Facultative Crassulacean Acid Metabolism Plant Research Articles

Defining Mechanisms of C3 to CAM Photosynthesis Transition toward Enhancing Crop Stress Resilience.

Global climate change and population growth are persistently posing threats to natural resources (e.g., freshwater) and agricultural production. Crassulacean acid metabolism (CAM) evolved from C3 photosynthesis as an adaptive form of photosynthesis in hot and arid regions. It features the nocturnal opening of stomata for CO2 assimilation, diurnal closure of stomata for water conservation, and high water-use efficiency. To cope with global climate challenges, the CAM mechanism has attracted renewed attention. Facultative CAM is a specialized form of CAM that normally employs C3 or C4 photosynthesis but can shift to CAM under stress conditions. It not only serves as a model for studying the molecular mechanisms underlying the CAM evolution, but also provides a plausible solution for creating stress-resilient crops with facultative CAM traits. This review mainly discusses the recent research effort in defining the C3 to CAM transition of facultative CAM plants, and highlights challenges and future directions in this important research area with great application potential.

Mechanisms Underlying the C3–CAM Photosynthetic Shift in Facultative CAM Plants

Crassulacean acid metabolism (CAM), one of three kinds of photosynthesis, is a water-use efficient adaptation to an arid environment. CAM is characterized by CO2 uptake via open stomata during the nighttime and refixation CO2 via the Calvin cycle during the daytime. Facultative CAM plants can shift the photosynthesis from C3 to CAM and exhibit greater plasticity in CAM expression under different environments. Though leaf thickness is an important anatomical feature of CAM plants, there may be no anatomical feature changes during the C3–CAM transition for all facultative CAM plants. The shift from C3 photosynthesis to CAM in facultative CAM plants is accompanied by significant changes in physiology including stomata opening, CO2 gas exchange and organic acid fluxes; the activities of many decarboxylating enzymes increase during the shift from C3 to CAM; the molecular changes occur during the photosynthesis C3–CAM shift involved DNA hypermethylation, transcriptional regulation, post-transcriptional regulation and protein level regulation. Recently, omics approaches were used to discover more proceedings underling the C3–CAM transition. However, there are few reviews on the mechanisms involved in this photosynthetic shift in facultative CAM plants. In this paper, we summarize the progress in the comparative analysis of anatomical, physiological, metabolic and molecular properties of facultative CAM plants between C3 and CAM photosynthesis. Facultative CAM plants also show the potential for sustainable food crop and biomass production. We also discuss the implications of the photosynthesis transition from C3 to CAM on horticultural crops and address future directions for research.

Physiological and transcriptomic analysis uncovers salinity stress mechanisms in a facultative crassulacean acid metabolism plant Dendrobium officinale.

Dendrobium officinale is a precious medicinal Chinese herb that employs facultative crassulacean acid metabolism (CAM) and has a high degree of abiotic stress tolerance, but the molecular mechanism underlying the response of this orchid to abiotic stresses is poorly understood. In this study, we analyzed the root microstructure of D. officinale plantlets and verified the presence of chloroplasts by transmission electron microscopy. To obtain a more comprehensive overview of the molecular mechanism underlying their tolerance to abiotic stress, we performed whole‐transcriptome sequencing of the roots of 10-month-old plantlets exposed to salt (NaCl) treatment in a time‐course experiment (0, 4 and 12 h). The total of 7376 differentially expressed genes that were identified were grouped into three clusters (P < 0.05). Metabolic pathway analysis revealed that the expression of genes related to hormone (such as auxins, cytokinins, abscisic acid, ethylene and jasmonic acid) biosynthesis and response, as well as the expression of genes related to photosynthesis, amino acid and flavonoid metabolism, and the SOS pathway, were either up- or down-regulated after salt treatment. Additionally, we identified an up-regulated WRKY transcription factor, DoWRKY69, whose ectopic expression in Arabidopsis promoted seed germination under salt tress. Collectively, our findings provide a greater understanding of the salt stress response mechanisms in the roots of a facultative CAM plant. A number of candidate genes that were discovered may help plants to cope with salt stress when introduced via genetic engineering.

