Crassulacean Research Articles

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.

Kinetic properties of recombinant phosphomimic mutant of Zea mays phosphoenolpyruvate carboxylase (ZmPEPCS15D)

Phosphoenolpyruvate carboxylase (PEPC) is the key enzyme for fixing atmospheric carbon dioxide in C4 and CAM plants, and in anaplerotic metabolism in most plants. It is regulated by reversible phosphorylation, and allosteric activation or inhibition. The regulatory role of phosphorylation at N-terminal serine residue (S15) is documented earlier. In this study PEPC was cloned from maize and engineered to generate phosphomimic mutant enzyme by conversion of the codon for Serine 15 to aspartate (PEPCS15D). In vitro study with recombinant wild-type PEPC and phosphomimic PEPCS15D showed that both the enzymes are active catalytically. Kinetics analysis showed that PEPCS15D enzyme has comparatively lower Km value over WT PEPC, thus implying higher affinity towards its substrate PEP than the wild type PEPC. However, both WT PEPC and mutant PEPCS15D showed similar regulation with the allosteric activator (Glycine) and allosteric inhibitors (l-Aspartate or l-Malate). Thus, the engineered phosphomimic mutant PEPCS15D with high affinity for PEP will be useful to engineer rice and other C3 plants for enhancing photosynthesis and minimizing respiratory CO2 loss.

Diurnal modulation of PEPCK decarboxylation activity impacts photosystem II light-energy use in a drought-induced CAM species

Crassulacean acid metabolism (CAM) is a photosynthetic pathway characterized by nocturnal CO2 fixation into organic acids followed by their diurnal decarboxylation. Decarboxylation activity is time regulated, usually presenting higher activity in the middle of the day. This process can be performed by phosphoenolpyruvate carboxykinase (PEPCK) and/or by malic enzyme, resulting in an increase in internal CO2 concentration which may act to suppress photorespiration. It is suggested that the CO2 provided by the decarboxylation can improve the photochemical use of energy in CAM plants. This photosynthetic pathway also contributes to improve water use efficiency, and, in an interesting way, some species can engage this photosynthesis when subjected to water deficit. Guzmania monostachia is a bromeliad that occupies habitats with intermittent water availability and CAM can be induced by water deficit. To test the light-time modulation of decarboxylation activity and light-energy use in G. monostachia, we analyzed the activity of PEPCK, the main decarboxylating enzyme for this bromeliad, as well as the photochemical and non-photochemical quenching and organic acids contents at three times of the day. Results showed a diurnal modulation where the highest PEPCK decarboxylation activity coincided with an increase in the photochemical quenching and a decrease in the non-photochemical quenching. In addition, a reduction in acid content was observed, strongly indicating the interface between CO2 generation through acid decarboxylation and its effect on PSII photochemical activity and photorespiration in CAM-induced plants. The same was not observed for well-watered plants. Also, PEPCK activity modulation may be related to transcriptional and post-translational regulation, since an increase in transcript abundance and a modulation in the dephosphorylated enzyme activity was noted. Moreover, we suggest that the time modulation of PEPCK plays an important role in regulating PSII energy use in CAM plants.

Recent developments in mesophyll conductance in C3, C4, and crassulacean acid metabolism plants.

The conductance of carbon dioxide (CO2 ) from the substomatal cavities to the initial sites of CO2 fixation (gm ) can significantly reduce the availability of CO2 for photosynthesis. There have been many recent reviews on: (i) the importance of gm for accurately modelling net rates of CO2 assimilation, (ii) on how leaf biochemical and anatomical factors influence gm , (iii) the technical limitation of estimating gm , which cannot be directly measured, and (iv) how gm responds to long- and short-term changes in growth and measurement environmental conditions. Therefore, this review will highlight these previous publications but will attempt not to repeat what has already been published. We will instead initially focus on the recent developments on the two-resistance model of gm that describe the potential of photorespiratory and respiratory CO2 released within the mitochondria to diffuse directly into both the chloroplast and the cytosol. Subsequently, we summarize recent developments in the three-dimensional (3-D) reaction-diffusion models and 3-D image analysis that are providing new insights into how the complex structure and organization of the leaf influences gm . Finally, because most of the reviews and literature on gm have traditionally focused on C3 plants we review in the final sections some of the recent developments, current understanding and measurement techniques of gm in C4 and crassulacean acid metabolism (CAM) plants. These plants have both specialized leaf anatomy and either a spatially or temporally separated CO2 concentrating mechanisms (C4 and CAM, respectively) that influence how we interpret and estimate gm compared with a C3 plants.

