The organ-specific differential roles of rice DXS and DXR, the first two enzymes of the MEP pathway, in carotenoid metabolism in Oryza sativa leaves and seeds.

BACKGROUND: Deoxyxylulose 5-phosphate synthase (DXS) and deoxyxylulose 5-phosphate reductoisomerase (DXR) are the enzymes that catalyze the first two enzyme steps of the methylerythritol 4-phosphate (MEP) pathway to supply the isoprene building-blocks of carotenoids. Plant DXR and DXS enzymes have been reported to function differently depending on the plant species. In this study, the differential roles of rice DXS and DXR genes in carotenoid metabolism were investigated. RESULTS: The accumulation of carotenoids in rice seeds co-expressing OsDXS2 and stPAC was largely enhanced by 3.4-fold relative to the stPAC seeds and 315.3-fold relative to non-transgenic (NT) seeds, while the overexpression of each OsDXS2 or OsDXR caused no positive effect on the accumulation of either carotenoids or chlorophylls in leaves and seeds, suggesting that OsDXS2 functions as a rate-limiting enzyme supplying IPP/DMAPPs to seed carotenoid metabolism, but OsDXR doesn't in either leaves or seeds. The expressions of OsDXS1, OsPSY1, OsPSY2, and OsBCH2 genes were upregulated regardless of the reductions of chlorophylls and carotenoids in leaves; however, there was no significant change in the expression of most carotenogenic genes, even though there was a 315.3-fold increase in the amount of carotenoid in rice seeds. These non-proportional expression patterns in leaves and seeds suggest that those metabolic changes of carotenoids were associated with overexpression of the OsDXS2, OsDXR and stPAC transgenes, and the capacities of the intermediate biosynthetic enzymes might be much more important for those metabolic alterations than the transcript levels of intermediate biosynthetic genes are. Taken together, we propose a 'Three Faucets and Cisterns Model' about the relationship among the rate-limiting enzymes OsDXSs, OsPSYs, and OsBCHs as a "Faucet", the biosynthetic capacity of intermediate metabolites as a "Cistern", and the carotenoid accumulations as the content of "Cistern". CONCLUSION: Our study suggests that OsDXS2 plays an important role as a rate-limiting enzyme supplying IPP/DMAPPs to the seed-carotenoid accumulation, and rice seed carotenoid metabolism could be largely enhanced without any significant transcriptional alteration of carotenogenic genes. Finally, the "Three Faucets and Cisterns model" presents the extenuating circumstance to elucidate rice seed carotenoid metabolism.

The signal metabolite trehalose-6-phosphate inhibits the sucrolytic activity of sucrose synthase from developing castor beans.

In plants, trehalose 6-phosphate (T6P) is a key signaling metabolite that functions as both a signal and negative feedback regulator of sucrose levels. The mode of action by which T6P senses and regulates sucrose is not fully understood. Here, we demonstrate that the sucrolytic activity of RcSUS1, the dominant sucrose synthase isozyme expressed in developing castor beans, is allosterically inhibited by T6P. The feedback inhibition of SUS by T6P may contribute to the control of sink strength and sucrolytic flux in heterotrophic plant tissues.

Trehalose biosynthesis in response to abiotic stresses.

Trehalose is a non-reducing disaccharide that is present in diverse organisms ranging from bacteria and fungi to invertebrates, in which it serves as an energy source, osmolyte or protein/membrane protectant. The occurrence of trehalose and trehalose biosynthesis pathway in plants has been discovered recently. Multiple studies have revealed regulatory roles of trehalose-6-phosphate, a precursor of trehalose, in sugar metabolism, growth and development in plants. Trehalose levels are generally quite low in plants but may alter in response to environmental stresses. Transgenic plants overexpressing microbial trehalose biosynthesis genes have been shown to contain increased levels of trehalose and display drought, salt and cold tolerance. In-silico expression profiling of all Arabidopsis trehalose-6-phosphate synthases (TPSs) and trehalose-6-phosphate phosphatases (TPPs) revealed that certain classes of TPS and TPP genes are differentially regulated in response to a variety of abiotic stresses. These studies point to the importance of trehalose biosynthesis in stress responses.

Trehalose 6-phosphate: a signal of sucrose status.

