Publikationen - Molekulare Signalverarbeitung

Although RNase-based self-incompatibility (SI) is suspected to operate in a wide group of plant families, it has been characterized as the molecular genetic basis of SI in only three distantly related families, Solanaceae, Plantaginaceae, and Rosaceae, all described over a decade ago. Previous studies found that gametophytic SI, controlled by a multi-allelic S-locus, operates in the coffee family (Rubiaceae). The molecular genetic basis of this mechanism remains unknown, despite the immense importance of coffee as an agricultural commodity. Here, we isolated ten sequences with features of T2-S-type RNases from two Coffea species. While three of the sequences were identified in both species and clearly do not appear to be S-locus products, our data suggest that six sequences may be S-alleles in the self-incompatible C. canephora, and one may be a relict in the self-compatible C. arabica. We demonstrate that these sequences show style-specific expression, display polymorphism in C. canephora, and cluster with S-locus products in a phylogenetic analysis that includes other plant families with RNase-based SI. Although our results are not definitive, in part because the available plant materials were limited and data patterns relatively complex, our results strongly hint that RNase-based SI mechanism operates in the Rubiaceae family.

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.

Coffea arabica is susceptible to several pests and diseases, some of which affect the leaves and roots. Systemic acquired resistance (SAR) is the main defence mechanism activated in plants in response to pathogen attack. Here, we report the effects of benzo(1,2,3)thiadiazole-7-carbothioic acid-s-methyl ester (BTH), a SAR chemical inducer, on the expression profile of C. arabica. Two cDNA libraries were constructed from the mRNA isolated from leaves and embryonic roots to create 1587 nonredundant expressed sequence tags (ESTs). We developed a cDNA microarray containing 1506 ESTs from the leaves and embryonic roots, and 48 NBS-LRR (nucleotide-binding site leucine-rich repeat) gene fragments derived from 2 specific genomic libraries. Competitive hybridization between untreated and BTH-treated leaves resulted in 55 genes that were significantly overexpressed and 16 genes that were significantly underexpressed. In the roots, 37 and 42 genes were over and underexpressed, respectively. A general shift in metabolism from housekeeping to defence occurred in the leaves and roots after BTH treatment. We observed a systemic increase in pathogenesis-related protein synthesis, in the oxidative burst, and in the cell wall strengthening processes. Moreover, responses in the roots and leaves varied significantly.