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Publications - Cell and Metabolic Biology

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Publications

Druege, U.; Franken, P.; Lischewski, S.; Ahkami, A. H.; Zerche, S.; Hause, B.; Hajirezaei, M. R.; Transcriptomic analysis reveals ethylene as stimulator and auxin as regulator of adventitious root formation in petunia cuttings Front. Plant Sci. 5, 494, (2014) DOI: 10.3389/fpls.2014.00494

Adventitious root (AR) formation in the stem base (SB) of cuttings is the basis for propagation of many plant species and petunia is used as model to study this developmental process. Following AR formation from 2 to 192 hours post-excision (hpe) of cuttings, transcriptome analysis by microarray revealed a change of the character of the rooting zone from SB to root identity. The greatest shift in the number of differentially expressed genes was observed between 24 and 72 hpe, when the categories storage, mineral nutrient acquisition, anti-oxidative and secondary metabolism, and biotic stimuli showed a notable high number of induced genes. Analyses of phytohormone-related genes disclosed multifaceted changes of the auxin transport system, auxin conjugation and the auxin signal perception machinery indicating a reduction in auxin sensitivity and phase-specific responses of particular auxin-regulated genes. Genes involved in ethylene biosynthesis and action showed a more uniform pattern as a high number of respective genes were generally induced during the whole process of AR formation. The important role of ethylene for stimulating AR formation was demonstrated by the application of inhibitors of ethylene biosynthesis and perception as well as of the precursor aminocyclopropane-1-carboxylic acid, all changing the number and length of AR. A model is proposed showing the putative role of polar auxin transport and resulting auxin accumulation in initiation of subsequent changes in auxin homeostasis and signal perception with a particular role of Aux/IAA expression. These changes might in turn guide the entrance into the different phases of AR formation. Ethylene biosynthesis, which is stimulated by wounding and does probably also respond to other stresses and auxin, acts as important stimulator of AR formation probably via the expression of ethylene responsive transcription factor genes, whereas the timing of different phases seems to be controlled by auxin.
Publications

Mallona, I.; Lischewski, S.; Weiss, J.; Hause, B.; Egea-Cortines, M.; Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida BMC Plant Biol. 10, 4, (2010) DOI: 10.1186/1471-2229-10-4

BackgroundIdentification of genes with invariant levels of gene expression is a prerequisite for validating transcriptomic changes accompanying development. Ideally expression of these genes should be independent of the morphogenetic process or environmental condition tested as well as the methods used for RNA purification and analysis.ResultsIn an effort to identify endogenous genes meeting these criteria nine reference genes (RG) were tested in two Petunia lines (Mitchell and V30). Growth conditions differed in Mitchell and V30, and different methods were used for RNA isolation and analysis. Four different software tools were employed to analyze the data. We merged the four outputs by means of a non-weighted unsupervised rank aggregation method. The genes identified as optimal for transcriptomic analysis of Mitchell and V30 were EF1α in Mitchell and CYP in V30, whereas the least suitable gene was GAPDH in both lines.ConclusionsThe least adequate gene turned out to be GAPDH indicating that it should be rejected as reference gene in Petunia. The absence of correspondence of the best-suited genes suggests that assessing reference gene stability is needed when performing normalization of data from transcriptomic analysis of flower and leaf development.
Publications

Ahkami, A. H.; Lischewski, S.; Haensch, K.-T.; Porfirova, S.; Hofmann, J.; Rolletschek, H.; Melzer, M.; Franken, P.; Hause, B.; Druege, U.; Hajirezaei, M. R.; Molecular physiology of adventitious root formation in Petunia hybrida cuttings: involvement of wound response and primary metabolism New Phytol. 181, 613-625, (2009) DOI: 10.1111/j.1469-8137.2008.02704.x

Adventitious root formation (ARF) in the model plant Petunia hybrida cv. Mitchell has been analysed in terms of anatomy, gene expression, enzymatic activities and levels of metabolites. This study focuses on the involvement of wound response and primary metabolism.Microscopic techniques were complemented with targeted transcript, enzyme and metabolite profiling using real time polymerase chain reaction (PCR), Northern blot, enzymatic assays, chromatography and mass spectrometry.Three days after severance from the stock plants, first meristematic cells appeared which further developed into root primordia and finally adventitious roots. Excision of cuttings led to a fast and transient increase in the wound‐hormone jasmonic acid, followed by the expression of jasmonate‐regulated genes such as cell wall invertase. Analysis of soluble and insoluble carbohydrates showed a continuous accumulation during ARF. A broad metabolite profiling revealed a strong increase in organic acids and resynthesis of essential amino acids.Substantial changes in enzyme activities and metabolite levels indicate that specific enzymes and metabolites might play a crucial role during ARF. Three metabolic phases could be defined: (i) sink establishment phase characterized by apoplastic unloading of sucrose and being probably mediated by jasmonates; (ii) recovery phase; and (iii) maintenance phase, in which a symplastic unloading occurs.
Publications

Hause, G.; Lischewski, S.; Wessjohann, L. A.; Hause, B.; Epothilone D affects cell cycle and microtubular pattern in plant cells J. Exp. Bot. 56, 2131-2137, (2005) DOI: 10.1093/jxb/eri211

Epothilones, macrocyclic lactones from culture filtrates of the myxobacterium Sorangium cellulosum, are known as taxol-like microtubular drugs in human medicine. To date, nothing is known about the effect of epothilones on microtubules (MTs) in plant cells and/or on the plant cell cycle. As shown in this report, the treatment of tomato cell suspension cultures with epothilone D produced a continuous increase in the mitotic index. Dose–response curves revealed that epothilone D alters the mitotic index at concentrations as low as 1.5 μM. Mitotic arrest was already visible after only 2 h of treatment, and 55% of the cells were arrested after 24 h. As shown by immunocytological methods, abnormal spindles are formed during metaphase, which leads to a random distribution of chromosomes in the whole cell and prevents the formation of a metaphase plate. The process of chromosome decondensation does not seem to be affected, because micronuclei form at the same place with the distributed chromosomes. This suggests that epothilone D influences the stability of plant MTs mainly during metaphase of the mitotic cycle. In metaphase, the effects of epothilone D seem to be irreversible, because cells with an abnormal spindle could not be recovered after removal of the drug.
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