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Publications - Cell and Metabolic Biology

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Publications

Klopotek, Y.; Haensch, K.-T.; Hause, B.; Hajirezaei, M.-R.; Druege, U.; Dark exposure of petunia cuttings strongly improves adventitious root formation and enhances carbohydrate availability during rooting in the light J. Plant Physiol. 167, 547-554, (2010) DOI: 10.1016/j.jplph.2009.11.008

The effect of temporary dark exposure on adventitious root formation (ARF) in Petunia×hybrida ‘Mitchell’ cuttings was investigated. Histological and metabolic changes in the cuttings during the dark treatment and subsequent rooting in the light were recorded. Excised cuttings were exposed to the dark for seven days at 10°C followed by a nine-day rooting period in perlite or were rooted immediately for 16 days in a climate chamber at 22/20°C (day/night) and a photosynthetic photon flux density (PPFD) of 100 μmol m−2 s−1. Dark exposure prior to rooting increased, accelerated and synchronized ARF. The rooting period was reduced from 16 days (non-treated cuttings) to 9 days (treated cuttings). Under optimum conditions, despite the reduced rooting period, dark-exposed cuttings produced a higher number and length of roots than non-treated cuttings. An increase in temperature to 20 °C during the dark treatment or extending the cold dark exposure to 14 days caused a similar enhancement of root development compared to non-treated cuttings. Root meristem formation had already started during the dark treatment and was enhanced during the subsequent rooting period. Levels of soluble sugars (glucose, fructose and sucrose) and starch in leaf and basal stem tissues significantly decreased during the seven days of dark exposure. This depletion was, however, compensated during rooting after 6 and 24h for soluble sugars in leaves and the basal stem, respectively, whereas the sucrose level in the basal stem was already increased at 6 h. The association of higher carbohydrate levels with improved rooting in previously dark-exposed versus non-treated cuttings indicates that increased post-darkness carbohydrate availability and allocation towards the stem base contribute to ARF under the influence of dark treatment and provide energy for cell growth subject to a rising sink intensity in the base of the cutting.
Publications

Handrick, V.; Vogt, T.; Frolov, A.; Profiling of hydroxycinnamic acid amides in Arabidopsis thaliana pollen by tandem mass spectrometry Anal. Bioanal. Chem. 398, 2789-2801, (2010) DOI: 10.1007/s00216-010-4129-2

Phenylpropanoid polyamine conjugates are widespread in plant species. Their presence has been established in seeds, flower buds, and pollen grains. A biosynthetic pathway proposed for hydroxycinnamoyl spermidine conjugates has been suggested for the model plant Arabidopsis thaliana with a central acyl transfer reaction performed by a BAHD-like hydroxycinnamoyl transferase. A detailed liquid chromatography (LC)–electrospray ionization–mass spectrometry- and tandem-mass-spectrometry (MS/MS)-based survey of wild-type and spermidine hydroxycinnamoyl transferase (SHT) mutants identified more than 30 different bis- and tris-substituted spermidine conjugates, five of which were glycosylated, in the methanol-soluble fraction of the pollen exine. On the basis of characterized fragmentation patterns, a high-throughput LC–MS/MS method for highly sensitive HCAA relative quantification (targeted profiling) was developed. Only minor qualitative and quantitative differences in the pattern of bis-acyl spermidine conjugates in the SHT mutant compared to wild-type plants provide strong evidence for the presence of multiple BAHD-like acyl transferases and suggest a much more complex array of enzymatic steps in the biosynthesis of these conjugates than previously anticipated.
Publications

Frolov, A.; Hoffmann, R.; Identification and relative quantification of specific glycation sites in human serum albumin Anal. Bioanal. Chem. 397, 2349-2356, (2010) DOI: 10.1007/s00216-010-3810-9

Glycation (or non-enzymatic glycosylation) is a common non-enzymatic covalent modification of human proteins. Glucose, the highest concentrated monosaccharide in blood, can reversibly react with amino groups of proteins to form Schiff bases that can rearrange to form relatively stable Amadori products. These can be further oxidized to advanced glycation end products (AGEs). Here, we analyzed the glycation patterns of human serum albumin (HSA) in plasma samples obtained from five patients with type 2 diabetes mellitus. Therefore, glycated peptides from a tryptic digest of plasma were enriched with m-aminophenylboronic acid (mAPBA) affinity chromatography. The glycated peptides were then further separated in the second dimension by RP-HPLC coupled on-line to an electrospray ionization (ESI) tandem mass spectrometer (MS/MS). Altogether, 18 Amadori peptides, encompassing 40% of the HSA sequence, were identified. The majority of the peptides were detected and relatively quantified in all five samples with a high reproducibility among the replicas. Eleven Lys-residues were glycated at similar quantities in all samples, with glycation site Lys549 (KAm(Glc)QTALVELVK) being the most abundant. In conclusion, the established mAPBA/nanoRP-HPLC-ESI-MS/MS approach could reproducibly identify and quantify glycation sites in plasma samples, potentially useful in diagnosis and therapeutic control.
Publications

