Photosynthetic acclimation to changing environments.

Plants are exposed to environments that fluctuate of timescales varying from seconds to months. Leaves that develop in one set of conditions optimise their metabolism to the conditions experienced, in a process called developmental acclimation. However, when plants experience a sustained change in conditions, existing leaves will also acclimate dynamically to the new conditions. Typically this process takes several days. In this review, we discuss this dynamic acclimation process, focussing on the responses of the photosynthetic apparatus to light and temperature. We briefly discuss the principal changes occurring in the chloroplast, before examining what is known, and not known, about the sensing and signalling processes that underlie acclimation, identifying likely regulators of acclimation.

Mass Spectrometry Imaging for Spatial Chemical Profiling of Vegetative Parts of Plants.

The detection of chemical species and understanding their respective localisations in tissues have important implications in plant science. The conventional methods for imaging spatial localisation of chemical species are often restricted by the number of species that can be identified and is mostly done in a targeted manner. Mass spectrometry imaging combines the ability of traditional mass spectrometry to detect numerous chemical species in a sample with their spatial localisation information by analysing the specimen in a 2D manner. This article details the popular mass spectrometry imaging methodologies which are widely pursued along with their respective sample preparation and the data analysis methods that are commonly used. We also review the advancements through the years in the usage of the technique for the spatial profiling of endogenous metabolites, detection of xenobiotic agrochemicals and disease detection in plants. As an actively pursued area of research, we also address the hurdles in the analysis of plant tissues, the future scopes and an integrated approach to analyse samples combining different mass spectrometry imaging methods to obtain the most information from a sample of interest.

Cytosolic fumarase acts as a metabolic fail-safe for both high and low temperature acclimation of Arabidopsis thaliana.

Plants acclimate their photosynthetic capacity (Pmax) in response to changing environmental conditions. In Arabidopsis thaliana, photosynthetic acclimation to cold requires the accumulation of the organic acid fumarate, catalysed by a cytosolically localized fumarase, FUM2. However, the role of this accumulation is currently unknown. Here, we use an integrated experimental and modelling approach to examine the role of FUM2 and fumarate across the physiological temperature range. We have studied three genotypes: Col-0; a fum2 mutant in a Col-0 background; and C24, an accession with reduced FUM2 expression. While low temperature causes an increase in Pmax in the Col-0 plants, this parameter decreases following exposure of plants to 30 °C for 7 d. Plants in which fumarate accumulation is partially (C24) or completely (fum2) abolished show a reduced acclimation of Pmax across the physiological temperature range (i.e. Pmax changes less in response to changing temperature). To understand the role of fumarate accumulation, we have adapted a reliability engineering technique, Failure Mode and Effect Analysis (FMEA), to formalize a rigorous approach for ranking metabolites according to the potential risk that they pose to the metabolic system. FMEA identifies fumarate as a low-risk metabolite, while its precursor, malate, is shown to be high risk and liable to cause system instability. We propose that the role of FUM2 is to provide a fail-safe in order to control malate concentration, maintaining system stability in a changing environment. We suggest that FMEA is a technique that is not only useful in understanding plant metabolism but can also be used to study reliability in other systems and synthetic pathways.

Acclimation of Photosynthesis to Changes in the Environment Results in Decreases of Oxidative Stress in Arabidopsis thaliana.

The dynamic acclimation of photosynthesis plays an important role in increasing the fitness of a plant under variable light environments. Since acclimation is partially mediated by a glucose-6-phosphate/phosphate translocator 2 (GPT2), this study examined whether plants lacking GPT2, which consequently have defective acclimation to increases in light, are more susceptible to oxidative stress. To understand this mechanism, we used the model plant Arabidopsis thaliana [accession Wassilewskija-4 (Ws-4)] and compared it with mutants lacking GPT2. The plants were then grown at low light (LL) at 100 µmol m-2 s-1 for 7 weeks. For the acclimation experiments, a set of plants from LL was transferred to 400 µmol m-2 s-1 conditions for 7 days. Biochemical and physiological analyses showed that the gpt2 mutant plants had significantly greater activity for ascorbate peroxidase (APX), guiacol peroxidase (GPOX), and superoxide dismutase (SOD). Furthermore, the mutant plants had significantly lower maximum quantum yields of photosynthesis (Fv/Fm). A microarray analysis also showed that gpt2 plants exhibited a greater induction of stress-related genes relative to wild-type (WT) plants. We then concluded that photosynthetic acclimation to a higher intensity of light protects plants against oxidative stress.

