The interplay of light and the circadian clock. Independent dual regulation of clock-controlled gene ccg-2(eas).

Ambient light is the major agent mediating entrainment of circadian rhythms and is also a major factor influencing development and morphogenesis. We show that in Neurospora crassa the expression of clock-controlled gene 2 (ccg-2), a gene under the control of the circadian clock and allelic to the developmental gene easy wettable (eas), is regulated by light in wild-type strains. Light elicits a direct and important physiological effect on ccg-2(eas) expression as demonstrated using several mutant Neurospora strains. In white collar mutants (wc-1 and wc-2) that are "blind" to blue light, ccg-2(eas) mRNA shows no variation following illumination with saturating light. By contrast, ccg-2(eas) mRNA is photoinduced in clock-null strains such as frequency (bd;frq). The results in the clock mutants show that an intact circadian oscillator is not required for light induction of ccg-2(eas). Thus, ccg-2(eas) is subject to a dual regulation that involves separable regulation by light and circadian rhythm.     

Blue light negatively regulates the sexual filamentation via the Cwc1 and Cwc2 proteins in Cryptococcus neoformans

Cryptococcus neoformans is a heterothallic basidiomycetous yeast that primarily infects immunocompromised individuals. Dikaryotic hyphae resulting from the fusion of the MATa and MATalpha mating type strains represent the filamentous stage in the sexual life cycle of C. neoformans. In this study we demonstrate that the production of dikaryotic filaments is inhibited by blue light. To study blue light photoresponse in C. neoformans, we have identified and characterized two genes, CWC1 and CWC2, which are homologous to Neurospora crassa wc-1 and wc-2 genes. Conserved domain analyses indicate that the functions of Cwc1 and Cwc2 proteins may be evolutionally conserved. To dissect their roles in the light response, the CWC1 gene deletion mutants are created in both mating type strains. Mating filamentation in the bilateral cross of cwc1 MATa and MATalpha strains is not sensitive to light. The results indicate that Cwc1 may be an essential regulator of light responses in C. neoformans. Furthermore, overexpression of the CWC1 or CWC2 gene requires light activation to inhibit sexual filamentation, suggesting both genes may function together in the early step of blue light signalling. Taken together, our findings illustrate blue light negatively regulates the sexual filamentation via the Cwc1 and Cwc2 proteins in C. neoformans.     

Genetic analysis of signal transduction through light-induced protein phosphorylation in Neurospora crassa perithecia

We have previously demonstrated that blue light induces the phosphorylation of a 15-kDa protein in crude membrane fractions of Neurospora crassa mycelia. Here we report the isolation and characterization of a mutant (?psp; phosphorylation of small proteins) that is completely defective for phosphorylation of that protein, as assayed in both crude membrane and soluble fractions. This mutation defines a unique locus that maps to linkage group VR between al-3 and his-6. To elucidate the photobiological significance of the phosphorylation of the protein, we analyzed known photobiological phenomena and discovered that the positioning of beaks on the perithecia, defined as perithecial polarity, was light-dependent in the wild type. In the psp mutant, beaks were phototropic as in the wild type, but their position was random. In a wc-1 mutant, however, beaks were positioned at random and were not phototropic. Thus light-induced perithecial polarity and phototropism of perithecial beaks are controlled differently. A psp; wc-1 double mutant showed the same phenotype as that of wc-1 with respect to these two photomorphogenetic characters. These results indicate that the wc-1 gene is epistatic to psp in the light-signal transduction pathway that controls both phototropism and perithecial polarity.     

Genetic analysis of signal transduction through light-induced protein phosphorylation in Neurospora crassa perithecia

