Phosphate Translocator Research Articles

Overexpression of the triose phosphate translocator (TPT) complements the abnormal metabolism and development of plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase mutants.

The presence of two glycolytic pathways working in parallel in plastids and cytosol has complicated the understanding of this essential process in plant cells, especially the integration of the plastidial pathway into the metabolism of heterotrophic and autotrophic organs. It is assumed that this integration is achieved by transport systems, which exchange glycolytic intermediates across plastidial membranes. However, it is unknown whether plastidial and cytosolic pools of 3-phosphoglycerate (3-PGA) can equilibrate in non-photosynthetic tissues. To resolve this question, we employed Arabidopsis mutants of the plastidial glycolytic isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPCp) that express the triose phosphate translocator (TPT) under the control of the 35S (35S:TPT) or the native GAPCp1 (GAPCp1:TPT) promoters. TPT expression under the control of both promoters complemented the vegetative developmental defects and metabolic disorders of the GAPCp double mutants (gapcp1gapcp2). However, as the 35S is poorly expressed in the tapetum, full vegetative and reproductive complementation of gapcp1gapcp2 was achieved only by transforming this mutant with the GAPCp1:TPT construct. Our results indicate that the main function of GAPCp is to supply 3-PGA for anabolic pathways in plastids of heterotrophic cells and suggest that the plastidial glycolysis may contribute to fatty acid biosynthesis in seeds. They also suggest a 3-PGA deficiency in the plastids of gapcp1gapcp2, and that 3-PGA pools between cytosol and plastid do not equilibrate in heterotrophic cells.

Phosphate Uptake and Allocation - A Closer Look at Arabidopsis thaliana L. and Oryza sativa L.

This year marks the 20th anniversary of the discovery and characterization of the two Arabidopsis PHT1 genes encoding the phosphate transporter in Arabidopsis thaliana. So far, multiple inorganic phosphate (Pi) transporters have been described, and the molecular basis of Pi acquisition by plants has been well-characterized. These genes are involved in Pi acquisition, allocation, and/or signal transduction. This review summarizes how Pi is taken up by the roots and further distributed within two plants: A. thaliana and Oryza sativa L. by plasma membrane phosphate transporters PHT1 and PHO1 as well as by intracellular transporters: PHO1, PHT2, PHT3, PHT4, PHT5 (VPT1), SPX-MFS and phosphate translocators family. We also describe the role of the PHT1 transporters in mycorrhizal roots of rice as an adaptive strategy to cope with limited phosphate availability in soil.

Targeting of a Transporter to the Outer Apicoplast Membrane in the Human Malaria Parasite Plasmodium falciparum.

Apicoplasts are vestigial plastids in apicomplexan parasites like Plasmodium, the causative agent of malaria. Apicomplexan parasites are dependant on their apicoplasts for synthesis of various molecules that they are unable to scavenge in sufficient quantity from their host, which makes apicoplasts attractive drug targets. Proteins known as plastid phosphate translocators (pPTs) are embedded in the outer apicoplast membrane and are responsible for the import of carbon, energy and reducing power to drive anabolic synthesis in the organelle. We investigated how a pPT is targeted into the outer apicoplast membrane of the human malaria parasite P. falciparum. We showed that a transmembrane domain is likely to act as a recessed signal anchor to direct the protein into the endomembrane system, and that a tyrosine in the cytosolic N-terminus of the protein is essential for targeting, but one or more, as yet unidentified, factors are also essential to direct the protein into the outer apicoplast membrane.

Functional Relationship between a Dinoflagellate Host and Its Diatom Endosymbiont.

While we know much about the evolutionary patterns of endosymbiotic organelle origins, we know less about how the actual process unfolded within each system. This is partly due to the massive changes endosymbiosis appears to trigger, and partly because most organelles evolved in the distant past. The dinotoms are dinoflagellates with diatom endosymbionts, and they represent a relatively recent but nevertheless obligate endosymbiotic association. We have carried out deep sequencing of both the host and endosymbiont transcriptomes from two dinotoms, Durinskia baltica and Glenodinium foliaceum, to examine how the nucleocytosolic compartments have functionally integrated. This analysis showed little or no functional reduction in either the endosymbiont or host, and no evidence for genetic integration. Rather, host and endosymbiont seem to be bound to each other via metabolites, such as photosynthate exported from the endosymbiont to the host as indicated by the presence of plastidic phosphate translocators in the host transcriptome. The host is able to synthesize starch, using plant-specific starch synthases, as a way to store imported photosynthate.

