Ethylene inhibits photosynthesis via temporally distinct responses in tomato plants.

Ethylene is a volatile plant hormone that regulates many developmental processes and responses toward (a)biotic stress. Studies have shown that high levels of ethylene repress vegetative growth in many important crops, including tomato (Solanum lycopersicum), possibly by inhibiting photosynthesis. We investigated the temporal effects of ethylene on young tomato plants using an automated ethylene gassing system to monitor the physiological, biochemical, and molecular responses through time course RNA-seq of a photosynthetically active source leaf. We found that ethylene evokes a dose-dependent inhibition of photosynthesis, which can be characterized by 3 temporally distinct phases. The earliest ethylene responses that marked the first phase and occurred a few hours after the start of the treatment were leaf epinasty and a decline in stomatal conductance, which led to lower light perception and CO2 uptake, respectively, resulting in a rapid decline of soluble sugar levels (glucose, fructose). The second phase of the ethylene effect was marked by low carbohydrate availability, which modulated plant energy metabolism to adapt by using alternative substrates (lipids and proteins) to fuel the TCA cycle. Long-term continuous exposure to ethylene led to the third phase, characterized by starch and chlorophyll breakdown, which further inhibited photosynthesis, leading to premature leaf senescence. To reveal early (3 h) ethylene-dependent regulators of photosynthesis, we performed a ChIP-seq experiment using anti-ETHYLENE INSENSITIVE 3-like 1 (EIL1) antibodies and found several candidate transcriptional regulators. Collectively, our study revealed a temporal sequence of events that led to the inhibition of photosynthesis by ethylene and identified potential transcriptional regulators responsible for this regulation.

How to resolve the enigma of diurnal malate remobilisation from the vacuole in plants with crassulacean acid metabolism?

Opening of stomata in plants with crassulacean acid metabolism (CAM) is mainly shifted to the night period when atmospheric CO2 is fixed by phosphoenolpyruvate carboxylase and stored as malic acid in the vacuole. As such, CAM plants ameliorate transpirational water losses and display substantially higher water-use efficiency compared with C3 and C4 plants. In the past decade significant technical advances have allowed an unprecedented exploration of genomes, transcriptomes, proteomes and metabolomes of CAM plants and efforts are ongoing to engineer the CAM pathway in C3 plants. Whilst research efforts have traditionally focused on nocturnal carboxylation, less information is known regarding the drivers behind diurnal malate remobilisation from the vacuole that liberates CO2 to be fixed by RuBisCo behind closed stomata. To shed more light on this process, we provide a stoichiometric analysis to identify potentially rate-limiting steps underpinning diurnal malate mobilisation and help direct future research efforts. Within this remit we address three key questions: Q1 Does light-dependent assimilation of CO2 via RuBisCo dictate the rate of malate mobilisation? Q2: Do the enzymes responsible for malate decarboxylation limit daytime mobilisation from the vacuole? Q3: Does malate efflux from the vacuole set the pace of decarboxylation?

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.

Light quality affects light harvesting and carbon sequestration during the diel cycle of crassulacean acid metabolism in Phalaenopsis.

Crassulacean acid metabolism (CAM) is a specialized photosynthetic pathway present in a variety of genera including many epiphytic orchids. CAM is under circadian control and can be subdivided into four discrete phases during a diel cycle. Inherent to this specific mode of metabolism, carbohydrate availability is a limiting factor for nocturnal CO2 uptake and biomass production. To evaluate the effects of light quality on the photosynthetic performance and diel changes in carbohydrates during the CAM cycle. Phalaenopsis plants were grown under four different light qualities (red, blue, red + blue and full spectrum white light) at a fluence of 100 µmol m-2 s-1 and a photoperiod of 12 h for 8 weeks. In contrast to monochromatic blue light, plants grown under monochromatic red light showed already a significant decline of the quantum efficiency (ΦPSII) after 5 days and of the maximum quantum yield (Fv/Fm) after 10 days under this treatment. This was also reflected in a compromised chlorophyll and carotenoid content and total diel CO2 uptake under red light in comparison with monochromatic blue and full spectrum white light. In particular, CO2 uptake during nocturnal phase I was affected under red illumination resulting in a reduced amount of vacuolar malate. In addition, red light caused the rate of decarboxylation of malate during the day to be consistently lower and malic acid breakdown persisted until 4 h after dusk. Because the intrinsic activity of PEPC was not affected, the restricted availability of storage carbohydrates such as starch was likely to cause these adverse effects under red light. Addition of blue to the red light spectrum restored the diel fluxes of carbohydrates and malate and resulted in a significant enhancement of the daily CO2 uptake, pigment concentration and biomass formation.

