Triose Phosphates Research Articles

Remodeling of Carbon Metabolism during Sulfoglycolysis in Escherichia coli.

Sulfoquinovose (SQ) is a major metabolite in the global sulfur cycle produced by nearly all photosynthetic organisms. One of the major pathways involved in the catabolism of SQ in bacteria such as Escherichia coli is a variant of the glycolytic Embden-Meyerhof-Parnas (EMP) pathway termed the sulfoglycolytic EMP (sulfo-EMP) pathway, which leads to the consumption of three of the six carbons of SQ and the excretion of 2,3-dihydroxypropanesulfonate (DHPS). Comparative metabolite profiling of aerobically glucose (Glc)-grown and SQ-grown E. coli cells was undertaken to identify the metabolic consequences of the switch from glycolysis to sulfoglycolysis. Sulfoglycolysis was associated with the diversion of triose phosphates (triose-P) to synthesize sugar phosphates (gluconeogenesis) and an unexpected accumulation of trehalose and glycogen storage carbohydrates. Sulfoglycolysis was also associated with global changes in central carbon metabolism, as indicated by the changes in the levels of intermediates in the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway (PPP), polyamine metabolism, pyrimidine metabolism, and many amino acid metabolic pathways. Upon entry into stationary phase and the depletion of SQ, E. coli cells utilize their glycogen, indicating a reversal of metabolic fluxes to allow glycolytic metabolism. IMPORTANCE The sulfosugar sulfoquinovose is estimated to be produced on a scale of 10 billion metric tons per annum, making it a major organosulfur species in the biosulfur cycle. The microbial degradation of sulfoquinovose through sulfoglycolysis allows the utilization of its carbon content and contributes to the biomineralization of its sulfur. However, the metabolic consequences of microbial growth on sulfoquinovose are unclear. We use metabolomics to identify the metabolic adaptations that Escherichia coli undergoes when grown on sulfoquinovose versus glucose. This revealed the increased flux into storage carbohydrates through gluconeogenesis and the reduced flux of carbon into the TCA cycle and downstream metabolism. These changes are relieved upon entry into stationary phase and reversion to glycolytic metabolism. This work provides new insights into the metabolic consequences of microbial growth on an abundant sulfosugar.

Photorespiration is the solution, not the problem

The entry of carbon dioxide from the atmosphere into the biosphere is mediated by the enzyme Rubisco, which catalyzes the carboxylation of ribulose 1,5-bisphosphate (RuBP) as the entry reaction of the Calvin Benson Bassham cycle (CBBC), leading to the formation of 2 molecules of 3-phosphoglyceric acid (3PGA) per CO2 fixed. 3PGA is reduced to triose phosphates at the expense of NADPH + H+ and ATP that are provided by the photosynthetic light reactions. Triose phosphates are the principal products of the CBBC and the precursors for almost any compound in the biosphere.

Sufficient potassium improves inorganic phosphate-limited photosynthesis in Brassica napus by enhancing metabolic P fractions and Rubisco activity.

Crop photosynthesis (A) and productivity are often limited by a combination of nutrient stresses, such that changes in the availability of one nutrient may affect that of another nutrient, in turn influencing A. In this study, we examined the synergistic effects of phosphorus (P) and potassium (K) on leaf A in a nutrient amendment experiment, in which P and K were added individually or in combination to Brassica napus grown under P and K co-limitation. The data revealed that P addition gradually removed the dominant limiting factor (i.e., P) and improved leaf A. Strikingly, the addition of K synergistically improved gross P uptake, mainly by boosting plant growth, and compensated the physiological demand for P by prioritising investment in metabolic P pools (P-containing metabolites and inorganic phosphate, Pi). The enlarged pool of metabolically active P was partially associated with upregulation of Pi regeneration through release from triose phosphates rather than replacement of P-containing lipids. This process mitigated P restrictions on A by maintaining the ATP/NADPH and NADPH/NADP+ ratios and increasing the content and activity of Rubisco. Our findings demonstrate that sufficient K increased Pi-limited A by enhancing metabolic P fractions and Rubisco activity. Thus, ionic synergism may be exploited to mitigate nutrient-limiting factors to improve crop productivity.

