Crassulacean Acid Metabolism Plant Kalanchoe Research Articles

Enhancing Abiotic Stress Response via a Kalanchoe fedtschenkoi NF‐Y Transcription Factor in C3 Plants

Global crop production is expected to decrease because of increasing abiotic stressors including extremes in temperature, water, light intensity, nutrients, and radiation due to climate change and anthropogenic activities. Increased abiotic stressors will alter the metabolism in C3 plants and thus negatively affect their growth and productivity. Developing C3 crop varieties with improved abiotic stress tolerances emerges as the most sustainable solution towards ensuring global food security under the predicted climate change scenario. Crassulacean Acid Metabolism (CAM) plants are considered well‐adapted to drought, heat, increased light intensities, higher UV irradiation, and are far more water‐use efficient compared to C3 photosynthesis plants. The use of abiotic stress‐responsive transcription factors (TFs) from CAM plants is one feasible approach to improve crop production under adverse abiotic stress conditions. We have identified a NF‐YB TF from the heat‐tolerant obligate CAM plant Kalanchoe fedtschenkoi as a possible regulator of abiotic stresses. This NF‐Y is a ubiquitous transcription factor that is expressed as one of three possible isoforms NF‐YA, NF‐YB, or NF‐YC. Their isoforms interact to form a heterotrimeric complex with each subunit playing key roles in the abiotic stress responses including drought, heat, freezing, and salinity. We are functionally characterizing this KfNF‐YB via overexpression in Arabidopsis thaliana to determine its role in abiotic stress responses. Preliminary results following in vitro salinity stress assay of T2 transgenic lines after 12 days in MS media with 200 mM NaCl indicate increased root growth, whereas the wild type had retarded root growth. In addition, transgenic lines had higher biomass dry weight compared to wild type after 12 days. T2 lines also have increased rosette size in two‐week old transgenic A. thaliana compared to wild type. We will discuss the implications of the preliminary results as a potential strategy to enhance multiple abiotic stress tolerance responses in C3 plants.Support or Funding InformationThis work is funded by South Dakota Board of Regents Grant #A18‐0015‐001 to BWM Wone and supported in part by U. S. Department of Energy, Office of Science, Genomic Science Program under award #DE‐SC0008834 to JC Cushman.

A photosynthesis continuous monitoring system for CAM plants

Diurnal CO2 exchanges in crassulacean acid metabolism (CAM) plants are significantly different from those in C3 and C4 plants. The instantaneous short-time CO2 exchange of a single leaf measured by commercial portable photosynthesis measuring systems with a small leaf chamber cannot reflect the plant photosynthetic capacity for CAM plants because of the CO2 fixation property. Therefore, a photosynthesis continuous monitoring system with two canopy cuvettes was developed for measuring diurnal net CO2 exchange rates for CAM plants. To evaluate stability and applicability of the photosynthesis continuous monitoring system, continuous measurement of net CO2 exchange rates of plants with different photosynthetic pathways were conducted. An obligate CAM plant (Kalanchoe daigremontiana), four facultative CAM plants (Dendrobium officinale, D. chrysotoxum, D. nobile, and D. primulinum), a C3 plant (Strawberry, Fragaria ananassa), and a C4 plant (Corn, Zea mays) were selected as model plants. K. daigremontiana had a significant CO2 absorption during the dark period and its net CO2 exchange rates fluctuated around 0 μmol/(m2•s) during the photoperiod in a growth chamber. Net CO2 exchange rates of F. ananassa and Z. mays in a greenhouse gradually increased after sunrise, reaching a maximum at about 12:00, and then gradually decreased to negative values during the night time. It is interesting to observe that D. officinale in the greenhouse and growth chamber absorbed CO2 during both day and night times. The photosynthetic pathways of D. chrysotoxum, D. nobile, and D. primulinum were also well distinguished by this photosynthesis continuous monitoring system. The results showed that the photosynthesis continuous monitoring system is capable for quantitative evaluation of diurnal net CO2 exchange characteristics not only in the CAM plants but also in small size C3 and C4 plants with low net photosynthetic rates for long-time and high-accuracy measurements. Keywords: diurnal net CO2 exchanges, infrared CO2 analyzer, mass airflow meter, net photosynthetic rate, photosynthetic pathway DOI: 10.25165/j.ijabe.20191203.4885 Citation: Cheng Y S, He D X. A photosynthesis continuous monitoring system for CAM plants. Int J Agric & Biol Eng, 2019; 12(3): 141–146.