Hierarchical clustering reveals unique features in the diel dynamics of metabolites in the CAM orchid Phalaenopsis.

Crassulacean acid metabolism (CAM) is a major adaptation of photosynthesis that involves temporally separated phases of CO2 fixation and accumulation of organic acids at night, followed by decarboxylation and refixation of CO2 by the classical C3 pathway during the day. Transitory reserves such as soluble sugars or starch are degraded at night to provide the phosphoenolpyruvate (PEP) and energy needed for initial carboxylation by PEP carboxylase. The primary photosynthetic pathways in CAM species are well known, but their integration with other pathways of central C metabolism during different phases of the diel light-dark cycle is poorly understood. Gas exchange was measured in leaves of the CAM orchid Phalaenopsis 'Edessa' and leaves were sampled every 2 h during a complete 12-h light-12-h dark cycle for metabolite analysis. A hierarchical agglomerative clustering approach was employed to explore the diel dynamics and relationships of metabolites in this CAM species, and compare these with those in model C3 species. High levels of 3-phosphoglycerate (3PGA) in the light activated ADP-glucose pyrophosphorylase, thereby enhancing production of ADP-glucose, the substrate for starch synthesis. Trehalose 6-phosphate (T6P), a sugar signalling metabolite, was also correlated with ADP-glucose, 3PGA and PEP, but not sucrose, over the diel cycle. Whether or not this indicates a different function of T6P in CAM plants is discussed. T6P levels were low at night, suggesting that starch degradation is regulated primarily by circadian clock-dependent mechanisms. During the lag in starch degradation at dusk, carbon and energy could be supplied by rapid consumption of a large pool of aconitate that accumulates in the light. Our study showed similarities in the diel dynamics and relationships between many photosynthetic metabolites in CAM and C3 plants, but also revealed some major differences reflecting the specialized metabolic fluxes in CAM plants, especially during light-dark transitions and at night.

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.

Light quality modulates metabolic synchronization over the diel phases of crassulacean acid metabolism.

Temporal compartmentation of carboxylation processes is a defining feature of crassulacean acid metabolism and involves circadian control of key metabolic and transport steps that regulate the supply and demand for carbon over a 24h cycle. Recent insights on the molecular workings of the circadian clock and its connection with environmental inputs raise new questions on the importance of light quality and, by analogy, certain photoreceptors for synchronizing the metabolic components of CAM. The present work tested the hypothesis that optimal coupling of stomatal conductance, net CO2 uptake, and the reciprocal turnover of carbohydrates and organic acids over the diel CAM cycle requires both blue and red light input signals. Contrasting monochromatic wavelengths of blue, green, and red light (i.e. 475, 530, 630nm) with low fluence rates (10 µmol m(-2) s(-1)) were administered for 16 hours each diel cycle for a total treatment time of 48 hours to the obligate CAM bromeliad, Aechmea 'Maya'. Of the light treatments imposed, low-fluence blue light was a key determinant in regulating stomatal responses, organic acid mobilization from the vacuole, and daytime decarboxylation. However, the reciprocal relationship between starch and organic acid turnover that is typical for CAM was uncoupled under low-fluence blue light. Under low-fluence red or green light, the diel turnover of storage carbohydrates was orchestrated in line with the requirements of CAM, but a consistent delay in acid consumption at dawn compared with plants under white or low-fluence blue light was noted. Consistent with the acknowledged influences of both red and blue light as input signals for the circadian clock, the data stress the importance of both red and blue-light signalling pathways for synchronizing the metabolic and physiological components of CAM over the day/night cycle.

