Lipid synthesis depends on the correct spatial and temporal activity of many gene products. These genes execute their function in three stages: fatty acid synthesis in the plastid, triacylglycerol (TAG) synthesis in the endoplasmic reticulum (ER), and assembly into an oil body. Significant improvements in oil accumulation must be accompanied by changes in activity of the genes involved in fatty acid biosynthesis in developing seeds. Identification of these genes and their regulatory pathway would provide not only new genetic information for understanding soybean seed development, but also for controlling gene expression in developing seeds to alter oil accumulation.
This study has provided a new data set identifying the expression of DEGs during oil accumulation in developing seeds. Massive parallel sequencing identified 11592, 16594 and 16255 differentially expressed unigenes from three contrasting libraries covering four stages of seed development. We examined gene expression levels in detail, and found significant differences among the four growth stages. Among the differentially expressed genes, more were down-regulated than up-regulated, and some showed a differential expression pattern in all three contrast groups, indicating that there were overlaps at the transcriptional level. The fact that there were many down-regulated genes indicates that there are more negatively regulated genes than positively regulated ones with functions in the fatty acid pathway. However, it does not mean that lower expression of these genes leads to lower oil contents, because of the complexity of the lipid biosynthetic pathway. To identify the genes associated with lipid biosynthesis, we determined gene expression patterns at specific stages of seed development, and conducted a GO analysis. To explore the genes with unknown functions, the expression patterns of 124 unigenes were analyzed by hierarchical clustering according to similarities in expression profiles across all conditions.
In the lipid biosynthetic pathway, acetyl CoA carboxylase (ACCase) plays an important regulatory role. ACCase catalyzes the carboxylation of acetyl-CoA to malonyl-CoA , which is then transacylated by malonyl-CoA-acyl carrier protein transacylase (FabD or MCAT,[EC 22.214.171.124]) to the acyl carrier protein (ACP), forming malonyl ACP. The latter adds a 2-carbon acetyl unit to the nascent or growing fatty acyl chain. The basic reaction of fatty acid synthesis is to combine molecules composed of two carbon units into longer chains to form fatty acids. The initial condensation of 2-carbon units is catalyzed by β-ketoacyl-ACP synthase III (KASIII, EC126.96.36.199) which uses acetyl-CoA and malonyl-ACP as substrates. After the two reductions and dehydration reactions, a 4-carbon fatty acid, butyrate, is produced. This is poorly condensed by KASIII but is a good substrate for KASI, which elongates 4-carbon chains to 14-carbon chains.
In this study, we studied in detail the genes with key roles in lipid biosynthesis. Very frequently, the same enzymatic function is redundantly encoded by several unigenes. This may be the result of different proteins referenced with the same EC number or it may represent different transcripts encoding specific enzyme subunits. This situation was significant in the present study. Most plants have two forms of ACCase, the homomeric form in the cytosol, composed of a single large polypeptide catalyzing the individual carboxylation reactions , and the heteromeric form in plastids, composed of four subunits; biotin carboxylase (BC) , biotin carboxyl carrier protein (BCCP) , α-carboxyltransferase (α-CT)  and β-carboxyltransferase (β-CT) . In the present study, seven co-expressed unigenes (GenBank accession nos: BT094733, AK245399, U40979, AF165159, CX703216, BM188175, AW830581) encoding ACCases were identified as DEGs in the three contrast groups. All of them were down-regulated (log2ratio ≤ 1) in the three contrast groups except one gene (AK245356) encoding an ACCase was up-regulated (log2ratio = 1.4) at 35 DAF. Three co-expressed unigenes (GenBank accession nos: BM188175, CX703216, AW830581, EC188.8.131.52) that are associated with the transformation of BCCP to carboxybiotin-carboxyl-carrier protein were down-regulated during seed development.
The next enzyme in fatty acid pathway is FabD, which catalyzes the transfer of malonyl-CoA to the holo acyl carrier protein (ACP), generating malonyl-ACP . In the present study, the co-expressed unigene (AW34878) encoding FabD was down-regulated during seed development.
