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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 first 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 a flavin chromophore. Upon illumination with blue light, VIVID undergoes a photocycle indicative of the formation of a flavin-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 flavin-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.

Carotenoids are ubiquitous and essential components of photosynthetic tissues in plants, algae and cyanobacteria. They participate in the light harvesting process and prevent photooxidative damage of the photosynthetic apparatus. Although de-etiolation and growth under different light conditions were reported to have pronounced effects on carotenoid contents in higher plants and algae, very little is known about the light regulation of carotenogenesis on a molecular level. In the present study, we chose the unicellular green alga Chlamydomonas reinhardtii to investigate the regulation of carotenoid biosynthesis genes in response to light. The carotenoid genes phytoene synthase and phytoene desaturase were selected for gene expression studies. Both phytoene synthase and phytoene desaturase revealed a fast up-regulation in response to light, which seemed to be due to transcriptional control. Only blue light was effective whereas illumination with red light did not lead to elevated transcript levels of phytoene synthase and phytoene desaturase. The inhibition of photosynthesis did not abolish the light induction of carotenoid genes. Comparison with published results showed that the carotenoid genes are simultaneously expressed with other genes involved in chlorophyll biosynthesis and light harvesting. This simultaneous expression may represent one mechanism for the coordinated biosynthesis of carotenoids, chlorophylls and the proteins of the photosynthetic apparatus.

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.

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 fungus Neurospora crassa has been shown to be a paradigm for photobiological, biochemical, and genetic studies of blue light perception and signal transduction. Several different developmental and morphological processes of Neurospora are regulated by blue light and can be divided into early and late blue light responses. The characterization of two central regulator proteins of blue light signal transduction in Neurospora crassa, WC1 and WC2, and the isolation of light-regulated genes, indicate transcriptional control as a central step in blue light signalling.

In situ location of phytoene desaturase, a key enzyme in the carotenoid biosynthesis pathway, has been investigated in chloroplasts from higher plants. For this purpose, an antiserum has been raised against the phytoene desaturase from the cyanobacterium Synechococcus PCC 7942 overexpressed in E. coli. The specifity of this antiserum was demonstrated by inhibition of the enzymatic desaturation reaction in vitro. The antiserum was further purified and immunoabsorbed with E. coli proteins. The resulting IgG-fraction was tested by western blotting against membrane proteins from chloroplasts of tobacco (Nicotiana tabacum L. cv. Samsun) and spinach (Spinacia oleracea L. cv. Atlanta). Apparent molecular masses of immunoreactive proteins were 62 and 64 kDa. A western blot of different membrane fractions of spinach chloroplasts (inner and outer envelopes, and thylakoids) indicated a localization of the phytoene desaturase in thylakoids. A post embedding immunogold microscopy procedure was employed. In these experiments the main labelling (79%) was associated with thylakoid membranes of tobacco chloroplasts. Of the counted colloidal gold particles, 16% were found in the stroma. Only 5% were detected in the envelope membranes. These results give clear evidence that at least the majority of phytoene desaturase molecules is localized within thylakoid membranes of higher plant chloroplasts and that the presence of the enzyme in the envelope is of minor significance.

The Synechococcus phytoene desaturase has been isolated from an overexpressing strain of Escherichia coli. The plasma pPDSde135 mediated the overexpression of the full-length polypeptide directly. The recombinant protein comprised 5% of the total cellular protein and was found predominantly in the inclusion body fraction. Urea was used to solubilize the recombinant protein from the inclusion fraction and the protein was subsequently purified to homogeneity on a DEAE-cellulose column. The purification scheme yielded 4.0 mg of homogeneous desaturase protein after a 20-fold purification, recovering 40% of the original protein from a 100 ml suspension culture of E. coli. The recombinant desaturase had an apparent molecular mass of 53 kDa on SDS/PAGE and crossreacted with an antiserum raised against the expressed protein. Desaturase activity was restored upon the removal of urea. The enzyme catalysed the conversion of phytoene to zeta-carotene via phytofluene. These products of the desaturase reaction existed predominantly in a cis configuration. Lipid replenishment enhanced activity. NAD+ and NADP+ were observed to be involved, whilst FAD was an ineffective electron acceptor.

