Phytochemistry 117 (2015) 34–42
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Diacylglyceryltrimethylhomoserine content and gene expression changes triggered by phosphate deprivation in the mycelium of the basidiomycete Flammulina velutipes Svetlana V. Senik a,⇑, Liliya G. Maloshenok c, Ekaterina R. Kotlova a, Alexey L. Shavarda a, Konstantin V. Moiseenko b, Sergey A. Bruskin c, Olga V. Koroleva b, Nadezhda V. Psurtseva a a
Komarov Botanical Institute, Russian Academy of Sciences, 2 Professor Popov str., St. Petersburg 197376, Russia A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, 33 Leninsky pr., Moscow 117071, Russia c Vavilov Institute of General Genetics, Russian Academy of Sciences, 3 Gubkina str., Moscow 119991, Russia b
a r t i c l e
i n f o
Article history: Received 26 November 2014 Received in revised form 16 May 2015 Accepted 27 May 2015
Keywords: Flammulina velutipes Basidiomycetes Betaine lipid Glycerolipid metabolism Phosphate starvation Gene expression BTA1 CHO2 CPT1 Phylogenetic analysis
a b s t r a c t Diacylglyceryltrimethylhomoserines (DGTS) are betaine-type lipids that are phosphate-free analogs of phosphatidylcholines (PC). DGTS are abundant in some bacteria, algae, primitive vascular plants and fungi. In this study, we report inorganic phosphate (Pi) deﬁciency-induced DGTS synthesis in the basidial fungus Flammulina velutipes (Curt.: Fr.) Sing. We present results of an expression analysis of the BTA1 gene that codes for betaine lipid synthase and two genes of PC biosynthesis (CHO2 and CPT1) during phosphate starvation of F. velutipes culture. We demonstrate that FvBTA1 gene has increased transcript abundance under phosphate starvation. Despite depletion in PC, both CHO2 and CPT1 were determined to have increased expression. We also describe the deduced amino acid sequence and genomic structure of the BTA1 gene in F. velutipes. Phylogenetic relationships between putative orthologs of BTA1 proteins of basidiomycete fungi are discussed. Ó 2015 Published by Elsevier Ltd.
1. Introduction Betaine lipids are non-phosphorous glycerolipids that are structurally similar to the phospholipid PC. Both phospho- and betaine lipids have positively charged trimethylammonium group and similar phase transition temperatures (Sato and Murata, 1991). Diacylglyceryl-N,N,N-trimethylhomoserines (DGTS) are the most widespread class of betaine lipids. DGTS are abundant in several
Abbreviations: AdoMet, S-adenosylmethionine; CDP, cytidine diphosphate; DAG, diacylglycerols; DGTS, diacylglyceryltrimethylhomoserines; DGTA, diacylglycerylhydroxymethyltrimethylalanines; DGCC, diacylglycerylcarboxyhydroxymethylcholines; DPG, diphosphatidylglycerols; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ITS, internal transcribed spacer; Pi, inorganic phosphate; PC, phosphatidylcholines; PE, phosphatidylethanolamines; PI, phosphatidylinositols; PiC, inorganic phosphate containing (medium); PiF, inorganic phosphate-free (medium); PS, phosphatidylserines; RACE, Rapid Ampliﬁcation of cDNA Ends; RTqPCR, real time quantitative polymerase chain reaction. ⇑ Corresponding author. E-mail address: [email protected]
(S.V. Senik). http://dx.doi.org/10.1016/j.phytochem.2015.05.021 0031-9422/Ó 2015 Published by Elsevier Ltd.
groups of bacteria (Benning et al., 1995; Geiger et al., 1999), green algae (Eichenberger, 1982; Sato and Furuya, 1985; Vaskovsky et al., 1996; Künzler and Eichenberger, 1997), primitive vascular plants such as mosses (Sato and Furuya, 1985; Künzler and Eichenberger, 1997), lycophytes and ferns (Künzler and Eichenberger, 1997; Rozentsvet et al., 2000; Rozentsvet, 2004), as well as lichens (Künzler and Eichenberger, 1997) and fungi (Künzler and Eichenberger, 1997; Dembitsky, 1996; Vaskovsky et al., 1998; Kotlova and Popov, 2005). Biosynthesis of DGTS has been studied in detail only in bacterial, algal and yeast cells. Two enzymes named BtaA and BtaB are required for DGTS production in bacteria (Klug and Benning, 2001; Riekhof et al., 2005). BtaA transfers a four-carbon backbone from S-adenosylmethionine to the diglyceride moiety, forming the intermediate diacylglycerylhomoserine. BtaB catalyses a threestep N-methylation of the amino group on the intermediate to form the ﬁnal DGTS product. The green algae Chlamydomonas reinhardtii has a single polypeptide, BTA1, containing BtaA- and BtaB-like domains that carry out all steps in DGTS biosynthesis
