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

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

Phosphorolytic degradation of leaf starch via plastidic α-glucan phosphorylase leads to optimized plant growth and water use efficiency over the diel phases of Crassulacean acid metabolism.

In plants with Crassulacean acid metabolism (CAM), it has been proposed that the requirement for nocturnal provision of phosphoenolpyruvate as a substrate for CO2 uptake has resulted in a re-routing of chloroplastic starch degradation from the amylolytic route to the phosphorolytic route. To test this hypothesis, we generated and characterized four independent RNAi lines of the obligate CAM species Kalanchoë fedtschenkoi with a >10-fold reduction in transcript abundance of plastidic α-glucan phosphorylase (PHS1). The rPHS1 lines showed diminished nocturnal starch degradation, reduced dark CO2 uptake, a reduction in diel water use efficiency (WUE), and an overall reduction in growth. A re-routing of starch degradation via the hydrolytic/amylolytic pathway was indicated by hyperaccumulation of maltose in all rPHS1 lines. Further examination indicated that whilst operation of the core circadian clock was not compromised, plasticity in modulating net dark CO2 uptake in response to changing photoperiods was curtailed. The data show that phosphorolytic starch degradation is critical for efficient operation of the CAM cycle and for optimizing WUE. This finding has clear relevance for ongoing efforts to engineer CAM into non-CAM species as a means of boosting crop WUE for a warmer, drier future.

Exploring C4-CAM plasticity within the Portulaca oleracea complex.

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

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

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

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.

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.

The Kalanchoë genome provides insights into convergent evolution and building blocks of crassulacean acid metabolism.

Crassulacean acid metabolism (CAM) is a water-use efficient adaptation of photosynthesis that has evolved independently many times in diverse lineages of flowering plants. We hypothesize that convergent evolution of protein sequence and temporal gene expression underpins the independent emergences of CAM from C3 photosynthesis. To test this hypothesis, we generate a de novo genome assembly and genome-wide transcript expression data for Kalanchoë fedtschenkoi, an obligate CAM species within the core eudicots with a relatively small genome (~260 Mb). Our comparative analyses identify signatures of convergence in protein sequence and re-scheduling of diel transcript expression of genes involved in nocturnal CO2 fixation, stomatal movement, heat tolerance, circadian clock, and carbohydrate metabolism in K. fedtschenkoi and other CAM species in comparison with non-CAM species. These findings provide new insights into molecular convergence and building blocks of CAM and will facilitate CAM-into-C3 photosynthesis engineering to enhance water-use efficiency in crops.

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

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

Emerging model systems for functional genomics analysis of Crassulacean acid metabolism.

Crassulacean acid metabolism (CAM) is one of three main pathways of photosynthetic carbon dioxide fixation found in higher plants. It stands out for its ability to underpin dramatic improvements in plant water use efficiency, which in turn has led to a recent renaissance in CAM research. The current ease with which candidate CAM-associated genes and proteins can be identified through high-throughput sequencing has opened up a new horizon for the development of diverse model CAM species that are amenable to genetic manipulations. The adoption of these model CAM species is underpinning rapid advances in our understanding of the complete gene set for CAM. We highlight recent breakthroughs in the functional characterisation of CAM genes that have been achieved through transgenic approaches.

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

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

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

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

Parallel recruitment of multiple genes into c4 photosynthesis.

During the diversification of living organisms, novel adaptive traits usually evolve through the co-option of preexisting genes. However, most enzymes are encoded by gene families, whose members vary in their expression and catalytic properties. Each may therefore differ in its suitability for recruitment into a novel function. In this work, we test for the presence of such a gene recruitment bias using the example of C4 photosynthesis, a complex trait that evolved recurrently in flowering plants as a response to atmospheric CO2 depletion. We combined the analysis of complete nuclear genomes and high-throughput transcriptome data for three grass species that evolved the C4 trait independently. For five of the seven enzymes analyzed, the same gene lineage was recruited across the independent C4 origins, despite the existence of multiple copies. The analysis of a closely related C3 grass confirmed that C4 expression patterns were not present in the C3 ancestors but were acquired during the evolutionary transition to C4 photosynthesis. The significant bias in gene recruitment indicates that some genes are more suitable for a novel function, probably because the mutations they accumulated brought them closer to the characteristics required for the new function.

Adaptive evolution of C(4) photosynthesis through recurrent lateral gene transfer.

C(4) photosynthesis is a complex trait that confers higher productivity under warm and arid conditions. It has evolved more than 60 times via the co-option of genes present in C(3) ancestors followed by alteration of the patterns and levels of expression and adaptive changes in the coding sequences, but the evolutionary path to C(4) photosynthesis is still poorly understood. The grass lineage Alloteropsis offers unparalleled opportunities for studying C(4) evolution, because it includes a C(3) taxon and five C(4) species that vary significantly in C(4) anatomy and biochemistry. Using phylogenetic analyses of nuclear genes and leaf transcriptomes, we show that fundamental elements of the C(4) pathway in the grass lineage Alloteropsis were acquired via a minimum of four independent lateral gene transfers from C(4) taxa that diverged from this group more than 20 million years ago. The transfer of genes that were already fully adapted for C(4) function has occurred periodically over at least the last 10 million years and has been a recurrent source for the optimization of the C(4) pathway. This report shows that plant-plant lateral nuclear gene transfers can be a potent source of genetic novelty and adaptation in flowering plants.

Conservation and divergence of circadian clock operation in a stress-inducible Crassulacean acid metabolism species reveals clock compensation against stress.

One of the best-characterized physiological rhythms in plants is the circadian rhythm of CO(2) metabolism in Crassulacean acid metabolism (CAM) plants, which is the focus here. The central components of the plant circadian clock have been studied in detail only in Arabidopsis (Arabidopsis thaliana). Full-length cDNAs have been obtained encoding orthologs of CIRCADIAN CLOCK-ASSOCIATED1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION1 (TOC1), EARLY FLOWERING4 (ELF4), ZEITLUPE (ZTL), FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1), EARLY FLOWERING3 (ELF3), and a partial cDNA encoding GIGANTEA in the model stress-inducible CAM plant, Mesembryanthemum crystallinum (Common Ice Plant). TOC1 and LHY/CCA1 are under reciprocal circadian control in a manner similar to their regulation in Arabidopsis. ELF4, FKF1, ZTL, GIGANTEA, and ELF3 are under circadian control in C(3) and CAM leaves. ELF4 transcripts peak in the evening and are unaffected by CAM induction. FKF1 shows an abrupt transcript peak 3 h before subjective dusk. ELF3 transcripts appear in the evening, consistent with their role in gating light input to the circadian clock. Intriguingly, ZTL transcripts do not oscillate in Arabidopsis, but do in M. crystallinum. The transcript abundance of the clock-associated genes in M. crystallinum is largely unaffected by development and salt stress, revealing compensation of the central circadian clock against development and abiotic stress in addition to the well-known temperature compensation. Importantly, the clock in M. crystallinum is very similar to that in Arabidopsis, indicating that such a clock could control CAM without requiring additional components of the central oscillator or a novel CAM oscillator.