At the Edges of Photosynthetic Metabolic Plasticity-On the Rapidity and Extent of Changes Accompanying Salinity Stress-Induced CAM Photosynthesis Withdrawal.

The common ice plant (Mesembryanthemum crystallinum L.) is a facultative crassulacean acid metabolism (CAM) plant, and its ability to recover from stress-induced CAM has been confirmed. We analysed the photosynthetic metabolism of this plant during the 72-h response period following salinity stress removal from three perspectives. In plants under salinity stress (CAM) we found a decline of the quantum efficiencies of PSII (Y(II)) and PSI (Y(I)) by 17% and 15%, respectively, and an increase in nonphotochemical quenching (NPQ) by almost 25% in comparison to untreated control. However, 48 h after salinity stress removal, the PSII and PSI efficiencies, specifically Y(II) and Y(I), elevated nonphotochemical quenching (NPQ) and donor side limitation of PSI (YND), were restored to the level observed in control (C3 plants). Swelling of the thylakoid membranes, as well as changes in starch grain quantity and size, have been found to be components of the salinity stress response in CAM plants. Salinity stress induced an over 3-fold increase in average starch area and over 50% decline of average seed number in comparison to untreated control. However, in plants withdrawn from salinity stress, during the first 24 h of recovery, we observed chloroplast ultrastructures closely resembling those found in intact (control) ice plants. Rapid changes in photosystem functionality and chloroplast ultrastructure were accompanied by the induction of the expression (within 24 h) of structural genes related to the PSI and PSII reaction centres, including PSAA, PSAB, PSBA (D1), PSBD (D2) and cp43. Our findings describe one of the most flexible photosynthetic metabolic pathways among facultative CAM plants and reveal the extent of the plasticity of the photosynthetic metabolism and related structures in the common ice plant.

Comparative proteomics of Mesembryanthemum crystallinum guard cells and mesophyll cells in transition from C3 to CAM

Salinity can induce Mesembryanthemum crystallinum to shift its photosynthesis from C3 to crassulacean acid metabolism (CAM), leading to enhanced plant water use efficiency. Studying how M. crystallinum changes its carbon fixation pathways is important for potential translation into crops and enhancing crop resilience. In this study, we examined proteomic changes in guard cells and mesophyll cells in the course of the C3 to CAM transition. We collected enriched guard cells and mesophyll cells during a short period of transition. A total of 1153 proteins were identified and quantified in the two cell-types. During the transition, proteins in the guard cells and mesophyll cells exhibited differential changes. For example, we observed nocturnal carbon fixation in mesophyll cells and proteins involved in cell growth in the two cell-types. Proteins involved in osmotic adjustment, ion transport, energy metabolism and light response may play important roles in the C3 to CAM transition. Real-time PCR experiments were conducted to determine potential correlations between transcript and protein levels. These results have highlighted potential molecular mechanisms underlying the C3 to CAM transition of guard cells and mesophyll cells of the important facultative CAM plant. Biological significanceFresh water resource for agricultural food production is a global challenge. Nature has evolved crassulacean acid metabolism (CAM) plants with enhanced water use efficiency. Using single cell-type proteomics, this study revealed molecular changes taking place in guard cells and mesophyll cells during the shift of ice plant photosynthesis from C3 to CAM. The results have provided important insights into the CAM transition and may facilitate effort toward enhancing crop resilience for global food security.

Photo-Protective Mechanisms and the Role of Poly (ADP-Ribose) Polymerase Activity in a Facultative CAM Plant Exposed to Long-Term Water Deprivation.

The Crassulacean acid metabolism (CAM) pathway helps plants to alleviate the oxidative stress under drought, but the shift to CAM-idling may expose plants to the overproduction of reactive oxygen species causing cell damages. The facultative CAM species Portulacaria afra L., was subjected to long-term water deprivation to assess the photo-protective strategies and the poly (ADP-ribose) polymerase (PARP) activity during water stress and plant capability to recover from the stress. Measurements of titratable acidity, chlorophyll fluorescence emission, and antioxidant activity were performed during the stress and rewatering. Under water deprivation, plants shifted from C3 to CAM metabolism, reaching the CAM-idling status at the end of the stress period. The daily variation of the titratable acidity and PARP activity increased at the beginning of stress and declined with stress progression, reaching the lowest value at the end of stress treatment. H2O2 content, superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities increased with the severity of water stress. The photochemical processes remained high during the entire stress period indicating the presence of alternative sinks to CO2 fixation. The elevated activity of catalase under severe water stress suggests the occurrence of photorespiration in sustaining the photosynthetic electron transport under CAM-idling condition. The overall data indicate that scavenger enzymes, photorespiration and PARP activity modulation contribute to the strong resistance of P. afra to severe water stress, preserving the functioning of photosynthetic apparatus and ensuring plant recovery with rewatering.