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consecutively (Riekhof et al., 2005). Analyses of whole-genome sequences have revealed that CrBTA1 orthologs are abundant among eukaryotic organisms, and their distribution correlates with the distribution of DGTS. However, eukaryotic DGTS synthases, aside from BTA1 of C. reinhardtii, have been functionally studied only in the ascomycete yeast Kluyveromyces lactis (Riekhof et al., 2014). The amino acid sequence of KlBta1 was shown to be similar to CrBTA1 and contains conserved residues that have been implicated in the binding of AdoMet. Distribution of DGTS in basidiomycete fungi has been demonstrated to be heterogeneous. In certain fungal taxons, such as Agaricales, Polyporales and Russulales, there are species that synthesize and species that do not synthesize DGTS that belong to the same order or even family (Dembitsky, 1996; Vaskovsky et al., 1998). The unstable presence of betaine lipids in some groups of Basidiomycetes suggests a regulatory mechanism for the synthesis of DGTS in fungi. Phosphate deﬁciency is considered to be one a condition that triggers the synthesis of DGTS. The ability to compensate for reduced phospholipid content by producing phosphorus-free betaine lipids during Pi starvation has been shown in the photosynthetic bacteria Rhodobacter sphaeroides (Benning et al., 1995), the symbiotic soil bacteria Sinorhizobium meliloti (Geiger et al., 1999; López-Lara et al., 2003; Zavaleta-Pastor et al., 2010), the mycelial ascomycete Neurospora crassa and in the yeast K. lactis (Riekhof et al., 2014). It should be noted that several authors have suggested that there is a negative correlation between the presence and abundance of betaine lipids and PC (Eichenberger, 1982; Sato, 1992; Benning et al., 1995; Dembitsky, 1996; Vaskovsky et al., 1998). However, the mechanism behind the reciprocity between DGTS and PC remains unclear. PC biosynthesis in mushrooms has been extensively studied. In ascomycete yeasts, as in most eukaryotes, two pathways for PC synthesis have been found. One method for PC synthesis is by the methylation of phosphatidylethanolamine (PE), where PE is converted to PC by a three-step S-adenosylmethionine (AdoMet)-dependent methylation reaction. The ﬁrst methylation reaction is catalyzed by the CHO2-encoded PE methyltransferase (Kodaki and Yamashita, 1987; Summers et al., 1988) and the ﬁnal two methylations are catalyzed by the OPI3-encoded phospholipid methyltransferase (Kodaki and Yamashita, 1987; McGraw and Henry, 1989). When choline is present in the growth media, PC may also be synthesized by the Kennedy pathway from CDP-choline that reacts with DAG in reactions catalyzed by the CPT1-encoded choline phosphotransferase (Hjelmstad and Bell, 1987, 1990). Previous studies have suggested that the contribution of the methylation pathway for PC synthesis in Saccharomyces cerevisiae is more important but that the Kennedy pathway for PC synthesis assumes a critical role when the enzymes in the CDP-DAG pathway are defective or repressed (Carman and Henry, 1989; Greenberg and Lopes, 1996). However, it is not clear what the relative contributions of the CDP-DAG and Kennedy pathways in basidiomycetes are and whether their balance changes during adaptation to phosphate starvation. Basidiomycete xylotrophic fungus Flammulina velutipes (Curt.: Fr.) Sing. is an edible and medicinal mushroom commercially cultivated all over the world. According to the early data, fruit bodies of F. velutipes do not contain DGTS (Vaskovsky et al., 1998). In a previous report, we demonstrated that surface cultures of F. velutipes do synthesize DGTS when they are deprived of a complex of nutrients, including phosphorus, nitrogen, potassium, and some trace elements (Senik et al., 2012). The present study provides evidence that phosphorus deﬁciency alone induces DGTS synthesis by this fungus. This study focuses on mechanisms of reciprocity between DGTS and PC in fungi during Pi starvation. We report changes in expression
