1 Introduction

Seed development proceeds through a series of spatiotemporal controls of gene expression. Different stages of seed development feature specific molecular and metabolic events that define the course of physiological changes. Complex regulatory interactions, involving transcriptional, biochemical, and metabolic processes define the seed development program (Xiang et al. 2008). Therefore, a genome-wide view of gene expression profiles in developing seeds is needed to understand the underlying molecular mechanisms governing seed development. Knowledge of gene expression patterns associated with a specific stage of seed development is crucial to understanding the molecular and biochemical events characteristic of that stage. A good understanding of the genomic and transcriptomic makeup of the seed will facilitate the development of suitable tools for altering seed development and seed quality traits, such as oil content and quality, using molecular genetics approaches.

Like most other dicot plants, Brassica napus (also called rapeseed, oilseed rape, or canola) seed is composed of a seed coat, endosperm, and embryo. The cruciferous rapeseeds are exalbuminous, where the endosperm is eventually consumed and its space is occupied by the embryo during seed development. The main constituent of the early developing seeds is the liquid endosperm, which serves as a significant carbon resource for the ensuing stages of seed development from the embryo through to the endosperm. The endosperm is depleted to a single aleuronic layer in mature rapeseeds as a result of the significant cellular and biochemical changes in the endosperm during seed development (Huang et al. 2009b).

In general, three main stages mark seed development in higher plants: pattern formation/cell proliferation, maturation (including the seed filling), and desiccation (Goldberg et al. 1989; Thomas 1993). Rapeseeds undergo active cell proliferation during the 10–12 DAP, in which they simultaneously establish metabolic networks for subsequent seed maturation (Dong et al. 2004). Seed reserves (such as carbohydrates, oils, and proteins) accumulate rapidly during the seed-filling phase of maturation stage, which is followed by seed desiccation and acquisition of dormancy (Close 1996). The LEA genes play an important role in establishing tolerance to desiccation during maturation, as well as tolerance to osmotic stress (Xu et al. 1996).

2 Differential Expression of Genes During Seed Development

Analysis of differential gene expression is a commonly used approach to determine gene expression profiles during different stages of plant development. Several studies have been conducted to identify genes that are expressed preferentially during seed development, as well as the patterns of their expression at various seed developmental stages (Dong et al. 2004; Huang et al. 2009b; Niu et al. 2009; Xiang et al. 2008; Yu et al. 2010). Some of these studies revealed conserved metabolic processes in seeds (Niu et al. 2009), while others revealed developmental stage-specific genes and some differences (Dong et al. 2004; Yu et al. 2010). The cell proliferation is an important stage in seed development owing to its critical role in determining the pattern of embryo formation. This stage also signifies establishment of a network for the biosynthesis of storage products in the embryo. A sufficient number of ESTs derived from a given tissue can provide a reasonable picture of tissue-specific gene-expression profile (Girke et al. 2000; Park et al. 1993). A comparison of gene expression profiles between rapeseed embryo and leaf tissues identified 2,561 differentially-expressed genes with changes ranging from 1.5 to 65 folds (Xiang et al. 2008). Based on SAGE and further biometric analysis ∼35,000 expressed transcripts were identified during rapeseed early seed development (http://rapeseed.plantsignal.cn; Wu et al. 2008). Dong et al. (2004) analyzed a rapeseed cDNA library derived from seeds at 15 DAP and identified 104 differentially expressed and 54 unique genes. These genes indicated a diverse array of functions ranging from cell structure and development to unknown function. Twenty-five genes were found to be expressed only in seeds, of which 11 were expressed as early as 5–10 DAP. Four of the seed-specific genes were expressed only in the seed coat and another five in both the embryos and seed coats.

