During the first few weeks of seed development, the seed coat expanded until the seed was almost full size. At this stage, the seed was somewhat translucent and resembled a water-filled balloon. It was difficult to dissect ovules from the pod at 5 DAP(days after pollination) but it became possible after 10 DAP. The seed's embryo now began development and grew rapidly within the seed coat to fill the space previously occupied by fluid. The mature seed was harvested at 45 DAP.

In the ZS9 cultivar, the total fatty acid content continued to increase and peaked at about 40 DAP, followed by a gentle drop with seed maturation (Fig. 1A). In contrast, ZY821 showed a similar pattern of accumulation of total fatty acids except that it had higher levels at 30 DAP (Fig. 1A). Nine fatty acids were found in ZY821 (Fig. 1B–J) but only five in ZS9 (Fig. 1B–F). Among the five common fatty acids, only oleic acid (C18:1) showed different accumulation patterns (Fig. 1D), whereas the other fatty acids accumulated in similar patterns in both cultivars (Fig. 1B–F). In ZS9, the oleic acid content increased continuously and became a major component at the end (Fig. 1D), whereas in ZY821, oleic acid ascended in the early stages (10–25 DAP) but decreased from 25 DAP to 35 DAP and then remained constant until the end (Fig. 1D). The initial content of all saturated fatty acids (C16:0, C18:0, C20:0 and C22:0) was relatively high but decreased from 15 DAP and reached a minimum at about 25–30 DAP, ultimately comprising a minor proportion of the fatty acids in mature seeds (Fig. 1B, C, G, I). The content of C18:2 was as high as 35–42% during the initial stages (10–15 DAP) and decreased continuously to a final content of about 20% (Fig. 1E). The C18:3 content fluctuated from 6% to 11.4% and was fixed at about 7% in mature seeds (Fig. 1F).

Different genes had different transcript levels but with very similar tendencies between the two cultivars in general (Table 2 and Fig. 2). In both cultivars, ZY821 and ZS9, the top 7 genes, including Napin, Oleosin, β-CT, Caleosin, β-actin, FAE1 and Cruciferin, accounted for over 95% of the transcript levels from the 32 genes (Table 2). The genes coding for storage proteins alone accounted for 87%, of which the Napin transcript was 77~78% of the total, Oleosin 6~8%, and Caleosin and Cruciferin about 1~2%. For other non-storage protein genes, β-CT, GAPDH, FAE1 and SAD showed relatively higher transcript levels and accounted for 11% of the total transcripts (Table 2). The other 23 genes accounted for only about 3% of the total transcripts, of which AAPT1, KAS3, MCAT, FatB, SUC1 and α-CT had the lowest transcript levels (Table 2).

For most of the genes investigated, the transcript levels changed with time during seed development (Fig. 2). Comparison of the mean transcript levels of ZS9 and ZY821 revealed that 11 of the 32 genes (FAD3, ACCase, FAE1, GKTP, Caleosin, GAPDH, PEPC, KACD, KAS1, LPAAT and MCAT) were upregulated by 20% to 109%, whereas 10 of the 32 genes (KAS3, β-CT, BcRK6, P450, FatA, Oleosin, FAD6, FatB, α-CT and SUC1) were downregulated by between 20% and 50% (Table 2 and Fig. 2).

Among the upregulated genes, FAD3, ACCase and FAE1 were the most remarkable. During 20–30 DAPs, the transcripts of FAD3 were 130~180% more abundant in ZS9 than in ZY821 (Fig. 2M). In ZY821 the ACCase transcript stayed stable at low levels, whereas in ZS9 it displayed high and low peaks at 25–30 DAPs and 40 DAP, about 4 fold and 2 fold the levels in ZY821, respectively (Fig. 2A). FAE1 gene transcripts were 150%–480% more abundant in ZS9 than in ZY821 during 15–35 DAPs. At 30 DAP, more than 4-fold higher FAE1 transcript levels were observed in ZS9 than in ZY821 (Fig. 2I). Caleosin and GKTP transcripts were more abundant only during the final stage of seed development (Fig. 2U, AC).

Among the downregulated genes, KAS3 showed significantly lower expression at 10 and 20 DAPs (Fig. 2H), FatA at 10–20 DAPs (Fig. 2O), BcRK6 and P450 at 20 DAP (Fig. 2AD, Y), β-CT at 20–40 DAPs (Fig. 2D) and Oleosin at 40–45 DAPs (Fig. 2T).