A Holistic Approach to Study Photosynthetic Acclimation Responses of Plants to Fluctuating Light.

Plants in natural environments receive light through sunflecks, the duration and distribution of these being highly variable across the day. Consequently, plants need to adjust their photosynthetic processes to avoid photoinhibition and maximize yield. Changes in the composition of the photosynthetic apparatus in response to sustained changes in the environment are referred to as photosynthetic acclimation, a process that involves changes in protein content and composition. Considering this definition, acclimation differs from regulation, which involves processes that alter the activity of individual proteins over short-time periods, without changing the abundance of those proteins. The interconnection and overlapping of the short- and long-term photosynthetic responses, which can occur simultaneously or/and sequentially over time, make the study of long-term acclimation to fluctuating light in plants challenging. In this review we identify short-term responses of plants to fluctuating light that could act as sensors and signals for acclimation responses, with the aim of understanding how plants integrate environmental fluctuations over time and tailor their responses accordingly. Mathematical modeling has the potential to integrate physiological processes over different timescales and to help disentangle short-term regulatory responses from long-term acclimation responses. We review existing mathematical modeling techniques for studying photosynthetic responses to fluctuating light and propose new methods for addressing the topic from a holistic point of view.

Metabolic flux from the chloroplast provides signals controlling photosynthetic acclimation to cold in Arabidopsis thaliana.

Photosynthesis is especially sensitive to environmental conditions, and the composition of the photosynthetic apparatus can be modulated in response to environmental change, a process termed photosynthetic acclimation. Previously, we identified a role for a cytosolic fumarase, FUM2 in acclimation to low temperature in Arabidopsis thaliana. Mutant lines lacking FUM2 were unable to acclimate their photosynthetic apparatus to cold. Here, using gas exchange measurements and metabolite assays of acclimating and non-acclimating plants, we show that acclimation to low temperature results in a change in the distribution of photosynthetically fixed carbon to different storage pools during the day. Proteomic analysis of wild-type Col-0 Arabidopsis and of a fum2 mutant, which was unable to acclimate to cold, indicates that extensive changes occurring in response to cold are affected in the mutant. Metabolic and proteomic data were used to parameterize metabolic models. Using an approach called flux sampling, we show how the relative export of triose phosphate and 3-phosphoglycerate provides a signal of the chloroplast redox state that could underlie photosynthetic acclimation to cold.

Contrasting Responses to Stress Displayed by Tobacco Overexpressing an Algal Plastid Terminal Oxidase in the Chloroplast.

The plastid terminal oxidase (PTOX) - an interfacial diiron carboxylate protein found in the thylakoid membranes of chloroplasts - oxidizes plastoquinol and reduces molecular oxygen to water. It is believed to play a physiologically important role in the response of some plant species to light and salt (NaCl) stress by diverting excess electrons to oxygen thereby protecting photosystem II (PSII) from photodamage. PTOX is therefore a candidate for engineering stress tolerance in crop plants. Previously, we used chloroplast transformation technology to over express PTOX1 from the green alga Chlamydomonas reinhardtii in tobacco (generating line Nt-PTOX-OE). Contrary to expectation, growth of Nt-PTOX-OE plants was more sensitive to light stress. Here we have examined in detail the effects of PTOX1 on photosynthesis in Nt-PTOX-OE tobacco plants grown at two different light intensities. Under 'low light' (50 µmol photons m-2 s-1) conditions, Nt-PTOX-OE and WT plants showed similar photosynthetic activities. In contrast, under 'high light' (125 µmol photons m-2 s-1) conditions, Nt-PTOX-OE showed less PSII activity than WT while photosystem I (PSI) activity was unaffected. Nt-PTOX-OE grown under high light also failed to increase the chlorophyll a/b ratio and the maximum rate of CO2 assimilation compared to low-light grown plants, suggesting a defect in acclimation. In contrast, Nt-PTOX-OE plants showed much better germination, root length, and shoot biomass accumulation than WT when exposed to high levels of NaCl and showed better recovery and less chlorophyll bleaching after NaCl stress when grown hydroponically. Overall, our results strengthen the link between PTOX and the resistance of plants to salt stress.

From empirical to theoretical models of light response curves - linking photosynthetic and metabolic acclimation.

