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.

Compartmentation of metabolism in developing seeds is poorly understood due to the lack of data on metabolite distributions at the subcellular level. In this report, a non-aqueous fractionation method is described that allows subcellular concentrations of metabolites in developing barley endosperm to be calculated. (i) Analysis of subcellular volumes in developing endosperm using micrographs shows that plastids and cytosol occupy 50.5% and 49.9% of the total cell volume, respectively, while vacuoles and mitochondria can be neglected. (ii) By using non-aqueous fractionation, subcellular distribution between the cytosol and plastid of the levels of metabolites involved in sucrose degradation, starch synthesis, and respiration were determined. With the exception of ADP and AMP which were mainly located in the plastid, most other metabolites of carbon and energy metabolism were mainly located outside the plastid in the cytosolic compartment. (iii) In developing barley endosperm, the ultimate precursor of starch, ADPglucose (ADPGlc), was mainly located in the cytosol (80–90%), which was opposite to the situation in growing potato tubers where ADPGlc was almost exclusively located in the plastid (98%). This reflects the different subcellular distribution of ADPGlc pyrophosphorylase (AGPase) in these tissues. (iv) Cytosolic concentrations of ADPGlc were found to be close to the published Km values of AGPase and the ADPGlc/ADP transporter at the plastid envelope. Also the concentrations of the reaction partners glucose-1-phosphate, ATP, and inorganic pyrophosphate were close to the respective Km values of AGPase. (v) Knock-out of cytosolic AGPase in Riso16 mutants led to a strong decrease in ADPGlc level, in both the cytosol and plastid, whereas knock-down of the ADPGlc/ADP transporter led to a large shift in the intracellular distribution of ADPGlc. (v) The thermodynamic structure of the pathway of sucrose to starch was determined by calculating the mass–action ratios of all the steps in the pathway. The data show that AGPase is close to equilibrium, in both the cytosol and plastid, whereas the ADPGlc/ADP transporter is strongly displaced from equilibrium in vivo. This is in contrast to most other tissues, including leaves and potato tubers. (vi) Results indicate transport rather than synthesis of ADPGlc to be the major regulatory site of starch synthesis in barley endosperm. The reversibility of AGPase in the plastid has important implications for the regulation of carbon partitioning between different biosynthetic pathways.