of the BTA1 gene and two PC biosynthesis genes during phosphate starvation of F. velutipes culture. We describe the deduced amino acid sequence and genomic structure of the FvBTA1 gene coding for DGTS synthase in F. velutipes. We show that the FvBTA1 gene has increased transcript abundance under phosphate starvation. Despite PC depletion, expression of both PC biosynthesis genes was determined to increase. Phylogenetic relationships between putative orthologs of the BTA1 gene are also discussed. 2. Results 2.1. Strain veriﬁcation We veriﬁed our strain by a ribosomal DNA internal transcribed spacer (ITS)1, 5.8S and ITS2 (rDNA ITS) ampliﬁcation using the basidiomycete-speciﬁc primers ITS1-F and ITS4-B (Gardes and Bruns, 1993). The partial nucleotide sequence of the 18S-ITS15.8S-ITS2-28S region (921 bp) was obtained (GenBank accession number KM668876) (Fig. S1). A BLAST search of this fragment against the whole GenBank database revealed 100% identity with F. velutipes (e.g., GenBank accession number EU191062.1 and FJ889514.1). 2.2. Changes in membrane lipid content in F. velutipes culture under phosphate starvation To analyze the effect of phosphate deprivation on the lipid content of F. velutipes, we compared cultures that were grown on inorganic phosphate-free (PiF) media with control cultures grown on media containing phosphate salts (PiC media). As shown in Fig. 1, PC and PE were the primary membrane glycerolipids of F. velutipes grown on PiC media. Minor phospholipids included phosphatidic acids (PA), phosphatidylserines (PS), phosphatidylinositols (PI), and diphosphatidylglycerol (DPG). During growth of the culture on PiC media, the level of PA increased from 3% to 12% of the total membrane glycerolipids. The culture grown under phosphate-limiting conditions exhibited a lower biomass accumulation rate and a lesser density of the aerial mycelium compared with control colonies (data not shown). These changes were accompanied by induction of DGTS synthesis and its accumulation up to 42% of the total membrane lipids. DGTS accumulation was concomitant with PC depletion from 31% to 14%. Special attention is given to the fact that in culture starved for phosphorus PS was elevated 2-fold compared with one growing on complete medium. The culture grown under phosphate starvation showed a similar but less pronounced trend than that observed for the PiC culture with PA increasing from 0.5% to 6% of the total membrane glycerolipids. 2.3. Ampliﬁcation and analysis of BTA1 gene ortholog coding by genome of F. velutipes The full-length sequence (2954 bp) of the FvBTA1 gene was found to contain an open reading frame of 2241 bp encoding a protein product of 747 amino acids (GenBank accession number KM668875) (Fig. S2). A comparison of the genomic and cDNA sequences of FvBTA1 indicated the presence of 14 introns varying in size from 47 to 56 bp with splicing junctions adhering to the GT-AG rule (Padgett et al., 1984). The deduced amino acid sequence of the FvBTA1 gene product revealed a 37% identity and 58% similarity with a previously characterized BTA1 protein from K. lactis (NCBI Accession # XM_456071.2), with the regions of highest conservation being observed in predicted methyltransferase motifs (Fig. 2). We found that regions corresponding to AdoMet binding sites predicted by means of a Conserved Domain
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PS DPG 7% 3% PA 3%
B DPG 1% PA 7%
PS 6% DPG 1%
PE 24% PE 47%
PI 5% PS 11%
C DPG 1% PA 8%
PC 17% DGTS 35%
PE 25% PE 45%
PI 4% PS 10%
D PS DPG 5% 1%
PI 6% PC 14%
PE 41% PI 3%
PA 6% PS 10%
Fig. 1. Membrane lipid composition of F. velutipes: A – Day 8. B – Day 15. C – Day 20. D – Day 32. PC – phosphatidylcholines, PE – phosphatidylethanolamines, DGTS – diacylglyceryltrimethylhomoserines, PA – phosphatidic acids, PS – phosphatidylserines, PI – phosphatidylinositols, DPG – diphosphatidylglycerins, Pi – Pi starvation, Pi+ – complete media.
Database (Marchler-Bauer et al., 2011) were conserved between proteins from both fungus species. 2.4. Phylogenetic analysis of basidiomycete BTA1 protein orthologs Searching with the KlBta1 protein sequence in the wholegenome sequences deposited in GenBank, we found 32 putative
BTA1 orthologs of basidiomycetes. These orthologs included proteins from wood-decaying fungi that cause white rot (F. velutipes, Stereum hirsutum, Phanerochaete carnosa, Ceriporiopsis subvermispora, Trametes versicolor, Fomitiporia mediterranea, Auricularia delicata, Tremella mesenterica, and Dichomitus squalens) and brown rot (Serpula lacrymans, Coniophora puteana, Dacryopinax sp., Gloeophyllum trabeum, and Jaapia argillacea),
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Fig. 2. A comparison of the amino acid sequence deduced for the F. velutipes BTA1 protein with the BTA1 protein sequence of Kluyveromyces lactis NRRL Y-1140 (NCBI Accession # XP_456071.2). Gray shading and black shading indicate conserved and identical residues, respectively; dashes indicate gaps. Arrow heads indicate the border between BTA A-like (carboxypropyltransferase) and Bta B-like (N-methyltransferase) domains. Asterisks (⁄) indicate AdoMet binding sites predicted by a Conserved Domain Database.
