Regulation Of Crassulacean Acid Metabolism Research Articles

High-quality Cymbidium mannii genome and multifaceted regulation of crassulacean acid metabolism in epiphytes

Epiphytes with crassulacean acid metabolism (CAM) photosynthesis are widespread among vascular plants, and repeated evolution of CAM photosynthesis is a key innovation for micro-ecosystem adaptation. However, we lack a complete understanding of the molecular regulation of CAM photosynthesis in epiphytes. Here, we report a high-quality chromosome-level genome assembly of a CAM epiphyte, Cymbidium mannii (Orchidaceae). The 2.88-Gb orchid genome with a contig N50 of 22.7 Mb and 27 192 annotated genes was organized into 20 pseudochromosomes, 82.8% of which consisted of repetitive elements. Recent expansions of long terminal repeat retrotransposon families have made a major contribution to the evolution of genome size in Cymbidium orchids. We reveal a holistic scenario of molecular regulation of metabolic physiology using high-resolution transcriptomics, proteomics, and metabolomics data collected across a CAM diel cycle. Patterns of rhythmically oscillating metabolites, especially CAM-related products, reveal circadian rhythmicity in metabolite accumulation in epiphytes. Genome-wide analysis of transcript and protein level regulation revealed phase shifts during the multifaceted regulation of circadian metabolism. Notably, we observed diurnal expression of several core CAM genes (especially βCA and PPC) that may be involved in temporal fixation of carbon sources. Our study provides a valuable resource for investigating post-transcription and translation scenarios in C. mannii, an Orchidaceae model for understanding the evolution of innovative traits in epiphytes.

Transcriptome and Degradome Profiling Reveals a Role of miR530 in the Circadian Regulation of Gene Expression in Kalanchoë marnieriana.

Crassulacean acid metabolism (CAM) is an important photosynthetic pathway for plant adaptation to dry environments. CAM plants feature a coordinated interaction between mesophyll and epidermis functions that involves refined regulations of gene expression. Plant microRNAs (miRNAs) are crucial post-transcription regulators of gene expression, however, their roles underlying the CAM pathway remain poorly investigated. Here, we present a study characterizing the expression of miRNAs in an obligate CAM species Kalanchoë marnieriana. Through sequencing of transcriptome and degradome in mesophyll and epidermal tissues under the drought treatments, we identified differentially expressed miRNAs that were potentially involved in the regulation of CAM. In total, we obtained 84 miRNA genes, and eight of them were determined to be Kalanchoë-specific miRNAs. It is widely accepted that CAM pathway is regulated by circadian clock. We showed that miR530 was substantially downregulated in epidermal peels under drought conditions; miR530 targeted two tandem zinc knuckle/PLU3 domain encoding genes (TZPs) that were potentially involved in light signaling and circadian clock pathways. Our work suggests that the miR530-TZPs module might play a role of regulating CAM-related gene expression in Kalanchoë.

RNA-seq transcriptomic profiling of crassulacean acid metabolism pathway in Dendrobium catenatum

The regulation of crassulacean acid metabolism (CAM) pathway has recently become a topic of intensive research and has been explored in terms of several aspects, including phylogenetics, genomics, and transcriptomics. Orchidaceae, which contains approximately 9,000 CAM species, is one of the largest lineages using this special photosynthetic pathway. However, no comprehensive transcriptomic profiling focused on CAM regulation in orchid species had previously been performed. In this report, we present two Illumina RNA-seq datasets, including a total of 24 mature leaf samples with 844.4 million reads, from Dendrobium catenatum (Orchidaceae), a facultative CAM species. The first dataset was generated from a time-course experiment based on the typical CAM phases in a diel. The second was derived from an experiment on drought stress and stress removal. A series of quality assessments were conducted to verify the reliability of the datasets. These transcriptomic profiling datasets will be useful to explore and understand the essence of CAM regulation.

Shifts in gene expression profiles are associated with weak and strong Crassulacean acid metabolism.