Ethylene is involved in root phosphorus remobilization in rice (Oryza sativa) by regulating cell-wall pectin and enhancing phosphate translocation to shoots.

Background and aims Plants are able to grow under phosphorus (P)-deficient conditions by coordinating Pi acquisition, translocation from roots to shoots and remobilization within the plant. Previous reports have demonstrated that cell-wall pectin contributes greatly to rice cell-wall Pi re-utilization under P-deficient conditions, but whether other factors such as ethylene also affect the pectin-remobilizing capacity remains unclear. Methods Two rice cultivars, 'Nipponbare' (Nip) and 'Kasalath' (Kas) were cultured in the +P (complete nutrient solution), -P (withdrawing P from the complete nutrient solution), +P+ACC (1-amino-cyclopropane-1-carboxylic acid, an ethylene precursor, adding 1 μm ACC to the complete nutrient solution) and -P+ACC (adding 1 μm ACC to -P nutrient solution) nutrient solutions for 7 d. Key Results After 7 d -P treatment, there was clearly more soluble P in Nip root and shoot, accompanied by additional production of ethylene in Nip root compared with Kas. Under P-deficient conditions, addition of ACC significantly increased the cell-wall pectin content and decreased cell-wall retained P, and thus more soluble P was released to the root and translocated to the shoot, which was mediated by the expression of the P deficiency-responsive gene OsPT2, which also strongly induced by ACC treatment under both P-sufficient and P-deficient conditions. Conclusions Ethylene positively regulates pectin content and expression of OsPT2, which ultimately makes more P available by facilitating the solubilization of P fixed in the cell wall and its translocation to the shoot.

Localization and Evolution of Putative Triose Phosphate Translocators in the Diatom Phaeodactylum tricornutum.

The establishment of a metabolic connection between host and symbiont is a crucial step in the evolution of an obligate endosymbiotic relationship. Such was the case in the evolution of mitochondria and plastids. Whereas the mechanisms of metabolite shuttling between the plastid and host cytosol are relatively well studied in Archaeplastida—organisms that acquired photosynthesis through primary endosymbiosis—little is known about this process in organisms with complex plastids. Here, we focus on the presence, localization, and phylogeny of putative triose phosphate translocators (TPTs) in the complex plastid of diatoms. These proteins are thought to play an essential role in connecting endosymbiont and host metabolism via transport of carbohydrates generated by the photosynthesis machinery of the endosymbiont. We show that the complex plastid localized TPTs are monophyletic and present a model for how the initial metabolic link between host and endosymbiont might have been established in diatoms and other algae with complex red plastids and discuss its implications on the evolution of those lineages.

English

This study was designed to characterize morphological, physiological and molecular responses of sugarcane genotypes to a simulated water deficit stress. Two genotypes (TSP05-4: Drought-tolerant; TCP02-4589: Drought-sensitive) were subjected to a 20-day water deficit treatment and an 8-day recovery period. Leaf photosynthesis (Pn), transpiration rate (E), stomatal conductance (Gs), leaf greenness index (SPAD) and variable-to-maximum chlorophyll a fluorescence ratio (Fv/Fm), were evaluated before, during and after water deficit. Root-to-shoot ratio (R/S), stalk height (SH), diameter (SD) and stalk weight (SW) were evaluated at the end of the experiment. Real-time RT-PCR confirmed seven differentially-expressed transcript-derived fragments (TDFs) identified by cDNA-AFLP. Pn rates were similar between the genotypes under well-watered conditions. However, under water deficit, TSP05-4 had higher Pn rates. SPAD, Fv/Fm and R/S were also generally higher in TSP05-4, regardless of soil moisture status. Water deficit-induced reductions in SH and SW were greater in TCP02-4589 than in TSP05-4. Three TDFs showing sequence similarities to genes encoding a putative expressed pentatricopeptide, a protein kinase CK2 regulatory subunit CK2β3, and a glucose-6-phosphate/phosphate translocator 2 were identified in TCP02-4589. One TDF similar to a drought-inducible protein was identified in TSP05-4. Recovery of physiological processes and gene expression patterns to the water stress levels was fast. Key words: Differential gene expression, water deficit stress, re-watering, Saccharum spp.