Sedoheptulose accumulation under CO2 enrichment in leaves of Kalanchoë pinnata: a novel mechanism to enhance C and P homeostasis?

In contrast to the well-documented roles of its mono- and bisphosphate esters, the occurrence of free sedoheptulose in plant tissues remains a matter of conjecture. The present work sought to determine the origin of sedoheptulose formation in planta, as well as its physiological importance. Elevated CO2 and sucrose induction experiments were used to study sedoheptulose metabolism in the Crassulacean acid metabolism (CAM) plants Kalanchoë pinnata and Sedum spectabile. Experimental evidence suggested that sedoheptulose is produced from the oxidative pentose phosphate pathway intermediate sedoheptulose-7-phosphate, by a sedoheptulose-7-phosphate phosphatase. Carbon flux through this pathway was stimulated by increased triose-phosphate levels (elevated CO2, compromised sink availability, and sucrose incubation of source leaves) and attenuated by ADP and inorganic phosphate (Pi). The accumulation of free sedoheptulose is proposed to act as a mechanism contributing to both C and P homeostasis by serving as an alternative carbon store under elevated CO2 or a compromised sink capacity to avoid sucrose accumulation, depletion of inorganic phosphate, and suppression of photosynthesis. It remains to be established whether this acclimation-avoiding mechanism is confined to CAM plants, which might be especially vulnerable to Pi imbalances, or whether some C3 and C4 plants also dispose of the genetic capacity to induce and accelerate sedoheptulose synthesis upon CO2 elevation.

Effect of ethylene pretreatment on tomato plant responses to salt, drought, and waterlogging stress.

Salinity, drought, and waterlogging are common environmental stresses that negatively impact plant growth, development, and productivity. One of the responses to abiotic stresses is the production of the phytohormone ethylene, which induces different coping mechanisms that help plants resist or tolerate stress. In this study, we investigated if an ethylene pretreatment can aid plants in activating stress-coping responses prior to the onset of salt, drought, and waterlogging stress. Therefore, we measured real-time transpiration and CO2 assimilation rates and the impact on biomass during and after 3 days of abiotic stress. Our results showed that an ethylene pretreatment of 1 ppm for 4 h did not significantly influence the negative effects of waterlogging stress, while plants were more sensitive to salt stress as reflected by enhanced water losses due to a higher transpiration rate. However, when exposed to drought stress, an ethylene pretreatment resulted in reduced transpiration rates, reducing water loss during drought stress. Overall, our findings indicate that pretreating tomato plants with ethylene can potentially regulate their responses during the forthcoming stress period, but optimization of the ethylene pre-treatment duration, timing, and dose is needed. Furthermore, it remains tested if the effect is related to the stress duration and severity and whether an ethylene pretreatment has a net positive or negative effect on plant vigor during stress recovery. Further investigations are needed to elucidate the mode of action of how ethylene priming impacts subsequent stress responses.

Effects of chilling on the photosynthetic performance of the CAM orchid Phalaenopsis.

