Pyrophosphate-dependent Phosphofructokinase Research Articles

Characterization of ATP-dependent phosphofructokinase genes during ripening and their modulation by phytohormones during postharvest storage of citrus fruits (Citrus reticulata Blanco.)

The level of sweetness in citrus fruit is crucial for consumer appeal and market competitiveness, determined mainly by soluble sugars and organic acids. ATP-dependent 6-phosphofructokinase is central to regulating sugar metabolism, yet its role in citrus fruit ripening and postharvest storage remains underexplored. We characterized phosphofructokinase genes in citrus, identifying eight genes classified into pyrophosphate-dependent phosphofructokinase (PFP) and ATP-dependent 6-phosphofructokinase (PFK) subgroups using phylogenetic analysis, genomic architectures, and protein motifs. Comparative genomic analysis with other plants highlighted significant protein homology among CitPFKs. The motif analysis indicated conserved phosphofructokinase domains in CitPFK sequences, with upstream promoter regions containing diverse cis-regulatory elements, most notably light-responsive (LREs). The gene expression profiling throughout fruit development and ripening revealed differential patterns, with responses to gibberellic acid and salicylic acid phytohormones during postharvest indicating their roles in regulating CitPFK genes. The analysis of the transcriptome showed high expression of ATP-dependent 6-phosphofructokinase 3 (CitPFK3) during fruit development, indicating a positive role in fruit maturation. Consequently, silencing CitPFK3 through virus-induced gene silencing (VIGS) increased hexose sugar content, suggesting its function in sugar accumulation. These findings improve our understanding of PFKs in citrus, particularly CitPFK3's pivotal role in regulating hexose sugar dynamics and their modulation by exogenous phytohormones after harvest. This study provides a foundation for optimizing soluble sugar regulation to enhance fruit quality and postharvest handling in citrus production.

ClSnRK2.3 negatively regulates watermelon fruit ripening and sugar accumulation.

Watermelon (Citrullus lanatus) as non-climacteric fruit is domesticated from the ancestors with inedible fruits. We previously revealed that the abscisic acid (ABA) signaling pathway gene ClSnRK2.3 might influence watermelon fruit ripening. However, the molecular mechanisms are unclear. Here, we found that the selective variation of ClSnRK2.3 resulted in lower promoter activity and gene expression level in cultivated watermelons than ancestors, which indicated ClSnRK2.3 might be a negative regulator in fruit ripening. Overexpression (OE) of ClSnRK2.3 significantly delayed watermelon fruit ripening and suppressed the accumulation of sucrose, ABA and gibberellin GA4 . Furthermore, we determined that the pyrophosphate-dependent phosphofructokinase (ClPFP1) in sugar metabolism pathway and GA biosynthesis enzyme GA20 oxidase (ClGA20ox) could be phosphorylated by ClSnRK2.3 and thereby resulting in accelerated protein degradation in OE lines and finally led to low levels of sucrose and GA4 . Besides that, ClSnRK2.3 phosphorylated homeodomain-leucine zipper protein (ClHAT1) and protected it from degradation to suppress the expression of the ABA biosynthesis gene 9'-cis-epoxycarotenoid dioxygenase 3 (ClNCED3). These results indicated that ClSnRK2.3 negatively regulated watermelon fruit ripening by manipulating the biosynthesis of sucrose, ABA and GA4 . Altogether, these findings revealed a novel regulatory mechanism in non-climacteric fruit development and ripening.

Auxenochlorella protothecoides populations adapted to low phosphate conditions accumulated more non-phosphorus glycerolipids and biomass than wild type progenitors

