Light-regulated modification and nuclear translocation of cytosolic G-box binding factors in parsley.

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 GT- and 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 light-modulated 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.     

Phytochrome and UV signal transduction pathways

The phytochromes are the best studied plant photoreceptors, controlling a wide variety of responses at both whole plant and single cell levels. Three signal transduction pathways, dependent on cGMP and/or calcium, have been found to be utilized by phytochrome to control the expression of genes required for chloroplast development (e.g., CAB and FNR) and anthocyanin biosynthesis (e.g., CHS). In particular, cGMP is a second messenger positively regulating CHS gene expression whilst calcium and calmodulin act as negative regulators. In addition to phytochrome regulation of CHS we have begun to examine the signal transduction pathways utilized by UV photoreceptors. In contrast to phytochrome-mediated responses, results indicate a role for calcium and calmodulin as positive regulators of CHS gene expression in UV light.     

Plant RanGAPs are localized at the nuclear envelope in interphase and associated with microtubules in mitotic cells

In 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-immunofluorescence 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.     

Millisecond UV-B irradiation evokes prolonged elevation of cytosolic-free Ca2+ and stimulates gene expression in transgenic parsley cell cultures

Chalcone synthase (CHS) is a key enzyme leading to the generation of protective flavonoids in plants under environmental stress. Expression of the CHS gene is strongly upregulated by exposures to UV light, a response also observed in heterotrophic parsley cell cultures. Although there are hints that the stimulus for CHS expression may be coupled to UV-B irradiation through a rise in cytosolic-free Ca2+ ([Ca2+]i), the temporal relationship of these events has never been investigated critically. To explore this question, we have used a CHS promoter/luciferase (CHS/LUC) reporter gene fusion and recorded its expression and [Ca2+]i elevation in a transgenic parsley cell culture following millisecond light pulses. Luciferase expression was enhanced maximally seven- (+/- 2) fold by 30 10 ms flashes of UV-B light. The response was specific to wavelengths of 300-330 nm and could be inhibited in the presence of the Ca2+ channel blocker nifedipine. In parallel measurements, using Fura-2 fluorescence ratio microphotometry, we found that 10 ms UV-B flashes also evoked a gradual and prolonged rise of [Ca2+]i in the parsley cells which was irreversible within the timescale of these experiments, but could be prevented by prior treatment with nifedipine. These, and additional results, indicate a remarkably high temporal sensitivity to, and specificity for, UV-B light in CHS gene expression independent of UV-mediated DNA damage by thymine dimerization. The ability of transient UV-B stimulation to evoke prolonged elevations of [Ca2+]i suggests a functional coupling between the initial light stimulus and subsequent gene expression that takes place many tens of minutes later.     

Secretome profiling of Propionibacterium freudenreichii reveals highly variable responses even among the closely related strains

This study compared the secretomes (proteins exported out of the cell) of Propionibacterium freudenreichii of different origin to identify plausible adaptation factors. Phylosecretomics indicated strain‐specific variation in secretion of adhesins/invasins (SlpA, InlA), cell‐wall hydrolysing (NlpC60 peptidase, transglycosylase), protective (RpfB) and moonlighting (DnaK, GroEL, GaPDH, IDH, ENO, ClpB) enzymes and/or proteins. Detailed secretome comparison suggested that one of the cereal strains (JS14) released a tip fimbrillin (FimB) in to the extracellular milieu, which was in line with the electron microscopy and genomic analyses, indicating the lack of surface‐associated fimbrial‐like structures, predicting a mutated type‐2 fimbrial gene cluster (fimB‐fimA‐srtC2) and production of anchorless FimB. Instead, the cereal strain produced high amounts of SlpB that tentatively mediated adherent growth on hydrophilic surface and adherence to hydrophobic material. One of the dairy strains (JS22), producing non‐covalently bound surface‐proteins (LspA, ClpB, AraI) and releasing SlpA and InlA into the culture medium, was found to form clumps under physiological conditions. The JS22 strain lacked SlpB and displayed a non‐clumping and biofilm‐forming phenotype only under conditions of increased ionic strength (300 mM NaCl). However, this strain cultured under the same conditions was not adherent to hydrophobic support, which supports the contributory role of SlpB in mediating hydrophobic interactions. Thus, this study reports significant secretome variation in P. freudenreichii and suggests that strain‐specific differences in protein export, modification and protein–protein interactions have been the driving forces behind the adaptation of this bacterial species.