The role of lysophosphatidic acid acyltransferase 1 in reproductive growth of Arabidopsis thaliana.

Lysophosphatidic acid acyltransferase1 (LPAT1) catalyzes the second step of de novo glycerolipid biosynthesis in chloroplasts. However, the embryonic-lethal phenotype of the knockout mutant suggested an unknown role for LPAT1 in non-photosynthetic reproductive organs. Reciprocal genetic crossing of the lpat1-1 heterozygous line suggested a female gametophytic defect of the lpat1-1 knockout mutant. By suppressing LPAT1 specifically during seed development, we showed that LPAT1 suppression affected silique growth and seed production. Glycerolipid analysis of the LPAT1 knockdown lines revealed a pronounced decrease of phosphatidylcholine (PC) content in mature siliques along with an altered polyunsaturation level of the polar glycerolipids. In seeds, the acyl composition of triacylglycerol (TAG) was altered albeit not the content. These results indicate that plastidic LPAT1 plays an important role in reproductive growth and extraplastidic glycerolipid metabolism involving PC and TAG.

Plastid LPAT1 is an integral inner envelope membrane protein with the acyltransferase domain located in the stroma.

KEY MESSAGE: The N-terminal transmembrane domain of LPAT1 crosses the inner membrane placing the N terminus in the intermembrane space and the C-terminal enzymatic domain in the stroma. Galactolipids mono- and di-galactosyl diacylglycerol are the major and vital lipids of photosynthetic membranes. They are synthesized by five enzymes hosted at different sub-chloroplast locations. However, localization and topology of the second-acting enzyme, lysophosphatidic acid acyltransferase 1 (LPAT1), which acylates the sn-2 position of glycerol-3-phosphate (G3P) to produce phosphatidic acid (PA), remain unclear. It is not known whether LPAT1 is located at the outer or the inner envelope membrane and whether its enzymatic domain faces the cytosol, the intermembrane space, or the stroma. Even the size of mature LPAT1 in chloroplasts is not known. More information is essential for understanding the pathways of metabolite flow and for future engineering endeavors to modify glycerolipid biosynthesis. We used LPAT1 preproteins translated in vitro for import assays to determine the precise size of the mature protein and found that the LPAT1 transit peptide is at least 85 residues in length, substantially longer than previously predicted. A construct comprising LPAT1 fused to the Venus fluorescent protein and driven by the LPAT1 promoter was used to complement an Arabidopsis lpat1 knockout mutant. To determine the sub-chloroplast location and topology of LPAT1, we performed protease treatment and alkaline extraction using chloroplasts containing in vitro-imported LPAT1 and chloroplasts isolated from LPAT1-Venus-complemented transgenic plants. We show that LPAT1 traverses the inner membrane via an N-terminal transmembrane domain, with its N terminus protruding into the intermembrane space and the C-terminal enzymatic domain residing in the stroma, hence displaying a different membrane topology from its bacterial homolog, PlsC.

LYSOPHOSPHATIDIC ACID ACYLTRANSFERASE 2 (LPAT2) is required for de novo glycerolipid biosynthesis, growth, and development in vegetative and reproductive tissues of Arabidopsis.

The Kennedy pathway is a highly conserved de novo glycerolipid biosynthesis pathway in prokaryotes and eukaryotes. In Arabidopsis, LYSOPHOSPHATIDIC ACID ACYLTRANSFERASE 2 (LPAT2) was assumed to catalyze a crucial reaction step of the endoplasmic reticulum (ER)-localized Kennedy pathway because of lethality in the lpat2-1 knockout mutant. However, whether this lethal phenotype was due to the essential role of the Kennedy pathway or LPAT2 as the key enzyme of the Kennedy pathway was unclear. By creating non-lethal LPAT2-knockdown mutants in Arabidopsis, we found that LPAT2 is required for phospholipid content and plant development in vegetative and reproductive growth. Functional in vivo reporter assays revealed that LPAT2 was ubiquitously expressed and localized to the ER, where de novo phospholipid biosynthesis takes place. Intriguingly, our lipid analysis revealed that LPAT2 suppression had different effects among the organs examined: phospholipid levels were decreased both in leaves and flowers and the effect was more pronounced in flowers, a non-photosynthetic organ enriched with phospholipids. Although seed size was reduced in the LPAT2 suppression lines, no remarkable effect was observed in the lipid content of mature siliques. Our results show that LPAT2 is involved in the ER-localized Kennedy pathway, and suggest that its contribution to de novo phospholipid biosynthesis may have organ selectivity.