The relative ease of high throughput sequencing is facilitating comprehensive phylogenomic and gene expression studies, even for nonmodel groups. To date, however, these two approaches have not been merged; while phylogenomic methods might use transcriptome sequences to resolve relationships, assessment of gene expression patterns in a phylogenetic context is less common. Here we analyzed both carbon assimilation and gene expression patterns of closely related species within the Agavoideae (Asparagaceae) to elucidate changes in gene expression across weak and strong phenotypes for Crassulacean acid metabolism (CAM). Gene expression patterns were compared across four genera: Agave (CAM), which is paraphyletic with Polianthes (weak CAM) and Manfreda (CAM), and Beschorneria (weak CAM). RNA-sequencing was paired with measures of gas exchange and titratable acidity. Climate niche space was compared across the four lineages to examine abiotic factors and their correlation to CAM. Expression of homologous genes showed both shared and variable patterns in weak and strong CAM species. Network analysis highlights that despite shared expression patterns, highly connected genes differ between weak and strong CAM, implicating shifts in regulatory gene function as key for the evolution of CAM. Variation in carbohydrate metabolism between weak and strong CAM supports the importance of sugar turnovers for CAM physiology. Integration of phylogenetics and RNA-sequencing provides a powerful tool to study the evolution of CAM photosynthesis across closely related but photosynthetically variable species. Our findings regarding shared or shifted gene expression and regulation of CAM via carbohydrate metabolism have important implications for efforts to engineer the CAM pathway into C3 food and biofuel crops.

Temporal and spatial transcriptomic and microRNA dynamics of CAM photosynthesis in pineapple.

The altered carbon assimilation pathway of crassulacean acid metabolism (CAM) photosynthesis results in an up to 80% higher water-use efficiency than C3 photosynthesis in plants making it a potentially useful pathway for engineering crop plants with improved drought tolerance. Here we surveyed detailed temporal (diel time course) and spatial (across a leaf gradient) gene and microRNA (miRNA) expression patterns in the obligate CAM plant pineapple [Ananas comosus (L.) Merr.]. The high-resolution transcriptome atlas allowed us to distinguish between CAM-related and non-CAM gene copies. A differential gene co-expression network across green and white leaf diel datasets identified genes with circadian oscillation, CAM-related functions, and source-sink relations. Gene co-expression clusters containing CAM pathway genes are enriched with clock-associated cis-elements, suggesting circadian regulation of CAM. About 20% of pineapple microRNAs have diel expression patterns, with several that target key CAM-related genes. Expression and physiology data provide a model for CAM-specific carbohydrate flux and long-distance hexose transport. Together these resources provide a list of candidate genes for targeted engineering of CAM into C3 photosynthesis crop species.

Effect of light and CO2 on inorganic carbon uptake in the invasive aquatic CAM-plant Crassula helmsii

Crassula helmsii (T. Kirk) Cockayne is an invasive aquatic plant in Europe that can suppress many native species because it can grow at a large range of dissolved inorganic carbon concentrations and light levels. One reason for its ecological success may be the possession of a regulated Crassulacean Acid Metabolism (CAM), which allows aquatic macrophytes to take up CO2 in the night in addition to the daytime. The effect of light and CO2 on the regulation of CAM and photosynthesis in C. helmsii was investigated to characterise how physiological acclimation may confer this ecological flexibility. After 3 weeks of growth at high light (230 µmol photon m–2 s–1), C. helmsii displayed 2.8 times higher CAM at low compared with high CO2 (22 v. 230 mmol m–3). CAM was absent in plants grown at low light (23 µmol photon m–2 s–1) at both CO2 concentrations. The observed regulation patterns are consistent with CAM acting as a carbon conserving mechanism. For C. helmsii grown at high light and low CO2, mean photosynthetic rates were relatively high at low concentrations of CO2 and were on average 80 and 102 µmol O2 g–1 DW h–1 at CO2 concentrations of 3 and 22 mmol m–3 CO2, which, together with mean final pH values of 9.01 in the pH drift, indicate a low CO2 compensation point (<3 mmol m–3) but do not indicate use of bicarbonate as an additional source of exogenous inorganic carbon. The relatively high photosynthetic rates during the entire daytime were caused by internally derived CAM-CO2 and uptake from the external medium. During decarboxylation, CO2 generated from CAM contributed up to 29% to photosynthesis, whereas over a day the contribution to the carbon balance was ≤13%. The flexible adjustment of CAM and the ability to maintain photosynthesis at very low external CO2 concentrations, partly by making use of internally generated CO2 via CAM, may contribute to the broad ecological niche of C. helmsii.

