Phylogenomics and plastomics offer new evolutionary perspectives on Kalanchoideae (Crassulaceae).

BACKGROUND AND AIMS: Kalanchoideae is one of three subfamilies within Crassulaceae and contains four genera. Despite previous efforts, the phylogeny of Kalanchoideae remains inadequately resolved with persistent issues including low support, unstructured topologies and polytomies. This study aimed to address two central objectives: (1) resolving the pending phylogenetic questions within Kalanchoideae by using organelle-scale 'barcodes' (plastomes) and nuclear data; and (2) investigating interspecific diversity patterns among Kalanchoideae plastomes. METHODS: To explore the plastome evolution in Kalanchoideae, we newly sequenced 38 plastomes representing all four constituent genera (Adromischus, Cotyledon, Kalanchoe and Tylecodon). We performed comparative analyses of plastomic features, including GC and gene contents, gene distributions at the IR (inverted repeat) boundaries, nucleotide divergence, plastomic tRNA (pttRNA) structures and codon aversions. Additionally, phylogenetic inferences were inferred using both the plastomic dataset (79 genes) and nuclear dataset (1054 genes). KEY RESULTS: Significant heterogeneities were observed in plastome lengths among Kalanchoideae, strongly correlated with LSC (large single copy) lengths. Informative diversities existed in the gene content at SSC/IRa (small single copy/inverted repeat a), with unique patterns individually identified in Adromischus leucophyllus and one major Kalanchoe clade. The ycf1 gene was assessed as a shared hypervariable region among all four genera, containing nine lineage-specific indels. Three pttRNAs exhibited unique structures specific to Kalanchoideae and the genera Adromischus and Kalanchoe. Moreover, 24 coding sequences revealed a total of 41 lineage-specific unused codons across all four constituent genera. The phyloplastomic inferences clearly depicted internal branching patterns in Kalanchoideae. Most notably, by both plastid- and nuclear-based phylogenies, our research offers the first evidence that Kalanchoe section Eukalanchoe is not monophyletic. CONCLUSIONS: This study conducted comprehensive analyses on 38 newly reported Kalanchoideae plastomes. Importantly, our results not only reconstructed well-resolved phylogenies within Kalanchoideae, but also identified highly informative unique markers at the subfamily, genus and species levels. These findings significantly enhance our understanding of the evolutionary history of Kalanchoideae.

Genomic data provide a robust phylogeny backbone for Rhodiola L. (Crassulaceae) and reveal extensive reticulate evolution during its rapid radiation.

The Tibetan Plateau and adjacent mountain regions (TP; including the Tibetan Plateau, Himalaya, Hengduan Mountains and Mountains of Central Asia) harbor great biodiversity, some lineages on which may have undergone rapid radiations. However, only a few studies have investigated the evolutionary pattern of such diversification in depth using genomic data. In this study, we reconstructed a robust phylogeny backbone of Rhodiola, a lineage that may have undergone rapid radiation in the TP, using Genotyping-by-sequencing data, and conducted a series of gene flow and diversification analyses. The concatenation and coalescent-based methods yield similar tree topologies, and five well-supported clades were revealed. Potential gene flow and introgression events were detected, both between species from different major clades and closely related species, suggesting pervasive hybridization and introgression. An initial rapid and later slowdown of the diversification rate was revealed, indicating niche filling. Molecular dating and correlation analyses showed that the uplift of TP and global cooling in the mid-Miocene might have played an important role in promoting the rapid radiation of Rhodiola. Our work demonstrates that gene flow and introgression might be an important contributor to rapid radiation possibly by quickly reassembling old genetic variation into new combinations.

Genetic Diversity in Natural Populations of Rhodiola Species of Different Adaptation Strategies.