KASIII (FabH) catalyzes the subsequent condensation and transacylation of acetyl-CoA with malonyl-ACP and has a universal role in fatty acid biosynthesis. Transgenic B. napus seeds overexpressing KASIII driven by napin also contained lower oil levels compared to what was found in the wild-type . In the present study, the co-expressed unigene (AF260565) encoding KASIII was significantly down-regulated in the fatty acid pathway with a log2ratio of −1.10, -2.05, and −2.09 in the three contrast groups. So compared to 15 DAF, the differentially expressed levels of the gene encoding KASIII at 35DAF, 55DAF, 65DAF negatively correlated to fatty acid synthesis during the seed development, which is consistent with previous research . The same down-regulated patterns were also observed for FabF (AF243183, CX702997, AK244605, AK285347), FabZ (BT098415), FabI (BI970856), FatB (AK244101) and oleoyl-[acyl-carrier-protein] hydrolase (EC 184.108.40.206, AK244101).
In most plant tissues, Acyl-ACP thioesterase is the major determinant of chain length and level of saturated fatty acids . It plays an important role by influencing the fatty acid composition of the produced oil and then mainly the ratio of 16 C to 18 C fatty acids and the level of saturated fatty acid . Two distinct but related thioesterase gene classes exist in higher plants: FatA is an acyl-specific thioesterase, with specificity for 18:1> > 18:0> > 16:0 fatty acids , which is considered an essential “housekeeping” enzyme in all plant cells; FatB is a thioesterase, which shows specificity for 16:0 > 18:1 > 18:0 fatty acids . In this study, Solexa sequencing analysis showed that the expression of gene (CX706542) encoding FatA was unchanged at 35 DAF, but down-regulated at 55 DAF and 65 DAF with log2ratios of −1.7 and −1.5. While differentially co-expressed unigene (AK244101) encoding FatB showed constant decreased expressed levels with log2ratios of −1.4, -2.4, -2.7, respectively. Therefore, the expression level variations of CX706542 and AK244101 would influence the 16 and 18 carbon fatty acids synthesis, which needs to be confirmed by experiment.
Soybean seeds contain three lipoxygenase isozymes; lipoxygenases 1, 2, and 3. Lipoxygenases (linoleate: oxygen oxidoreductase; EC 220.127.116.11) catalyze the oxidation of unsaturated fatty acids to produce conjugated unsaturated fatty acid hydroperoxides, which are converted to volatile compounds associated with undesirable flavors . Eliminating this enzyme from seeds could lead to better quality soybean protein and oil products. Three co-expressed lipoxygenase genes (X67304, U50081, D13949) identified in this study were among those with the highest expression levels. To our knowledge, this is the first report of a close correlation between lipoxygenase expression and fatty acid accumulation.
The P450 family is a large and diverse group of isozymes that mediate a diverse array of oxidative reactions. The activities of most of these enzymes are associated with biosynthetic processes such as phenylpropanoid, terpenoid, and fatty acid biosyntheses. Ten alkane-inducible P450 genes from Candida tropicalis (ATCC20336), which were responsible for omega-hydroxylation of n-alkanes and fatty acids, were cloned . In their research, these enzymes were believed to be at least in part limiting in the conversion of fatty acid to diacids, but their relative oxidative activity toward other fatty acids was not known. The two unigenes (AF022464, DQ340246) encoding soybean P450 monooxygenase were identified in this research. Both of them showed down-regulated expressions during seed development, which indicated that they are negatively correlated to the fatty acid accumulation. For the other differentially co-expressed unigenes shown in Table 2, there is little information available about their relationship with lipid accumulation. Their log2ratios indicate that they have significant functions in regulating soybean seed development. However, our results cannot validate a direct relationship between these unigenes and oil accumulation. This topic requires further research.
Because we are interested in oil production in soybeans, we selected the fatty acid biosynthesis pathway for deep analysis from among the 124 biochemical pathways identified by Solexa sequencing. Twenty four co-expressed unigenes in the fatty acid pathway, including 13 that overlapped with the unigenes identified in the GO analysis, showed significant correlations with fatty acid accumulation (Table 3).
The up-regulated genes that were significantly correlated with fatty acid accumulation included ACCase and lipoxygenase. The down-regulated genes that were significantly correlated with fatty acid accumulation included FabD, KAS III, FabF, FabZ, FabI, FatB, FatA, P450, and oleoyl-[acyl-carrier-protein] hydrolase genes. FabG genes were both up- and down-regulated during seed development.
The analysis of the genes involved in the fatty acid biosynthetic pathway provides a basis to identify key regulatory processes controlling oil accumulation in soybean. However, biosynthetic pathways involve the cooperation of multiple genes. It is difficult to increase seed oil content by overexpressing a single gene. The large-scale characterization of unigenes described in this study shows comprehensive correlations between DEGs and fatty acid accumulation in soybean.