Abstract
Cotton (Gossypium hirsutum L.) provides a major source of oil for food and feed industries, but little was known about the enzymes in the oil biosynthesis pathway in cottonseed. We are interested in a better understanding of enzymatic components for oil accumulation in cottonseed. The objective of this study was to identify one key enzyme in oil biosynthesis pathway: phosphatidic acid phosphatase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4). PAP hydrolyzes the phosphomonoester bond in phosphatidate yielding diacylglycerol and Pi. PAPs are generally categorized into Mg2+-dependent soluble PAP and Mg2+-independent membrane-associated PAP. Cottonseed from 25 - 30 days post anthesis was used for the study. The results showed that an Mg2+-independent soluble PAP activity was identified from the cottonseed. While the microsomal fraction of the extract provided only 9% of the PAP activity, 69% of the PAP activity was associated with the cytosol. The PAP activity correlated well with enzyme concentration and incubation time. The pH and temperature optima of the enzyme were pH 5 and 55℃, respectively. Under optimized assay conditions, the Vmax and Km values of cottonseed PAP for dioleoyl phosphatidic acid as the substrate were 2.8 nkat/mg of protein and 539 μM, respectively. Inclusion of the detergent Triton X-100 (0% - 0.5%) or magnesium chloride (1 mM) in the reaction mix did not alter activity to a significant degree. This is the first report of a PAP activity in the seeds of Gossipium hirsutum. This study should provide a basis for purification and characterization of this important enzyme from cottonseed in the future.
Highlights
Cotton plant provides two economically important products, fiber as the major product and cottonseed as the minor product
95% of the cotton crop grown world-wide is derived from Gossypium hirsutum and about 4% is derived from Gossypium barbadense. 5.6 million tons of cottonseed was produced in the United States in 2012 [1]
Towards better understanding the enzymatic steps responsible for oil accumulation in cottonseed, we initially focused our studies on Phosphatidic acid phosphatase (PAP) (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) that dephosphorylates phosphatidic acid (PA, called Phosphatidate or phosphatidic acid (PtdOH)) to generate DAG and Inorganic phosphate (Pi)
Summary
Cotton plant provides two economically important products, fiber as the major product and cottonseed as the minor product. Cotton plant produces more seed in terms of quantity than fiber with a seed/fiber ratio of 1.5 - 1.7. Cottonseed typically accounts for 15% - 25% of the crop value. 95% of the cotton crop grown world-wide is derived from Gossypium hirsutum (short staple cotton varieties) and about 4% is derived from Gossypium barbadense (long staple cotton varieties). 5.6 million tons of cottonseed was produced in the United States in 2012 [1]. Three million tons of cottonseed was used to feed dairy cows and 2.5 million tons of cottonseed was used for oil production, which generated 1.3 million tons of cottonseed meal and 0.4 million tons of vegetable oil. The amount of oil in the seed varies from 20% to 24% (dry-weight basis) [1]
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