Model approaches to advance crassulacean acid metabolism system integration.

This review summarises recent progress in understanding crassulacean acid metabolism (CAM) systems and the integration of internal and external stimuli to maximise water-use efficiency. Complex CAM traits have been reduced to their minimum and captured as computational models, which can now be refined using recently available data from transgenic manipulations and large-scale omics studies. We identify three key areas in which an appropriate choice of modelling tool could help capture relevant comparative molecular data to address the evolutionary drivers and plasticity of CAM. One focus is to identify the environmental and internal signals that drive inverse stomatal opening at night. Secondly, it is important to identify the regulatory processes required to orchestrate the diel pattern of carbon fluxes within mesophyll layers. Finally, the limitations imposed by contrasting succulent systems and associated hydraulic conductance components should be compared in the context of water-use and evolutionary strategies. While network analysis of transcriptomic data can provide insights via co-expression modules and hubs, alternative forms of computational modelling should be used iteratively to define the physiological significance of key components and informing targeted functional gene manipulation studies. We conclude that the resultant improvements of bottom-up, mechanistic modelling systems can enhance progress towards capturing the physiological controls for phylogenetically diverse CAM systems in the face of the recent surge of information in this omics era.

The potential of δ2Hn-alkanes and δ18Osugar for paleoclimate reconstruction - A regional calibration study for South Africa.

The hydrogen isotopic composition of leaf wax-derived n-alkanes (δ2Hn-alkanes) is a widely applied proxy for (paleo)climatic changes. It has been suggested that the coupling with the oxygen isotopic composition of hemicellulose-derived sugars (δ18Osugar) - an approach dubbed 'paleohygrometer' - might allow more robust and quantitative (paleo)hydrological reconstructions. However, the paleohygrometer remains to be evaluated and tested regionally. In this study, topsoil samples from South Africa, covering extensive environmental gradients, are analysed. δ2Hn-alkanes correlates significantly with the isotopic composition of precipitation (δ2Hp), whereas no significant correlation exists between δ18Osugar and δ18Op. The apparent fractionation (εapp) is the difference between δ2Hn-alkanes and δ2Hp (εapp 2H) and δ18Osugar and δ18Op (εapp 18O), respectively, and integrates i) isotopic enrichment due to soil water evaporation, ii) leaf (and xylem) water transpiration and iii) biosynthetic fractionation. We find no correlation of εapp 18O nor for εapp 2H with temperature, and no correlation of εapp 2H with potential evapotranspiration and an aridity index. By contrast, εapp 18O correlates significantly with both potential evapotranspiration and the aridity index. This highlights the strong effect of evapotranspirative enrichment on δ18Osugar. In study areas without plant predominance using Crassulacean Acid Metabolism (CAM), coupling δ18Osugar and δ2Hn-alkanes enables to reconstruct δ2Hp and δ18Op with an offset of Δδ2H = 6 ± 27‰ and Δδ18O = 0.8 ± 3.7‰, respectively, as well as relative humidity (RH) with an offset of ΔRH = 6 ± 17%. The paleohygrometer does, however, not work well for our study areas where CAM plants prevail (reconstructed δ18Op, δ2Hp and RH are off by 3.1‰, 27.2‰ and 31.7%). This probably reflects plant-specific (phenological) adaptations and/or post-photosynthetic exchange reactions related to CAM metabolism. Overall, our findings corroborate that δ2Hn-alkanes and δ18Osugar are valuable proxies, and the paleohygrometer is a promising approach for paleoclimate reconstructions in southern Africa.

Are thick leaves, large mesophyll cells and small intercellular air spaces requisites for CAM?

