Photoregulation of the Carotenoid Biosynthetic Pathway in Albino and White Collar Mutants of Neurospora crassa

The conversion of isopentenyl pyrophosphate to phytoene in Neurospora crassa requires both a soluble and a particulate fraction. Soluble and particulate enzyme fractions obtained from light-treated and dark-grown wild type, albino-1, albino-2, albino-3, and white collar-1 strains were mixed in various combinations, and the activity for conversion of [1-¹⁴C]isopentenyl pyrophosphate to phytoene was assayed. From such experiments it can be concluded that: (a) albino-3 is defective in the soluble fraction; (b) albino-2 is defective in the particulate fraction; (c) the in vivo light treatment increases the enzyme activity in the particulate fraction; (d) this light effect occurs in wild type, albino-1, and albino-3 strains; and (e) enzyme activity is present in the particulate fraction obtained from the white collar-1 mutant, but the in vivo light treatment does not cause an increase in this activity. To measure directly the level of particulate enzyme activity, [¹⁴C]geranylgeranyl pyrophosphate was used as a substrate. This compound, which is not available commercially, was synthesized enzymically using extracts of pea cotyledons. Particulate enzyme fractions obtained from wild type, albino-1, and albino-3 strains incorporate [¹⁴C]geranylgeranyl pyrophosphate into phytoene, and this activity is higher in extracts obtained from light-treated cultures. The particulate fraction obtained from the white collar-1 mutant also incorporates [¹⁴C]geranylgeranyl pyrophosphate into phytoene, but the in vivo light treatment does not cause an increase in this activity. No incorporation occurs when particulate fractions obtained from either dark-grown or light-treated albino-2 cultures are assayed. The soluble enzyme fraction obtained from the albino-3 mutant was shown to be almost totally defective in enzyme activity required for the biosynthesis of [¹⁴C]geranylgeranyl pyrophosphate from [1-¹⁴C]isopentenyl pyrophosphate. An in vivo light treatment increases the level of this activity in wild type, albino-1, albino-2, and albino-3 strains, but not in the white collar-1 mutant. A model is presented to account for all of the results obtained in this investigation. It is proposed that the white collar-1 strain is a regulatory mutant blocked in the light induction process, whereas the albino-1, albino-2, and albino-3 strains are each defective for a different enzyme in the carotenoid biosynthetic pathway.     

Genetic Analysis of Phototropism of Neurospora crassa Perithecial Beaks Using White Collar and Albino Mutants

Positive phototropism of perithecial beaks in the fungus Neurospora crassa has been demonstrated. The effect was shown to be mediated by blue light. When mutants (white collar-1 and white collar-2) which are blocked in the light induction of enzymes in the carotenoid biosynthetic pathway were used as the protoperithecial parent in crosses, the resulting perithecial beaks did not show a phototropic response. However, when wild type, albino-1, albino-2, or albino-3 strains were used as the protoperithecial parent, phototropism occurred.

The results show that both photoinduced carotenogenesis and phototropism in N. crassa are controlled by the white collar-1 and white collar-2 loci. Thus, the sensory transduction pathways for the two photoresponses must have some steps in common. The results further support the proposal that the white collar strains are regulatory mutants blocked in the light induction process, whereas the albino-1, albino-2, and albino-3 strains can carry out light induction but have the albino phenotype because they are each defective for a different enzyme in the carotenoid biosynthetic pathway.

     

Subcellular site of carotenoid biosynthesis in Neurospora crassa

A cell-free system prepared from Neurospora crassa mycelia was capable of incorporating radioactivity from [¹⁴C]-mevalonic acid into phytoene and to a much lesser extent into more unsaturated carotenoids. Whereas carotenogenic activities were only minimal in extracts from dark-grown cultures, they were several-fold increased following a short in vivo illumination; this photo-induced increase was inhibited by cycloheximide. Subcellular localization of carotenogenic enzyme activities was investigated using incubations of particular isolated fractions, together with [¹⁴C]-mevalonic acid and a geranylgeranyl-pyrophosphate-synthesizing system provided by the endosperm of maturing pumpkin seeds. Maximum carotenogenic activity (ca 80%) was localized in two membrane fractions previously shown to contain plasma membranes and, in particular, membranes of the endoplasmic reticulum. The lipid layer, containing the bulk of carotenoid pigments, possesses only trace amounts of enzyme activities.     

