Three Genes Involved in Different Signaling Pathways, carS, wcoA, and acyA, Participate in the Regulation of Fusarin Biosynthesis in Fusarium fujikuroi.

The phytopathogenic fungus Fusarium fujikuroi has a rich secondary metabolism which includes the synthesis of very different metabolites in response to diverse environmental cues, such as light or nitrogen. Here, we focused our attention on fusarins, a class of mycotoxins whose synthesis is downregulated by nitrogen starvation. Previous data showed that mutants of genes involved in carotenoid regulation (carS, encoding a RING finger protein repressor), light detection (wcoA, White Collar photoreceptor), and cAMP signaling (AcyA, adenylate cyclase) affect the synthesis of different metabolites. We studied the effect of these mutations on fusarin production and the expression of the fus1 gene, which encodes the key polyketide synthase of the pathway. We found that the three proteins are positive regulators of fusarin synthesis, especially WcoA and AcyA, linking light regulation to cAMP signaling. Genes for two other photoreceptors, the cryptochrome CryD and the Vivid flavoprotein VvdA, were not involved in fusarin regulation. In most cases, there was a correspondence between fusarin production and fus1 mRNA, indicating that regulation is mainly exerted at the transcriptional level. We conclude that fusarin synthesis is subject to a complex control involving regulators from different signaling pathways.

Effects of 4 Different Velocity-Based Resistance-Training Programming Models on Physical Performance.

PURPOSE: To examine the effects of 4 programming models (linear [LP], undulating [UP], reverse [RP], and constant [CP]) on physical performance. METHODS: Forty-eight moderately strength-trained men were randomly assigned to LP, UP, RP, and CP groups according to their 1-repetition maximum (1RM) in the full-squat exercise (SQ) and followed an 8-week training intervention using the SQ and monitoring movement velocity for every repetition. All groups trained with similar mean relative intensity (65% 1RM), number of repetitions (240), sets (3), and interset recovery (4 min) throughout the training program. Pretraining and posttraining measurements included, in the SQ, 1RM load, the average velocity attained for all absolute loads common to pretests and posttests (AV), and the average velocity for loads that were moved faster (AV > 1) and slower (AV < 1) than 1 m·s-1 at pretraining tests. Moreover, countermovement jump height and 20-m running sprint time were measured. RESULTS: A significant time effect was found for all variables analyzed (P < .05), except for 20-m running sprint time. Significant group × time interactions were observed for 1RM, AV > 1, and AV (P < .05). After training, all groups attained significant strength gains on 1RM, AV, AV > 1, and AV < 1 (P < .001-.01). LP and RP groups improved their countermovement jump height (P < .01), but no significant changes were observed for UP and CP. No significant improvements were achieved in 20-m running sprint time for any groups. CONCLUSIONS: These different programming models are all suitable for improving physical performance. LP and RP induce similar or greater gains in physical performance than UP and CP.

Bioavailability and provitamin A activity of neurosporaxanthin in mice.

Various species of ascomycete fungi synthesize the carboxylic carotenoid neurosporaxanthin. The unique chemical structure of this xanthophyll reveals that: (1) Its carboxylic end and shorter length increase the polarity of neurosporaxanthin in comparison to other carotenoids, and (2) it contains an unsubstituted ß-ionone ring, conferring the potential to form vitamin A. Previously, neurosporaxanthin production was optimized in Fusarium fujikuroi, which allowed us to characterize its antioxidant properties in in vitro assays. In this study, we assessed the bioavailability of neurosporaxanthin compared to other provitamin A carotenoids in mice and examined whether it can be cleaved by the two carotenoid-cleaving enzymes: ß-carotene-oxygenase 1 (BCO1) and 2 (BCO2). Using Bco1-/-Bco2-/- mice, we report that neurosporaxanthin displays greater bioavailability than ß-carotene and ß-cryptoxanthin, as evidenced by higher accumulation and decreased fecal elimination. Enzymatic assays with purified BCO1 and BCO2, together with feeding studies in wild-type, Bco1-/-, Bco2-/-, and Bco1-/-Bco2-/- mice, revealed that neurosporaxanthin is a substrate for either carotenoid-cleaving enzyme. Wild-type mice fed neurosporaxanthin displayed comparable amounts of vitamin A to those fed ß-carotene. Together, our study unveils neurosporaxanthin as a highly bioavailable fungal carotenoid with provitamin A activity, highlighting its potential as a novel food additive.

