Isopentenyl diphosphate (IPP) is the biological C 5 precursor of isoprenoids. By labeling experiments using [1-13C)glucose, higher plants were shown to possess two distinct biosynthetic routes for IPP biosynthesis: while the cytoplasmic sterols were formed via the acetate/mevalonate pathway, the chloroplast-bound isoprenoids (~-carotene, lutein, prenyl chains of chlorophylls and plastoquinone-9) were synthesized via a novel IPP biosynthesis pathway (glyceraldehyde phosphate/pyruvate pathway) which was first found in eubacteria and a green alga. The dichotomy in isoprenoid biosynthesis in higher plants allows a reasonable interpretation of previous odd and inconclusive results concerning the biosynthesis of chloroplast isoprenoids, which so far had mainly been interpreted in the frame of models using compartmentation of the mevalonate pathway.

Efficient storage of carbon in seeds is crucial to plant fitness and to agricultural productivity. Oil is a major reserve material in most seeds, and these oils provide the largest source of renewable reduced carbon chains available from nature. However, the conversion of carbohydrate to oil through glycolysis results in the loss of one-third of the carbon as CO2. Here we show that, in developing embryos of Brassica napus L. (oilseed rape), Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) acts without the Calvin cycle and in a previously undescribed metabolic context to increase the efficiency of carbon use during the formation of oil. In comparison with glycolysis, the metabolic conversion we describe provides 20% more acetyl-CoA for fatty-acid synthesis and results in 40% less loss of carbon as CO2. Our conclusions are based on measurements of mass balance, enzyme activity and stable isotope labelling, as well as an analysis of elementary flux modes.

The metabolism of developing plant seeds is directed toward transforming primary assimilatory products (sugars and amino acids) into seed storage compounds. To understand the role of mitochondria in this metabolism, metabolic fluxes were determined in developing embryos of Brassica napus. After labeling with [1,2-13 C 2 ]glucose ؉ [U- 13 N]glutamine, the resulting labeling patterns in protein amino acids and in fatty acids were analyzed by gas chromatography-mass spectrometry. Fluxes through mitochondrial metabolism were quantified using a steady state flux model. Labeling information from experiments using different labeled substrates was essential for model validation and reliable flux estimation. The resulting flux map shows that mitochondrial metabolism in these developing seeds is very different from that in either heterotrophic or autotrophic plant tissues or in most other organisms: (i) flux around the tricarboxylic acid cycle is absent and the small fluxes through oxidative reactions in the mitochondrion can generate (via oxidative phosphorylation) at most 22% of the ATP needed for biosynthesis; (ii) isocitrate dehydrogenase is reversible in vivo; (iii) about 40% of mitochondrial pyruvate is produced by malic enzyme rather than being imported from the cytosol; (iv) mitochondrial flux is largely devoted to providing precursors for cytosolic fatty acid elongation; and (v) the uptake of amino acids rather than anaplerosis via PEP carboxylase determines carbon flow into storage proteins.Directly or indirectly plant seeds provide most of the food consumed by humans. Although the metabolism of developing seeds has been extensively studied, quantitative understanding of fluxes through central metabolism is still quite limited. Brassica napus (canola, oilseed rape) is a major oil crop and is amenable to detailed quantitative flux analysis under conditions that closely mimic in planta seed development (1). The main storage compounds in seeds of B. napus are oil (triacylglycerols) and proteins, which are synthesized by the developing embryo from sugars and amino acids taken up from the surrounding endosperm liquid.Developing seeds of B. napus have also been the subject of numerous biochemical studies and are a model for oil accumulating seeds (2-13). In previous studies we have used intact developing embryos to make a quantitative analysis of steadystate metabolic fluxes during the conversion of carbohydrates to fatty acids in vivo (1, 14 -17). These studies introduced a labeling approach using multiple carbon sources (1), quantified the contribution of the oxidative pentose phosphate pathway to biosynthetic NADPH demands (14), and revealed that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) 2 operates in a novel context of carbohydrate conversion to fatty acids, bypassing glycolytic reactions and increasing the efficiency of seed carbon metabolism (16). However, a detailed description of fluxes through mitochondrial metabolism in B. napus or other developing seeds is lacking.Several functions of mitoc...