ectomycorrhizal species (Laccaria bicolor, and Galerina marginata), basidiomycete yeasts – human pathogens (Cryptococcus gattii, Cryptococcus neoformans, Rhodotorula glutinis, Malassezia globosa, Malassezia sympodialis, and Trichosporon asahii), plant pathogens (Moniliophthora roreri, Melanopsichium pennsylvanicum, Ustilago maydis, Ustilago hordei, and Sporisorium reilianum) and non-pathogenic basidiomycete yeasts (Pseudozyma antarctica, Pseudozyma brasiliensis, Pseudozyma ﬂocculosa, Pseudozyma hubeiensis, and Rhodosporidium toruloides). Phylogenetic analysis of identiﬁed putative BTA1 orthologs of basidiomycete fungi was done. Multiple sequence alignment of 32 basidiomycete proteins and rooted phylogenetic tree were obtained (Supplementary Fig. S3 and Fig. 3). The topology of the phylogenetic tree generally agrees with the currently accepted view of the organismal phylogeny of basidiomycete fungi according to the system of Hibbett et al. (2007). However, some exceptions to this organization are observed; for example, the sequence of the putative BTA1 ortholog in S. reilianum, a fungus of the Pucciniomycotina subdivision, is closer to proteins from the Ustilaginomycotina fungi. Unexpectedly, putative BTA1 orthologs from T. versicolor, C. subvermispora and D. squalens, fungi from order Polyporales (class Agaricomycetes), are clustered together but generate a branch separated from all other basidiomycete proteins. The ortholog sequence from P. carnosa – another member of the Polyporales – also extends beyond the limits of
Agaricomycetes. It is unlikely that proteins from Polyporales fungi have a function other than betaine lipid synthesis because it has previously been shown that T. versicolor synthesizes DGTS in response to phosphate deﬁciency similar to F. velutipes (unpublished data) but other BTA1 orthologs were not found in the genome of T. versicolor. This phylogenetic analysis allows the assumption that betaine lipids are likely to have a particular evolutionary signiﬁcance for species from Polyporales and therefore that the betaine lipid synthase gene can be a target of natural selection for these fungi. Deeper analyses of the function, physiological and ecological roles of polypores-speciﬁc BTA1 orthologs is needed to explain its relatively low sequence similarity with putative orthologs from other basidiomycetes. 2.5. Expression of FvBTA1 gene To analyze the effect of phosphate starvation on DGTS biosynthesis gene expression, the FvBTA1 gene transcripts were measured by qRT-PCR. Fig. 4 indicates that FvBTA1 gene is markedly upregulated in the mycelia grown under phosphate-limiting conditions when compared to the mycelia grown on PiC media. Interestingly, FvBTA1 gene expression is detectable in both conditions, even though DGTS did not accumulate to a detectable level in the PiC media grown culture. Evidently, the transcription of this gene occurs permanently at some minimal level. FvBTA1 gene
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Pseudozyma flocculosa Ustilago hordei 99
Ustilago maydis 100
Pseudozyma antarctica Pseudozyma brasiliensis
Pseudozyma hubeiensis 38
Trichosporon asahii 100
Polyporales Hymenochaetales Dacrymycetales Auriculariales Russulales
Fomitiporia mediterranea 66
Dacryopinax sp. 72
Auricularia delicata 98
Stereum hirsutum 64
Gloeophyllum trabeum Jaapia argillacea
Boletales Gloeophyllales Jaapiales
Moniliophthora roreri Galerina marginata
Ceriporiopsis subvermispora Dichomitus squalens
Kluyveromyces lactis 0.1
Fig. 3. A rooted phylogenetic tree of putative BTA1 orthologs in fungi (basidiomycetes). The tree was produced using an amino acid sequence alignment of the 32 basidiomycete and 1 ascomycete (an outgroup) proteins using the maximum likelihood method based on CLUSTAL W multiple sequence alignments. A bar represents the number of substitution events (0.1 substitution per position). Taxon names of basidiomycete fungi are indicated according to the system of Hibbett et al. (2007) with supplements from Binder et al. (2010).
2.6. Expression of CHO2 and CPT1 genes
Fig. 4. Changes in the expression of the FvBTA1 gene at different developmental stages of F. velutipes grown under phosphate-limiting conditions (grey bars) compared to cultures grown in complete media (white bars). The results were normalized to GAPDH mRNA. The experiments were performed in triplicate and the data are presented as means ± SD.
expression in young mycelium (8 day) grown on PiC media can be intensiﬁed in response to stress associated with mycelium injuring during inoculum transplanting. A small increase of BTA1 expression in PiC media at day 32 can be related to phosphate exhaustion from the medium by the end of experiment.
To test the hypothesis that PC depletion under Pi-limiting conditions is speciﬁcally regulated by coordinated expression of PC biosynthesis genes, the expression of two genes necessary for PC metabolism, cholinephosphotransferase (CPT1) and phosphatidylethanolamine N-methyltransferase (CHO2), were analyzed. The results of the qRT-PCR analysis are depicted in Fig. 5. Both genes coding for PE methyltransferase and cholinephosphotransferase, catalyzing the PC biosynthesis via CDP-DAG and the Kennedy pathways, respectively, displayed increased transcript abundance under phosphate starvation. In the early stages of growth (day 8) transcript level in control samples was almost equal to samples subjected to phosphate depletion. After 15 days, however, the transcripts in the phosphate-starved culture exceeded control levels by 20-fold. By 32 days of growth on PiF media, both CPT1 and CHO2 decreased their expression to the control levels. The transcript abundance of both the CPT1 and CHO2 genes decreased during culture growth, regardless of phosphate availability. This observation correlates with data showing the predominance of PC in the early stages of growth (day 8) (Fig. 1). Similar results were observed in previous studies of surface cultures of F. velutipes and Lentinula edodes grown on complete organic media (Sakai and Kajiwara, 2004; Kotlova et al., 2009). In both studies the PC/PE ratio in young and actively growing mycelium was higher than at later stages of development. The results of our
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Fig. 5. The expression of CPT1 (A) and CHO2 (B) genes at different developmental stages of F. velutipes grown under phosphate-limiting conditions (gray bars) and on complete media (white bars). The results are normalized to GAPDH mRNA. The experiment was performed in triplicate and the data are presented as mean ± SD.
present study suggest that changes in PC levels during the growth of a culture are under control of PC biosynthesis gene expression. It is interesting to note that throughout the experiment, CPT1 was expressed at signiﬁcantly higher levels than CHO2. This evidence suggests the predominance of the Kennedy pathway in PC biosynthesis in F. velutipes and is in accordance with our previous data on the effect of PC synthesis inhibitors on the growth of F. velutipes. We found that treatment with 200 lM farnesol, a cholinephosphotransferase (CPT1) inhibitor, signiﬁcantly reduced the intensity of mycelium growth and a 1 mM farnesol treatment almost completely arrested growth. At the same time cloﬁbric acid and bezaﬁbrate, inhibitors of PE methyltransferase, did not affect the growth rate and morphology of the fungus (unpublished data).