Nutraceutical Prevention of Diabetic Complications-Focus on Dicarbonyl and Oxidative Stress.

Oxidative and dicarbonyl stress, driven by excess accumulation of glycolytic intermediates in cells that are highly permeable to glucose in the absence of effective insulin activity, appear to be the chief mediators of the complications of diabetes. The most pathogenically significant dicarbonyl stress reflects spontaneous dephosphorylation of glycolytic triose phosphates, giving rise to highly reactive methylglyoxal. This compound can be converted to harmless lactate by the sequential activity of glyoxalase I and II, employing glutathione as a catalyst. The transcription of glyoxalase I, rate-limiting for this process, is promoted by Nrf2, which can be activated by nutraceutical phase 2 inducers such as lipoic acid and sulforaphane. In cells exposed to hyperglycemia, glycine somehow up-regulates Nrf2 activity. Zinc can likewise promote glyoxalase I transcription, via activation of the metal-responsive transcription factor (MTF) that binds to the glyoxalase promoter. Induction of glyoxalase I and metallothionein may explain the protective impact of zinc in rodent models of diabetic complications. With respect to the contribution of oxidative stress to diabetic complications, promoters of mitophagy and mitochondrial biogenesis, UCP2 inducers, inhibitors of NAPDH oxidase, recouplers of eNOS, glutathione precursors, membrane oxidant scavengers, Nrf2 activators, and correction of diabetic thiamine deficiency should help to quell this.

Vertical profile of photosynthetic CO<sub>2</sub> response within rice canopy

Leaf-specific Farquhar-von Caemmerer-Berry (FvCB) model was fitted to characterize the vertical profile of photosynthetic CO2 response within rice canopy. Leaf-position-specific and canopy average FvCB models were fitted to study a suitable leaf representing photosynthetic parameters at the canopy scale. The results showed that leaf photosynthesis was limited by Rubisco activity or ribulose-1,5-bisphosphate regeneration under field conditions. The maximum rate of carboxylation, maximum rate of electron transport, rate of triose phosphates utilization, and light respiration rate in the FvCB model reached the highest values for the top second leaf and then decreased, while the mesophyll diffusion conductance kept decreased in downward leaves. The integrated photosynthetic CO2-response curves for the top fourth and fifth leaves were appropriate for estimating parameters in the FvCB model at the canopy scale.

Effect of sugar metabolite methylglyoxal on equine lamellar explants: An ex vivo model of laminitis.

Laminitis is one of the most devastating diseases in equine medicine, and although several etiopathogenetic mechanisms have been proposed, few clear answers have been identified to date. Several lines of evidence point towards its underlying pathology as being metabolism-related. In the carbonyl stress pathway, sugars are converted to methylglyoxal (MG)-a highly reactive α-oxoaldehyde, mainly derived during glycolysis in eukaryotic cells from the triose phosphates: D-glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. One common hypothesis is that MG could be synthesized during the digestive process in horses, and excessive levels absorbed into peripheral blood could be delivered to the foot and lead to alterations in the hoof lamellar structure. In the present study, employing an ex vivo experimental design, different concentrations of MG were applied to hoof explants (HE), which were then incubated and maintained in a specific medium for 24 and 48 h. Macroscopic and histological analyses and a separation force test were performed at 24 and 48 h post-MG application. Gene expression levels of matrix metalloproteinase (MMP)-2 and -14 and tissue inhibitor of metalloproteinase (TIMP)-2 were also measured at each time point for all experimental conditions. High concentrations of MG induced macroscopic and histological changes mimicking laminitis. The separation force test revealed that hoof tissue samples incubated for 24 h in a high concentration of MG, or with lower doses but for a longer period (48 h), demonstrated significant weaknesses, and samples were easily separated. All results support that high levels of MG could induce irreversible damage in HEs, mimicking laminitis in an ex vivo model.