Light response curves (LRCs) describe how the rate of photosynthesis varies as a function of light. They provide information on the maximum photosynthetic capacity, quantum yield, light compensation point and leaf radiation use efficiency of leaves. Light response curves are widely used to capture photosynthetic phenotypes in response to changing environmental conditions. However, models describing these are predominantly empirical and do not attempt to explain behaviour at a mechanistic level. Here, we use modelling to understand the metabolic changes required for photosynthetic acclimation to changing environmental conditions. Using a simple kinetic model, we predicted LRCs across the physiological temperature range of Arabidopsis thaliana and confirm these using experimental data. We use our validated metabolic model to make novel predictions about the metabolic changes of temperature acclimation. We demonstrate that NADPH utilization are enhanced in warm-acclimated plants, whereas both NADPH and CO2 utilization is enhanced in cold-acclimated plants. We demonstrate how different metabolic acclimation strategies may lead to the same photosynthetic response across environmental change. We further identify that certain metabolic acclimation strategies, such as NADPH utilization, are only triggered when plants are moved beyond a threshold high or low temperature.

Flux sampling is a powerful tool to study metabolism under changing environmental conditions.

The development of high-throughput 'omic techniques has sparked a rising interest in genome-scale metabolic models, with applications ranging from disease diagnostics to crop adaptation. Efficient and accurate methods are required to analyze large metabolic networks. Flux sampling can be used to explore the feasible flux solutions in metabolic networks by generating probability distributions of steady-state reaction fluxes. Unlike other methods, flux sampling can be used without assuming a particular cellular objective. We have undertaken a rigorous comparison of several sampling algorithms and concluded that the coordinate hit-and-run with rounding (CHRR) algorithm is the most efficient based on both run-time and multiple convergence diagnostics. We demonstrate the power of CHRR by using it to study the metabolic changes that underlie photosynthetic acclimation to cold of Arabidopsis thaliana plant leaves. In combination with experimental measurements, we show how the regulated interplay between diurnal starch and organic acid accumulation defines the plant acclimation process. We confirm fumarate accumulation as a requirement for cold acclimation and further predict γ-aminobutyric acid to have a key role in metabolic signaling under cold conditions. These results demonstrate how flux sampling can be used to analyze the feasible flux solutions across changing environmental conditions, whereas eliminating the need to make assumptions which introduce observer bias.

Metabolic acclimation-a key to enhancing photosynthesis in changing environments?

Plants adjust their photosynthetic capacity in response to their environment in a way that optimizes their yield and fitness. There is growing evidence that this acclimation is a response to changes in the leaf metabolome, but the extent to which these are linked and how this is optimized remain poorly understood. Using as an example the metabolic perturbations occurring in response to cold, we define the different stages required for acclimation, discuss the evidence for a metabolic temperature sensor, and suggest further work towards designing climate-smart crops. In particular, we discuss how constraint-based and kinetic metabolic modelling approaches can be used to generate targeted hypotheses about relevant pathways, and argue that a stronger integration of experimental and in silico studies will help us to understand the tightly regulated interplay of carbon partitioning and resource allocation required for photosynthetic acclimation to different environmental conditions.

Plastid terminal oxidase requires translocation to the grana stacks to act as a sink for electron transport.

The plastid terminal oxidase (PTOX) has been shown to be an important sink for photosynthetic electron transport in stress-tolerant plants. However, overexpression studies in stress-sensitive species have previously failed to induce significant activity of this protein. Here we show that overexpression of PTOX from the salt-tolerant brassica species Eutrema salsugineum does not, alone, result in activity, but that overexpressing plants show faster induction and a greater final level of PTOX activity once exposed to salt stress. This implies that an additional activation step is required before activity is induced. We show that that activation involves the translocation of the protein from the unstacked stromal lamellae to the thylakoid grana and a protection of the protein from trypsin digestion. This represents an important activation step and opens up possibilities in the search for stress-tolerant crops.

Drought neutralises plant-soil feedback of two mesic grassland forbs.