It is commonly accepted that the leaf of a crassulacean acid metabolism (CAM) plant is thick, with large mesophyll cells and vacuoles that can accommodate the malic acid produced during the night. The link between mesophyll characteristics and CAM mode, whether obligate or C3/CAM, was evaluated. Published values of the carbon isotopic ratio (δ 13C) as an indicator of CAM, leaf thickness, leaf micrographs and other evidence of CAM operation were used to correlate cell density, cell area, the proportion of intercellular space in the mesophyll (IAS) and the length of cell wall facing the intercellular air spaces (Lmes/A) with CAM mode. Based on 81 species and relatively unrelated families (15) belonging to nine orders, neither leaf thickness nor mesophyll traits helped explain the degree of CAM expression. A strong correlation was found between leaf thickness and δ 13C in some species of Crassulaceae and between leaf thickness and nocturnal acid accumulation in a few obligate CAM species of Bromeliaceae but, when all 81 species were pooled together, no significant changes with δ 13C were observed in cell density, cell area, IAS or Lmes/A. An influence of phylogeny on leaf anatomy was evidenced in a few cases but this precluded generalization for widely separate taxa containing CAM species. The possible relationships between leaf anatomy and CAM mode should be interpreted cautiously.

Exploring molecular evolution of Rubisco in C3 and CAM Orchidaceae and Bromeliaceae

BackgroundThe CO2-concentrating mechanism associated to Crassulacean acid metabolism (CAM) alters the catalytic context for Rubisco by increasing CO2 availability and provides an advantage in particular ecological conditions. We hypothesized about the existence of molecular changes linked to these particular adaptations in CAM Rubisco. We investigated molecular evolution of the Rubisco large (L-) subunit in 78 orchids and 144 bromeliads with C3 and CAM photosynthetic pathways. The sequence analyses were complemented with measurements of Rubisco kinetics in some species with contrasting photosynthetic mechanism and differing in the L-subunit sequence.ResultsWe identified potential positively selected sites and residues with signatures of co-adaptation. The implementation of a decision tree model related Rubisco specific variable sites to the leaf carbon isotopic composition of the species. Differences in the Rubisco catalytic traits found among C3 orchids and between strong CAM and C3 bromeliads suggested Rubisco had evolved in response to differing CO2 concentration.ConclusionsThe results revealed that the variability in the Rubisco L-subunit sequence in orchids and bromeliads is composed of coevolving sites under potential positive adaptive signal. The sequence variability was related to δ13C in orchids and bromeliads, however it could not be linked to the variability found in the kinetic properties of the studied species.

Structural basis for C4 photosynthesis without Kranz anatomy in leaves of the submerged freshwater plant Ottelia alismoides.

Ottelia alismoides (Hydrocharitaceae) is a freshwater macrophyte that, unusually, possesses three different CO2-concentrating mechanisms. Here we describe its leaf anatomy and chloroplast ultrastructure, how these are altered by CO2 concentration and how they may underlie C4 photosynthesis. Light and transmission electron microscopy were used to study the anatomy of mature leaves of O. alismoides grown at high and low CO2 concentrations. Diel acid change and the activity of phosphoenolpyruvate carboxylase were measured to confirm that CAM activity and C4 photosynthesis were present. When O. alismoides was grown at low CO2, the leaves performed both C4 and CAM photosynthesis whereas at high CO2 leaves used C4 photosynthesis. The leaf comprised an upper and lower layer of epidermal cells separated by a large air space occupying about 22 % of the leaf transverse-section area, and by mesophyll cells connecting the two epidermal layers. Kranz anatomy was absent. At low CO2, chloroplasts in the mesophyll cells were filled with starch even at the start of the photoperiod, while epidermal chloroplasts contained small starch grains. The number of chloroplasts in the epidermis was greater than in the mesophyll cells. At high CO2, the structure was unchanged but the thicknesses of the two epidermal layers, the air space, mesophyll and the transverse-section area of cells and air space were greater. Leaves of O. alismoides have epidermal and mesophyll cells that contain chloroplasts and large air spaces but lack Kranz anatomy. The high starch content of mesophyll cells suggests they may benefit from an internal source of CO2, for example via C4 metabolism, and are also sites of starch storage. The air spaces may help in the recycling of decarboxylated or respired CO2. The structural similarity of leaves at low and high CO2 is consistent with the constitutive nature of bicarbonate and C4 photosynthesis. There is sufficient structural diversity within the leaf of O. alismoides to support dual-cell C4 photosynthesis even though Kranz anatomy is absent.

