Photosynthesis is the basis for life, and its optimization is a key biotechnological aim given the problems of population explosion and environmental deterioration. We describe a method to resolve intracellular fluxes in intact Arabidopsis thaliana rosettes based on time-dependent labeling patterns in the metabolome. Plants photosynthesizing under limiting irradiance and ambient CO 2 in a custom-built chamber were transferred into a 13 CO 2 -enriched environment. The isotope labeling patterns of 40 metabolites were obtained using liquid or gas chromatography coupled to mass spectrometry. Labeling kinetics revealed striking differences between metabolites. At a qualitative level, they matched expectations in terms of pathway topology and stoichiometry, but some unexpected features point to the complexity of subcellular and cellular compartmentation. To achieve quantitative insights, the data set was used for estimating fluxes in the framework of kinetic flux profiling. We benchmarked flux estimates to four classically determined flux signatures of photosynthesis and assessed the robustness of the estimates with respect to different features of the underlying metabolic model and the time-resolved data set.

Many plants accumulate substantial starch reserves in their leaves during the day and remobilize them at night to provide carbon and energy for maintenance and growth. In this paper, we explore the role of a sugar-signaling metabolite, trehalose-6-phosphate (Tre6P), in regulating the accumulation and turnover of transitory starch in Arabidopsis (Arabidopsis thaliana) leaves. Ethanol-induced overexpression of trehalose-phosphate synthase during the day increased Tre6P levels up to 11-fold. There was a transient increase in the rate of starch accumulation in the middle of the day, but this was not linked to reductive activation of ADP-glucose pyrophosphorylase. A 2-to 3-fold increase in Tre6P during the night led to significant inhibition of starch degradation. Maltose and maltotriose did not accumulate, suggesting that Tre6P affects an early step in the pathway of starch degradation in the chloroplasts. Starch granules isolated from induced plants had a higher orthophosphate content than granules from noninduced control plants, consistent either with disruption of the phosphorylation-dephosphorylation cycle that is essential for efficient starch breakdown or with inhibition of starch hydrolysis by b-amylase. Nonaqueous fractionation of leaves showed that Tre6P is predominantly located in the cytosol, with estimated in vivo Tre6P concentrations of 4 to 7 mM in the cytosol, 0.2 to 0.5 mM in the chloroplasts, and 0.05 mM in the vacuole. It is proposed that Tre6P is a component in a signaling pathway that mediates the feedback regulation of starch breakdown by sucrose, potentially linking starch turnover to demand for sucrose by growing sink organs at night.

SUMMARYA platform using reverse-phase liquid chromatography coupled to tandem mass spectrometry was developed to measure 28 metabolites from photosynthetic metabolism. It was validated by comparison with authentic standards, with a requirement for distinct and clearly separated peaks, high sensitivity and repeatability in Arabidopsis rosette extracts. The recovery of authentic standards added to the plant material before extraction was 80-120%, demonstrating the reliability of the extraction and analytic procedures. Some metabolites could not be reliably measured, and were extracted and determined by other methods. Measurements of 37 metabolites in Arabidopsis rosettes after 15 min of illumination at different CO 2 concentrations showed that most Calvin cycle intermediates remain unaltered, or decrease only slightly (<30%), at compensation point CO 2 , whereas dedicated metabolites in end-product synthesis pathways decrease strongly. The inhibition of end-product synthesis allows high levels of metabolites to be retained in the Calvin cycle to support a rapid cycle with photorespiration.

Fundamental plant functions such as control of the membrane potential, osmo-regulation, and turgor-driven growth and movements are based on the availability to gain high cellular potassium concentrations (1). The absorption of this inorganic osmolyte from the soil by the root therefore represents a pivotal process for plant life. Classical experiments by Epstein et al. in 1963 (2) described K ϩ root uptake as a biphasic process mediated by two uptake mechanisms: high affinity potassium transport with apparent affinities of ϳ20 M and a low affinity transport system with K m values in the millimolar range. During the last decades several molecular components of potassium transport systems have been identified and functionally characterized in plants (3, 4). Mutant analyses, heterologous expression, as well as radiotracer uptake experiments characterized the K ϩ channels AKT1⅐AtKC1 and members of the HAK⅐KT⅐KUP family as major components of the Arabidopsis thaliana root-localized potassium transport system (5-9). In this study we focused on AKT1 and AtKC1, members of the Arabidopsis Shaker-like K ϩ channel family. AKT1 is a voltagedependent inward-rectifying K ϩ channel mediating potassium uptake over a wide range of external potassium concentrations (10 -15). Root cells of the akt1-1 loss-of-function mutant completely lack inward rectifying K ϩ currents (12). As a consequence the growth of akt1-1 seedlings is strongly impaired on low potassium medium (100 M and less) (11,12,15). Rescue of yeast growth on 20 M K ϩ and patch clamp experiments (16, 17) directly demonstrated that plant inward rectifying K ϩ channels are capable of serving as high affinity potassium uptake transporters. AtKC1 shares its expression pattern with . AtKC1 ␣-subunits, however, neither form functional channels in akt1-1 knock-out plants nor in heterologous expression systems. In contrast to root cells of akt1-1 loss of function mutants, root protoplasts of AtKC1 null mutants (atkc1-f) still exhibit inward rectifying potassium currents most likely derived from homomeric AKT1 tetramers (20). Inward K ϩ currents in this atkc1-f mutant were characterized by a more positive activation voltage. These data suggested that the AtKC1 ␣-subunits do not form K ϩ channels per se but modulate the properties of the AKT1⅐AtKC1 heterocomplex (20 -22

We investigated the systems response of metabolism and growth after an increase in irradiance in the nonsaturating range in the algal model Chlamydomonas reinhardtii. In a three-step process, photosynthesis and the levels of metabolites increased immediately, growth increased after 10 to 15 min, and transcript and protein abundance responded by 40 and 120 to 240 min, respectively. In the first phase, starch and metabolites provided a transient buffer for carbon until growth increased. This uncouples photosynthesis from growth in a fluctuating light environment. In the first and second phases, rising metabolite levels and increased polysome loading drove an increase in fluxes. Most Calvin-Benson cycle (CBC) enzymes were substrate-limited in vivo, and strikingly, many were present at higher concentrations than their substrates, explaining how rising metabolite levels stimulate CBC flux. Rubisco, fructose-1,6-biosphosphatase, and seduheptulose-1,7-bisphosphatase were close to substrate saturation in vivo, and flux was increased by posttranslational activation. In the third phase, changes in abundance of particular proteins, including increases in plastidial ATP synthase and some CBC enzymes, relieved potential bottlenecks and readjusted protein allocation between different processes. Despite reasonable overall agreement between changes in transcript and protein abundance (R 2 = 0.24), many proteins, including those in photosynthesis, changed independently of transcript abundance.