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First published online April 20, 2007; 10.1104/pp.107.095414 Plant Physiology 144:904-914 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Deficiency in Phosphatidylserine Decarboxylase Activity in the psd1 psd2 psd3 Triple Mutant of Arabidopsis Affects Phosphatidylethanolamine Accumulation in Mitochondria1,[OA]Max Planck Institute of Molecular Plant Physiology, Department of Molecular Physiology, 14476 Potsdam, Germany (A.N., P.D.); Institute of Plant Physiology, Free University of Berlin, 14195 Berlin, Germany (M.v.O.); Librophyt, Centre de Cadarache, 13115 St. Paul-Lez-Durance, France (D.R.); and Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611–0690 (A.D.H.)
Phosphatidylserine (PS) decarboxylase is involved in the synthesis of the abundant phospholipid phosphatidylethanolamine (PE), particularly in mitochondria, in many organisms, including yeast (Saccharomyces cerevisiae) and animals. Arabidopsis (Arabidopsis thaliana) contains three genes with sequence similarity to PS decarboxylases, and the respective gene products were functionally characterized after heterologous expression in yeast and Escherichia coli. While the PSD1 protein localizes to mitochondria, PSD2 and PSD3 are found in the endomembrane system. To study the role of PSD genes in plant phospholipid metabolism, Arabidopsis insertional mutants for psd1, psd2, and psd3 were obtained. The single mutants were decreased in PS decarboxylase activity to various extents, but mutant plants showed no obvious growth or morphological phenotype. A triple mutant, psd1 psd2 psd3, was generated that was totally devoid of PS decarboxylase activity. While the phospholipid composition in whole leaves was unchanged, the PE content in isolated mitochondria of psd1 psd2 psd3 was decreased. Therefore, the predominant proportion of PE in Arabidopsis is synthesized by alternative pathways, but a significant amount of mitochondrial PE is derived from the PS decarboxylase reaction. These results imply that, similar to yeast and animal cells, a specific phospholipid transfer from the endoplasmic reticulum to mitochondria exists in plants.
In contrast to chloroplasts of higher plants where galactolipids are predominant, phospholipids (e.g. phosphatidylcholine and phosphatidylethanolamine [PE]) are the most abundant lipids in extraplastidial membranes. Phospholipid metabolism in plants involves a complex network of biosynthetic pathways, some of which are localized to different subcellular compartments. Two phospholipids, PE and phosphatidyl-Ser (PS), which can be interconverted in many organisms, are characterized by the presence of an ammonium moiety in their head group. PE represents the most abundant nonbilayer-forming lipid in extraplastidial membranes (Webb and Green, 1991  ), and its deficiency was shown to result in severe developmental defects in a number of organisms (yeast [Saccharomyces cerevisiae]: Trotter and Voelker, 1995; Birner et al., 2001; hamster ovary cells: Emoto and Umeda, 2000; Drosophila: Pavlidis et al., 1994; mouse: Steenbergen et al., 2005). The aminophospholipid PS is of very low abundance in most membranes, and it serves as a precursor for PE synthesis. PS in animal cells is confined to the inner leaflet of the plasma membrane, and this distinct distribution is maintained by specific transporters. Exposure of PS to the outer leaflet of the plasma membrane serves as signal for apoptosis in animal cells and results in phagocytosis of affected cells (Vance and Steenbergen, 2005). In plants, distinct pools of PS seem to exist that localize to different membranes. In contrast to other membrane lipids, the PS pool of the endoplasmic reticulum (ER) and of the plasma membranes was shown to be enriched in very long chain fatty acids (Bohn et al., 2001; Vincent et al., 2001).
