Publikationen - Molekulare Signalverarbeitung

BackgroundThe formation of flowers is one of the main model systems to elucidate the molecular mechanisms that control developmental processes in plants. Although several studies have explored gene expression during flower development in the model plant Arabidopsis thaliana on a genome-wide scale, a continuous series of expression data from the earliest floral stages until maturation has been lacking. Here, we used a floral induction system to close this information gap and to generate a reference dataset for stage-specific gene expression during flower formation.ResultsUsing a floral induction system, we collected floral buds at 14 different stages from the time of initiation until maturation. Using whole-genome microarray analysis, we identified 7,405 genes that exhibit rapid expression changes during flower development. These genes comprise many known floral regulators and we found that the expression profiles for these regulators match their known expression patterns, thus validating the dataset. We analyzed groups of co-expressed genes for over-represented cellular and developmental functions through Gene Ontology analysis and found that they could be assigned specific patterns of activities, which are in agreement with the progression of flower development. Furthermore, by mapping binding sites of floral organ identity factors onto our dataset, we were able to identify gene groups that are likely predominantly under control of these transcriptional regulators. We further found that the distribution of paralogs among groups of co-expressed genes varies considerably, with genes expressed predominantly at early and intermediate stages of flower development showing the highest proportion of such genes.ConclusionsOur results highlight and describe the dynamic expression changes undergone by a large number of genes during flower development. They further provide a comprehensive reference dataset for temporal gene expression during flower formation and we demonstrate that it can be used to integrate data from other genomics approaches such as genome-wide localization studies of transcription factor binding sites.

The phytohormone auxin is a potent regulator of plant development. Since its discovery in the beginning of the twentieth century many aspects of auxin biology have been extensively studied, ranging from biosynthesis and metabolism to the elucidation of molecular components of downstream signaling. With the identification of the F-box protein TIR1 as an auxin receptor a major breakthrough in understanding auxin signaling has been achieved and recent modeling approaches have shed light on the putative mechanisms underlying the establishment of auxin gradients and maxima essential for many auxin-regulated processes. Here, we review these and other recent advances in unraveling the entanglement of biosynthesis, polar transport and cellular signaling events that allow small auxinic molecules to facilitate their complex regulatory action.

Genetic linkage maps, constructed from multi-locus recombination data, are the basis for many applications of molecular markers. For the successful employment of a linkage map, it is essential that the linear order of loci on a chromosome is correct. The objectives of this theoretical study were to (1) investigate the occurrence of incorrect locus orders caused by duplicate marker loci, (2) develop a statistical test for the detection of duplicate markers, and (3) discuss the implications for practical applications of linkage maps. We derived conditions, under which incorrect locus orders do or do not occur with duplicate marker loci for the general case of n markers on a chromosome in a BC1 mapping population. We further illustrated these conditions numerically for the special case of four markers. On the basis of the extent of segregation distortion, an exact test for the presence of duplicate marker loci was suggested and its power was investigated numerically. Incorrect locus orders caused by duplicate marker loci can (1) negatively affect the assignment of target genes to chromosome regions in a map-based cloning experiment, (2) hinder indirect selection for a favorable allele at a quantitative trait locus, and (3) decrease the efficiency of reducing the length of the chromosome segment attached to a target gene in marker-assisted backcrossing.

Quantitative trait loci (QTLs) and bulked segregant analyses (BSA) identified the major genes Scmv1 on chromosome 6 and Scmv2 on chromosome 3, conferring resistance against sugarcane mosaic virus (SCMV) in maize. Both chromosome regions were further enriched for SSR and AFLP markers by targeted bulked segregant analysis (tBSA) in order to identify and map only markers closely linked to either Scmv1 or Scmv2. For identification of markers closely linked to the target genes, symptomless individuals of advanced backcross generations BC5 to BC9 were employed. All AFLP markers, identified by tBSA using 400 EcoRI/MseI primer combinations, mapped within both targeted marker intervals. Fourteen SSR and six AFLP markers mapped to the Scmv1 region. Eleven SSR and 18 AFLP markers were located in the Scmv2 region. Whereas the linear order of SSR markers and the window size for the Scmv2 region fitted well with publicly available genetic maps, map distances and window size differed substantially for the Scmv1 region on chromosome 6. A possible explanation for the observed discrepancies is the presence of two closely linked resistance genes in the Scmv1 region.

The resistance gene analogue (RGA) pic19 in maize, a candidate for sugarcane mosaic virus (SCMV) resistance gene (R gene) Scmv1, was used to screen a maize BAC library to identify homologous sequences in the maize genome and to investigate their genomic organisation. Fifteen positive BAC clones were identified and could be classified into five physically independent contigs consisting of overlapping clones. Genetic mapping clustered three contigs into the same genomic region as Scmv1 on chromosome 6S. The two remaining contigs mapped to the same region as a QTL for SCMV resistance on chromosome 1. Thus, RGAs mapping to a target region can be successfully used to identify further-linked candidate sequences. The pic19 homologous sequences of these clones revealed a sequence similarity of 94–98% on the nucleotide level. The high sequence similarity reveals potential problems for the use of RGAs as molecular markers. Their application in marker-assisted selection (MAS) and the construction of high-density genetic maps is complicated by the existence of closely linked homologues resulting in 'ghost' marker loci analogous to 'ghost' QTLs. Therefore, implementation of genomic library screening, including genetic mapping of potential homologues, seems necessary for the safe application of RGA markers in MAS and gene isolation.