Representatives of the Crassulaceae family's genus Rhodiola are succulents, making them distinctive in a changing environment. One of the most significant tools for analyzing plant resources, including numerous genetic processes in wild populations, is the analysis of molecular genetic polymorphism. This work aimed to look at the polymorphisms of allelic variations of the superoxide dismutase (SOD) and auxin response factor (ARF) gene families, as well as the genetic diversity of five Rhodiola species, using the retrotransposons-based fingerprinting approach. The multi-locus exon-primed intron-crossing (EPIC-PCR) profiling approach was used to examine allelic variations in the SOD and ARF gene families. We implemented the inter-primer binding site (iPBS) PCR amplification technique for genome profiling, which demonstrated a significant level of polymorphism in the Rhodiola samples studied. Natural populations of Rhodiola species have a great capacity for adaptation to unfavorable environmental influences. The genetic variety of wild populations of Rhodiola species leads to their improved tolerance of opposing environmental circumstances and species evolutionary divergence based on the diversity of reproductive systems.

Chromosome Number, Ploidy Level, and Nuclear DNA Content in 23 Species of Echeveria (Crassulaceae).

Echeveria is a polyploid genus with a wide diversity of species and morphologies. The number of species registered for Echeveria is approximately 170; many of them are native to Mexico. This genus is of special interest in cytogenetic research because it has a variety of chromosome numbers and ploidy levels. Additionally, there are no studies concerning nuclear DNA content and the extent of endopolyploidy. This work aims to investigate the cytogenetic characteristics of 23 species of Echeveria collected in 9 states of Mexico, analyzing 2n chromosome numbers, ploidy level, nuclear DNA content, and endopolyploidy levels. Chromosome numbers were obtained from root tips. DNA content was obtained from the leaf parenchyma, which was processed according to the two-step protocol with Otto solutions and propidium iodide as fluorochrome, and then analyzed by flow cytometry. From the 23 species of Echeveria analyzed, 16 species lacked previous reports of 2n chromosome numbers. The 2n chromosome numbers found and analyzed in this research for Echeveria species ranged from 24 to 270. The range of 2C nuclear DNA amounts ranged from 1.26 pg in E. catorce to 7.70 pg in E. roseiflora, while the 1C values were 616 Mbp and 753 Mbp, respectively, for the same species. However, differences in the level of endopolyploidy nuclei were found, corresponding to 4 endocycles (8C, 16C, 32C and 64C) in E. olivacea, E. catorce, E. juarezensis and E. perezcalixii. In contrast, E. longiflora presented 3 endocycles (8C, 16C and 32C) and E. roseiflora presented 2 endocycles (8C and 16C). It has been suggested that polyploidization and diploidization processes, together with the presence of endopolyploidy, allowed Echeveria species to adapt and colonize new adverse environments.

Hybridization, ecogeographical displacement and the emergence of new lineages - A genotyping-by-sequencing and ecological niche and species distribution modelling study of Sempervivum tectorum L. (Houseleek).

Ecogeographical displacement of homoploid hybrid lineages from their parents is well documented and considered an important mechanism to achieve reproductive isolation. In this study, we investigated the origin of the flowering plant species Sempervivum tectorum in the Massif Central (France) through homoploid hybridization between lineages of the species from the Rhine Gorge area (Germany) and the Pyrenees (France). We used genotyping-by-sequencing genetic data as evidence for the hybrid origin of the Massif Central lineage, and WorldClim climatic data and soil pH and soil temperature data collected by us for ecological niche and species distribution modelling. We could show that the Massif Central lineage shows hybrid admixture and that the niche of this lineage is significantly different from those of the parental lineages. In comparison with the parental niches, different variables of the niche of the hybrid lineage are intermediate, parental-combined or extreme. The different niche of the Massif Central populations thus can plausibly be interpreted as hybridization-derived. Our species distribution modelling for the Last Glacial Maximum and Mid-Holocene showed that the potential distribution of the hybrid lineage at the likely time of its origin in the Quaternary possibly was parapatric in relation to the largely sympatric distributions of the parental lineages. We hypothesize that reproductive isolation of the hybrid lineage from the parental lineages resulted from the segregation of distribution ranges by a differential response of the three lineages to a warming climate.