The Development of Crassulacean Acid Metabolism (CAM) Photosynthesis in Cotyledons of the C4 Species, Portulaca grandiflora (Portulacaceae).

Portulaca grandiflora simultaneously utilizes both the C4 and Crassulacean acid metabolism (CAM) photosynthetic pathways. Our goal was to determine whether CAM developed and was functional simultaneously with the C4 pathway in cotyledons of P. grandiflora. We studied during development whether CAM would be induced with water stress by monitoring the enzyme activity, leaf structure, JO2 (rate of O2 evolution calculated by fluorescence analysis), and the changes in titratable acidity of 10 and 25 days old cotyledons. In the 10 days old cotyledons, C4 and CAM anatomy were evident within the leaf tissue. The cotyledons showed high titratable acid levels but a small CAM induction. In the 25 days old cotyledons, there was a significant acid fluctuation under 7 days of water stress. The overall enzyme activity was reduced in the 10 days old plants, while in the 25 days old plants CAM activity increased under water-stressed conditions. In addition to CAM, the research showed the presence of glycine decarboxylase in the CAM tissue. Thus, it appears both pathways develop simultaneously in the cotyledons but the CAM pathway, due to anatomical constraints, may be slower to develop than the C4 pathway. Cotyledons showed the ancestral Atriplicoid leaf anatomy, which leads to the question: Could a CAM cell be the precursor to the C4 pathway? Further study of this may lead to understanding into the evolution of C4 photosynthesis in the Portulaca.

Analysis of MADS-box genes revealed modified flowering gene network and diurnal expression in pineapple

BackgroundPineapple is the most important crop with CAM photosynthesis, but its molecular biology is underexplored. MADS-box genes are crucial transcription factors involving in plant development and several biological processes. However, there is no systematic analysis of MADS-box family genes in pineapple (Ananas comosus).ResultsForty-eight MADS-box genes were identified in the pineapple genome. Based on the phylogenetic studies, pineapple MADS-box genes can be divided into type I and type II MADS-box genes. Thirty-four pineapple genes were classified as type II MADS-box genes including 32 MIKC-type and 2 Mδ-type, while 14 type I MADS-box genes were further divided into Mα, Mβ and Mγ subgroups. A majority of pineapple MADS-box genes were randomly distributed across 19 chromosomes. RNA-seq expression patterns of MADS-box genes in four different tissues revealed that more genes were highly expressed in flowers, which was confirmed by our quantitative RT-PCR results. There is no FLC and CO orthologs in pineapple. The loss of FLC and CO orthologs in pineapple indicated that modified flowering genes network in this tropical plant compared with Arabidopsis. The expression patterns of MADS-box genes in photosynthetic and non-photosynthetic leaf tissues indicated the potential roles of some MADS-box genes in pineapple CAM photosynthesis. The 23% of pineapple MADS-box genes showed diurnal rhythm, indicating that these MADS-box genes are regulated by circadian clock.ConclusionsMADS-box genes identified in pineapple are closely related to flowering development. Some MADS-box genes are involved in CAM photosynthesis and regulated by the circadian clock. These findings will facilitate research on the development of unusual spiral inflorescences on pineapple fruit and CAM photosynthesis.

Biosystems Design to Accelerate C3-to-CAM Progression.

