Bellis perennis: a useful tool for protein localization studies.

Fluorescent fusion proteins together with transient transformation techniques are commonly used to investigate intracellular protein localisation in vivo. Biolistic transfection is reliable, efficient and avoids experimental problems associated with producing and handling fragile protoplasts. Onion epidermis pavement cells are frequently used with this technique, their excellent properties for microscopy resulting from their easy removal from the underlying tissues and large size. They also have advantages over mesophyll cells for fluorescence microscopy, as they are devoid of chloroplasts whose autofluorescence can pose problems. The arrested plastid development is peculiar to epidermal cells, however, and stands in the way of studies on protein targeting to plastids. We have developed a system enabling studies of in vivo protein targeting to organelles including chloroplasts within a photosynthetically active plant cell with excellent optical properties using a transient transformation procedure. We established biolistic transfection in epidermal pavement cells of the lawn daisy (Bellis perennis L., cultivar "Galaxy red") which unusually contain a moderate number of functional chloroplasts. These cells are excellent objects for fluorescence microscopy using current reporters, combining the advantages of the ease of biolistic transfection, the excellent optical properties of a single cell layer and access to chloroplast protein targeting. We demonstrate chloroplast targeting of plastid-localised heme oxygenase, and two further proteins whose localisation was equivocal. We also demonstrate unambiguous targeting to mitochondria, peroxisomes and nuclei. We thus propose that the Bellis system represents a valuable tool for protein localisation studies in living plant cells.

Cytoplasmic phytochrome action.

Phytochrome photoperception is a common mechanism for the detection of red and far-red light in bacteria, cyanobacteria, fungi and plants. However, the responses following phytochrome activation appear to be quite diverse between species. Lower plants, such as mosses, show phytochrome-mediated directional responses, namely phototropism and polarotropism. These cannot be explained by nuclear gene regulation and are thought to be triggered by phytochromes in the cytoplasm or at the plasma membrane. In higher plants, similar directional responses are mediated via phototropin, a blue light receptor, with phytochromes mainly controlling morphogenetic responses through gene regulation. However, cytoplasmic phytochrome responses exist in higher plants too, which appear to be intertwined with directional blue light perception. By summarizing the respective findings, a possible conservation of cytoplasmic phytochrome function in higher and lower plants is addressed here.

Arabidopsis fhl/fhy1 double mutant reveals a distinct cytoplasmic action of phytochrome A.

Phytochrome A (phyA) plays an important role during germination and early seedling development. Because phyA is the primary photoreceptor for the high-irradiance response and the very-low-fluence response, it can trigger development not only in red and far-red (FR) light but also in a wider range of light qualities. Although phyA action is generally associated with translocation to the nucleus and regulation of transcription, there is evidence for additional cytoplasmic functions. Because nuclear accumulation of phyA has been shown to depend on far-red-elongated hypocotyl 1 (FHY1) and FHL (FHY1-like), investigation of phyA function in a double fhl/fhy1 mutant might be valuable in revealing the mechanism of phyA translocation and possible cytoplasmic functions. In fhl/fhy1, the FR-triggered nuclear translocation of phyA could no longer be detected but could be restored by transgenic expression of CFP:FHY1. Whereas the fhl/fhy1 mutant showed a phyA phenotype in respect to hypocotyl elongation and cotyledon opening under high-irradiance response conditions as well as a typical phyA germination phenotype under very-low-fluence response conditions, fhl/fhy1 showed no phenotype with respect to the phyA-dependent abrogation of negative gravitropism in blue light and in red-enhanced phototropism, demonstrating clear cytoplasmic functions of phyA. Disturbance of phyA nuclear import in fhl/fhy1 led to formation of FR-induced phyA:GFP cytoplasmic foci resembling the sequestered areas of phytochrome. FHY1 and FHL play crucial roles in phyA nuclear translocation and signaling. Thus the double-mutant fhl/fhy1 allows nuclear and cytoplasmic phyA functions to be separated, leading to the novel identification of cytoplasmic phyA responses.