The value and time of a transcript's peak levels are an indication of gene activity and regulation. ZY821 and ZS9 had different peak values and times for the expression of many of the genes investigated. The peak value was 338%, 288% and 162% higher in ZS9 than in ZY821 for FAD3, ACCase and GKTP, respectively (Table 2 and Fig. 2M, A, AC), and 40–60% higher in ZS9 than in ZY821 for KAS2, MCAT, AGPase, PEPC, KACD, FAE1, Caleosin, KAS1 and GAPDH (Table 2 and Fig. 2G, E, X, Z, J, I, U, F, AE). The peak value was over 60% lower for AAPT1 and 20–40% lower for Oleosin, KAS3, FatA, BcRK6, SAD and P450 in ZS9 than in ZY821 (Fig. 2S, T, H, O, AD, K, Y).

According to the peak time of gene expression, the 32 genes can be clustered into 4 groups. In ZS9, 12 genes (P450, KCR2, SUC1, FatB, BC, KAS2, PEPC, KACD, AAPT1, FatA, BcRK6, KAS3) peaked at 10 or 15 DAPs, 8 genes (α-CT, MCAT, GAPDH, SAD, FAD2, LPAAT, AGPase, FAD3) at 20–25 DAPs, 4 genes (FAE1, KAS1, ACCase and Napin) at 30–35 DAPs and 4 genes (Oleosin, Cruciferin, Caleosin, and GKTP) at 40–45 DAPs. In ZY821, 15 genes (P450, FatB, BC, KAS2, KACD, AAPT1, FatA, KCR2, PEPC, β-CT, DGAT2, FAD6, GAPDH, LPAAT and AGPase) peaked at 10 or 15 DAPs, 8 genes (SUC1, KAS3, BcRK6, α-CT, MCAT, SAD, FAD2, KAS1) at 20–25 DAPs, 2 genes (ACCase and FAD3) at 20–25 DAPs, and 7 genes (FAE1, Oleosin, Caleosin, β-actin, Napin, Cruciferin and GKTP) at 40–45 DAPs. In most cases, the difference in peak times between the two cultivars was no more than 5 days, but LPAAT and AGPase peaked 10 days later in ZS9 than in ZY821, whereas SUC1 and Napin peaked 10 days earlier.

Different genes showed different degrees of variation in their transcript levels over the course of seed development, as shown by the variation coefficients in Table 2. In ZY821, AAPT1, Oleosin, Cruciferin, NapinB, Caleosin, FAE1, P450 and KAS3 had relatively higher variation, whereas in ZS9, Cruciferin, NapinB, P450, GKTP, Caleosin, Oleosin, BcRK6 and SUC1 did. β-actin had the lowest variation in both cultivars, serving as a satisfactory control gene in this study.

The 32 genes can be divided into five groups according to their expression patterns.

The first group is characterized by a basic transcript level at the initiation of seed formation, a rapid rise to a peak at 35–40 DAP, and then by a gradual decline. The genes for storage proteins, such as Napin, Caleosin, Cruciferin, and Oleosin, possessed this pattern (Fig. 2T, U, V, W). The expression patterns of these genes were much like the accumulation pattern of the total fatty acids, C18:1 in ZS9 (Fig. 1D) and C22:1 in ZY821 (Fig. 1J).

The second group is characterized by a bell-shape transcript curve, with a moderate level of expression at the initial stage followed by a gradual increase in transcripts at the middle stage and a decline to the initial level at the final stage. The genes SAD, FAD2, FAD3, KAS1, AGPase, MCAT and FAE1 possessed this pattern (Fig. 2K, L, M, F, X, E, I). These enzymes may function in fatty acid synthesis. The accumulation of the fatty acids C18:1 and C20:1 in ZY821 (Fig. 1D, H) also fell into such a pattern.

The third group is characterized by a low transcript level at the beginning, followed by further decreasing expression throughout seed development, as exhibited by FAD6, FatA, FatB, α-CT, BC, PEPC, KCR2, and KACD (Fig. 2N, O, P, C, B, Z, J). The fatty acids C16:0, C18:0, C18:2, C20:0, and C22:0, which are minor components in mature seeds once synthesized in early seed development, also showed such a pattern (Fig. 1B, C, E, G, I).