Introduction: Crassulacean acid metabolism (CAM) is one of the three main metabolic adaptations for CO2 fixation found in plants. A striking feature for these plants is nocturnal carbon fixation and diurnal decarboxylation of malic acid to feed Rubisco with CO2 behind closed stomata, thereby saving considerable amounts of water. Compared to the effects of high temperatures, drought, and light, much less information is available about the effects of chilling temperatures on CAM plants. In addition a lot of CAM ornamentals are grown in heated greenhouses, urging for a deeper understanding about the physiological responses to chilling in order to increase sustainability in the horticultural sector. Methods: The present study focuses on the impact of chilling temperatures (10°C) for 3 weeks on the photosynthetic performance of the obligate CAM orchid Phalaenopsis 'Edessa'. Detailed assessments of the light reactions were performed by analyzing chlorophyll a fluorescence induction (OJIP) parameters and the carbon fixation reactions by measuring diel leaf gas exchange and diel metabolite patterns. Results and Discussion: Results showed that chilling already affected the light reactions after 24h. Whilst the potential efficiency of photosystem II (PSII) (Fv/Fm) was not yet influenced, a massive decrease in the performance index (PIabs) was noticed. This decrease did not depict an overall downregulation of PSII related energy fluxes since energy absorption and dissipation remained uninfluenced whilst the trapped energy and reduction flux were upregulated. This might point to the presence of short-term adaptation mechanisms to chilling stress. However, in the longer term the electron transport chain from PSII to PSI was affected, impacting both ATP and NADPH provision. To avoid over-excitation and photodamage plants showed a massive increase in thermal dissipation. These considerations are also in line with carbon fixation data showing initial signs of cold adaptation by achieving comparable Rubisco activity compared to unstressed plants but increasing daytime stomatal opening in order to capture a higher proportion of CO2 during daytime. However, in accordance with the light reactions data, Rubisco activity declined and stomatal conductance and CO2 uptake diminished to near zero levels after 3 weeks, indicating that plants were not successful in cold acclimation on the longer term.

Elucidating the microbiome of the sustainable peat replacers composts and nature management residues.

Sustainable peat alternatives, such as composts and management residues, are considered to have beneficial microbiological characteristics compared to peat-based substrates. Studies comparing microbiological characteristics of these three types of biomass are, however, lacking. This study examined if and how microbiological characteristics of subtypes of composts and management residues differ from peat-based substrates, and how feedstock and (bio)chemical characteristics drive these characteristics. In addition, microbiome characteristics were evaluated that may contribute to plant growth and health. These characteristics include: genera associated with known beneficial or harmful microorganisms, microbial diversity, functional diversity/activity, microbial biomass, fungal to bacterial ratio and inoculation efficiency with the biocontrol fungus Trichoderma harzianum. Bacterial and fungal communities were studied using 16S rRNA and ITS2 gene metabarcoding, community-level physiological profiling (Biolog EcoPlates) and PLFA analysis. Inoculation with T. harzianum was assessed using qPCR. Samples of feedstock-based subtypes of composts and peat-based substrates showed similar microbial community compositions, while subtypes based on management residues were more variable in their microbial community composition. For management residues, a classification based on pH and hemicellulose content may be relevant for bacterial and fungal communities, respectively. Green composts, vegetable, fruit and garden composts and woody composts show the most potential to enhance plant growth or to suppress pathogens for non-acidophilic plants, while grass clippings, chopped heath and woody fractions of compost show the most potential for blends for calcifuge plants. Fungal biomass was a suitable predictor for inoculation efficiency of composts and management residues.

The photosynthetic plasticity of crassulacean acid metabolism: an evolutionary innovation for sustainable productivity in a changing world.

SUMMARY: The photosynthetic specialization of crassulacean acid metabolism (CAM) has evolved many times in response to selective pressures imposed by water limitation. Integration of circadian and metabolite control over nocturnal C4 and daytime C3 carboxylation processes in CAM plants provides plasticity for optimizing carbon gain and water use by extending or curtailing the period of net CO2 uptake over any 24-h period. Photosynthetic plasticity underpins the ecological diversity of CAM species and contributes to the potential for high biomass production in water-limited habitats. Perceived evolutionary constraints on the dynamic range of CO2 acquisition strategies in CAM species can be reconciled with functional anatomical requirements and the metabolic costs of maintaining the enzymatic machinery required for C3 and C4 carboxylation processes. Succulence is highlighted as a key trait for maximizing biomass productivity in water-limited habitats by serving to buffer water availability, by maximizing the magnitude of nocturnal CO2 uptake and by extending the duration of C4 carboxylation beyond the night period. Examples are discussed where an understanding of the diverse metabolic and ecological manifestations of CAM can be exploited for the sustainable productivity of economically and ecologically important species.

Crassulacean acid metabolism under severe light limitation: a matter of plasticity in the shadows?

