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.

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.

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.

Acclimation of metabolism to light in Arabidopsis thaliana: the glucose 6-phosphate/phosphate translocator GPT2 directs metabolic acclimation.

Mature leaves of plants transferred from low to high light typically increase their photosynthetic capacity. In Arabidopsis thaliana, this dynamic acclimation requires expression of GPT2, a glucose 6-phosphate/phosphate translocator. Here, we examine the impact of GPT2 on leaf metabolism and photosynthesis. Plants of wild type and of a GPT2 knockout (gpt2.2) grown under low light achieved the same photosynthetic rate despite having different metabolic and transcriptomic strategies. Immediately upon transfer to high light, gpt2.2 plants showed a higher rate of photosynthesis than wild-type plants (35%); however, over subsequent days, wild-type plants acclimated photosynthetic capacity, increasing the photosynthesis rate by 100% after 7 d. Wild-type plants accumulated more starch than gpt2.2 plants throughout acclimation. We suggest that GPT2 activity results in the net import of glucose 6-phosphate from cytosol to chloroplast, increasing starch synthesis. There was clear acclimation of metabolism, with short-term changes typically being reversed as plants acclimated. Distinct responses to light were observed in wild-type and gpt2.2 leaves. Significantly higher levels of sugar phosphates were observed in gpt2.2. We suggest that GPT2 alters the distribution of metabolites between compartments and that this plays an essential role in allowing the cell to interpret environmental signals.

GPT2: a glucose 6-phosphate/phosphate translocator with a novel role in the regulation of sugar signalling during seedling development.

BACKGROUND AND AIMS: GPT2, a glucose 6-phosphate/phosphate translocator, plays an important role in environmental sensing in mature leaves of Arabidopsis thaliana. Its expression has also been detected in arabidopsis seeds and seedlings. In order to examine the role of this protein early in development, germination and seedling growth were studied. METHODS: Germination, greening and establishment of seedlings were monitored in both wild-type Arabidopsis thaliana and in a gpt2 T-DNA insertion knockout line. Seeds were sown on agar plates in the presence or absence of glucose and abscisic acid. Relative expression of GPT2 in seedlings was measured using quantitative PCR. KEY RESULTS: Plants lacking GPT2 expression were delayed (25-40 %) in seedling establishment, specifically in the process of cotyledon greening (rather than germination). This phenotype could not be rescued by glucose in the growth medium, with greening being hypersensitive to glucose. Germination itself was, however, hyposensitive to glucose in the gpt2 mutant. CONCLUSIONS: The expression of GPT2 modulates seedling development and plays a crucial role in determining the response of seedlings to exogenous sugars during their establishment. This allows us to conclude that endogenous sugar signals function in controlling germination and the transition from heterotrophic to autotrophic growth, and that the partitioning of glucose 6-phosphate, or related metabolites, between the cytosol and the plastid modulates these developmental responses.

Dynamic acclimation of photosynthesis increases plant fitness in changing environments.

Plants growing in different environments develop with different photosynthetic capacities--developmental acclimation of photosynthesis. It is also possible for fully developed leaves to change their photosynthetic capacity--dynamic acclimation. The importance of acclimation has not previously been demonstrated. Here, we show that developmental and dynamic acclimation are distinct processes. Furthermore, we demonstrate that dynamic acclimation plays an important role in increasing the fitness of plants in natural environments. Plants of Arabidopsis (Arabidopsis thaliana) were grown at low light and then transferred to high light for up to 9 d. This resulted in an increase in photosynthetic capacity of approximately 40%. A microarray analysis showed that transfer to high light resulted in a substantial but transient increase in expression of a gene, At1g61800, encoding a glucose-6-phosphate/phosphate translocator GPT2. Plants where this gene was disrupted were unable to undergo dynamic acclimation. They were, however, still able to acclimate developmentally. When grown under controlled conditions, fitness, measured as seed output and germination, was identical, regardless of GPT2 expression. Under naturally variable conditions, however, fitness was substantially reduced in plants lacking the ability to acclimate. Seed production was halved in gpt2- plants, relative to wild type, and germination of the seed produced substantially less. Dynamic acclimation of photosynthesis is thus shown to play a crucial and previously unrecognized role in determining the fitness of plants growing in changing environments.