HmbC, a Protein of the HMG Family, Participates in the Regulation of Carotenoid Biosynthesis in Fusarium fujikuroi.

In the fungus Fusarium fujikuroi, carotenoid production is up-regulated by light and down-regulated by the CarS RING finger protein, which modulates the mRNA levels of carotenoid pathway genes (car genes). To identify new potential regulators of car genes, we used a biotin-mediated pull-down procedure to detect proteins capable of binding to their promoters. We focused our attention on one of the proteins found in the screening, belonging to the High-Mobility Group (HMG) family that was named HmbC. The deletion of the hmbC gene resulted in increased carotenoid production due to higher mRNA levels of car biosynthetic genes. In addition, the deletion resulted in reduced carS mRNA levels, which could also explain the partial deregulation of the carotenoid pathway. The mutants exhibited other phenotypic traits, such as alterations in development under certain stress conditions, or reduced sensitivity to cell wall degrading enzymes, revealed by less efficient protoplast formation, indicating that HmbC is also involved in other cellular processes. In conclusion, we identified a protein of the HMG family that participates in the regulation of carotenoid biosynthesis. This is probably achieved through an epigenetic mechanism related to chromatin structure, as is frequent in this class of proteins.

Water treatment membranes embedded with a stable and bactericidal nanodiamond material.

Filtration has emerged as a critical technology to reduce waterborne diseases caused by poor water quality. Filtration technology presents key challenges, such as membrane selectivity, permeability and biofouling. Nanomaterials can offer solutions to these challenges by varying the membranes' mechanical and bactericidal properties. This research uses nanodiamond particles with facile surface functionality and biocompatibility properties that are added to membranes used for filtration treatments. Scanning and transmission electron microscopy (SEM and TEM) and Fourier transform infrared spectroscopy (FTIR) were performed to study the membrane surface. FTIR spectra confirms an increase in oxygen functional groups onto the ultradispersed diamond's (UDD) surface following acid treatment. SEM images show particle deagglomeration of functionalized UDD at the membrane surface. Tensile strength tests were done to measure the UDD mechanical properties and Coliscan membrane filtration characterization was performed to determine the filter effectiveness. Polyether sulfone (PES) and polyvinylidene (PVDF) membranes expressed a change in their yield point when UDD was incorporated into the porous matrix. A significant microorganism reduction was obtained and confirmed using t-test analysis at a 95% level of confidence. UDD-embedded membranes exhibit a significant bactericidal reduction compared to commercial membranes suggesting these membranes have the potential to enhance current membrane filtration systems.

Fusarium Photoreceptors.

Light is an important modulating signal in fungi. Fusarium species stand out as research models for their phytopathogenic activity and their complex secondary metabolism. This includes the synthesis of carotenoids, whose induction by light is their best known photoregulated process. In these fungi, light also affects other metabolic pathways and developmental stages, such as the formation of conidia. Photoreceptor proteins are essential elements in signal transduction from light. Fusarium genomes contain genes for at least ten photoreceptors: four flavoproteins, one photolyase, two cryptochromes, two rhodopsins, and one phytochrome. Mutations in five of these genes provide information about their functions in light regulation, in which the flavoprotein WcoA, belonging to the White Collar (WC) family, plays a predominant role. Global transcriptomic techniques have opened new perspectives for the study of photoreceptor functions and have recently been used in Fusarium fujikuroi on a WC protein and a cryptochrome from the DASH family. The data showed that the WC protein participates in the transcriptional control of most of the photoregulated genes, as well as of many genes not regulated by light, while the DASH cryptochrome potentially plays a supporting role in the photoinduction of many genes.