Pentose Phosphate Pathway Reactions in Photosynthesizing Cells

The pentose phosphate pathway (PPP) is divided into an oxidative branch that makes pentose phosphates and a non-oxidative branch that consumes pentose phosphates, though the non-oxidative branch is considered reversible. A modified version of the non-oxidative branch is a critical component of the Calvin–Benson cycle that converts CO2 into sugar. The reaction sequence in the Calvin–Benson cycle is from triose phosphates to pentose phosphates, the opposite of the typical direction of the non-oxidative PPP. The photosynthetic direction is favored by replacing the transaldolase step of the normal non-oxidative PPP with a second aldolase reaction plus sedoheptulose-1,7-bisphosphatase. This can be considered an anabolic version of the non-oxidative PPP and is found in a few situations other than photosynthesis. In addition to the strong association of the non-oxidative PPP with photosynthesis metabolism, there is recent evidence that the oxidative PPP reactions are also important in photosynthesizing cells. These reactions can form a shunt around the non-oxidative PPP section of the Calvin–Benson cycle, consuming three ATP per glucose 6-phosphate consumed. A constitutive operation of this shunt occurs in the cytosol and gives rise to an unusual labeling pattern of photosynthetic metabolites while an inducible shunt in the stroma may occur in response to stress.

Differential expression of starch and sucrose metabolic genes linked to varying biomass yield in Miscanthus hybrids

BackgroundMiscanthus is a commercial lignocellulosic biomass crop owing to its high biomass productivity and low chemical input requirements. Within an interspecific Miscanthus cross, progeny with high biomass yield were shown to have low concentrations of starch and sucrose but high concentrations of fructose. We performed a transcriptional RNA-seq analysis between selected Miscanthus hybrids with contrasting values for these phenotypes to clarify how these phenotypes are genetically controlled.ResultsWe observed that genes directly involved in the synthesis and degradation of starch and sucrose were down-regulated in high-yielding Miscanthus hybrids. At the same time, glycolysis and export of triose phosphates were up-regulated in high-yielding Miscanthus hybrids. These differentially expressed genes and biological functions were regulated by a well-connected network of less than 25 co-regulated transcription factors.ConclusionsOur results evidence a direct relationship between high expression of essential enzymatic genes in the starch and sucrose pathways and co-expression with their transcriptional regulators, with high starch concentrations and lower biomass production. The strong interconnectivity between gene expression and regulators, chemotype and agronomic traits opens the door to use the expression of well-characterised genes associated with carbohydrate metabolism, particularly in the starch and sucrose pathway, for the early selection of high biomass-yielding genotypes from large Miscanthus populations.

A process-based coupled model of stomatal conductance-photosynthesis-transpiration during leaf ontogeny for water-saving irrigated rice.

Process-based coupled model of stomatal conductance-photosynthesis-transpiration was developed to estimate simultaneously stomatal conductance gsw, photosynthetic rate Pn, and transpiration rate Tr during leaf ontogeny. The modified Jarvis model was constructed by superposing the influence of leaf age LA on gsw in traditional Jarvis model. And the modified Farquhar model was constructed by incorporating the relationships of the LA with parameters in Farquhar model into traditional Farquhar model. The average and leaf-age-based coupled models were constructed, respectively, by combining traditional Farquhar and Penman-Monteith models with traditional Jarvis, and combining modified Farquhar and Penman-Monteith models with modified Jarvis. The results showed that the gsw, the maximum rate of carboxylation, maximum rate of electron transport, rate of triose phosphates utilization, and mitochondrial respiration rate varied in a positive skew pattern, while the mesophyll diffusion conductance decreased linearly with increase in LA. The average coupled model underestimated gsw, Pn, and Tr for young leaves and overestimated gsw, Pn, and Tr for old leaves. And the leaf-age-based coupled model generally perfected well in estimating gsw, Pn, and Tr for all leaves during leaf ontogeny. The study will provide basic information for either modeling leaf gsw, Pn, and Tr continuously, or upscaling them from leaf to canopy scale by considering the variation of LA within canopy.

Triose phosphate utilization limitation: an unnecessary complexity in terrestrial biosphere model representation of photosynthesis

Triose phosphates are the principal product of photosynthesis. They are used within the chloroplast for starch synthesis, or translocated to the cytosol where they are used to fuel sucrose synthesis. Use of triose phosphate releases inorganic phosphate, and is under strict metabolic control that matches the supply of triose phosphate from the Calvin-Benson cycle to the demand for carbon by sinks (Heldt & Heldt, 2011; McClain & Sharkey, 2019). However, a low rate of triose phosphate utilization (TPU) can deplete the phosphate pool, restrict ATP synthesis and reduce the availability of ATP to power the Calvin-Benson cycle, thereby limiting photosynthesis (Sharkey, 1985).

