Ageing of cucumber and onion seeds: phospholipase D, lipoxygenase activity and changes in phospholipid content

Cucumber (Cucumis sativus L. cv. Mesa) and onion (Allium cepa L. cv. Rijnsburger Heldis) seeds were rapidly aged at 40 degrees C and 74% relative humidity. Onion seeds were also slowly aged at 40 degrees C with 15% relative humidity for 11 months and one more month at 28% relative humidity. Significant loss of total and individual phospholipids was an early event during both storage treatments. With slow ageing of onion, loss of phosphatidylcholine occurred several months before loss of viability and vigour was detected. Phosphatidic acid, the lipid product of phospholipase D action, increased during rapid ageing of both cucumber and onion. Phosphatidic acid was present in onion seeds before the ageing treatments and its content remained unchanged in the slowly aged seeds. There was 1600 (cucumber) and 2000 (onion) times more phospholipase D activity (6 X 10(5) and 2.9 X 10(5) nmol g-1 d-1 in cucumber and onion, respectively) in crude extracts from non-aged seeds than was required to account for the fastest fall in phospholipids (72, 372 and 144 nmol g-1 d-1 for cotyledons and radicles of cucumber and onion, respectively, over the first 9 d [cucumber] or 1 d [onion] of ageing) and fastest increase in phosphatidic acid (7, 162 and 37 nmol g-1 d-1). How accurate a guide the in vitro activity of phospholipase D was to the in vivo activity was unclear. However, the considerable excess activity seen with the formation of phosphatidic acid supports the proposal that hydrolysis of phospholipids by phospholipase D is a first step in deterioration during ageing. Substantial lipoxygenase activity was also detected (58 X 10(3) and 54 X 10(3) nmol g-1 d-1, respectively, for non-aged cucumber and onion seeds). However, the increase in conjugated dienes (an early product of peroxidation) in ageing cucumber seeds was comparatively small (90 nmol g-1 d-1 over 21 d ageing), and increase in malondialdehyde could not be detected, indicating that peroxidation may not have been a major factor in cucumber. The increase in conjugated dienes during rapid ageing of onion seeds was larger 1.5 X 10(3) nmol g-1 d-1 over days 0-2 of rapid ageing), much greater than the decrease in phospholipid acyl groups (260 nmol g-1 d-1 over days 0-2 of rapid ageing) indicating the occurrence of peroxidation of fatty acids released from reserve as well as from membrane lipids. This higher level of conjugated dienes during onion ageing was the main difference between cucumber and onion, indicating that the level of peroxidation could be an important difference between fast and slow ageing seeds. However, peroxidation is not the only possible deleterious process since hydrolysis of the normal membrane phospholipids to phosphatidic acid increased the content of non-bilayer-forming lipids and this too could be a direct membrane-destabilizing consequence of phospholipase D action during ageing.