Physiological Changes in Mesembryanthemum crystallinum During the C3 to CAM Transition Induced by Salt Stress.

Salt stress impedes plant growth and development, and leads to yield loss. Recently, a halophyte species Mesembryanthemum crystallinum has become a model to study plant photosynthetic responses to salt stress. It has an adaptive mechanism of shifting from C3 photosynthesis to crassulacean acid metabolism (CAM) photosynthesis under stresses, which greatly enhances water usage efficiency and stress tolerance. In this study, we focused on investigating the morphological and physiological changes [e.g., leaf area, stomatal movement behavior, gas exchange, leaf succulence, and relative water content (RWC)] of M. crystallinum during the C3 to CAM photosynthetic transition under salt stress. Our results showed that in M. crystallinum seedlings, CAM photosynthesis was initiated after 6 days of salt treatment, the transition takes place within a 3-day period, and plants became mostly CAM in 2 weeks. This result defined the transition period of a facultative CAM plant, laid a foundation for future studies on identifying the molecular switches responsible for the transition from C3 to CAM, and contributed to the ultimate goal of engineering CAM characteristics into C3 crops.

Drought does not induce crassulacean acid metabolism (CAM) but regulates photosynthesis and enhances nutritional quality of Mesembryanthemum crystallinum.

Physiology and nutritional quality of a facultative CAM plant Mesembryanthemum crystallinum under drought stress alone are poorly understood. To induce drought, M. crystallinum was cultured aeroponically with different nutrient spraying intervals such as 5, 30, 60 and 240 min. The long spraying interval such as 240 min resulted in lower mass of root and shoot, shorter total root length with less tips and smaller surface area, compared to short interval of 5 min. Grown under the longest spraying interval of 240 min, M. crystallinumalso had significantly higher leaf dry matter content but lower leaf succulence. However, CAM acidity was undetectable for any plants. Although M. crystallinum grown under extended spraying intervals had higher photosynthetic pigments, they utilized lesser light energy and did not dissipate heat as effectively as those grown under 5 min. Compare to other shorter spraying intervals, photosynthetic gas exchange rates were significantly reduced under 240 min spraying interval, indicating signs of water deficit stress. Shoot nitrate, total reduced nitrogen, total soluble protein and Rubisco concentrations were similar for all plants. For phytochemicals and dietary minerals, plants grown under 240 min spraying interval had significantly higher values than the other plants. Therefore, drought does not result in the induction of CAM but regulates photosynthetic performance and enhances nutritional quality of M. crystallinum.

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.

Effect of Light/Dark Cycle on Photosynthetic Pathway Switching and CO2 Absorption in Two Dendrobium Species.

Many Dendrobium species are both ornamental and medicinal plants in China. Several wild species have been exploited to near extinction, and facility cultivation has become an important way to meet the great market demand. Most Dendrobium species have evolved into crassulacean acid metabolism (CAM) pathways in adapting to harsh epiphytic environment, leading to low daily net CO2 absorption. Photosynthetic pathways of many facultative CAM plants are regulated by various environmental factors. Light/dark cycle plays an important role in regulating the photosynthetic pathway of several CAM species. The aims of this study were to investigate whether the photosynthetic pathway of Dendrobium species could be regulated between C3 and CAM by changing light/dark cycles and the daily net CO2 absorption could be enhanced by shortening light/dark cycle. In this study, net CO2 exchange rates of D. officinale and D. primulinum were monitored continuously during two different light/dark cycles conversion compared to Kalanchoe daigremontiana as an obligate CAM plant. The net CO2 exchange pattern and stomatal behavior of D. officinale and D. primulinum were switched from CAM to C3-like by changing the light/dark cycle from 12/12 h to 4/4 h. However, this switching was not completely reversible. Compared to the original 12/12 h light/dark cycle, the dark, light, and daily net CO2 exchange amount of D. officinale were significantly increased after the light/dark cycle was changed from 4/4 h to 12/12 h, but those in D. primulinum was opposite and those in K. daigremontiana was not affected. Daily net CO2 exchange amount of D. officinale increased by 47% after the light/dark cycle was changed from 12/12 h to 4/4 h, due to the sharp increase of light net CO2 exchange amount. However, the large decrease of dark net CO2 exchange amount could not be offset by increased light net CO2 exchange amount, leading to reduced daily net CO2 exchange amount of D. primulinum. In conclusion, the 4/4 h light/dark cycle can induce the photosynthetic pathway of D. officinale and D. primulinum to C3-like, and improve the daily CO2 absorption of D. officinale.