Cytoplasmic Streaming as an Intracellular Conveyer: Effect on Photosynthesis and H+ Fluxes in Chara Cells

Giant-sized cells, such as the internodes of characean algae, demonstrate rapid (up to 100 μm/s) rotational cytoplasmic streaming that participates in long-distance intracellular interactions and coordinates the functional activity of organelles under a nonuniform light environment. The specific functions of intense cytoplasmic streaming remain poorly investigated. The lateral transport of photometabolites can be detected by combining pinhole illumination with measurements of chlorophyll fluorescence and external pH on microscopic cell regions located downstream of the locally illuminated area. The involvement of cyclosis in regulation of photosynthesis and plasmalemmal H+ transport was revealed by this means. In regions exposed to bright local light, the chloroplasts export reducing equivalents and triose phosphates into the streaming cytoplasm that spread over the cell and induce a transient rise of chlorophyll fluorescence in shaded cell areas located far from the site of photostimulus application. This review highlights the properties of cyclosis-mediated fluorescence changes, including the photoinduction of long-distance transmission, sensitivity to metabolic inhibitors, its nonuniform spatial distribution in illuminated cells, and gradual (1–5 min) inactivation of long-range signaling after transferring the cell to darkness. A stimulatory influence of the action potential on long-distance signal transmission is shown. The new method is suitable for studying the intercellular transport of metabolites and the permeability of plasmodesmata.

Substrate specificity of plastid phosphate transporters in a non-photosynthetic diatom and its implication in evolution of red alga-derived complex plastids

The triose phosphate transporter (TPT) is one of the prerequisites to exchange metabolites between the cytosol and plastids. In this study, we demonstrated that the four plastid TPT homologues in the non-photosynthetic diatom Nitzschia sp. NIES-3581 were highly likely integrated into plastid envelope membranes similar to counterparts in the model photosynthetic diatom Phaeodactylum tricornutum, in terms of target membranes and C-terminal orientations. Three of the four Nitzschia TPT homologues are capable of transporting various metabolites into proteo-liposomes including triose phosphates (TPs) and phosphoenolpyruvate (PEP), the transport substrates sufficient to support the metabolic pathways retained in the non-photosynthetic diatom plastid. Phylogenetic analysis of TPTs and closely related transporter proteins indicated that diatoms and other algae with red alga-derived complex plastids possess only TPT homologues but lack homologues of the glucose 6-phosphate transporter (GPT), xylulose 5-phosphate transporter (XPT), and phosphoenolpyruvate transporter (PPT). Comparative sequence analysis suggests that many TPT homologues of red alga-derived complex plastids potentially have the ability to transport mainly TPs and PEP. TPTs transporting both TPs and PEP highly likely mediate a metabolic crosstalk between a red alga-derived complex plastid and the cytosol in photosynthetic and non-photosynthetic species, which explains the lack of PPTs in all the lineages with red alga-derived complex plastids. The PEP-transporting TPTs might have emerged in an early phase of endosymbiosis between a red alga and a eukaryote host, given the broad distribution of that type of transporters in all branches of red alga-derived complex plastid-bearing lineages, and have probably played a key role in the establishment and retention of a controllable, intracellular metabolic connection in those organisms.

Application of Stable Isotope Tracing to Elucidate Metabolic Dynamics During Yarrowia lipolytica α-Ionone Fermentation.

SummaryTargeted metabolite analysis in combination with 13C-tracing is a convenient strategy to determine pathway activity in biological systems; however, metabolite analysis is limited by challenges in separating and detecting pathway intermediates with current chromatographic methods. Here, a hydrophilic interaction chromatography tandem mass spectrometry approach was developed for improved metabolite separation, isotopologue analysis, and quantification. The physiological responses of a Yarrowia lipolytica strain engineered to produce ∼400 mg/L α-ionone and temporal changes in metabolism were quantified (e.g., mevalonate secretion, then uptake) indicating bottleneck shifts in the engineered pathway over the course of fermentation. Dynamic labeling results indicated limited tricarboxylic acid cycle label incorporation and, combined with a measurable ATP shortage during the high ionone production phase, suggested that electron transport and oxidative phosphorylation may limit energy supply and strain performance. The results provide insights into terpenoid pathway metabolic dynamics of non-model yeasts and offer guidelines for sensor development and modular engineering.

