a b s t r a c tThe Arabidopsis thaliana gene At1g74030 codes for a putative plastid phosphoenolpyruvate (PEP) enolase (ENO1). The recombinant ENO1 protein exhibited enolase activity and its kinetic properties were determined. ENO1 is localized to plastids and expressed in most heterotrophic tissues including trichomes and non-root-hair cells, but not in the mesophyll of leaves. Two T-DNA insertion eno1 mutants exhibited distorted trichomes and reduced numbers of root hairs as the only visible phenotype. The essential role of ENO1 in PEP provision for anabolic processes within plastids, such as the shikimate pathway, is discussed with respect to plastid transporters, such as the PEP/phosphate translocator.

Restriction of phosphoenolpyruvate (PEP) supply to plastids causes lethality of female and male gametophytes in Arabidopsis thaliana defective in both a phosphoenolpyruvate/phosphate translocator (PPT) of the inner envelope membrane and the plastid-localized enolase (ENO1) involved in glycolytic PEP provision. Homozygous double mutants of cue1 (defective in PPT1) and eno1 could not be obtained, and homozygous cue1 heterozygous eno1 mutants [cue1/eno1 (+/2)] exhibited retarded vegetative growth, disturbed flower development, and up to 80% seed abortion. The phenotypes of diminished oil in seeds, reduced flavonoids and aromatic amino acids in flowers, compromised lignin biosynthesis in stems, and aberrant exine formation in pollen indicate that cue1/eno1(+/2) disrupts multiple pathways. While diminished fatty acid biosynthesis from PEP via plastidial pyruvate kinase appears to affect seed abortion, a restriction in the shikimate pathway affects formation of sporopollonin in the tapetum and lignin in the stem. Vegetative parts of cue1/eno1(+/2) contained increased free amino acids and jasmonic acid but had normal wax biosynthesis. ENO1 overexpression in cue1 rescued the leaf and root phenotypes, restored photosynthetic capacity, and improved seed yield and oil contents. In chloroplasts, ENO1 might be the only enzyme missing for a complete plastidic glycolysis. INTRODUCTIONPhosphoenolpyruvate (PEP) plays a central role in plant metabolism. As an intermediate of glycolysis, PEP is indispensable for energy metabolism in the cytosol and delivers ATP and pyruvate by the action of cytosolic pyruvate kinase (PK) (Plaxton, 1996;Givan, 1999). Pyruvate can be fed into the citric acid cycle, yielding NADH for respiratory ATP generation (Fernie et al., 2004). Inside the plastids, PEP may act as a precursor for at least four metabolic pathways ( Figure 1A).Together with erythrose 4-phosphate, PEP is fed into the shikimate pathway, which delivers essential aromatic amino acids and a large number of secondary plant products. The initial steps of the shikimate pathway are exclusively localized within the plastid stroma (Herrmann, 1995;Schmid and Amrhein, 1995;Herrmann and Weaver, 1999). Inside the stroma, PEP can also be sequentially metabolized to pyruvate and acetyl-CoA by plastid PK and the pyruvate dehydrogenase complex (Reid et al., 1977;Elias and Givan, 1979; Lernmark and Gardeströ m, 1994) and thus enter the biosynthesis of fatty acids (Dennis, 1989;Ohlrogge and Jaworski, 1997), which are quantitatively important for triacylglycerol production in oil seeds (e.g., Voelker and Kinney, 2001;Rawsthorne, 2002;Ruuska et al., 2002). Like the shikimate pathway, the de novo biosynthesis of fatty acids for membranes and storage lipids is localized to the plastids (Ohlrogge et al., 1979;Ohlrogge and Jaworski, 1997). Moreover, stromal pyruvate can act as a precursor for the synthesis of branched-chain amino acids (Schulze-Siebert et al., 1984) and together with glyceraldehyde 3-phosphate for the mevalonate-independent way (2-C-methyl-...