Inoculation of sugarcane with Pantoea sp. increases amino acid contents in shoot tissues; serine, alanine, glutamine and asparagine permit concomitantly ammonium excretion and nitrogenase activity of the bacterium.

Pantoea sp. is an endophytic nitrogen-fixing bacterium isolated from sugarcane tissues. The aim of the present study was to determine the contents of amino acids in sugarcane as a result of inoculation of nodes and nodal roots with Pantoea sp. strain 9C and to evaluate the effects of amino acids on growth, nitrogenase activity and ammonium excretion of the bacterium. Content of almost all amino acids increased in 30-day-old plantlets by root inoculation. The most abundant amino acids in shoot tissues were asparagine and proline, and those in nodal roots were asparagine, proline, aspartic acid, glutamic acid and serine. The bacterium was able to grow on all tested amino acids except histidine, isoleucine and leucine. Nitrogenase Pantoea sp. was partially inhibited by 1, 2 or 5mmolL(-1) and completely inhibited by 10mmolL(-1) of NH(4)(+) in the media. Pantoea sp. showed nitrogenase activity in 5mmolL(-1) of serine, asparagine, threonine, alanine, proline, tyrosine, valine, methionine, lysine, phenylalanine, cysteine, tryptophan, citrulline and ornithine. Pantoea sp. did not excrete ammonium when it grew in vivo conditions favoring nitrogen fixation; however, ammonium was detected in the supernatant when 5mmolL(-1) asparagine, aspartic acid, alanine, serine or glutamine was added to the medium. The highest ammonium concentration in the supernatant was detected, when Pantoea grew on serine. Ammonium in the supernatant and nitrogenase activity were only detectable concomitantly when the medium was supplemented with serine, alanine, glutamine or asparagine. We discuss roles of amino acids on plant-bacteria interaction during the colonization of sugarcane plants.

Cloning and characterization of the gene cluster for palatinose metabolism from the phytopathogenic bacterium Erwinia rhapontici.

Erwinia rhapontici is able to convert sucrose into isomaltulose (palatinose, 6-O-alpha-D-glucopyranosyl-D-fructose) and trehalulose (1-O-alpha-D-glucopyranosyl-D-fructose) by the activity of a sucrose isomerase. These sucrose isomers cannot be metabolized by plant cells and most other organisms and therefore are possibly advantageous for the pathogen. This view is supported by the observation that in vitro yeast invertase activity can be inhibited by palatinose, thus preventing sucrose consumption. Due to the lack of genetic information, the role of sucrose isomers in pathogenicity has not been evaluated. Here we describe for the first time the cloning and characterization of the palatinose (pal) genes from Erwinia rhapontici. To this end, a 15-kb chromosomal DNA fragment containing nine complete open reading frames (ORFs) was cloned. The pal gene products of Erwinia rhapontici were shown to be homologous to proteins involved in uptake and metabolism of various sugars from other microorganisms. The palE, palF, palG, palH, palK, palQ, and palZ genes were oriented divergently with respect to the palR and palI genes, and sequence analysis suggested that the first set of genes constitutes an operon. Northern blot analysis of RNA extracted from bacteria grown under various conditions implies that the expression of the palI gene and the palEFGHKQZ genes is oppositely regulated at the transcriptional level. Genes involved in palatinose uptake and metabolism are down regulated by sucrose and activated by palatinose. Palatinose activation is inhibited by sucrose. Functional expression of palI and palQ in Escherichia coli revealed sucrose isomerase and palatinase activity, respectively.

Impact of elevated cytosolic and apoplastic invertase activity on carbon metabolism during potato tuber development.

During tuberization in Solanum tuberosum var. Desirée maximal catalytic activities of invertase(s) and sucrose synthase are inversely correlated. During the early stages, invertase activity is high and declines during maturation. The decrease in invertase activity is accompanied by a decrease in the hexose to sucrose ratio and an increase in sucrose synthase activity. This switch is paralleled by the onset of the storage phase as shown by the accumulation of starch and storage proteins. Biochemical and genetic evidence suggests that sucrose synthase activity is positively correlated with sink strength. To explore the possibility of enhancing sink strength in potato tubers by elevating the sucrolytic capacity, transgenic potato plants expressing either cytosolic or apoplastic yeast invertase in their tubers were made. Surprisingly, cytosolic invertase led to a decrease and apoplastic invertase to an increase in tuber yield. To understand the causes of the observed phenotypes, carbon metabolism in tubers of transgenic and control plants was analysed during different stages of tuber development. Both cytosolic and apoplastic invertase resulted in decreased sucrose and elevated glucose contents, indicating that sucrose is accessible in both compartments. Metabolic perturbation, however, was found to be compartment specific. Elevated cytosolic invertase activity led to increased carbon flux towards glycolysis and accumulation of phosphorylated intermediates. The phosphorylated intermediates were not used to build up starch. In contrast, apoplastic invertase does not lead to a significant increase in hexose phosphates compared to untransformed controls. Thus, hexoses originating in the apoplast are not efficiently phosphorylated during potato tuber development, which might be explained by an endocytotic uptake of sucrose and/or hexoses from the apoplast into the vacuole bypassing the cytosolic compartment.