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.

The effect of nitrogen availability and water conditions on competition between a facultative CAM plant and an invasive grass.

Plants with crassulacean acid metabolism (CAM) are increasing their abundance in drylands worldwide. The drivers and mechanisms underlying the increased dominance of CAM plants and CAM expression (i.e., nocturnal carboxylation) in facultative CAM plants, however, remain poorly understood. We investigated how nutrient and water availability affected competition between Mesembryanthemum crystallinum (a model facultative CAM species) and the invasive C3 grass Bromus mollis that co‐occur in California's coastal grasslands. Specifically we investigated the extent to which water stress, nutrients, and competition affect nocturnal carboxylation in M. crystallinum. High nutrient and low water conditions favored M. crystallinum over B. mollis, in contrast to high water conditions. While low water conditions induced nocturnal carboxylation in 9‐week‐old individuals of M. crystallinum, in these low water treatments, a 66% reduction in nutrient applied over the entire experiment did not further enhance nocturnal carboxylation. In high water conditions M. crystallinum both alone and in association with B. mollis did not perform nocturnal carboxylation, regardless of the nutrient levels. Thus, nocturnal carboxylation in M. crystallinum was restricted by strong competition with B. mollis in high water conditions. This study provides empirical evidence of the competitive advantage of facultative CAM plants over grasses in drought conditions and of the restricted ability of M. crystallinum to use their photosynthetic plasticity (i.e., ability to switch to CAM behavior) to compete with grasses in well‐watered conditions. We suggest that a high drought tolerance could explain the increased dominance of facultative CAM plants in a future environment with increased drought and nitrogen deposition, while the potential of facultative CAM plants such as M. crystallinum to expand to wet environments is expected to be limited.

Plant Growth and Photosynthetic Characteristics of Mesembryanthemum crystallinum Grown Aeroponically under Different Blue- and Red-LEDs.

Mesembryanthemum crystallinum is a succulent, facultative crassulacean acid metabolism (CAM) plant. Plant growth and photosynthetic characteristics were studied when M. crystallinum plants were grown indoor under light emitting diodes (LED)-lighting with adequate water supply. Plants were cultured aeroponically for a 16-h photoperiod at an equal photosynthetic photon flux density of 350 μmol m-2 s-1 under different red:blue LED ratios: (1) 100:0 (0B); (2) 90:10 (10B); (3) 80:20 (20B); (4) 70:30 (30B); (5) 50:50 (50B); and (6)100:0 (100B). M. crystallinum grown under 10B condition had the highest shoot and root biomass and shoot/root ratio while those grown under 0B condition exhibited the lowest values. Compared to plants grown under 0B condition, all other plants had similar but higher total chlorophyll (Chl) and carotenoids (Car) contents and higher Chl a/b ratios. However, there were no significant differences in Chl/Car ratio among all plants grown under different red- and blue-LEDs. Photosynthetic light use efficiency measured by photochemical quenching, non-photochemical quenching, and electron transport rate, demonstrated that plants grown under high blue-LED utilized more light energy and had more effective heat dissipation mechanism compared to plants grown under 0B or lower blue-LED. Statistically, there were no differences in photosynthetic O2 evolution rate, light-saturated CO2 assimilation rate (Asat), and light-saturated stomatal conductance (gssat) among plants grown under different combined red- and blue-LEDs but they were significantly higher than those of 0B plants. No statistically differences in total reduced nitrogen content were found among all plants. For the total soluble protein, all plants grown under different combined red- and blue-LEDs had similar values but they were significantly higher than that of plants grown under 0B condition. However, plants grown under higher blue-LEDs had significant higher ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) protein than those plants grown under lower blue-LED. High Asat and gssat but very low CAM acidity of all M. crystallinum plants during light period, imply that this facultative CAM plant performed C3 photosynthesis when supplied with adequate water. Results of this study suggest that compared to red- or blue-LED alone, appropriate combination of red- and blue-LED lighting enhanced plant growth and photosynthetic capacities of M. crystallinum.