PS metabolism in different organisms was recently reviewed by Vance and Steenbergen (2005)
In castor bean endosperm, radioactive label from [14C]Ser was incorporated into PS and PE, suggesting that PS was synthesized via the PE-PS base exchange enzyme and that a fraction of PS was decarboxylated (Moore, 1975
The first molecular study on PS decarboxylases was the isolation of the temperature-sensitive Escherichia coli mutant EH150, which accumulates large amounts of PS when grown at 42°C (Hawrot and Kennedy, 1975
Two PS decarboxylase proteins were found in yeast: PSD1 localizes to the mitochondria (Clancey et al., 1993
Additional PS decarboxylases have been isolated from bacteria (Bacillus subtilis; Matsumoto et al., 1998
The first molecular study on higher plant PS decarboxylases was the isolation and characterization of the PSD1 cDNAs from tomato (Solanum lycopersicum) and Arabidopsis (Rontein et al., 2003b
Isolation and Functional Characterization of PS Decarboxylases from Arabidopsis
In many eukaryotic organisms, mitochondrial PS decarboxylase is involved in the synthesis of a major proportion of cellular PE. Mitochondrial PS decarboxylases were identified in yeast, Chinese hamster, tomato, and Arabidopsis (Kuge et al., 1991
To functionally characterize the putative PS decarboxylases PSD2 and PSD3 from Arabidopsis, the respective cDNAs were heterologously expressed in psd mutants of yeast and E. coli. A yeast double mutant (psd1 psd2) deficient in both PSD genes is unable to synthesize PE via the PS decarboxylase pathway. This mutant is auxotroph for ethanolamine because PE can be synthesized from ethanolamine via the Kennedy pathway. Transformation of the yeast psd1 psd2 mutant with plasmids harboring the Arabidopsis PSD2 or PSD3 cDNAs restored growth on ethanolamine-free medium, indicating that these two sequences encode functional PS decarboxylases (Fig. 2A ). An alternative strategy to study PS decarboxylase activity is the complementation of the E. coli EH150 mutant, which carries a temperature-sensitive mutation in its single psd gene. To address the question whether the C-terminal part of PSD3 carrying the PS decarboxylase sequence domain is sufficient to encode a functionally active enzyme, the entire PSD3 cDNA or its C-terminal part (starting at Ser-352; Fig. 1) was transferred into the E. coli mutant EH150. Proteins were isolated from the EH150 mutant carrying the Arabidopsis PSD3 constructs, and PS decarboxylase activity measured at 42°C with radioactively labeled PS. Figure 2B shows that PS decarboxylase activity in EH150 cells transformed with an empty vector or with the full-length Arabidopsis PSD3 cDNA was low as compared to cells harboring the N-terminally truncated form. Therefore, the C-terminal, PSD-like part of Arabidopsis PSD3 encodes a functionally active PS decarboxylase, and the long N terminal is not required for enzymatic activity but has an inhibitory effect when expressed in E. coli.
The fact that the two Arabidopsis PS decarboxylases PSD2 and PSD3 contain a long N-terminal extension might suggest that these two proteins do not localize to mitochondria. The full-length sequences of PSD2 and PSD3 were N-terminally fused to the sequence of the GFP and transiently expressed in Arabidopsis leaves. As a control, a GFP fusion construct of the mitochondrial PS decarboxylase (PSD1) from Arabidopsis was employed. Analysis by confocal microscopy and comparison to control constructs revealed that the PSD1-GFP fusion protein accumulated in mitochondria in agreement with previous results (Fig. 3, A and B
; Rontein et al., 2003b
Expression of PS decarboxylase genes in different plant organs was analyzed by northern hybridization and semiquantitative PCR analysis. In agreement with previous results (Rontein et al., 2003b ), the mitochondrial PSD1 was found to be expressed in roots and to a lesser extent in stems and leaves (Fig. 4A
). PSD1 expression was highest in flowers but very low in siliques. PSD3 expression was comparable in roots, stems, leaves, and flowers, but very low in siliques (Fig. 4C). Because of the high sequence similarity with PSD3 and the low expression level, it was not possible to detect PSD2 expression by northern hybridization (data not shown). Therefore, a specific PSD2 primer pair was designed for semiquantitative reverse transcription (RT)-PCR to distinguish between PSD2 and PSD3 mRNA expression. As shown in Figure 4B, PSD2 expression was highest in flowers and lower in leaves. Expression in roots, shoots, and siliques was very low when measured by RT-PCR.
Isolation of Arabidopsis Mutants Defective in PS Decarboxylase Activity To study the role of PS decarboxylase in plant lipid metabolism, a reverse genetic approach was chosen. To this end, T-DNA insertion mutant lines for the three PSD genes were selected from different Arabidopsis mutant populations (Fig. 5A ). One mutant line, psd1, was found in the SALK population. Screening of the Madison T-DNA insertion population resulted in the identification of two mutant lines, psd2-1 and psd3-1. Two additional mutant alleles, psd2-2 and psd2-3, were obtained from Syngenta. Sequencing of PCR fragments revealed that the insertions in all mutant plants are localized in exons or introns between the start and stop codons (Fig. 5A). Homozygous lines for all mutant alleles were identified by PCR, segregation analysis, and Southern-blot hybridization using genomic PSD fragments as probes (data not shown). PSD mRNA abundance in psd mutants was analyzed to unravel whether there still was residual expression (Fig. 5B). For psd1, northern hybridization was done with total RNA from flowers because PSD1 expression was high in flowers (see Fig. 4). PSD1 expression in the psd1 mutant was not detectable, in contrast to wild type, where a clear band was identified. Northern hybridization of leaf RNA revealed that one band corresponding to PSD3 was detectable in wild type, but this band was absent from the psd3-1 mutant (Fig. 5B). PSD2 expression was analyzed by RT-PCR of leaf RNA and PCR fragments separated by agarose gel electrophoresis. A band for PSD2 was observed in wild type, but this band was absent from psd2-1. In conclusion, the mRNA of the respective PSD genes was shown to be absent from the psd1, psd2-1, and psd3-1 mutant lines, indicating that these insertion lines represent null alleles.