Chloroplast DNA insights into the phylogenetic position and anagenetic speciation of Phedimus takesimensis (Crassulaceae) on Ulleung and Dokdo Islands, Korea.

Phedimus takesimensis (Ulleungdo flat-leaved stonecrop) is endemic to Ulleung and Dokdo Islands off the east coast of the Korean Peninsula. It was suggested that P. takesimensis originated via anagenetic speciation from the continental progenitor species P. kamtchaticus or P. aizoon. However, little is known of the phylogenetic relationships and population genetic structure among species of Phedimus in the Korean Peninsula and Ulleung/Dokdo Islands. We inferred the phylogenetic relationships among congeneric species in Korea based on nuclear ribosomal DNA internal transcribed spacer and chloroplast noncoding regions. We also sampled extensively for P. takesimensis on Ulleung Island and the continental species, P. kamtschaticus and P. aizoon, to assess the genetic consequences of anagenetic speciation. We found (1) the monophyly of P. takesimensis, (2) no apparent reduction in genetic diversity in anagenetically derived P. takesimensis compared to the continental progenitor species, (3) apparent population genetic structuring of P. takesimensis, and (4) two separate colonization events for the origin of the Dokdo Island population. This study contributes to our understanding of the genetic consequences of anagenetic speciation on Ulleung Island.

Conservation and innovation: Plastome evolution during rapid radiation of Rhodiola on the Qinghai-Tibetan Plateau.

The amount of plastome sequence data available has soared in the last decade, but the nature of plastome evolution during rapid radiations is largely unknown. Moreover, although there is increasing evidence showing that plastomes may have undergone adaptive evolution in order to allow adaptation to various environments, few studies have systematically investigated the role of the plastome in alpine adaptation. To address these questions, we sequenced and analyzed 12 representative species of Rhodiola, a genus which includes ca. 70 perennial herbs mainly growing in alpine habitats in the Qinghai-Tibet Plateau and the Hengduan Mountains. Rapid radiation in this genus was triggered by the uplift of the Qinghai-Tibet Plateau. We also included nine species of Crassulaceae as the outgroups. All plastomes were conserved with respect to size, structure, and gene content and order, with few variations: each contained 134 genes, including 85 protein-coding genes, 37 tRNAs, 8 rRNAs, and 4 potential pseudogenes. Four types of repeat sequence were detected. Slight contraction and expansion of the inverted repeats were also revealed. Both the genome-wide alignment and sequence polymorphism analyses showed that the inverted repeats and coding regions were more conserved than the single-copy regions and the non-coding regions. Positive selection analyses identified three genes containing sites of positive selection (rpl16, ndhA, ndhH), and one gene with a faster than average rate of evolution (psaA). The products of these genes may be involved in the adaptation of Rhodiola to alpine environments such as low CO2 concentration and high-intensity light.

Nitric oxide modulating ion balance in Hylotelephium erythrostictum roots subjected to NaCl stress based on the analysis of transcriptome, fluorescence, and ion fluxes.

Soil salinization is one of the main stress factors that affect both growth and development of plants. Hylotelephium erythrostictum exhibits strong resistance to salt, but the underlying genetic mechanisms remain unclear. In this study, hydroponically cultured seedlings of H. erythrostictum were exposed to 200 mM NaCl. RNA-Seq was used to determine root transcriptomes at 0, 5, and 10 days, and potential candidate genes with differential expression were analyzed. Transcriptome sequencing generated 89.413 Gb of raw data, which were assembled into 111,341 unigenes, 82,081 of which were annotated. Differentially expressed genes associated to Na+ and K+ transport, Ca2+ channel, calcium binding protein, and nitric oxide (NO) biosynthesis had high expression levels in response to salt stress. An increased fluorescence intensity of NO indicated that it played an important role in the regulation of the cytosolic K+/Na+ balance in response to salt stress. Exogenous NO donor and NO biosynthesis inhibitors significantly increased and decreased the Na+ efflux, respectively, thus causing the opposite effect for K+ efflux. Moreover, under salt stress, exogenous NO donors and NO biosynthesis inhibitors enhanced and reduced Ca2+ influx, respectively. Combined with Ca2+ reagent regulation of Na+ and K+ fluxes, this study identifies how NaCl-induced NO may function as a signaling messenger that modulates the K+/Na+ balance in the cytoplasm via the Ca2+ signaling pathway. This enhances the salt resistance in H. erythrostictum roots.

