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Germinating pea (Pisum sativum L.) roots were divided into five sequential 0.5 cm segments from the root tip. Pooled segments were analyzed for their protein, starch and lipid content as an indirect indication of plastid function. Fresh weights of root segments were lowest in the tips (4.45mug per segment) and progressively higher up to the fifth segment (11.09mug per segment). Total protein, starch and lipid content, on a per segment basis, were all highest in zone 1 (tip segment) and progressively lower up to zone 5. Plastids were isolated from each of the five root segments and analyzed for their capacity for lipid biosynthesis under several different in vitro conditions. Collectively, the observations presented here suggest that the relative contributions of plastids to the overall physiology of germinating pea roots gradually diminishes as root development proceeds, and that plastids isolated from progressively older root zones have increased capacity for glycolytic and/or pentose phosphate metabolism. (Abstract shortened by UMI.)

The interaction between the glycolytic metabolism and fatty acid and glycerolipid biosynthesis in pea root (Pisum sativum L.) plastids was assessed in this study. When various glycolytic intermediates were used to substitute for the APT requirement for fatty acid synthesis from acetate, phosphoenolpyruvate, 2-phosphoglycerate, fructose-6-phosphate and glucose-6-phosphate each gave 48, 17, 23 and 17%, respectively, of the ATP-control activity. Similarly, in the absence of exogenously supplied ATP, the optimized triose-phosphate shuttle, which consists of 2 mM dihydroxyacetone phosphate, 2 mM oxaloacetic acid and 4 mM inorganic phosphate, gave up to 44% the ATP-control activity in promoting fatty acid synthesis from acetate. These results suggest that 3-phosphoglycerate kinase and pyruvate kinase in these plastids can function in intraplastidic ATP production through substrate level phosphorylation. However, in all cases, exogenously supplied ATP gave the greatest rates of fatty acid and glycerolipid synthesis. Radiolabeled pyruvate, glucose, glucose-6-phosphate, and malate in comparison to acetate were all variously utilized for fatty acid and glycerolipid biosynthesis by the root plastid. At the highest concentrations tested (3-5 mM), the rates of incorporation of pyruvate, glucose-6-phosphate and acetate into fatty acids were 183, 154, 125 nd 99 nmol $ rm cdot h sp{-1} cdot mg sp{-1}$, respectively. Malate was the least effective precursor, giving less than 55 nmol $ rm cdot h sp{-1} cdot mg sp{-1}$. Acetate incorporation was approximately 55% dependent on exogenously supplied reduced nuclotides (NADPH and NADH), whereas the utilization of the remaining precursors was only approximately 10-20% dependent on NAD(P)H. These results indicate that the entire pathway of carbon flow from glycolysis, including pyruvate dehydrogenase (PDHase), to fatty acids is operating in pea root plastids. Further, the intraplastidic glycolytic pathway plays an important role in provi

Fatty acid biosynthesis from (1-$ sp{14}$C) acetate was optimized in plastids isolated from primary root tips of 7-day-old germinating pea seeds. Fatty acid synthesis was maximum at 82.3 nmol/hr/mg protein in the presence of 200$ mu$M acetate, 0.5mM each of NADH, NADPH and CoA, 6mM each of ATP and MgCl$ sb2$, 1mM each of MnCl$ sb2$ and glycerol-3-phosphate (G3P), 15mM KHCO$ sb3$, and 0.1M Bis tris propane, pH 8.0 incubated at 35C. At the standard incubation temperature of 25C, fatty acid synthesis was linear for up to 6 hours with 80 to 120 $ mu$g/ml plastid protein. ATP and CoA were absolute requirements, whereas divalent cations, potassium bicarbonate and reduced nucelotides all improved activity by 2 to 10 fold. Mg$ sp{2+}$ and NADH were the preferred cation and nucleotide, respectively. G3P and dihydroxyacetone phosphate had little effect, and dithiothreitol and detergents generally inhibited incorporation of $ sp{14}$C-acetate into fatty acid. / Glycerolipid synthesis was obtained from $ sp{14}$C-acetate, (U-$ sp{14}$C) G3P and (U-$ sp{14}$C) glycerol at relative rates of 3.7:1.0:0.1, respectively. (Abstract shortened by UMI.)