WRKY42 modulates phosphate homeostasis through regulating phosphate translocation and acquisition in Arabidopsis.

The Arabidopsis (Arabidopsis thaliana) WRKY transcription factor family has more than 70 members, and some of them have been reported to play important roles in plant response to biotic and abiotic stresses. This study shows that WRKY42 regulated phosphate homeostasis in Arabidopsis. The WRKY42-overexpressing lines, similar to the phosphate1 (pho1) mutant, were more sensitive to low-inorganic phosphate (Pi) stress and had lower shoot Pi content compared with wild-type plants. The PHO1 expression was repressed in WRKY42-overexpressing lines and enhanced in the wrky42 wrky6 double mutant. The WRKY42 protein bound to the PHO1 promoter under Pi-sufficient condition, and this binding was abrogated during Pi starvation. These data indicate that WRKY42 modulated Pi translocation by regulating PHO1 expression. Furthermore, overexpression of WRKY42 increased root Pi content and Pi uptake, whereas the wrky42 mutant had lower root Pi content and Pi uptake rate compared with wild-type plants. Under Pi-sufficient condition, WRKY42 positively regulated PHOSPHATE TRANSPORTER1;1 (PHT1;1) expression by binding to the PHT1;1 promoter, and this binding was abolished by low-Pi stress. During Pi starvation, the WRKY42 protein was degraded through the 26S proteasome pathway. Our results showed that AtWRKY42 modulated Pi homeostasis by regulating the expression of PHO1 and PHT1;1 to adapt to environmental changes in Pi availability.

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 A rabidopsis 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.

Reticulate leaves and stunted roots are independent phenotypes pointing at opposite roles of the phosphoenolpyruvate/phosphate translocator defective in cue1 in the plastids of both organs.

Phosphoenolpyruvate (PEP) serves not only as a high energy carbon compound in glycolysis, but it acts also as precursor for plastidial anabolic sequences like the shikimate pathway, which produces aromatic amino acids (AAA) and subsequently secondary plant products. After conversion to pyruvate, PEP can also enter de novo fatty acid biosynthesis, the synthesis of branched-chain amino acids, and the non-mevalonate way of isoprenoid production. As PEP cannot be generated by glycolysis in chloroplasts and a variety of non-green plastids, it has to be imported from the cytosol by a phosphate translocator (PT) specific for PEP (PPT). A loss of function of PPT1 in Arabidopsis thaliana results in the chlorophyll a/b binding protein underexpressed1 (cue1) mutant, which is characterized by reticulate leaves and stunted roots. Here we dissect the shoot- and root phenotypes, and also address the question whether or not long distance signaling by metabolites is involved in the perturbed mesophyll development of cue1. Reverse grafting experiments showed that the shoot- and root phenotypes develop independently from each other, ruling out long distance metabolite signaling. The leaf phenotype could be transiently modified even in mature leaves, e.g. by an inducible PPT1RNAi approach or by feeding AAA, the cytokinin trans-zeatin (tZ), or the putative signaling molecule dehydrodiconiferyl alcohol glucoside (DCG). Hormones, such as auxins, abscisic acid, gibberellic acid, ethylene, methyl jasmonate, and salicylic acid did not rescue the cue1 leaf phenotype. The low cell density1 (lcd1) mutant shares the reticulate leaf-, but not the stunted root phenotype with cue1. It could neither be rescued by AAA nor by tZ. In contrast, tZ and AAA further inhibited root growth both in cue1 and wild-type plants. Based on our results, we propose a model that PPT1 acts as a net importer of PEP into chloroplast, but as an overflow valve and hence exporter in root plastids.