3. Discussion Cell responds to ﬂuctuating environmental factors by activating a set of compensatory mechanisms including changes in the lipid proﬁle. Compensatory reactions occurring in membranes in response to phosphate deprivation include replacement of phospholipids by phosphorus-free lipids such as galactolipids (Härtel et al., 2000), sulfolipids (Benning et al., 1993; Essigmann et al., 1998) or betaine lipids (Benning et al., 1995; Riekhof et al., 2014). The present study increases our understanding of this biochemical strategy aimed at maintaining Pi homeostasis. This is
the ﬁrst report of Pi deﬁciency-induced DGTS synthesis in basidial fungi. Another feature to be shown for basidiomycete F. velutipes grown under phosphate starvation is elevated level of PS. PS is synthesized from CDP-DAG by CHO1-encoded PS synthase and is regulated by genetic and biochemical mechanisms. The elevated expression of the S. cerevisiae CHO1 gene results in an increase in CHO1 mRNA abundance, PS synthase protein, and its activity (Bailis et al., 1987; Iwanyshyn et al., 2004). Another mechanism by which PS synthase activity is regulated is control of mRNA stability, namely its rate of decay (Choi et al., 2004). Finally, phosphatidylserine synthase from S. cerevisiae is inhibited by phosphorylation (Kinney and Carman, 1988; Choi et al., 2010). The latter mechanism can clarify interaction between Pi deﬁciency and elevating level of PS. However, the effect of Pi starvation on PS can be more complicated. There is evidence that the concentrations of most PS species with very long chain fatty acids were higher in phosphorus-starved leaves of Arabidopsis than in normally grown ones whereas the concentrations of other major molecular species in phospholipid classes were lower in phosphorus-starved leaves (Li et al., 2006). Authors suggest that higher concentrations of individual PS species may serve as signaling molecules to modulate the survival of stressed cells. The ability of fungi to regulate the synthesis of betaine lipids depending on nutrient availability can provide an explanation for the heterogeneous distribution of DGTS in the fruiting bodies of some groups of Basidiomycetes (Dembitsky, 1996; Vaskovsky et al., 1998). It seems unlikely that the absence of DGTS in natural occurring fruiting bodies can be a permanent chemotaxonomic marker and is instead due to the transient downregulation of BTA1 gene expression. Patterns in DGTS distribution in certain fungal orders, such as Boletales and Russulales, may be evidence of differences in regulatory mechanisms of DGTS synthesis or ecological association of these taxons with ecotopes with low or high Pi availability. BLAST analysis of BTA1 genes from C. reinhardtii and K. lactis show that databases contain sequences of BTA1 orthologs from more than 30 species of basidiomycetes, all from fungal wholegenome projects. None of them has been functionally characterized. We demonstrated that BTA1 gene of basidiomycete F. velutipes is inducible by Pi deﬁciency. Unfortunately, the regulatory mechanism of BTA1 induction in basidiomycetes has not yet been studied. It is known that DGTS synthesis during Pi limitation in ascomycete fungi is under the control of the PHO regulon, mediated by the transcription factor referred to as Pho4p in S. cerevisiae and NUC-1 in N. crassa (Riekhof et al., 2014). While the PHO pathway is widespread among ascomycete fungi (Tomar and Sinha, 2014), no information is available about the function of this pathway in basidiomycetes. Based on similarity searches, we could not identify clear orthologs of Pho4 and Pho2 proteins in complete genomes of basidiomycetes but found putative orthologs of other components of the PHO pathway: cyclin Pho80, cyclin-dependent kinase Pho85 and cyclin-dependent kinase inhibitor Pho81. However, the role of these proteins in phosphate starvation response in basidiomycete fungi has not been elucidated. Additionally, there is evidence that in some ascomycete fungi, orthologs of the S. cerevisiae PHO pathway components can have another functions, for example, the cyclin-CDK complex SpPHO80-SpPHO85 in S. pombe (Henry et al., 2011). Thus, while the basidiomycete F. velutipes and the ascomycetes N. crassa and K. lactis demonstrate a similar phosphate starvation response by the replacement of phospholipid PC with non-phosphorus DGTS, it is unclear how the Pi starvation response is regulated in fungi. Another question concerns the mechanism of PC reduction during the phosphate starvation response. There are at least three main mechanisms of PC depletion: suppression of PC synthesis
