| Lipids are the major structural components of all cellular membranes of livingorganisms. Oil-producing crop plants such as soybean (Glycine max L.) and rapeseedsalso synthesize and store energy in the form of TAGs, which are composed of aglycerol backbone molecule esterified by three saturated and/or unsaturated fattyacid acyl groups. The relative composition of saturated and unsaturated fatty acids inseed TAGs is one of the major factors influencing the quality of edible oils. Forexample, oils in high 18:1 and low in polyunsaturated fatty acids appear to haveimproved nutritional benefits and increased stability. Therefore, the food industry hasa major commercial interest in understanding how to regulate desaturation of fattyacids within storage in oil seed crops.In plants, microsomal and plastidialω-6 fatty acid desaturases are the twoenzymes responsible for converting oleic acid to linoleic acid through the eukaryoticand prokaryotic pathway, respectively. In soybean, one of the most importantresources of vegetable oils, two different microsomal oleate desaturase genes havepreviously been reported: a seed-specific gene FAD2-1 and a constitutively expressedgene FAD2-2. The seed-specifically expressed FAD2-1 gene is likely to play a majorrole in controlling conversion of oleic acid to linoleic acid within storage lipids duringseed development. The FAD2-2 gene-encodedω-6 desaturase appears to beresponsible for production of polyunsaturated fatty acids within membrane lipids inboth vegetative tissues and developing seeds. As a partial work of this study, weisolated two novel microsomal oleate desaturase genes (FAD2) in soybean andcharacterized their function by expression in yeast. The expression patterns of bothgenes were investigated during soybean seed development and in different tissues. Inaddition, the transcript accumulation levels of the two constitutively expressed genesin response to low temperature were also analyzed. We isolated a novel gene encoding FAD2 isoform, designated as FAD2-3. Thededuced amino acid sequences of the FAD2-3 displayed the typical three-histidineboxes characteristic of all membrane-bound desaturases, and possessed a C-terminalsignal for endoplasmic reticulum retention. Phylogenetic analysis showed thatFAD2-3 is grouped within plant house-keeping FAD2 sequences. Yeast cellstransformed with a plasmid construct containing the FAD2-3 coding regionaccumulated a considerable amount of linoleic acid (18:2), normally not present inwild-type yeast cells, suggesting that the isolated gene encodes a functional FAD2enzyme. Semi-quantitative RT-PCR and in silico analysis showed that FAD2-3 gene isconstitutively expressed in both vegetative tissues and developing seeds. In soybeanleaves, the level of linolenic acid (18:3) increases with the decrease of linoleic aicd(18:2) under cold treatment. However, no significant change of transcript levels ofFAD2-2 and FAD2-3 genes was detected. These results indicated that there may be adifferent mechanism to regulate the fatty acid composition of soybean leaf lipidsunder cold treatment from low grown temperature. The low growth temperaturecaused to an increase in the 18:3 content but maintaining the relatively constant levelof 18:2, which may exist a mechanism to retain a certain level of 18:2 by balancingthe flux through 18:1 and 18:2 fatty acids. Another mechanism for the shift to lowtemperature may be due to△-15 FAD converting 18:2 to 18:3 to maintain membranefunction.We isolated a seed-specific isoform of microsomal omega-6 fatty acid desaturasegene (FAD2-1B) sharing high sequence similarity with FAD2-1. Several potentialpromoter elements, including seed-specific motif, occur in the 5'-flanking region. TheFAD2-1B coding region is 1134bp and would encode a protein of 378 amino acids.The polypeptide has three histidine boxes and four putative membrane-spanninghelices, indicative of an integral membrane-bound desaturase, and possesses aC-terminal endoplasmic reticulum retention signal. Yeast cells transformed with aplasmid construct containing the soybean FAD2-1B coding region accumulate anappreciable amount of linoleic acid (18:2), not normally present in wild-type yeastcell, indicating that the gene encodes a functional FAD2 enzyme. Semi-quantitativeRT-PCR and in silico analysis showed that FAD2-1B gene is strongly expressed indeveloping seeds.Fatty acid hydroperoxides derived from linolenic acid are precursors for an arrayof oxylipins that function in diverse aspects of plant growth and development. Allene oxide synthase (AOS) and fatty acid hydroperoxide lyase (HPL) are plant-specificcytochrome P450s that commit fatty acid hedroperoxides to different branches ofoxylipin metabolism. The products of one branch of oxylipin metabolism, referred toas the AOS pathway, are essential signals for plant defense against pest attack,mechanical responses, and some development processes. The products of analternative pathway of oxylipin metabolism, initiated by fatty acid hydroperoxidelyase (HPL), are important volatile constituents of the characteristic odor of fruits,vegetables, and green leaves. In addition, P450s are found in other major plantbiosynthetic pathways, including those for UV protectants, pigments, defensecompounds, hormones, signaling molecules, accessory pigments and structuralpolymers. However, only a few of P450 genes have been identified in Medicagotruncatula and Glycine max. As another part of this study, we identified, cloned andannotated the putatively functional P450 encoding sequences in M. truncatula andGlycine max using bioinformatic tools and in silico resources. The phylogeneticanalysis has allowed us to identify paralogous genes and clusters of orthologousgroups as the basis for further characterization of P450 genes of unknown function.We then evaluated the relationship between the structures and functions, and analyzedESTs-derived expression profiles of these P450 genes. The comparative genomicanalysis of P450 gene superfamily in Arabidopsis, Medicago truncatula and Glycinemax were also carried out.In the model legume M. truncatula genome, we identified 152 putative P450genes using data mining methods in the TIGR consensus contigs in the TIGR GeneIndex for M. truncatula (Release 8.0) and in the nr and GSS section of GenBank.These genes were classified into 9 clans and 45 families by sequence similarity, forwhich 4 clans and 22 families not reported in legumes previously. The representativeprotein sequences of these putative P450s were aligned, and the secondary elementswere assigned based on the known structure P450BM3. Putative substrate recognitionsites (SRSs) and substrate binding sites were also identified in these sequences. TheESTs-derived expression profiles (digital northern) of these putative P450 genes inwere also analyzed. To confirm the digital northern data, semi-quantitative RT-PCRanalyses of several selected P450 genes were carried out. These results provide a basisfor catalogue information on P450 genes in M. truncatula and for further functionalanalysis of P450 gene superfamily in legumes.In the most important legume crop Glycine max genome, we identified 56 putative P450 genes using data mining method. We named the unannotated sequencesbased on P450 standarized nomenclature system. The G max P450 sequences and therepresentative members of known P450 families in plants were carried outphylogenetic analysis, and all the sequences were classified into two major branches,termed the A-type, Non-A-type. All the identified P450 sequences in G max weredistributed among these branches. Comparison of these G max genes with the 246P450 genes in the Arabidopsis genome and 151 P450 genes in the M. truncatulagenome has indicated that all the identified G max P450 families exist in Arabidopsisand M. truncatula genome, and the CYP93C is specific-legume subfamily.Comparative analysis of ESTs-derived expression profiles (digital Northern) foundthat some P450 subfamilies have similar expression patterns in M. truncatula and Gmax organs.
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