Mild RIP — an alternative method for in vivo mutagenesis of thealbino-3 gene inNeurospora crassa

We have used a biological phenomenon that occurs inNeurospora crassa, termed Repeat-Induced Point mutation (RIP), to create partially functional mutant alleles of thealbino-3 (al-3) gene encoding geranylgeranyl pyrophosphate synthase, an enzyme involved in the biosynthesis of carotenoids and diverse prenylated compounds. A total of 70 RIP-inducedal- 3 mutants were identified by their pale albino phenotype, resulting from inactivation of carotenoid biosynthesis. Nucleotide sequence analysis of theal-3 gene in five of the RIP-induced mutants revealed that in each case RIP had introduced no more than six point mutations. The low frequency of RIP mutants (0.42%) and the isolation of only leaky mutants with very few mutations suggest that ascospores containing a heavily mutatedal-3 gene do not survive. These results are evidence that the RIP phenomenon, used to inactivate and silence duplicated genes inN. crassa, may be exploited in its mild version as a method of sequence-specific in vivo mutagenesis to obtain functional mutant alleles ofNeurospora genes. This mild form of mutagenesis may be particularly advantageous in selecting for leaky mutations in essentialNeurospora genes.     

The partial purification and properties of a phytoene synthesizing enzyme system.

An enzyme system catalyzing the synthesis of phytoene from isopentenyl pyrophosphate has been isolated from tomato fruit plastids and purified approximately 350-fold in specific activity. This enzyme system has a molecular weight of approximately 200,000. The rate of phytoene formation is maximal at pH 7.0 and 23 °C and the apparent K_m for isopentenyl pyrophosphate is 10 μm The rates of phytoene synthesis when geranylgeranyl pyrophosphate and isopentenyl pyrophosphate were used as substrates were 0.08 and 0.17 nmol of phytoene/mg of protein/h, respectively. The enzyme complex showed an absolute requirement for Mn²⁺, but not for NADP⁺. At a concentration of 2 mm, NADP⁺ produced only a 1.5- to 3-fold stimulation, and this effect varied from preparation to preparation. The addition of NADPH to the incubation mixture produced inhibition of phytoene synthesis and there was no evidence for the concomitant accumulation of lycopersene. The acid labiles produced on acid treatment of the incubation mixture indicated that geranylgeranyl pyrophosphate was formed by the enzyme complex. The enzyme system is stabilized in the presence of 30% glycerol and 10 mm dithiothreitol and it can be stored at ?20 °C for over 1 month without significant loss of activity. However, the enzyme activity for phytoene formation is heat labile, and it is not stable when attempts are made to purify it further by ion-exchange chromatography.     

Accumulation of carotenoids in structural and regulatory mutants of the bacterium Myxococcus xanthus

Summary Accumulation of carotenoids in Myxococcus xanthus is absolutely dependent on illumination with blue light. We report the analysis of the carotenoids of dark- and light-grown cultures of the wild type and several previously characterized mutants. A carR mutant produces the same carotenoids in the dark as the wild type grown in the light. This agrees with previous evidence indicating that the carR gene codes for a general negative regulator of the system. A cis-dominant mutation in the gene carA causes constitutive expression of the light-inducible gene carB, which is linked to carA. In the dark, the carA mutant produces high levels of phytoene, the first C₄₀ colourless carotenoid precursor; in the light, it produces the same carotenoids as the wild type. Since a mutation in carB blocks accumulation of phytoene, we propose that carB, and probably other linked genes also controlled by carA, code for enzymes involved in the synthesis of phytoene. This is virtually the only carotene accumulated by strains mutated in the gene carC, which is unlinked to the others. Thus carC codes for phytoene dehydrogenase, the enzyme that converts phytoene into coloured carotenoids. The results presented here also provide evidence for control of carotenogenesis by an endproduct that is independent of the blue light effect.     

