Gravitropism in lateral roots of Arabidopsis pgm-1 mutants is indistinguishable from that of wild-type

The majority of understanding of root gravity responses comes from the study of primary roots, even though lateral roots make a far greater contribution to root system architecture. The focus of this report is the analysis of gravitropic responses in lateral roots of wild-type background and pgm-1 mutants. Despite the significant reduction in gravitropic response of primary roots of pgm-1 mutants, the lateral roots of this mutant demonstrate wild-type rates of gravitropism, suggesting a significant difference in gravity signal transduction between primary and lateral roots.Key words: gravitropism, lateral roots, pgm-1, root system architecturePlants are extremely sensitive to numerous environmental stimuli, including touch, gravity, light and humidity, among many others. As a pervasive signal on Earth, gravity exerts a persistent influence on plant morphogenesis by directing the primary roots and shoots of most species to align parallel with the gravity vector. The vertical orientations obtained by primary organs has provided for a simple assay of gravitropic responses, and much of our understanding of gravity stimulus perception, signal transduction and differential growth response has been gained by a focus on primary organ systems.With respect to gravity stimulus perception, there is strong evidence that the movement of starch-filled plastids plays a primary role in the detection of a change in the orientation of an organ relative to gravity.¹ Consistent with this evidence, we have recently demonstrated that roots of the starchless mutant of Arabidopsis, pgm-1, respond to gravity at approximately 30% the rate of wild-type roots, and that they lack the wild-type relationship between cap angle and response rate.² Furthermore, pgm-1 roots lack the gravity-induced gradient of auxin reported by DR5-GFP expression, found in wild-type roots, linking plastid sedimentation with the differential auxin transport thought to mediate the differential growth response.³While our understanding of root gravitropism has grown in sophistication and detail, the emerging picture has been compiled almost entirely from observations of primary organ behavior. The degree to which our model of signaling involved in primary root gravitropic responses applies to the behavior of lateral roots is an almost entirely open question, with only a handful of studies investigating lateral root gravitropic responses.^4–6 Toward that end, we have begun to explore the question of lateral root gravitropism in the overall context of root system architecture, and wish to report here on the gravitropic response of lateral roots in wild-type and pgm-1 genetic backgrounds.     

In vitro activation of phosphoglucomutase by fructose 2,6-bisphosphate

The hexose bisphosphate activation of phosphoglucomutase was investigated with both plant (pea and mung bean) and animal (rabbit muscle) sources of the enzyme. Plant phosphoglucomutase was purified about 50-fold from seeds, and to a lesser extent, from seedlings of Pisum sativum L. cv Grenadier and seedlings of Phaseolus aureus. It was found that the plant enzyme was isolated in a mostly dephosphorylated form while commercial rabbit muscle phosphoglucomutase was predominantly in the phosphorylated form. Activation studies were done using the dephosphorylated enzymes. The range of activation constant (K_a) values were obtained for each bisphosphate were: for glucose 1-6-P₂, 0.5 to 1.8; fructose 2,6-P₂, 6 to 11.7; and fructose 1,6-P₂, 7 micromolar, respectively. Fructose 2,6-P₂ is known to occur in both plant and animal tissues at changing levels encompassing the K_a values found in this study; hence, these results implicate fructose 2,6-P₂ as a natural activator of phosphoglucomutase, particularly in plants. Also, glucose 1,6-P₂ has not been found in plants, and the method for measuring glucose 1,6-P₂ by monitoring the activation of phosphoglucomutase is not specific.     

Molecular characterization reveals that YMR278w encoded protein is environmental stress response homologue of Saccharomyces cerevisiae PGM2

The uncharacterized ORF YMR278w of Saccharomyces cerevisiae is a member of D-phosphohexomutase super family, annotated as phosphoribomutase. In order to evaluate its functional role, we cloned, over-expressed and purified YMR278w protein. The protein product of YMR278w exhibits phosphoglucomutase activity. S158T mutant derivative of YMR278w protein lost phosphoglucomutase activity. Purified YMR278w protein has higher K_m for glucose-1-phosphate compared to other known phosphoglucomutases. Trehalose content was reduced in YMR278w disruptant as compared to the wild type strain. Based on the above results we suggest that YMR278w encodes phosphoglucomutase and not phosphoribomutase.     

