker genes followed the opposite pattern. A gradient of morphological traits and drought-induced nighttime malate accumulation was observed across genotypes. Therefore, different combinations of CAM expression levels, plant sizes and shapes are available within the P. oleracea complex, which can be a valuable tool in the context of C4/CAM photosynthesis research.

Ecological implications of plants ability to tell the time.

The circadian clock (the endogenous mechanism that anticipates diurnal cycles) acts as a central coordinator of plant activity. At the molecular and organism level, it regulates key traits for plant fitness, including seed germination, gas exchange, growth and flowering, among others. In this article, we explore current evidence on the effect of the clock for the scales of interest to ecologists. We begin by synthesizing available knowledge on the effect of the clock on biosphere-atmosphere interactions and observe that, at least in the systems where it has been tested, the clock regulates gas exchange from the leaf to the ecosystem level, and we discuss its implications for estimates of the carbon balance. Then, we analyse whether incorporating the action of the clock may help in elucidating the effects of climate change on plant distributions. Circadian rhythms are involved in regulating the range of temperatures a species can survive and affects plant interactions. Finally, we review the involvement of the clock in key phenological events, such as flowering time and seed germination. Because the clock may act as a common mechanism affecting many of the diverse branches of ecology, our ultimate goal is to stimulate further research into this pressing, yet unexplored, topic.

Exploiting the potential of plants with crassulacean acid metabolism for bioenergy production on marginal lands.

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.