Involvement of protein kinase C in the response of Neurospora crassa to blue light

As a first step towards understanding the process of blue light perception, and the signal transduction mechanisms involved, in Neurospora crassa we have used a pharmacological approach to screen a wide range of second messengers and chemical compounds known to interfere with the activity of well-known signal transducing molecules in vivo. We tested the influence of these compounds on the induction of the al-3 gene, a key step in light-induced carotenoid biosynthesis. This approach has implicated protein kinase C (PKC) as a component of the light transduction machinery. The conclusion is based on the effects of specific inhibitors (calphostin C and chelerythrine chloride) and activators of PKC (1,2-dihexanoyl-sn-glycerol). During vegetative growth PKC may be responsible for desensitization to light because inhibitors of the enzyme cause an increase in the total amount of mRNA transcribed after illumination. PKC is therefore proposed here to be an important regulator of transduction of the blue light signal, and may act through modification of the protein White Collar-1, which we show to be a substrate for PKC in N. crassa.     

Altered Protein Formation as a Result of Suppression in Neurospora Crassa

The mutant td201 of Neurospora crassa is mutated in the trp-3 locus and forms an altered tryptophan synthetase. A suppressor mutation, su2-6, in this mutant, unlinked to the trp-3 locus, results in the production of wild-type tryptophan synthetase activity, which accounts for the alleviation of the tryptophan or indole requirement. This enzyme activity is associated with a protein physically dissimilar to the wild-type enzyme. A second altered protein, a serologically cross-reacting material is also formed in the suppressed mutant, in addition to the altered enzyme normally formed by the td201 mutant. Normal growth, equivalent to that of wild type, is not restored in the suppressed mutant even with tryptophan supplementation. The relationship of the data to possible mechanisms of suppression is discussed.     

Regulation and function of glutamate synthase in Neurospora crassa

In Neurospora crassa two enzymes can provide glutamate: the NADPH dependent GDH and the NADH dependent GOGAT. An elevated GOGAT activity was found in Neurospora wild-type under ammonium limitation in contrast to a 4-fold lower activity on excess of a$\text{m}$ monium. Glutamate and glutamine repress this enzyme. On excess of ammonium the GDH-NADPH deficient mutant am-1 grows poorly with an elevated GOGAT activity. A GOGAT less mutant was found. It presented a lag-phase to grow on ammonium. It is concluded that N. crassa glutamate synthase provides glutamate from low am-monium concentrations. The enzyme was purified to homogeneity and shown to be composed of a single type of monomer with a molecular weight above 200,000.     

Modification of Blue Light Photoresponses by Riboflavin Analogs in Neurospora crassa

The effect of riboflavin analogs on blue light responses in a riboflavin mutant of Neurospora crassa was studied. The analogs 1-deazariboflavin and roseoflavin, which have red-shifted absorption, acted as photoreceptors for the photosuppression and phase shifting of circadian conidiation by 540 nm light, but were ineffective as photoreceptors for the induction of carotenoid synthesis. These results provide addtional evidence implicating a flavin photoreceptor for at least two blue light responses of Neurospora.     

Evidence for Transceptor Function of Cellodextrin Transporters in Neurospora crassa

Neurospora crassa colonizes burnt grasslands and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source to cellulose, N. crassa dramatically up-regulates expression and secretion of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Previously, we have shown that a N. crassa mutant carrying deletions of three β-glucosidase enzymes (Δ3βG) lacks β-glucosidase activity, but efficiently induces cellulase gene expression and cellulolytic activity in the presence of cellobiose as the sole carbon source. These observations indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression and activity in N. crassa. Here, we show that in N. crassa, two cellodextrin transporters, CDT-1 and CDT-2, contribute to cellulose sensing. A N. crassa mutant carrying deletions for both transporters is unable to induce cellulase gene expression in response to crystalline cellulose. Furthermore, a mutant lacking genes encoding both the β-glucosidase enzymes and cellodextrin transporters (Δ3βGΔ2T) does not induce cellulase gene expression in response to cellobiose. Point mutations that severely reduce cellobiose transport by either CDT-1 or CDT-2 when expressed individually do not greatly impact cellobiose induction of cellulase gene expression. These data suggest that the N. crassa cellodextrin transporters act as “transceptors” with dual functions - cellodextrin transport and receptor signaling that results in downstream activation of cellulolytic gene expression. Similar mechanisms of transceptor activity likely occur in related ascomycetes used for industrial cellulase production.     

