Plastid synthesis

One of the more exciting and recent advances in the field of plant biochemistry has been the discovery of the mevalonate-independent pathway for the biosynthesis of isoprenoids (Fig. 10.4). This new pathway, referred to a the methyl-erythritol-phosphate or MEP pathway for the first intermediate committed solely to the biosynthesis of isoprenoids, was first discovered in prokaryotes capable of accumulating hopenes, the equivalent of eukaryotic sterols. 6,17 The MEP pathway has since been confirmed in plants and, not surprisingly, has been localized to chloroplasts.18 Operation of the MEP pathway is intimately related to the reactions of CO2 fixation and photosynthesis, as evidenced by the two immediate precursors pyruvate and phosphoglyceraldehyde for this pathway. Two important features of this pathway are that mevalonate is not an intermediate in the plastidic synthesis of isopentenyl (IPP) and dimethylallyl diphosphate, (DMAPP), and this pathway... 

Fig. 1. Models for the light-harvesting complexes surrounding the PS-II reaction center in the thylakoid (A) Simplified model of the thylakoid in cross section, (B) More detailed model of PS II. (A) taken from Fig. 21 (A) in Chapter 1. The light-harvesting complex portion in (B) adapted from H-E Akerlund (1993) Function and organization of photosysfem II. In C Sundqvist and M Ryberg (eds) Pigment-Protein Complexes in Plastids Synthesis and Assembly, p 419. Acad Press.

Nieto-Sotelo, J. Ho, T.-H.D. (1987). Absence of heat shock protein synthesis in isolated mitochondria and plastids from maize. Journal of Biological Chemistry, 262, 12288-92. 

Poly(3HB) is synthesized in bacteria from acetyl-CoA by a three-step reaction (Fig. 1). The first enzyme of the pathway, 3-ketothiolase, catalyzes the condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA. Aceto-acetyl-CoA reductase subsequently reduces acetoacetyl-CoA to R-3-hydroxy-butyryl-CoA, which is then polymerized by the PHA synthase to produce poly(3HB). Since acetyl-CoA is present in plant cells in the cytosol, plastid, mitochondrion, and peroxisome, the synthesis of poly(3HB) in plants could, in... 

It has, thus, been demonstrated that redirecting the poly(3HB) biosynthetic pathway from the cytoplasm to the plastid resulted in an approximate 100-fold increase in poly(3HB) production [24]. However, it must be kept in mind that the rate of poly(3HB) biosynthesis in A thaliana leaves was relatively low, since poly(3HB) accumulated progressively over 40-60 days to reach 10-14% of the dry weight, whereas synthesis of starch can reach 17% dry weight for a 12 h photoperiod and seed storage lipids can reach 8% dry weight per day. 

The wild type ilvA gene was modified to target the protein to the plastid and expressed in A. thaliana. Transgenic plants showed a 20-fold increase in levels of 2-ketobutyrate as well as a large increase in 2-aminobutyrate, the transaminated product of 2-ketobutyrate [27, 41]. The levels of threonine remained stable whereas isoleucine concentration increased. Constitutive expression of the ilvA protein along with bktB, phaA, and phaC proteins in the plastids of A. thaliana led to the synthesis of poly(3HB-co-3HV) in the range of 0.2 - 0.8 % dry weight, with a HV level between 4-17 mol % [27,41]. Co-expression of the iso-... 

Modulation of the quantity and/or quality of poly(3HAMCL) synthesized in peroxisomes was also achieved by modifying the endogenous fatty acid biosynthetic pathway [58]. For example, expression of the peroxisomal PHA synthase in an A. thaliana mutant deficient in the synthesis of triunsaturated fatty acids [59] resulted in the synthesis of a PHA having an almost complete absence of triunsaturated 3-hydroxyacid monomers [58]. In a different strategy, expression of a fatty acyl-ACP thio esterase in the plastid was combined with the expression of a peroxisomal PHA synthase [58]. Fatty acyl-ACP thioesterases are... 

Poly(3HB) synthesis in various subcellular compartments could be used to study how plants adjust their metabolism and gene expression to accommodate the production of a new sink, and how carbon flux through one pathway can affect carbon flux through another. For example, one could study how modifying the flux of carbon to starch or lipid biosynthesis in the plastid affects the flux of carbon to acetyl-CoA and poly(3HB). Alternatively, one could study how plants adjust the activity of genes and proteins involved in isoprenoid and flavonoid biosynthesis to the creation of the poly(3HB) biosynthetic pathway in the cytoplasm, since these three pathways compete for the same building block, i. e., acetyl-CoA. 

Chloroplasts (29-36) are the sites of photosynthesis and their ribosomes can carry out protein synthesis. Chloroplasts that contain chlorophylls and carotenoids, are disc shaped and 4-6 pm in diameter. These plastids are comprised of a ground substance (stroma) and are traversed by thylakoids (flattened membranous sacs). The thylakoids are stacked as grana. In addition, the chloroplasts of green algae and plants contain starch grains, small lipid oil droplets, and DNA. 

The tightly regulated pathway specifying aromatic amino acid biosynthesis within the plastid compartment implies maintenance of an amino acid pool to mediate regulation. Thus, we have concluded that loss to the cytoplasm of aromatic amino acids synthesized in the chloroplast compartment is unlikely (13). Yet a source of aromatic amino acids is needed in the cytosol to support protein synthesis. Furthermore, since the enzyme systems of the general phenylpropanoid pathway and its specialized branches of secondary metabolism are located in the cytosol (17), aromatic amino acids (especially L-phenylalanine) are also required in the cytosol as initial substrates for secondary metabolism. The simplest possibility would be that a second, complete pathway of aromatic amino acid biosynthesis exists in the cytosol. Ample precedent has been established for duplicate, major biochemical pathways (glycolysis and oxidative pentose phosphate cycle) of higher plants that are separated from one another in the plastid and cytosolic compartments (18). Evidence to support the hypothesis for a cytosolic pathway (1,13) and the various approaches underway to prove or disprove the dual-pathway hypothesis are summarized in this paper. 

ADP-Glucose Is the Substrate for Starch Synthesis in Plant Plastids and for Glycogen Synthesis in Bacteria... 

Many of the metabolite uptake studies cited above rely on combined uptake and incorporation into starch. In order to separate uptake from incorporation, Schott et al.226 extracted amyloplast membrane proteins from potato tubers and reconstituted them into liposomes. These reconstituted liposomes transported Pi, triose phosphates and G6P in a counter-exchange mode. The liposomes were ineffective in the transfer of G1P uptake of ADP-Glc was not tested. Mohlmann et al.236 have used a proteoliposomic system to reconstitute plastid envelope proteins. In this system, ADP-Glc is transported in exchange for AMP. Thus the more widely studied plastid ATP/ADP transporter was not responsible for ADP-Glc uptake. More recently, Bowsher et al.237 reported that wheat endosperm amyloplasts membrane proteins reconstituted into proteoliposomes took up ADP-Glc in exchange for AMP and ADP. In addition, they showed that under conditions of ADP-Glc dependent starch biosynthesis, the efflux of ADP from intact amyloplasts was equal to that of ADP-Glc utilization by starch synthesis. The amyloplast membrane ADP-Glc/ADP transporter was a 38 000 molecular weight integral membrane protein.237... 

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