T6P (trehalose 6-phosphate), the precursor of trehalose, has come out of obscurity over 10 years to be appreciated as an important regulator of plant metabolism and development, quite possibly linking the two. This information has been gained from analysis of mutant and transgenic plants, which show strong, diverse and strategically important phenotypes. Plant genes that encode the trehalose pathway are numerous and highly regulated transcriptionally and post-translationally, responding sensitively to the environment in a developmentally programmed and tissue-specific manner further suggestive of a vital function. Yet the precise role of T6P has not been clear. In an article published in the Biochemical Journal in 2006, John Lunn and colleagues addressed a major obstacle to understanding the function of T6P through development of a method capable of resolving femtomolar quantities of T6P from very small amounts of tissue. Using this technology, the authors showed large changes in T6P content that reflect tissue sucrose status. Overall, this elegant work makes an important contribution towards our understanding of the function of T6P in plants.

Trehalose 6-phosphate.

Trehalose 6-phosphate (T6P) is a sugar signal of emerging significance. It is an essential component of the mechanisms that coordinate metabolism with plant growth adaptation and development. Its significance began to dawn when genetic modification of the trehalose pathway produced dramatic phenotypes, before the genetic proliferation of the trehalose pathway in plants was fully realised. T6P regulates sugar utilization and starch metabolism and interacts with other signalling pathways, including those mediated by plant hormones. Trehalose phosphate synthases (TPSs) and trehalose phosphate phosphatases are regulated at the gene level by sugars, nitrate, cytokinin and abscisic acid. TPSs are also regulated post-translationally. Mechanistic details of how T6P signals are emerging, but still sparse. Nevertheless, even at this stage, targeting central regulators such as T6P offers promise in crop improvement.

The MEP pathway: a new target for the development of herbicides, antibiotics and antimalarial drugs.

Isoprenoids, a diverse group of compounds derived from the five-carbon building units isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), are essential for survival in all organisms. Animals synthesize their isoprenoids from mevalonic acid (MVA), whereas most pathogenic bacteria and the malaria parasites utilize a completely different pathway for IPP and DMAPP synthesis, the methylerythritol phosphate (MEP) pathway. Plants use both pathways for the synthesis of isoprenoid precursors. The recent elucidation of the MEP pathway has opened the possibility to develop new strategies against microbial pathogens. Novel immunotherapeutic agents can be developed based on the MEP pathway intermediates known to activate the proliferation of human V-delta-9 V-gamma-2 T-cells after infection by many pathogenic bacteria and protozoa. Moreover, the design of specific inhibitors of MEP pathway enzymes (which are highly conserved but show no homology to mammalian proteins) should result in herbicides and drugs with broad-spectrum antimicrobial activity without mechanism-based toxicity to humans. A good example is the cure of bacterial infections and malaria with fosmidomycin, a highly stable inhibitor of the MEP pathway. The use of plants as test systems has led to the identification of additional inhibitors such as ketoclomazone. Biochemical, genetic and crystallographic approaches with the MEP pathway enzymes are now starting to characterize the inhibition kinetics and identify which residues play a structural or catalytic role. Current efforts should eventually contribute to an effective drug designed to fight against microbial pathogens that show resistance to currently available agents.

Cutting edge: human gamma delta T cells are activated by intermediates of the 2-C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis.

Activation of V gamma 9/V delta 2 T cells by small nonprotein Ags is frequently observed after infection with various viruses, bacteria, and eukaryotic parasites. We suggested earlier that compounds synthesized by the 2-C:-methyl-D-erythritol 4-phosphate (MEP) pathway of isopentenyl pyrophosphate synthesis are responsible for the V gamma 9/V delta 2 T cell reactivity of many pathogens. Using genetically engineered Escherichia coli knockout strains, we now demonstrate that the ability of E. coli extracts to stimulate gamma delta T cell proliferation is abrogated when genes coding for essential enzymes of the MEP pathway, dxr or gcpE, are disrupted or deleted from the bacterial genome.

Phosphorylation of recombinant human ATP:citrate lyase by cAMP-dependent protein kinase abolishes homotropic allosteric regulation of the enzyme by citrate and increases the enzyme activity. Allosteric activation of ATP:citrate lyase by phosphorylated sugars.

Recombinantly expressed human ATP:citrate lyase was purified from E. coli, and its kinetic behavior was characterized before and after phosphorylation. Cyclic AMP-dependent protein kinase catalyzed the incorporation of only 1 mol of phosphate per mole of enzyme homotetramer, and glycogen synthase kinase-3 incorporated an additional 2 mol of phosphate into the phosphorylated protein. Isoelectric focusing revealed that all of the phosphates were incorporated into only one of the four enzyme subunits. Phosphorylation resulted in a 6-fold increase in V(max) and the conversion of citrate dependence from sigmoidal, displaying negative cooperativity, to hyperbolic. The phosphorylated recombinant enzyme is more similar to the enzyme isolated from mammalian tissues than unphosphorylated enzyme with respect to the K(m) for citrate, CoA, and ATP, and the specific activity. Fructose 6-phosphate was found to be a potent activator (60-fold) of the unphosphorylated recombinant enzyme, with half-maximal activation at 0.16 mM, which results in a decrease in the apparent K(m) for citrate and ATP, as well as an increase in the V(max) of the reaction. Thus, human ATP:citrate lyase activity is regulated in vitro allosterically by phosphorylated sugars as well as covalently by phosphorylation.