Yu et al. (2010) identified a large number of genes with diverse functions that were differentially regulated during seed development. These genes were involved in photosynthesis, CW biosynthesis, lipid metabolism, secondary metabolism, hormone metabolism, protein metabolism, signaling, development, and transport. In their experiments, seeds at 35 days post anthesis (DPA) showed 10-fold higher number of upregulated genes involved in photosynthesis, including those implicated in light reactions. In addition, photosynthetic genes involved in Calvin cycle were downregulated at 20 DPA (Yu et al. 2010). Fei et al. (2007) noticed a decline in expression of a substantial number of genes during seed maturation. Many of the downregulated genes were associated with energy pathways, including carbohydrate metabolism, protein metabolism, and photosynthesis. The gene encoding a 12 S globulin (cruciferin) seed SP CRA1 was two-fold lower in seeds at 20 DPA relative to 35 DPA (Yu et al. 2010). However, Arabidopsis 12 S seed SP family was previously reported to be associated with embryo development with high abundance during the latter half of embryogenesis (Pang et al. 1988). Huang et al. (2009b) identified 429 unisequences across three endosperm development stages: globular, heart-shaped, and cotyledon. The most evident differential expression was detected in globular-shaped embryos relative to heart shaped and cotyledon at the early stages of endosperm development. However, they were unable to detect any obvious differences in gene expression between the heart-shaped embryo and cotyledon stages. In another study to understand gene expression during seed maturation in rapeseed in relation to the induction of secondary dormancy, the majority of genes were found to be downregulated during maturation in cultivars DH12075 and AC Excel (Fei et al. 2007). Over 300 genes each in DH12075 and AC Excel cultivars were found to have significantly altered expression during seed maturation (Fei et al. 2007). Differences in gene expression patterns between cultivars were more evident during the transition from full-size embryo to mature seed.

3 Genes Involved in Lipid Metabolism

Increased knowledge on the regulation of lipid metabolism in B. napus helps plant breeders and biotechnologists to modify seed oil composition to meet market needs. Our ability to enhance lipid quality and quantity in rapeseed could be aided by deciphering its genome. However, B. napus, an allotetraploid (AACC genome, ∼ 1200 Mb), is a combination of the genomes from B. rapa (AA, 700 Mb) and B. oleracea (CC, 500 Mb) that underwent duplication during evolution. The availability of a close relative model plant A. thaliana (125 Mb) has made it easy to use comparative genomics approaches to understand metabolic processes. The development of high-throughput technologies, such as transcriptomics, SAGE, and next generation whole genome sequencing (Schena et al. 1995; Velculescu et al. 1995), makes it possible to perform a comprehensive gene expression profiling during B. napus seed development and to identify regulatory factors for FA metabolism. For example, ESTs obtained from developing Arabidopsis seeds served as the useful resource to investigate the conversion of carbohydrates to seed oil in higher plants (White et al. 2000). So far, approximately 700 genes encoding lipid metabolism-related proteins have been identified in Arabidopsis (Beisson et al. 2003) and this could serve as an important resource to understand the regulation of lipid metabolism in rapeseed.

The B. napus oilseed is rich in oleic and linoleic acids, and is a widely used source of oil for food and other industrial purposes. TAGs are the major storage oil in B. napus (Chia et al. 2005). Genes responsible for active FA metabolism were reported to express as early as nine DAF (O’Hara et al. 2002). Elaborate studies on seed development in Arabidopsis revealed that seed weight and lipid content were relatively low during early embryo development, whereas carbohydrate accumulation was high (Baud et al. 2002). It was also found that seed weight and FA accumulation occurred concurrently and accumulation of different types of FAs occurred at different periods of development. Moreover, 93 % of FAs were acetylated by 19 DAF. Weselake et al. (1993) reported that the appearance of DAGAT activity coincided well with the onset of lipid accumulation, and its activity was directly related to lipid accumulation. According to them, the DAGAT activity was increased during 14–33 DAF and then decreased rapidly as the lipid content stabilized in seeds.

