Phylogenetic relationships of Fusarium poae based on EF-1 alpha and mtSSU sequences.

A molecular phylogenetic analysis of Fusarium poae isolates from South America (Argentina) and Europe (mainly England, Germany, Italy) was performed using 98 F. poae, four Fusarium culmorum, two Fusarium sporotrichioides and one Fusarium langsethiae isolates. Phylogenetic analyses were performed using nuclear (translation elongation factor 1-alpha, EF-1 alpha) and mitochondrial (mitochondrial small subunit rDNA, mtSSU) sequences. Partitioned (each dataset separately) and combined (EF-1 alpha+mtSSU) analyses did not reveal any clear correlations from the inferred branching topology, between the distribution of observed haplotypes and the geographic origin and/or host species. Results from the present study confirmed that isolates from F. poae form a monophyletic group, and the low variability within isolates from a broad geographic range suggests a common lineage history. Among F. poae isolates from Argentina, however, some were found to possess an insert within mtSSU with structural similarities to group IC2 introns. F. poae isolates differing by the presence/absence of a mtSSU insertion were characterized further by analysis of a portion of the Tri5 gene, but this sequence was unable to reveal variability. The presence of this insert only within isolates from Argentina suggests that evolutionary events (insertions/deletions) are probably taking place within the Argentinian F. poae isolates, and that the acquisition of this insert occurred after geographic isolation of the Argentinian and European populations.

Mating-type distribution and fertility status in Magnaporthe grisea populations from Argentina.

Isolates of Magnaporthe grisea causing gray leaf spot on rice were collected in Argentina and analyzed for mating distribution and fertility. One hundred and twenty-five isolates of M. grisea were collected from rice plants between 2000 and 2003. Each isolate was tested for mating type through a polymerase chain reaction based assay. All M. grisea isolates from Argentina belonged to a single mating type, MAT1.1. The fertility status of isolates was determined using controlled crosses in vitro, pairing each isolate with GUY11 and KA9 (MAT1.2 standard hermaphroditic testers). Production of perithecia was scarce among isolates of the blast pathogen since a low percentage of them (7.2%) developed perithecia with only one of the fertile tester (KA9); all crosses failed with the other tester strain. Asci and ascospores were not observed. The presence of only one mating type and the absence of female fertile isolates indicate that sexual reproduction is rare or absent in M. grisea populations associated with rice in Argentina.

Sucrose-phosphate phosphatase from Anabaena sp. strain PCC 7120: isolation of the protein and gene revealed significant structural differences from the higher-plant enzyme.

The present study describes the first isolation and characterization of a prokaryotic protein and gene for sucrose-phosphate phosphatase (SPP), the enzyme that catalyzes the terminal step in sucrose synthesis. For gene isolation, a 2,015-bp DNA fragment containing an open reading frame with about 31% amino acid identity to Synechocystis SPS was amplified from Anabaena sp. PCC 7120 DNA. Surprisingly, expression of the putative gene in Escherichia coli demonstrated that it encoded an SPP protein. The expressed protein cross-reacted with antibodies against the native form of Anabaena SPP and its biochemical properties were identical to those of the enzyme purified from the cyanobacterial cells. Comparisons of the Anabaena SPP with the higher-plant enzyme revealed important differences in the C-terminal region, molecular mass, subunit composition and immunoreactivity. Nevertheless, two conserved motifs, including four invariant aspartate residues similar to those found in members of the phosphohydrolase superfamily, were identified in the Anabaena SPP deduced amino acid sequence.

A prokaryotic sucrose synthase gene (susA) isolated from a filamentous nitrogen-fixing cyanobacterium encodes a protein similar to those of plants.

Sucrose synthase (SS), a key enzyme in plant carbohydrate metabolism, has recently been isolated from Anabaena sp. strain PCC 7119, and biochemically characterized; two forms (SS-I and SS-II) were detected (Porchia et al. 1999, Planta 210: 34-40). The present study describes the first isolation and characterization of a prokaryotic SS gene, susA, encoding SS-II from that strain of Anabaena. A 7 kbp DNA fragment containing an open reading frame (EMBL accession number AJ010639) with about 30-40% amino acid identity with plant SSs was isolated from an Anabaena subgenomic library. The putative SS gene was demonstrated to encode an SS protein by expression in Escherichia coli. The biochemical properties of the recombinant enzyme were identical to those of the enzyme purified from the cyanobacterial cells. The deduced amino acid sequence of the Anabaena SS diverged from every plant SS reported. The occurrence of SS in cyanobacteria of different taxonomic groups was investigated. The enzyme occurs in several filamentous nitrogen-fixing cyanobacteria but not in two species of unicellular, non-diazotrophic cyanobacteria.