In vitro carotenogenesis by wild type and mutants of Phycomyces blakesleeanus

Cell extracts from shake cultures of the wild type and six mutant strains of Phycomyces converted [2-¹⁴C] MVA into carotenes, squalene and prenyl phosphates. Oxygen was required for the desaturation of phytoene. When compared with the wild type, cells extracts of carB and carR mutants are much less effective in phytoene dehydrogenation and lycopene cyclization, respectively. This confirms previous conclusions about the biochemical functions of the carB and carR genes, which were based on genetic and in vivo studies. CarA strain mutants accumulate, in vivo, much less β-carotene than the wild type. This correlates with a 10-fold decrease in carotenogenesis in vitro. The addition of retinol to incubations of cell extracts of the wild type and C2 strains stimulated β-carotene formation. Both carB and carR mutants show enhanced total carotenogenic activities in vitro and the carS mutant shows a higher β-carotene-synthesizing activity than the wild type. It is suggested that the feed-back regulatory mechanism known to control this pathway operates at the level of enzyme synthesis.     

Blue-light requirement for the biosynthesis of an NO2− transport system in the Chlamydomonas reinhardtii nitrate transport mutant S10*

The blue-light requirement for the biosynthesis of nitrite reductase and an NO₂^– transport system was studied in Chlamydomonas reinhardtii mutant S10. The only oxidized nitrogen species that could be taken up by this mutant was NO₂^–, due to the presence of NO₂^– transport systems and the absence of high-affinity NO₃^– transporters. NH₄⁺-grown cells required illumination with blue light to recover the ability to take up NO₂^– when resuspended in an NO₂^–-containing NH₄⁺-deprived medium. This blue-light- dependent recovery, which took 1 h, could be suppressed by cycloheximide, indicating that protein biosynthesis was involved. The biosynthesis of nitrite reductase took place in cell suspensions irradiated with red light, even in the absence of NO₂^–, thus suggesting that the process requiring blue light was the biosynthesis of an NO₂^– transport system. Nitrite reductase-containing cells (pre-irradiated with red light) took 1 h to start consuming NO₂^– when they were additionally irradiated with blue light in the presence of this anion, and this process was also cycloheximide-sensitive. The NO₂^– transport system operated either under red plus blue light or red light only. Thus, in C. reinhardtii mutant S10 cells, blue light was only required for the biosynthesis of an NO₂^– transport system and not for its activity.     

delta-Aminolevulinic Acid Biosynthesis from Glutamatein Euglena gracilis: Photocontrol of Enzyme Levels in a Chlorophyll-Free Mutant

Wild-type Euglena gracillis cells synthesize the key chlorophyll precursor, δ-aminolevulinic acid (ALA), from glutamate in their plastids. The synthesis requires transfer RNA^Glu (tRNA^Glu) and the three enzymes, glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde aminotransferase. Non-greening mutant Euglena strain W₁₄ZNaIL does not synthesize ALA from glutamate and is devoid of the required tRNA^Glu. Other cellular tRNA^Glus present in the mutant cells were capable of being charged with glutamate, but the resulting glutamyl-tRNAs did not support ALA synthesis. Surprisingly, the mutant cells contain all three of the enzymes, and their cell extracts can convert glutamate to ALA when supplemented with tRNA^Glu obtained from wild-type cells. Activity levels of the three enzymes were measured in extracts of cells grown under a number of light conditions. All three activities were diminished in extracts of cells grown in complete darkness, and full induction of activity required 72 hours of growth in the light. A light intensity of 4 microeinsteins per square meter per second was sufficient for full induction. Blue light was as effective as white light, but red light was ineffective, in inducing extractable enzyme activity above that of cells grown in complete darkness, indicating that the light control operates via the nonchloroplast blue light receptor in the mutant cells. Of the three enzyme activities, the one that is most acutely affected by light is glutamate-1-semialdehyde aminotransferase, as has been previously shown for wild-type Euglena cells. These results indicate that the enzymes required for ALA synthesis from glutamate are present in an active form in the nongreening mutant cells, even though they cannot participate in ALA formation in these cells because of the absence of the required tRNA^Glu, and that the activity of all three enzymes is regulated by light. Because the absence of plastid tRNA^Glu precludes the synthesis of proteins within the plastids, the three enzymes must be synthesized in the cytoplasm and their genes encoded in the nucleus in Euglena.