The fourth group is characterized by a stable expression or a gently rise and fall in expression pattern. GKTP, ACCase, KAS2, DGAT2 and β-actin are included in this group (Fig. 2AC, A, G, R). The fatty acid α-C18:3 had a similar pattern to this group (Fig. 1F). The fifth group is characterized by a high level of expression at the initiation of seed development, peaking at 10 or 15 DAP, followed by an abrupt decline, as exhibited by β-CT, BcRK6, KAS3, AAPT1, SUC1 and P450 (Fig. 2D, AD, H, S, AA).

A comparison of ZS9 and ZY821 indicated that most of the genes displayed similar expression patterns, although a few of the genes, such as ACCase, FAE1 and GKTP, had obvious differences between ZY821 and ZS9 (Fig. 2A, I, AC). ACCase had a bell-shape expression pattern in ZS9, putting it in the second group, whereas it had a flat pattern in ZY821, putting it in the third group (Fig. 2A). FAE1 was in the first group of expression pattern in ZY821 but had a bell-shape pattern in ZS9, putting it in the second group (Fig. 2I). GKTP also showed a significant difference, with a significant rise in transcript levels at the mature stage of seed development in ZS9, placing it in third group, although it had a very low level throughout all stages of seed development in ZY821 (Fig. 2AC).

Correlation analysis was performed between transcript profiles of the genes and accumulation patterns of the fatty acids. The results showed that the two cultivars had different correlation patterns (Table 3). For the Low Erucic Acid Rapeseed (LEAR) ZS9, the total amount of fatty acids was highly correlated with Caleosin and correlated with Cruciferin and Oleosin; the amount of C16:0 was highly correlated with BC, PEPC and FAD6 and correlated with Oleosin, α-CT, KAS2, β-CT, BcRK6, SUC1, DGAT2, FatA, P450, FatB, AAPT1, and KCR2; C18:0 was highly correlated with SUC1, β-CT, BC, PEPC, FAD6, FatA, P450, FatB, and AAPT1, positively correlated with KCR2, DGAT2, KAS3, BcRK6, KAS2, and α-CT, and negatively correlated with Oleosin; C18:1 was highly negatively correlated with BC, FAD6 and PEPC, negatively correlated with AAPT1, FatB, SUC1, P450, β-CT, FatA, KCR2, BcRK6, KAS3, DGAT2, α-CT, and KAS2 and positively correlated with Caleosin and Oleosin; C18:2 was highly positively correlated with BC, FatB, AAPT1, FAD6, SUC1, PEPC, P450 and KCR2 and correlated with β-CT, FatA, BcRK6, KAS3, DGAT2 and α-CT; C18:3 was highly positively correlated with ACCase and correlated with FAD3. For High Erucic Acid Rapeseed (HEAR) ZY821, the total amount of fatty acids was highly negatively correlated with KCR2, negatively correlated with ACCase and positively correlated with FAE1. C16:0 was highly positively correlated with KCR2, positively correlated with AGPase, PEPC, FAD6, ACCase, FatA, GKTP, LPAAT, and FatB and negatively correlated with FAE1; no significant correlation for C18:1 was found with any gene; C18:2 was highly positively correlated with KCR2 and highly negatively correlated with FAE1, positively correlated with FatB, ACCase, and PEPC and negatively correlated with Caleosin; C18:3 was correlated only with FAD3; C20:0 was highly positively correlated with KCR2 and correlated with AGPase, ACCase, and GKTP; C20:1 was highly positively correlated with FAE1, highly negatively correlated with KCR2 and negatively correlated with PEPC; C22:1 was highly positively correlated with Caleosin and Cruciferin, highly negatively correlated with KAS1, BC, KCR2 and β-CT, positively correlated with Napin, Oleosin, and FAE1, and negatively correlated with KACD, FAD6, FatA, FatB, and LPAAT.

The genes Napin, KACD, AGPase, GKTP, and LPAAT in LEAR ZS9 and the genes α-CT, KAS2, BcRK6, KAS3, SUC1, DGAT2, and P450 in HEAR ZY821 showed no significant correlation with any fatty acids or with the total amount of fatty acids.

Lipid synthesis depends on the correct spatial and temporal activity of many gene products 78. 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 10.