A screening procedure for carotenoid genes involving heterologous complementation with two different plasmid constructs was developed. The plasmids contained the crtE and crtB genes from Erwinia unredovora together with the phytoene desaturase gene from either Rhodobacter capsulatus or Synechococcus PCC 7942. Transformation in E. coli led to the accumulation of neurosporene and ζ-carotene, respectively. Co-transformation with an Anabaena plasmid library resulted in the isolation of the two plasmids, pZDS1 and pZDS1. Their gene products showed the ability to convert neurosporene and ζ-carotene into lycopene. In contrast, accumulated phytoene could not be converted. We conclude that the cloned gene codes for the carotenoid biosynthesis gene ζ-carotene desaturase (zds).

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 Escherichia coli that expressed the tomato pds gene could convert phytoene to zeta-carotene. This result suggests that one polypeptide, the product of the pds gene, can carry out phytoene desaturation in the carotenoid biosynthetic pathway. Transcripts of the pds gene accumulate in orange tomato fruit, indicating transcriptional control of pds expression during fruit ripening. The deduced amino acid sequence of phytoene desaturase indicates that this enzyme in tomato contains 583 amino acids that are highly conserved with respect to the homologous enzymes in cyanobacteria and algae. The deduced amino acid sequences of the phytoene desaturases from other microorganisms (purple bacteria and fungi) appear to be evolutionarily unrelated to those from green photosynthetic organisms.

Many bleaching herbicides with different core structures inhibit phytoene desaturase (PD), a membranebound enzyme in the carotenogenic pathway catalyzing the hydrogen abstraction step at the first C40 precursor of n-carotene. Prospects are good that new PD-active herbicides will be discovered by screening for bleaching activity. Accordingly, interest in PD enzymology and molecular genetics has increased. Although active carotenogenic cell-free systems are available, no isolation of PD has been achieved since the enzyme cannot be detected in its isolated form due to complete loss of activity. A portion of the Rhodobacter PD gene was incorporated into an appropriate plasmid which could be expressed in E. coli. This system was used to produce an antibody specific against PD from higher plants as well as Rhodobacter. All PDs assayed had an apparent molecular weight of 52 to 55 kDa. A Rhodobacter gene probe hybridized with a 3.1 kbBamH I fragment from Aphanocapsa which allowed us to sequence the PD gene from this cyanobacterium. Its DNA sequence matched with the apparent molecular weight of the PD band in the western blot, and a fusion-gene product was found to be immunoreactive with the Rhodobacter PD antibody, Anacystis mutants were produced exhibiting cross-resistance against norflurazon and fluorochloridone. Apparently, this resistance is due to an altered PD with concurrent decrease of inhibitor binding affinity. Cloning of the resistant gene into the wild type is in progress. Additional index words. Herbicide resistance, cell-free carotenogenic assays, phytoene desaturase. THE CAROTENOGENIC PATHWAY Chlorophylls and carotenoids are formed by plant-specific pathways, and interruption of biosynthesis by inhibitors causes bleaching of cells and plant foliage (4, 25). Carotenogenesis is affected by many agrochemicals. Carotenoids, namely carotenes and xanthophylls, play an essential role in photosynthesis by protecting chlorophyll against photooxidative destruction by singlet oxygen. This highly reactive oxygen species is formed in excess through excited chlorophyll when the photosynthetic machinery (electron transport and subsequent CO2 assimilation) operates too slowly to accommodate strong light. Protection against singlet 02 is achieved when nine or more double bonds are 'Received for publication February 22, 1990, and in revised form January 15, 1991. 2Assoc. Prof., former Grad. Student, Grad. Student, and Prof. of Plant Physiol. and Biochem., Univ. Konstanz, Konstanz, Germany. Present address of senior author Lehrstuhl fUr Physiologie und Biochemie der Pflanzen, Universitit Konstanz, D-7750 Konstanz, Germany. present in the carotenoid molecule (13) as is the case with lycopene, 5-carotene, or with the xanthophylls. These molecules physically interact with triplet state chlorophyll and dissipate its energy as heat. If a herbicide prevents carotene and xanthophyll formation, chlorophyll although formed will not accumulate either. Consequently leaves emerging after herbicide treatment will be depleted of all colored plastidic pigments. The mode of action of bleaching herbicides is inhibition of formation of carotenes. Thylakoids having an intact pigment inventory before herbicide treatment are not affected by application of the bleaching herbicide. Typically, bleaching occurs only in newly formed leaves as is well known to herbicide "screeners" in the greenhouse. This phenomenon is in contrast to peroxidative, degradative herbicides like p