A coupled model of photosynthesis and stomatal conductance for the ice plant (Mesembryanthemum crystallinum L.), a facultative CAM plant

The ice plant (Mesembryanthemum crystallinum L.), a medicinal plant with well-known effects on retarding diabetes mellitus, is increasingly being produced in plant factories in Asia. The ice plant is a Crassulacean Acid Metabolism (CAM) plant, but performs C3 photosynthesis during the juvenile period. The objective of this study was to develop a photosynthetic model of ice plants growing under plant factory conditions. C3 photosynthesis was observed in juvenile plants in plant factory growth conditions and a conversion from C3 to CAM photosynthesis was observed under salt-stressed condition at an electrical conductivity (EC) of 6.0 dS·m-1. The light saturation and compensation points, determined by a regression analysis of C3 light curves for the ice plant leaves, were 609.4 and 53.2 μmol·m-2·s-1, respectively. The accuracy of the light response was compared between negative exponential and non-rectangular hyperbolic function models. The non-rectangular hyperbola was more accurate with complicated parameters while the negative exponential function was more practical with simple parameters in the light response curves. Measurement of net CO2 assimilation rate (A) and intercellular CO2 concentration (C i ) allowed construction of the A-Ci curve and regression analysis of this curve revealed the CO2 saturation and compensation points as 632.9 and 117.2 μmol·mol-1, respectively. A coupled photosynthetic model was developed for the simultaneous prediction of photosynthesis, stomatal conductance, transpiration, and temperature of the ice plant leaves. Sharkey’s regression method was used to determine the photosynthetic parameters of maximum carboxylation rate, the potential rate of electron transport, and the rate of triose phosphate utilization, which were 222.3, 234.9, and 13.0 μmol·m-2·s-1, respectively. The parameters of minimum stomatal conductance of water vapor at the light compensation point (b) and the empirical coefficient (m) for the sensitivity of stomatal conductance and relative humidity in the Ball, Woodrow and Berry model could be solved as b = 0.0487 and m = 0.0012 by linear regression analysis using the measured A-Ci values. Although the A-Ci curve of the negative exponential function had higher accuracy than the biochemical model, the coupled biochemical model could physiologically explain the photosynthesis of the ice plant leaves under plant factory conditions.

Concomitant CAM and C3 Photosynthetic Pathways in Dendrobium officinale Plants

Dendrobium officinale, endemic to China, is a rare and endangered medicinal herb. As a result of its high economic value, slow growth, and diminishing wild population, protected cultivation is preferred. However, little information is available on its growing environment and photosynthetic characteristics. In this study, the photosynthetic patterns of D. officinale were investigated under various environmental conditions by measuring the net CO2 exchange rates continuously for several days or weeks. Under non-stressed growth chamber conditions with 12-hour light and 12-hour dark periods, D. officinale had concomitance of C3 and crassulacean acid metabolism (CAM) photosynthesis patterns. Different degrees of CAM in D. officinale, expressed as the percentage of CO2 exchanges in the dark period to the daily amount of CO2 exchanges, were observed depending on environmental conditions. With decreasing substrate water content, a typical CAM pattern was found, and concomitance of C3 and CAM patterns was found again when plants were rewatered. The accumulation of leaf titratable acidity during a dark period increased as substrate dried out but decreased again as plants were rewatered. A shorter light–dark cycle (4-hour light and 4-hour dark periods) led to a C3 pattern alone. The substrate moisture and light–dark cycle were inducible factors for switching between C3 and CAM patterns in D. officinale. These results indicate that D. officinale is a facultative CAM plant and the C3 pathway can be induced by controlling the growing environment. Further studies are needed to identify the optimal environmental conditions to enhance the growth of D. officinale.