Transcriptional profiling of the CAM plant Agave salmiana reveals conservation of a genetic program for regeneration.

In plants, the best characterized plant regeneration process is de novo organogenesis. This type of regeneration is characterized by the formation of a multicellular structure called callus. Calli are induced via phytohormone treatment of plant sections. The callus formation in plants like Agave species with Crassulacean Acid Metabolism (CAM) is poorly studied. In this study, we induced callus formation from Agave salmiana leaves and describe cell arrangement in this tissue. Moreover, we determined and analyzed the transcriptional program of calli, as well as those of differentiated root and leaf tissues, by using RNA-seq. We were able to reconstruct 170,844 transcripts of which 40,644 have a full Open Reading Frame (ORF). The global profile obtained by Next Generation Sequencing (NGS) reveals that several callus-enriched protein coding transcripts are orthologs of previously reported factors highly expressed in Arabidopsis calli. At least 62 genes were differentially expressed in Agave calli, 50 of which were up-regulated. Several of these are actively involved in the perception of, and response to, auxin and cytokinin. Not only are these the first results for the A. salmiana callus, but they provide novel data from roots and leaves of this Agave species, one of the largest non-tree plants in nature.

Phylogeography of Rhodiola kirilowii (Crassulaceae): a story of Miocene divergence and quaternary expansion.

The evolution and current distribution of the Sino-Tibetan flora have been greatly affected by historical geological events, such as the uplift of the Qinghai-Tibetan Plateau (QTP), and Quaternary climatic oscillations. Rhodiola kirilowii, a perennial herb with its distribution ranging from the southeastern QTP and the Hengduan Mountains (HM) to adjacent northern China and central Asia, provides an excellent model to examine and disentangle the effect of both geological orogeny and climatic oscillation on the evolutionary history of species with such distribution patterns. We here conducted a phylogeographic study using sequences of two chloroplast fragments (trnL-F and trnS-G) and internal transcribed spacers in 29 populations of R. kirilowii. A total of 25 plastid haplotypes and 12 ITS ribotypes were found. Molecular clock estimation revealed deep divergence between the central Asian populations and other populations from the HM and northern China; this split occurred ca. 2.84 million year ago. The majority of populations from the mountains of northern China were dominated by a single haplotype or ribotype, while populations of the HM harbored both high genetic diversity and high haplotype diversity. This distribution pattern indicates that HM was either a diversification center or a refugium for R. kirilowii during the Quaternary climatic oscillations. The present distribution of this species on mountains in northern China may have resulted from a rapid glacial population expansion from the HM. This expansion was confirmed by the mismatch distribution analysis and negative Tajima's D and Fu's FS values, and was dated to ca. 168 thousand years ago. High genetic diversity and population differentiation in both plastid and ITS sequences were revealed; these imply restricted gene flow between populations. A distinct isolation-by-distance pattern was suggested by the Mantel test. Our results show that in old lineages, populations may harbour divergent genetic forms that are sufficient to maintain or even increase overall genetic diversity despite fragmentation and low within-population variation.

[Molecular phylogeny and systematics of flowering plants of the family Crassulaceae DC].

Crassulaceae is the most species rich (ca. 1400) family in the order Saxifragales. Most members of the family are succulent plants. Phenotypic diversity and a large number of species complicate systematics of the family and reconstruction of relationship within it. Phylogenetic analyses based on morphological and molecular markers placed Crassulaceae as one of the crown clades of Saxifragales. In this contribution a review of phylogenetic studies of the family Crassulaceae, based on DNA nucleotide sequence comparisons is presented; major clades established in the family are characterised; their structure and polyphylesis of some genera related to it are discussed. It was shown that the traditional taxonomic structure of Crassulaceae contradicts pattern of phylogenetic relationships between its members. We critically analysed recent taxonomic systems of the family and stress that homoplasy of morphological characters does not allow to use them to reconstruct relationships between crassulacean taxa even at the low taxonomic levels.