Sucrose metabolism in cyanobacteria: sucrose synthase from Anabaena sp. strain PCC 7119 is remarkably different from the plant enzymes with respect to substrate affinity and amino-terminal sequence.

The pathway of sucrose metabolism in cyanobacteria is just starting to be elucidated. The present study describes the first isolation and biochemical characterization of a prokaryotic sucrose synthase (SS, EC 2.4.1.13). Two SS forms (SS-I and SS-II) were detected in Anabaena sp. strain PCC 7119. The isoform SS-II was purified 457-fold and its amino-terminal portion sequenced. Substrate specificity, kinetic constants, native protein and subunit molecular masses, and the effect of different ions and metabolites were studied for SS-II. Anabaena SS was shown to be a tetramer with a 92-kDa polypeptide that was recognized by maize SS polyclonal antibodies. Some striking differences from plant enzymes were demonstrated with respect to substrate affinities, regulation by metal ions and ATP, and the amino-acid sequence of the N-terminal region.

Studies on sucrose-phosphate synthase from rice leaves.

Sucrose-phosphate synthase (SPS, EC 2.4.1.14) biochemical properties and peptide composition have been analyzed in rice leaf seedlings. SPS was purified using DEAE-Sephacel chromatography, gel filtration on Sepharose 6B and anion exchange chromatography on Mono Q. At this stage two enzyme forms (SPS-I and -II) were separated. SPS-II was purified 90-fold; however, SPS-I presented a lower specific activity regarding the previous purification step and an unstable activity. Both enzyme forms had similar apparent Km values for Fru-6P but the SPS-I Km for UDP-Glc was ca. 10-fold higher than the SPS-II one. In addition, they differentiate in the capacity of being modulated by Glc-6-P and Pi: while SPS-II activity was inhibited by Pi and activated by Glc-6-P, SPS-I was not affected by either effectors. A native molecular mass of ca. 420 kDa was found by gel filtration. In SPS expression analysis using leaf rice and wheat germ SPS antibodies, a 116 kDa polypeptide was revealed in rice leaf extracts and no polypeptide was immunoactive in rice roots.

Sucrose biosynthesis in a prokaryotic organism: Presence of two sucrose-phosphate synthases in Anabaena with remarkable differences compared with the plant enzymes.

Biosynthesis of sucrose-6-P catalyzed by sucrose-phosphate synthase (SPS), and the presence of sucrose-phosphate phosphatase (SPP) leading to the formation of sucrose, have both been ascertained in a prokaryotic organism: Anabaena 7119, a filamentous heterocystic cyanobacterium. Two SPS activities (SPS-I and SPS-II) were isolated by ion-exchange chromatography and partially purified. Four remarkable differences between SPSs from Anabaena and those from higher plants were shown: substrate specificity, effect of divalent cations, native molecular mass, and oligomeric composition. Both SPS-I and SPS-II accept Fru-6-P (K(m) for SPS-I = 0.8 +/- 0.1 mM; K(m) for SPS-II = 0.7 +/- 0.1 mM) and UDP-Glc as substrates (K(m) for SPS-I = 1.3 +/- 0.4 mM; K(m) for SPS-II = 4.6 +/- 0.4 mM), but unlike higher plant enzymes, they are not specific for UDP-Glc. GDP-Glc and TDP-Glc are also SPS-I substrates (K(m) for GDP-Glc = 1.2 +/- 0.2 mM and K(m) for TDP-Glc = 4.0 +/- 0.4 mM), and ADP-Glc is used by SPS-II (K(m) for ADP-Glc = 5.7 +/- 0.7 mM). SPS-I has an absolute dependence toward divalent metal ions (Mg2+ or Mn2+) for catalytic activity, not found in plants. A strikingly smaller native molecular mass (between 45 and 47 kDa) was determined by gel filtration for both SPSs, which, when submitted to SDS/PAGE, showed a monomeric composition. Cyanobacteria are, as far as the authors know, the most primitive organisms that are able to biosynthesize sucrose as higher plants do.