Identification of the 64 kilodalton chloroplast stromal phosphoprotein as phosphoglucomutase

Phosphorylation of the 64 kilodalton stromal phosphoprotein by incubation of pea (Pisum sativum) chloroplast extracts with [γ-³²P]ATP decreased in the presence of Glc-6-P and Glc-1,6-P₂, but was stimulated by glucose. Two-dimensional gel electrophoresis following incubation of intact chloroplasts and stromal extracts with [γ-³²P]ATP, or incubation of stromal extracts and partially purified phosphoglucomutase (EC 2.7.5.1) with [³²P]Glc-1-P showed that the identical 64 kilodalton polypeptide was labeled. A 62 kilodalton polypeptide was phosphorylated by incubation of tobacco (Nicotiana sylvestris) stromal extracts with either [γ-³²P]ATP or [³²P]Glc-1-P. In contrast, an analogous polypeptide was not phosphorylated in extracts from a tobacco mutant deficient in plastid phosphoglucomutase activity. The results indicate that the 64 (or 62) kilodalton chloroplast stromal phosphoprotein is phosphoglucomutase.     

Engineering of Carbon Distribution between Glycolysis and Sugar Nucleotide Biosynthesis in Lactococcus lactis

We describe the effects of modulating the activities of glucokinase, phosphofructokinase, and phosphoglucomutase on the branching point between sugar degradation and the biosynthesis of sugar nucleotides involved in the production of exopolysaccharide biosynthesis by Lactococcus lactis. This was realized by using a described isogenic L. lactis mutant with reduced enzyme activities or by controlled expression of the well-characterized genes for phosphoglucomutase or glucokinase from Escherichia coli or Bacillus subtilis, respectively. The role of decreased metabolic flux was studied in L. lactis strains with decreased phosphofructokinase activities. The concomitant reduction of the activities of phosphofructokinase and other enzymes encoded by the las operon (lactate dehydrogenase and pyruvate kinase) resulted in significant changes in the concentrations of sugar-phosphates. In contrast, a >25-fold overproduction of glucokinase resulted in 7-fold-increased fructose-6-phosphate levels and 2-fold-reduced glucose-1-phosphate and glucose-6-phosphate levels. However, these increased sugar-phosphate concentrations did not affect the levels of sugar nucleotides. Finally, an ~100-fold overproduction of phosphoglucomutase resulted in 5-fold-increased levels of both UDP-glucose and UDP-galactose. While the increased concentrations of sugar-phosphates or sugar nucleotides did not significantly affect the production of exopolysaccharides, they demonstrate the metabolic flexibility of L. lactis.     

Phosphoglucomutase in Helianthus debilis: a polymorphism for isoenzyme number

Helianthus debilis is an annual, outcrossing sunflower species comprising five subspecies. Subspecies cucumerifolius and silvestris are characterized by two chloroplastic and one cytosolic phosphoglucomutase (PGM) isoenzymes, whereas the remaining three subspecies, debilis, tradiflorus. and vestitus exhibit two chloroplastic and two cystosolic PGM isoenzymes. Polymorphism for isoenzyme number within a plant species has rarely been reported.     

Amylopectin degradation in pea chloroplast extracts

Phosphorolysis rather than phosphorylation of amylolysis products was found to be the major pathway of sugar phosphate formation from amylopectin by pea (Pisum sativum L.) chloroplast stromal proteins. The K_m for inorganic phosphate incorporation was 2.5 mm, and ATP did not stimulate amylopectin-dependent phosphate incorporation. Arsenate (10 mm) inhibited phosphate incorporation into glucose monophosphates up to 46% and phosphoglucomutase activity 96%, resulting in glucose 1-phosphate accumulation as a product of amylopectin degradation. The intracellular distribution of enzymes of starch utilization was determined. Phosphorylase, phosphoglucomutase, and hexokinase were found in the chloroplast and cytoplasm, while β-amylase was restricted to the cytoplasm. Maltase was not detectable; maltose phosphorylase was active in the chloroplast.     