Cloning and biochemical characterisation of Aspergillus niger hexokinase--the enzyme is strongly inhibited by physiological concentrations of trehalose 6-phosphate.

The Aspergillus niger hexokinase gene hxkA has been cloned by heterologous hybridisation using the Aspergillus nidulans hexokinase gene as a probe. The DNA sequence of the gene was determined, and the deduced amino acid sequence showed significant similarity to other eukaryotic hexokinase and glucokinase proteins, in particular to those of the budding yeasts. The encoded protein was purified from a multicopy hxkA transformant, and extensively characterised. The hexokinase protein has a molecular mass of 54090, a pI of 4.9 and is a homodimer. D-Glucose, the glucose analogue 2-deoxy-D-glucose, D-fructose, D-mannose and D-glucosamine are phosphorylated by hexokinase, whereas the hexoses D-galactose, L-sorbose, methyl alpha-D-glucoside and the pentoses L-arabinose and D-xylose are not. The enzyme has high affinity for glucose (Km = 0.35 mM at pH 7.5) and for fructose (Km = 2.0 mM at pH 7.5) and is inhibited by ADP. The enzyme is strongly inhibited by physiological concentrations (0.1-0.2 mM) of trehalose 6-phosphate, which may be of importance for in vivo regulation of the enzyme. Inhibition of A. niger hexokinase by trehalose 6-phosphate is competitive towards the sugar substrate (Ki = 0.01 mM). Based on the kinetic constants of hexokinase and glucokinase their relative contribution to in vivo glucose phosphorylation was calculated and found to be strongly dependent on intracellular pH and glucose concentration. At pH 7.5 glucokinase is predominant, whereas at pH 6.5 hexokinase is predominant at glucose concentrations higher than 0.5 mM. Expression of the hexokinase and the glucokinase gene requires active carbon metabolism. Also on carbon sources which are not substrates for hexokinase or glucokinase, clear expression is observed. The hexokinase and glucokinase enzymes are quite stable in vivo. Even in the absence of transcription, active glucokinase and hexokinase remain present in the cells at almost the same level for at least 3-4 h after depletion of the carbon source.

Opine catabolism and conjugal transfer of the nopaline Ti plasmid pTiC58 are coordinately regulated by a single repressor.

The Ti plasmids of Agrobacterium tumefaciens are conjugal elements whose transfer is strongly repressed. Transfer is induced by the conjugal opines, a group of unique carbon compounds synthesized in crown gall tumors. The opines also induce Ti plasmid-encoded genes required by the bacteria for opine catabolism. We have cloned and sequenced a gene from the Ti plasmid pTiC58, whose product mediates the opine-dependent regulation of conjugal transfer and catabolism of the conjugal opines, agrocinopines A and B. The gene, accR, is closely linked to the agrocinopine catabolic locus. A spontaneous mutant Ti plasmid, pTiC58Trac, which constitutively expresses conjugal transfer and opine catabolism, was complemented in trans by a clone of wild-type accR. Comparative sequence analysis identified a 5-base-pair deletion close to the 5' end of the mutant accR allele from pTiC58Trac. Analysis of lacZ fusions in conjugal transfer and opine catabolic structural genes demonstrated that the accR-encoded function is a transcriptional repressor. accR can encode a 28-kDa protein. This protein is related to a class of repressor proteins that includes LacR, GutR, DeoR, FucR, and GlpR that regulate sugar catabolic systems in several bacterial genera.

Suppression of focus formation by bovine papillomavirus-transformed cells by contact with non-transformed cells: involvement of sugar(s) and phosphorylation.