The first fatty acid synthesis stage commences with ACCase, catalyzing the carboxylation of acetyl-CoA to malonyl-CoA 11. Most plants have two forms of ACCase, the homomeric form in the cytosol, composed of a single large polypeptide catalyzing the individual carboxylation reactions 12, and the heteromeric form in plastids, composed of four subunits, including Biotin Carboxylase (BC) 13, Biotin Carboxyl Carrier Protein (BCCP) 14, α-Carboxyltransferase (α-CT) 15 and β-Carboxyltransferase (β-CT) 16.

The next enzyme is MCAT (Malonyl-CoA-acyl carrier protein transacylase), which catalyzes the transfer of malonyl-CoA to the holo acyl carrier protein (ACP), generating malonyl-ACP 17. Beta-ketoacyl-acyl carrier protein synthase III (KAS3) catalyzes the subsequent condensation and transacylation of acetyl-CoA with malonyl-ACP 18. KAS1 catalyzes the subsequent condensation from C4- to C14-ACP in the plastid 19, and KAS2 elongates 16:0-ACP to 18:0-ACP in the plastid 20.

The synthesized 18:0-ACP is converted to oleic acid (18:1) by SAD 21. In HEAR cultivars such as ZY821, the oleic acids (18:1) continue the elongation process to synthesize C20- to C22- fatty acids such as erucic acid (22:1), which involves four enzymatic steps: condensation by 3-ketoacyl-CoA synthase (FAE1) 22, reduction by 3-ketoacyl-CoA reductase (KCR) 23, dehydration by 3-hydroxyacyl-CoA dehydratase (KACD) 24 and finally reduction by trans-2,3-enoyl-CoA reductase. In LEAR cultivars, such as ZS9, fatty acids do not elongate any more due to the disfunction of FAE1 2526.

Oleic acid (18:1) can be further desaturated to oleate acids (18:2) by FAD2 27 and FAD6 28 in the cytosol and the plastid, respectively. Cytosolic and plastid ω-3 desaturations that result in the production of linolenic acids (18:3) are controlled by FAD3 29 and FAD7 30, respectively.

The final step in the plastid is the release of fatty acids from ACP by thioesterase, resulting in the formation of dissociated fatty acids 31. Two distinct but related thioesterase gene classes exist in higher plants. FatA is an acyl specificity 18:1>>18:0>>16:0 thioesterase 32. In contrast, FatB, encoding thioesterases, has a specificity for 16:0>18:1>18:0 fatty acids 33. Fatty acids are exported to the cytosol and activated to their acyl-CoA derivatives.

In the following stage, fatty acids are incorporated into glycerolipids through the acylation of glycerol-3-phosphate by acyltransferases, producing TAG. De novo phosphatidic acid biosynthesis in plants occurs in three steps: first, the acylation of the sn-1 position of glycerol-3-phosphate gives rise to lysophosphatidic acid, which is catalyzed by Glycerol-Phosphate Acyltransferase (GPAT) 34; second, the acylation of the sn-2 position forms phosphatidic acid, which is catalyzed by a lysophosphatidic acid acyltransferase (LPAAT) 35. Diacylglycerol acyltransferase (DGAT) catalyzes the final step of the TAG synthesis pathway 36.

The TAG assembles with oil body proteins to form oil bodies. There are four predominant seed storage proteins: Cruciferin, Caleosin, Oleosin and Napin. Cruciferin is a high molecular weight neutral complex of 12S proteins, composed of six subunit pairs 37. Napin is a low molecular weight basic 1.7S protein, composed of two disulfide-linked polypeptide chains 38. Caleosin is a Ca²⁺-binding oil-body surface protein 39 playing a role in both the normal modification of storage vacuole membranes and the interaction of oil bodies with vacuoles 40. Oleosins are oil body-associated proteins that cover and stabilize oil bodies during TAG accumulation 41.

According to DeLisle and Crouch 42, mRNA is about 1% of the total RNA in B. napus embryos. Cruciferin and Napin represent 11 and 8%, respectively, of embryonic mRNA based on mass 42. Cruciferin and Napin constitute 20% and 60% of the total protein content of mature seeds, respectively 43. Moreover, Napin (12.5–14.5 kDa) is about 25 fold smaller in mass (2S albumin) than Cruciferin (12S globulin, 300–310 kDa) 43. All of this implies that Napin mRNA is the most abundant and is much higher in copy number than Cruciferin mRNA during rapeseed development, in agreement with our results. With the strongest expression in seed, the Napin promoter has been popularly used for genetic engineering of seed components.