Biodiesel produced by microalgae has great potential as a portable energy that can replace traditional hydrocarbon sources. One limitation of scaling up algal cultivation is the availability of macronutrients, particularly inorganic phosphate (Pi), which is a finite and dwindling resource. Here, Auxenochlorella protothecoides was adapted to low Pi conditions by continuous cultivation in growth media containing 100 times less Pi (17.15 µM PO4) than replete media for ∼ 40 generations. The low Pi-adapted A. protothecoides populations showed significantly higher growth rates, compared to wild type (WT) (natural, non-mutated) progenitor populations in batch experiments, with average maximum growth rates of 0.72 d−1 and 0.54 d−1, respectively. Total lipid profiling of the adapting A. protothecoides populations indicated a shift to non-phosphorus glycerolipids, based on UPLC/MS analyzes. The proportions of monogalactosyldiacylglycerol (MGDG) and sulfoquinovosyldiacyglycerol (SQDG) fluctuated during adaptation, accumulating 305% and 317% of the WT levels respectively by the final sampling. Time-course transcriptome profiling of A. protothecoides across all adaptation stages revealed initial increases in transcript levels, followed by global decreased expression. The short-term transcriptomic changes, prior to ∼ 11 generations, were associated with major metabolic pathways. The long-term changes indicated increased fatty acid turnover and a decrease in photosynthesis-related gene expression. Transcripts predicted to encode alternative oxidase and pyrophosphate-dependent phosphofructokinase fluctuated during adaptation. The selection of A. protothecoides under low Pi conditions resulted in a microalga variant that after only ∼40 generations utilized Pi more efficiently for growth than its wild type progenitor population, while also producing 1.22 times more biomass. The adaptive processes described herein produced commercially relevant strain material and provide avenues for future, targeted engineering of molecular pathways for increased Pi use efficiency in similar organisms.

MdbHLH3 modulates apple soluble sugar content by activating phosphofructokinase gene expression.

Sugars are involved in plant growth, fruit quality, and signaling perception. Therefore, understanding the mechanisms involved in soluble sugar accumulation is essential to understand fruit development. Here, we report that MdPFPβ, a pyrophosphate-dependent phosphofructokinase gene, regulates soluble sugar accumulation by enhancing the photosynthetic performance and sugar-metabolizing enzyme activities in apple (Malus domestica Borkh.). Biochemical analysis revealed that a basic helix-loop-helix (bHLH) transcription factor, MdbHLH3, binds to the MdPFPβ promoter and activates its expression, thus promoting soluble sugar accumulation in apple fruit. In addition, MdPFPβ overexpression in tomato influenced photosynthesis and carbon metabolism in the plant. Furthermore, we determined that MdbHLH3 increases photosynthetic rates and soluble sugar accumulation in apple by activating MdPFPβ expression. Our results thus shed light on the mechanism of soluble sugar accumulation in apple leaves and fruit: MdbHLH3 regulates soluble sugar accumulation by activating MdPFPβ gene expression and coordinating carbohydrate allocation.

Essential role of pyrophosphate homeostasis mediated by the pyrophosphate-dependent phosphofructokinase in Toxoplasma gondii.

Many biosynthetic pathways produce pyrophosphate (PPi) as a by-product, which is cytotoxic if accumulated at high levels. Pyrophosphatases play pivotal roles in PPi detoxification by converting PPi to inorganic phosphate. A number of apicomplexan parasites, including Toxoplasma gondii and Cryptosporidium parvum, express a PPi-dependent phosphofructokinase (PPi-PFK) that consumes PPi to power the phosphorylation of fructose-6-phosphate. However, the physiological roles of PPi-PFKs in these organisms are not known. Here, we report that Toxoplasma expresses both ATP- and PPi-dependent phosphofructokinases in the cytoplasm. Nonetheless, only PPi-PFK was indispensable for parasite growth, whereas the deletion of ATP-PFK did not affect parasite proliferation or virulence. The conditional depletion of PPi-PFK completely arrested parasite growth, but it did not affect the ATP level and only modestly reduced the flux of central carbon metabolism. However, PPi-PFK depletion caused a significant increase in cellular PPi and decreased the rates of nascent protein synthesis. The expression of a cytosolic pyrophosphatase in the PPi-PFK depletion mutant reduced its PPi level and increased the protein synthesis rate, therefore partially rescuing its growth. These results suggest that PPi-PFK has a major role in maintaining pyrophosphate homeostasis in T. gondii. This role may allow PPi-PFK to fine-tune the balance of catabolism and anabolism and maximize the utilization efficiency for carbon nutrients derived from host cells, increasing the success of parasitism. Moreover, PPi-PFK is essential for parasite propagation and virulence in vivo but it is not present in human hosts, making it a potential drug target to combat toxoplasmosis.