A search for growth related genes in Kalanchoë blossfeldiana.

Differential display of mRNA from four sets of contrasting phenotypes were carried out in order to identify and isolate genes associated with elongating growth of Kalanchoë blossfeldiana. A total of 17 unique differential expressed cDNA fragments were sequenced and 12 showed homology to genes in other plant species. Three genes were subsequently tested for growth related activity by Virus Induced Gene Silencing (VIGS) in Nicotiana benthamiana. One gene fragment (13C) resulted in plants with significantly reduced growth (N = 20, P = 0.05, one-tailed students t-test) from day 25 after virus infection. Full-length cDNA and genomic DNA sequences were obtained by inverse PCR and thermal asymmetric interlaced (TAIL) PCR and the gene was named KbORF1. The predicted gene is 2244 bp long with three exons of 411 bp in total encoding a protein of 137 amino acid residues with homologs widespread among plants. The protein has no known function, but its expression has been confirmed in a proteomic study of Arabidopsis. Southern blot analysis shows two hybridizing fragments in agreement with the tetraploid nature of K. blossfeldiana. Fragment 13C comprises 446 bp of the gene, and the portion of 13C conferring growth retardation by VIGS is located 10 bp into the second intron indicating a regulatory function of this part of the KbORF1 mRNA. Differential display in combination with VIGS as a screening method proved to be a good functional approach not only to search for genes of interest, but also to isolate expressed genetic regulatory domains.

Molecular phylogeny of the Acre clade (Crassulaceae): dealing with the lack of definitions for Echeveria and Sedum.

The phylogenetic relationships within many clades of the Crassulaceae are still uncertain, therefore in this study attention was focused on the "Acre clade", a group comprised of approximately 526 species in eight genera that include many Asian and Mediterranean species of Sedum and the majority of the American genera (Echeveria, Graptopetalum, Lenophyllum, Pachyphytum, Villadia, and Thompsonella). Parsimony and Bayesian analyses were conducted with 133 species based on nuclear (ETS, ITS) and chloroplast DNA regions (rpS16, matK). Our analyses retrieved four major clades within the Acre clade. Two of these were in a grade and corresponded to Asian species of Sedum, the rest corresponded to a European-Macaronesian group and to an American group. The American group included all taxa that were formerly placed in the Echeverioideae and the majority of the American Sedoideae. Our analyses support the monophyly of three genera--Lenophyllum, Thompsonella, and Pachyphytum; however, the relationships among Echeveria, Sedum and the various segregates of Sedum are largely unresolved. Our analyses represents the first broad phylogenetic framework for Acre clade, but further studies are necessary on the groups poorly represented here, such as the European and Asian species of Sedum and the Central and South American species of Echeveria.

Resolving an ancient, rapid radiation in Saxifragales.