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due to substrate exhaustion, downregulation of PC synthase expression by suppression of gene transcription or intensiﬁcation of mRNA degradation as well as intensive hydrolysis of PC by phospholipases. We supposed that substitution of PC by DGTS is compensatory reaction directed on phosphate release for vitally important cell processes and regulated at the level of both DGTS and PC gene transcription. Therefore we expected the decrease of mRNA abundance of PC synthesis genes. However our data provides the ﬁrst evidence that the PC biosynthesis genes CPT1 and CHO2 increase their expression under phosphate-limiting conditions. These results indicate that regulation by mRNA abundance does not contribute to PC depletion during Pi-starvation in basidial fungi. What is the mechanism of elevating of mRNA abundance of PC synthesis genes – activation of synthesis or increase of stability – are the questions for future studies. The present work broadens the scope of our knowledge on the adaptive mechanism that induce DGTS synthesis in response to Pi-deﬁciency. 4. Experimental 4.1. Fungal strain and growth conditions In this study, we used F. velutipes (Curt.: Fr.) Sing. dikaryotic strain 1483 obtained from the Basidiomycetes Culture Collection of the Komarov Botanical Institute, RAS (St. Petersburg, Russia). For lipid extraction and RNA preparation the strain was grown on the agarized medium [0.3% bacteriological peptone (w/v), 0.05% MgSO4 (w/v), 0.005% CaCl2 (w/v), 0.0001% ZnSO4 (w/v), 0.05% FeSO4 (w/v), 0.06% KH2PO4 (w/v), 0.04% K2HPO4 (w/v), and 2% agar] in the dark at 25 °C. Mycelium was sampled on days 8, 15, 20 and 32. For growth under phosphate deﬁciency, the strain was cultivated on the same media but lacking of phosphate salts. 4.2. Lipid extraction and quantitative lipid analysis After harvesting the mycelium, lipid extracts were prepared with isopropanol according to the Nichols method (Nichols, 1963) with modiﬁcations as previously described (Kotlova et al., 2009). Concentrated lipid preparations were separated on TLC silica gel 60 10 10 cm plates (Merck, Germany) in two dimensions in a solvent system of chloroform–methanol–water (65:25:4) in the ﬁrst direction and chloroform–acetone–methanol–acetic acid–water (50:20:10:10:5) in the second direction (Benning et al., 1995). PC used for a standard on TLC was obtained from Sigma (UK). The amounts of glycero- and sphingolipids were determined densitometrically using a Denscan device (Lenchrom,
Russia) after visualization by heating with a 5% H2SO4 solution in methanol. 4.3. DNA and RNA extraction For both DNA and RNA extraction, F. velutipes mycelium (obtained from the same Petri dish used for lipid extraction) was disrupted by grinding in liquid nitrogen with a mortar and pestle and stored at 80 °C. Total RNA was isolated using the RNeasy Plant Mini kit (Qiagen, USA). Additionally, during RNA puriﬁcation on columns, each sample was treated by DNase from RNase Free DNase Set (Qiagen, USA). RNA samples were stored at 80 °C. DNA was isolated using the AxyPrep Multisource Genomic DNA Miniprep kit (Axygen, USA). All kits were used according to the manufacturer’s instructions. The integrity of the extracted total RNA and DNA samples was veriﬁed via electrophoresis on a 1.4% agarose gel followed by ethidium bromide staining. For RNA after staining with ethidium bromide, two sharp bands of 28S and 18S rRNA were seen. Purity and concentration were determined using spectrophotometer (NanoDrop Lite, Thermo Scientiﬁc, USA) and Qubit ﬂuorometer (Invitrogen). Isolated RNA was checked for gDNA contamination by performing PCR reaction with all primers in the presence of RNA instead of cDNA. After 35 cycles ampliﬁcation was not observed. 4.4. ITS ampliﬁcation To verify the strain, ampliﬁcation of the ITS regions of the rRNA gene was carried out using the ITS1-F and ITS4-B primers (Gardes and Bruns, 1993). The nucleotide sequences of the primers used in our study are shown in Table 1. The ﬁnal PCR reaction mixture contained 10 lL of PCR IQ SuperMix (Bio-Rad, USA) with hot-start iTaq DNA polymerase, 0.3 lM of each primer and up to 20 lL of deionized water. The PCR thermocycler parameters were 94 °C for 5 min, followed by 35 cycles of 94 °C for 1 min, 52 °C for 1 min and 72 °C for 1 min, and a ﬁnal step at 72 °C for 3 min. The PCR products were directly puriﬁed from the reaction mixture using AxyPrep PCR Clean-Up Kit (Axygen, USA) and sequenced by the Sanger method. 4.5. Ampliﬁcation of partial Fv BTA1, CHO2, CPT1 and GAPDH genes For ampliﬁcation of each gene fragment, primers were designed (Table 1) based on conserved regions in alignments of homologues genes from closely related fungal species. The ﬁnal PCR reaction mixture contained 10 lL of PCR IQ SuperMix (Bio-Rad, USA) with hot-start iTaq DNA polymerase, 0.3 lM of each primer and up to