No estimate of the relative mRNA abundance has been reported for Oleosin and Caleosin in B. napus thus far, but it has been reported that Oleosins are low molecular weight (15–26 kDa) alkaline proteins that represent about 2–8% of the total seed proteins 44, and Caleosins are present at relatively low levels and are mainly bound to microsomal membrane fractions 45. Our data are the first on the relative abundance of Oleosin and Caleosin transcripts in rapeseed development, indicating that Oleosin transcripts are the second most abundant in copy number after Napin among the 4 storage proteins (Table 2).

No significant difference was found in the relative abundance of seed mRNA between ZY821 and ZS9, indicating that the genes are expressed in a highly organized and coordinated way and in a stable molar amount during seed development.

PEPC, upregulated by 39% in ZS9 compared with ZY821, is an enzyme in the family of carboxylyases that catalyzes the addition of CO₂ to phosphoenolpyruvate (PEP) to form the four-carbon compound oxaloacetate. Seeds of transgenic bean plants expressing Corynebacterium glutamicum PEPC in a seed-specific manner had a more rapid shift in metabolic fluxes from sugars/starch into organic acids and free amino acids 46, resulting in an accumulation of up to 20% more protein per gram dry seed weight in the transgenic seeds and a 20–30% higher seed dry weight 46. We show that PEPC upregulation may result in increased C16:0, C18:0 and C18: 2 but decreased C18:1 (Table 3).

ACCase, upregulated by 105% in ZS9 compared with ZY821, is the homomeric ACCase that catalyzes the first committed step of fatty acid synthesis, the carboxylation of acetyl-CoA to malonyl-CoA. Targeting a homomeric ACCase to rapeseed plastids under the control of a seed-specific promoter resulted in higher ACCase activity and increased oil yield by 3–5% on a dry seed weight basis 12. Over-expression of ACCase in the amyloplasts of potato tubers led to an increase in fatty acid synthesis and a more than 5-fold increase in the amount of TAG 47. ACCase is a key target to increase seed oil by genetic engineering 1247. In the present study, increased ACCase activity was accompanied with increased C18:3 in ZS9 and with increased C16:0, C18:0, C20:2 and C18:2 and decreased total fatty acids in ZY821. Considering that C18:3 does not change much with seed development, increased C18:3 must be consumed at a similar amount during seed development.

FAE1, upregulated by 80% in ZS9 compared with ZY821, is a key gene responsible for fatty acid elongation from 18:1 to 22:1 or erucic acid biosynthesis in Brassica. Seed-specific expression in Arabidopsis thaliana resulted in up to a 12-fold increase in the proportion of erucic acid. On the other hand, in transgenic high-erucic Brassica carinata plants, the proportion of erucic acid was as high as 51.9% in the best transgenic line, a net increase of 40% compared with wild type 48. FAE1 mRNA was normally transcribed in both HEAR and LEAR cultivars but a 57 kDa protein was found only in HEAR seeds, whereas it was absent in all LEAR cultivars using an antibody against FAE1 2649. It was beyond our expectation that the FAE1 transcript was more abundant in LEAR ZS9 than in HEAR ZY821 (Fig. 2I). The upregulated FAE1 transcript observed in this study (Fig. 2I) might be due to synthesized FAE1 mRNA that was not used for the blocked synthesis of functional FAE1 enzyme.

FAD3 was upregulated by 109% in ZS9 compared with ZY821 (Table 2) and was significantly correlated only with C18:3 in the present study (Table 3). No significant difference in C18:3 content is found during seed development between ZY821 and ZS9 (Fig. 1D). Why the FAD3 transcript level was up-regulated by so much and no more C18:3 was found in ZS9 needs further investigation. It is worthwhile to notice that the FAD3 and FAE1 changed coodinately (Fig. 2I, M), consistent with previous reports that they are both regulated by abscisic acid (ABA) 5. Some key transcription factors involved in lipid metabolism are under the influence of ABA signaling 5. The upregulation of FAD3 found in this study may be linked through some regulation cofactors with the upregulation of FAE1 in ZS9.