Niu et al. (2009) classified the B. napus ESTs involved in lipid metabolism into four subprocesses: (i) metabolic pathways converting the photosynthate into seed oil; (ii) FA elongation and degradation; (iii) lipid metabolism in the ER and mitochondria; and (iv) proteins involved in lipid metabolism. These authors observed that most FA synthesis-related genes and some genes related to the OPPP were highly expressed at the advanced stages of seed development (21 DAF through 31 DAF) compared to sugar or starch metabolism-related genes. It has been proposed that during lipid synthesis, sugars are transported from the source tissues to the endosperm and then absorbed by the embryo (Fischer and Weber 2002). The cleavage product of sucrose by SUS, glc-6-P is utilized by both the cytosolic and plastidic glycolytic pathways. A glycolytic pathway in oilseeds helps conversion of the intermediates to pyruvate. Acetyl-CoA generated from pyruvate is then used as the source of carbon for the synthesis of FAs. This process requires combined action of acetyl-CoA carboxylase and FA synthase (Eastmond and Rawsthorne 2000). The glycoxylate cycle, b-oxidation, and gluconeogenesis enzymes are active during embryo development and their activities increase during embryo maturation and desiccation (Chia et al. 2005). The activities of these enzymes were also detected in the cotyledon, where the majority of lipid accumulation occurs (Chia et al. 2005).

Proteomics studies showed that FA synthesis-related proteins were expressed prominently at the midpoint of seed filling (bell-shaped pattern) and were highly expressed at four or five weeks after flowering (WAF; then decrease after six WAF; Hajduch et al. 2006). This finding is consistent with the findings reported by Niu et al. (2009). It was reported that types I and II genes related to FA synthesis started to show increased expression at 21 or 25 DAF and reached their maximum expression at 31 DAF. An independent transcriptomics study also reported that numerous genes including those involved in FA synthesis, FA elongation, triglyceride synthesis, and lipid transfer proteins (LTPs) were differentially expressed in seeds at 20 DPA compared to 35 DPA (Yu et al. 2010).

Numerous other factors, including plant hormones, were found to be involved in regulating lipid biosynthesis and FA accumulation. Expression of genes involved in wax FA metabolism, including CER1, KCS1, and CER2, were found to be regulated by WIN1, an Arabidopsis ethylene response factor (ERF)-type TF (Broun et al. 2004). While ABA is known to induce lipid biosynthesis in the developing rapeseed embryo (Zou et al. 1995), ABI4 served as a repressor of lipid breakdown during Arabidopsis seed germination (Penfield et al. 2006).

4 Biology of Starch and Sugar Metabolism

Embryos accumulate lipid as a major storage product and generally contain little or no starch at maturity. However, some oilseeds were reported to accumulate starch at the intermediate stages of development (da Silva et al. 1997; Kang and Rawsthorne 1994; Niu et al. 2009; Yu et al. 2010). There appears an active metabolic flux from photoassimilation to primary carbon metabolism in the endosperm. Given that starch is a carbon reserve, it could be utilized at later stages of seed development and in the synthesis of lipids (Norton and Harris 1975). Starch may also serve as the carbon source for synthesis of sugars, such as sucrose and stachyose, which have been proposed to enhance desiccation tolerance during the dry-out phase (Leprince et al. 1990). Active starch synthesis and decomposition occur during the early stages of embryo development, and typically peaks at the early to mid-stages of development (Niu et al. 2009). Expression of genes involved in carbohydrate production was found to be higher at 20 DPA compared to 35 DPA and was consistent with peak starch accumulation during 32–33 DPA (Yu et al. 2010).