Fedorova, M.; Frolov, A.; Hoffmann, R.; Fragmentation behavior of Amadori-peptides obtained by non-enzymatic glycosylation of lysine residues with ADP-ribose in tandem mass spectrometry J. Mass Spectrom. 45, 664-669, (2010) DOI: 10.1002/jms.1758

Mono‐ and poly‐adenosine diphosphate (ADP)‐ribosylation are common post‐translational modifications incorporated by sequence‐specific enzymes at, predominantly, arginine, asparagine, glutamic acid or aspartic acid residues, whereas non‐enzymatic ADP‐ribosylation (glycation) modifies lysine and cysteine residues. These glycated proteins and peptides (Amadori‐compounds) are commonly found in organisms, but have so far not been investigated to any great degree. In this study, we have analyzed their fragmentation characteristics using different mass spectrometry (MS) techniques. In matrix‐assisted laser desorption/ionization (MALDI)‐MS, the ADP‐ribosyl group was cleaved, almost completely, at the pyrophosphate bond by in‐source decay. In contrast, this cleavage was very weak in electrospray ionization (ESI)‐MS. The same fragmentation site also dominated the MALDI‐PSD (post‐source decay) and ESI‐CID (collision‐induced dissociation) mass spectra. The remaining phospho‐ribosyl group (formed by the loss of adenosine monophosphate) was stable, providing a direct and reliable identification of the modification site via the b‐ and y‐ion series. Cleavage of the ADP‐ribose pyrophosphate bond under CID conditions gives access to both neutral loss (347.10 u) and precursor‐ion scans (m/z 348.08), and thereby permits the identification of ADP‐ribosylated peptides in complex mixtures with high sensitivity and specificity. With electron transfer dissociation (ETD), the ADP‐ribosyl group was stable, providing ADP‐ribosylated c‐ and z‐ions, and thus allowing reliable sequence analyses.
Publications

Ehrlich, H.; Hanke, T.; Simon, P.; Born, R.; Fischer, C.; Frolov, A.; Langrock, T.; Hoffmann, R.; Schwarzenbolz, U.; Henle, T.; Bazhenov, V. V.; Worch, H.; Carboxymethylation of the fibrillar collagen with respect to formation of hydroxyapatite J. Biomed. Mater. Res. B 92B, 542-551, (2010) DOI: 10.1002/jbm.b.31551

Control over crystal growth by acidic matrix macromolecules is an important process in the formation of many mineralized tissues. Highly acidic macromolecules are postulated intermediates in tissue mineralization, because they sequester many calcium ions and occur in high concentrations at mineralizing foci in distantly related organisms. A prerequisite for biomineralization is the ability of cations like calcium to bind to proteins and to result in concert with appropriate anions like phosphates or carbonates in composite materials with bone‐like properties. For this mineralization process the proteins have to be modified with respect to acidification. In this study we modified the protein collagen by carboxymethylation using glucuronic acid. Our experiments showed unambigously, that Nε‐carboxymethyllysine is the major product of the in vitro nonenzymatic glycation reaction between glucuronic acid and collagen. We hypothesized that the function of biomimetically carboxymethylated collagen is to increase the local concentration of corresponding ions so that a critical nucleus of ions can be formed, leading to the formation of the mineral. Thus, the self‐organization of HAP nanocrystals on and within collagen fibrils was intensified by carboxymethylation.
Publications

Breuillin, F.; Schramm, J.; Hajirezaei, M.; Ahkami, A.; Favre, P.; Druege, U.; Hause, B.; Bucher, M.; Kretzschmar, T.; Bossolini, E.; Kuhlemeier, C.; Martinoia, E.; Franken, P.; Scholz, U.; Reinhardt, D.; Phosphate systemically inhibits development of arbuscular mycorrhiza in Petunia hybrida and represses genes involved in mycorrhizal functioning Plant J. 64, 1002-1017, (2010) DOI: 10.1111/j.1365-313X.2010.04385.x

Most terrestrial plants form arbuscular mycorrhiza (AM), mutualistic associations with soil fungi of the order Glomeromycota. The obligate biotrophic fungi trade mineral nutrients, mainly phosphate (Pi), for carbohydrates from the plants. Under conditions of high exogenous phosphate supply, when the plant can meet its own P requirements without the fungus, AM are suppressed, an effect which could be interpreted as an active strategy of the plant to limit carbohydrate consumption of the fungus by inhibiting its proliferation in the roots. However, the mechanisms involved in fungal inhibition are poorly understood. Here, we employ a transcriptomic approach to get insight into potential shifts in metabolic activity and symbiotic signalling, and in the defence status of plants exposed to high Pi levels. We show that in mycorrhizal roots of petunia, a similar set of symbiosis‐related genes is expressed as in mycorrhizal roots of Medicago, Lotus and rice. Pi acts systemically to repress symbiotic gene expression and AM colonization in the root. In established mycorrhizal roots, Pi repressed symbiotic gene expression rapidly, whereas the inhibition of colonization followed with a lag of more than a week. Taken together, these results suggest that Pi acts by repressing essential symbiotic genes, in particular genes encoding enzymes of carotenoid and strigolactone biosynthesis, and symbiosis‐associated phosphate transporters. The role of these effects in the suppression of symbiosis under high Pi conditions is discussed.
Publications

