Strong Crassulacean Acid Metabolism Research Articles

Diel dynamics of multi-omics in elkhorn fern provide new insights into weak CAM photosynthesis

Crassulacean acid metabolism (CAM) has high water-use efficiency (WUE) and is widely recognized to have evolved from C3 photosynthesis. Different plant lineages have convergently evolved CAM, but the molecular mechanism that underlies C3-to-CAM evolution remains to be clarified. Platycerium bifurcatum (elkhorn fern) provides an opportunity to study the molecular changes underlying the transition from C3 to CAM photosynthesis because both modes of photosynthesis occur in this species, with sporotrophophyll leaves (SLs) and cover leaves (CLs) performing C3 and weak CAM photosynthesis, respectively. Here, we report that the physiological and biochemical attributes of CAM in weak CAM-performing CLs differed from those in strong CAM species. We investigated the diel dynamics of the metabolome, proteome, and transcriptome in these dimorphic leaves within the same genetic background and under identical environmental conditions. We found that multi-omic diel dynamics in P. bifurcatum exhibit both tissue and diel effects. Our analysis revealed temporal rewiring of biochemistry relevant to the energy-producing pathway (TCA cycle), CAM pathway, and stomatal movement in CLs compared with SLs. We also confirmed that PHOSPHOENOLPYRUVATE CARBOXYLASE KINASE (PPCK) exhibits convergence in gene expression among highly divergent CAM lineages. Gene regulatory network analysis identified candidate transcription factors regulating the CAM pathway and stomatal movement. Taken together, our results provide new insights into weak CAM photosynthesis and new avenues for CAM bioengineering.

Updating the occurrence of crassulacean acid metabolism (CAM) in the genus Clusia through carbon isotope analysis of species from Colombia

Clusia is a widely distributed neotropical genus with 321 currently described species. This remarkable genus is the only one known to contain trees sensu stricto with CAM photosynthesis. To survey the occurrence of CAM in Clusia species from Colombia, we determined the leaf stable carbon isotope composition (δ13C) of 568 specimens from 114 species deposited in 12 Colombian herbaria. In the vast majority of specimens, δ13C values indicated that C3 photosynthesis was the principal contributor to carbon gain. δ13C values typical of strong CAM (less negative than -20‰) were observed in only five species, in four of them for the first time. All samples with CAM-type isotopic signatures were collected below 1,000 m a.s.l., whereas species with predominantly C3 occurred from sea level to 3,500 m a.s.l. Together with information already available in the literature, we conclude that CAM is present in 22% (35/156) of the species of Clusia investigated thus far.

Diversification Slowdown in the Cirrhopetalum Alliance (Bulbophyllum, Orchidaceae): Insights From the Evolutionary Dynamics of Crassulacean Acid Metabolism.

Evolutionary slowdowns in diversification have been inferred in various plant and animal lineages. Investigation based on diversification models integrated with environmental factors and key characters could provide critical insights into this diversification trend. We evaluate diversification rates in the Cirrhopetalum alliance (Bulbophyllum, Orchidaceae subfam. Epidendroideae) using a time-calibrated phylogeny and assess the role of Crassulacean acid metabolism (CAM) as a hypothesised key innovation promoting the spectacular diversity of orchids, especially those with an epiphytic habit. An explosive early speciation in the Cirrhopetalum alliance is evident, with the origin of CAM providing a short-term advantage under the low atmospheric CO2 concentrations (pCO2) associated with cooling and aridification in the late Miocene. A subsequent slowdown of diversification in the Cirrhopetalum alliance is possibly explained by a failure to keep pace with pCO2 dynamics. We further demonstrate that extinction rates in strong CAM lineages are ten times higher than those of C3 lineages, with CAM not as evolutionarily labile as previously assumed. These results challenge the role of CAM as a “key innovation” in the diversification of epiphytic orchids.

Changes in crassulacean acid metabolism expression, chloroplast ultrastructure, photochemical and antioxidant activity in the Aloe vera during acclimation to combined drought and salt stress.