Plant-soil feedbacks (PSFs) describe the effect of a plant species on soil properties, which affect the performance of future generations. Here we test the hypothesis that drought alters PSFs by reducing plant-microbe associations and nutrient uptake. We chose two grassland forb species, previously shown to respond differently to soil conditioning and drought, to test our hypothesis. We conditioned unsterilised grassland soil with one generation of each species, and left a third soil unconditioned. We grew a second generation consisting of each combination of plant species, soil, and drought in a full factorial design, and measured soil microbial community and nutrient availability. Scabiosa columbaria displayed negative PSF (smaller plants) under non-droughted conditions, but neutral under drought, suggesting that drought disrupts plant-soil interactions and can advantage the plant. Photosynthetic efficiency of S. columbaria was reduced under drought, but recovered on rewetting regardless of soil conditioning, indicating that PSFs do not impede resilience of this species. Sanguisorba minor showed positive PSFs (larger plants), probably due to an increase in soil N in conspecific soil, but neutral PSF under drought. PSF neutralisation appeared to occur through drought-induced change in the soil microbial community for this species. When S. minor was planted in conspecific soil, photosynthetic efficiency declined to almost zero, with no recovery following rewetting. We attributed this to increased demand for water through higher demand for nutrients with positive PSF. Here we show that drought neutralises PSFs of two grassland forbs, which could have implications for plant communities under climate change.

Dissecting the Native Architecture and Dynamics of Cyanobacterial Photosynthetic Machinery.

The structural dynamics and flexibility of cell membranes play fundamental roles in the functions of the cells, i.e., signaling, energy transduction, and physiological adaptation. The cyanobacterial thylakoid membrane represents a model membrane that can conduct both oxygenic photosynthesis and respiration simultaneously. In this study, we conducted direct visualization of the global organization and mobility of photosynthetic complexes in thylakoid membranes from a model cyanobacterium, Synechococcus elongatus PCC 7942, using high-resolution atomic force, confocal, and total internal reflection fluorescence microscopy. We visualized the native arrangement and dense packing of photosystem I (PSI), photosystem II (PSII), and cytochrome (Cyt) b6f within thylakoid membranes at the molecular level. Furthermore, we functionally tagged PSI, PSII, Cyt b6f, and ATP synthase individually with fluorescent proteins, and revealed the heterogeneous distribution of these four photosynthetic complexes and determined their dynamic features within the crowding membrane environment using live-cell fluorescence imaging. We characterized red light-induced clustering localization and adjustable diffusion of photosynthetic complexes in thylakoid membranes, representative of the reorganization of photosynthetic apparatus in response to environmental changes. Understanding the organization and dynamics of photosynthetic membranes is essential for rational design and construction of artificial photosynthetic systems to underpin bioenergy development. Knowledge of cyanobacterial thylakoid membranes could also be extended to other cell membranes, such as chloroplast and mitochondrial membranes.

Cyclic decomposition explains a photosynthetic down regulation for Chlamydomonas reinhardtii.

The regulation of metabolic networks has been shown to be distributed and shared through the action of metabolic cycles. Biochemical cycles play important roles in maintaining flux and substrate availability for multiple pathways to supply cellular energy and contribute to dynamic stability. By understanding the cyclic and acyclic flows of matter through a network, we are closer to understanding how complex dynamic systems distribute flux along interconnected pathways. In this work, we have applied a cycle decomposition algorithm to a genome-scale metabolic model of Chlamydomonas reinhardtii to analyse how acetate supply affects the distribution of fluxes that sustain cellular activity. We examined the role of metabolic cycles which explain the down regulation of photosynthesis that is observed when cells are grown in the presence of acetate. Our results suggest that acetate modulates changes in global metabolism, with the pentose phosphate pathway, the Calvin-Benson cycle and mitochondrial respiration activity being affected whilst reducing photosynthesis. These results show how the decomposition of metabolic flux into cyclic and acyclic components helps to understand the impact of metabolic cycling on organismal behaviour at the genome scale.

Dynamic Acclimation to High Light in Arabidopsis thaliana Involves Widespread Reengineering of the Leaf Proteome.

Leaves of Arabidopsis thaliana transferred from low to high light increase their capacity for photosynthesis, a process of dynamic acclimation. A mutant, gpt2, lacking a chloroplast glucose-6-phosphate/phosphate translocator, is deficient in its ability to acclimate to increased light. Here, we have used a label-free proteomics approach, to perform relative quantitation of 1993 proteins from Arabidopsis wild type and gpt2 leaves exposed to increased light. Data are available via ProteomeXchange with identifier PXD006598. Acclimation to light is shown to involve increases in electron transport and carbon metabolism but no change in the abundance of photosynthetic reaction centers. The gpt2 mutant shows a similar increase in total protein content to wild type but differences in the extent of change of certain proteins, including in the relative abundance of the cytochrome b6f complex and plastocyanin, the thylakoid ATPase and selected Benson-Calvin cycle enzymes. Changes in leaf metabolite content as plants acclimate can be explained by changes in the abundance of enzymes involved in metabolism, which were reduced in gpt2 in some cases. Plants of gpt2 invest more in stress-related proteins, suggesting that their reduced ability to acclimate photosynthetic capacity results in increased stress.