Relation between CarS expression and activation of carotenogenesis by stress in Fusarium fujikuroi.

Fusarium fujikuroi, a model organism for secondary metabolism in fungi, produces carotenoids, terpenoid pigments with antioxidant activity. Previous results indicate that carotenoid synthesis in F. fujikuroi is stimulated by light or by different stress conditions and downregulated by a RING finger protein encoded by carS gene. Here, we have analyzed the effects of three stressors, nitrogen scarcity, heat shock, and oxidative stress. We compared them with the effect of light in the wild type, a carS mutant that overproduces carotenoids, and its complemented strain. The assayed stressors increase the synthesis of carotenoids in the three strains, but mRNA levels of structural genes of carotenogenesis, carRA and carB, are only enhanced in the presence of a functional carS gene. In the wild-type strain, the four conditions affect in different manners the mRNA levels of carS: greater in the presence of light, without significant changes in nitrogen starvation, and with patent decreases after heat shock or oxidative stress, suggesting different activation mechanisms. The spores of the carS mutant are more resistant to H2O2 than those of the wild type; however, the mutant shows a greater H2O2 sensitivity at the growth level, which may be due to the participation of CarS in the regulation of genes with catalase domains, formerly described. A possible mechanism of regulation by heat stress has been found in the alternative splicing of the intron of the carS gene, located close to its 3' end, giving rise to the formation of a shorter protein. This action could explain the inducing effect of the heat shock, but not of the other inducing conditions, which may involve other mechanisms of action on the CarS regulator, either transcriptionally or post-transcriptionally.

The carP lncRNA Is a carS-Related Regulatory Element with Broad Effects on the Fusarium fujikuroi Transcriptome.

Carotenoid biosynthesis in the fungus Fusarium fujikuroi is regulated by environmental factors, with light being the main stimulating signal. The CarS RING-finger protein plays an important role in the downregulation of structural genes of the carotenoid pathway. A recent transcriptomic analysis on the effect of carS mutation identified a gene for a long non-coding RNA (lncRNA) upstream of carS, called carP, the deletion of which results in increased carS mRNA levels and lack of carotenoid production. We have investigated the function of carP by studying the transcriptomic effect of its deletion and the phenotypes resulting from the reintroduction of carP to a deletion strain. The RNA-seq data showed that the loss of carP affected the mRNA levels of hundreds of genes, especially after illumination. Many of these changes appeared to be cascade effects as a result of changes in carS expression, as suggested by the comparison with differentially expressed genes in a carS mutant. Carotenoid production only recovered when carP was integrated upstream of carS, but not at other genomic locations, indicating a cis-acting mechanism on carS. However, some genes hardly affected by CarS were strongly upregulated in the carP mutant, indicating that carP may have other regulatory functions as an independent regulatory element.

Neurosporaxanthin Overproduction by Fusarium fujikuroi and Evaluation of Its Antioxidant Properties.

Neurosporaxanthin (NX) is a carboxylic carotenoid produced by some filamentous fungi, including species of the genera Neurospora and Fusarium. NX biosynthetic genes and their regulation have been thoroughly investigated in Fusarium fujikuroi, an industrial fungus used for gibberellin production. In this species, carotenoid-overproducing mutants, affected in the regulatory gene carS, exhibit an upregulated expression of the NX pathway. Based on former data on a stimulatory effect of nitrogen starvation on carotenoid biosynthesis, we developed culture conditions with carS mutants allowing the production of deep-pigmented mycelia. With this method, we obtained samples with ca. 8 mg NX/g dry mass, in turn the highest concentration for this carotenoid described so far. NX-rich extracts obtained from these samples were used in parallel with carS-complemented NX-poor extracts obtained under the same conditions, to check the antioxidant properties of this carotenoid in in vitro assays. NX-rich extracts exhibited higher antioxidant capacity than NX-poor extracts, either when considering their quenching activity against [O2(1g)] in organic solvent (singlet oxygen absorption capacity (SOAC) assays) or their scavenging activity against different free radicals in aqueous solution and in liposomes. These results make NX a promising carotenoid as a possible feed or food additive, and encourage further studies on its chemical properties.

