Analysis of subcellular metabolite levels of potato tubers (Solanum tuberosum) displaying alterations in cellular or extracellular sucrose metabolism

The expression of a heterologous invertase in potato tubers (Solanum tuberosum) in either the cytosol or apoplast leads to a decrease in total sucrose content and to an increase in glucose. Depending on the targeting of the enzyme different changes in phenotype and metabolism of the tubers occur: the cytosolic invertase expressing tubers show an increase in the glycolytic flux, accumulation of amino acids and organic acids, and the appearance of novel disaccharides; however, these changes are not observed when the enzyme is expressed in the apoplast [Roessner et al. (2001). Plant Cell, 13, 11-29]. The analysis of these lines raised several questions concerning the regulation of compartmentation of metabolites in potato tubers. In the current study we addressed these questions by performing comparative subcellular metabolite profiling. We demonstrate that: (i) hexoses accumulate in the vacuole independently of their site of production, but that the cytosolic invertase expression led to a strong increase in the cytosolic glucose concentration and decrease in cytosolic sucrose, whereas these effects were more moderate in the apoplastic expressors; (ii) three out of four of the novel compounds found in the cytosolic overexpressors accumulate in the same compartment; (iii) despite changes in absolute cellular content the subcellular distribution of amino acids was invariant in the invertase overexpressing tubers. These results are discussed in the context of current models of the compartmentation of primary metabolism in heterotrophic plant tissues

Subcellular concentrations of sugar alcohols and sugars in relation to phloem translocation in Plantago major, Plantago maritima, Prunus persica, and Apium graveolens

Sugar and sugar alcohol concentrations were analyzed in subcellular compartments of mesophyll cells, in the apoplast, and in the phloem sap of leaves of Plantago major (common plantain), Plantago maritima (sea plantain), Prunus persica (peach) and Apium graveolens (celery). In addition to sucrose, common plantain, sea plantain, and peach also translocated substantial amounts of sorbitol, whereas celery translocated mannitol as well. Sucrose was always present in vacuole and cytosol of mesophyll cells, whereas sorbitol and mannitol were found in vacuole, stroma, and cytosol in all cases except for sea plantain. The concentration of sorbitol, mannitol and sucrose in phloem sap was 2- to 40-fold higher than that in the cytosol of mesophyll cells. Apoplastic carbohydrate concentrations in all species tested were in the low millimolar range versus high millimolar concentrations in symplastic compartments. Therefore, the concentration ratios between the apoplast and the phloem were very strong, ranging between 20- to 100-fold for sorbitol and mannitol, and between 200- and 2000-fold for sucrose. The woody species, peach, showed the smallest concentration ratios between the cytosol of mesophyll cells and the phloem as well as between the apoplast and the phloem, suggesting a mixture of apoplastic and symplastic phloem loading, in contrast to the herbal plant species (common plantain, sea plantain, celery) which likely exhibit an active loading mode for sorbitol and mannitol as well as sucrose from the apoplast into the phloem

Silencing of Vlaro2 for chorismate synthase revealed that the phytopathogen Verticillium longisporum induces the cross-pathway control in the xylem

The first leaky auxotrophic mutant for aromatic amino acids of the near-diploid fungal plant pathogen Verticillium longisporum (VL) has been generated. VL enters its host Brassica napus through the roots and colonizes the xylem vessels. The xylem contains little nutrients including low concentrations of amino acids. We isolated the gene Vlaro2 encoding chorismate synthase by complementation of the corresponding yeast mutant strain. Chorismate synthase produces the first branch point intermediate of aromatic amino acid biosynthesis. A novel RNA-mediated gene silencing method reduced gene expression of both isogenes by 80% and resulted in a bradytrophic mutant, which is a leaky auxotroph due to impaired expression of chorismate synthase. In contrast to the wild type, silencing resulted in increased expression of the cross-pathway regulatory gene VlcpcA (similar to cpcA/GCN4) during saprotrophic life. The mutant fungus is still able to infect the host plant B. napus and the model Arabidopsis thaliana with reduced efficiency. VlcpcA expression is increased in planta in the mutant and the wild-type fungus. We assume that xylem colonization requires induction of the cross-pathway control, presumably because the fungus has to overcome imbalanced amino acid supply in the xylem