Influence of tubificid worms on nutrient fluxes across water-sediment interface in fish farm settling ponds

Key-words: tubificids, phosphorus, ammonia, nitrate, fish farming, settling ponds The influence of tubificid worms on nutrient translocation from water to fish farm sediments accumulating in settling ponds was addressed under laboratory conditions. Small microcosms of 0.5 L were filled up with 35 g of sludge from a fish farm settling pond and 0.15 L of filtered settling pond water. The experimental set up consisted of one control line (no worms added), a second experimental line with 1 mg of tubificid worms·g −1 fresh sediment (550 individuals·m −2 ) and a third experimental line with 40 mg of tubificid worms·g −1 fresh sediment (22000 individuals·m −2 ). Nutrients translocation was determined by monitoring overlaying water concentration of ammonia, nitrate and phosphate for ten days. Results showed that abundances of 550 individuals·m −2 had no significant influence on the fluxes of nutrients here considered. However, the influence of higher abundances of tubificids (22000 individuals·m −2 ) was of significant extent on the translocation of nitrate and phosphate. Accordingly, bioturbation of tubificids caused 55% lower nitrate uptake by the sediment when compared to control conditions. Phosphorus released by the sediments of the control condition was ca. 90% higher than that recorded under abundances of tubificids (22000 individuals·m −2 ). Results obtained allowed us to estimate that fish farm settling ponds highly colonized by tubificid worms (22000 individuals·m −2 ) may contribute to decrease phosphorus discharge (in terms of soluble phosphorus) in ca. 5go f P·ton −1 of fish produced.

The SLC34 family of sodium-dependent phosphate transporters

The SLC34 family of sodium-driven phosphate cotransporters comprises three members: NaPi-IIa (SLC34A1), NaPi-IIb (SLC34A2), and NaPi-IIc (SLC34A3). These transporters mediate the translocation of divalent inorganic phosphate (HPO4 (2-)) together with two (NaPi-IIc) or three sodium ions (NaPi-IIa and NaPi-IIb), respectively. Consequently, phosphate transport by NaPi-IIa and NaPi-IIb is electrogenic. NaPi-IIa and NaPi-IIc are predominantly expressed in the brush border membrane of the proximal tubule, whereas NaPi-IIb is found in many more organs including the small intestine, lung, liver, and testis. The abundance and activity of these transporters are mostly regulated by changes in their expression at the cell surface and are determined by interactions with proteins involved in scaffolding, trafficking, or intracellular signaling. All three transporters are highly regulated by factors including dietary phosphate status, hormones like parathyroid hormone, 1,25-OH2 vitamin D3 or FGF23, electrolyte, and acid-base status. The physiological relevance of the three members of the SLC34 family is underlined by rare Mendelian disorders causing phosphaturia, hypophosphatemia, or ectopic organ calcifications.

Characterization of the Plastidic Phosphate Translocators in the Inducible Crassulacean Acid Metabolism PlantMesembryanthemum crystallinum

In plant Mesembryanthemum crystallinum, which has the inducible crassulacean acid metabolism (CAM), isoforms of plastidic phosphate translocators (pPTs) are categorized into three subfamilies: the triose phosphate/phosphate translocator (McTPT1), the phosphoenolpyruvate/phosphate translocator (McPPT1), and the glucose 6-phosphate/phosphate translocator (McGPT1 and McGPT2). In order to elucidate the physiological roles of these pPTs in M. crystallinum, we determined the substrate specificity of each pPT isoform. The substrate specificities of McTPT1, McPPT1, and McGPT1 showed overall similarities to those of orthologs that have been characterized. In contrast, for glucose 6-phosphate, McGPT2 showed higher selectivity than McGPT1 and other GPT orthologs. Because the expression of McGTP2 is specific to CAM while that of McGTP1 is constitutively expressed in both the C3- and the CAM-state in M. crystallinum, we propose that McGPT2 functions as a CAM system-specific GPT in this plant.

Molecular cloning, phylogenetic analysis, and expression profiling of a grape CMP-sialic acid transporter-like gene induced by phytohormone and abiotic stress