Crassulacean Acid Metabolism Abiotic Stress-Responsive Transcription Factors: a Potential Genetic Engineering Approach for Improving Crop Tolerance to Abiotic Stress.

This perspective paper explores the utilization of abiotic stress-responsive transcription factors (TFs) from crassulacean acid metabolism (CAM) plants to improve abiotic stress tolerance in crop plants. CAM is a specialized type of photosynthetic adaptation that enhances water-use efficiency (WUE) by shifting CO2 uptake to all or part of the nighttime when evaporative water losses are minimal. Recent studies have shown that TF-based genetic engineering could be a useful approach for improving plant abiotic stress tolerance because of the role of TFs as master regulators of clusters of stress-responsive genes. Here, we explore the use of abiotic stress-responsive TFs from CAM plants to improve abiotic stress tolerance and WUE in crops by controlling the expression of gene cohorts that mediate drought-responsive adaptations. Recent research has revealed several TF families including AP2/ERF, MYB, WRKY, NAC, NF-Y, and bZIP that might regulate water-deficit stress responses and CAM in the inducible CAM plant Mesembryanthemum crystallinum under water-deficit stress-induced CAM and in the obligate CAM plant Kalanchoe fedtschenkoi. Overexpression of genes from these families in Arabidopsis thaliana can improve abiotic stress tolerance in A. thaliana in some instances. Therefore, we propose that TF-based genetic engineering with a small number of CAM abiotic stress-responsive TFs will be a promising strategy for improving abiotic stress tolerance and WUE in crop plants in a projected hotter and drier landscape in the 21st-century and beyond.

Contribution of carbon fixed by Rubisco and PEPC to phloem export in the Crassulacean acid metabolism plant Kalanchoe daigremontiana.

Crassulacean acid metabolism (CAM) plants exhibit a complex interplay between CO2 fixation by phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco), and carbon demand for CAM maintenance and growth. This study investigated the flux of carbon from PEPC and direct Rubisco fixation to different leaf carbon pools and to phloem sap over the diurnal cycle. Concentrations and carbon isotope compositions of starch, soluble sugars, and organic acids were determined in leaves and phloem exudates of Kalanchoë daigremontiana Hamet et Perr., and related to CO2 fixation by PEPC and Rubisco. Three types of leaf carbon pools could be distinguished. (i) Starch and malate pools were dominant and showed a pattern of reciprocal mobilization and accumulation (85/54 and 13/48 mg C g−1 DW, respective, at the beginning/end of phase I). The carbon isotope composition of these pools was compatible with predominant PEPC fixation (δ13C values of –13 and –11‰ for starch and malate compared to –11‰ of PEPC fixed carbon). (ii) Isotopic composition (–17‰ and –14‰) and concentration of glucose and fructose (2 and 3 mg C g−1 DW, respectively) were not affected by diurnal metabolism, suggesting a low turnover. (iii) Sucrose (1–3 mg C g−1 DW), in contrast, exhibited large diurnal changes in δ13C values (from –17‰ in the evening to –12‰ in the morning), which were not matched by net changes in sucrose concentration. This suggests a high sucrose turnover, fed by nocturnal starch degradation and direct Rubisco fixation during the day. A detailed dissection of the carbon fixation and mobilization pattern in K. daigremontiana revealed that direct fixation of Rubisco during the light accounted for 30% of phloem sucrose, but only 15% of fixed carbon, indicating that carbon from direct Rubisco fixation was preferentially used for leaf export.