Single-Stage Astaxanthin Production Enhances the Nonmevalonate Pathway and Photosynthetic Central Metabolism in Synechococcus sp. PCC 7002.

The natural pigment astaxanthin is widely used in aquaculture, pharmaceutical, nutraceutical, and cosmetic industries due to superior antioxidant properties. The green alga Haematococcus pluvialis is currently used for commercial production of astaxanthin pigment. However, slow growing H.pluvialis requires a complex two-stage stress-induced process with high light intensity leading to increased contamination risks. In contrast, the fast-growing euryhaline cyanobacterium Synechococcus sp. PCC 7002 (Synechococcus 7002) is able to reach high density under stress-free phototrophic conditions, and is therefore a promising metabolic engineering platform for astaxanthin production. In the present study, genes encoding β-carotene hydroxylase and β-carotene ketolase, from the marine bacterium Brevundimonas sp. SD212, are integrated into the endogenous plasmid of Synechococcus 7002, and then expressed to biosynthesize astaxanthin. Although Synechococcus 7002 does not inherently produce astaxanthin, the recombinant ZW strain yields 3 mg/g dry cell weight astaxanthin from CO2 as the sole carbon source, with significantly higher astaxanthin content than previous cyanobacteria reports. Synechococcus 7002 astaxanthin productivity reached 3.35 mg/L/day after just 2 days in a continuous autotrophic process, which is comparable to the best H.pluvialis astaxanthin productivities when factoring in growth times. Metabolomics analysis reveals increases in fractions of hexose-, pentose-, and triose phosphates along with intermediates involved in the nonmevalonate pathway. Dynamic metabolomics analysis of 13C labeled metabolites clearly indicates flux enhancements in the Calvin cycle and glycolysis resulting from the overexpression of astaxanthin biosynthetic genes. This study suggests that cyanobacteria may enhance central metabolism as well as the nonmevalonate pathway in an attempt to replenish depleted pigments such as β-carotene and zeaxanthin.

Health outcomes of a high fructose intake: the importance of physical activity.

Fructose metabolism is generally held to occur essentially in cells of the small bowel, the liver, and the kidneys expressing fructolytic enzymes (fructokinase, aldolase B and a triokinase). In these cells, fructose uptake and fructolysis are unregulated processes, resulting in the generation of intracellular triose phosphates proportionate to fructose intake. Triose phosphates are then processed into lactate, glucose and fatty acids to serve as metabolic substrates in other cells of the body. With small oral loads, fructose is mainly metabolized in the small bowel, while with larger loads fructose reaches the portal circulation and is largely extracted by the liver. A small portion, however, escapes liver extraction and is metabolized either in the kidneys or in other tissues through yet unspecified pathways. In sedentary subjects, consumption of a fructose‐rich diet for several days stimulates hepatic de novo lipogenesis, increases intrahepatic fat and blood triglyceride concentrations, and impairs insulin effects on hepatic glucose production. All these effects can be prevented when high fructose intake is associated with increased levels of physical activity. There is also evidence that, during exercise, fructose carbons are efficiently transferred to skeletal muscle as glucose and lactate to be used for energy production. Glucose and lactate formed from fructose can also contribute to the re‐synthesis of muscle glycogen after exercise. We therefore propose that the deleterious health effects of fructose are tightly related to an imbalance between fructose energy intake on one hand, and whole‐body energy output related to a low physical activity on the other hand.

Patients With Aldolase B Deficiency Are Characterized by Increased Intrahepatic Triglyceride Content.