Electrophoretic studies on glucosephosphate isomerase and phosphoglucomutase in two types of Anisakis larvae

Agatsuma T. 1982. Electrophoretie studies on glucosephosphate isornerase and phosphoglucomutase in two types of Anisakis larvae. International Journal for Parasitology12: 35–39. Enzyme electrophoresis was carried out between the larval forms. Type I and II, of Anisakis using starch gel. In glucose-phosphate isomerase, considerable polymorphisms were found in each type. At least 5 alleles appear to occur at this enzyme locus in natural populations of both types. Out of 5 alleles, 3 were common to both types. No significant difference was found in their frequencies. However, each larval form can be easily distinguished by the electrophoretic mobility of phosphoglucomutase. It was concluded that enzyme electrophoresis is an alternative useful tool for the identification of larval forms of Anisakis.     

Comparisons of Isozyme Phenotypes in Five Meloidogyne spp. with Isoelectric Focusing

Meloidogyne incognita race 1, M. javanica, M. arenaria race 1, M. hapla, and an undescribed Meloidogyne sp. were analyzed by comparing isozyme phenotypes of esterase, malate dehydrogenase, phosphoglucomutase, isocitrate dehydrogenase, and α-glycerophosphate dehydrogenase. Isozyme phenotypes were obtained from single mature females by isoelectric focusing electrophoresis. Of these five isozymes, only esterase and phosphoglucomutase could be used to separate all five Meloidogyne spp.; however, the single esterase electromorphs were similar for M. incognita and M. hapla. Yet when both nematodes were run on the same gel, differences in their esterase phenotypes were detectable. Isozyme phenotypes from the other three isozymes revealed a great deal of similarity among M. incognita, M. javanica, M. arenaria, and the undescribed Meloidogyne sp.     

Genetics of glucose phosphate isomerase and phosphoglucomutase in Aedes albopictus (diptera: culicidae)

Summary Glucose phosphate isomerase (E.C. 5.3.1.9) and phosphoglucomutase (E.C. 2.7.5.1) were found to be polymorphic in a laboratory colony of Aedes albopictus. The glucose phosphate isomerase locus is represented by two alleles resulting in three genotypes, while the phosphoglucomutase locus is represented by at least five alleles giving rise to a total of 15 genotypes. The inheritance of these two enzymes is of the Mendelian type with codominant alleles. Present data indicate that these genes are not linked.Of 105 mosquitoes analysed for these two gene-enzyme systems, the frequencies for glucose phosphate isomerase alleles are Gpi ^S=0.68 and Gpi ^F=0.32, while the frequencies for phosphoglucomutase alleles are Pgm ^A=0.16, Pgm ^B=0.11, Pgm ^C=0.19, Pgm ^D=0.30 and Pgm ^F= 0.24. The frequencies of the three glucose phosphate isomerase genotypes are in accord with Hardy-Weinberg expectations (X ₁ ² =2.74). Similarly, the frequencies of the 15 phosphoglucomutase genotypes probably do not differ significantly from Hardy-Weinberg expectations (X ₁₀ ² = 18.45).     

Proteins encoded by the trans-acting replication and maintenance regions of broad host range plasmid RK2

The broad host range plasmid RK2 has previously been found to contain three separate regions of the genome involved in replication and maintenance in Escherichia coli (C. M. Thomas, R. Meyer, D. R. Helinski, 1980, J. Bacteriol.141, 213–222). They include the origin of replication (ori_RK2) and the trfA region which encodes a trans-acting function required for replication. The third region (trfB), although not essential for replication, supplies a function involved in the maintenance of plasmid RK2. Using the maxicell system of labeling plasmid-specific proteins, we have identified all of the proteins encoded by two miniplasmid derivatives of RK2 which contain only the regions ori_RK2, trfA, and trfB. To determine which region specifies each protein, RK2/mini-ColE1 hybrid plasmids were used which contain various restriction fragments of the mini-RK2 replicon. The trfA region appears to encode three proteins designated A1 (39,000 MW), A2 (31,000 MW), and A3 (14,000 MW). Analysis of proteins synthesized by plasmids containing deleted forms of the trfA region indicates that the A2 protein is the essential trfA-encoded replication protein of plasmid RK2. The proteins A1 and A3 may be the products specified by the genes tra3 (involved in transmissibility) and kilB1 (involved in host-cell viability) which also map in the trfA region. The trfB region specifies two proteins designated B1 (36,000 MW) and B2 (30,000 MW). These may be the products of the two kil-override (kor) genes located in the trfB region which have been implicated in plasmid maintenance.     

