ABSTRACT

Over 75% of fatty acids in plants are unsaturated by desaturase enzymes in chloroplast and endoplasmic reticulum membranes. Two fatty acid desaturases designated FAD2 and FAD3 primarily desaturate extrachloroplast lipids and occur as integral membrane proteins in the endoplasmic reticulum. The FAD2 gene has been shown by several laboratories to be important in the chilling sensitivity of plants, since polyunsaturated phospholipids are essential for maintaining plant viability at lowered temperatures. Cotton genomic libraries harbored in lambda phage were screened to isolate prospective FAD2 genes, in order to study their regulation of gene expression. The structure and expression of a cotton FAD2 gene (called the FAD2-3 gene) was previously analyzed. Recently, the structure and functional expression of a second cotton FAD2 gene (called the FAD2-4 gene) was analyzed for comparison with that of the FAD2-3 gene, to compare the similarities/differences of the genes in the 5'-flanking introns, the promoter elements, and the protein-coding regions. This is to gain insight into how the two cotton FAD2 genes are regulated for gene expression, and if the 5'-flanking introns really have any bearing on transcriptional regulation of the genes. The FAD2-4 gene is distinctly different from the FAD2-3 gene, with minor differences in the coding regions and major differences in the flanking regions. The FAD2-4 gene is expressed in cotton, since its cDNA was amplified from a cotton cDNA library by PCR. The putative cotton FAD2-4 polypeptide likely is an integral membrane protein in the cellular endoplasmic reticulum, since it has four tentative membrane-spanning domains corresponding to the predicted membrane-spanning domains in desaturase integral membrane protein models. The 5'-flanking region of the FAD2-4 gene has a number of prospective promoter elements that also occur in the 5'-flanking region of the FAD2-3 gene. A large intron occurs in the 5'-flanking region of the FAD2-4 gene, similar in size and location to the large intron in the 5'-flanking region of the FAD2-3 gene. However, there are substantial differences in the nucleotide sequences of the two introns, indicating that the genes are probably orthologs. These 5'-flanking introns could be important in the transcriptional regulation of expression of the genes. The protein-coding region of the FAD2-4 gene is 1,155 bp, and is continuous with no introns. The deduced amino acid sequence of 384 amino acids of the putative cotton FAD2 polypeptide has a high identity (about 75%) with the deduced amino acid sequences of other plant FAD2 enzymes. Yeast cells transformed with a plasmid construct containing the cotton FAD2-4 coding region have an appreciable accumulation of linoleic acid (18:2), not normally present in wild-type yeast cells. Thus, this cotton FAD2 gene is truly functional, since it encodes an enzyme that catalyses the desaturation of oleic acid (18:1) into linoleic acid (18:2). Two binary plasmid constructs containing the two FAD2 genes are being generated for transformation of model Arabidopsis plants, before beginning transformation of cotton plants. An understanding of the expression patterns of the FAD2 genes should allow us to genetically engineer these genes in cotton, and possibly modify the membrane fatty acid compositions to improve viability of this crop plant.