There is an ongoing debate about whether and how fructose is involved in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). A recent experimental study showed an increased intrahepatic triglyceride (IHTG) content in mice deficient for aldolase B (aldo B-/-), the enzyme that converts fructose-1-phosphate to triose phosphates. To translate these experimental findings to the human situation. Case-control study. Outpatient clinic for inborn errors of metabolism. Patients with hereditary fructose intolerance, a rare inborn error of metabolism caused by a defect in aldolase B (n = 15), and healthy persons matched for age, sex, and body mass index (BMI) (n =15). IHTG content, assessed by proton magnetic resonance spectroscopy. IHTG content was higher in aldo B-/- patients than controls (2.5% vs 0.6%; P = 0.001) on a background of lean body mass (median BMI, 20.4 and 21.8 kg/m2, respectively). Glucose excursions during an oral glucose load were higher in aldo B-/- patients (P = 0.043). Hypoglycosylated transferrin, a surrogate marker for hepatic fructose-1-phosphate concentrations, was more abundant in aldo B-/- patients than in controls (P < 0.001). Finally, plasma β-hydroxybutyrate, a biomarker of hepatic β-oxidation, was lower in aldo B-/- patients than controls (P = 0.009). This study extends previous experimental findings by demonstrating that aldolase B deficiency also results in IHTG accumulation in humans. It suggests that the accumulation of fructose-1-phosphate and impairment of β-oxidation are involved in the pathogenesis.

Triose phosphate utilization and beyond: from photosynthesis to end product synthesis.

During photosynthesis, plants fix CO2 from the atmosphere onto ribulose-bisphosphate, producing 3-phosphoglycerate, which is reduced to triose phosphates (TPs). The TPs are then converted into the end products of photosynthesis. When a plant is photosynthesizing very quickly, it may not be possible to commit photosynthate to end products as fast as it is produced, causing a decrease in available phosphate and limiting the rate of photosynthesis to the rate of triose phosphate utilization (TPU). The occurrence of an observable TPU limitation is highly variable based on species and especially growth conditions, with TPU capacity seemingly regulated to be in slight excess of typical photosynthetic rates the plant might experience. The physiological effects of TPU limitation are discussed with an emphasis on interactions between the Calvin-Benson cycle and the light reactions. Methods for detecting TPU-limited data from gas exchange data are detailed and the impact on modeling of some physiological effects are shown. Special consideration is given to common misconceptions about TPU.

Heterologous expression of a Glyoxalase I gene from sugarcane confers tolerance to several environmental stresses in bacteria.

Glyoxalase I belongs to the glyoxalase system that detoxifies methylglyoxal (MG), a cytotoxic by-product produced mainly from triose phosphates. The concentration of MG increases rapidly under stress conditions. In this study, a novel glyoxalase I gene, designated as SoGloI was identified from sugarcane. SoGloI had a size of 1,091 bp with one open reading frame (ORF) of 885 bp encoding a protein of 294 amino acids. SoGloI was predicted as a Ni2+-dependent GLOI protein with two typical glyoxalase domains at positions 28–149 and 159–283, respectively. SoGloI was cloned into an expression plasmid vector, and the Trx-His-S-tag SoGloI protein produced in Escherichia coli was about 51 kDa. The recombinant E. coli cells expressing SoGloI compared to the control grew faster and tolerated higher concentrations of NaCl, CuCl2, CdCl2, or ZnSO4. SoGloI ubiquitously expressed in various sugarcane tissues. The expression was up-regulated under the treatments of NaCl, CuCl2, CdCl2, ZnSO4 and abscisic acid (ABA), or under simulated biotic stress conditions upon exposure to salicylic acid (SA) and methyl jasmonate (MeJA). SoGloI activity steadily increased when sugarcane was subjected to NaCl, CuCl2, CdCl2, or ZnSO4 treatments. Sub-cellular observations indicated that the SoGloI protein was located in both cytosol and nucleus. These results suggest that the SoGloI gene may play an important role in sugarcane’s response to various biotic and abiotic stresses.

The Xylulose 5-Phosphate/Phosphate Translocator Supports Triose Phosphate, but Not Phosphoenolpyruvate Transport Across the Inner Envelope Membrane of Plastids in Arabidopsis thaliana Mutant Plants.