Effects of temperature on circadian rhythm of crassulacean acid metabolism

Crassulacean acid metabolism (CAM) is known to exhibit endogenous circadian rhythm under continuous light conditions. The rhythm, however, disappears at higher temperatures above approximately 30°C. We investigated effects of the temperature on the rhythm of phosphorylation of phosphoenolpyruvate carboxylase (PEP-C), the key enzyme in the regulation of CAM, with a CAM plant Graptopetalum paraguayense. We also analyzed expression of chlorophyll a/b binding protein gene (CAB) as an indicator of the state of the central oscillator, because CCA1 is a transcription factor of CAB in Arabidopsis thaliana and is thought to be a component of the oscillator or a component closely associated with it. The expression of CAB is expected to be under direct control of the clock. Under the continuous light condition, the rhythm in the phosphorylation of PEP-C, which was estimated by measuring the inhibition by 2 mM malate of the activity in leaf extract, did not persist at 32°C. The rhythm in the mRNA level of CAB, which persisted at 20°C, also disappeared at 32°C, suggesting that the circadian clock stops at high temperatures. Decreasing the temperature from 32°C to 25°C reset the rhythm of the phosphrylation of PEP-C. On the other hand, temperature shift from 27°C, at which the rhythm persisted, to 20°C had no effect on the phase of the rhythm.

Recycling of respiratory CO2 during Crassulacean acid metabolism: alleviation of photoinhibition in Pyrrosia piloselloides

The regulation of Crassulacean acid metabolism (CAM) in the fern Pyrrosia piloselloides (L.) Price was investigated in Singapore on two epiphytic populations acclimated to sun and shade conditions. The shade fronds were less succulent and had a higher chlorophyll content although the chlorophyll a:b ratio was lower and light compensation points and dark-respiration rates were reduced. Dawn-dusk variations in titratable acidity and carbohydrate pools were two to three times greater in fronds acclimated to high photosynthetically active radiation (PAR), although water deficits were also higher than in shade fronds. External and internal CO2 supply to attached fronds of the fern was varied so as to regulate the magnitude of CAM activity. A significant proportion of titratable acidity was derived from the refixation of respiratory CO2 (27% and 35% recycling for sun and shade populations, respectively), as measured directly under CO2-free conditions. Starch was shown to be the storage carbodydrate for CAM in Pyrrosia, with a stoichiometric reduction of "C3-skeleton" units in proportion to malic-acid accumulation. Measurements of photosynthetic O2 evolution under saturating CO2 were used to compare the light responses of sun and shade fronds for each CO2 supply regime, and also following the imposition of a photoinhibitory PAR treatment (1600 μmol·m(-2)·s(-1) for 3 h). Apparent quantum yield declined following the high-PAR treatment for sun- and shade-adapted plants, although for sun fronds CAM activity derived from respiratory CO2 prevented any further reduction in photosynthetic efficiency. Recycling of respiratory CO2 by shade plants could only partly prevent photoinhibitory damage. These observations provide experimental evidence that respiratory CO2 recycling, ubiquitous in CAM plants, may have developed so as to alleviate photoinhibition.

Carbon balance during CAM: an assessment of respiratory CO2 recycling in the epiphytic bromeliads Aechmea nudicaulis and Aechmea fendleri

Abstract The regulation of crassulacean acid metabolism (CAM) under controlled environmental conditions has been investigated for two tropical epiphytes, relating plant water and carbon balance to growth form and habitat preference under natural conditions. Aechmea fendleri is restricted to wet, upper montane regions of Trinidad, while A. nudicaulis has a wider distribution extending into more arid regions of the island. Morphological characteristics of these plants are related to habitat preference in terms of leaf succulence (0.44 and 0.94 kg m−2 for the two species respectively) and a distinct layer of water storage parenchyma in A. nudicaulis In contrast, the thinner leaves of A. fendleri contain little water‐storage parenchyma and less chlorenchyma per unit area, but the plants have a more open leaf rosette. The two species differ in expression of CAM, since the proportion of respiratory CO2 recycled as part of CAM had been found to be much lower in A. fendleri This study compared the efficiency of water use and role of respiratory CO2 recycling under two PAR regimes (300 and 120 μnol m−2 s−1) and three night temperatures (12, 18 and 25 °C). Dark CO2 uptake rates for both species were comparable to plants in the field (maximum of 2.3 ± 0.2 μmol m−2s−1± SD, n= 3). Total net CO2 uptake at night increased on leaf area basis with temperature for both species under higher PAR, although under the low PAR regime CO2 uptake was maximal at 18 °C. Water‐use efficiency (WUE) increased at 18 °C and 25 °C during dark CO2 uptake (Phase I) and also during late afternoon photosynthesis (Phase IV) in both species. For A. fendleri, dawn to dusk changes in titrable acidity (ΔH +) were similar under high and low PAR, although ΔH+ was correlated to night temperature and PAR in A. nudicaulis. The proportion of ΔH+ derived from respiratory CO2 also varied with experimental conditions. Thus percentage recycling was lower in A. fendleri under high PAR (0–10%), but was only reduced at 18 °C under low PAR. Recycling by A. nudicaulis ranged from 32–42% under high PAR, but was also reduced to 6% under low PAR at 18 °C; at 12 °C and 25 °C, recycling was 37% and 52% respectively. Previous studies have suggested a relationship between the proportion of recycling and degree of water stress. This study indicated that CAM as a CO2 concentrating mechanism regulates both water‐use efficiency and plant carbon balance in these epiphytes, in response to PAR and night temperature. However, the precise relationship between respiratory processes and the balance between external and internal sources of CO2 is as yet unresolved.