Three previously published resistance gene analogues (RGAs), pic13, pic21 and pic19, were mapped in relation to sugarcane mosaic virus (SCMV) resistance genes (Scmv1, Scmv2) in maize. We cloned these RGAs from six inbreds including three SCMV-resistant lines (D21, D32, FAP1360A) and three SCMV-susceptible lines (D145, D408, F7). Pairwise sequence alignments among the six inbreds revealed a frequency of one single nucleotide polymorphism (SNP) per 33 bp for the three RGAs, indicating a high degree of polymorphism and a high probability of success in converting RGAs into codominant cleaved amplified polymorphic sequence (CAPS) markers compared to other sequences. SNPs were used to develop CAPS markers for mapping of the three RGAs in relation to Scmv1 (chromosome 6) and Scmv2 (chromosome 3), and for pedigree analyses of resistant inbred lines. By genetic mapping pic21 was shown to be different from Scmv2, whereas pic19 and pic13 are still candidates for Scmv1 and Scmv2, respectively, due to genetic mapping and consistent restriction patterns of ancestral lines.

In a previous study, bulked segregant analysis with amplified fragment length polymorphisms (AFLPs) identified several markers closely linked to the sugarcane mosaic virus resistance genes Scmv1 on chromosome 6 and Scmv2 on chromosome 3. Six AFLP markers (E33M61-2, E33M52, E38M51, E82M57, E84M59 and E93M53) were located on chromosome 3 and two markers (E33M61-1 and E35M62-1) on chromosome 6. Our objective in the present study was to sequence the respective AFLP bands in order to convert these dominant markers into more simple and reliable polymerase chain reaction (PCR)-based sequence-tagged site markers. Six AFLP markers resulted either in complete identical sequences between the six inbreds investigated in this study or revealed single nucleotide polymorphisms within the inbred lines and were, therefore, not converted. One dominant AFLP marker (E35M62-1) was converted into an insertion/deletion (indel) marker and a second AFLP marker (E33M61-2) into a cleaved amplified polymorphic sequence marker. Mapping of both converted PCR-based markers confirmed their localization to the same chromosome region (E33M61-2 on chromosome 3; E35M62-1 on chromosome 6) as the original AFLP markers. Thus, these markers will be useful for marker-assisted selection and facilitate map-based cloning of SCMV resistance genes.

The role of systemin in Pin2 gene expression was analyzed in wild-type plants of potato (Solanum tuberosum L.) and tomato (Lycopersicon esculentum Mill.), as well as in abscisic acid (ABA)-deficient tomato (sitiens) and potato (droopy) plants. The results showed that systemin initiates Pin2 mRNA accumulation only in wildtype tomato and potato plants. As in the situation after mechanical wounding,Pin2 gene expression in ABA-deficient plants was not activated by systemin. Increased endogenous levels of jasmonic acid (JA) and accumulation of Pin2 mRNA were observed following treatment with α-linolenic acid, the precursor of JA biosynthesis, suggesting that these ABA mutants still have the capability to synthesize de novo JA. Measurement of endogenous levels of ABA and JA showed that systemin leads to an increase of both phytohormones (ABA and JA) only in wild-type but not in ABA-deficient plants.

The accumulation of abundant proteins and their respective transcripts, induced by 10−4 M cisabscisic acid or 10−5 M jasmonic acid methyl ester, was studied in barley (Hordeum vulgare L.) leaf segments and compared to that resulting from osmotic stress caused by floating the segments on solutions of sorbitol, glucose, polyethyleneglycol (PEG)-6000 or NaCl. Osmotic stress or treatment with abscisic acid led to the synthesis of novel proteins which were identical to jasmonateinduced proteins (JIPs) with respect to immunological properties and molecular masses. The most prominent polypeptides were characterized by molecular masses of 66, 37 and 23 kDa and were newly synthesized. Whereas sorbitol, mannitol, sucrose, glucose and PEG provoked the synthesis of JIPs, 2deoxyglucose and NaCl did not. We provide evidence that the synthesis of JIPs induced by osmotic stress is directly correlated with a preceding rise in endogenous jasmonates. These jasmonates, quantified by an enzyme immunoassay specific for (−)jasmonic acid and its aminoacid conjugates, increased remarkably in leaf segments treated with sorbitol, glucose or other sugars. In contrast, no increase in jasmonates could be observed in tissues exposed to salts (NaCl). The results strengthen the hypothesis that the accumulation of jasmonates, probably by de-novo synthesis, is an intermediate and essential step in a signalling pathway between (osmotic) stress and activation of genes coding for polypeptides of high abundance.