An alternative pentose phosphate pathway in human gut bacteria for the degradation of C5 sugars in dietary fibers.

The microbial degradation of pentoses in the human gut is a crucial factor for the utilization of plant-based dietary fibers. A vast majority of gut microbes are able to use these C5-sugars as a carbon and energy source. However, the underlying metabolic pathways are not fully understood. Bioinformatic analysis showed that a large number of abundant gut bacteria lack genes encoding a transaldolase as a key enzyme of the pentose phosphate pathway. Among them was the important human gut microbe Prevotellacopri, which was able to grow in minimal media containing xylose or hemicelluloses as the sole carbon source. Therefore, we looked for an alternative pathway for pentose conversion in P.copri using bioinformatics, enzyme activity assays, and the detection of intermediates of pentose metabolism. It became evident that the organism converted C5-sugars via the sedoheptulose-1,7-bisphosphate pathway (SBPP) to connect pentose metabolism with glycolysis. To circumvent the transaldolase reaction, P.copri uses the combined catalysis of a pyrophosphate-dependent phosphofructokinase and a fructose-bisphosphate aldolase. Furthermore, we present strong evidence that the SBPP is widely distributed in important gut bacteria, including members of the phyla Bacteroides, Firmicutes, Proteobacteria, Verrucomicrobia, and Lentisphaerae.

Metabolic role of pyrophosphate-linked phosphofructokinase pfk for C1 assimilation in Methylotuvimicrobium alcaliphilum 20Z

BackgroundMethanotrophs is a promising biocatalyst in biotechnological applications with their ability to utilize single carbon (C1) feedstock to produce high-value compounds. Understanding the behavior of biological networks of methanotrophic bacteria in different parameters is vital to systems biology and metabolic engineering. Interestingly, methanotrophic bacteria possess the pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) instead of the ATP-dependent 6-phosphofructokinase, indicating their potentials to serve as promising model for investigation the role of inorganic pyrophosphate (PPi) and PPi-dependent glycolysis in bacteria. Gene knockout experiments along with global-omics approaches can be used for studying gene functions as well as unraveling regulatory networks that rely on the gene product.ResultsIn this study, we performed gene knockout and RNA-seq experiments in Methylotuvimicrobium alcaliphilum 20Z to investigate the functional roles of PPi-PFK in C1 metabolism when cells were grown on methane and methanol, highlighting its metabolic importance in C1 assimilation in M. alcaliphilum 20Z. We further conducted adaptive laboratory evolution (ALE) to investigate regulatory architecture in pfk knockout strain. Whole-genome resequencing and RNA-seq approaches were performed to characterize the genetic and metabolic responses of adaptation to pfk knockout. A number of mutations, as well as gene expression profiles, were identified in pfk ALE strain to overcome insufficient C1 assimilation pathway which limits the growth in the unevolved strain.ConclusionsThis study first revealed the regulatory roles of PPi-PFK on C1 metabolism and then provided novel insights into mechanism of adaptation to the loss of this major metabolic enzyme as well as an improved basis for future strain design in type I methanotrophs.

The pentose phosphate pathway of cellulolytic clostridia relies on 6-phosphofructokinase instead of transaldolase