Purification and Properties of Phosphoenolpyruvate Carboxylase From Plants With Crassulacean Acid Metabolism

Phosphoenolpyruvate (PEP) carboxylase was purified from three species of crassulacean acid metabolism (CAM) plants. There was no evidence for isoenzymes of PEP carboxylase in these plants and the purified protein was an active dimer of Mr 220 000-250 000 which dissociated to a monomer of Mr 110 000 after treatment with sodium dodecyl sulfate. Active, higher aggregates could be obtained on Sepharose 6B but the functional significance, if any, of these remains to be assessed. In the absence of effectors, normal Michaelis-Menten kinetics were obtamed with the substrates HCO3- and PEP. The purified enzyme shows a preference for HCO3-, rather than CO2, at pH 6.1 and 8.1, with a Km (HCO3-) of 10-20 �M. The Vmax was relatively independent of pH between pH 5.5 and 8.5, but the Km (PEP) (like most other kinetic properties) was pH dependent with a minimum of about 0.1 mM PEP at pH 6.8. Malate inhibition was more effective at pH 6.2 than at pH 8.2, and the inhibition evidently involved a slow binding of malate which increased the Km (PEP) and resulted in non-hyperbolic kinetics. The Km (PEP) was lowered about 5-10-fold by 1.0 mM glucose 6-phosphate which also overcame malate inhibition and restored hyperbolic kinetic relationships in the presence of malate. Possible roles for these properties in the regulation of CAM are discussed.

The activity and malate inhibition/stimulation of phosphoenolpyruvate-carboxylase in crassulacean-acid-metabolism plants in their natural environment

The effect of environmental conditions, temperature, relative humidity, and light, together with the regulation of PEPC (phosphoenolpyruvate-carboxylase) activity by malate and pH on CAM (crassulacean acid metabolism), was studied in members of the Mesembryanthemaceae in their natural environment, the southern Namib desert. It was found that during a 24 h period the characteristics of PEPC change. Before sunrise the activity is higher when measured at pH 7 than 8. With bright sunlight the activity measured at pH 7 drops to 20% of its pre-sunrise value, the activity only recovers gradually after malate disappearance and stays constant throughout the night. When measured at pH 8, PEPC shows an opposite behavior, i.e., activity increases in bright sunlight and declines as the pH 7 activity increases. A day-night oscillation in the capacity of malate to stimulate or inhibit PEPC was found. During the day malate inhibits about 90% of the PEPC activity at both pH 7 and 8. After sunset there is a sudden decrease in this inhibition and, at pH 8, malate stimulates the activity by 50%. At pH 7 the stimulation was less.Both stomatal conductance and malate formation were found to increase only when the relative humidity at night rose to 80%. Changes in the properties of the PEPC coincided with the exposure to bright sunlight and changes in leaf temperature. The importance of these metabolic and environmental controls on the regulation of CAM in the Mesembryanthemaceae will be discussed.

Proton and malate fluxes in cells of Bryophyllum daigremontianum leaf slices in relation to potential osmotic pressure of the medium

Summary Leaf slices obtained from the CAM plant Bryophyllum daigremontianum at the end of the dark phase were used to study proton und malate fluxes. Increasing potential osmotic pressure (π*) of the medium decreases loss of H + and malate from the leaf cells to the medium and increases the amounts of H+ and malate retained in the cells. As compared to the initial H + and malic acid levels, after the efflux experiments a certain amount of H+ and malate is not accounted for by efflux and retainment in the tissue. This amount is thought to be due to metabolic utilization of malic acid, it is smaller in the dark than in the light, and it is not clearly correlated to π * of the medium. Changes in potential osmotic pressure of extracts (Δπ * ) of the leaf slices are highly correlated to changes in malate content of the extracts. Malic acid accounts for about 55% of Δπ * of the extracts. It is suggested that net efflux of malic acid from the vacuoles of B. daigremontianum depends on turgor of the cells and that this mechanism plays a role in regulation of crassulacean acid metabolism (CAM) in vivo .