Plastid ancestors lacked a complete Entner-Doudoroff pathway, limiting plants to glycolysis and the pentose phosphate pathway.

The Entner-Doudoroff (ED) pathway provides an alternative to glycolysis. It converts 6-phosphogluconate (6-PG) to glyceraldehyde-3-phosphate and pyruvate in two steps consisting of a dehydratase (EDD) and an aldolase (EDA). Here, we investigate its distribution and significance in higher plants and determine the ED pathway is restricted to prokaryotes due to the absence of EDD genes in eukaryotes. EDDs share a common origin with dihydroxy-acid dehydratases (DHADs) of the branched chain amino acid pathway (BCAA). Each dehydratase features strict substrate specificity. E. coli EDD dehydrates 6-PG to 2-keto-3-deoxy-6-phosphogluconate, while DHAD only dehydrates substrates from the BCAA pathway. Structural modeling identifies two divergent domains which account for their non-overlapping substrate affinities. Coupled enzyme assays confirm only EDD participates in the ED pathway. Plastid ancestors lacked EDD but transferred metabolically promiscuous EDA, which explains the absence of the ED pathway from the Viridiplantae and sporadic persistence of EDA genes across the plant kingdom.

Arabidopsis TGA256 Transcription Factors Suppress Salicylic-Acid-Induced Sucrose Starvation.

Salicylic acid (SA) is produced by plants in response to pathogen infection. SA binds the NONEXPRESSOR OF PATHOGENESIS-RELATED GENES (NPR) family of receptors to regulate both positive (NPR1) and negative (NPR3/4) plant immune responses by interacting with the clade II TGACG (TGA) motif-binding transcription factors (TGA2, TGA5, and TGA6). Here, we report that the principal metabolome-level response to SA treatment in Arabidopsis is a reduction in sucrose and other free sugars. We observed nearly identical effects in the tga256 triple mutant, which lacks all clade II TGA transcription factors. The tga256 mutant presents reduced leaf blade development and elongated hypocotyls, roots, and petioles consistent with sucrose starvation. No changes were detected in auxin levels, and mutant seedling growth could be restored to that of wild-type by sucrose supplementation. Although the retrograde signal 2-C-methyl-D-erythritol-2,4-cyclodiphosphate is known to stimulate SA biosynthesis and defense signaling, we detected no negative feedback by SA on this or any other intermediate of the 2-C-methyl-D-erythritol-4-phosphate pathway. Trehalose, a proxy for the sucrose regulator trehalose-6-phosphate (T6P), was highly reduced in tga256, suggesting that defense-related reductions in sugar availability may be controlled by changes in T6P levels. We conclude that the negative regulatory roles of TGA2/5/6 include maintaining sucrose levels in healthy plants. Disruption of TGA2/5/6-NPR3/4 inhibitory complexes by mutation or SA triggers sucrose reductions in Arabidopsis leaves, consistent with the 'pathogen starvation' hypothesis. These findings highlight sucrose availability as a mechanism by which TGA2/5/6 balance defense and development.

An Arabidopsis GCMS chemical ionization technique to quantify adaptive responses in central metabolism.

We present a methodology to survey central metabolism in 13CO2-labeled Arabidopsis (Arabidopsis thaliana) rosettes by ammonia positive chemical ionization-gas chromatography-mass spectrometry. This technique preserves the molecular ion cluster of methyloxime/trimethylsilyl-derivatized analytes up to 1 kDa, providing unambiguous nominal mass assignment of >200 central metabolites and 13C incorporation rates into a subset of 111 from the tricarboxylic acid (TCA) cycle, photorespiratory pathway, amino acid metabolism, shikimate pathway, and lipid and sugar metabolism. In short-term labeling assays, we observed plateau labeling of ∼35% for intermediates of the photorespiratory cycle except for glyoxylate, which reached only ∼4% labeling and was also present at molar concentrations several fold lower than other photorespiratory intermediates. This suggests photorespiratory flux may involve alternate intermediate pools besides the generally accepted route through glyoxylate. Untargeted scans showed that in illuminated leaves, noncyclic TCA cycle flux and citrate export to the cytosol revert to a cyclic flux mode following methyl jasmonate (MJ) treatment. MJ also caused a block in the photorespiratory transamination of glyoxylate to glycine. Salicylic acid treatment induced the opposite effects in both cases, indicating the antagonistic relationship of these defense signaling hormones is preserved at the metabolome level. We provide complete chemical ionization spectra for 203 Arabidopsis metabolites from central metabolism, which uniformly feature the unfragmented pseudomolecular ion as the base peak. This unbiased, soft ionization technique is a powerful screening tool to identify adaptive metabolic trends in photosynthetic tissue and represents an important advance in methodology to measure plant metabolic flux.

Design and fabrication of an improved dynamic flow cuvette for 13CO2 labeling in Arabidopsis plants.

BACKGROUND: Stable isotope labeling is a non-invasive, sensitive means of monitoring metabolic flux in plants. The most physiologically meaningful information is obtained from experiments that take advantage of the natural photosynthetic carbon assimilation pathway to introduce a traceable marker with minimal effects on the physiology of the organism. The fundamental substrate in isotopic labeling experiments is 13CO2, which can reveal the earliest events in carbon assimilation and realistically portray downstream metabolism when administered under conditions suitable for making kinetic inferences. Efforts to improve the accuracy and resolution of whole plant labeling techniques have focused on improvements in environmental control, air flow characteristics, and harvesting methods. RESULTS: Here we present a dynamic flow cuvette designed for single Arabidopsis thaliana labeling experiments. We have also verified its suitability for labeling Nicotiana benthamiana and essential oils in Pelargonium graveolens. Complete plans for fabrication of this device are included. The design includes three important innovations. First, uniform, circular air flow over the rosette surface is accomplished by a fan and deflector that creates a mini-cyclone effect within the chamber interior. Second, a network of circulating canals connected to a water bath provides temperature control to within ± 0.1 ºC under variable irradiance, humidity, and air flow conditions. When photosynthetically active radiation (PAR) was varied over a range of 1000 µEinsteins m-2 s-1 with no adjustment to the external temperature control system, the abaxial leaf temperature changed by < 3 ºC/1000 PAR. Third, the device is fully compatible with liquid nitrogen quenching of metabolic activity without perturbation of the light environment. For short labeling experiments (< 10 s), the most critical variable is the half-life (t1/2) of the atmosphere within the chamber, which determines the maximum resolution of the labeling system. Using an infrared gas analyzer, we monitored the atmospheric half-life during the transition from 12CO2 to 13CO2 air at different flow rates and determined that 3.5 L min-1 is the optimal flow rate to initiate labeling (t1/2 ~ 5 s). Under these conditions, we observed linear incorporation of 13C into triose phosphate with labeling times as short as 5 s. CONCLUSIONS: Advances in our ability to conduct short term labeling experiments are critical to understanding of the rates and control of the earliest steps in plant metabolism. Precise kinetic measurements in whole plants using 13CO2 inform metabolic models and reveal control points that can be exploited in agricultural or biotechnological contexts. The dynamic labeling cuvette presented here is suitable for studying early events in carbon assimilation and provides high resolution kinetic data for studies of metabolism in intact plants under physiologically realistic scenarios.