Global demand for food and bioenergy production has increased rapidly, while the area of arable land has been declining for decades due to damage caused by erosion, pollution, sea level rise, urban development, soil salinization, and water scarcity driven by global climate change. In order to overcome this conflict, there is an urgent need to adapt conventional agriculture to water-limited and hotter conditions with plant crop systems that display higher water-use efficiency (WUE). Crassulacean acid metabolism (CAM) species have substantially higher WUE than species performing C3 or C4 photosynthesis. CAM plants are derived from C3 photosynthesis ancestors. However, it is extremely unlikely that the C3 or C4 crop plants would evolve rapidly into CAM photosynthesis without human intervention. Currently, there is growing interest in improving WUE through transferring CAM into C3 crops. However, engineering a major metabolic plant pathway, like CAM, is challenging and requires a comprehensive deep understanding of the enzymatic reactions and regulatory networks in both C3 and CAM photosynthesis, as well as overcoming physiometabolic limitations such as diurnal stomatal regulation. Recent advances in CAM evolutionary genomics research, genome editing, and synthetic biology have increased the likelihood of successful acceleration of C3-to-CAM progression. Here, we first summarize the systems biology-level understanding of the molecular processes in the CAM pathway. Then, we review the principles of CAM engineering in an evolutionary context. Lastly, we discuss the technical approaches to accelerate the C3-to-CAM transition in plants using synthetic biology toolboxes.

Crassulacean acid metabolism.

Gilman and Edwards introduce crassulacean acid metabolism and highlight how recent advances in molecular biology are deepening our knowledge of CAM evolution.

Adaptive features of In vitro-derived plantlets of MD2 pineapple during acclimatization process

Tissue culture-derived planting materials require optimal acclimatization period to ensure high survival rates in the nursery and to save labor and production cost. However, there is little information on how the morphology and physiology of in vitro-derived plantlets can be affected by culture conditions and acclimatization. We compared different types of media for shoot multiplication using thirty pineapple suckers as explants. Morphological and physiological changes of thirty field-grown plants and in vitro-derived plantlets during ex vitro acclimatization at 0, 14, 28, 42, 56 and 84 days were evaluated. Experiments were conducted in triplicate. Observations were based on the leaf morphology (number, width, ratio value of dry weight to fresh weight and succulence index of the leaves) and physiological characteristics (photosynthetic rate, stomatal conductance, transpiration rate, stomata opening, trichomes and histological examination). We found that the in vitro plantlets undergoing acclimatization showed C3 characteristics and adapted to CAM characteristics after 42 days of acclimatization. The in vitro plantlets were compared with field-grown plants and observations were made based on the morphological and physiological characteristics of the leaves. In conclusion, our findings indicated that in vitro-derived pineapple plantlets started to develop and achieve similar characteristics with the field-grown CAM plants after 42 days of acclimatization, suggesting that this could be the suitable period prior to transfer into the fields.

What does the RuBisCO activity tell us about a C3-CAM plant?

Plants that perform the Crassulacean acid metabolism (CAM), which obtain CO2 overnight and convert it mainly in malic acid, successfully grow in environments with water and nutrient shortages, that is partly associated with their higher water- and nitrogen-use efficiencies. Water and nutrient limitations can impair photosynthesis through the reduction of RuBisCO and increment of photorespiration, disturbing the plant carbon balance. In this context, we conducted a controlled experiment with the epiphytic C3-CAM bromeliad Guzmania monostachia to investigate how the combined water and nutritional deficits affect the activity of RuBisCO and its activation state (RAS), and to evaluate the efficiency of photosynthesis during the transition from C3 to CAM. Apart from an increase in CAM activity, bromeliads submitted to both water and nutritional deficits showed higher RAS values and unaltered RuBisCO activity compared to C3 bromeliads and, surprisingly, the maximum quantum efficiency of photosynthesis increased. Glucose, fructose and starch levels were maintained, while sucrose concentrations increased over time. These results, combined with the high RAS values, suggest an increased efficiency of RuBisCO functioning. Our results reinforce the ability of epiphytic bromeliads to deal with stressful habitats by a higher efficiency of RuBisCO during the transition to CAM, another feature that may allow their evolution in the epiphytic environment.

The 18 O-signal transfer from water vapour to leaf water and assimilates varies among plant species and growth forms.