The genomes of most cellulolytic clostridia do not contain genes annotated as transaldolase. Therefore, for assimilating pentose sugars or for generating C5 precursors (such as ribose) during growth on other (non-C5) substrates, they must possess a pathway that connects pentose metabolism with the rest of metabolism. Here we provide evidence that for this connection cellulolytic clostridia rely on the sedoheptulose 1,7-bisphosphate (SBP) pathway, using pyrophosphate-dependent phosphofructokinase (PPi-PFK) instead of transaldolase. In this reversible pathway, PFK converts sedoheptulose 7-phosphate (S7P) to SBP, after which fructose-bisphosphate aldolase cleaves SBP into dihydroxyacetone phosphate and erythrose 4-phosphate. We show that PPi-PFKs of Clostridium thermosuccinogenes and Clostridium thermocellum indeed can convert S7P to SBP, and have similar affinities for S7P and the canonical substrate fructose 6-phosphate (F6P). By contrast, (ATP-dependent) PfkA of Escherichia coli, which does rely on transaldolase, had a very poor affinity for S7P. This indicates that the PPi-PFK of cellulolytic clostridia has evolved the use of S7P. We further show that C. thermosuccinogenes contains a significant SBP pool, an unusual metabolite that is elevated during growth on xylose, demonstrating its relevance for pentose assimilation. Last, we demonstrate that a second PFK of C. thermosuccinogenes that operates with ATP and GTP exhibits unusual kinetics toward F6P, as it appears to have an extremely high degree of cooperative binding, resulting in a virtual on/off switch for substrate concentrations near its K½ value. In summary, our results confirm the existence of an SBP pathway for pentose assimilation in cellulolytic clostridia.

Characterization of pyrophosphate-dependent phosphofructokinase α-subunit gene from sugarcane showing gene without intron

Pyrophosphate-dependent Phosphofructokinase or PFP is an enzyme that regulate sucrose metabolism. It consists of α- and β-subunits, which encoded by PFPα and PFPβ genes, respectively. Sugarcane PFPa has a strong function in glycolysis and has the potency to be engineered to increase sugarcane yield. Hence, the purpose of this work was to isolate, clone and characterize the sugarcane PFPα gene. Total RNA was isolated from leafrolls of TD 91 sugarcane variety. cDNA synthesis followed by DNA amplification of PFPα gene were performed using degenerate primers. cDNA and DNA fragments were ligated into pGEM-T Easy vector, which were subsequently introduced to E. coli competent cells. EcoRI were used to cut the plasmids for sequencing. Finally, homology searching was conducted using BLASTn, and then the nucleotide sequence was translated to a protein sequence using Bioedit. The results showed that PFPα cDNA fragment was 900 bp in length. The translated PFPa protein showed binding sites for fructose-6-phosphate and fructose-1,6-biphosphate, which are conserved in all family members of PFP. In silico analysis of the DNA fragment showed gene without intron. In conclusion, the PFPα gene from sugarcane has been successfully isolated, cloned and characterized.

Evidence that tolerance of Eutrema salsugineum to low phosphate conditions is hard-wired by constitutive metabolic and root-associated adaptations.

The extremophyte Eutrema salsugineum (Yukon ecotype) has adapted to an environment low in available phosphate through metabolic and root-associated traits that enables it to efficiently retrieve, use, and recycle phosphorus. Efficient phosphate (Pi) use by plants would increase crop productivity under Pi-limiting conditions and reduce our reliance on Pi applied as fertilizer. An ecotype of Eutrema salsugineum originating from the Yukon, Canada, shows no evidence of decreased relative growth rate or biomass under low Pi conditions and, as such, offers a promising model for identifying mechanisms to improve Pi use by crops. We evaluated traits associated with efficient Pi use by Eutrema (Yukon ecotype) seedlings and 4-week-old plants, including acquisition, remobilization, and the operation of metabolic bypasses. Relative to Arabidopsis, Eutrema was slower to remobilize phosphorus (P) from senescing leaves, primary and lateral roots showed a lower capacity for rhizosphere acidification, and root acid phosphatase activity was more broadly distributed and not Pi responsive. Both species produced long root hairs on low Pi media, whereas Arabidopsis root hairs were well endowed with phosphatase activity. This capacity was largely absent in Eutrema. In contrast to Arabidopsis, maximal in vitro rates of pyrophosphate-dependent phosphofructokinase and phosphoenolpyruvate carboxylase activities were not responsive to low Pi conditions suggesting that Eutrema has a constitutive and likely preferential capacity to use glycolytic bypass enzymes. Rhizosphere acidification, exudation of acid phosphatases, and rapid remobilization of leaf P are unlikely strategies used by Eutrema for coping with low Pi. Rather, equipping an entire root system for Pi acquisition and utilizing a metabolic strategy suited to deficient Pi conditions offer better explanations for how Eutrema has adapted to thrive on alkaline, highly saline soil that is naturally low in available Pi.

