Publikationen - Molekulare Signalverarbeitung

The F-box protein superfamily represents one of the largest families in the plant kingdom. F-box proteins phylogenetically organize into numerous subfamilies characterized by their carboxyl (C)-terminal protein-protein interaction domain. Among the largest F-box protein subfamilies in plant genomes are those with C-terminal kelch repeats. In this study, we analyzed the phylogeny and evolution of F-box kelch proteins/genes (FBKs) in seven completely sequenced land plant genomes including a bryophyte, a lycophyte, monocots, and eudicots. While absent in prokaryotes, F-box kelch proteins are widespread in eukaryotes. Nonplant eukaryotes usually contain only a single FBK gene. In land plant genomes, however, FBKs expanded dramatically. Arabidopsis thaliana, for example, contains at least 103 F-box genes with well-conserved C-terminal kelch repeats. The construction of a phylogenetic tree based on the full-length amino acid sequences of the FBKs that we identified in the seven species enabled us to classify FBK genes into unstable/stable/superstable categories. In contrast to superstable genes, which are conserved across all seven species, kelch domains of unstable genes, which are defined as lineage specific, showed strong signatures of positive selection, indicating adaptational potential. We found evidence for conserved protein features such as binding affinities toward A. thaliana SKP1-like adaptor proteins and subcellular localization among closely related FBKs. Pseudogenization seems to occur only rarely, but differential transcriptional regulation of close relatives may result in subfunctionalization.

Natural variation has been observed for various traits in Arabidopsis thaliana. Here, we investigated natural variation in the context of physiological and transcriptional responses to the phytohormone auxin, a key regulator of plant development. A survey of the general extent of natural variation to auxin stimuli revealed significant physiological variation among 20 genetically diverse natural accessions. Moreover, we observed dramatic variation on the global transcriptome level after induction of auxin responses in seven accessions. Although we detect isolated cases of major-effect polymorphisms, sequencing of signaling genes revealed sequence conservation, making selective pressures that favor functionally different protein variants among accessions unlikely. However, coexpression analyses of a priori defined auxin signaling networks identified variations in the transcriptional equilibrium of signaling components. In agreement with this, cluster analyses of genome-wide expression profiles followed by analyses of a posteriori defined gene networks revealed accession-specific auxin responses. We hypothesize that quantitative distortions in the ratios of interacting signaling components contribute to the detected transcriptional variation, resulting in physiological variation of auxin responses among accessions.

Three chimeric gene constructs were designed comprising the full length cDNA of a lipoxygenase (LOX) from barley (LOX2:Hv:1) including its chloroplast targeting sequence (cTP) under control of either (1) CaMV35S- or (2) polyubiquitin-1-promoter, whereas the third plasmid contains 35S promoter and the cDNA without cTP. Transgenic barley plants overexpressing LOX2:Hv:1 were generated by biolistics of scutella from immature embryos. Transformation frequency for 35S::LOX with or without cTP was in a range known for barley particle bombardment, whereas for Ubi::cTP-LOX no transgenic plants were detected. In general, a high number of green plantlets selected on bialaphos became yellow and finally died either in vitro or after potting. All transgenic plants obtained were phenotypically indistinguishable from wild type plants and all of them set seeds. The corresponding protein (LOX-100) in transgenic T0 and T1 plants accumulated constitutively to similar levels as in the jasmonic acid methyl ester (JAME)-treated wild type plants. Moreover, LOX-100 was clearly detectable immunocytochemically within the chloroplasts of untreated T0 plants containing the LOX-100-cDNA with the chloroplast target sequence. In contrast, an exclusive localization of LOX-100 in the cytoplasm was detectable when the target sequence was removed. In comparison to sorbitol-treated wild type leaves, analysis of oxylipin profiles in T2 progenies showed higher levels of jasmonic acid (JA) for those lines that displayed elevated levels of LOX-100 in the chloroplasts and for those lines that harboured LOX-100 in the cytoplasm, respectively. The studies demonstrate for the first time the constitutive overexpression of a cDNA coding for a 13-LOX in a monocotyledonous species and indicate a link between the occurrence of LOX-100 and senescence.

Immature scutella of barley were transformed with cDNA coding for a 13-lipoxygenase of barley (LOX-100) via particle bombardment. Regenerated plants were tested by PAT-assay, Western-analysis and PCR-screening. Immunocytochemical assay of T0 plants showed expression of the LOX cDNA both in the chloroplasts and in the cytosol, depending on the presence of the chloroplast signal peptide sequences in the cDNA. A few transgenic plants containing higher amounts of LOX-derived products have been found. These are the candidates for further analysis concerning pathogen resistance.

The most abundant jasmonate-induced protein (JIP) in barley leaves is a 23 kDa protein (JIP23). Its function, however, is unknown. In order to analyze its function by homologous transformation, new plasmid vectors have been constructed. They carry the cDNA coding for JIP23 in sense or antisense orientation under the control of the Ubi-1-promoter as well as the pat resistance gene under the control of the 35S promoter. Barley mesophyll protoplasts were transiently transformed with the sense constructs. PAT activity and immunological detection of JIP23 could be achieved in transformed protoplasts but not in untransformed protoplasts indicating that the construct was active. Thus, these new vectors are suitable for stable transformation of barley. Carrying a multiple cloning site (MCS), these vectors can be used now in a wide range of transformation of barley.