Growth and morphology of the different mutant lines was indistinguishable from the respective wild-type ecotypes, indicating that single mutations in PSD genes have no effect on overall plant physiology. To address the question whether the different PS decarboxylase forms are functionally redundant, double mutants were generated by crosses between the psd2-1 and psd3-1 as well as the psd2-2 and psd3-2 mutant lines. PSD2 and PSD3 share a high degree of sequence similarity, and the two proteins localize to the endomembrane system, suggesting that they might have redundant functions. However, similar to the psd single mutants, growth and morphology of the two double mutant lines, psd2-1 psd3-1 and psd2-2 psd3-2, were very similar to wild type. To obtain plants carrying T-DNA insertions in all three PS decarboxylase genes, triple mutant lines (psd1 psd2-1 psd3-1 and psd1 psd2-2 psd3-2) were generated by crossing the double mutant lines psd2-1 psd3-1 or psd2-2 psd3-2 with the psd1 single mutant. Growth of the two lines psd1 psd2-1 psd3-1 and psd1 psd2-2 psd3-2 was not different from wild type (data not shown). PS decarboxylase activity was measured in microsomal membranes isolated from leaves. The enzyme activities in wild type, psd2-1, and psd2-2 were very similar, indicating that the PSD2 enzyme is responsible for only a minor fraction of the total PS decarboxylase activity in leaves (Fig. 6A ; data not shown). This is in agreement with the low expression level of PSD2 (Fig. 4; data not shown). Microsomal PSD activities in psd3-1, psd3-2, and the psd2-1 psd3-1 double mutant were decreased to about one-third of wild-type activity (Fig. 6A; data not shown). Therefore, PSD3 represents the major PS decarboxylase activity in Arabidopsis leaves. PSD activity in microsomal fractions of the psd1 psd2-1 psd3-1 triple mutant was below detection limit. The residual amount of PSD activity found in the psd2-1 psd3-1 double mutant is derived from PSD1, suggesting that the microsomal fraction contained some mitochondrial membranes. The complete absence of PSD activity from the psd1 psd2-1 psd3-1 triple mutant is in agreement with the assumption that this mutant plant carries three null mutations in the PSD genes and that Arabidopsis contains no other membrane-associated protein capable of decarboxylating PS. Mitochondria were isolated from dark-grown seedlings and employed for PSD activity assays (Fig. 6B). PSD activity in mitochondria of the psd1 mutant was decreased to less than 10% of wild-type activity. The very low activity in psd1 mitochondria presumably is derived from PSD2 and PSD3 activities still associated with the mitochondrial preparation.
Phospholipid Composition in PS Decarboxylase-Deficient Mutants
PS decarboxylase activity was shown to be required for the synthesis of a large proportion of PE in yeast and animals. Therefore, a block in PSD gene expression in Arabidopsis mutants was expected to affect the PE content, particularly in mitochondria, possibly accompanied with an increase of the substrate, PS. Total lipids were isolated from leaves of wild type and the single, double, and triple mutant lines (psd1, psd2-1, psd3-1, psd2-1 psd3-1, and psd1 psd2-1 psd3-1) and after separation via two-dimensional thin-layer chromatography (TLC), quantified by gas chromatography (GC) of fatty acid methyl esters. Lipid contents were unchanged in all lines analyzed (Fig. 7A
). Therefore, partial or total deficiency in PSD activity in Arabidopsis mutants has no measurable effect on PE synthesis in leaves. The contents of phospholipids in leaves are considerably low because leaves are rich in galactolipids. Therefore, lipid analysis of nongreen plant organs (e.g. flowers) that contain higher proportions of phospholipids might reveal small differences in phospholipid composition that would not be detectable in leaves. Membrane lipid composition of flowers of the psd1 psd2-1 psd3-1 triple mutant was also not changed as compared to wild type (Fig. 7B). Fatty acid composition of PE and PS in plants is characterized by a high proportion of linoleic acid (18:2) and
In yeast, mitochondria represent an essential site for PE synthesis via the PS decarboxylase pathway. To elucidate the role of PS decarboxylase in plants for mitochondrial PE synthesis, mitochondria were isolated from dark-grown seedlings of the Arabidopsis psd1 and psd1 psd2-1 psd3-1 triple mutant and employed for lipid analysis. The amount of PE was slightly decreased in psd1 (data not shown), and it was even further reduced in psd1 psd2-1 psd3-1 (Fig. 7C). Concomitantly, the amount of PS was increased in mitochondria of psd1 psd2-1 psd3-1.