We have previously demonstrated that blue light induces the phosphorylation of a 15-kDa protein in crude membrane fractions of Neurospora crassa mycelia. Here we report the isolation and characterization of a mutant ( psp; phosphorylation of small proteins) that is completely defective for phosphorylation of that protein, as assayed in both crude membrane and soluble fractions. This mutation defines a unique locus that maps to linkage group VR between al-3 and his-6. To elucidate the photobiological significance of the phosphorylation of the protein, we analyzed known photobiological phenomena and discovered that the positioning of beaks on the perithecia, defined as perithecial polarity, was light-dependent in the wild type. In the psp mutant, beaks were phototropic as in the wild type, but their position was random. In a wc-1 mutant, however, beaks were positioned at random and were not phototropic. Thus light-induced perithecial polarity and phototropism of perithecial beaks are controlled differently. A psp; wc-1 double mutant showed the same phenotype as that of wc-1 with respect to these two photomorphogenetic characters. These results indicate that the wc-1 gene is epistatic to psp in the light-signal transduction pathway that controls both phototropism and perithecial polarity. Received: 30 January 1997 / Accepted: 30 July 1997     

A new wc-1 mutant of Neurospora crassa shows unique light sensitivity in the circadian conidiation rhythm

A new clock mutant ( rhy-2) was isolated by DNA insertion mutagenesis using a plasmid that contains a region located upstream of the cmd gene in the genome of Neurospora crassa. This mutant is arrhythmic with regard to conidiation in continuous darkness but rhythmic under a light-dark cycle. After plasmid rescue from genomic DNA of the rhy-2 strain, the insertion was localized to the gene white collar-1 ( wc-1). Plasmid DNA was inserted 3' to the sequence encoding the polyglutamine region of the WC-1 gene product, and an mRNA encoding a truncated WC-1 protein must be synthesized under the control of the cmd promoter. The new wc-1 mutant, rhy-2, is still sensitive to light, although only weakly. Since the circadian rhythm of conidiation in continuous darkness is eliminated in the mutant, the polyglutamine region in WC-1 may be essential for both clock function and light-induced carotenogenesis in Neurospora.     

Medicago truncatula Mt-ZFP1 encoding a root enhanced zinc finger protein is regulated by cytokinin, abscisic acid and jasmonate, but not cold.

A Medicago truncatula zinc finger protein cDNA (Mt-ZFP1) was isolated from a M.truncatula seedling cDNA library using RT-PCR product as a probe. The predicted amino acid sequence of Mt-ZFP1 is over 79% similar to S-SCOF-1 from soybean, a novel cold-inducible zinc finger protein involved in cold stress signal transduction mediated by abscisic acid (ABA). The secondary structure of Mt-ZFP1 protein was almost identical to that of S-SCOF-1. Mt-ZFP1 also contained a typical C2H2-type zinc finger domain and a putative nuclear located signal. RNA gel blot hybridization demonstrated that the Mt-ZFP1 gene was actively expressed in roots, with a lower abundance in leaf and stem tissues. Cold treatment did not induce the expression of Mt-ZFP1 in either leaves and stems or roots. Exogenous application of cytokinins marginally increased the accumulation of Mt-ZFP1 mRNA, while ABA and jasmonate treatments decreased the levels of Mt-ZFP1 mRNA. DNA gel-blot analysis demonstrated that Mt-ZFP1 is present as a single copy gene in the M. truncatula genome. These data suggest that Mt-ZFP1 is a novel zinc finger protein with different physiological functions to that of S-SCOF-1. The similar cold-inducible factor like S-SCOF-1 might not exist in M. truncatula.     

High pigment1 mutation negatively regulates phototropic signal transduction in tomato seedlings

Phototropins and phytochromes are the major photosensory receptors in plants and they regulate distinct photomorphogenic responses. The molecular mechanisms underlying functional interactions of phototropins and phytochromes remain largely unclear. We show that the tomato (Lycopersicon esculentum) phytochrome A deficient mutant fri lacks phototropic curvature to low fluence blue light, indicating requirement for phytochrome A for expression of phototropic response. The hp1 mutant that exhibits hypersensitive responses to blue light and red light reverses the impairment of second-positive phototropic response in tomato in phytochrome A-deficient background. Physiological analyses indicate that HP1 functions as a negative regulator of phototropic signal transduction pathway, which is removed via action of phytochrome A. The loss of HP1 gene product in frihp1 double mutant allows the unhindered operation of phototropic signal transduction chain, obviating the need for the phytochrome action. Our results also indicate that the role of phytochrome in regulating phototropism is restricted to low fluence blue light only, and at high fluence blue light, the phytochrome A-deficient fri mutant shows the normal phototropic response.