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SummaryIn animals and yeast, the small GTP-binding protein Ran has multiple functions ± it is involved in mediating (i) the directional passage of proteins and RNA through the nuclear pores in interphase cells; and (ii) the formation of spindle asters, the polymerization of microtubules, and the re-assembly of the nuclear envelope in mitotic cells. Nucleotide binding of Ran is modulated by a series of accessory proteins. For instance, the hydrolysis of RanGTP requires stimulation by the RanGTPase protein RanGAP. Here we report the complementation of the yeast RanGAP mutant rna1 with Medicago sativa and Arabidopsis thaliana cDNAs encoding RanGAP-like proteins. Confocal laser microscopy of Arabidopsis plants overexpressing chimeric constructs of GFP with AtRanGAP1 and 2 demonstrated that the fusion protein is localized to patchy areas at the nuclear envelope of interphase cells. In contrast, the cellular distribution of RanGAPs in synchronized tobacco cells undergoing mitosis is characteristically different. Double-immuno¯uorescence shows that RanGAPs are co-localized with spindle microtubules during anaphase, with the microtubular phragmoplast and the surface of the daughter nuclei during telophase. Co-assembly of RanGAPs with tubulin correlates with these in vivo observations. The detected localization pattern is consistent with the postulated function of plant RanGAPs in the regulation of nuclear transport during interphase, and suggests a role for these proteins in the organization of the microtubular mitotic structures.

UV irradiation stimulates expression of the gene encoding the key enzyme chalcone synthase ( CHS ), which leads to the generation of protective flavonoids in parsley cell cultures. CHS transcripts increase after 3 to 4 hr, and early genes are involved in the signal transduction to the CHS promoter. By using the fluorescent differential display technique in a large-scale screening, several early UV light-induced genes were isolated. Of these, a novel glutathione S -transferase (PcGST1) is induced within 2 hr and precedes CHS expression. Overexpression of PcGST1 in transformed cell lines containing a CHS promoter/luciferase reporter ( CHS-LUC ) affected the onset of LUC transcription. Supplementing these cell lines with glutathione immediately stimulated CHS-LUC expression within 2 hr in dark-incubated cells and resulted in a biphasic induction profile in UV-irradiated cells. Our data indicate the involvement of glutathione and PcGST1 in early events of a UV light-dependent signal transduction pathway to CHS . In this context, the oxidative status of a cell acts as a central regulating element. INTRODUCTIONDuring photomorphogenesis, the expression of numerous genes is controlled by several photoreceptors that absorb in the visible and UV range of sunlight. In addition to the family of photoreversible phytochromes, several photoreceptors absorbing blue and UV-A light have recently been isolated from higher plants (Furuya and Schaefer, 1996;Cashmore et al., 1999). Although phytochrome-mediated responses have been extensively studied, only a few elements involved in signal transduction have been isolated so far (Neff et al., 2000). UV-B irradiation of higher plants also specifically induces various responses that are attributed to the action of a hypothesized UV-B photoreceptor (Jordan, 1996). Of these responses, the induction of phenylpropanoid and flavonoid glycoside biosynthetic pathways and the consequential accumulation of UV-protective flavonoids in UVirradiated cells of a parsley suspension culture have been analyzed in detail (Hahlbrock and Scheel, 1989). As a key enzyme of flavonoid biosynthesis, chalcone synthase (CHS) is transcriptionally stimulated by UV light in this cell culture but also in leaves of adult parsley plants (Chappell and Hahlbrock, 1984;Frohnmeyer et al., 1992). CHS mRNA is strongly and exclusively induced by irradiation with low fluences of short-wavelength light with an apparent lag phase of a few hours (Chappell and Hahlbrock, 1984). This CHS induction is independent of the formation of dimerized DNA produced by irradiation with high fluences of UV-B (Frohnmeyer et al., 1999). Other external signals are ineffective in stimulating CHS expression, rendering the parsley cell culture a suitable system for the study of UV-dependent signal transduction.The parsley CHS promoter contains four cis -acting elements that mediate the light response (Schulze-Lefert et al., 1989). Several trans -acting factors binding to the CHS promoter have been isolated (Weisshaar et al., 1991; Feldbruegge et al....

Functional cell-free systems may be excellent tools with which to investigate light-dependent signal transduction mechanisms in plants. By evacuolation of parsley protoplasts and subsequent silicon oil gradient centrifugation of lysed evacuolated protoplasts, we obtained a highly pure and concentrated plasma membrane-containing cytosol. Using GTand G-box DNA elements, we were able to demonstrate a specific localization of a pool of G-box binding activity and factors (GBFs) but not one of GT-box binding activity in this cytosolic fraction. The DNA binding activity of the cytosolic GBFs is modulated in vivo as well as in vitro by light and phosphorylation/dephosphorylation activities. The regulation of cytosolic G-box binding activity by irradiation with continuous white light and phosphorylation correlates with a lightmodulated transport of GBFs to the nucleus. This was shown by a GBF-antibody cotranslocation assay in permeabilized, cell-free evacuolated parsley protoplasts. We propose that a light-regulated subcellular displacement of cytosolic GBFs to the nucleus may be an important step in the signal transduction pathway coupling photoreception to light-dependent gene expression.