Despite the increased energetic costs of CAM compared with C(3) photosynthesis, it is hypothesized that the inherent photosynthetic plasticity of CAM allows successful acclimation to light-limiting conditions. The present work sought to determine if CAM presented any constraints to short and longer term acclimation to light limitation and to establish if and how metabolic and photosynthetic plasticity in the deployment of the four phases of CAM might facilitate acclimation to conditions of deep shade. Measurements of leaf gas exchange, organic acids, starch and soluble sugar (glucose, fructose, and sucrose) contents were made in the leaves of the constitutive CAM bromeliad Aechmea 'Maya' over a three month period under severe light limitation. A. 'Maya' was not particularly tolerant of severe light limitation in the short term. A complete absence of net CO(2) uptake and fluctuations in key metabolites (i.e. malate, starch or soluble sugars) indicated a dampened metabolism whilst cell death in the most photosynthetically active leaves was attributed to an over-acidification of the cytoplasm. However, in the longer term, plasticity in the use of the different phases of gas exchange and different storage carbohydrate pools, i.e. a switch from starch to sucrose as the major carbohydrate source, ensured a positive carbon balance for this CAM species under extremely low levels of irradiance. As such, co-ordinated plasticity in the use of C(3) and C(4) carboxylases and different carbohydrate pools together with an increase in the abundance of light-harvesting complexes, appear to underpin the adaptive radiation of the energetically costly CAM pathway within light-limiting environments such as wet cloud forests and shaded understoreys of tropical forests.

Understanding the Shift in the Microbiome of Composts That Are Optimized for a Better Fit-for-Purpose in Growing Media.

Three characteristics are considered key for optimal use of composts in growing media: maturity, pH and organic matter content. Maturation is a critical step in the processing of composts contributing to compost quality. Blending of composts with chopped heath biomass, sieving out the larger fraction of composts and acidification of composts by adding elemental sulfur may be used either to increase organic matter content or to reduce pH for a better fit in growing media. While several studies have shown the effectiveness of these treatments to improve the use of composts in growing media, the effect of these treatments on the compost microbiome has merely been assessed before. In the present study, five immature composts were allowed to mature, and were subsequently acidified, blended or sieved. Bacterial and fungal communities of the composts were characterized and quantified using 16S rRNA and ITS2 gene metabarcoding and phospholipid fatty acid analysis. Metabolic biodiversity and activity were analyzed using Biolog EcoPlates. Compost batch was shown to be more important than maturation or optimization treatments to determine the compost microbiome. Compost maturation increased microbial diversity and favored beneficial microorganisms, which may be positive for the use of composts in growing media. Blending of composts increased microbial diversity, metabolic diversity, and metabolic activity, which may have a positive effect in growing media. Blending may be used to modify the microbiome to a certain degree in order to optimize microbiological characteristics. Acidification caused a decrease in bacterial diversity and microbial activity, which may be negative for the use in growing media, although the changes are limited. Sieving had limited effect on the microbiome of composts. Because of the limited effect on the microbiome, sieving of composts may be used flexible to improve (bio)chemical characteristics. This is the first study to assess the effects of maturation and optimization treatments to either increase organic matter content or lower pH in composts on the compost microbiome.

Seasonal influences on carbohydrate metabolism in the CAM bromeliad Aechmea 'Maya': consequences for carbohydrate partitioning and growth.