Intracellular positioning of nucleus and mitochondria with clumping of chloroplasts in the succulent CAM plant Kalanchoe blossfeldiana: an investigation using fluorescence microscopy

Summary: In leaves of some succulent crassulacean-acid-metabolism(CAM)plants, including Kalanchoe species, we previously discovered the phenomenon of chloroplast clumping. A combination of light and water stress induced chloroplasts to form spherical bodies within mesophyll cells. In this study we focused on the positioning of nuclei and mitochondria with clumping of chloroplasts in mesophyll cells in leaves of the succulent CAM plant Kalanchoe blossfeldiana using fluorescence microscopy. Under both well-watered and water-stressed conditions, there were several mitochondria around each chloroplast. When subjected to water-stress, chloroplasts gathered around a nucleus and formed a clumped chloroplasts. Three-dimensional analysis of the clump showed that the nucleus was localized within the clump. Our findings suggest that the chloroplast movement in succulent CAM plants is closely connected with the nucleus. We discuss the significance of the organelle interactions in the leaves of succulent CAM plant under drought conditions.

Redundancy of Stomatal Control for the Circadian Photosynthetic Rhythm in Kalanchoë daigremontiana Hamet et Perrier

In continuous light, the Crassulacean acid metabolism plant Kalanchoe daigremontiana Hamet et Perrier has a circadian rhythm of gas exchange with peaks occurring during the subjective night. The rhythm of gas exchange is coupled to a weak, reverse phased rhythm of quantum yield of photosystem II (Phi (PSII)). To test if the rhythm of Phi (PSII) persists in the absence of stomatal control, leaves were coated with a thin layer of translucent silicone grease which prevented CO2 and H2O exchange. In spite of this treatment, the rhythm of Phi (PSII) occurred with close to normal phase timing and with a much larger amplitude than in uncoated leaves. The mechanism underlying the Phi (PSII) rhythm in coated leaves can be explained by a circadian activity of phosphoenolpyruvate carboxylase (PEPC). At peaks of PEPC activity, the small amount of CO2 contained in the coated leaf could have become depleted, preventing the carboxylase activity of Rubisco and causing decreases in electron transport rates (observed as deep troughs of Phi (PSII) at 23-h in LL and at ca. 24-h intervals afterwards). Peaks of Phi (PSII) would be caused by a downregulation of PEPC leading to improved supply of CO2 to Rubisco. Substrate limitation of photochemistry at 23 h (trough of Phi (PSII)) was also suggested by the weak response of ETR in coated leaves to stepwise light enhancement. These results show that photosynthetic rhythmicity in K. daigremontiana is independent of stomatal regulation and may originate in the mesophyll.

Research note: Large gene family of phosphoenolpyruvate carboxylase in the crassulacean acid metabolism plant Kalanchoe pinnata (Crassulaceae) characterised by partial cDNA sequence analysis.

Clones coding for a 1100-bp cDNA sequence of phosphoenolpyruvate carboxylase (PEPC) of the constitutive crassulacean acid metabolism (CAM) plant Kalanchoe pinnata (Lam.) Pers., were isolated by reverse transcription-polymerase chain reaction (RT-PCR) and characterised by restriction fragment length polymorphism analysis and DNA sequencing. Seven distinct PEPC isogenes were recovered, four in leaves and three in roots (EMBL accession numbers: AJ344052-AJ344058). Sequence similarity comparisons and distance neighbour-joining calculations separate the seven PEPC isoforms into two clades, one of which contains the three PEPCs found in roots. The second clade contains the four isoforms found in leaves and is divided into two branches, one of which contains two PEPCs most similar with described previously CAM isoforms. Of these two isoforms, however, only one exhibited abundant expression in CAM-performing leaves, but not in very young leaves, which do not exhibit CAM, suggesting this isoform encodes a CAM-specific PEPC. Protein sequence calculations suggest that all isogenes are likely derived from a common ancestor gene, presumably by serial gene duplication events. To our knowledge, this is the most comprehensive identification of a PEPC gene family from a CAM plant, and the greatest number of PEPC isogenes reported for any vascular plant to date.

Contribution of C3 carboxylation to the circadian rhythm of carbon dioxide uptake in a Crassulacean acid metabolism plant Kalanchoë daigremontiana.

