| dc.description.abstract | The developing seeds of groundnut (Arachis hypogaea) show a triphasic pattern in lipid and protein accumulation.
In the first phase extending from 0–15 DAF, there is no significant accumulation of lipids and proteins.
During the second phase (15–35 DAF), active lipid accumulation occurs with a low rate of protein synthesis.
In the next phase lasting from 40–60 DAF, lipid synthesis continues at a much reduced rate while protein synthesis increases markedly.
Lipids are stored exclusively as triacylglycerols. Phospholipids are present in very small amounts at all stages of development.
[U ¹ C]Glucose and [U ¹ C]fructose were incorporated with equal efficiency into lipids of seed slices incubated in vitro, with preferential incorporation into polar lipids. The rate of incorporation did not show a discernible developmental pattern.
The incorporation of [³H]glycerol and [1 ¹ C]acetate was 10 fold higher than hexoses in seed slices at 30 DAF. These were incorporated preferentially into triacylglycerols. The incorporation of these precursors increased progressively during the active period of lipid accumulation, suggesting a change in the biosynthetic ability of the seeds during development.
In experiments on the incorporation of [³H]glycerol into seed slices, formation of sn glycerol 3 phosphate was demonstrated. The rate of formation correlated with the period of active lipid accumulation.
Glycerol phosphate dehydrogenase could not be detected in developing groundnut seeds using the conventional spectrophotometric assay.
To improve assay sensitivity, additional procedures using conversion of [³²P]G 3 P to [³²P]DHAP (or the reverse) were tested, but glycerol phosphate dehydrogenase activity could not be detected, suggesting its absence in developing seeds.
The absence of glycerol phosphate dehydrogenase and active incorporation of [³H]glycerol suggests that sn glycerol 3 phosphate may be formed by direct phosphorylation of glycerol.
An active glycerokinase was demonstrated in the cytosolic fraction of developing seeds.
The enzyme had a pH optimum of 8.6, similar to alkaline optima observed in other systems.
The enzyme exhibited positive cooperativity with respect to both glycerol and ATP and may therefore represent a regulatory site.
Glycerokinase specific activity increased from 15–35 DAF, correlating with rapid lipid accumulation.
A pathway for glycerol formation involving triose phosphate isomerase, glyceraldehyde 3 phosphate phosphatase, and aldehyde reductase was tested.
Active triose phosphate isomerase was demonstrated in the cytosolic fraction. By this reaction, DHAP can also be utilized for lipid synthesis after conversion to glyceraldehyde 3 phosphate.
This enzyme had a broad pH range for activity, as observed in other systems.
Triose phosphate isomerase exhibited Michaelis–Menten kinetics toward glyceraldehyde 3 phosphate, with a Km of 0.6 mM.
High activity was maintained between 20–35 DAF; thereafter it decreased to almost 50% at maturity.
Glyceraldehyde 3 phosphate phosphatase activity was observed in the cytosolic fraction.
The enzyme showed positive cooperativity for glyceraldehyde 3 phosphate, suggesting regulatory significance.
It was specific for glyceraldehyde 3 phosphate. Only 12–16% activity was observed with DHAP and 3 phosphoglyceric acid. Hexose 6 phosphates were cleaved at ~20% relative activity. Fructose 1 phosphate was not cleaved.
The enzyme activity increased from 15–30 DAF and declined thereafter, matching lipid accumulation patterns.
An active glyceraldehyde reductase was demonstrated in mitochondria, microsomes, and cytosol.
This enzyme reduced several aldoses, similar to aldose reductases in other systems. It was specific for NADPH, with no activity using NADH.
It exhibited Michaelis–Menten kinetics with NADPH (Km = 30.8 mM) and glyceraldehyde (Km = 2 mM).
Mitochondrial and microsomal enzyme fractions peaked at 30 DAF, while cytosolic activity declined during development.
A comparison of specific activities of triose phosphate isomerase, glyceraldehyde 3 phosphate phosphatase, glyceraldehyde reductase, and glycerokinase at 30 DAF suggested that glycerokinase is the rate limiting enzyme for sn glycerol 3 phosphate formation. However, its maximum activity was more than adequate to support observed lipid synthesis rates.
It is proposed that in developing groundnut seeds, sn glycerol 3 phosphate formation for triacylglycerol synthesis occurs essentially via the following pathway: | |