Cyclic electron flow: facts and hypotheses.

Over the last 15 years, research into the process of cyclic electron flow in photosynthesis has seen a huge resurgence. Having been considered by some in the early 1990s as a physiologically unimportant artefact, it is now recognised as essential to normal plant growth. Here, we provide an overview of the major developments covered in this special issue of photosynthesis research.

FUM2, a Cytosolic Fumarase, Is Essential for Acclimation to Low Temperature in Arabidopsis thaliana.

Although cold acclimation is a key process in plants from temperate climates, the mechanisms sensing low temperature remain obscure. Here, we show that the accumulation of the organic acid fumaric acid, mediated by the cytosolic fumarase FUM2, is essential for cold acclimation of metabolism in the cold-tolerant model species Arabidopsis (Arabidopsis thaliana). A nontargeted metabolomic approach, using gas chromatography-mass spectrometry, identifies fumarate as a key component of the cold response in this species. Plants of T-DNA insertion mutants, lacking FUM2, show marked differences in their response to cold, with contrasting responses both in terms of metabolite concentrations and gene expression. The fum2 plants accumulated higher concentrations of phosphorylated sugar intermediates and of starch and malate. Transcripts for proteins involved in photosynthesis were markedly down-regulated in fum2.2 but not in wild-type Columbia-0. Plants of fum2 show a complete loss of the ability to acclimate photosynthesis to low temperature. We conclude that fumarate accumulation plays an essential role in low temperature sensing in Arabidopsis, either indirectly modulating metabolic or redox signals or possibly being itself directly involved in cold sensing.

Biochemical Analyses of Sorghum Varieties Reveal Differential Responses to Drought.

We have examined the biochemical responses of two sorghum cultivars of differing drought tolerance, Samsorg 17 (more drought tolerant) and Samsorg 40 (less drought tolerant), to sustained drought. Plants were exposed to different degrees of drought and then maintained at that level for five days. Responses were examined in terms of metabolic changes and the expression of drought induced proteins-Heat Shock Proteins (HSPs) and dehydrins (DHNs). Generalised phenotypic changes were studied using Fourier transform infrared (FT-IR) Spectroscopy and non-targeted Gas Chromatography Mass Spectrometry (GC-MS) was employed to detect changes in metabolites, while changes in protein expression were examined using Western blot analysis. Different response profiles of metabolites, HSPs and DHNs were observed in the two cultivars. Metabolic changes involved variation in amino acids, polysaccharides and their derivatives. A total of 188 compounds, with 142 known metabolites and 46 unknown small molecules, were detected in the two sorghum varieties. Under water deficit conditions, Samsorg 17 accumulated sugars and sugar alcohols, while in Samsorg 40 amino acids increased in concentration. This study suggest that the two Sorghum varieties adopt distinct approaches in response to drought, with Samsorg 17 being better able to maintain leaf function under severe drought conditions.

Corrigendum: Flux balance analysis reveals acetate metabolism modulates cyclic electron flow and alternative glycolytic pathways in Chlamydomonas reinhardtii.

[This corrects the article on p. 474 in vol. 6, PMID: 26175742.].

Plastid Terminal Oxidase as a Route to Improving Plant Stress Tolerance: Known Knowns and Known Unknowns.

A plastid-localized terminal oxidase, PTox, was first described due to its role in chloroplast development, with plants lacking PTox producing white sectors on their leaves. This phenotype is explained as being due to PTox playing a role in carotenoid biosynthesis, as a cofactor of phytoene desaturase. Co-occurrence of PTox with a chloroplast-localized NADPH dehydrogenase (NDH) has suggested the possibility of a functional respiratory pathway in plastids. Evidence has also been found that, in certain stress-tolerant plant species, PTox can act as an electron acceptor from PSII, making it a candidate for engineering stress-tolerant crops. However, attempts to induce such a pathway via overexpression of the PTox protein have failed to date. Here we review the current understanding of PTox function in higher plants and discuss possible barriers to inducing PTox activity to improve stress tolerance.