Hohnjec, N.; Lenz, F.; Fehlberg, V.; Vieweg, M. F.; Baier, M. C.; Hause, B.; Küster, H.; The Signal Peptide of the Medicago truncatula Modular Nodulin MtNOD25 Operates as an Address Label for the Specific Targeting of Proteins to Nitrogen-Fixing Symbiosomes Mol. Plant Microbe Interact. 22, 63-72, (2009) DOI: 10.1094/MPMI-22-1-0063

The nodule-specific MtNOD25 gene of the model legume Medicago truncatula encodes a modular nodulin composed of different repetitive modules flanked by distinct N- and C-termini. Although similarities are low with respect to all repetitive modules, both the N-terminal signal peptide (SP) and the C-terminus are highly conserved in modular nodulins from different legumes. On the cellular level, MtNOD25 is only transcribed in the infected cells of root nodules, and this activation is mediated by a 299-bp minimal promoter containing an organ-specific element. By expressing mGFP6 translational fusions in transgenic nodules, we show that MtNOD25 proteins are exclusively translocated to the symbiosomes of infected cells. This specific targeting only requires an N-terminal MtNOD25 SP that is highly conserved across a family of legume-specific symbiosome proteins. Our finding sheds light on one possible mechanism for the delivery of host proteins to the symbiosomes of infected root nodule cells and, in addition, defines a short molecular address label of only 24 amino acids whose N-terminal presence is sufficient to translocate proteins across the peribacteroid membrane.
Publications

Hause, B.; Schaarschmidt, S.; The role of jasmonates in mutualistic symbioses between plants and soil-born microorganisms Phytochemistry 70, 1589-1599, (2009) DOI: 10.1016/j.phytochem.2009.07.003

Many plants are able to develop mutualistic interactions with arbuscular mycorrhizal fungi and/or nitrogen-fixing bacteria. Whereas the former is widely distributed among most of the land plants, the latter is restricted to species of ten plant families, including the legumes. The establishment of both associations is based on mutual recognition and a high degree of coordination at the morphological and physiological level. This requires the activity of a number of signals, including jasmonates. Here, recent knowledge on the putative roles of jasmonates in both mutualistic symbioses will be reviewed. Firstly, the action of jasmonates will be discussed in terms of the initial signal exchange between symbionts and in the resulting plant signaling cascade common for nodulation and mycorrhization. Secondly, the putative role of jasmonates in the autoregulation of the endosymbioses will be outlined. Finally, aspects of function of jasmonates in the fully established symbioses will be presented. Various processes will be discussed that are possibly mediated by jasmonates, including the redox status of nodules and the carbohydrate partitioning of mycorrhizal roots.
Publications

Grunwald, U.; Guo, W.; Fischer, K.; Isayenkov, S.; Ludwig-Müller, J.; Hause, B.; Yan, X.; Küster, H.; Franken, P.; Overlapping expression patterns and differential transcript levels of phosphate transporter genes in arbuscular mycorrhizal, Pi-fertilised and phytohormone-treated Medicago truncatula roots Planta 229, 1023-1034, (2009) DOI: 10.1007/s00425-008-0877-z

A microarray carrying 5,648 probes of Medicago truncatula root-expressed genes was screened in order to identify those that are specifically regulated by the arbuscular mycorrhizal (AM) fungus Gigaspora rosea, by Pi fertilisation or by the phytohormones abscisic acid and jasmonic acid. Amongst the identified genes, 21% showed a common induction and 31% a common repression between roots fertilised with Pi or inoculated with the AM fungus G. rosea, while there was no obvious overlap in the expression patterns between mycorrhizal and phytohormone-treated roots. Expression patterns were further studied by comparing the results with published data obtained from roots colonised by the AM fungi Glomus mosseae and Glomus intraradices, but only very few genes were identified as being commonly regulated by all three AM fungi. Analysis of Pi concentrations in plants colonised by either of the three AM fungi revealed that this could be due to the higher Pi levels in plants inoculated by G. rosea compared with the other two fungi, explaining that numerous genes are commonly regulated by the interaction with G. rosea and by phosphate. Differential gene expression in roots inoculated with the three AM fungi was further studied by expression analyses of six genes from the phosphate transporter gene family in M. truncatula. While MtPT4 was induced by all three fungi, the other five genes showed different degrees of repression mirroring the functional differences in phosphate nutrition by G. rosea, G. mosseae and G. intraradices.
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