Nucleotide sugars are synthesized in the cytosol and nucleus and transported into the lumen of the endoplasmic reticulum and the Golgi apparatus via nucleotide sugar transporters (NSTs). Because NSTs exhibit high similarities with triose phosphate translocators (TPTs), they are classified into the NST/TPT superfamily. Here, we identified 38 members of the NST/TPT family by screening the grapevine genome and proteome 12× database. Vitis vinifera NST/TPT proteins can be classified into two groups on the basis of their phylogenetic relationships. From these, we isolated a full-length cDNA encoding a putative NST and named it VvCSTLP1. VvCSTLP1 comprises 15 exons and 14 introns and exhibits high similarities with OsCSTLP2. A search for cis-regulatory elements in the promoter region of VvCSTLP1 revealed that this protein is probably regulated by phytohormones and abiotic stresses. The VvCSTLP1 cDNA encodes an open reading frame of 1065 bp, and the predicted polypeptide is 354 amino acids long with a molecular mass of 39.11 kDa. Expression of VvCSTLP1 was elevated during early berry development, and dramatically decreased after the initiation of ripening. VvCSTLP1 was highly expressed in old leaves and mature leaves, and at low levels in young leaves, pollen, roots, and tendrils. Finally, VvCSTLP1 expression was induced in response to 1-naphthaleneacetic acid, salicylic acid (SA), and boric acid treatments, but was decreased by drought stress. The regulation of VvCSTLP1 expression led us to conclude that it may play a role in cell wall composition and structure and in the cross-talk between the auxin, SA, and abiotic stress signaling pathways.

Molecular Adaptation of Peanut Metabolic Pathways to Wide Variations of Mineral Ion Composition and Concentration

Plant evolution, nutritional genomics, and mineral nutrition have been well documented but no studies have focused on the molecular adaptation of crop metabolism to wide variations of mineral ion composition and concentration. Diversification of peanut species from primary centers of domestication in South America depended on metabolic adaptation to the mineral ion conditions of the newer habitats. Understanding the diversification molecular biology of peanut metabolic pathways will permit the synthesis of the best mineral ion combinations for doubling CO2 assimilation. Valencia and Virginia cultivars belong to different subspecies of the tetraploid Arachis hypogaea. They were planted in the absence and presence of up to 99 mM (equivalent to 166 moles per hectare) of different mineral ions. Molecular properties of the primary metabolic pathways were studied by Northern analyses using Valencia GDH-synthesized RNAs as probes for Virginia mRNA and GDH-synthesized RNAs. Messenger RNAs are silenced by homologous RNAs synthesized by GDH. Peanut cellulose was analyzed by gravimetry; and fatty acids by HPLC. Complementary DNA probes made from Valencia GDH-synthesized RNAs hybridized perfectly to Virginia mRNAs and GDH-synthesized RNAs. Wide variations in mineral ion compositions and concentrations induced the GDHs of Valencia and Virginia to synthesize RNAs that differentially down-regulated the mRNAs encoding phosphate translocator, granule-bound starch synthase, phosphoglucomutase, glucosyltransferase, acetyl CoA carboxylase, nitrate reductase, and NADH-glutamate synthase so that the percent weights of oil (41.53 ± 8.75) and cellulose (30.29 ± 3.12) were similar in the control and mineral-treated peanuts. Therefore, RNA sequences that defined the molecular adaptation of mRNAs encoding the enzymes of primary metabolism were the same in the varietal types of A. hypogaea, in agreement with genetic data suggesting that tetraploid Arachis evolved relatively recently from the wild diploid ancestral species. Another molecular adaptation was to phosphate with or without K+ ion, and it prevented the silencing by GDH-synthesized RNAs of the mRNA encoding phosphate translocator resulting to doubling of cellulosic biomass yield (41323 kg/ha) compared with the N + P + K + S-treated positive control peanut (19428 kg/ha). Molecular adaptation of primary metabolic pathways at the mRNA level to SO42- ion with or without SO42- ion did not increase cellulosic biomass yields (27057 kg/ha) compared with negative control peanut because the mRNAs encoding granule-bound starch synthase, and NADH-glutamate synthase were not silenced by GDH-synthesized RNA in the N + S, SO42-, and N + P + K + S-treated peanuts. These results could contribute towards further modeling at the mRNA level for improved mineral nutrient management of peanut production for fuel, fiber, feed, and food.

Molecular cloning, characterization and expression analysis of two members of the Pht1 family of phosphate transporters in Glycine max.