We determined time course changes of photochemical and antioxidant activity during the induction of strong crassulacean acid metabolism (CAM) in Aloe vera L. plants grown under salt and drought stress. We found that the strong CAM was induced during 25-30days of drought alone treatment. After 25-30days, we showed the withdrawal of strong CAM back to constitutive CAM background under the combination of simultaneous drought and salt stress, which coincided with the accumulation of malondialdehyde, and the decrease in the contents of endogenous nitric oxide (NO) and non-enzymatic antioxidants. At the same time, the chloroplast ultrastructure was damaged with a parallel accumulation of reactive oxygen species, and the whole photosynthetic electron transport flux was impaired by combined stress treatment. In conclusion, the changes in CAM expression parameters was attended by a similar pattern of antioxidant and photochemical change in Aloe plants subjected to only drought or combined stress.

Leaf anatomy is not correlated to CAM function in a C3+CAM hybrid species, Yucca gloriosa.

Crassulacean acid metabolism (CAM) is often considered to be a complex trait, requiring orchestration of leaf anatomy and physiology for optimal performance. However, the observation of trait correlations is based largely on comparisons between C3 and strong CAM species, resulting in a lack of understanding as to how such traits evolve and the level of intraspecific variability for CAM and associated traits. To understand intraspecific variation for traits underlying CAM and how these traits might assemble over evolutionary time, we conducted detailed time course physiological screens and measured aspects of leaf anatomy in 24 genotypes of a C3+CAM hybrid species, Yucca gloriosa (Asparagaceae). Comparisons were made to Y. gloriosa's progenitor species, Y. filamentosa (C3) and Y. aloifolia (CAM). Based on gas exchange and measurement of leaf acids, Y. gloriosa appears to use both C3 and CAM, and varies across genotypes in the degree to which CAM can be upregulated under drought stress. While correlations between leaf anatomy and physiology exist when testing across all three Yucca species, such correlations break down at the species level in Y. gloriosa. The variation in CAM upregulation in Y. gloriosa is a result of its relatively recent hybrid origin. The lack of trait correlations between anatomy and physiology within Y. gloriosa indicate that the evolution of CAM, at least initially, can proceed through a wide combination of anatomical traits, and more favourable combinations are eventually selected for in strong CAM plants.

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.

The Evolution of CAM Photosynthesis in Australian Calandrinia Reveals Lability in C3+CAM Phenotypes and a Possible Constraint to the Evolution of Strong CAM.

Australian Calandrinia has radiated across the Australian continent during the last 30 Ma, and today inhabits most Australian ecosystems. Given its biogeographic range and reports of facultative Crassulacean acid metabolism (CAM) photosynthesis in multiple species, we hypothesized (1) that CAM would be widespread across Australian Calandrinia and that species, especially those that live in arid regions, would engage in strong CAM, and (2) that Australian Calandrinia would be an important lineage for informing on the CAM evolutionary trajectory. We cultivated 22 Australian Calandrinia species for a drought experiment. Using physiological measurements and δ13C values we characterized photosynthetic mode across these species, mapped the resulting character states onto a phylogeny, and characterized the climatic envelopes of species in their native ranges. Most species primarily utilize C3 photosynthesis, with CAM operating secondarily, often upregulated following drought. Several phylogenetically nested species are C3, indicating evolutionary losses of CAM. No strong CAM was detected in any of the species. Results highlight the limitations of δ13C surveys in detecting C3+CAM phenotypes, and the evolutionary lability of C3+CAM phenotypes. We propose a model of CAM evolution that allows for lability and reversibility among C3+CAM phenotypes and C3 and suggest that an annual life-cycle may preclude the evolution of strong CAM.

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.

Multiple isoforms of phosphoenolpyruvate carboxylase in the Orchidaceae (subtribe Oncidiinae): implications for the evolution of crassulacean acid metabolism.