Two classes of PS decarboxylases can be distinguished in different organisms based on sequence similarity (Fig. 1). Eukaryotic organisms contain PS decarboxylases that localize to the endomembrane system and carry a long N-terminal extension. A second class of PS decarboxylases includes prokaryotic and mitochondrial enzymes. The close relationship between mitochondrial and bacterial PS decarboxylases can be explained by the common origin according to the endosymbiont hypothesis. Here, we show that in addition to PSD1, which localizes to the mitochondria (Rontein et al., 2003b ), Arabidopsis contains two further PS decarboxylases (PSD2 and PSD3) of eukaryotic type that localize to the tonoplast and ER, respectively (Fig. 3). All three enzymes encode functionally active PS decarboxylases (Fig. 2). Analysis of single and multiple T-DNA insertion mutants for all three genes revealed that in leaves, about two-thirds of total PS decarboxylase activity is associated with PSD3, while PSD2 shows only very low activity. PSD1 is the major PS decarboxylase enzyme of mitochondria, and it also contributes to about one-third of total leaf PSD activity (Fig. 6).
PE represents one of the major phospholipids in all organisms. It is abundant in the plasma membrane, ER, tonoplast, nuclear membrane, and mitochondria but is absent from plastids. Three pathways have been described to contribute to PE synthesis: (1) the transfer of phosphoethanolamine from CDP-ethanolamine onto diacylglycerol by aminoalcoholphospho-transferase (Kennedy pathway); (2) decarboxylation of PS by PS decarboxylase; and (3) base exchange reaction of PS with ethanolamine. In yeast, mitochondria are essential for PE production for the entire cell because PS synthesized at the ER is transported to the mitochondria for subsequent decarboxylation by PSD. PE produced in yeast mitochondria is redistributed to the entire cell. No change of PE content was observed in leaves or flowers of the Arabidopsis psd1 psd2 psd3 triple mutant (Fig. 7), indicating that in contrast to other organisms, PS decarboxylase is not a major pathway for PE synthesis in plants. These results are in line with data recently obtained by Mizoi et al. (2006)
Origin of PS Decarboxylase cDNAs The PSD1 (At4g16700) full-length open reading frame was amplified from first-strand cDNA derived from Arabidopsis (Arabidopsis thaliana) leaf mRNA (Superscript cDNA synthesis kit; Invitrogen) by PCR using the primers PD288 and PD246 (Table II ). The PCR product was ligated into the SphI, HindIII sites of pQE30 (Qiagen), resulting in the vector pQE30-PSD1. The coding region of PSD2 (At5g57190) was amplified from single-strand cDNA by PCR with the primers PD97 and PD98, thereby introducing SphI and KpnI sites at the 5' end and 3' end, respectively. The PSD2 PCR fragment was subcloned into pGEMTeasy (Promega), resulting in the construct pGEMTeasy-PSD2 and sequenced on both strands. The EST (GenBank accession no. AV527283) of PSD3 (At4g25970) was obtained from the Kazusa DNA Research Institute. Sequencing revealed that this clone contained the authentic PSD3 cDNA in the EcoRI and XhoI sites of pBluescriptIISK–. The full-length coding sequences for At-PSD2 (At5g57190) and At-PSD3 (At4g25970) were deposited in GenBank with the accession numbers EF203902 and EF203901, respectively.