Table 1 List of gene-speciﬁc primers used in this study. Name
ITS1-F ITS4-B FvBTA1-Fw1 FvBTA1-Fw2 FvBTA1-Rev1 FvBTA1-Fw3 FvBTA1-Rev2 FvBTA1-Fw FvBTA1-Rev CHO2-Fw CHO2-Rev CPT1-Fw CPT1-Rev GAPDH-Fw GAPDH-Rev
50 -CTTGGTCATTTAGAGGAAGTAA-30 50 -CAGGAGACTTGTACACGGTCCAG-30 50 -CGAAATAGAAGGAGGTAAC-30 50 -CCTCTCCCAGTATCTCAG-30 50 -CCGAAGAGAAGGAAGTAA-30 50 -ATTATGCCATTCGAGCAGGACC-30 50 -AGTAAGATACGCTGGACAGGAC-30 50 -GTTGAAGAGTCCTGTGTTCTGTTG-30 50 -CTGTTGTATAGTGTCCGAGTAATGC-30 50 -TGTTCAACCTGTCTCTTCTGG-30 50 -CGTTTCTTGCTCTGCTTGG-30 50 -TTCGTGCCGTTCCTCTGC-30 50 -ATACTCCATATCCACATCCAATCC-30 50 -CCTCGACAAGTATGACCCCAAATTCA-30 50 -CCACCACGCCAGTCCTTGTTG-30
ITS ampliﬁcation ITS ampliﬁcation RACE-PCR RACE-PCR RACE-PCR RACE-PCR RACE-PCR qRT-PCR, for FvBTA1 gene qRT-PCR, for FvBTA1 gene qRT-PCR, for CHO2 gene qRT-PCR, for CHO2 gene qRT-PCR, for CPT1 gene qRT-PCR, for CPT1 gene qRT-PCR, for GAPDH gene qRT-PCR, for GAPDH gene
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20 lL of deionized water. The PCR thermocycler parameters were 94 °C for 5 min, followed by 35 cycles of 95 °C for 0.5 min, 48 °C for 0.5 min and 72 °C for 0.5 min, and ending with 72 °C for 3 min. The PCR products were directly puriﬁed from the reaction mixture using the AxyPrep PCR Clean-Up Kit (Axygen, USA) and sequenced using the Sanger method. Sequences were assembled manually and aligned using the BioEdit software (Hall, 1999). 4.6. Ampliﬁcation of full-length FvBTA1 gene sequence Double stranded complementary DNA (cDNA) was obtained using a MINT cDNA synthesis kit (Evrogen, Russia) according to the manufacturer’s protocol. The cDNA was then used in a 50 /30 RACE (Rapid Ampliﬁcation of cDNA Ends) PCR reaction according to the instructions in the Mint RACE primer set kit (Evrogen, Russia). FvBTA1 gene speciﬁc primers were designed based on the previously obtained (2.6) partial gene region (Table 1). The PCR products were gel puriﬁed and sequenced using the Sanger method. The full gene sequence, containing introns, was obtained by PCR using total DNA. 4.7. Phylogenetic study of BTA1 orthologs A search for BTA1 orthologs was performed using the BLAST program (Altschul et al., 1990) on the NCBI website for all of the databases available as of September 20, 2014. Sequences used in this analysis are listed in Supplementary Fig. 3. Sequences were aligned using the ClustalW interface and used for tree building by the maximum likelihood method with the Mega 6.06 software (Tamura et al., 2013). 4.8. Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) cDNA templates for all quantitative real-time reverse transcriptase polymerase chain reactions (qRT-PCR) were synthesized from 100 ng of total RNA using the MMLV RT kit (Evrogen, Russia) following the manufacturer’s protocol. All gene speciﬁc primers (Table 1) were designed based on sequences (2.6 & 2.7) using the Beacon Designer software and synthesized from Evrogen, Russia. All qRT-PCR experiments were done on an Eco Real-Time PCR System (Illumina, Inc., USA). Ampliﬁcation efﬁciency for each primer set was calculated by serially diluting exponential-phase cDNA template. Efﬁciency values for each primer set showed high linearity in the investigated range of 0.1–100 ng cDNA. The ﬁnal qRT-PCR reaction mixture contained 1 lL of cDNA, 0.2 lM of each primer, 2 lL of qPCR mix-HS SYBR (Evrogen, Russia) and up to 10 lL of sterile PCR water. A multiwell plate was sealed with sealing foil, centrifuged at 1500g for 1 min and loaded into the Eco Real-Time PCR System instrument. Three technical replicates were provided in each qPCR experiment and the mean Ct values were used for quantiﬁcation. No template controls were used as negative control for each set of primer pairs. Real-time PCR analysis was performed using the following optimized assay conditions: denaturation program (95 °C for 10 min); ampliﬁcation and quantiﬁcation program repeated for 40 cycles (95 °C for 10 s, 58 °C for 20 s, 72 °C for 15 s with a single ﬂuorescence measurement); melting curve program (from 95 °C to 55 °C with continuous ﬂuorescence measurement at 95 °C); and ﬁnally a cooling step at 4 °C for 30 s. Melting curve analysis was performed after each run in a Eco Real-Time PCR System instrument to conﬁrm the speciﬁcity of the primers. All qRT-PCR experiments were done on an Eco Real-Time PCR System (Illumina, Inc., USA). The mRNA level was normalized to expression of GAPDH (glyceraldehyde-3-phosphate