BackgroundPhosphorus is one of the macronutrients essential for plant growth and development. The acquisition and translocation of phosphate are pivotal processes of plant growth. In a large number of plants, phosphate uptake by roots and translocation within the plant are presumed to occur via a phosphate/proton cotransport mechanism.Principal FindingsWe cloned two cDNAs from soybean (Glycine max), GmPT1 and GmPT2, which show homology to the phosphate/proton cotransporter PHO84 from the budding yeast Saccharomyces cerevisiae. The amino acid sequence of the products predicted from GmPT1 and GmPT2 share 61% and 63% identity, respectively, with the PHO84 in amino acid sequence. The deduced structure of the encoded proteins revealed 12 membrane-spanning domains with a central hydrophilic region. The molecular mass values are ∼58.7 kDa for GmPT1 and ∼58.6 kDa for GmPT2. Transiently expressed GFP–protein fusions provide direct evidence that the two Pi transporters are located in the plasma membrane. Uptake of radioactive orthophosphate by the yeast mutant MB192 showed that GmPT1 and GmPT2 are dependent on pH and uptake is reduced by the addition of uncouplers of oxidative phosphorylation. The K m for phosphate uptake by GmPT1 and GmPT2 is 6.65 mM and 6.63 mM, respectively. A quantitative real time RT-PCR assay indicated that these two genes are expressed in the roots and shoots of seedlings whether they are phosphate-deficient or not. Deficiency of phosphorus caused a slight change of the expression levels of GmPT1 and GmPT2.ConclusionsThe results of our experiments show that the two phosphate transporters have low affinity and the corresponding genes are constitutively expressed. Thereby, the two phosphate transporters can perform translocation of phosphate within the plant.

Discovery of polymorphisms in starch‐related genes in rice germplasm by amplification of pooled DNA and deeply parallel sequencing†

High-throughput sequencing of pooled DNA was applied to polymorphism discovery in candidate genes involved in starch synthesis. This approach employed semi- to long-range PCR (LR-PCR) followed by next-generation sequencing technology. A total of 17 rice starch synthesis genes encoding seven classes of enzymes, including ADP-glucose pyrophosphorylase (AGPase), granule starch synthase (GBSS), soluble starch synthase (SS), starch branching enzyme (BE), starch debranching enzyme (DBE) and starch phosphorylase (SPHOL) and phosphate translocator (GPT1) from 233 genotypes were PCR amplified using semi- to long-range PCR. The amplification products were equimolarly pooled and sequenced using massively parallel sequencing technology (MPS). By detecting single nucleotide polymorphism (SNP)/Indels in both coding and noncoding areas of the genes, we identified genetic differences and characterized the SNP/Indel variation and distribution patterns among individual starch candidate genes. Approximately, 60.9 million reads were generated, of which 54.8 million (90%) mapped to the reference sequences. The average coverage rate ranged from 12,708 to 38,300 times for SSIIa and SSIIIb, respectively. SNPs and single/multiple-base Indels were analysed in a total assembled length of 116,403 bp. In total, 501 SNPs and 113 Indels were detected across the 17 starch-related loci. The ratio of synonymous to nonsynonymous SNPs (Ka/Ks) test indicated GBSSI and isoamylase 1 (ISA1) as the least diversified (most purified) and conservative genes as the studied populations have been through cycles of selection. This report demonstrates a useful strategy for screening germplasm by MPS to discover variants in a specific target group of genes.

Renoprotective antioxidant effect of alagebrium in experimental diabetes

Despite the beneficial effects of alagebrium (ALA), a putative advanced glycation end-product (AGE) breaker, on diabetic nephropathy, its renoprotective mechanisms are incompletely understood. Since oxidative stress exacerbates diabetic renal injury through interaction with AGE, the present study examined the antioxidative property of ALA in db/db mice, mesangial cells cultured under high glucose or H(2)O(2) and a test tube. ALA (2 mg/kg/day) was administered intraperitoneally for 12 weeks to 8-week-old db/m and db/db (D(ALA)E) mice or for 4 weeks to 16-week-old db/db mice (D(ALA)L). Oxidative stress markers (nitrotyrosine accumulation, expression and translocation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits, cellular DCF-DA fluorescence) together with urinary albumin excretion and histological changes including mesangial expansion were measured. The concentration of H(2)O(2) in the presence and absence of ALA was measured by iodometric analysis in a test tube. ALA significantly reduced not only urinary albumin excretion and renal pathological changes but also accumulation of pentosidine and nitrotyrosine and expression of NADPH oxidase subunits in db/db mice regardless of treatment protocol. In mesangial cells, ALA effectively prevented not only high glucose- but also H(2)O(2)-induced membrane translocation of NADPH oxidase subunit (p47 phox, p67 phox and rac1) and protein kinase C isoform (α, βI and βII) and Nox4 messenger RNA expression concomitant with cellular reactive oxygen species. Furthermore, ALA directly decreased H(2)O(2) in a test tube. ALA has both direct and indirect antioxidant effects that may play important roles in ALA's renoprotective effect in diabetic kidneys.