Focus formation by bovine papilloma virus-transformed C127 cells was inhibited by direct contact with non-transformed C127 cells. The suppressive capacity of C127 cells was abolished by introduction of the neomycin resistance gene (neor) but not by that of the hygromycin resistance gene (hygrr). Though both genes code for phosphotransferase which inactivates the aminoglycoside antibiotics, their substrates are different, i.e., there is no cross-resistance between them. As the neomycin phosphotransferase phosphorylates the specific hydroxyl group of the sugar in the aminoglycosides, such as 3'OH of the glucose residue of kanamycin A, some specific sugar(s) on the molecules exposed on the cell surface must be responsible for the suppressive signal and their phosphorylation must have resulted in the loss of that signal. The sugar must have the structure shared by kanamycin, neomycin or G418 but not by hygromycin B. Involvement of sugar was also suggested by the observation that concanavalin A partially abrogated the suppressive capacity of C127 cells.

The Albert Lasker Medical Awards. Inositol trisphosphate, calcium, lithium, and cell signaling.

Inositol lipids play a major role in cell signaling by functioning as precursors of second messengers. Of the three common inositol-containing lipids found in the plasma membrane, phosphatidylinositol (4,5)-bisphosphate is hydrolyzed to give diacylglycerol, which stimulates protein kinase C, and inositol 1,4,5-trisphosphate, which diffuses into the cell to release intracellular calcium. Inositol 1,4,5-trisphosphate is metabolized to give free inositol by two separate pathways. Lithium inhibits the final dephosphorylation step of both pathways, thus reducing the supply of the free inositol required to maintain the lipid precursors used for signaling. An inositol-depletion hypothesis may explain both the teratogenic effects of lithium and its therapeutic action in controlling manic-depressive illness. One of the metabolic pathways generates inositol tetrakisphosphate, which may also play a messenger role by expanding the size of the inositol 1,4,5-trisphosphate-sensitive pool of calcium. Calcium imaging of single cells has begun to reveal that this inositol 1,4,5-trisphosphate/calcium signaling system is organized in complex patterns, which include localization of calcium signals to discrete regions of cells and the generation of both calcium waves and calcium oscillations.

Inositol phosphates and cell signalling.

Inositol 1,4,5-trisphosphate is a second messenger which regulates intracellular calcium both by mobilizing calcium from internal stores and, perhaps indirectly, by stimulating calcium entry. In these actions it may function with its phosphorylated metabolite, inositol 1,3,4,5-tetrakisphosphate. The subtlety of calcium regulation by inositol phosphates is emphasized by recent studies that have revealed oscillations in calcium concentration which are perhaps part of a frequency-encoded second-messenger system.

Diacylglycerol, but not inositol 1,4,5-trisphosphate, accounts for platelet-derived growth factor-stimulated proliferation of BALB 3T3 cells.

Recently we found that an intracellular event related to phosphatidylinositol 4,5-bisphosphate (PIP2) is crucial for platelet-derived growth factor (PDGF)-induced mitogenesis in fibroblastic cells (Matuoka, K., et al.: Science 239:640-643, 1988). In the present study we examined the mitogenic effects of PIP2 and its hydrolysis products introduced into the cytoplasm of BALB 3T3 cells by micro-injection to confirm the role of PIP2 hydrolysis in PDGF stimulation of cell proliferation. Injection of 1,2-dioleylglycerol (diolein) into serum-deprived quiescent cells induced DNA synthesis with the same time course as that induced by exposure of the cells to PDGF and, in the presence of PDGF, caused no additional increase in the cell population entering S phase. The injection of PIP2, inositol 1,4,5-trisphosphate, or 1,2-dioleylphosphatidic acid into the cells did not induce mitogenesis. Consistent results were obtained in experiments in which the cells were exposed to 1-oleyl-2-acetylglycerol (OAG) and ionomycin; namely, OAG stimulated proliferation of BALB 3T3 cells, but ionomycin did not induce any mitogenesis. Desensitization of the protein kinase C pathway by prolonged exposure of the cells to phorbol ester abolished the induction of cell proliferation by subsequent injection of diolein or exposure to phorbol ester or OAG as well as by PDGF challenge. These findings strongly suggest that activation of the protein kinase C system following formation of diacylglycerol by PIP2 hydrolysis is mainly responsible for the mitogenic action of PDGF on BALB 3T3 cells.

Dissociation of alpha-adrenergic and cholinergic stimulated inositol trisphosphate-dependent calcium mobilization at the "post-receptor" level.

Carbachol stimulated inositol trisphosphate (IP3) production and subsequent calcium mobilization in parotid cells are almost completely inhibited by neomycin. In contrast epinephrine stimulated IP3 production and calcium mobilization are much less sensitive to such inhibition. Since neomycin exerts its effects primarily at the level of inositol phosphate production and action, cholinergic and alpha adrenergic stimulation of IP3 dependent calcium mobilization may proceed through different "post-receptor" signal transduction mechanisms in parotid cells.