Maltose metabolism of Lactobacillus sanfranciscensis: cloning and heterologous expression of the key enzymes, maltose phosphorylase and phosphoglucomutase

The maltose degradation operon containing genes encoding maltose phosphorylase mapA and phosphoglucomutase pgmA from Lactobacillus sanfranciscensis DSM20451^T were cloned and expressed in Escherichia coli. These genes represent the first genetic data available for this species beyond taxonomic classification. MapA encodes a 754-amino acid polypeptide representing maltose phosphorylase, MapA, with a calculated molecular mass of 85.7 kDa. Comparative sequence analysis showed that mapA is of a new type distinct from other α-glucosidase genes sequenced so far. Putatively, pyridoxal 5′-phosphate is required as cofactor. The deduced amino acid sequence of pgmA shows an overall similarity of 39% to the phosphoglucomutase of Lactococcus lactis. pgmA is separated by a single nucleotide from the preceding mapA gene indicating effective translation by translational coupling. Upon subcloning mapA was heterologously expressed in E. coli. Additionally, upstream of the maltose-degrading operon ORF1 and ORF2 are located in the opposite direction. These genes show homology to fabZ and accB from E. coli and Bacillus subtilis, respectively, both involved in fatty acids biosynthesis.     

Biosynthèse de l'udpglucose par les microsomes des hépatocytes de ratBiosynthesis of UDPglucose by rat liver microsomes

Evidence is presented to show that all enzymes and all intermediary metabolites of a UDPglucose biosynthesis pathway are present in the microsomal membranes of rat liver. Glucose 6-phosphate, glucose 1-phosphate and UDPglucose are characterized by chromatography.The properties of phosphoglucomutase and UTP: D-Glucose-1-phosphate uridyltransferase are studied. The K_m values of phosphoglucomutase at pH 7.2 and 42°C were 0.26 · 10^?3 mM for glucose 1,6-diphosphate and 80 · 10^?3 mM for glucose 1-phosphate. The K_m values of UTP: D-glucose-1-phosphate uridyltransferase at pH 8.5 and 37°C were 220 · 10^?3 mM for UTP and 166 · 10^?3 mM for glucose 1-phosphate. These values are compared to the given values for enzymes from different species, and to those found for soluble enzymes. The significance of this membranous pathway is discussed.     

Phosphomannomutase and phosphoglucomutase in developing Cassia corymbosa seeds

Phosphomannomutase and phosphoglucomutase in developing Cassia corymbosa seeds have been completely separated from each other and from glucose phosphate and mannose phosphate isomerases by a series of chromatographic procedures that included affinity elution chromatography. Some properties, including the K_m for d-mannose 1,6-biphosphate with phosphomannomutase, are described. The activities of phosphoglucomutase and phosphomannomutase in some other plant tissues are also compared. The significance of these enzymes and the pathway of galactomannan synthesis are discussed.     

Properties and intracellular distribution of two phosphoglucomutases from spinach leaves

Two isoenzymes of phosphoglucomutase from spinach (Spinacia oleracea L.) leaves can be separated by ammonium-sulfate gradient solubilization or DEAE-cellulose ion exchange chromatography. They were designated as phosphoglucomutase 1 and 2, according to decreasing electrophoretic mobility towards the anode at pH 8.9. Phosphoglucomutase 1 is localized in the stroma of the chloroplasts, phosphoglucomutase 2 is a cytosolic enzyme as judged from aqueous cell fractionation studies. Both isoenzymes have very similar properties such as dependence on MgCl₂, pH activity profile, and K_m for glucose-1-phosphate and glucose-1,6-bisphosphate. From sedimentation-velocity analysis a molecular weight of 60,000 was estimated for either isoenzyme.     

Multifunctional Properties of Beef Liver Phosphoglucomutase

Liver phosphoglucomutase was found to catalyze also the reaction of Glc-1,6-P₂ formation from Glc-1-P and Fru-1,6-P_z or Glc-1-P and glycerate-1,3-P₂. The specific activity of Glc-1,6-P₂ formation from Glc-1-P and Fru-1,6-P₂ was 1/9200 of that of the mutase activity. The activity of Glc-1,6-P₂ formation from Glc-1-P and glycerate-1,3-P₂ was 1/122,000 of the mutase activity. From the results of the kinetics and the thermal inactivation experiments, the reaction of the mutase and Glc-1,6-P₂ synthesis were strongly suggested to occur at the same active site of liver phosphoglucomutase.