Activation of sucrose-phosphate synthase by a protein factor/sucrose-phosphate phosphatase.

The possible presence of a sucrose-phosphate synthase (SPS) activating/stabilizing factor (SAF) presumably lost during SPS purification was investigated. Rice leaf protein extracts were chromatographed in a DEAE-Sephacel column. SPS activity of previously purified rice enzyme was enhanced to different extent by aliquots of fractions from such column. The activating capacity could not be replaced by albumin, but was nullified by EDTA. When the fractions were boiled or treated with TCA, the activating capacity disappeared suggesting its proteinaceous nature. The presence of 10 microM okadaic acid had no effect on the stimulatory action of SAF on SPS denying the possibility to SAF to be a SPS-phosphatase. Although it overlaps somehow with sucrose synthase (SS) in DEAE-Sephacel fractions, the activating protein factor and SS eluted separately during Sephadex G-200 chromatography. The activating ability was saturable at a fixed SPS concentration and was able to enhance SPS activity from other plant sources. Simultaneous studies on the activities of SPS and sucrose-phosphate phosphatase (SPP), closely linked to SPS, allowed us to suggest that SAF could be SPP. The presence of SAF/SPP did not alter the affinity of SPS for its substrates but helped to reverse the Pi inhibition at low Fru-6-P concentrations. We conclude that SPS may possibly interact with SPP, contributing to a more effective sucrose synthesis.

Sucrose Synthase Expression during Cold Acclimation in Wheat.

When wheat (Triticum aestivum) seedlings are exposed to a cold temperature (2-4 degrees C) above 0 degrees C, sucrose accumulates and sucrose synthase activity increases. The effect of a cold period on the level of sucrose synthase (SS) was investigated. Using antibodies against wheat germ SS, Western blots studies showed that the amount of the SS peptide increased during 14 days in the cold, when plants were moved from 23 degrees C to 4 degrees C. The level of SS diminished when plants were moved back to 23 degrees C. Northern blots of poly(A)(+) RNA, confirmed a five- to sixfold induction of SS in wheat leaves during cold acclimation. These results indicate that SS is involved in the plant response to a chilling stress.

Raffinose Synthesis in Chlorella vulgaris Cultures after a Cold Shock.

Chlorella vulgaris cultures have been submitted to a chilling shock, bringing down the growing temperature from to 24 degrees C to 4 degrees C. Growth was stopped immediately, and concomitantly there was an accumulation of sucrose and a decrease in the starch content. The enzymes involved in sucrose metabolism were differentially affected by the chilling shock. Sucrose phosphate synthase activity increased while sucrose synthase was not affected. Simultaneously with the chilling shock, raffinose began to accumulate. When algal cultures were returned at 24 degrees C, raffinose disappeared. The presence of raffinose in algal cells has not been reported before.

Sucrose phosphate synthetase : Separation from sucrose synthetase and a study of its properties.

A method for the complete separation of sucrose phosphate synthetase (EC 2.4.1.14) and sucrose synthetase (EC 2.4.1.13) from wheat (Triticum aestivum L.) germ is described. The separation is achieved by chromatography on DEAE-cellulose at pH 6.5. The sucrose phosphate synthetase obtained can be further purified by gel filtration. Disc electrophoresis of sucrose phosphate preparations reveals the presence of isoenzymes. Molecular weight estimates of sucrose phosphate synthetase by gel filtration and sedimentation velocity give a value of 380,000. The enzyme is inhibited by various anions, particularly citrate, maleate, and phosphate. Activity estimate should be carried out with Good's buffers in order to avoid inhibition. Nucleoside triphosphates are competitive inhibitors toward UDP-glucose. The enzyme is sensitive to sulfhydryl reagents, but activity can be restored with DTT or ß-mercapto ethanol. The fact that the enzyme is inhibited by δ-gluconolactone suggests that the reaction occurs through the formation of an unstable glucose-enzyme complex. Mg(2+) can restore enzyme activity to control values when inhibited by nucleoside triphosphates, citrate, or phosphate.