[Secondary messenger level of inositol triphosphate in fibroblasts after treatment with papaverine]. / Az inozitol-trifoszfát másodlagos messenger szintje fibroblasztokban papaverin kezelés után.

Ten per cent foetal bovine serum had been added to human embryonic fibroblasts which had previously been incubated in serum free cultures. The effect has been studied on quantity of synthetized DNA in next 30 hours and on inositol-1,4,5-phosphate contents of cells. On the effect of foetal bovine serum synthesis of DNA has increased four times and 1 minute after its addition inositol-1,4,5-phosphate level has doubled. Papaverine given together with FBS has suspended inductive effect of FBS on DNA synthesis and its increasing effect on level of inositol-1,4,5-phosphate.

Inositolphospholipid-linked glutamate receptors mediate cerebellar parallel-fiber-Purkinje-cell synaptic transmission.

In slices of adult rat cerebellum inositolphospholipid turnover is stimulated markedly by glutamate and its rigid analogues quisqualate and ibotenate. The drug and amino acid specificity of the response reflects a quisqualate-preferring excitatory amino acid receptor. The absence of glutamate-enhanced inositolphospholipid turnover in mice with Purkinje-cell degeneration indicates that the inositolphospholipid-linked quisqualate receptor mediates parallel fiber-Purkinje cell synaptic transmission. The quantitative prominence of this synapse accounts for the massive enrichment of elements of the inositolphospholipid system in cerebellar Purkinje cells.

[Effect of vasopressin on the osmotic permeability of the bladder wall in the frog and its content of cAMP, cGMP and inositol triphosphate]. / Vliianie vazopressina na osmoticheskuiu pronitsaemost' stenki mochevogo puzyria liagushki i soderzhanie v nei tsAMF, tsGMF i inozittrifosfata

An increase in the water permeability of the frog urinary bladder due to vasopressin, correlates with an increase of cAMP content in the bladder tissue. The osmotic permeability reached its maximum in 15-20 min. The sharp increase of inositol triphosphate content was observed within 20 sec after vasopressin administration, whereas cGMP content significantly decreased within 5 min. The augmentation of cAMP content seems to lead to a rise in water permeability white inositol triphosphate and cGMP acted, probably, as modulators of the vasopressin effect.

Chemoattractant receptor promotion of Ca2+ influx across the plasma membrane of HL-60 cells. A role for cytosolic free calcium elevations and inositol 1,3,4,5-tetrakisphosphate production.

UNLABELLED: The mechanisms by which the chemotactic peptide formyl-methyl-leucyl-phenyl-alanine stimulates Ca2+ influx across the plasma membrane were investigated in the human promyelocytic cell line HL-60, induced to differentiate with dimethyl sulfoxide. Ca2+ influx was determined: (a) from the initial rate of Mn2+ influx, apparent from the quenching of intracellular quin2 or fura-2 fluorescence; (b) from the rate of the elevation of cytosolic free calcium, [Ca2+]i, upon readdition of Ca2+ to cells previously stimulated in the absence of extracellular Ca2+. [3H]Inositol tris-, tetrakis-, and pentakisphosphates were analyzed by a high performance liquid chromatography procedure which was optimized for the separation of inositol tetrakisphosphates, yielding three predominant isomers: inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), inositol 1,4,5,6-tetrakisphosphate, and inositol 1,3,4, 6-tetrakisphosphate. Both the kinetics and agonist dose dependence of Ca2+ influx stimulation correlated closely with the corresponding receptor-mediated variations of [Ca2+]i either in the presence or in the absence of extracellular Ca2+. Of the different inositol phosphates determined in parallel and under the same conditions, accumulation of [3H]Ins(1,3,4,5)P4 correlated best with Ca2+ influx both temporally and in its dose dependence in the presence or in the absence of extracellular Ca2+; inositol 1,3,4-trisphosphate was also correlated but to a lesser extent. Attenuations of [Ca2+]i elevations by decreasing extracellular Ca2+ or by increasing the cytosolic Ca2+ buffering capacity with quin2 led to parallel inhibition of Ca2+ influx and Ins(1,3,4,5)P4 production. IN CONCLUSION: 1) activation of Ca2+ influx by formyl-methionyl-leucyl-phenylalanine depends on the elevation of [Ca2+]i, the latter being initiated by Ca2+ mobilization from intracellular stores; 2) Ins(1,3, 4,5)P4 is a strong candidate for maintaining receptor-mediated activation of Ca2+ influx in differentiated HL-60 cells.