Diacylglyceryltrimethylhomoserine content and gene expression changes triggered by phosphate deprivation in the mycelium of the basidiomycete Flammulina velutipes

Highlights

• The basidial fungus Flammulina velutipes synthesizes DGTS under P_i starvation.

• DGTS accumulation is concomitant with PC depletion.

• Transcript levels of BTA1 gene, coding DGTS synthase increase under P_i starvation.

• Genes of PC biosynthesis CHO2 and CPT1 have increased expression under P_i starvation.

• Phylogenetic relationships between basidiomycete BTA1 orthologs are discussed.

Abstract

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 (P_i) 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.

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 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 three-step 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 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 P_i 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, Hjelmstad and Bell, 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 P_i 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.