PS Decarboxylase Assay
PS decarboxylase activity was measured by incubating protein extracts with radioactive PS (sn-1,2-dioleoyl-3-phosphoryl-L-[3-14C]Ser, Amersham). Each reaction (total volume of 200 µL) contained 1 mg protein in 50 mM Tris-HCl, pH 7.0, 0.1% Triton X-100, 0.05 µCi (130 ng) radioactive PS, and 1.3 µg nonradioactive PS. Reactions were incubated at 28°C for 10 min. Lipids were extracted after addition of 100 µL 1 M KCl, 0.2 M H3PO4, and 200 µL chloroform:methanol (2:1) and separated by TLC on silica plates according to Hawrot and Kennedy (1975)
Lipids were extracted from leaves and flowers with chloroform-methanol and separated via two-dimensional TLC with chloroform:methanol:water (65:25:4) and chloroform:acetone:methanol:acetic acid:water (50:20:10:10:5) according to Benning et al. (1995)
The yeast (Saccharomyces cerevisiae) strains were RYY51 (ura3 his3 trp1 leu2 lys2 psd1-1::TRP1 psd2-1::HIS3) and the corresponding wild type, SEY6210 (ura3 his3 trp1 leu2 lys2; Trotter and Voelker, 1995
The full-length PSD3 cDNA was released from pBluescriptIISK– (see above) with XbaI and KpnI and ligated into pKK-MCS. This vector is a derivative of pKK233-2 (GenBank accession no. U02439; Amann and Brosius, 1985
Arabidopsis leaves were homogenized in 50 mM Tris-HCl, pH 7.0, at 4°C by grinding in a mortar with sand. Cell debris was separated by centrifugation (1,200g, 1 min). After centrifugation of the supernatant at 100,000g for 1 h, a microsomal membrane fraction was obtained by resuspending the pellet in 50 mM Tris-HCl, pH 7.0.
A simplified method of Klein et al. (1998)
Arabidopsis plants were grown on soil at 120 µmol m–2 s–1, 16 h light/day, 60% humidity, and 20°C. A mutant carrying a T-DNA insertion in the PSD1 gene (Salk_064716) was obtained from the SALK collection (Arabidopsis Biological Resource Center, The Ohio State University, Columbus, OH; Alonso et al., 2003
The
Additional mutant alleles carrying T-DNA insertions in the genes PSD2 and PSD3 were obtained from Syngenta (SAIL 93b_G04.b.1a and SAIL 851_E09.b.1a, respectively, ecotype Col-0; Sessions et al., 2002 The position of the insertion in the genomic region was confirmed by sequencing of the PCR fragments. Homozygous mutant lines were obtained by segregation and PCR analysis. PCR amplification of homozygous plants using the gene-specific and insert-specific primers resulted in the synthesis of the fragment containing the genomic DNA/T-DNA junction, but due to the large size of the insertion, no band was obtained when using the two gene-specific primers. Furthermore, Southern-blot hybridization using genomic probes confirmed that mutant plants carried homozygous insertions.
Northern blotting was done with total RNA isolated from different Arabidopsis organs (Sambrook et al., 1989
The PSD1 cDNA was amplified from clone pQE30-PSD1 with the primers PD243 and PD244 (Table II) and ligated into the EcoRI and BamHI sites of pEZR-K-LN (Gert-Jan de Boer, Carnegie Institution, Stanford, California) as an N-terminal, translational fusion with GFP. The PCR fragment of PSD2 obtained by PCR amplification of pGEMTeasy-PSD2 with the primers ZP2F and ZP2R was digested with EcoRI and BclI and ligated into the EcoRI and BamHI sites of pEZR-K-LN, resulting in the plasmid pEZR-PSD2. The PSD3 cDNA was amplified by PCR using the primers ZP1F and ZP1R from clone AV527283, introducing new KpnI and BclI restriction sites. This fragment was ligated into the KpnI, BamHI sites of pEZR-K-LN, resulting in the plasmid pEZR-PSD3.
Plasmid DNA was coated onto 1.0-µm gold micro-carriers (Bio-Rad). Transfer into Arabidopsis leaves was achieved by bombardment in a Biolistic PSD-1000/He particle delivery system (Bio-Rad) using 1,100 psi rupture discs according to the manufacturer's protocol. Leaves were incubated on Murashige and Skoog (1962) Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers EF203901 and EF203902.
We thank Professor Elmar Hartmann (Free University of Berlin) for his generous support of this project in its initial phase. The constructs pre-101-GFP and pTPK1-DsRed2 were kindly provided by Katrin Czempinski and Bernd Müller-Röber, University of Potsdam, Germany. Received January 3, 2007; accepted April 10, 2007; published April 20, 2007.
1 This work was supported by the Deutsche Forschungsgemeinschaft (grant no. SFB 429, B6) and by the Max Planck Society.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Peter Dörmann (doermann{at}mpimp-golm.mpg.de).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.095414 * Corresponding author; e-mail doermann{at}mpimp-golm.mpg.de; fax 49–331–567–8259.
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TOP
ABSTRACT
RESULTS
-subunit carrying an N-terminal pyruvoyl prosthetic group, and the N-terminal, 28-kD 
-subunit (Li and Dowhan, 1988