Liver phosphoglucomutase exhibited the Glc-1,6-P₂ phosphatase activity only in the presence of xylose 1-phosphate. The specific activity of phosphatase was only 1/154,000 of that of the mutase activity.     

Defects in leaf carbohydrate metabolism compromise acclimation to high light and lead to a high chlorophyll fluorescence phenotype in Arabidopsis thaliana

Background

We have studied the impact of carbohydrate-starvation on the acclimation response to high light using Arabidopsis thaliana double mutants strongly impaired in the day- and night path of photoassimilate export from the chloroplast. A complete knock-out mutant of the triose phosphate/phosphate translocator (TPT; tpt-2 mutant) was crossed to mutants defective in (i) starch biosynthesis (adg1-1, pgm1 and pgi1-1; knock-outs of ADP-glucose pyrophosphorylase, plastidial phosphoglucomutase and phosphoglucose isomerase) or (ii) starch mobilization (sex1-3, knock-out of glucan water dikinase) as well as in (iii) maltose export from the chloroplast (mex1-2).

Results

All double mutants were viable and indistinguishable from the wild type when grown under low light conditions, but - except for sex1-3/tpt-2 - developed a high chlorophyll fluorescence (HCF) phenotype and growth retardation when grown in high light. Immunoblots of thylakoid proteins, Blue-Native gel electrophoresis and chlorophyll fluorescence emission analyses at 77 Kelvin with the adg1-1/tpt-2 double mutant revealed that HCF was linked to a specific decrease in plastome-encoded core proteins of both photosystems (with the exception of the PSII component cytochrome b₅₅₉), whereas nuclear-encoded antennae (LHCs) accumulated normally, but were predominantly not attached to their photosystems. Uncoupled antennae are the major cause for HCF of dark-adapted plants. Feeding of sucrose or glucose to high light-grown adg1-1/tpt-2 plants rescued the HCF- and growth phenotypes. Elevated sugar levels induce the expression of the glucose-6-phosphate/phosphate translocator2 (GPT2), which in principle could compensate for the deficiency in the TPT. A triple mutant with an additional defect in GPT2 (adg1-1/tpt-2/gpt2-1) exhibited an identical rescue of the HCF- and growth phenotype in response to sugar feeding as the adg1-1/tpt-2 double mutant, indicating that this rescue is independent from the sugar-triggered induction of GPT2.

Conclusions

We propose that cytosolic carbohydrate availability modulates acclimation to high light in A. thaliana. It is conceivable that the strong relationship between the chloroplast and nucleus with respect to a co-ordinated expression of photosynthesis genes is modified in carbohydrate-starved plants. Hence carbohydrates may be considered as a novel component involved in chloroplast-to-nucleus retrograde signaling, an aspect that will be addressed in future studies.     

Linkage relationships of nine enzyme Loci in sunfishes (lepomis; centrarchidae)

Linkage relationships of nine enzyme loci; aconitase (Acon ), esterase (Est), glucosephosphate isomerase A and B ( Gpi), glycerate-2-dehydrogenase (G2dh), malic enzyme (Me ), phosphoglycerate kinase (Pgk), phosphoglucomutase (Pgm ) and superoxide dismutase (Sod), were investigated in sunfishes (Lepomis, Centrarchidae). Reciprocal F₁ hybrids produced from crosses between green sunfish (Lepomis cyanellus) and redear sunfish ( L. microlophus) were backcrossed with each of the two parental species. A three-point linkage map comprising G2dh, Pgk and Sod is reported. The frequencies of recombination between G2dh and Pgk and between Pgk and Sod are estimated as 45.3 and 24.7%. The remaining six loci assort independently. Possible linkage conservation and homology of this linkage group with those of other vertebrate species are discussed.     

Genetic Difference Between Two Field Populations of Plutella xylostella (Linnée) Based on Four Polymorphic Allozymes

We analyzed the breeding structure of the field diamondback moth, Plutella xylostella (Linnée), populations. Four polymorphic allozyme loci of acid phosphatase, esterase-1, hexokinase, and phosphoglucomutase were used for the population genetic markers. The estimated genetic distance between Andong and Sangju populations (ca. 50km apart) ranged from 0.0215 by fixation index (Fst) to 0.0550 by Nei's genetic distance (D). Such a little genetic differentiation between these two populations is explained by the significant migrants (11.5 per generation per population) which are estimated by Wright's method (N_em).