Genetic and environmental factors affecting the fatty acid composition of polar and non-polar lipids of developing cottonseed
Cotton (Gossypium hirsutum L.) is native to the semi-arid tropics of Southern Mexico and Central America. During its evolution, it has rarely been exposed to temperatures below 20°C. The optimum temperature for seed germination is in excess of 30°C, and the absolute minimum temperature is around 12°C. However, in most of the US Cotton Belt, planting begins when the soil temperature averages 15®C for 5 or more days. On the Texas High Plains, the imbibed seed or germinating seedlings are often exposed to temperatures below lO°C resulting in chilling injury. The current hypothesis on cold tolerance of plants is based on the fatty acid composition of the lipid components. This hypothesis states that unsaturated fatty acids, fatty acids with double bond(s) (C=C), allow the lipids to remain functional at lower temperatures compare to saturated fatty acids (no double bonds). The fatty acid composition of cottonseed lipids allows phase transition to occur around 15°C(59°F). When temperatures drop below this point during germination and seedling establishment, significant disruptions in lipid metabolism occurs. The oil, which is the primary source of carbon and energy for the emerging seedling, tends to solidify making it unavailable for hydrolysis and subsequent metabolism. The phospholipid components of the membranes tend to crystallize causing disruption of the transport functions, loss of cellular integrity and loss of solutes. Depending upon the intensity and duration of the cool temperature exposure, the extent of damage may vary from simply extending the emergence time to loss of vigor and even seedling death. Improving cotton's cool tolerance requires improving our current understanding of its fatty acid composition. The objective of this project was to determine the genetic and environmental factors affecting the fatty acid composition of polar and non-polar lipids of developing cottonseed, and then determine whether or not the fatty acid composition affects the germination of that cottonseed under cool conditions. Five commercial varieties including 2 High Plains stripper types, 2 Delta picker types, and 1 Delta stripper type were chosen to evaluate genetic variability. White flowers were tagged once a week over a four-week blooming period. Ten developing fruit (bolls) were collected at 10-day intervals for 40 days from each tagging. The final collection (open bolls) was done before harvest. The seed were separated from the bolls by hand, and a differential extraction using hexane/ether and chloroform/methanol was used to separate the non-polar fraction from the polar fraction. The fatty acids from each fraction were methylated to form methyl esters that were analyzed by gas/liquid chromatography to define the fatty acid composition Percent oil was determined for 20-, 30-, 40-day-old and open boll seed. Determining percent oil allowed for quantifying the amount of triacylglycerol fatty acids, as well as indicating the developmental progress of the seed. Oil from 20-, 30-, 40-day old, and OB seed was extracted by using a Soxhlet extraction procedure. 100 open boll seed (mature) from each variety at each week of bloom were germinated at cool (18.5°C) and warm (25°C) temperatures in rolled germination paper to determine percent germination and vigor indexes. The number of radicals greater than 50mni at 7 days warm and 10 days cool were used to evaluate percent germination. The G.C. defined fatty acid profile (unsaturated/saturated ratio) were assessed and correlated against germination to see if the degree of unsaturation was a factor that affected percent germination. Boll age (maturity) was the major factor affecting the fatty acid composition of cottonseed lipids. Young developing seed (immature) were high in unsaturated fatty acids, mainly linolenic acid (18:3). As the cottonseed developed (aged), linolenic and palmitic fatty acid concentrations decreased while oleic and linoleic fatty acid concentrations increased. Linolenic acid speared to be saturated to linoleic acid while the seed continued to synthesize more fatty acids. Mature seed from open bolls contained -22-23% palmitic acid, -1-2% stearic acid, -17-18% oleic acid, -54-56% linoleic acid, and -0-0.5% linolenic acid. A decreasing temperature gradient across weeks of bloom resulted in significant weeks of bloom effects. The main week of bloom effect was between the first and fourth weeks of bloom, which had the greatest difference in heat unit accumulation The first week of bloom accumulated more heat units that resulted in lower 18:3 and higher 18:2 percents compared to the fourth week of bloom that accumulated less heat units. Therefore, week of bloom had an effect on fatty acid desaturation/saturation and synthesis rates due to differences in temperature. By the time bolls opened and seed has reached ^'maturity,'* there were no differences in fatty acid composition across weeks of bloom. Varieties grown within the same location showed no significant biological differences in fatty acid composition during development. Therefore there was little genetic variability in fatty acid composition among varieties grown under the same environmental conditions. There was no major difference in fatty acid composition of non-polar and polar lipids. Throughout development of cottonseed, the fatty acid composition of non-polar (storage oils) and polar lipids (membranes) were very similar. Polar lipid fatty acids were used to describe boll ages 10 and 20, and non-polar Lipid fatty acids were used to describe boll ages 30-OB based on the developmental stages of oil seed. Embryogenesis (10-20 DAP) is characterized by synthesis of tissues (membranes), and 30-OB is characterized by rapid accumulation of oil lipids. Fatty acid profiles were used based on the physiology of developing cottonseed so that polar Lipid fatty acids (membranes) were described when membranes were prominent and non-polar Lipid fatty acids (oil) were described when oil was prominent in developing cottonseed. Determining percent oil (g oil/l00g seed) indicated the exponential rate of accumulation from 20 DAP to 40 DAP, and then there was a lag phase as the seed was drying down between 40 DAP to open bolls. Twenty-day-old seed consisted of 3. 5± 1.7% oil, 30-day-old seed contained 7.7±2.4% oil, 40-day-old seed contained 17.4±2.4% oil, and mature seed from open boll consisted of 21.4± 1.8% oil. Weeks of bloom temperature difference not only affected the fatty acid composition of developing cottonseed, but also affected the rate at which seed synthesized oil. Seed from the fourth week of bloom accumulated less growing degree heat units that slowed development. Since development was slowed, then the synthesis of oil was also slowed. By the time bolls opened and seed had reached "maturity," there were no differences in percent oil across weeks of bloom. Delta Pine & Land 2156 seed contained significantly higher levels of oil than the other varieties at 20, 30, and 40 days after flowering. This would indicate that DP&L 2156 had a significantly higher rate of oil synthesis. Although DP&L 2156 accumulated oil at a higher rate, it did not contain significantly more oil in mature seed over the other varieties. However, DP&L 50 did contained significantly less oil in mature seed versus the other varieties. Warm and cool germination tests were conducted to try and correlate some of the seeds' chemical properties to cold tolerance. Week of bloom was not a significant factor affecting warm germination percents. DP&L 50 significantly had the highest warm germination percent, while PM 200 significantly had the lowest warm germination percent. Week of bloom was a significant factor affecting cool germination percents. Seed that developed from the first week of bloom had a significantly higher germination percent (32.5±13.1%) under cool conditions than seed that developed from the third (22.1±9.9%) and fourth (22.9±14.0%) weeks of bloom. So, seed that developed earlier in the season under warmer conditions had some seed "quality" that could be related to cool tolerance compared to seed that develop later in the season under cooler conditions. There was no significant difference in the fatty acid composition of mature cottonseed determined across weeks of bloom. Consequently, variations within cool germination percents conducted on germination paper could not be correlated to the unsaturated/saturated fatty acid ratios. This indicated that dry seed fatty acid composition was not related to cool tolerance.