During the endogenous circadian rhythm of carbon dioxide uptake in continuous light by a Crassula cean acid metabolism plant, Kalanchoë daigremontiana, the two carboxylating enzymes, phosphoenolpyruvate carboxylase (PEPC) and ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco), are active simultaneously, although, until now, only the role of PEPC in generating the rhythm has been acknowledged. According to the established model, the rhythm is primarily regulated at the PEPC activity level, modulated by periodic compartmentation of its inhibitor, malate, in the vacuole and controlled by tension/relaxation of the tonoplast. However, the circadian accumulation of malic acid (the main indicator of PEPC activity) dampened significantly within the first few periods without affecting the rhythm's amplitude. Moreover, the amount of malate accumulated during a free-running oscillation was several-fold lower than the amount expected if PEPC were the key carboxylating enzyme, based on a 1:1 stoichiometry of CO(2) and malate. Together with the observation that rates of CO(2) uptake under continuous light were higher than in darkness, the evidence shows that C(3) carboxylation greatly contributes to the generation of rhythmic CO(2) uptake in continuous light in this 'obligate' CAM plant. Because the shift from predominantly CAM to predominantly C(3) carboxylation is smooth and does not distort the trajectory of the rhythm, its control probably arises from a robust network of oscillators, perhaps also involving stomata.

Day-to-night variations of cytoplasmic pH in a crassulacean acid metabolism plant.

In crassulacean acid metabolism (CAM) large amounts of malic acid are redistributed between vacuole and cytoplasm in the course of night-to-day transitions. The corresponding changes of the cytoplasmic pH (pHcyt) were monitored in mesophyll protoplasts from the CAM plant Kalanchoe daigremontiana Hamet et Perrier by ratiometric fluorimetry with the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein as a pHcyt indicator. At the beginning of the light phase, pHcyt was slightly alkaline (about 7.5). It dropped during midday by about 0.3 pH units before recovering again in the late-day-to-early-dark phase. In the physiological context the variation in pHcyt may be a component of CAM regulation. Due to its pH sensitivity, phosphoenolpyruvate carboxylase appears as a likely target enzyme. From monitoring delta pHcyt in response to loading the cytoplasm with the weak acid salt K-acetate a cytoplasmic H(+)-buffer capacity in the order of 65 mM H+ per pH unit was estimated at a pHcyt of about 7.5. With this value, an acid load of the cytoplasm by about 10 mM malic acid can be estimated as the cause of the observed drop in pHcyt. A diurnal oscillation in pHcyt and a quantitatively similar cytoplasmic malic acid is predicted from an established mathematical model which allows simulation of the CAM dynamics. The similarity of model predictions and experimental data supports the view put forward in this model that a phase transition of the tonoplast is an essential functional element in CAM dynamics.

Partial purification and characterization of a protein inhibitor of phosphoenolpyruvate carboxylase kinase.

The activity of phosphoenolpyruvate carboxylase (PEPCase) kinase in leaf extracts increased markedly on dilution. This was shown to be caused by the presence of a protein that inhibits the kinase. The inhibitor protein was separated from the kinase and purified partially. It inhibited the kinase reversibly, presumably by a direct interaction; it was neither a protease nor a protein phosphatase. The amounts of kinase and inhibitor in leaves were estimated following separation by hydrophobic chromatography. The amount of inhibitor in the crassulacean acid metabolism plant Kalanchoe fedtschenkoi Hamet et Perrier was sufficient to inhibit the basal level of kinase activity present during the light period and the early stages of the dark period. Similarly, the amount of inhibitor in the C4 plant Zea mays L. was sufficient to inhibit the low amount of kinase activity present in the dark and at moderate light intensity. Analogous to the role of the protein inhibitor of mammalian cyclic AMP-dependent protein kinase, the function of the PEPCase kinase inhibitor may be to inhibit the basal level of kinase present in conditions under which rapid flux through PEPCase is not required.