Fructose metabolism in Chromohalobacter salexigens: interplay between the Embden\u2013Meyerhof\u2013Parnas and Entner\u2013Doudoroff pathways

BackgroundThe halophilic bacterium Chromohalobacter salexigens metabolizes glucose exclusively through the Entner–Doudoroff (ED) pathway, an adaptation which results in inefficient growth, with significant carbon overflow, especially at low salinity. Preliminary analysis of C. salexigens genome suggests that fructose metabolism could proceed through the Entner–Doudoroff and Embden–Meyerhof–Parnas (EMP) pathways. In order to thrive at high salinity, this bacterium relies on the biosynthesis and accumulation of ectoines as major compatible solutes. This metabolic pathway imposes a high metabolic burden due to the consumption of a relevant proportion of cellular resources, including both energy molecules (NADPH and ATP) and carbon building blocks. Therefore, the existence of more than one glycolytic pathway with different stoichiometries may be an advantage for C. salexigens. The aim of this work is to experimentally characterize the metabolism of fructose in C. salexigens.ResultsFructose metabolism was analyzed using in silico genome analysis, RT-PCR, isotopic labeling, and genetic approaches. During growth on fructose as the sole carbon source, carbon overflow was not observed in a wide range of salt concentrations, and higher biomass yields were reached. We unveiled the initial steps of the two pathways for fructose incorporation and their links to central metabolism. While glucose is metabolized exclusively through the Entner–Doudoroff (ED) pathway, fructose is also partially metabolized by the Embden–Meyerhof–Parnas (EMP) route. Tracking isotopic label from [1-13C] fructose to ectoines revealed that 81% and 19% of the fructose were metabolized through ED and EMP-like routes, respectively. Activities of enzymes from both routes were demonstrated in vitro by 31P-NMR. Genes encoding predicted fructokinase and 1-phosphofructokinase were cloned and the activities of their protein products were confirmed. Importantly, the protein encoded by csal1534 gene functions as fructose bisphosphatase, although it had been annotated previously as pyrophosphate-dependent phosphofructokinase. The gluconeogenic rather than glycolytic role of this enzyme in vivo is in agreement with the lack of 6-phosphofructokinase activity previously described.ConclusionsOverall, this study shows that C. salexigens possesses a greater metabolic flexibility for fructose catabolism, the ED and EMP pathways contributing to a fine balancing of energy and biosynthetic demands and, subsequently, to a more efficient metabolism.

Assessing Cofactor Usage in Pseudoclostridium thermosuccinogenes via Heterologous Expression of Central Metabolic Enzymes.

Pseudoclostridium thermosuccinogenes and Hungateiclostridium thermocellum are being studied for their potential to contribute to a more sustainable bio-based economy. Both species were shown previously to rely on GTP or pyrophosphate instead of ATP as cofactors in specific reactions of central energy metabolism for reasons that are not well understood yet. Since it is often impossible to predict cofactor specificity from the primary protein structure, thirteen enzymes from P. thermosuccinogenes were cloned and heterologous expressed in Escherichia coli to assess the cofactor usage in vitro and paint a more complete picture of the cofactor usage in the central metabolism of P. thermosuccinogenes. The assays were conducted with heat-treated E. coli cell-free extract devoid of background activity to allow the quick assessment of a relatively large number of (thermophilic) enzymes. Selected enzymes were also purified to allow the determination of the enzyme kinetics for competing cofactors. Following the results of the glucokinase (GK), galactokinase, xylulokinase (XK), and ribokinase assays, it seems that phosphorylation of monosaccharides by and large is mainly GTP-dependent. Some possible implications of this relating to the adenylate/guanylate energy charge are discussed here. Besides the highly expressed pyrophosphate-dependent 6-phosphofructokinase, another 6-phosphofructokinase was found to be equally dependent on ATP and GTP, while no 6-phosphofructokinase activity could be demonstrated for a third. Both type I glyceraldehyde 3-phosphate dehydrogenases were found to be NAD+-dependent, and further, acetate kinase, isocitrate dehydrogenase, and three enzymes predicted to be responsible for the interconversion of phosphoenolpyruvate and pyruvate (i.e., pyruvate kinase; pyruvate, phosphate dikinase; phosphoenolpyruvate synthase), were also assessed.