Osmotolerance as a determinant of microbial ecology: A study of phylogenetically diverse fungi.

Osmotic stress induced by high solute concentration can prevent fungal metabolism and growth due to alterations in properties of the cytosol, changes in turgor, and the energy required to synthesize and retain compatible solutes. We used germination to quantify tolerance/sensitivity to the osmolyte KCl (0.1-4.5 M, in 0.1 M increments) for 71 strains (40 species) of ecologically diverse fungi. These include 11 saprotrophic species (17 strains, including two xerophilic species), five mycoparasitic species (five strains), six plant-pathogenic species (13 strains), and 19 entomopathogenic species (36 strains). A dendrogram obtained from cluster analyses, based on KCl inhibitory concentrations 50 % and 90 % calculated by Probit Analysis, revealed three groups of fungal isolates accordingly to their osmotolerance. The most-osmotolerant group (Group 3) contained the majority of saprotrophic fungi, and Aspergillus niger (F19) was the most tolerant. The highly xerophilic Aspergillus montevidense and Aspergillus pseudoglaucus were the second- and third-most tolerant species, respectively. All Aspergillus and Cladosporium species belonged to Group 3, followed by the entomopathogens Colletotrichum fioriniae, Simplicillium lanosoniveum, and Trichothecium roseum. Group 2 exhibited a moderate osmotolerance, and included plant-pathogens such as Colletotrichum and Fusarium, mycoparasites such as Clonostachys spp, some saprotrophs such as Mucor and Penicillium spp., and some entomopathogens such as Isaria, Lecanicillium, Mariannaea, Simplicillium, and Torrubiella. Group 1 contained the osmo-sensitive strains: the rest of the entomopathogens and the mycoparasitic Gliocladium and Trichoderma. Although stress tolerance did not correlate with their primary ecological niche, classification of these 71 fungal strains was more closely aligned with their ecology than with their phylogenetic relatedness. We discuss the implications for both microbial ecology and fungal taxonomy.

A novel lncRNA as a positive regulator of carotenoid biosynthesis in Fusarium.

The fungi Fusarium oxysporum and Fusarium fujikuroi produce carotenoids, lipophilic terpenoid pigments of biotechnological interest, with xanthophyll neurosporaxanthin as the main end product. Their carotenoid biosynthesis is activated by light and negatively regulated by the RING-finger protein CarS. Global transcriptomic analysis identified in both species a putative 1-kb lncRNA that we call carP, referred to as Fo-carP and Ff-carP in each species, upstream to the gene carS and transcribed from the same DNA strand. Fo-carP and Ff-carP are poorly transcribed, but their RNA levels increase in carS mutants. The deletion of Fo-carP or Ff-carP in the respective species results in albino phenotypes, with strong reductions in mRNA levels of structural genes for carotenoid biosynthesis and higher mRNA content of the carS gene, which could explain the low accumulation of carotenoids. Upon alignment, Fo-carP and Ff-carP show 75-80% identity, with short insertions or deletions resulting in a lack of coincident ORFs. Moreover, none of the ORFs found in their sequences have indications of possible coding functions. We conclude that Fo-carP and Ff-carP are regulatory lncRNAs necessary for the active expression of the carotenoid genes in Fusarium through an unknown molecular mechanism, probably related to the control of carS function or expression.

Controlled Transcription of Regulator Gene carS by Tet-on or by a Strong Promoter Confirms Its Role as a Repressor of Carotenoid Biosynthesis in Fusarium fujikuroi.

