Pollen viability and membrane lipid composition.

In this thesis membrane lipid composition is studied in relation to pollen viability during storage. Chapter 1 reviews pollen viability, membranes in the dry state and membrane changes associated with cellular aging. This chapter is followed by a study of age-related changes in phospholipid composition in Typha latifolia L. pollen.Typha pollen was stored at room temperature, with internal moisture contents of respectively 0.06 and 0.15 g H ₂ O g <sup>-1</SUP>dry weight. In the course of the storage period, imbibition was accompanied by increased leakage of endogenous K <sup>+</SUP>and germination declined. Simultaneously, phospholipids were deesterified in situ, leading to the accumulation of lysophospholipids and free fatty acids in the membranes. Viability declined much faster at the higher internal moisture content. An analysis of the fatty acid composition of the pollen phospholipids after aging indicated that the membrane degradation was not mediated by phospholipase A2. When stored with 0.06 g H ₂ O g <sup>-1</SUP>dry weight, the level of polyunsaturated fatty acids remained constant, at the higher internal moisture content a slight decline in the level of linolenic acid content was found. The observed phospholipid degradation could be mediated by an unspecific lipid acyl hydrolase, but free-radical activity is more likely because of the low metabolic activity at the studied moisture levels. The pollen lipids were purified for liposome studies. These revealed that the lysophospholipids and the free fatty acids, accumulated during aging, enhanced the leakage of entrapped solutes from the liposomes (Chapter 2).The phase behaviour of liposomal membranes containing lysophospholipids was studied after drying, to investigate the effect of these compounds in dried and in reimbibed phospholipid bilayers. Liposome studies, using Differential scanning calorimetry and Fourier transform infrared spectroscopy, showed that the lysophospholipids caused a dehydration dependent lateral phase separation. Membrane phase behaviour was also studied in Typha pollen and isolated pollen membranes. After aging the pollen membranes also exhibited lateral phase separation. The phase separation clearly coincided with the above described deesterification of membrane lipids in situ after aging, and caused an extremely fast leakage of endogenous K <sup>+</SUP>upon imbibition (Chapter 3).Two other pollen species were studied to assess whether the observed phospholipid deesterification is a general characteristic of pollen aging. In the course of storage with an internal moisture level 0.06 g H ₂ O g <sup>-1</SUP>dry weight, germination declined and the leakage of endogenous K <sup>+</SUP>upon imbibition was increased in pollen from Papaver rhoeas L. and Narcissus poeticus L. At the same time phospholipid deesterification was observed in these species and lysophospholipids and free fatty acids accumulated. The degradation was again accelerated when the pollen was stored with an internal moisture content of 0.15 g H ₂ O g <sup>-1</SUP>dry weight. In pollen species with high levels of linolenic acid such as Papaver and Narcissus the deesterification occurred at a higher rate than in pollen species such as Typha in which linoleic acid is predominant. However, no selective loss of polyunsaturated fatty acids was observed. The degradation of the phospholipids in the pollen during dry storage was again most likely free radical-mediated (Chapter 4).Although there was no large preferential degradation of linolenic acid, longevity was somehow correlated with the linolenic acid content. To study whether membrane fluidity in general determines longevity, attempts were undertaken to manipulate the level of linolenic acid in situ . Unfortunately catalytic hydrogenation, using Pd-alizarine as the catalyst, failed to result in saturation of the phospholipids in intact pollen, owing to an inhibition of the activity of the catalyst in viable pollen (Chapter 5).Because catalytic hydrogenation was unsuccessful, an in situ modification of the level of linolenic acid during the germination process was not possible. So, to study the role of linolenic acid in relation to pollen germination, Arabidopsis thaliana L. Heynh. mutants exhibiting desaturase-deficiencies were used. Pollen tube growth rate was severely decreased in pollen from the mutant fad2, in which the activity of the 16:0/18:1 desaturase located in the endoplasmatic reticulum is suppressed. In pollen from this mutant line the level of polyunsaturated fatty acids was decreased, confirming the correlation between membrane fluidity and pollen tube growth rate. In the other mutants in which chloroplast located enzymes were affected, pollen tube growth rate was unaffected (Chapter 6).The results from these studies show that with respect to aging and storage of pollen, the key factor in controling viability is the fluidity of the phospholipid bilayer. When the pollen membranes are in the high fluidity liquid crystalline phase during storage, aging is much more rapid than when the membranes are in the low fluidity gel phase. So for storage gel phase is preferable, but for rapid tube growth, on the contrary the liquid crystalline phase is required. Thus for both proper storage and germination conditions, membrane fluidity has to be carefully monitored, for instance with the aid of Fourier transform infrared spectroscopy (Chapter 7)