Heterologous expression and kinetic characterization of the α, β and αβ blend of the PPi-dependent phosphofructokinase from Citrus sinensis

This work reports the molecular cloning and heterologous expression of the genes coding for α and β subunits of pyrophosphate-dependent phosphofructokinase (PPi-PFK) from orange. When expressed individually, both recombinant subunits were produced as highly purified monomeric proteins able to phosphorylate fructose-6-phosphate at the expenses of PPi (specific activity of 0.075 and 0.017 units. mg−1 for α and β subunits, respectively). On the other hand, co-expression rendered a α3β3 hexamer with specific activity three orders of magnitude higher than the single subunits. All the conformations of the enzyme were characterized with respect to its kinetic properties and sensitivity to the regulator fructose-2,6-bisphosphate. A thorough review of current knowledge on the matter indicates that this is the first report of the recombinant production of active plant PPi-PFK and the characterization of its different conformations. This is a main contribution for future studies focused to better understand the enzyme properties and how it accomplishes its relevant role in plant metabolism.

Comparative Proteomics of Three Species of Ammonia-Oxidizing Bacteria.

Ammonia-oxidizing bacteria (AOB) are important members of terrestrial, marine, and industrial microbial communities and play a fundamental role in the Nitrogen cycle within these systems. They are responsible for the first step of nitrification, ammonia oxidation to nitrite. Although AOB are widespread and essential to environmental and industrial systems, where they regularly experience fluctuations in ammonia availability, no comparative studies of the physiological response of diverse AOB species at the protein level exist. In the present study, we used 1D-LC-MS/MS proteomics to compare the metabolism and physiology of three species of ammonia AOB, Nitrosomonas europaea, Nitrosospira multiformis, and Nitrosomonas ureae, under ammonia replete and ammonia starved conditions. Additionally, we compared the expression of orthologous genes to determine the major differences in the proteome composition of the three species. We found that approximately one-third of the predicted proteome was expressed in each species and that proteins for the key metabolic processes, ammonia oxidation and carbon fixation, were among the most abundant. The red copper protein, nitrosocyanin was highly abundant in all three species hinting toward its possible role as a central metabolic enzyme in AOB. The proteomic data also allowed us to identify pyrophosphate-dependent 6-phosphofructokinase as the potential enzyme replacing the Calvin-Benson-Bassham cycle enzyme Fructose-1,6-bisphosphatase missing in N. multiformis and N. ureae. Additionally, between species, there were statistically significant differences in the expression of many abundant proteins, including those related to nitrogen metabolism (nitrite reductase), motility (flagellin), cell growth and division (FtsH), and stress response (rubrerythrin). The three species did not exhibit a starvation response at the proteome level after 24 h of ammonia starvation, however, the levels of the RuBisCO enzyme were consistently reduced after the starvation period, suggesting a decrease in capacity for biomass accumulation. This study presents the first published proteomes of N. ureae and N. multiformis, and the first comparative proteomics study of ammonia-oxidizing bacteria, which gives new insights into consistent metabolic features and differences between members of this environmentally and industrially important group.

Measurement of glycolysis reactants by high-throughput solid phase extraction with tandem mass spectrometry: Characterization of pyrophosphate-dependent phosphofructokinase as a case study

Glycolysis is a 10-step metabolic pathway involved in producing cellular energy. Many tumors exhibit accelerated glycolytic rates, and enzymes that participate in this pathway are focal points of cancer research. Here, a novel method for the measurement of glycolysis reactants from in vitro samples is presented. Fast and direct measurement is achieved by an automated system that couples on-line solid phase extraction (SPE) with tandem mass spectrometry (MS/MS). The single analytical method enables multiple reactants to be measured concurrently, sustains a cycle time of 8s, and permits the measurement of up to 10,000 samples per day. Concentration–response curves were conducted using standards for 10 metabolic intermediates, and the results demonstrate that the detection strategy has excellent sensitivity (average limit of detection=5.4nM), dynamic range (nanomolar to micromolar), and linear response (average R2=0.998). To test the analysis method on reactions, pyrophosphate-dependent phosphofructokinase (PPi–PFK) was used as a model system. Data that corroborate the activation and inhibition of PPi–PFK are presented, and the ways in which SPE–MS/MS simplifies experimental design and interpretation are highlighted. In summary, the method for measuring metabolic intermediates described here demonstrates unprecedented speed, performance, and versatility.

