Blue light regulates many physiological and developmental processes in fungi. Most of the blue light responses in the ascomycete Neurospora crassa are dependent on the two blue light regulatory proteins White Collar (WC)-1 and -2. WC-1 has recently been shown to be the ®rst fungal blue light photoreceptor. In the present study, we characterize the Neurospora protein VIVID. VIVID shows a partial sequence similarity with plant blue light photoreceptors. In addition, we found that VIVID non-covalently binds ā avin chromophore. Upon illumination with blue light, VIVID undergoes a photocycle indicative of the formation of a¯avin-cysteinyl adduct. VVD is localized in the cytoplasm and is only present after light induction. A loss-of-function vvd mutant was insensitive to increases in light intensities. Furthermore, mutational analysis of the photoactive cysteine indicated that the formation of a¯avin-cysteinyl adduct is essential for VIVID functions in vivo. Our results show that VIVID is a second fungal blue light photoreceptor which enables Neurospora to perceive and respond to daily changes in light intensity.

A saturating genetic dissection of ‘blind’ mutants in Neurospora crassa has identified a total of two non‐redundant loci (wc‐1 and wc‐2) each of which is required for blue‐light perception/signal transduction. Previously, we demonstrated that WC1 is a putative zinc finger transcription factor able to bind specifically to a light‐regulated promoter. Here, we present the cloning and characterization of the wc‐2 gene. We demonstrate using mutation analysis and in vitro DNA‐binding assays that WC2, the second partner of this light signal transduction system, encodes a functional zinc finger DNA‐binding protein with putative PAS dimerization and transcription activation domains. This molecular genetic dissection of the second of two components of this light signal transduction system has enabled us to devise a model whereby WC1 and WC2 are proposed to interact via homologous PAS domains, bind to promoters of light‐regulated genes and activate transcription. As such, this study provides the first insight into two co‐operating partners in blue‐light signal transduction in any organism and describes the molecular tools with which to dissect this enigmatic process.

Mutations in either white collar-1 (wc-1) or white collar-2 (wc-2) lead to a loss of most blue-light-induced phenomena in Neurospora crassa. Sequence analysis and in vitro experiments show that WC-1 and WC-2 are transcription factors regulating the expression of light-induced genes. The WC proteins form homo-and heterodimers in vitro; this interaction could represent a fundamental step in the control of their activity. We demonstrate in vivo that the WC proteins are assembled in a white collar complex (WCC) and that WC-1 undergoes a change in mobility due to light-induced phosphorylation events. The phosphorylation level increases progressively upon light exposure, producing a hyperphosphorylated form that is degraded and apparently replaced in the complex by a newly synthesized WC-1. WC-2 is unmodified and also does not change quantitatively in the time frame examined. Light-dependent phosphorylation of WC-1 also occurs in a wc-2 mutant, suggesting that a functional WC-2 is dispensable for this light-specific event. These results suggest that light-induced phosphorylation and degradation of WC-1 could play a role in the transient expression of blue-light-regulated genes. Our findings suggest a mechanism by which WC-1 and WC-2 mediate light responses in Neurospora.

The genes coding for white collar-1 and white collar-2 (wc-1 and wc-2) have been isolated previously, and their products characterized as Zn-finger transcription factors involved in the control of blue light-induced genes. Here, we show that the PAS dimerization domains present in both proteins enable the WC-1 and WC-2 proteins to dimerize in vitro. Homodimers and heterodimers are formed between the white collar (WC) proteins. A computer analysis of WC-1 reveals a second domain, called LOV, also identified in NPH1, a putative blue light photoreceptor in plants and conserved in redox-sensitive proteins and in the phytochromes. The WC-1 LOV domain does not dimerize with canonical PAS domains, but it is able to self-dimerize. The isolation of three blind wc-1 strains, each with a single amino acid substitution only in the LOV domain, reveals that this region is essential for blue light responses in Neurospora. The demonstration that the WC-1 proteins in these LOV mutants are still able to self-dimerize suggests that this domain plays an additional role, essential in blue light signal transduction.

