| Diacylglycerols (DAG) are important second messengers in the phosphatidylinositol 4,5-bisphosphate (PIP{dollar}sb2{dollar})-mediated signal transduction pathway as well as being key intermediates in the biosynthesis of glycerolipids. The DAG content and molecular species composition of Dunaliella salina whole cells and their cellular fractions were measured by a combined HPLC and GC method which we devised. At 4.2 nmol/100 nmol lipid phosphorus, the whole cell DAG level was high in comparison with that of most animals, but similar to that of other plants. DAG in chloroplasts, the endoplasmic reticulum (ER)-enriched fraction, and the plasma membrane were found to account for 45, 49, and 6 mol %, respectively, of the total recovered cellular DAG. The predominant molecular species of DAG in all cellular fractions were of the prokaryotic type, which is synthesized in the chloroplasts. Hypoosmotic shock in D. salina induced a biphasic accumulation of DAG at 0.5, and 40 min in the plasma membrane. Whereas hypoosmotic shock induced a monophasic rise at 40 min in the ER-enriched fraction. It is the first time that biphasic changes of DAG have been reported in plants. The initial DAG increase at 0.5 min was induced mainly by the hydrolysis of PIP{dollar}sb2{dollar}, whose major molecular species, 18:1/16:0, was a prominent component of the DAG. Within 8 min following hypoosmotic shock, the molecular species composition of DAG in the plasma membrane reflected a de novo synthesis of 18:1/18:1 and 18:1/16:0 and a rise in 18:2/16:0 and 18:3/16:0 derived from phosphatidylcholine (PC) hydrolysis. After 8 min, de novo synthesis of DAG contributed heavily to the increase of DAG without any further hydrolysis of PC. This is the first clear evidence for de novo synthesis as a major source of DAG following PIP{dollar}sb2{dollar}-mediated signaling. Cells briefly preincubated with {dollar}sp{lcub}32{rcub}{dollar}Pi sustained a more pronounced labeling of an approximately 29 kDa protein during the first 30 sec following hypoosmotic shock than did non-stressed cells during a same time period. The difference in labeling intensity between stressed and non-stressed cells was less marked during the second phase of DAG production. |