Mutants from the cyanobacterium Synechococcus PCC 7942 have been generated by treatment with ethyl methanesulfonate and selected against the bleaching herbicide norflurazon. Several strains with different degrees of norflurazon resistance have been obtained and assayed. Biochemical characterization of norflurazon interaction with phytoene desaturation showed similar inhibition properties of carotene biosynthesis in vivo and in vitro using four different mutants. This result indicates that in the mutants assayed the affinities for binding of norflurazon to phytoene desaturase are decreased. Concurrently with norflurazon resistance, decreased rates of the phytoenedesaturase reaction and lower carotenoid concentrations in the mutants are found. Strain NFZ 49 also exhibited resistance against fluorochloridone. The other three mutants assayed did not show cross-resistance against various phytoene-desaturase inhibitors.

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.

A cDNA homologous to beta-carotene hydroxylase from Arabidopsis thaliana was isolated from the green alga Haematococcus pluvialis. The predicted amino acid sequence for this enzyme shows homology to the three known plant beta-carotene hydroxylases from Arabidopsis thaliana and from Capsicum annuum (38% identity) and to prokaryote carotenoid hydroxylases (32-34% identities). Heterologous complementation using E. coli strains which were genetically engineered to produce carotenoids indicated that the H. pluvialis beta-carotene hydroxylase was able to catalyse not only the conversion of beta-carotene to zeaxanthin but also the conversion of canthaxanthin to astaxanthin. Furthermore, Northern blot analysis revealed increased beta-carotene hydroxylase mRNA steady state levels after induction of astaxanthin biosynthesis. In accordance with the latter results, it is proposed that the carotenoid hydroxylase characterized in the present publication is involved in the biosynthesis of astaxanthin during cyst cell formation of H. pluvialis.

A novel enzyme, zeta-carotene desaturase from the cyanobacterium Anabaena, which catalyzes the last two steps in a series of desaturations, was overexpressed in Escherichia coli. For the first time, this allowed the purification of this enzyme and subsequent enzyme kinetic studies. The enzyme was solubilized from the E. coli membranes by Chaps and purified to homogeneity by ammonium sulfate precipitation, ion-exchange and hydrophobic interaction chromatography. The correct translational start was confirmed by N-terminal protein sequencing. Substrates for zeta-carotene desaturase apart from zeta-carotene are those carotenes which partially resemble the latter, like neurosporene and beta-zeacarotene yielding lycopene and gamma-carotene, respectively as reaction products. Also cis isomers like pro-zeta-carotene were converted to the corresponding products. Km values of 10 microM were determined for both substrates zeta-carotene and neurosporene. The enzyme was inhibited to some extent by the experimental herbicides J852 and LS80707 and also by diphenylamine which is a well-known inhibitor of the bacterial-type phytoene desaturase.