Carotenoid biosynthesis is a frequent trait in fungi. In the ascomycete Fusarium fujikuroi, the synthesis of the carboxylic xanthophyll neurosporaxanthin (NX) is stimulated by light. However, the mutants of the carS gene, encoding a protein of the RING finger family, accumulate large NX amounts regardless of illumination, indicating the role of CarS as a negative regulator. To confirm CarS function, we used the Tet-on system to control carS expression in this fungus. The system was first set up with a reporter mluc gene, which showed a positive correlation between the inducer doxycycline and luminescence. Once the system was improved, the carS gene was expressed using Tet-on in the wild strain and in a carS mutant. In both cases, increased carS transcription provoked a downregulation of the structural genes of the pathway and albino phenotypes even under light. Similarly, when the carS gene was constitutively overexpressed under the control of a gpdA promoter, total downregulation of the NX pathway was observed. The results confirmed the role of CarS as a repressor of carotenogenesis in F. fujikuroi and revealed that its expression must be regulated in the wild strain to allow appropriate NX biosynthesis in response to illumination.

Impact of the White Collar Photoreceptor WcoA on the Fusarium fujikuroi Transcriptome.

The proteins of the White Collar 1 family (WC) constitute a major class of flavin photoreceptors, widely distributed in fungi, that work in cooperation with a WC 2 protein forming a regulatory complex. The WC complex was investigated in great detail in Neurospora crassa, a model fungus in photobiology studies, where it controls all its major photoresponses. The fungus Fusarium fujikuroi, a model system in the production of secondary metabolites, contains a single WC-1 gene called wcoA. The best-known light response in this fungus is the photoinduction of the synthesis of carotenoids, terpenoid pigments with antioxidant properties. Loss of WcoA in F. fujikuroi results in a drastic reduction in the mRNA levels of the carotenoid genes, and a diversity of morphological and metabolic changes, including alterations in the synthesis of several secondary metabolites, suggesting a complex regulatory role. To investigate the function of WcoA, the transcriptome of F. fujikuroi was analyzed in the dark and after 15-, 60- or 240-min illumination in a wild strain and in a formerly investigated wcoA insertional mutant. Using a threshold of four-fold change in transcript levels, 298 genes were activated and 160 were repressed in the wild strain under at least one of the light exposures. Different response patterns were observed among them, with genes exhibiting either fast, intermediate, and slow photoinduction, or intermediate or slow repression. All the fast and intermediate photoresponses, and most of the slow ones, were lost in the wcoA mutant. However, the wcoA mutation altered the expression of a much larger number of genes irrespective of illumination, reaching at least 16% of the annotated genes in this fungus. Such genes include many related to secondary metabolism, as well as others related to photobiology and other cellular functions, including the production of hydrophobins. As judged by the massive transcriptomic changes exhibited by the wcoA mutant in the dark, the results point to WcoA as a master regulatory protein in F. fujikuroi, in addition to a central function as the photoreceptor responsible for most of the transcriptional responses to light in this fungus.

Modulation of Activity of a Carotenoid Pathway Through the Use of the TeT-on Regulatory System: Application in the Fungus Fusarium fujikuroi.

Carotenoids are widespread pigments in photosynthetic species, but they are also found in nonphotosynthetic microorganisms, such as bacteria and fungi. The amenability of fungi to genetic studies have made some fungal species advantageous models in the study of the genetics and biochemistry of carotenoid biosynthesis, while others have been used for biotechnological carotenoid production. The availability of molecular techniques that allow modulating the expression of target genes is a powerful tool in the manipulation of carotenoid synthesis. An example of an adjustable gene expression is based on the tetracycline-controlled transcriptional activation system, known as Tet-on. We describe here the material and protocols for the construction of a Tet-on regulated gene, its introduction in the filamentous fungus F. fujikuroi, and its use to modulate the expression of a negative regulator of carotenoid biosynthesis.

Comparative transcriptomic analysis unveils interactions between the regulatory CarS protein and light response in Fusarium.