Despite the prior use of approximately 9000 bp, deep-level relationships within the angiosperm clade, Saxifragales remain enigmatic, due to an ancient, rapid radiation (89.5 to 110 Ma based on the fossil record). To resolve these deep relationships, we constructed several new data sets: (1) 16 genes representing the three genomic compartments within plant cells (2 nuclear, 10 plastid, 4 mitochondrial; aligned, analyzed length = 21,460 bp) for 28 taxa; (2) the entire plastid inverted repeat (IR; 26,625 bp) for 17 taxa; (3) "total evidence" (50,845 bp) for both 17 and 28 taxa (the latter missing the IR). Bayesian and ML methods yielded identical topologies across partitions with most clades receiving high posterior probability (pp = 1.0) and bootstrap (95% to 100%) values, suggesting that with sufficient data, rapid radiations can be resolved. In contrast, parsimony analyses of different partitions yielded conflicting topologies, particularly with respect to the placement of Paeoniaceae, a clade characterized by a long branch. In agreement with published simulations, the addition of characters increased bootstrap support for the putatively erroneous placement of Paeoniaceae. Although having far fewer parsimony-informative sites, slowly evolving plastid genes provided higher resolution and support for deep-level relationships than rapidly evolving plastid genes, yielding a topology close to the Bayesian and ML total evidence tree. The plastid IR region may be an ideal source of slowly evolving genes for resolution of deep-level angiosperm divergences that date to 90 My or more. Rapidly evolving genes provided support for tip relationships not recovered with slowly evolving genes, indicating some complementarity. Age estimates using penalized likelihood with and without age constraints for the 28-taxon, total evidence data set are comparable to fossil dates, whereas estimates based on the 17-taxon data are much older than implied by the fossil record. Hence, sufficient taxon density, and not simply numerous base pairs, is important in reliably estimating ages. Age estimates indicate that the early diversification of Saxifragales occurred rapidly, over a time span as short as 6 million years. Between 25,000 and 50,000 bp were needed to resolve this radiation with high support values. Extrapolating from Saxifragales, a similar number of base pairs may be needed to resolve the many other deep-level radiations of comparable age in angiosperms.

Biosystematics and conservation: a case study with two enigmatic and uncommon species of Crassula from New Zealand.

BACKGROUND AND AIMS: Crassula hunua and C. ruamahanga have been taxonomically controversial. Here their distinctiveness is assessed so that their taxonomic and conservation status can be clarified. METHODS: Populations of these two species were analysed using morphological, chromosomal and DNA sequence data. KEY RESULTS: It proved impossible to differentiate between these two species using 12 key morphological characters. Populations were found to be chromosomally variable with 11 different chromosome numbers ranging from 2n = 42 to 2n = 100. Meiotic behaviour and levels of pollen stainability were both variable. Phylogenetic analyses showed that differences exist in both nuclear and plastid DNA sequences between individual plants, sometimes from the same population. CONCLUSIONS: The results suggest that these plants are a species complex that has evolved through interspecific hybridization and polyploidy. Their high levels of chromosomal and DNA sequence variation present a problem for their conservation.

[Identification and phylogenetic application of unique nucleotide sequence of nad7 intron2 in Rhodiola (Crassulaceae) species].

Genetic characterization of 9 populations of Rhodiola crenulata, R. fastigiata and R. sachalinensis (Crassulaceae) species from Sichuan and Jilin Provinces of China, was investigated using the conserved primer of nad7 intron 2. All PCR products about 800 bp long were shorter than other Crassulaceae plants, which were used as molecular markers to identify the Rhodiola species. The sequence of the products indicated that total exon of 53 bp and intron of 738 bp exhibit only 9 nucleotide variations. Blasting the nad7 sequences to GenBank and the phylogenetic analysis showed that the sequence of Rhodiola species was clusted independently, and the length was smaller than all the registered sequences of higher plants. The result suggests that the Rhiodola species had a unique sequence in this gene region, which might be related to the special growth condition.

[Phylogenetic relationships among members of the subfamily sedoideae (Crassulaceae) inferred from the ITS region sequences of nuclear rDNA].

Nucleotide sequences of the nuclear rDNA ITS regions were determined in 20 species of the subfamily Sedoideae (Crassulaceae). The phylogenetic relationships of these species with other members of the subfamily, occurring mainly in Southeast Asia, were analyzed. It was shown that the genus Orostachys was not monophyletic; its typical subsection was reliably included into the clade of the genus Hylotelephium. Synapomorphic substitutions and indels, specific for the subsection Orostachys, were detected in ITS1. Sister relationships were established between clades Aizopsis and Phedimus, based on which they can be recognized as isolated genera.

Ecophysiology of Crassulacean Acid Metabolism (CAM).