Phosphoenolpyruvate carboxylase (PEPC) catalyses the initial fixation of atmospheric CO2 into oxaloacetate and subsequently malate. Nocturnal accumulation of malic acid within the vacuole of photosynthetic cells is a typical feature of plants that perform crassulacean acid metabolism (CAM). PEPC is a ubiquitous plant enzyme encoded by a small gene family, and each member encodes an isoform with specialized function. CAM-specific PEPC isoforms probably evolved from ancestral non-photosynthetic isoforms by gene duplication events and subsequent acquisition of transcriptional control elements that mediate increased leaf-specific or photosynthetic-tissue-specific mRNA expression. To understand the patterns of functional diversification related to the expression of CAM, ppc gene families and photosynthetic patterns were characterized in 11 closely related orchid species from the subtribe Oncidiinae with a range of photosynthetic pathways from C3 photosynthesis (Oncidium cheirophorum, Oncidium maduroi, Rossioglossum krameri, and Oncidium sotoanum) to weak CAM (Oncidium panamense, Oncidium sphacelatum, Gomesa flexuosa and Rossioglossum insleayi) and strong CAM (Rossioglossum ampliatum, Trichocentrum nanum, and Trichocentrum carthagenense). Phylogenetic analysis revealed the existence of two main ppc lineages in flowering plants, two main ppc lineages within the eudicots, and three ppc lineages within the Orchidaceae. Our results indicate that ppc gene family expansion within the Orchidaceae is likely to be the result of gene duplication events followed by adaptive sequence divergence. CAM-associated PEPC isoforms in the Orchidaceae probably evolved from several independent origins.

Environmental regulation of carbon isotope composition and crassulacean acid metabolism in three plant communities along a water availability gradient.

Expression of crassulacean acid metabolism (CAM) is characterized by extreme variability within and between taxa and its sensitivity to environmental variation. In this study, we determined seasonal fluctuations in CAM photosynthesis with measurements of nocturnal tissue acidification and carbon isotopic composition (δ13C) of bulk tissue and extracted sugars in three plant communities along a precipitation gradient (500, 700, and 1,000 mm year−1) on the Yucatan Peninsula. We also related the degree of CAM to light habitat and relative abundance of species in the three sites. For all species, the greatest tissue acid accumulation occurred during the rainy season. In the 500 mm site, tissue acidification was greater for the species growing at 30% of daily total photon flux density (PFD) than species growing at 80% PFD. Whereas in the two wetter sites, the species growing at 80% total PFD had greater tissue acidification. All species had values of bulk tissue δ13C less negative than −20‰, indicating strong CAM activity. The bulk tissue δ13C values in plants from the 500 mm site were 2‰ less negative than in plants from the wetter sites, and the only species growing in the three communities, Acanthocereus tetragonus (Cactaceae), showed a significant negative relationship between both bulk tissue and sugar δ13C values and annual rainfall, consistent with greater CO2 assimilation through the CAM pathway with decreasing water availability. Overall, variation in the use of CAM photosynthesis was related to water and light availability and CAM appeared to be more ecologically important in the tropical dry forests than in the coastal dune.

The incidence of crassulacean acid metabolism in Orchidaceae derived from carbon isotope ratios: a checklist of the flora of Panama and Costa Rica

Leaf carbon stable isotopic composition data for 1002 orchid species representing 61% of the total number of orchid species described for Panama and Costa Rica were obtained from herbarium specimens to survey the occurrence of crassulacean acid metabolism (CAM). Carbon isotopic composition of leaf material showed a bimodal distribution with modes at -28‰, indicating C3 photosynthesis, and at -15‰, indicating pronounced CAM photosynthesis. Strong CAM was present in 9.5% of species and in 31 of 162 genera studied. Twelve of these genera were not previously known to contain species exhibiting CAM. A checklist of orchids of Panama and Costa Rica with their d 13 C values and an updated list of all known orchid genera that possess species with the ability to perform CAM are presented. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 163, 194–222.

Carbon isotope composition and mode of photosynthesis in Clusia species from Mexico

The neotropical genus Clusia comprises arborescent species exhibiting Crassulacean Acid Metabolism (CAM) as was first reported for a Mexican species, Clusia lundellii. Here, the occurrence of CAM photosynthesis was studied in 20 species of Clusia, 18 from Mexico, and 2 from Guatemala, using leaf carbon isotopic composition. In most species, samples from individuals collected in different locations were analyzed. CAM was present in at least 11 species, eight of which contained specimens with δ13C values less negative than -20.0 ‰, indicating strong CAM (C. chanekiana, C. flava, C. lundellii, C. mexicana, C. quadrangula, C. rosea, C. suborbicularis, and C. tetra-trianthera). δ13C was highly variable in some species, but CAM expression was not correlated to life form (epiphytic, hemiepiphytic, terrestrial) or habitat. CAM specimens were not collected at altitudes above 1 700 m a.s.l.

Crassulacean acid metabolism and epiphytism linked to adaptive radiations in the Orchidaceae.