The cDNA of the gene pds from tomato, encoding the carotenoid biosynthesis enzyme phytoene desaturase, was cloned, and its nucleotide sequence was determined.Cells of Eschenchia coli that expressed the tomato pds gene could convert phytoene to C-carotene. This result sug s that one polypeptide, the product of the pds gene, can carry out phytoene desaturation in the carotenoid biosynthetic pathway.

In Neurospora crassa only two white collar (wc) mutants, wc-1 and wc-2, have been described that seem to be insensitive to light. The pleiotropic phenotypes of these mutants suggest that they represent two central components of blue light signal transduction. The WC proteins have several characteristics of transcription factors consistent with an involvement in transcriptional control of light-regulated genes. Here, we present a biochemical analysis of WC1 and WC2 polypeptides in N. crassa. Using specific antisera against WC1 and WC2, respectively, the subcellular localization of the WC polypeptides was investigated. The WC1 protein was localized exclusively in the nucleus, whereas WC2 was detected in both the nuclear and cytoplasmic fractions. The nuclear localization of WC1 and WC2 was shown to be independent of light and dimerization between the two proteins. In addition, WC1 and WC2 are phosphorylated in response to light. The phosphorylation of WC1 and WC2 was dependent on functional WC1 and WC2 proteins, respectively, which clearly indicated a correlation between the light-dependent phosphorylation and the function of WC1 and WC2 in blue light signaling. However, the light-specific phosphorylation of the WC proteins revealed different kinetics. The phosphorylation of WC1 was transient whereas the WC2 phosphorylation was shown to be stable under constant light conditions. The analysis of the light-dependent phosphorylation of WC1 and WC2 in wc-2 and wc-1 mutants revealed an epistatic relationship for WC1 and WC2 with WC2 acting downstream of WC1 in the signal transduction pathway of blue light.

Astaxanthin is a high-value carotenoid used as a pigmentation source in fish aquaculture. In addition, a beneficial role of astaxanthin as a food supplement for humans is becoming evident. The unicellular green alga Haematococcus pluvialis seems to be a suitable source for natural astaxanthin. Astaxanthin accumulation in H. pluvialis occurs in response to environmental stress such as high light and salt stress. Here, the isolation of the H. pluvialis carotenoid biosynthesis gene phytoene synthase is reported. Furthermore, the expression of phytoene synthase and carotenoid hydroxylase, two key enzymes in astaxanthin biosynthesis, was investigated at the transcriptional level. The application of environmental stress resulted in increased steady-state mRNA levels of both genes. High-light intensity led to a transient increase in carotenoid hydroxylase mRNA followed by moderate astaxanthin accumulation. In contrast, salt stress in combination with high light resulted in a sustained increase in both transcripts. The addition of compounds inducing reactive oxygen species did not influence transcript levels of phytoene synthase and carotenoid hydroxylase. The application of an inhibitor of photosynthesis, 3-(3, 4-dichlorophenyl)-1,1-dimethylurea, indicated that the light-induced expression of these carotenoid biosynthesis genes may be under photosynthetic control.

The ascomycete Neurospora crassa has the capacity of adapting to a given light quantity, leading to transient blue light responses under continuous light conditions. Here, we present an investigation of this photoadaptation phenomenon. We demonstrated previously that two proteins of the Neurospora blue light signal transduction chain, WC1 and WC2, are subject to light‐dependent phosphorylation. WC1 was phosphorylated in parallel with the transient increase in transcript levels of light‐regulated genes. Using the light‐dependent phosphorylation of WC1 as a marker for an active signalling state of WC1, we show that the transiency of Neurospora blue light responses results from desensitization of the photoreceptor and/or the signalling cascade. Furthermore, a Neurospora mutant was characterized that revealed a specific defect in photoadaptation. In this mutant, the transient expression of light‐regulated genes under continuous light, the temporary insensitivity after a light pulse and the capability of differentiating between and adapting to low and high light intensities were abolished. The corresponding protein seems to represent a central component of a negative feedback desensitization mechanism. This negative feedback regulation requires continuous and light‐dependent protein de novo biosynthesis.