'OMICS' AND CHEMICAL APPROACHES USED TO MONITOR IRON-DEFICIENCY IN CITRUS ROOTSTOCKS

Two different ‘omics’ approaches were performed to a better comprehension of biological mechanisms involved in citrus iron (Fe) deficiency. Tips roots from Swingle citrumelo and Carrizo citrange (sensitive and tolerant rootstocks, respectively), growing in pots with control and chlorotic soil, were used for transcriptomic and proteomic analysis. CombiMatrix array was performed to isolate differential genes, among which glutathione peroxidase (GPX), SAUR gene and glutamate dehydrogenase (GDH) showed to be the most involved ones. They were switched on Swingle grown on calcareous conditions compared to Carrizo (in the same soil) and to the same stock in the control soil. The over-expression of GPX could be the effort of plants to neutralize the oxidative environment produced by stress. The involvement of auxin (and as consequence of SAUR gene) in the regulation of Fe deficiency responses is also well known. Both genes were considered in association to peroxidase, ferric chelate reductase activities, iron and chlorophyll content, to monitor the degree of suffering of rootstocks. Among differentially expressed proteins, identified by means of 2D-PAGE and RP-HPLC/nESI-MSMS, a strong down-regulation of cytosolic pyrophosphate-dependent phosphofructokinase I²-subunit and NADPH-isocitrate dehydrogenase could produce plant inability to sustain the energetic request of cell roots.

Characterisation of the subunit genes of pyrophosphate-dependent phosphofructokinase from loquat (Eriobotrya japonica Lindl.)

Pyrophosphate-dependent phosphofructokinase (PFP) catalyses the reversible phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate in the glycolysis pathway. Two full-length complementary DNAs encoding putative PFP α- and β-subunits, named EjPFPa and EjPFPb1, were isolated by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of the cDNA ends (RACE) from Eriobotrya japonica Lindl. (loquat). The other β-subunit gene was identified from transcriptome data obtained by Illumina sequencing, designated as EjPFPb2. They share up to 88 % identity with other plant PFP α/β-subunits. EjPFPa and EjPFPb clustered separately in clades of plant PFP α- or β-subunits, respectively. EjPFPa and EjPFPb were both shown to be mainly localised in the cell membrane using confocal microscopy of GFP fusion proteins 35S:EjPFPa/b-GFP. Monitoring the dynamic changes of transcripts and proteins demonstrated that EjPFPa and EjPFPb did not show coordinated expression during fruit development in loquat. The transcript levels of EjPFPa and EjPFPb in the leaves of loquat seedlings were significantly enhanced after 3 h of treatment with 0.5, 1.0 and 1.5 M fructose or glucose, which indicates that EjPFPa and EjPFPb are modulated by fructose and glucose in vivo. Transgenic tobacco plants overexpressed EjPFPa did not show visible phenotype changes, while plants overexpressing EjPFPb1 grew faster at the cost of reduced leaf size and sucrose content. Meanwhile, fructose content increased in growing plants compared with wild-type plants. The results confirm that genes of the two subunits from loquat PFP have different transcriptional regulation systems and play different roles in carbohydrate metabolism. Elevation of the PFP β-subunit gene expression can partly impact the glycolytic carbon allocation in growing plants.

Carbon partitioning in sugarcane (Saccharum species).

