Arabidopsis PSD2 and PSD3 encode functional PS decarboxylases. A, Arabidopsis PSD2 and PSD3 complement ethanolamine auxotrophy of the PS decarboxylase-deficient yeast psd1 psd2 double mutant. Wild type and psd1 psd2 double mutant yeast strains harboring different plasmids were grown in the presence or absence of ethanolamine, as indicated. 1, Wild-type yeast (strain SEY6210); 2, double mutant psd1 psd2 (RYY51); 3, psd1 psd2 transformed with empty vector; 4, psd1 psd2 transformed with Arabidopsis PSD3; 5, psd1 psd2 transformed with Arabidopsis PSD2. B, The full-length and the C-terminal domain of the PSD3 cDNA of Arabidopsis harbor PS decarboxylase activity. The temperature-sensitive E. coli strain EH150 deficient in PS decarboxylase activity was transformed with different Arabidopsis PSD3 cDNA constructs. After growth at 42°C, cells were harvested and PS decarboxylase activity measured with ¹⁴C-labeled PS. The figure shows the conversion of PS to PE after lipid separation by TLC and autoradiography. 1, E. coli XL1-Blue (control); 2, EH150; 3, EH150 carrying empty vector; 4, EH150 transformed with the full-length Arabidopsis PSD3 cDNA; and 4, EH150 carrying the C-terminal PS decarboxylase domain of Arabidopsis PSD3 (starting at Ser-352).

Expression of PSD1, PSD2, and PSD3 in different Arabidopsis tissues. Expression of PS decarboxylase was determined with mRNA isolated from different plant tissues using northern hybridization (PSD1 and PSD3) or RT-PCR (PSD2). The top segments for PSD1 and PSD2 show the hybridization signals, and the bottom segments show a photo of the 26 ribosomal RNA bands stained with ethidium bromide. Because of its low expression, PSD2 mRNA was determined by semiquantitative RT-PCR. The top segment shows the band for the PSD2 RT-PCR product in agarose gel electrophoresis and the bottom segment for ubiquitin (control). 1, Roots; 2, stems; 3, leaves; 4, flowers; and 5, siliques.

PS decarboxylase activity in psd mutants. Plant protein was incubated with [¹⁴C]PS, and after lipid extraction [¹⁴C]PE production was quantified after TLC and autoradiography. A, PS decarboxylase activity in microsomal fractions. B, PS decarboxylase activity in mitochondria.

PS decarboxylases in Arabidopsis. A, Amino acid sequence comparison of Arabidopsis PSD1 (At4g16700), PSD2 (At5g57190), and PSD3 (At4g25970). Arabidopsis contains three genes with sequence similarities to PS decarboxylases. In contrast to the mitochondrial PS decarboxylase, PSD1, the PSD2 and PSD3 proteins contain an N-terminal extension of approximately 350 amino acids. A truncated form of PSD3 starting with the amino acid Ser-352 (indicated with an asterisk) was expressed in E. coli ( Fig. 2B). Identical amino acids are highlighted in black, and gaps are indicated with dashes. The conserved GSTV motif is marked with a box. B, Phylogenetic tree of PS decarboxylases. Amino acid sequences (the C-terminal 300 amino acids without N-terminal extensions) were compared using the ClustalW program of the Lasergene DNAstar software. Numbers indicate the nucleotide substitutions (×100). The sequences on the right depict the conserved sequence motif G-ST, at which the precursor protein is processed into the α and β polypeptides, constituting mature PS decarboxylase. The dashed line separates the two groups of PS decarboxylases containing the mitochondrial/bacterial and endomembrane forms. At-PSD1, AY189805 (At4g16700), Arabidopsis, and Le-PSD, AY093689, tomato, Rontein et al. (2003b); Sc-PSD1, L20973, yeast, Clancey et al. (1993); Cg-PSD, P27465, Chinese hamster, Kuge et al. (1991); Bs-PSD, P39822, B. subtilis, Matsumoto et al. (1998); Ec-PSD, J03916, E. coli, Li and Dowhan (1988); At-PSD2 (At5g57190) and At-PSD3, AV527283 (At4g25970), Arabidopsis, this study; Sc-PSD2, U19910, yeast, Trotter et al. (1995). 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.

Subcellular localization of Arabidopsis PS decarboxylases. N-terminal fusion constructs of full-length PSD proteins with GFP were transferred into Arabidopsis leaves by bombardment and subcellular localization analyzed by confocal fluorescence microscopy. A, PSD1; B, mitochondrial control (pre-101-GFP); C, PSD2; D, tonoplast control (KCO1-GFP); E, PSD3; F, ER control (KDEL-GFP). Bar = 20 μm.

PS decarboxylase mutants of Arabidopsis. A, Localization of T-DNA insertions in psd1, psd2, and psd3 mutants. Positions of insertions are indicated relative to the exon/intron structures of PSD genes. Exons are depicted by boxes and numbered from 5′ to 3′. Arrows show positions of oligonucleotides used for PCR analysis. For PSD1, one insertional mutant (psd1) was identified in the SALK collection. Two mutant alleles were found for PSD2, psd2-1 and psd2-2, from the Madison collection and from Syngenta, respectively. The psd3-1 and psd3-2 mutant alleles were derived from the Madison and from Syngenta, respectively. B, Expression of PSD genes in Arabidopsis psd1, psd2-1, and psd3-1 mutants. Expression of PSD1 and PSD3 were recorded by northern hybridization of total RNA isolated from flowers or leaves, respectively. The top segment shows the hybridization signal, and the bottom segment shows a photo of the 26S rRNA band stained with ethidium bromide (control). Because of its low expression, PSD2 was determined by semiquantitative RT-PCR. The top and bottom segments show the RT-PCR band for PSD2 and ubiquitin, respectively.

Lipid composition of the psd1 psd2-1 psd3-1 triple mutant. Total lipids were isolated from whole tissues or organelles, separated by TLC, and lipids quantified by GC analysis of fatty acid methyl esters. A, Lipid composition in leaves of wild-type Col-0 and psd1 psd2-1 psd3-1. B, Lipid composition in whole flowers of wild-type Col-0 and psd1 psd2-1 psd3-1. C, Mitochondria were isolated from dark-grown seedlings of wild-type Col-0 and psd1 psd2-1 psd3-1 and used for the determination of phospholipid composition.