Abscisic acid induces Oleosin expression 50, and FAD3 and FAE1 also are regulated by abscisic acid 515253. The upregulation of FAD3, FAE1 and Oleosin found in this study might be owing to the same regulation cofactor. The breeding pressure may have selected for the action of this cofactor.

Caleosin, one of the storage proteins, was upregulated by 60% in ZS9 compared with ZY821 (Table 2). It was significantly correlated with total fatty acids and C18:1 in ZS9 and with C22:1 in ZY821 (Table 3). Meanwhile, transcrips of other storage proteins (Cruciferin, Oleosin and Napin) were all downregulated. Unlike Oleosin, Caleosin may be involved in signal transduction via calcium binding or phosphorylation/dephosphorylation in processes such as membrane trafficking and lipid-body fusion 39. During germination Caleosin plays a role in the degradation of storage lipid in oil bodies 40. Upregulation of Caleosin implies enhanced oil deposition and improved seed germination in ZS9.

GKTP (synonym KAT2), upregulated by 66% in ZS9 compared with ZY821, codes for the precursor of glyoxysomal 3-ketoacyl-CoA thiolase, the last enzyme in the beta-oxidation of fatty acids in plant glyoxysomes. Expression of this enzyme is required for the timely onset of natural and dark-induced leaf senescence in Arabidopsis 54. In this study, the transcript abundance of GKTP is correlated with C16:0, C18:0 and C20:0 in ZY821 but not with any fatty acid in ZS9. The reason for this difference is not clear.

GAPDH, upregulated by 56% in ZS9 compared with ZY821, encodes glyceraldehyde 3-phosphate dehydrogenase, which catalyzes the sixth step of glycolysis and thus serves to break down glucose for energy and carbon molecules. In addition to this long established metabolic function, GAPDH has recently been implicated in several non-metabolic processes, including transcription activation, initiation of apoptosis, and ER to Golgi vesicle shuttling. In this study, GAPDH showed relatively stable and high expression among the non-storage protein genes during seed development. The activity of GAPDH was not found to be correlated with any fatty acid biosynthesis in this study (Table 3).

KAS3 was down regulated by 51% in ZS9 compared with ZY821 (Table 2). It was also found to be significantly correlated with C16:0, C18:0 and C18:2 in ZS9 but not in ZY821. Katayoon et al. indicated that overexpression of KAS3 in tobacco plants reduces the rate of lipid synthesis and increases the levels of C16:0 in leaves. The transgenic B. napus seeds overexpressing KAS3 driven by Napin also contained lower levels of oil compared with wild-type 55. In addition, the rate of lipid synthesis in transgenic rapeseed seeds was notably slower than that of the wild-type seeds 55. Therefore, down-regulation of KAS3 might result in reduced saturated fatty acids and increased C18:2 in ZS9 (Fig. 1B, C, G). The same situation is also found for P450, FatA, FatB, AAPT1, KCR2, α-CT, β-CT, BC, SUC1, BcRK6 and KAS2 in this study (Table 3).

FatA was downregulated 32% in ZS9 compared with ZY821 (Table 2). It was shown in this study that transcript levels of FatA were negatively correlated with C18:1 in ZS9 and with C22:1 in ZY821 (Table 3). In both cultivars, the transcripts of FatA peaked at 10 or 15 DAPs, and positively correlated with C16:0 and C18:0 (Table 3). Thioesterases FatA and FatB play an essential role in the partitioning of de novo-synthesized fatty acids between the prokaryotic and eukaryotic pathways. FatA determines the in vivo levels of C18:1 that move out from the plastid 31. Downregulated FatA means lower levels of C18:1 move out from the plastid. This is reasonable because C18:1 was not consumed for C22:1 in ZS9, high accumulation of C18:1 must inhibit transcription of FatA. Similar situations were found for FatB (Table 3).

As one of the 4 subunits of the multisubunit plastidial ACCase, β-CT was downregulated 36% in ZS9 compared with ZY821. The transcript abundance of β-CT was much higher than other subunits of the multisubunit plastidial ACCase and ranked the third just next to Napin and Oleosin in both cultivars (Table 2). This is probabally because β-CT gene is the only component of plant lipid metabolism known to be encoded by the plastid genome 11. We found downregulation of β-CT was accompanied by increased C18:1 in ZS9 and higher C22:1 in ZY821 (Table 3), while reduction of the expression of β-CT was reported to result in reduced fatty acid synthesis and even damage of embryo development 56.