The 18 O signature of atmospheric water vapour (δ18 OV ) is known to be transferred via leaf water to assimilates. It remains, however, unclear how the 18 O-signal transfer differs among plant species and growth forms. We performed a 9-hr greenhouse fog experiment (relative humidity ≥ 98%) with 18 O-depleted water vapour (-106.7‰) on 140 plant species of eight different growth forms during daytime. We quantified the 18 O-signal transfer by calculating the mean residence time of O in leaf water (MRTLW ) and sugars (MRTSugars ) and related it to leaf traits and physiological drivers. MRTLW increased with leaf succulence and thickness, varying between 1.4 and 10.8 hr. MRTSugars was shorter in C3 and C4 plants than in crassulacean acid metabolism (CAM) plants and highly variable among species and growth forms; MRTSugars was shortest for grasses and aquatic plants, intermediate for broadleaf trees, shrubs, and herbs, and longest for conifers, epiphytes, and succulents. Sucrose was more sensitive to δ18 OV variations than other assimilates. Our comprehensive study shows that plant species and growth forms vary strongly in their sensitivity to δ18 OV variations, which is important for the interpretation of δ18 O values in plant organic material and compounds and thus for the reconstruction of climatic conditions and plant functional responses.

Sansevieria trifasciata and Chlorophytum comosum botanical biofilter for cigarette smoke phytoremediation in a pilot-scale experiment—evaluation of multi-pollutant removal efficiency and CO2 emission

Botanical biofilters have been proposed as an effective technology for indoor air remediation. Plants, including Sansevieria trifasciata and Chlorophytum comosum, which remove VOCs effectively, can also reduce CO2 emission since S. trifasciata and C. comosum are CAM and C3 plant species, respectively. Therefore, a botanical biofilter using these plants together shows potential for use in contaminated sites. Herein, the potential of this mixed plant botanical biofilter was evaluated as a method of phytoremediation for multi-pollutants from cigarette smoke. The results showed that the combination of S. trifasciata and C. comosum in a botanical biofilter was highly effective in removing VOCs and PM2.5. In addition, this botanical biofilter can also successfully remove formaldehyde, acetone, benzene, and xylene, with low CO2 emission under indoor conditions of moderate light intensity (50 μmole PAR m−2 s−1). The system was also installed in a large volume room (24 m3) to test phytoremediation of multi-pollutants from cigarette smoke. The results showed that this mixed plant botanical biofilter can remediate indoor air pollution effectively under both light and dark conditions continuously for three cycles. The mixed plant botanical biofilter developed showed potential for use in real contamination sites.

New perspectives on crassulacean acid metabolism biology.

New perspectives on crassulacean acid metabolism biology.

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.

Maltose Processing and Not β-Amylase Activity Curtails Hydrolytic Starch Degradation in the CAM Orchid Phalaenopsis.

Crassulacean acid metabolism (CAM) is one of the three photosynthetic pathways in higher plants and is characterized by high water use efficiency. This mainly relies on major nocturnal CO2 fixation sustained by degradation of storage carbohydrate such as starch to provide phosphoenolpyruvate (PEP) and energy. In contrast to C3 plants where starch is mainly degraded by the hydrolytic route, different observations suggested the phosphorolytic route to be a major pathway for starch degradation in CAM plants. To elucidate the interplay and relevant contributions of the phosphorolytic and hydrolytic pathways for starch degradation in CAM, we assessed diel patterns for metabolites and enzymes implicated in both the hydrolytic route (β-amylase, DPE1, DPE2, maltase) and the phosphorolytic route (starch phosphorylase) of starch degradation in the CAM orchid Phalaenopsis “Edessa.” By comparing the catalytic enzyme activities and starch degradation rates, we showed that the phosphorolytic pathway is the major route to accommodate nocturnal starch degradation and that measured activities of starch phosphorylase perfectly matched calculated starch degradation rates in order to avoid premature exhaustion of starch reserves before dawn. The hydrolytic pathway seemed hampered in starch processing not by β-amylase but through insufficient catalytic capacity of both DPE2 and maltase. These considerations were further corroborated by measurements of enzyme activities in the CAM model plant Kalanchoë fedtschenkoi and strongly contradict with the situation in the C3 plant Arabidopsis. The data support the view that the phosphorolytic pathway might be the main route of starch degradation in CAM to provide substrate for PEP with additional hydrolytic starch breakdown to accommodate mainly sucrose synthesis.