The genome of Arabidopsis has been searched for sequences of genes involved in acyl lipid metabolism. Over 600 encoded proteins have been identified, cataloged, and classified according to predicted function, subcellular location, and alternative splicing. At least one-third of these proteins were previously annotated as "unknown function" or with functions unrelated to acyl lipid metabolism; therefore, this study has improved the annotation of over 200 genes. In particular, annotation of the lipolytic enzyme group (at least 110 members total) has been improved by the critical examination of the biochemical literature and the sequences of the numerous proteins annotated as "lipases." In addition, expressed sequence tag (EST) data have been surveyed, and more than 3,700 ESTs associated with the genes were cataloged. Statistical analysis of the number of ESTs associated with specific cDNA libraries has allowed calculation of probabilities of differential expression between different organs. More than 130 genes have been identified with a statistical probability Ͼ 0.95 of preferential expression in seed, leaf, root, or flower. All the data are available as a Web-based database, the Arabidopsis Lipid Gene database (http://www.plantbiology.msu.edu/lipids/genesurvey/index.htm). The combination of the data of the Lipid Gene Catalog and the EST analysis can be used to gain insights into differential expression of gene family members and sets of pathway-specific genes, which in turn will guide studies to understand specific functions of individual genes.Acyl lipids can be defined as fatty acids and their naturally occurring ester, ether, or amide derivatives. In plants, these include acylglycerols such as triacylglycerols (TAGs), phospholipids, galactolipids, and sulfolipids, plus sphingolipids, acylated steryl glycosides, oxylipins, cutins, suberins, estolides and wax, and sterol esters. The list may be extended if we consider molecules immediately derived from acyl groups, such as the epicuticular wax components (hydrocarbons, alcohols, ketones, and so on) or natural products such as anacardic acids that impart protection to predation. Polar lipids are amphipathic and as such self-associate in water to produce a variety of structures. Therefore, they provide the building blocks for biological membranes. There is substantial evidence indicating that the composition of acyl lipids in membranes influences the targeting, distribution, and functional properties of both integral and membrane-associated proteins (Sprong et al., 2001;Wallis and Browse, 2002). Furthermore, many polar lipids and the intermediates in their synthesis and degradation serve as signaling molecules. In summary, acyl lipids function in a wide range of biological processes, such as carbon and free energy storage, cell signaling, modulation of enzyme activity and protein localization, vesicle budding and fusion, waterproofing, and surface protection (Browse and Somerville, 1994).Some acyl lipids such as TAGs, the major constituent of vegetable oils, are ...

Computer simulation of the steady state distribution of isotopomers in intermediates of the glycolysis/OPPP network was used to fit metabolic flux parameters to the experimental data. The observed distribution of label in cytosolic and plastidic metabolites indicated that key intermediates of glycolysis and OPPP have similar labeling in these two compartments, suggesting rapid exchange of metabolites between these compartments compared with net fluxes into end products. Cycling between hexose phosphate and triose phosphate and reversible transketolase velocity were similar to net glycolytic flux, whereas reversible transaldolase velocity was minimal. Flux parameters were overdetermined by analyzing labeling in different metabolites and by using data from different labeling experiments, which increased the reliability of the findings. Net flux of glucose through the OPPP accounts for close to 10% of the total hexose influx into the embryo. Therefore, the reductant produced by the OPPP accounts for at most 44% of the NADPH and 22% of total reductant needed for fatty acid synthesis.Brassica napus (rapeseed, canola) is one of the world's major oilseed crops and is also a well studied model for oilseed metabolism (1-21). The main storage compounds in seeds of B. napus are oil (triacylglycerols) and storage proteins, which are derived from sugars and amino acids taken up from the surrounding endosperm liquid (11,12,22,23). Because of its high oil content and ease of genetic transformation, B. napus has also been a target for metabolic engineering of oil metabolism. However, some attempts to engineer plant oils have had limited success (for a review, see Ref. 24). In order to make advances in improving oil yield and quality, a more detailed understanding of metabolism during seed development is needed. In particular, a number of fundamental metabolic issues remain unresolved. These include the source(s) of reductant and ATP for fatty acid synthesis; the degree to which cytosolic, plastidial, and mitochondrial metabolic fluxes are integrated; and the chief metabolic and transport route(s) by which carbon flows from maternal sources to seed storage products.