A DNA fragment which has been isolated previously from an Anabaena DNA expression library was subcloned. The corresponding protein was overexpressed in Escherichia coli. The recombinant enzyme was fully active in converting zeta-carotene into lycopene in vitro with neurosporene as an intermediate. A smaller fragment which still contained the active enzyme was sequenced. An open reading frame of 1497 bp was found coding for a protein consisting of 499 amino acids with the calculated molecular weight of 56,740. In a computer search of nucleotide sequences contained in the EMBL nucleotide sequence library, all the best-fitting comparisons were carotenoid desaturases. The highest similarity was found with the crtI phytoene desaturase genes of bacteria and the al-1 gene from Neurospora crassa. A much lower similarity was found with the pds genes coding for phytoene desaturase from cyanobacteria and higher plants. It is shown in protein similarity plots that the amino acid similarity of zeta-carotene desaturase to the latter is mainly limited to the N terminus of the polypeptides. In contrast, the protein similarity plots and a comparison of a conserved region clearly demonstrate that there is a strong relationship between zeta-carotene desaturase and the phytoene desaturases from various bacteria and fungi. Therefore we propose that the zeta-carotene desaturase gene is homologous to the crt I phytoene desaturase genes of bacteria and fungi.

Three different phytoene desaturase genes, from Rhodobacter capsulatus, Erwinia uredovora, and Synechococcus PCC 7942, have been functionally complemented with a gene construct from E. uredovora which encodes all enzymes responsible for formation of 15-cis phytoene in Escherichia coli. As indicated by the contrasting reaction products detected in the pigmented E. coli cells after co-transformation, a wide functional diversity of these three different types of phytoene desaturases can be concluded. The carotenes formed by the phytoene desaturase from R. capsulatus were trans-neurosporene with three additional double bonds and two cis isomers. Furthermore, small amounts of three ζ-carotene isomers (2 double bonds more than phytoene) and phytofluene (15-cis and all-trans with + 1 double bond) were detected as inter- mediates. When the subsequent genes from E. uredovora which encode for lycopene cyclase and β-carotene hydroxylase were present, neurosporene, the phytoene desaturase product of R. capsulatus, was subsequently converted to the monocyclic β-zeacarotene and its mono- hydroxylation product. The most abundant carotene resulting from phytoene desaturation by the E. uredovora enzyme was trans-lycopene together with a cis isomer. In addition, bisdehy-drolycopene was also formed. The reaction products of Synechococcus phytoene desaturase were two cis isomers of ζ-carotene and only small amounts of trans-ζ-carotene including 15-cis. The I50 values for flurtamone and diphenylamine to inhibit phytoene desaturation were determined and differential inhibition was observed for diphenylamine.

A plasmid has been constructed by cloning the complete crtI gene encoding phytoene desaturase from Erwinia uredovora behind the lac Z promoter of pUC18 resulting in a reading frame for the full polypeptide with additional 9 amino acids at the N terminus. This plasmid mediated the overexpression of phytoene desaturase in transformed Escherichia coli. The overexpressed enzyme was sequestrated into inclusion bodies requiring urea treatment for solubilization. Purification to homogeneity was subsequently performed on a DEAE-cellulose column and by SDS-polyacrylamide gel electrophoresis. The purification scheme allowed the isolation of 5.3 mg of homogeneous desaturase protein from 100 ml of E. coli cell suspension. On SDS-polyacrylamide gel electrophoresis an apparent molecular mass of 56.2 kDa was determined. An antiserum raised against phytoene desaturase cross-reacted with the expressed protein and was employed to monitor the isolation steps. Upon removal of urea, desaturase activity was restored. The isolated desaturase catalyzed the conversion of 15-cis-phytoene to trans-lycopene as well as to bisdehydrolycopene. FAD was involved in desaturation, whereas NAD and NADP were inhibitory. This is the first time that a membrane-integrated carotenogenic enzyme has been purified and finally obtained in an active state.