Investigation of the metabolome and the transcriptome of pollen of lily (Lilium longiflorum) gave a comprehensive overview of metabolic pathways active during pollen germination and tube growth. More than 100 different metabolites were determined simultaneously by gas chromatography coupled to mass spectrometry, and expressed genes of selected metabolic pathways were identified by next-generation sequencing of lily pollen transcripts. The time-dependent changes in metabolite abundances, as well as the changes after inhibition of the mitochondrial electron transport chain, revealed a fast and dynamic adaption of the metabolic pathways in the range of minutes. The metabolic state prior to pollen germination differed clearly from the metabolic state during pollen tube growth, as indicated by principal component analysis of all detected metabolites and by detailed observation of individual metabolites. For instance, the amount of sucrose increased during the first 60 minutes of pollen culture but decreased during tube growth, while glucose and fructose showed the opposite behavior. Glycolysis, tricarbonic acid cycle, glyoxylate cycle, starch, and fatty acid degradation were activated, providing energy during pollen germination and tube growth. Inhibition of the mitochondrial electron transport chain by antimycin A resulted in an immediate production of ethanol and a fast rearrangement of metabolic pathways, which correlated with changes in the amounts of the majority of identified metabolites, e.g. a rapid increase in g-aminobutyric acid indicated the activation of a g-aminobutyric acid shunt in the tricarbonic acid cycle, while ethanol fermentation compensated the reduced ATP production after inhibition of the oxidative phosphorylation.

A mutant of Escherichia coli K-12 Bacterial cells are capable of taking up a variety of carboxylic acids from the surrounding medium. The translocation of carboxylic acids across the bacterial membrane is mediated by transport mechanisms such as facilitative diffusion, active transport, and vectorial acylation (for a review, see reference 11). Of all the carboxylate transport systems of bacteria studied, the tricarboxylic acid cycle dicarboxylates (1,9,13,16,17) and tricarboxylates (6,12,28) have received the most attention. The transport or uptake of a number of monocarboxylates by bacteria has also been reported, but the molecular mechanisms involved are less well established. Thus, specific transport systems have been suggested for the glycolytic intermediates D-and L-lactate (3, 19), pyruvate (4, 14, 19), glycolate (23), and phosphoenolpyruvate and 2-and 3-phosphoglycerate (26), as well as for monocarboxylates such as hexuronates (5), hexonates (24), acetate (29), and propionate (10).Preliminary studies by Kornberg and Smith (14) on the genetic control of intermediary metabolism revealed that a phosphoenolpyruvate synthase (pps)-deficient mutant with restored enzymatic activity still could not grow on pyruvate as the sole carbon source. Kornberg and Smith concluded that the cells must also have a pyruvate transport system and a defect in this system prevented growth on pyruvate. This purported pyruvate transport (usp) mutant could still take up lactate from the growth medium, indicating that pyruvate and lactate do not share the same transport system. Matin and Konings (19), on the basis of studies on the uptake of lactate and succinate in membrane vesicles of Escherichia coli K-12, concluded from inhibition studies that pyruvate and lactate may share the same transport system. In none of these studies was pyruvate transport thoroughly examined. Pyruvate has also been shown to be taken up by a system with high affinity in Rhodopseudomonas spheroides (4). This system has a wide substrate specificity, being competitively inhibited by a variety of short-chain fatty acids, as well as by lactate. * Corresponding author.To characterize the pyruvate transport system in E. coli K-12 whole cells, we chose a mutant strain deficient in pyruvate dehydrogenase and phosphoenolpyruvate synthase. Although these two major routes of pyruvate metabolism were blocked, pyruvate could still be metabolized in cells by a variety of enzymes including lactate dehydrogenase, pyruvate oxidase, and amino acid transaminases. To minimize this problem, pyruvate uptake was also studied in membrane vesicles devoid of cytoplasmic contents. The whole cell and vesicle results indicate that uptake of pyruvate in E. coli is due to a specific active transport system. MATERIALS AND METHODSBacterial strains and growth conditions. Whole cell studies were done with a mutant strain of E. coli K-12 [pps relAl thyA56 metBI azi-54 ton-54 tsx-97 A(aroP-ace)F73] kindly provided by the E. coli Genetic Stock Center (CGSC 5688). This mutant strain, designa...

The plasma membrane H(+) ATPase is a member of the P-ATPase family transporting H(+) from the cytosol to the extracellular space and thus energizing the plasma membrane for the uptake of ions and nutrients. As a housekeeping gene, this protein can be detected in almost every plant cell including the exclusive expression of specific isoforms in pollen grains and tubes where its activity is a prerequisite for successful germination and growth of pollen tubes. This review summarizes the current knowledge on pollen PM H(+) ATPases and hypothesizes a central role for pollen-specific isoforms of this protein in tube growth. External as well as cytosolic signals from signal transduction and metabolic pathways are integrated by the PM H(+) ATPase and directly translated to tube growth rates, allocating the PM H(+) ATPase to an essential node in the signalling network of pollen tubes in their race to the ovule.

Pollen grains of Lilium longiflorum are a long-established model system for pollen germination and tube tip growth. Due to their size, protein content and almost synchronous germination in synthetic media, they provide a simple system for physiological measurements as well as sufficient material for biochemical studies like protein purifications, enzyme assays, organelle isolation or determination of metabolites during germination and pollen tube elongation. Despite recent progresses in molecular biology techniques, sequence information of expressed proteins or transcripts in lily pollen is still scarce. Using a next generation sequencing strategy (RNAseq), the lily pollen transcriptome was investigated resulting in more than 50 million high quality reads with a length of 90 base pairs. Sequenced transcripts were assembled and annotated, and finally visualized with MAPMAN software tools and compared with other RNAseq or genome data including Arabidopsis pollen, Lilium vegetative tissues and the Amborella trichopoda genome. All lily pollen sequence data are provided as open access files with suitable tools to search sequences of interest.