BACKGROUND: The orange pigmentation of the agar cultures of many Fusarium species is due to the production of carotenoids, terpenoid pigments whose synthesis is stimulated by light. The genes of the carotenoid pathway and their regulation have been investigated in detail in Fusarium fujikuroi. In this and other Fusarium species, such as F. oxysporum, deep-pigmented mutants affected in the gene carS, which encodes a protein of the RING-finger family, overproduce carotenoids irrespective of light. The induction of carotenogenesis by light and its deregulation in carS mutants are achieved on the transcription of the structural genes of the pathway. We have carried out global RNA-seq transcriptomics analyses to investigate the relationship between the regulatory role of CarS and the control by light in these fungi. RESULTS: The absence of a functional carS gene or the illumination exert wide effects on the transcriptome of F. fujikuroi, with predominance of genes activated over repressed and a greater functional diversity in the case of genes induced by light. The number of the latter decreases drastically in a carS mutant (1.1% vs. 4.8% in the wild-type), indicating that the deregulation produced by the carS mutation affects the light response of many genes. Moreover, approximately 27% of the genes activated at least 2-fold by light or by the carS mutation are coincident, raising to 40% for an 8-fold activation threshold. As expected, the genes with the highest changes under both regulatory conditions include those involved in carotenoid metabolism. In addition, light and CarS strongly influence the expression of some genes associated with stress responses, including three genes with catalase domains, consistent with roles in the control of oxidative stress. The effects of the CarS mutation or light in the transcriptome of F. oxysporum were partially coincident with those of F. fujikuroi, indicating the conservation of the objectives of their regulatory mechanisms. CONCLUSIONS: The CarS RING finger protein down-regulates many genes whose expression is up-regulated by light in wild strains of the two investigated Fusarium species, indicating a regulatory interplay between the mechanism of action of the CarS protein and the control by light.

HPLC Analysis of Carotenoids in Neurosporaxanthin-Producing Fungi.

The ascomycetous fungi Fusarium fujikuroi and Neurospora crassa are widely used as research models in the study of secondary metabolism and photobiology, respectively. Both fungi exhibit a similar carotenoid pathway, for which all the genes and enzymes have been identified. Under standard laboratory conditions, either F. fujikuroi or N. crassa accumulate a mixture of neurosporaxanthin, a carboxylic apocarotenoid acid, and several of its carotene precursors. We formerly described methods for the identification and quantification of neurosporaxanthin. However, the differences in polarity between this acidic xanthophyll and neutral carotenes make their global analysis cumbersome. Here we propose a simple HPLC methodology for the efficient separation of neurosporaxanthin and earlier pathway intermediates in a single HPLC run. This method should be useful to check the abundance of neurosporaxanthin under different experimental conditions and to evaluate the relative proportions of their different carotene precursors. To assess the validity of the method, we have compared the carotenoid profiles in samples of mycelia of F. fujikuroi and conidia of N. crassa, in both cases obtained from surface cultures of a wild strain of each species.

A global perspective on carotenoids: Metabolism, biotechnology, and benefits for nutrition and health.

Carotenoids are lipophilic isoprenoid compounds synthesized by all photosynthetic organisms and some non-photosynthetic prokaryotes and fungi. With some notable exceptions, animals (including humans) do not produce carotenoids de novo but take them in their diets. In photosynthetic systems carotenoids are essential for photoprotection against excess light and contribute to light harvesting, but perhaps they are best known for their properties as natural pigments in the yellow to red range. Carotenoids can be associated to fatty acids, sugars, proteins, or other compounds that can change their physical and chemical properties and influence their biological roles. Furthermore, oxidative cleavage of carotenoids produces smaller molecules such as apocarotenoids, some of which are important pigments and volatile (aroma) compounds. Enzymatic breakage of carotenoids can also produce biologically active molecules in both plants (hormones, retrograde signals) and animals (retinoids). Both carotenoids and their enzymatic cleavage products are associated with other processes positively impacting human health. Carotenoids are widely used in the industry as food ingredients, feed additives, and supplements. This review, contributed by scientists of complementary disciplines related to carotenoid research, covers recent advances and provides a perspective on future directions on the subjects of carotenoid metabolism, biotechnology, and nutritional and health benefits.

Protein Activity of the Fusarium fujikuroi Rhodopsins CarO and OpsA and Their Relation to Fungus-Plant Interaction.