dehydrogenase) which was constantly expressed under all experimental conditions. The qRT-PCR results were elaborated using the 2DCT method (Livak and Schmittgen, 2001), where DCt = Ct (Target gene) Ct(GAPDH) and the RQ (relative transcript quantities) as: RQ = 2DCT. Acknowledgments The authors thank E.L. Illina, A.R. Kotsinyan, V.F. Malysheva, E.F. Malysheva, M.V. Okun and A.D. Zolotarenko for assistance with gene expression analysis and molecular phylogeny and S.V. Volobuev for assistance with fungal taxonomy and ecology. Phylogenetic analysis was carried out within the framework of the institutional research project (no. 01201255617) of the Komarov Botanical Institute of the Russian Academy of Sciences. Financial support was provided in part by the Russian Foundation for Basic Research Grants No. 14-04-01795 and 1504-06211. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2015. 05.021. References Altschul, S., Gish, W., Miller, W., Myers, E., Lipman, D., 1990. Basic local alignment search tool. J. Mol. Biol. 215 (3), 403–410. Bailis, A.M., Poole, M.A., Carman, G.M., Henry, S.A., 1987. The membrane-associated enzyme phosphatidylserine synthase is regulated at the level of mRNA abundance. Mol. Cell. Biol. 7 (1), 167–176. Benning, C., Beatty, J., Prince, R., Somerville, C., 1993. The sulpholipid sulphoquinovosyldiacylglycerol is not required for photosynthetic electron transport in Rhodobacter sphaeroides but enhances growth under phosphate limitation. Proc. Natl. Acad. Sci. USA 90, 1561–1565. Benning, C., Huang, Z.H., Gage, D.A., 1995. Accumulation of a novel glycolipid and a betaine lipid in cells of Rhodobacter sphaeroides grown under phosphate limitation. Arch. Biochem. Biophys. 317, 103–111. Binder, M., Larsson, K.-H., Matheny, P.B., Hibbett, D.S., 2010. Amylocorticiales ord. nov. and Jaapiales ord. nov.: early diverging clades of Agaricomycetidae dominated by corticioid forms. Mycologia 102 (4), 865–880. Carman, G.M., Henry, S.A., 1989. Phospholipid biosynthesis in yeast. Annu. Rev. Biochem. 58, 635–669. Choi, H.S., Han, G.S., Carman, G.M., 2010. Phosphorylation of yeast phosphatidylserine synthase by protein kinase A: identiﬁcation of Ser46 and Ser47 as major sites of phosphorylation. J. Biol. Chem. 285 (15), 11526–11536. Choi, H.S., Sreenivas, A., Han, G.S., Carman, G.M., 2004. Regulation of phospholipid synthesis in the yeast cki1Delta eki1Delta mutant defective in the Kennedy pathway. The Cho1-encoded phosphatidylserine synthase is regulated by mRNA stability. J. Biol. Chem. 279 (13), 12081–12087. Dembitsky, V.M., 1996. Betaine ether-linked glycerolipids: chemistry and biology. Prog. Lipid Res. 35, 1–51. Eichenberger, W., 1982. Distribution of diacylglyceryl.o-40 -n,n,n-trimethyl)homoserine in different algae. Plant Sci. Lett. 24, 91–95. Essigmann, B., Güler, S., Narang, R.A., Linke, D., Benning, C., 1998. Phosphate availability affects the thylakoid lipid composition and the expression of SQD1, a gene required for sulfolipid biosynthesis in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 95, 1950–1955. Gardes, M., Bruns, T.D., 1993. ITS primers with enhanced speciﬁcity for Basidiomycetes: application to the identiﬁcation of mycorrhizae and rusts. Mol. Ecol. 2, 113–118. Geiger, O., Röhrs, V., Weissenmayer, B., Finan, T.M., Thomas-Oates, J.E., 1999. The regulator gene phoB mediates phosphate stress-controlled synthesis of the membrane lipid diacylglyceryl-N,N,N-trimethylhomoserine in Rhizobium (Sinorhizobium) meliloti. Mol. Microbiol. 32, 63–73. Greenberg, M.L., Lopes, J.M., 1996. Genetic regulation of phospholipid biosynthesis in Saccharomyces cerevisiae. Microbiol. Rev. 60, 1–20. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95–98. Henry, T.C., Power, J.E., Kerwin, C.L., Mohammed, A., Weissman, J.S., Cameron, D.M., Wykoff, D.D., 2011. Systematic screen of Schizosaccharomyces pombe deletion collection uncovers parallel evolution of the phosphate signal transduction pathway in yeasts. Eukaryot. Cell 10, 198–206. Härtel, H., Dörmann, P., Benning, C., 2000. DGD1-independent biosynthesis of extraplastidic galactolipids following phosphate deprivation in Arabidopsis. Proc. Natl. Acad. Sci. USA 97, 10649–10654.
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