Species of the large family Orchidaceae display a spectacular array of adaptations and rapid speciations that are linked to several innovative features, including specialized pollination syndromes, colonization of epiphytic habitats, and the presence of Crassulacean acid metabolism (CAM), a water-conserving photosynthetic pathway. To better understand the role of CAM and epiphytism in the evolutionary expansion of tropical orchids, we sampled leaf carbon isotopic composition of 1,103 species native to Panama and Costa Rica, performed character state reconstruction and phylogenetic trait analysis of CAM and epiphytism, and related strong CAM, present in 10% of species surveyed, to climatic variables and the evolution of epiphytism in tropical regions. Altitude was the most important predictor of photosynthetic pathway when all environmental variables were taken into account, with CAM being most prevalent at low altitudes. By creating integrated orchid trees to reconstruct ancestral character states, we found that C3 photosynthesis is the ancestral state and that CAM has evolved at least 10 independent times with several reversals. A large CAM radiation event within the Epidendroideae, the most species-rich epiphytic clade of any known plant group, is linked to a Tertiary species radiation that originated 65 million years ago. Our study shows that parallel evolution of CAM is present among subfamilies of orchids, and correlated divergence between photosynthetic pathways and epiphytism can be explained by the prevalence of CAM in low-elevation epiphytes and rapid speciation of high-elevation epiphytes in the Neotropics, contributing to the astounding diversity in the Orchidaceae.

The occurrence of crassulacean acid metabolism in Cymbidium (Orchidaceae) and its ecological and evolutionary implications

Crassulacean acid metabolism (CAM) is one of the photosynthetic pathways regarded as adaptations to water stress in land plants. Little is known about correlations among the level of CAM activity, environment of habitat, life form, and phylogenetic relationship of a plant group from an evolutionary perspective. We examined these relationships in 18 species of Cymbidium (Orchidaceae) because the genus shows distinctive diversification of habitats and life forms. The photosynthetic type was classed into three categories, strong CAM, weak CAM, and C(3) on the basis of CAM activity. CAM expression in Cymbidium was confined to the epiphytic and lithophytic species. Especially, all of these species from tropical to subtropical rainforest exhibited CAM activity. On the other hand, the terrestrial species always exhibited C(3) metabolism irrespective of their varied habitats. Regarding the evolution of photosynthetic characters, weak CAM was the ancestral state in Cymbidium and strong CAM and C(3) metabolism occurred subsequently. The evolution of strong CAM likely enabled Cymbidium to extend to exposed sites in tropical lowland where marked water stress exists. Further, different levels of CAM activity characterized each species and such potential plasticity of CAM may realize the radiation of Cymbidium into sites with different environmental conditions.

Functional constraints of CAM leaf anatomy: tight cell packing is associated with increased CAM function across a gradient of CAM expression

Increased cell size, increased leaf succulence, reduced intercellular air space (IAS), and reduced surface of mesophyll exposed to IAS (L(mes)/area) are traits associated with the Crassulacean acid metabolism (CAM) photosynthetic pathway. An examination was carried out to determine whether these anatomical and structural traits are related to the degree of CAM function in eight CAM species, as measured by CO(2) assimilation during the CAM and C(3) phases. Increased cell size and leaf succulence were not closely related to the degree of CAM function, indicating that succulence beyond a certain threshold does not enhance CAM function. Reduced IAS and L(mes)/area were positively related to CAM function, and negatively related to C(3) function. These results indicate that reduced IAS and L(mes)/area are beneficial for CAM function through the reduction of CO(2) efflux and the improvement of carbon economy. However, reduced IAS and L(mes)/area limit C(3) photosynthesis, potentially mediating a bimodal distribution of weak and strong CAM species with high and low IAS and L(mes)/area values, respectively.

Distribution of crassulacean acid metabolism in orchids of Panama: evidence of selection for weak and strong modes.