The role of photorespiration during the evolution of C-4 photosynthesis in the genus Flaveria

Mallmann J, Heckmann D, Bräutigam A, et al. The role of photorespiration during the evolution of C-4 photosynthesis in the genus Flaveria. eLife. 2014;3: e02478.C-4 photosynthesis represents a most remarkable case of convergent evolution of a complex trait, which includes the reprogramming of the expression patterns of thousands of genes. Anatomical, physiological, and phylogenetic analyses as well as computational modeling indicate that the establishment of a photorespiratory carbon pump (termed C-2 photosynthesis) is a prerequisite for the evolution of C-4. However, a mechanistic model explaining the tight connection between the evolution of C-4 and C-2 photosynthesis is currently lacking. Here we address this question through comparative transcriptomic and biochemical analyses of closely related C-3, C-3-C-4, and C-4 species, combined with Flux Balance Analysis constrained through a mechanistic model of carbon fixation. We show that C-2 photosynthesis creates a misbalance in nitrogen metabolism between bundle sheath and mesophyll cells. Rebalancing nitrogen metabolism requires an anaplerotic carbon cycle that resembles at least parts of a basic C-4 cycle. Our findings thus show how C-2 photosynthesis represents a pre-adaptation for the C-4 system, where the evolution of the C-2 system establishes important C-4 components as a side effect

Aquatic Macroinvertebrate Communities and Water Quality at Buffered and Non-Buffered Wetland Sites on Federal Waterfowl Production Areas in the Rainwater Basin, Nebraska

Nebraska’s Rainwater Basin has an abundance of natural wetlands and is a focal point in the annual migration corridor used by millions of waterfowl and shorebirds. However, these wetlands are in a landscape dominated by agriculture and as a result, siltation and poor water quality are continual problems. We evaluated twelve wetland sites on federally managed Waterfowl Protection Areas from 2007 – 2009 for water quality, sediment quality, andmacroinvertebrate diversity. Six of the sites received agricultural runoff directly via culverts and drainage ditches (non-buffered sites) and six siteswere protected from agricultural runoff by a vegetated buffer (buffered sites). Mean total number of aquatic macroinvertebrates were significantly greater (

Contacts between the growing peptide chain and the 23S RNA in the 50S ribosomal subunit.

Peptides of defined length carrying a diazirine photoaffinity label attached either to the alpha-NH2 group of the N-terminal methionine residue, or to the epsilon-NH2 group of an immediately adjacent lysine residue, were prepared in situ on Escherichia coli ribosomes in the presence of a synthetic mRNA analogue. Peptide growth was stopped simply by withholding the aminoacyl-tRNA cognate to an appropriate downstream codon. After photo-activation at 350 nm the sites of cross-linking to ribosomal RNA were determined by our standard procedures; the C-terminal amino acid of each peptide was labelled with tritium, in order to confirm whether the individual cross-linked complexes contained the expected 'full-length' peptide, as opposed to shorter products. The shortest peptides became cross-linked to sites within the 'peptidyl transferase ring' of the 23S RNA, namely to positions 2062, 2506, 2585 and 2609. However, already when the peptide was three or four residues long, a new cross-link was observed several hundred nucleotides away in another secondary structural domain; this site, at position 1781, lies within one of several RNA regions which have been implicated in other studies as being located close to the peptidyl transferase ring. Further application of this approach, combined with model-building studies, should enable the path of the nascent peptide through the large ribosomal subunit to be definitively mapped

The epidermis of barley leaves is a dynamic intermediary storage compartment of carbohydrates, amino acids and nitrate

Dietz K-J, Hollenbach B, Hellwege E. The epidermis of barley leaves is a dynamic intermediary storage compartment of carbohydrates, amino acids and nitrate. Physiol. Plant. 1994;92(1):31-36