Fungi possess diverse photosensory proteins that allow them to perceive different light wavelengths and to adapt to changing light conditions in their environment. The biological and physiological roles of the green light-sensing rhodopsins in fungi are not yet resolved. The rice plant pathogen Fusarium fujikuroi exhibits two different rhodopsins, CarO and OpsA. CarO was previously characterized as a light-driven proton pump. We further analyzed the pumping behavior of CarO by patch-clamp experiments. Our data show that CarO pumping activity is strongly augmented in the presence of the plant hormone indole-3-acetic acid and in sodium acetate, in a dose-dependent manner under slightly acidic conditions. By contrast, under these and other tested conditions, the Neurospora rhodopsin (NR)-like rhodopsin OpsA did not exhibit any pump activity. Basic local alignment search tool (BLAST) searches in the genomes of ascomycetes revealed the occurrence of rhodopsin-encoding genes mainly in phyto-associated or phytopathogenic fungi, suggesting a possible correlation of the presence of rhodopsins with fungal ecology. In accordance, rice plants infected with a CarO-deficient F. fujikuroi strain showed more severe bakanae symptoms than the reference strain, indicating a potential role of the CarO rhodopsin in the regulation of plant infection by this fungus.

Transcriptional basis of enhanced photoinduction of carotenoid biosynthesis at low temperature in the fungus Neurospora crassa.

Stimulation by light of carotenoid biosynthesis in the mycelia of the fungus Neurospora crassa starts with transient transcriptional induction of the structural genes of the pathway triggered by the White Collar photoreceptor complex. Most studies on this process were carried out under standard growth conditions, but photoinduced carotenoid accumulation is more efficient if the fungus is incubated at low temperatures, from 6 to 12 °C. We have investigated the transcriptional photoresponse at 8 °C of the genes for proteins that participate in the carotenoid pathway. Exposure to light pulses of different light intensities revealed higher sensitivity if the mycelia were subsequently incubated at 8 °C compared to 30 °C. Illumination of precooled mycelia resulted in delayed kinetics of mRNA accumulation for the structural genes, and high mRNA accumulation for a longer time. Additionally, after a light pulse, stronger reduction in mRNAs for carotenoid genes was observed at 30 °C compared to 8 °C. A similar pattern was found for mRNAs of the photoreceptor genes wc-1 and vvd, the latter involved in photoadaptation. These results suggest that the increased efficiency in carotenoid photoinduction at low temperature is due to the higher mRNA levels of the structural genes under these conditions.

Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens.

In seed plants, strigolactones (SLs) regulate architecture and induce mycorrhizal symbiosis in response to environmental cues. SLs are formed by combined activity of the carotenoid cleavage dioxygenases (CCDs) 7 and 8 from 9-cis-ß-carotene, leading to carlactone that is converted by cytochromes P450 (clade 711; MAX1 in Arabidopsis) into various SLs. As Physcomitrella patens possesses CCD7 and CCD8 homologs but lacks MAX1, we investigated if PpCCD7 together with PpCCD8 form carlactone and how deletion of these enzymes influences growth and interactions with the environment. We investigated the enzymatic activity of PpCCD7 and PpCCD8 in vitro, identified the formed products by high performance liquid chromatography (HPLC) and LC-MS, and generated and analysed ΔCCD7 and ΔCCD8 mutants. We defined enzymatic activity of PpCCD7 as a stereospecific 9-cis-CCD and PpCCD8 as a carlactone synthase. ΔCCD7 and ΔCCD8 lines showed enhanced caulonema growth, which was revertible by adding the SL analogue GR24 or carlactone. Wild-type (WT) exudates induced seed germination in Orobanche ramosa. This activity was increased upon phosphate starvation and abolished in exudates of both mutants. Furthermore, both mutants showed increased susceptibility to phytopathogenic fungi. Our study reveals the deep evolutionary conservation of SL biosynthesis, SL function, and its regulation by biotic and abiotic cues.