The evolution of autotrophy in relation to phosphorus requirement

The evolution of autotrophy is considered in relation to the availability of phosphorus (P), the ultimate elemental resource limiting biological productivity through Earth's history. Work on microbes and plants is emphasized, dealing in turn with the main uses for P in cells, namely nucleic acids, phospholipids, and water-soluble low molecular mass phosphate esters plus metabolically active inorganic orthophosphate. There is a greater minimum gene number and minimum DNA content in autotrophic than in osmochemoorganotrophic archaea and bacteria, as well as a lower rate of biomass increase per unit P (P-use efficiency) in autotrophs than in osmochemoorganotrophs, in eukaryotes as well as bacteria. This may be due to the diversion of rRNA from producing proteins common to all organisms to producing highly expressed proteins specific to autotrophs. The P requirement for phospholipids is decreased in oxygenic photolithotrophs, and some anoxygenic photolithotrophs, by substituting galactolipids and sulpholipids for phospholipids in the photosynthetic, and some other, membranes. The six different autotrophic inorganic carbon assimilation pathways have varying requirements for low molecular mass water-soluble phosphate esters. In oxygenic photolithotrophs, there is no clear evidence of a different P requirement for growth in the absence (diffusive CO2 entry) relative to the presence of CO2-concentrating mechanisms (CCMs). P limitation increases the expression of crassulacean acid metabolism (CAM) in facultative CAM plants, decreases the extent of inorganic carbon accumulation in algae with CCMs, and (usually) their inorganic carbon affinity and the water-use efficiency of growth of terrestrial plants, and the light-use efficiency of photolithotrophs.

Isolation and Characterization of a Polyubiquitin Gene and Its Promoter Region fromMesembryanthemum crystallinum

Transcript levels of the polyubiquitin gene McUBI1 had been reported to be constant during Crassulacean acid metabolism (CAM) induction in the facultative CAM plant, Mesembryanthemum crystallinum. Here, we report the sequences of the full-length cDNA of McUBI1 and its promoter, and validation of the McUBI1 promoter as an internal control driving constitutive expression in transient assays using the dual-luciferase system to investigate the regulation of CAM-related gene expression. The McUBI1 promoter drove strong, constitutive expression during CAM induction. We compared the activities of this promoter with those of the cauliflower mosaic virus (CaMV) 35S promoter in detached C3- and CAM-performing M. crystallinum and tobacco leaves. We confirmed stable expression of the genes controlled by the McUBI1 promoter with far less variability than under the CaMV 35S promoter in M. crystallinum, whereas both promoters worked well in tobacco. We found the McUBI1 promoter more suitable than the CaMV 35S promoter as an internal control for transient expression assays in M. crystallinum.

Evolutionary physiology: the extent of C4 and CAM photosynthesis in the genera Anacampseros and Grahamia of the Portulacaceae

The Portulacaceae is one of the few terrestrial plant families known to have both C(4) and Crassulacean acid metabolism (CAM) species. There may be multiple origins of the evolution of CAM within the Portulacaceae but the only clear evidence of C(4) photosynthesis is found in members of the genus Portulaca. In the Portulaca, CAM succulent tissue is overlaid with the C(4) tissue in a unique fashion where both pathways are operating simultaneously. Earlier reports have shown that the clade containing the genera Anacampseros and Grahamia may also contain C(4) photosynthetic species similar to the Portulaca, which would indicate multiple origins of C(4) photosynthesis within the family. The aim of the present study was to ascertain the true photosynthetic nature of these genera. An initial survey of the carbon isotope composition of the Anacampseros ranged from -12.6 per thousand to -24.0 per thousand, indicating very little CAM activity in some species, with other values close to the C(4) range. Anacampseros (=Grahamia) australiana which had been previously identified as a C(4) species had a carbon isotope composition value of -24.0 per thousand, which is more indicative of a C(3) species with a slight contribution of CAM activity. Other Anacampseros species with C(4)-like values have been shown to be CAM plants. The initial isotope analysis of the Grahamia species gave values in the range of -27.1 per thousand to -23.6 per thousand, placing the Grahamia species well towards the C(3) photosynthetic range. Further physiological studies indicated increased night-time CO(2) uptake with imposition of water stress, associated with a large diurnal acid fluctuation and a marked increased phosphoenolpyruvate carboxylase activity. This showed that the Grahamia species are actually facultative CAM plants despite their C(3)-like carbon isotope values. The results indicate that the Grahamia and Anacampseros species do not utilize the C(4) photosynthetic pathway. This is the first to identify that the Grahamia species are facultative CAM plants where CAM can be induced by water stress. This work supports earlier physiological work that indicates that this clade containing Anacampseros and Grahamia species comprises predominantly facultative CAM plants. This report suggests there may be only one clade which contains C(4) photosynthetic members with CAM-like characteristics.