Spatio-temporal variation of photosynthetic activity during the endogenous circadian CAM rhythm of Kalanchoë daigremontiana: the biological clock as an assembly of coupled nonlinear oscillators

The complex dynamical properties of biological timing in organisms could never be fully accounted for, not even by increasingly precise characterization of oscillating units and their components. The circadian rhythm of CO2 exchange of the crassulacean acid metabolism plant Kalanchoe daigremontiana is regarded as a generic model system for exploration of endogenous rhythmicity in a well understood metabolic pathway. While the CAM-rhythm so far was thought to be solely time-dependent, we show here for the first time that the circadian rhythm of a metabolic cycle in a higher plant is expressed as independently initiated variations in photosynthetic efficiency (f PSII) over a single leaf. Non-invasive, highly sensitive chlorophyll fluorescence imaging under well controlled external conditions reveals dynamic clusters, moving wave fronts and clearly dephased regions of f PSII. This is the first demonstration that this biological clock is a spatio-temporal product of many weakly coupled oscillators. These oscillators are defined by the metabolic constraints of CAM but operate independently in space and time as a consequence of the dynamics of metabolic pools and limitations of CO2 diffusion between tightly packed cells. Several, especially long term aspects of this biological clock are strongly related to the self-organization of the interacting oscillators in space. We propose that synchronization of these individual oscillators is essential for the functioning of the biological clock.

Malate Dehydrogenase Forms a Complex with and Activates Phosphoenolpyruvate Carboxylase from Crassulacean Acid Metabolism Plants

Summary Gel chromatography and kinetic techniques give evidence for the association of malate dehydrogenase (MDH) and phosphoenolpyruvate carboxylase (PEPC) extracted from leaves of the Crassulacean Acid Metabolism plant Kalanchoe blossfeldiana . PEPC and one form of MDH co-eluted in the same peak from Ultrogel AcA 54, Ultrogel AcA 34 or Sephacryl 200. The complex was disrupted by gradient acrylamide gel electrophoresis and reconstituted in vitro by recovering the purified PEPC from the gel in the presence of MDH. This effect of MDH was not obtained with other proteins or enzymes at the same protein concentration. The molecular masses observed for the postulated complex are consistent with the association of one MDH dimer with one PEPC tetrameric form, or under higher ionic strength, one PEPC dimer. The PEPC-MDH interaction was shown to stimulate PEPC activity several times, or to depress it when MDH was in excess, which supports specificity of the effect. The inhibitory action of malate on PEPC activity was affected by the PEPC-MDH association.

Gradient in the degree of Crassulacean acid metabolism within leaves of Kalanchoe daigremontiana

Leaves of the Crassulacean acid metabolism plant Kalanchoe daigremontiana Hamet et Perr., about 3.3 mm thick, showed higher rates of net CO2 exchange through the lower than through the upper surface during day and night, although the lower surface received only a small fraction of the light which was incident on the upper surface. Nocturnal acidification was more pronounced in cells from the lower than from the upper portion of leaves. The lower activity of the exposed side of these long-lived succulent leaves may be related to the potentially adverse effects of excessive light.

Malate Accumulation in Leaf Slices of Mesembryanthemum crystallinum in Relation to Osmotic Gradients Between the Cells and the Medium

Leaf slices of two sets of M. crystallinum plants were used in the present study. The first set were plants grown in 400 mM NaCl and showing diurnal oscillations of malate levels typical of crassulacean acid metabolism (CAM). The second set were plants grown in non-saline media and exhibiting no CAM-like diurnal malate fluctuations. Both sets of leaf slices accumulated malate during a 12-h light or dark period, depending on the osmotic pressure of the incubation medium. Highest malate accumulations were obtained when media were isotonic or slightly hypertonic. These osmotic characteristics are similar to those of leaf slices of the CAM plant Kalanchoe daigremontiana as reported elsewhere. However, discrepancies are observed in light and temperature dependence. Unlike in K. daigremontiana leaf slices and in intact leaves with CAM (i.e. also in intact leaves of M. crystallinum grown on highly saline media), in both sets of M. crystallinum leaf slices used here light stimulated malate accumulation. Compared to 15°C, 25°C had either no effect on malate accumulation or stimulated malate accumulation. After leaf slices had accumulated malate in the dark in isotonic or slightly hypertonic media, malate accumulation continued in the light when the osmolarity of the medium remained unchanged. When the osmotic pressure of the medium was lowered considerably, however, malate accumulation in the light was much reduced or else there was a loss of malate from the tissue. Mechanisms different from CAM may be partially involved in the changes of malate levels in these experiments. The significance of the experimental results for the interpretation of the balance between net carbon gain via C3 pathway and CAM in M. crystallinum is discussed.