The xylulose 5-phosphate/phosphate translocator (PTs) (XPT) represents a link between the plastidial and extraplastidial branches of the oxidative pentose phosphate pathway. Its role is to retrieve pentose phosphates from the extraplastidial space and to make them available to the plastids. However, the XPT transports also triose phosphates and to a lesser extent phosphoenolpyruvate (PEP). Thus, it might support both the triose phosphate/PT (TPT) in the export of photoassimilates from illuminated chloroplasts and the PEP/PT (PPT) in the import of PEP into green or non-green plastids. In mutants defective in the day- and night-path of photoassimilate export from the chloroplasts (i.e., knockout of the TPT [tpt-2] in a starch-free background [adg1-1])the XPT provides a bypass for triose phosphate export and thereby guarantees survival of the adg1-1/tpt-2 double mutant. Here we show that the additional knockout of the XPT in adg1-1/tpt-2/xpt-1 triple mutants results in lethality when the plants were grown in soil. Thus the XPT can functionally support the TPT. The PEP transport capacity of the XPT has been revisited here with a protein heterologously expressed in yeast. PEP transport rates in the proteoliposome system were increased with decreasing pH-values below 7.0. Moreover, PEP transport determined in leaf extracts from wild-type plants showed a similar pH-response, suggesting that in both cases PEP2- is the transported charge-species. Hence, PEP import into illuminated chloroplasts might be unidirectional because of the alkaline pH of the stroma. Here the consequence of a block in PEP transport across the envelope was analyzed in triple mutants defective in both PPTs and the XPT. PPT1 is knocked out in the cue1 mutant. For PPT2 two new mutant alleles were isolated and established as homozygous lines. In contrast to the strong phenotype of cue1, both ppt2 alleles showed only slight growth retardation. As plastidial PEP is required e.g., for the shikimate pathway of aromatic amino acid synthesis, a block in PEP import should result in a lethal phenotype. However, the cue1-6/ppt2-1/ppt2-1 triple mutant was viable and even exhibited residual PEP transport capacity. Hence, alternative ways of PEP transport must exist and are discussed.

The Combined Loss of Triose Phosphate and Xylulose 5-Phosphate/Phosphate Translocators Leads to Severe Growth Retardation and Impaired Photosynthesis in Arabidopsis thaliana tpt/xpt Double Mutants.

The xylulose 5-phosphate/phosphate translocator (XPT) represents the fourth functional member of the phosphate translocator (PT) family residing in the plastid inner envelope membrane. In contrast to the other three members, little is known on the physiological role of the XPT. Based on its major transport substrates (i.e., pentose phosphates) the XPT has been proposed to act as a link between the plastidial and extraplastidial branches of the oxidative pentose phosphate pathway (OPPP). As the XPT is also capable of transporting triose phosphates, it might as well support the triose phosphate PT (TPT) in exporting photoassimilates from the chloroplast in the light (‘day path of carbon’) and hence in supplying the whole plant with carbohydrates. Two independent knockout mutant alleles of the XPT (xpt-1 and xpt-2) lacked any specific phenotype, suggesting that the XPT function is redundant. However, double mutants generated from crossings of xpt-1 to different mutant alleles of the TPT (tpt-1 and tpt-2) were severely retarded in size, exhibited a high chlorophyll fluorescence phenotype, and impaired photosynthetic electron transport rates. In the double mutant the export of triose phosphates from the chloroplasts is completely blocked. Hence, precursors for sucrose biosynthesis derive entirely from starch turnover (‘night path of carbon’), which was accompanied by a marked accumulation of maltose as a starch breakdown product. Moreover, pentose phosphates produced by the extraplastidial branch of the OPPP also accumulated in the double mutants. Thus, an active XPT indeed retrieves excessive pentose phosphates from the extra-plastidial space and makes them available to the plastids. Further metabolic profiling revealed that phosphorylated intermediates remained largely unaffected, whereas fumarate and glycine contents were diminished in the double mutants. The assessment of C/N-ratios suggested co-limitations of C- and N-metabolism as possible cause for growth retardation of the double mutants. Feeding of sucrose partially rescued the growth and photosynthesis phenotypes of the double mutants. Immunoblots of thylakoid proteins, spectroscopic determinations of photosynthesis complexes, and chlorophyll a fluorescence emission spectra at 77 Kelvin could only partially explain constrains in photosynthesis observed in the double mutants. The data are discussed together with aspects of the OPPP and central carbon metabolism.