BcRK6, downregulated 36% in ZS9 compared with ZY821, is a receptor kinase, catalyzing the key step in steroid perception and signaling in plants 57. This enzyme was active at 10–20 DAP and was reduced sharply afterward. Decreased BcRK6 transcripts were significantly accompanied by increased C18:1 accumulation in ZS9 (Table 3). The reason for the downregulation of BcRK6 in ZS9 observed at 20 DAP needs further investigation.

Cytochrome P450 catalyzes a monooxygenase reaction, e.g., insertion of one atom of oxygen into an organic substrate (RH), whereas the other oxygen atom is reduced to water using a plethora of both exogenous and endogenous compounds as substrates for enzymatic reactions, producing a wide cultivar of susceptibility to specific toxins. Cytochrome P450 enzymes are present in most other tissues of the body and play important roles in hormone synthesis and breakdown (including estrogen and testosterone synthesis and metabolism), cholesterol synthesis, and vitamin D metabolism. This gene showed significant correlations in ZS9 but correlations were not found in ZY821. The reasons for this difference are not clear.

Most of the genes investigated in this study showed significant correlation with fatty acid accumulation (Table 3). We found that the correlation of the genes in ZS9 was significantly different from that in ZY821. For example, significant correlation was not found with any fatty acid in ZS9 but it was found with 6 fatty acids in ZY821; ACCase is highly significantly correlated with C18:3 in the LEAR ZS9 but not in ZY821, whereas in ZY821, ACCase showed significant correlation with many other fatty acids, including total fatty acids C16:0, C18:0, C20:0, and C18:2. Exceptionally, in both cultivars FAD3 showed significant correlation only with C18:3, in accordance with its defined function for the production of C18:3 from C18:2 27.

In ZS9, more tighter correlations were found between gene transcript levels and fatty acid accumulation patterns. In ZS9, thirteen genes including P450, FatB, AAPT1, KCR2, DGAT2, PEPC, FAD6, FatA, BC, SUC1, Oleosin, α-CT, and β-CT, whereas only four genes in ZY821, i.e., KCR2, FAE1, ACCase and FatB, showed significant correlation with at least four fatty acids, indicating that they play important parts in the storage accumulation of seed development. However, in both cultivars, the genes SAD, MCAT and FAD2, which among the down regulated genes showed no significant correlation with either any fatty acids or the total amount of fatty acids, did not respond to the breeding progress for fatty acid composition in rapeseed.

According to the frequency of significant correlations, KCR2 seemed of the highest importance in the fatty acid accumulation process. Higher KCR2 activity is associated with higher C16:0, C18:0, and C18:2 in both cultivars, lower C22:1 and total fatty acids in ZY821, and lower C18:1 in ZS9. Bn-KCR was preferentially expressed in seeds and roots. The co-expression of Bn-FAE1 and Bn-KCR observed in the HEAR cultivar during seed development was different from that of the LEAR cultivar, suggesting that the expression of both genes was directly or indirectly linked 23. To our knowledge, this is the first time a close correlation between KCR2 and fatty acid accumulation has been shown.

Both ZY821 and ZS9 are conventional rapeseed cultivars (B. napus), bred by our Institute. ZS9 was the offspring of ZY821 by crossing. Because they are two Chinese representative rapeseed cultivars bred during the past 20 years, they show some major differences, as described in Table 1.

During the flowering stage, flower buds were self-pollinated and bagged after removal of flowered and young buds in the main inflorescence. Developing pods were harvested at 5-day intervals, 10 to 45 DAP. Dissected seeds were frozen immediately in liquid nitrogen and stored at -70°C until gas chromatographic (GC) analysis or RNA extraction.

