The Effect of Temperature on Fatty Acid Desaturase Gene Expression and Fatty Acid Composition in Developing Soybean Seeds.
BYFIELD, Grace E. The effect of temperature on fatty acid desaturase gene expression and fatty acid composition in developing soybean seeds (Under the direction of Dr. Robert G. Upchurch).
Soybean [Glycine max (L.) Merr.] is the largest oilseed crop produced and consumed worldwide. As an oilseed, soybean provides high quality protein for livestock and poultry feed, and the oil is used extensively in the cooking and food manufacturing industries across the world. Soybean producers have targeted improved oil traits as a priority area of research to enhance the market share of U.S. soybean. Soybean seed is approximately 18% oil and standard commodity soybean oil is a mixture of five fatty acids; palmitic (11%), stearic (4%), oleic (22%), linoleic (53%), and linolenic (8%). Oils high in monounsaturated fatty acids possess increased oxidative stability which negates the need for hydrogenation and eliminates the production of trans-fats. Food products containing trans-fatty acids are currently a major health concern and as such, soybean breeders and molecular geneticists have been challenged to efficiently alter the fatty acid composition to meet the specific needs of the various industries. Fatty acid desaturases are enzymes responsible for the insertion of double bonds (normally in the Z or cis conformation) into alkyl chains, following the abstraction of two hydrogen atoms. The physical properties and nutritional value of many animal and plant storage lipids are determined by desaturases. The quality of soybean oil is of paramount importance both economically and from a nutritional stand point and depends to a large extent on the ratio of polyunsaturated to monounsaturated fatty acid. Temperature also plays an important role in the final lipid content of oil seed plants. In this study, we designed and used gene-specific primers to the following fatty acid desaturases; stearoyl-ACP desaturase (SACPD), omega-6 fatty acid desaturase (FAD2-1), and omega-3 fatty acid desaturase (FAD3), to characterize soybean varieties. We further determined the effect of temperature on the expression of these desaturase genes by quantifying transcript accumulation at various stages of seed development.
We surveyed 51 soybean lines and found each contained two SACPD genes (A and B) with distinguishing amino acid variations in exon 3. The varieties also had two FAD2-1 (A and B) and three FAD3 (A, B, C) genes. Soluble ?9 stearoyl-ACP desaturases introduce the first double bond into stearoyl-ACP (18:0-ACP) between carbons 9 and 10 to produce oleoyl-ACP (18:1?9-ACP). Microsomal ù-6 desaturase catalyzes the first extra-plastidial desaturation and converts oleic acid to linoleic acid. Microsomal ù-3 fatty acid desaturases (FAD3s) catalyze the insertion of a third double bond into the linoleic (18:2) acid precursor to produce linolenic (18:3) acid.
An analysis of the effect of cold (22/18oC), normal (26/22oC), and warm (30/26oC) temperatures on the transcript accumulation of these desaturase genes revealed some differences. Transcript accumulation of SACPD-A and -B decreased by up to 69% with increasing temperature in cultivars Dare, A6 (a high stearate line), and N01-3544 (a mid-oleic line). The oleic acid content of these three lines was inversely related to the levels of SACPD expression at the warm and cold temperatures. This suggests that transcription control of SACPD may not be a crucial factor for regulating oleic acid content in soybean. FAD2-1A and FAD2-1B gene expression in stage 4 seeds was comparable at the normal temperature, but a change in growth temperature to either side of the norm resulted in increased expression of FAD2-1B over FAD2-1A, slight at the warm temperature, but more pronounced at the cold temperature. The three omega-3 fatty acid desaturase genes exhibited the highest levels of transcript accumulation in stage 4 seeds at the cold temperature, with FAD3A levels 1.3 to 1.8 fold higher than 3B and 3C.
The fatty acid composition of the seeds at different stages of development was determined in conjunction with steady state transcript levels. Results show that the stearic acid content of A6 had the most dramatic response of the three soybean lines to temperature manipulation. Stearic acid content increased at the warmer temperature for all stages of A6, but for Dare and N01-3544 the percentage change in 18:0 was slight and net negative. Conversely, growth at the cold temperature resulted in the most dramatic reduction (48%) of stearic acid content in A6. The oleate (18:1) concentration increased at the higher growth temperature compared to normal across all varieties and stages, with Dare showing the most dramatic (32 %) increase in oleic acid content at the warm temperature. The increase in FAD2-1B transcript accumulation with decreasing temperature was associated with increasing 18:2 content in two of the three varieties. Increased GmFAD3A transcript accumulation was accompanied by an increase in 18:3 in all three soybean varieties examined.
The differences in steady state mRNA levels we observed could be due to changes in transcription rate or mRNA stability. In light of this, further studies to measure desaturase activity under specific temperature regimes are needed in order to clarify the linkages of transcript level to enzyme activity and the fatty acid composition in developing soybean seeds.
Advisor:Dr. Robert G. Upchurch; Dr. Hosni Hassan; Dr. Ralph Dewey; Dr. Amy Grunden
School:North Carolina State University
School Location:USA - North Carolina
Source Type:Master's Thesis
Date of Publication:12/02/2005