BACKGROUND AND SCOPE: Crassulacean Acid Metabolism (CAM) as an ecophysiological modification of photosynthetic carbon acquisition has been reviewed extensively before. Cell biology, enzymology and the flow of carbon along various pathways and through various cellular compartments have been well documented and discussed. The present attempt at reviewing CAM once again tries to use a different approach, considering a wide range of inputs, receivers and outputs. INPUT: Input is given by a network of environmental parameters. Six major ones, CO(2), H(2)O, light, temperature, nutrients and salinity, are considered in detail, which allows discussion of the effects of these factors, and combinations thereof, at the individual plant level ('physiological aut-ecology'). RECEIVERS: Receivers of the environmental cues are the plant types genotypes and phenotypes, the latter including morphotypes and physiotypes. CAM genotypes largely remain 'black boxes', and research endeavours of genomics, producing mutants and following molecular phylogeny, are just beginning. There is no special development of CAM morphotypes except for a strong tendency for leaf or stem succulence with large cells with big vacuoles and often, but not always, special water storage tissues. Various CAM physiotypes with differing degrees of CAM expression are well characterized. OUTPUT: Output is the shaping of habitats, ecosystems and communities by CAM. A number of systems are briefly surveyed, namely aquatic systems, deserts, salinas, savannas, restingas, various types of forests, inselbergs and paramós. CONCLUSIONS: While quantitative census data for CAM diversity and biomass are largely missing, intuition suggests that the larger CAM domains are those systems which are governed by a network of interacting stress factors requiring versatile responses and not systems where a single stress factor strongly prevails. CAM is noted to be a strategy for variable, flexible and plastic niche occupation rather than lush productivity. 'Physiological syn-ecology' reveals that phenotypic plasticity constitutes the ecophysiological advantage of CAM.

Expression of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxylase kinase genes. Implications for genotypic capacity and phenotypic plasticity in the expression of crassulacean acid metabolism.

In plants with crassulacean acid metabolism (CAM), dark CO2 uptake is mediated by phosphoenolpyruvate carboxylase (PEPC), an enzyme that can be regulated at transcriptional and posttranslational levels. Reversible phosphorylation of PEPC is catalyzed by a dedicated PEPC kinase, which in turn is regulated at the transcriptional level over the 24-h cycle in CAM plants. PEPC kinase controls the day/night regulation of PEPC during the CAM cycle, thus facilitating plasticity for optimizing CO2 uptake under different environmental conditions. To understand the importance of PEPC kinase in relation to its target PEPC in terms of CAM performance, the expression of the genes encoding the two enzymes was investigated in four species of Clusia that have photosynthetic patterns ranging from C3 photosynthesis to constitutive CAM. By linking changes in the expression of PEPC and PEPC kinase to day/night patterns of leaf gas exchange, organic acid, and soluble sugar contents under different environmental conditions, the genetic and metabolic limitations to CAM plasticity were assessed. The results indicate that PEPC expression is a major factor underpinning the genotypic capacity for CAM and that PEPC kinase expression does not appear to limit CAM. The day/night regulation of Ppck transcript abundance was found to be a consequence of CAM and the day/night cycling of associated metabolites, rather than the primary controlling factor for the temporal separation of carboxylation processes.

Synchronization of metabolic processes in plants with Crassulacean acid metabolism.

In plants with Crassulacean acid metabolism, a diel separation of carboxylation processes mediated by phosphoenolpyruvate carboxylase (PEPC) and Rubisco optimizes photosynthetic performance and carbon gain in potentially limiting environments. This review considers the mechanisms that synchronize the supply and demand for carbon whilst maintaining photosynthetic plasticity over the 24 h CAM cycle. The circadian clock plays a central role in controlling many of the metabolic, transport and physiological components of CAM. The level of control exerted by the clock can range from transcriptional through to post-translational regulation, depending on the genes, proteins, and even plant species under consideration. A further layer of control is provided by metabolites, including organic acids and carbohydrates, which show substantial reciprocal fluctuations in content over the diel cycle. Mechanisms responsible for the sensing of metabolite contents are discussed, together with signalling requirements for the co-ordination of carbon fluxes. Evolutionary implications are considered in terms of how circadian and metabolic control of the CAM cycle may have been derived from C3 plants.