BACKGROUND AND AIMS: Photosynthetic plasticity in response to a range of environmental factors that include [CO(2)], water availability, light intensity and temperature, is ubiquitous among plants with crassulacean acid metabolism (CAM). The present study examined how seasonal changes in light availability, as experienced by greenhouse CAM crops in northern latitude regions, influence diel carboxylation patterns and impact on carbon gain and seasonal accumulation of biomass. METHODS: In the CAM bromeliad Aechmea 'Maya' integrated measurements of leaf gas exchange, diel metabolite dynamics (e.g. malate, soluble sugars and starch) and biomass accumulation were made four times a year, i.e. in winter, spring, summer and autumn. KEY RESULTS: During the brighter seasons (spring and summer) daytime Phases II and IV were dominated by C(4) carboxylation, whilst the higher diurnal uptake in the autumn and winter was characterized by equal contributions of both Rubisco and PEPC. As a consequence, net CO(2) uptake showed a significant depression at the end of the day in the darker months when supplementary illumination was turned off. Remarkable seasonal consistency was found in the amount of storage reserves available for nocturnal carboxylation, a consequence of predominantly daytime export of carbohydrate in spring and summer whilst nocturnal export was the major sink for carbohydrate in autumn and winter. CONCLUSIONS: Throughout the different seasons Aechmea 'Maya' showed considerable plasticity in the timing and magnitude of C(3) and C(4) carboxylation processes over the diel cycle. Under low PPFD (i.e. winter and autumn) it appears that there was a constraint on the amount of carbohydrate exported during the day in order to maintain a consistent pool of transient carbohydrate reserves. This gave remarkable seasonal consistency in the amount of storage reserves available at night, thereby optimizing biomass gain throughout the year. The data have important practical consequences for horticultural productivity of CAM plants and suggest a scenario for reconciling carbohydrate partitioning between competing sinks of nocturnal acidification and export for growth.

Differential usage of storage carbohydrates in the CAM bromeliad Aechmea 'Maya' during acclimation to drought and recovery from dehydration.

CAM requires a substantial investment of resources into storage carbohydrates to account for nocturnal CO(2) uptake, thereby restricting carbohydrate partitioning to other metabolic activities, including dark respiration, growth and acclimation to abiotic stress. Flexible modulation of carbon flow to the different competing sinks under changing environmental conditions is considered a key determinant for the growth, productivity and ecological success of the CAM pathway. The aim of the present study was to examine how shifts in carbohydrate partitioning could assure maintenance of photosynthetic integrity and a positive carbon balance under conditions of increasing water deprivation in CAM species. Measurements of gas exchange, leaf water relations, malate, starch and soluble sugar (glucose, fructose and sucrose) contents were made in leaves of the CAM bromeliad Aechmea 'Maya' over a 6-month period of drought and subsequently over a 2-month period of recovery from drought. Results indicated that short-term influences of water stress were minimized by elevating the level of respiratory recycling, and carbohydrate pools were maintained at the expense of export for growth while providing a comparable nocturnal carbon gain to that in well-watered control plants. Longer term drought resulted in a disproportionate depletion of key carbohydrate reserves. Sucrose, which was of minor importance for providing substrate for the dark reactions under well-watered conditions, became the major source of carbohydrate for nocturnal carboxylation as drought progressed. Flexibility in terms of the major carbohydrate source used to sustain dark CO(2) uptake is therefore considered a crucial factor in meeting the carbon and energy demands under limiting environmental conditions. Recovery from CAM-idling was found to be dependent on the restoration of the starch pool, which was used predominantly for provision of substrate for nocturnal carboxylation, while net carbon export was limited. The conservation of starch for the nocturnal reactions might be adaptive with regard to responding efficiently to a return of water stress.

Maltose Processing and Not ß-Amylase Activity Curtails Hydrolytic Starch Degradation in the CAM Orchid Phalaenopsis.

Crassulacean acid metabolism (CAM) is one of the three photosynthetic pathways in higher plants and is characterized by high water use efficiency. This mainly relies on major nocturnal CO2 fixation sustained by degradation of storage carbohydrate such as starch to provide phosphoenolpyruvate (PEP) and energy. In contrast to C3 plants where starch is mainly degraded by the hydrolytic route, different observations suggested the phosphorolytic route to be a major pathway for starch degradation in CAM plants. To elucidate the interplay and relevant contributions of the phosphorolytic and hydrolytic pathways for starch degradation in CAM, we assessed diel patterns for metabolites and enzymes implicated in both the hydrolytic route (ß-amylase, DPE1, DPE2, maltase) and the phosphorolytic route (starch phosphorylase) of starch degradation in the CAM orchid Phalaenopsis "Edessa." By comparing the catalytic enzyme activities and starch degradation rates, we showed that the phosphorolytic pathway is the major route to accommodate nocturnal starch degradation and that measured activities of starch phosphorylase perfectly matched calculated starch degradation rates in order to avoid premature exhaustion of starch reserves before dawn. The hydrolytic pathway seemed hampered in starch processing not by ß-amylase but through insufficient catalytic capacity of both DPE2 and maltase. These considerations were further corroborated by measurements of enzyme activities in the CAM model plant Kalanchoë fedtschenkoi and strongly contradict with the situation in the C3 plant Arabidopsis. The data support the view that the phosphorolytic pathway might be the main route of starch degradation in CAM to provide substrate for PEP with additional hydrolytic starch breakdown to accommodate mainly sucrose synthesis.