N-Acylethanolamines (NAEs) are endogenous plant lipids hydrolyzed by fatty acid amide hydrolase (FAAH). When wildtype Arabidopsis thaliana seeds were germinated and grown in exogenous NAE 12:0 (35 µM and above), growth was severely reduced in a concentration dependent manner. Wildtype A. thaliana seeds sown on exogenous abscisic acid (ABA) exhibited similar growth reduction to that seen with NAE treatment. AtFAAH knockouts grew and developed similarly to WT, but AtFAAH overexpressor lines show markedly enhanced sensitivity to ABA. When low levels of NAE and ABA, which have very little effect on growth alone, were combined, there was a dramatic reduction in seedling growth in all three genotypes, indicating a synergistic interaction between ABA and NAE. Notably, this synergistic arrest of seedling growth was partially reversed in the ABA insensitive (abi) mutant abi3-1, indicating that a functional ABA signaling pathway is required for the full synergistic effect. This synergistic growth arrest results in an increased accumulation of NAEs, but no concomitant increase in ABA levels. The combined NAE and ABA treatment induced a dose-dependent increase in ABI3 transcript levels, which was inversely related to growth. The ABA responsive genes AtHVA22B and RD29B also had increased expression in both NAE and ABA treatment. The abi3-1 mutant showed no expression of ABI3 and AtHVA22B, but RD29B expression remained similar to wildtype seedlings, suggesting an alternate mechanism for NAE and ABA interaction. Taken together, these data suggest that NAE metabolism acts through ABI3-dependent and independent pathways in the negative regulation of seedling development.

N-Acylethanolamines (NAEs) are fatty acid derivatives in plants that negatively influence seedling growth. N-Lauroylethanolamine (NAE 12:0), one type of NAE, inhibits root length, increases radial swelling of root tips and reduces root hair numbers in a dose dependent manner in Arabidopis thaliana L. (ecotype Columbia). A forward genetics approach was employed by screening a population of T-DNA “activation-tagged” developed by the Salk Institute lines for NAE resistance to identify potential genes involved in NAE signaling events in Arabidopsis thaliana L. (ecotype Columbia). Seeds of the activation tagged lines were grown at 0, 25, 30, 50, 75 and 100 µM N-lauroylethanolamime (NAE 12:0). Ten plants which displayed NAE tolerance (NRA) seedling phenotypes, compared with wildtype (Columbia, Col-0) seedlings were identified. I focused on one mutant line, identified as NRA 25, where the tolerance to NAE 12:0 appears to be mediated by a single dominant, nuclear gene. Thermal asymmetric interlaced (TAIL) PCR identified the location of the T-DNA insert as 3.86 kbp upstream of the locus At1g68510. Quantitative PCR indicated that the transcript level corresponding to At1g68510 is upregulated approximately 20 fold in the mutant relative to wildtype. To determine whether the NAE tolerance in NRA 25 is associated with overexpression of At1g68510 I created overexpressing lines of At1g68510 with and without GFP fusions behind the 2X35S CaMV promoter. As predicted, results with overexpressing lines of At1g68510 also exhibited enhanced resistance to NAE when compared with the wildtype. Confocal images of the fusion proteins suggest that GFP-At1g68510 is concentrated in the nucleus and this was confirmed by counterstaining with 4', 6-Diamidino-2-phenylindol (DAPI). Futhermore, At1g68510 overexpressing lines and NRA 25 line also exhibited tolerance to abscisic acid (ABA) during seedling germination. The findings suggests that At1g68510 overexpression mediates seedling tolerance to both ABA and NAE, a mechanism independent of fatty acid amide hydrolase in which its overexpression leads to NAE resistance but hypersensitivity to ABA. The next steps are to identify the association of At1g68510 with specific genomic regions or interacting proteins that may be additional components of NAE signaling in plants.

No description available.

No description available.