Cross-species approaches to seed dormancy and germination: conservation and biodiversity of ABA-regulated mechanisms and the Brassicaceae DOG1 genes.

Seed dormancy is genetically determined with substantial environmental influence mediated, at least in part, by the plant hormone abscisic acid (ABA). The ABA-related transcription factor ABI3/VP1 (ABA INSENSITIVE3/VIVIPAROUS1) is widespread among green plants. Alternative splicing of its transcripts appears to be involved in regulating seed dormancy, but the role of ABI3/VP1 goes beyond seeds and dormancy. In contrast, DOG1 (DELAY OF GERMINATION 1), a major quantitative trait gene more specifically involved in seed dormancy, was so far only known from Arabidopsis thaliana (AtDOG1) and whether it also has roles during the germination of non-dormant seeds was not known. Seed germination of Lepidium sativum ('garden cress') is controlled by ABA and its antagonists gibberellins and ethylene and involves the production of apoplastic hydroxyl radicals. We found orthologs of AtDOG1 in the Brassicaceae relatives L. sativum (LesaDOG1) and Brassica rapa (BrDOG1) and compared their gene structure and the sequences of their transcripts expressed in seeds. Tissue-specific analysis of LesaDOG1 transcript levels in L. sativum seeds showed that they are degraded upon imbibition in the radicle and the micropylar endosperm. ABA inhibits germination in that it delays radicle protrusion and endosperm weakening and it increased LesaDOG1 transcript levels during early germination due to enhanced transcription and/or inhibited degradation. A reduced decrease in LesaDOG1 transcript levels upon ABA treatment is evident in the late germination phase in both tissues. This temporal and ABA-related transcript expression pattern suggests a role for LesaDOG1 in the control of germination timing of non-dormant L. sativum seeds. The possible involvement of the ABA-related transcription factors ABI3 and ABI5 in the regulation of DOG1 transcript expression is discussed. Other species of the monophyletic genus Lepidium showed coat or embryo dormancy and are therefore highly suited for comparative seed biology.

Optimisation of a human neutrophil elastase assay and investigation of the effect of sesquiterpene lactones.

Neutrophil granulocytes are effector cells in innate and humoral immunity. They are involved in inflammatory processes by releasing pro-inflammatory enzymes, such as the human neutrophil elastase (HNE). We here report an optimisation of an HNE release assay using microplates. Special attention has been directed to overcome the often observed activation of neutrophils during isolation from fresh blood. This so-called basal stimulation can take place without addition of external stimulants and can cause severe problems during the assay. We demonstrated that bovine serum albumin (BSA), lipopolysaccharide (LPS), use of different blood donors, heparin and ethylenediaminetetraacetic acid (EDTA) do not cause basal stimulation, but may be due to mechanical stress and the immune system of the blood donor. Here, the number of eosinophils may play a role in the induction of activation. Basal stimulation was overcome when a hypertonic solution, such as sucrose- with N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid (HEPES) buffer, was used during centrifugation and the isolated granulocytes were left in phosphate buffered saline (PBS) for 30 min before stimulation. Sesquiterpene lactones (SLs), known for their anti-inflammatory activity were used for evaluation of the assay and were observed to inhibit HNE release at micromolar concentrations. Whereas N-formyl-methionyl-leucylphenylalanine (fMLP), platelet-activating factor (PAF) and basal stimulation resulted in similar IC50 values, phorbol-12-myristate-13-acetate (PMA) as a stimulant needed higher concentrations of SLs. The molecular inhibitory mechanism of SLs is discussed.