Performance Index and PSII Connectivity Under Drought and Contrasting Light Regimes in the CAM Orchid Phalaenopsis.

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis characterized by improved water use efficiency mediated by major nocturnal CO2 fixation. Due to its inherent metabolic plasticity CAM represents a successful physiological strategy for plant adaptation to abiotic stress. The present study reports on the impact of drought stress and different light intensities (PPFD 50 and 200 µmol m-2 s-1) on the photosynthetic performance of the obligate CAM orchid Phalaenopsis "Edessa" by integrating diel gas exchange patterns with assessments of the light reactions by analyzing fast chlorophyll a fluorescence induction. Parameters such as PIabs (performance index), different energy fluxes per active reaction centre (RC) reflecting the electron flow from photosystem II to photosystem I and the energetic communication between PSII complexes defined as connectivity were considered for the first time in a CAM plant. A higher PS II connectivity for plants grown under low light (p ∼ 0.51) compared to plants grown under high light (p ∼ 0.31) brought about similar specific energy fluxes of light absorbance, dissipation and processing through the electron transport chain, irrespective of the light treatment. With a 25% higher maximum quantum yield and comparable biomass formation, low light grown plants indeed proved to process light energy more efficiently compared to high light grown plants. The performance index was identified as a very reliable and sensitive parameter to indicate the onset and progress of drought stress. Under restricted CO2 availability (due to closed stomata) leaves showed higher energy dissipation and partial inactivation of PSII reaction centres to reduce the energy input to the electron transport chain and as such aid in avoiding overexcitation and photodamage. Especially during CAM idling there is a discrepancy between continuous input of light energy but severely reduced availability of both water and CO2, which represents the ultimate electron acceptor. Taken together, our results show a unique flexibility of CAM plants to optimize the light reactions under different environmental conditions in a dual way by either attenuating or increasing energy flux.

Glucuronoarabinoxylan structure in the walls of Aechmea leaf chlorenchyma cells is related to wall strength.

In CAM-plants rising levels of malic acid in the early morning cause elevated turgor pressures in leaf chlorenchyma cells. Under specific conditions this process is lethal for sensitive plants resulting in chlorenchyma cell burst while other species can cope with these high pressures and do not show cell burst under comparable conditions. The non-cellulosic polysaccharide composition of chlorenchyma cell walls was investigated and compared in three cultivars of Aechmea with high sensitivity for chlorenchyma cell burst and three cultivars with low sensitivity. Chlorenchyma layers were cut from the leaf and the non-cellulosic carbohydrate fraction of the cell wall fraction was analyzed by gas-liquid chromatography. Glucuronoarabinoxylans (GAXs) were the major non-cellulosic polysaccharides in Aechmea. The fine structure of these GAXs was strongly related to chlorenchyma wall strength. Chlorenchyma cell walls from cultivars with low sensitivity to cell burst were characterized by an A/X ratio of ca. 0.13 while those from cultivars with high sensitivity showed an A/X ratio of ca. 0.23. Xylose chains from cultivars with high cell burst sensitivity were ca. 40% more substituted with arabinose compared to cultivars with low sensitivity for cell burst. The results indicate a relationship in vivo between glucuronoarabinoxylan fine structure and chlorenchyma cell wall strength in Aechmea. The evidence obtained supports the hypothesis that GAXs with low degrees of substitution cross-link cellulose microfibrils, while GAXs with high degrees of substitution do not. A lower degree of arabinose substitution on the xylose backbone implies stronger cell walls and the possibility of withstanding higher internal turgor pressures without cell bursting.