Focus has centered on C-partitioning in stems of sugarcane (Saccharum sp.) due to their high-sucrose accumulation features, relevance to other grasses, and rising economic value. Here we review how sugarcane balances between sucrose storage, respiration, and cell wall biosynthesis. The specific topics involve (1) accumulation of exceptionally high sucrose levels (up to over 500 mM), (2) a potential, turgor-sensitive system for partitioning sucrose between storage inside (cytosol and vacuole) and outside cells, (3) mechanisms to prevent back-flow of extracellular sucrose to xylem or phloem, (4) apparent roles of sucrose-P-synthase in fructose retrieval and sucrose re-synthesis, (5) enhanced importance of invertases, and (6) control of C-flux at key points in cell wall biosynthesis (UDP-glucose dehydrogenase) and respiration (ATP- and pyrophosphate-dependent phosphofructokinases). A combination of emerging technologies is rapidly enhancing our understanding of these points and our capacity to shift C-flux between sucrose, cell wall polymers, or other C-sinks.

Differential expression of genes involved in alternative glycolytic pathways, phosphorus scavenging and recycling in response to aluminum and phosphorus interactions in Citrus roots

The objective was to determine the possible links between the expression levels of genes involved in alternative glycolytic pathways, phosphorus (P) scavenging and recycling and Citrus tolerance to aluminum (Al) and/or P-deficiency. 'Xuegan' (Citrus sinensis) and 'Sour pummelo' (Citrus grandis) seedlings were irrigated for 18weeks with nutrient solution containing 0 and 1.2mM AlCl(3)·6H(2)O×0, 50 and 200μM KH(2)PO(4). C. sinensis displayed more tolerant to Al and P-deficiency than C. grandis. Under Al stress, C. sinensis accumulated more Al in roots and less Al in shoots than C. grandis. P concentration was higher in C. sinensis shoots and roots than in C. grandis ones. C. sinensis roots secreted more malate and citrate than C. grandis ones when exposed to Al. Al-induced-secretion of malate and citrate by excised roots from Al-treated seedlings decreased with increasing P supply. Al-induced-secretion of malate and citrate from roots and Al precipitation by P in roots might be responsible for Al-tolerance of C. sinensis. qRT-PCR analysis showed that Al-activated malate transporter (ALMT1), ATP-dependent phosphofructokinase (ATP-PFK), pyrophosphate-dependent phosphofructokinase (PPi-PFK), tonoplast adenosine-triphosphatase subunit A (V-ATPase A), tonoplast pyrophosphatase (V-PPiase), pyruvate kinase (PK), acid phosphatase (APase), phosphoenolpyruvate carboxylase (PEPC), malic enzyme (ME) and malate dehydrogenase (MDH) genes might contribute to the tolerance of Citrus to Al and/or P-deficiency, but any single gene could not explain the differences between the two species. Citrus tolerance to Al and/or P-deficiency might be caused by the coordinated regulation of gene expression involved in alternative glycolytic pathways, P scavenging and recycling.

Status quo in physiological proteomics of the unculturedRiftia pachyptilaendosymbiont

Riftia pachyptila, the giant deep-sea tube worm, inhabits hydrothermal vents in the Eastern Pacific ocean. The worms are nourished by a dense population of chemoautotrophic bacterial endosymbionts. Using the energy derived from sulfide oxidation, the symbionts fix CO(2) and produce organic carbon, which provides the nutrition of the host. Although the endosymbionts have never been cultured, cultivation-independent techniques based on density gradient centrifugation and the sequencing of their (meta-) genome enabled a detailed physiological examination on the proteomic level. In this study, the Riftia symbionts' soluble proteome map was extended to a total of 493 identified proteins, which allowed for an explicit description of vital metabolic processes such as the energy-generating sulfide oxidation pathway or the Calvin cycle, which seems to involve a reversible pyrophosphate-dependent phosphofructokinase. Furthermore, the proteomic view supports the hypothesis that the symbiont uses nitrate as an alternative electron acceptor. Finally, the membrane-associated proteome of the Riftia symbiont was selectively enriched and analyzed. As a result, 275 additional proteins were identified, most of which have putative functions in electron transfer, transport processes, secretion, signal transduction and other cell surface-related functions. Integrating this information into complex pathway models a comprehensive survey of the symbiotic physiology was established.