Thirty-two genes involved in the biosynthesis of storage products in rapeseed (Table 4) were identified by exhaustive database searches and by referring to the comprehensive lipid gene catalog provided by Mekhedov et al. 7. The selected genes cover all of the major biochemical events in the biosynthesis of storage products in rapeseed 758. Seed oil is synthesized in three stages: de novo fatty acid synthesis from C2 to C16 in the plastid, fatty acid elongation from C16 to C22, fatty acid desaturation and TAG synthesis in the ER and assembly into the oil body 10. The following were identified: four eukaryotic ACCase and prokaryotic plastidial ACCase subunit genes, which catalyze the first reaction in the fatty acid biosynthetic pathway, seven fatty acid elongase genes, which catalyze fatty acid elongation from varied lengths of carbon chains, four fatty acid desaturase genes, which catalyze the subsequent desaturation of the lipids to highly unsaturated forms, two thioesterase genes, which release free fatty acid from an acyl-ACP, three glycerolipid synthesis genes, which catalyze the acylation reactions of free fatty acids with glycerol-3 phosphate, four storage protein biosynthesis genes, six basic metabolism genes, and two housekeeping genes (Table 4). The functions and roles of these genes have been explained in detail by Mekhedov et al. and Ohlrogge and Browse 758.

PCR efficiency was assayed for each gene. Plasmid DNA containing the gene of study was diluted in 5 serial ten-fold dilutions. An XY (scatter) plot was drawn and the linear regression equation was developed with the log input for the X values and the CT (Cycle of threshold) for the Y values. The results are listed in Table 4. The regression slope was used to calculate the PCR efficiency according to the following equation.

The relative expression ratio of each gene compared with the control gene β-actin was determined using the Pfaffl method 60 and defined as Transcript Level (TL) in this paper. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control 60. This model needs no calibration curve. Control levels were included to standardize each reaction run with respect to RNA integrity, sample loading and inter-PCR variations 6162.

The CT values recorded in the Opticon Monitor 2.0 (MJ Research) were downloaded and processed in EXCEL. The mean CT value for each gene was calculated from three replicates and used for further calculations of the Transcript Level (TL) using the following formula 60:

in which E_ref is the PCR efficiency of the control gene β-actin, E_target is the PCR efficiency of the gene under study, CT_ref is the CT value of the control gene β-actin, and CT_target is the CT value of the gene under study. The TL value determined by this formula represents a relative abundance of the transcripted copy number of a gene and allows us to make comparisons between the transcript abundances for different genes and between two cultivars for a specific gene.

The dissected seeds were dried by refrigeration until their weight remained constant and then the dried seeds were weighed and finely ground in tubes. After dissolution in 1 mL petroleum ether/aether (v/v = 1:1), each sample was saponified in 1 mL 0.4 N methanolic-KOH (10% (w/v) KOH, 5% (v/v) H₂O in methanol) at 60°C for 1 h. After saponification, samples were cooled on ice and added to 500 μg methy-nonadecanoate (C19:0) and 2 mL ddH₂O, and vortexed. After centrifugation, the supernatant was analyzed by GC (Gas Chromatograph).

GC analysis was performed with a gas chromatogram (Agilent 5890N) fitted with a 30 m FFAP capillary column (ID 0.25 mm narrow bore and film thickness 0.5 μm). The GC conditions were set as follows: injection volume, 1 μL; injector temperature and flame ionization detector temperature, 260°C; carrier gas, nitrogen at a split vent ratio of 50:1; column head pressure, 172.7 kPa (25 psi) to make up a total flow of 78.2 mL/min and a column flow of 1.5 mL/min (average velocity, 42 cm/s); hydrogen flow, 40 mL/min; air flow, 400 mL/min; running temperature program, 160°C for 1 min, and then increased 4°C per min to 240°C and held at this temperature for 10 min.

The result of GC analysis is displayed by its absolute value and relative value. The absolute value and relative value were calculated by

where A_i is the area of the fatty acid peak, A_s is the area of the control peak, m_s is the mass quantity of the control, and m is the dry weight of the sample. Every sample was analyzed three times. The average value and standard deviation were calculated for each sample.

As shown in Table 2, the mean (Mean TL), coefficient of variation (C.V. of TL) and relative value of the transcript levels (RTL) were determined in order to characterize the transcript abundance of the 32 genes in study over time.

The mean transcript level (Mean TL) for each gene was calculated as an average of 8 stages from 10 to 45 DAP, and the relative transcript level (RTL) was calculated as the mean transcript level of the gene divided by the sum of the mean transcript levels of 32 genes and expressed as a percentage. The coefficient of variation (C.V. of TL) was expressed as the standard deviation as a percentage of the mean to measure the variation in transcript abundance of a gene during the time course.

A correlation analysis was made between the fatty acid accumulation pattern and the transcript level time course for each gene using the software embedded in EXCEL (Table 3).