Overexpression of pyrophosphatase leads to increased sucrose degradation and starch synthesis, increased activities of enzymes for sucrose-starch interconversions, and increased levels of nucleotides in growing potato tubers.

Overexpression of inorganic pyrophosphatase (PPase) from Escherichia coli in the cytosol of plants (ppa 1 plants) leads to a decrease of inorganic pyrophosphate (PPi; U. Sonnewald, 1992, Plant J 2: 571-581). The consequences for sucrose-starch interconversions have now been studied in growing potato (Solanum tuberosum L. cv. Desirée) tubers. Sucrose is degraded via sucrose synthase and UDP-glucose pyrophosphorylase in growing tubers, and it was expected that the low PPi in the ppa 1 transformants would restrict the mobilisation of sucrose and conversion to starch. Over-expression of PPase resulted in an accumulation of sucrose and UDP-glucose, and decreased concentrations of hexose phosphates and glycerate-3-phosphate in growing ppa 1 tubers. Unexpectedly, the rate of degradation of [14C] sucrose was increased by up to 30%, the rate of starch synthesis was increased, and the starch content was increased by 20-30% in ppa 1 tubers compared to wild-type tubers. Reasons for this unexpectedly efficient conversion of sucrose to starch in the ppa 1 tubers were investigated. (i) The transformed tubers contained increased activities of several enzymes required for sucrose-starch interconversions including two- to three-fold more sucrose synthase and 60% more ADP-glucose pyrophosphorylase. They also contained 30-100% increased activities of several glycolytic enzymes and amylase, increased protein, and unaltered or slightly decreased starch phosphorylase, acid invertase and mannosidase. (ii) The transformants contained higher pools of uridine nucleotides. As a result, although the UDP-glucose pool is increased two- to threefold, this does not lead to a decrease of UTP or UDP. (iii) The transformants contained twofold larger pools of ATP and ADP, and ADP-glucose was increased by up to threefold. In stored ppa 1 tubers, there were no changes in the activities of glycolytic enzymes, and nucleotides did not increase. It is concluded that in growing tubers PPi has a wider-significance than just being an energy donor for specific reactions in the cytosol. Increased rates of PPi hydrolysis also affect general aspects of cell activity including the levels of nucleotides and protein. Possible ways in which PPi hydrolysis could affect these processes are discussed.

Combined expression of glucokinase and invertase in potato tubers leads to a dramatic reduction in starch accumulation and a stimulation of glycolysis.

The original aim of this work was to increase starch accumulation in potato tubers by enhancing their capacity to metabolise sucrose.We previously reported that specific expression of a yeast invertase in the cytosol of tubers led to a 95% reduction in sucrose content, but that this was accompanied by a larger accumulation of glucose and a reduction in starch. In the present paper we introduced a bacterial glucokinase from Zymomonas mobilis into an invertase-expressing transgenic line, with the intention of bringing the glucose into metabolism. Transgenic lines were obtained with up to threefold more glucokinase activity than in the parent invertase line and which did not accumulate glucose. Unexpectedly, there was a further dramatic reduction in starch content, down to 35% of wild-type levels. Biochemical analysis of growing tuber tissue revealed large increases in the metabolic intermediates of glycolysis, organic acids and amino acids,two- to threefold increases in the maximum catalytic activities of key enzymes in the respiratory pathways, and three- to fivefold increases in carbon dioxide production.These changes occur in the lines expressing invertase,and are accentuated following introduction of the second transgene, glucokinase. We conclude that the expression of invertase in potato tubers leads to an increased flux through the glycolytic pathway at the expense of starch synthesis and that heterologous overexpression of glucokinase enhances this change in partitioning.

Increased potato tuber size resulting from apoplastic expression of a yeast invertase.

The role of sucrose cleavage in determining sink strength in potato was investigated by generating transgenic potato plants that expressed a yeast invertase in either the cytosol or apoplast of tubers. Cytosolic localization gave rise to a reduction in tuber size and an increase in tuber number per plant whereas apoplastic targeting led to an increase in tuber size and a decrease in tuber number per plant. Sink organ size can be manipulated through modification of sucrose metabolism.