Na+/H+-transporter, H+-pumps and an aquaporin in light and heavy tonoplast membranes from organic acid and NaCl accumulating vacuoles of the annual facultative CAM plant and halophyte Mesembryanthemum crystallinum L.

Crassulacean acid metabolism (CAM) was induced in Mesembryanthemum crystallinum L. by either NaCl- or high light (HL)- stress. This generated in mesophyll cells predominantly of NaCl-stressed plants two different types of vacuoles: the generic acidic vacuoles for malic acid accumulation and additionally less acidic ("neutral") vacuoles for NaCl sequestration. To examine differences in the tonoplast properties of the two types of vacuoles, we separated microsomal membranes of HL- and NaCl-stressed M. crystallinum plants by centrifugation in sucrose density gradients. Positive immunoreactions of a set of antibodies directed against tonoplast specific proteins and tonoplast specific ATP- and PPi-hydrolytic activity were used as markers for vacuolar membranes. With these criteria tonoplast membranes were detected in both HL- and NaCl-stressed plants in association with the characteristic low sucrose density but also at an unusual high sucrose density. In HL-stressed plants most of the ATP- and PPi-hydrolytic activity and cross reactivity with antibodies including that directed against the Na+/H+-antiporter from Arabidopsis thaliana was detected with light sucrose density. This relationship was inverted in NaCl-stressed plants; they exhibited most pump activity and immunoreactivity in the heavy fraction. The relative abundance of the heavy membrane fraction reflects the relative occurrence of "neutral" vacuoles in either HL- or NaCl-stressed plants. This suggests that tonoplasts of the "neutral" vacuoles sediment at high sucrose densities. This is consistent with the view that this type of vacuoles serves for Na+ sequestration and is accordingly equipped with a high capacity of proton pumping and Na+ uptake via the Na+/H+-antiporter.

Redox changes in the chloroplast and hydrogen peroxide are essential for regulation of C(3)-CAM transition and photooxidative stress responses in the facultative CAM plant Mesembryanthemum crystallinum L.

Mesembryanthemum crystallinum, a facultative halophyte and C(3)-Crassulacean acid metabolism (CAM) intermediate plant, has become a favoured plant for studying stress response mechanisms during C(3)-CAM shifts. One hour of exposure to excess light (EL) caused inhibition of photosynthetic electron transport in M. crystallinum leaves as indicated by chlorophyll a fluorescence measurements. This was accompanied by an increase in NADP-malic enzyme (ME), one of the key cytosolic enzymes involved in CAM, and by a general increase in superoxide dismutase (SOD) activity. In contrast, NAD-ME activity (the mitochondrial form of ME) was not affected by EL. Exposure to EL and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) treatment of a whole plant in low light induced hydrogen peroxide (H(2)O(2)) and C(3) to CAM transition. In contrast, treatment with 3-3,4-dichlorophenyl-1,1-dimethyl urea (DCMU) has blocked high light-induced H(2)O(2) accumulation and C(3)-CAM transition. Moreover, the abundance of transcripts encoding different SODs, ascorbate peroxidase and SOD activity was differently regulated by DCMU and DBMIB. Results of applying EL or high light, H(2)O(2) and photosynthetic electron transport inhibitors suggest that the redox events in the vicinity of PSII and/or PSI and photo-produced H(2)O(2) play a major role in the regulation of C(3)-CAM transition and photooxidative stress responses in M. crystallinum.