Doubling of crop yield through permutation of metabolic pathways

Hunger and food insecurity can be minimized by doubling crop yield without increasing cultivated land area and fertilizer applied. Since plant breeding has not genetically doubled photosynthesis per unit leaf area, an approach for doubling crop yield would be through a biotechnology that reprograms metabolic pathways in favor of photosynthesis. The anchor of this biotechnology is glutamate dehydrogenase (GDH) including the RNAs it synthesizes. Peanut was treated with stoichiometric combinations of mineral salt solutions to synchronize the GDH subunit polypeptides. Matured seeds were analyzed for fats by HPLC; the RNA biosynthetic activity of GDH, and mRNAs encoding yield-specific enzymes by Northern hybridization. In the PK-treated peanut, the GDH-synthesized RNAs silenced the mRNAs encoding granule-bound starch synthase, phosphoglucomutase (glycolysis), glucosyltransferase (cellulose biosynthesis), and nitrate reductase leaving unaffected the mRNAs encoding acetylcoenzyme A carboxylase (fatty acid biosynthesis), phosphate translocator, and NADH-glutamate synthase resulting to double seed (4342 kg/ha), cellulose (1829 kg/ha), and fat (1381 kg/ha) yields compared with the controls. Down-regulation of phosphate translocator and acetylcoenzyme A carboxylase caused decreased pod yields. GDH-synthesized RNAs that were homologous to yield-specific mRNAs shared extensive plus/plus and plus/minus sequence similarities, and they reprogrammed metabolism by permuting the partially down-regulated, not down-regulated, and down-regulated yield-specific pathways. Control peanut produced 70, NPKS-treated produced 420, NS-treated produced 1680, and PK-treated produced 280 probable rearrangements of the pathways. Therefore, down-regulation of metabolic reactions followed by permutation of yield-related pathways, and redistribution of metabolite load to molecularly connected pathways controls crop yield. Operating as efficient bioreactor, peanut can be maximized to 10000 kg pod/ha, more than enough vegetable oil for nine billion people.

The role of plastidial glucose‐6‐phosphate/phosphate translocators in vegetative tissues of Arabidopsis thaliana mutants impaired in starch biosynthesis

Arabidopsis thaliana mutants impaired in starch biosynthesis due to defects in either ADP glucose pyrophosphorylase (adg1-1), plastidic phosphoglucose mutase (pgm) or a new allele of plastidic phosphoglucose isomerase (pgi1-2) exhibit substantial activity of glucose-6-phosphate (Glc6P) transport in leaves that is mediated by a Glc6P/phosphate translocator (GPT) of the inner plastid envelope membrane. In contrast to the wild type, GPT2, one of two functional GPT genes of A. thaliana, is strongly induced in these mutants during the light period. The proposed function of the GPT in plastids of non-green tissues is the provision of Glc6P for starch biosynthesis and/or the oxidative pentose phosphate pathway. The function of GPT in photosynthetic tissues, however, remains obscure. The adg1-1 and pgi1-2 mutants were crossed with the gpt2-1 mutant defective in GPT2. Whereas adg1-1/gpt2-1 was starch-free, residual starch could be detected in pgi1-2/gpt2-1 and was confined to stomatal guard cells, bundle sheath cells and root tips, which parallels the reported spatial expression profile of AtGPT1. Glucose content in the cytosolic heteroglycan increased substantially in adg1-1 but decreased in pgi1-2, suggesting that the plastidic Glc6P pool contributes to its biosynthesis. The abundance of GPT2 mRNA correlates with increased levels of soluble sugars, in particular of glucose in leaves, suggesting induction by the sugar-sensing pathway. The possible function of GPT2 in starch-free mutants is discussed in the background of carbon requirement in leaves during the light-dark cycle.