Crassulacean acid metabolism (CAM) is one of three metabolic pathways found in vascular plants for the assimilation of carbon dioxide. In this study, we investigate the occurrence of CAM photosynthesis in 200 native orchid species from Panama and 14 non-native species by carbon isotopic composition (δ13C) and compare these values with nocturnal acid accumulation measured by titration in 173 species. Foliar δ13C showed a bimodal distribution with the majority of species exhibiting values of approximately -28‰ (typically associated with the C3 pathway), or -15‰ (strong CAM). Although thick leaves were related to δ13C values in the CAM range, some thin-leaved orchids were capable of CAM photosynthesis, as demonstrated by acid titration. We also found species with C3 isotopic values and significant acid accumulation at night. Of 128 species with δ13C more negative than -22‰, 42 species showed nocturnal acid accumulation per unit fresh mass characteristic of weakly expressed CAM. These data suggest that among CAM orchids, there may be preferential selection for species to exhibit strong CAM or weak CAM, rather than intermediate metabolism.

? 13C values and crassulacean acid metabolism in Clusia species from Panama

The genus Clusia is notable in that it contains arborescent crassulacean acid metabolism (CAM) plants. As part of a study of CAM in Clusia, titratable acidities were measured in 25 species and δ 13C values were measured for 38 species from Panama, including seven undescribed species, and 11 species from Colombia, Costa Rica and Honduras. CAM was detected in 12 species. Clusia flava, C. rosea and C. uvitana exhibited δ 13C values or diurnal fluctuations in acidity indicative of strong CAM. In C. croatii, C. cylindrica, C. fructiangusta, C. lineata, C. odorata, C. pratensis, C. quadrangula, C. valerioi and C. sp. D diurnal fluctuations in acidity were consistent with weak CAM but the δ 13C values were C3-like. All of the species that exhibited strong or weak CAM were in the C. flava or C. minor species groups. CAM was not detected in any member of the C. multiflora species group. Strong CAM species were not collected at altitudes above 680 m a.s.l. On the basis of δ 13C values, the expression of CAM was similar in terrestrial, hemi-epiphytic and epiphytic species and did not differ between individuals of the same species that exhibited different life-forms. This study indicates that phylogenetic affiliation may be a predictor of an ability to exhibit CAM in Clusia species from the Panamanian region, and that weak CAM is probably a common photosynthetic option in many Clusia species. δ 13C value is not a particularly good indicator of a potential of Clusia species growing in the field to exhibit CAM because it appears that the contribution in most species of CAM to carbon gain is generally rather small when integrated over the life-time of leaves.

The evolution of crassulacean acid metabolism inAeonium inferred from carbon isotope composition and enzyme activities.

Variation in crassulacean acid metabolism (CAM) was investigated in thirtyAeonium species under controlled, well-watered conditions. Carbon isotope ratios and the specific activities of the CAM enzymes phosphoenolpyruvate carboxylase (PEPC) and malic enzyme ME_ are highly correlated and indicate a large variation in CAM. The δ13C values range from -13.6 to -27.1‰; PEPC and ME activities vary seven- and five-fold, respectively. Since these observations were made under controlled conditions, the differences are genetically determined and represent adaptations to different habitats. The CAM activity of the species is correlated with the characteristics of their natural habitats. CAM activity is also highly correlated with the growth- and life-forms of the species and accordingly agrees well with the infrageneric classifications which are partly based on these characters. Our results support the hypothesis that the Sempervivoideae have evolved from an EurasiaticSedum, which used C3 photosynthesis or weak CAM, and that evolution withinAeonium was from undifferentiatedSedum-like dwarf shrubs with C3 photosynthesis to woody candelabrum-shaped shrubs exhibiting strong CAM. The monocarpic, rosulate species with intermediate CAM may have evolved via an independent secondary route.

Variation in carbon isotope ratios of Sempervivoideae species from different habitats of Teneriffe in the spring.

According to carbon isotope ratios, species of the Sempervivoideae from Teneriffe show in general a tendency for increased participation of dark CO2 fixation via PEP-carboxylase in total carbon fixation as habitats become drier and warmer. Certain species are found in cool moist habitats and exhibit C3-like δ13C values. Other species occur in warm dry habitats and exhibit δ13C values which indicate strong Crassulacean Acid Metabolism. A third group of species shows intermediate δ13C values which are more C3-like in cool moist habitats and which indicate increased dark fixation in warmer and drier situations. Included in this group is Aeonium holochrysum, which of the Sempervivoideae of Teneriffe is thought to be most closely related to the common ancestor (Lems 1960). Comparison of CO2 gas exchange of several species under identical environmental conditions reveals differences among species in the ability to regulate CO2 fixation in the light and in the dark which may have arisen in the process of adaptive radiation.