Norton and Harris (1975) reported that oil content in seeds was five-fold higher than the peak starch content. King et al. (1997) suggested that even though starch was the predominant carbohydrate in seeds, it was not present in sufficient amounts to meet oil biosynthesis requirements. Starch synthesis in rapeseed embryos is known to occur in both the cotyledons and embryonic axis prior to and during the early phase of storage lipid accumulation (da Silva et al. 1997). According to that study, starch was not synthesized in the embryos by photosynthesis, but rather accumulated from carbon imported from various vegetative parts of the plant. They also showed that starch degradation starts after occurrence of one-third of the oil accumulation, and gradually disappears at maturity. Interestingly, blocking starch biosynthesis in the embryo was accompanied by a dramatic reduction in the importation of carbon source into the plastid (Vigeolas et al. 2004), and the impairment of formation of the embryo as a sink tissue. These findings indicate that imported sugars might be the main carbon source. Starch synthesis at the early stages may be essential in transforming the embryo into a sink organ prior to lipid synthesis (da Silva et al. 1997). In support of this, Niu et al. (2009) showed that the sucrose transporter (EL626999) gene was expressed two-fold higher at 19 DAF and later compared them to early stages (3 DAF through 9 DAF). A study on starch and FA accumulation in plastids of developing embryos of rapeseed showed that several cytosolic metabolites contribute to starch and/or FA synthesis during embryo development (Kang and Rawsthorne 1994). The gene encoding the phosphoenolpyruvate (PEP) translocator (PPT) was found to be highly expressed at all stages of seed development and was shown to be responsible for importing PEP, the main carbon source into the plastid during FA synthesis in Arabidopsis (Ruuska et al. 2002).

The process by which sugar and starch disappear at the later stages of seed maturity is worth noting. Imported sucrose is degraded by both SUS and invertase (Huang et al. 2009b). In developing rapeseed siliques, the switch to oil accumulation was accompanied by an increase in seed SUS activity (3.6-fold) and an approximately 76 % reduction in soluble acid invertase activity (King et al. 1997). Niu et al. (2009) also reported high expression of SUS throughout seed development. It is interesting to note that these authors also reported the isolation of a neutral invertase and found significant increase in its expression during seed development. Ruuska et al. (2002) reported increased expression of SUS genes after accumulation of storage products in the cotyledon. These independent studies also observed that the highest SUS activity coincided well with the formation of optimum seed size achieved by the accumulation of osmoticum (such as hexose), speculating that SUS accumulation might be related to seed size regulation (Huang et al. 2009b).

5 Storage Protein Accumulation in Rapeseed

The SPs are very useful in understanding developmental control of gene expression in seeds because of their abundance and developmental stage-specific accumulation (DeLisle and Crouch 1989). In Arabidopsis, protein deposition was reported to occur mainly at the seed-maturation stage following a sigmoid path (Baud et al. 2002). This report also indicated that protein synthesis continued at later maturity stages when oil content decreases on a whole seed basis, which could be due to the breakdown of lipids previously stored. Napin (2 S) and cruciferin (12 S) are the two major SPs of rapeseeds (Schwenke et al. 1983; Scofield and Crouch 1987). Napin and cruciferin were first detected in well-developed cotyledons. Cruciferin accumulation continues till the full mature seed stage, whereas napin accumulation ceases at the onset of embryo desiccation (Crouch and Sussex 1981). However, Ruuska et al. (2002) reported differential expression patterns of napin and cruciferin genes during seed development. In their study, napin genes were expressed abundantly between five DAF and 13 DAF with two-fold increase towards later stages of development. On the other hand, cruciferin genes were expressed weakly compared to napin genes and their expressions were increased by 10-fold at 8 DAF relative to 13 DAF. Napin gene, cruciferin gene CRB, cruciferin gene CRC, a putative cruciferin gene (Arabidopsis At 1g03890), and four seed SPs were identified by B. napus microarray analysis (Yu et al. 2010). This study showed that expression of CRA1 (Arabidopsis At 5g44120) was two-fold lower in B. napus seeds at 20 DPA relative to 35 DPA, supporting earlier reports that cruciferins are expressed strongly during later stages of seed development. A SAGE analysis of winter B. napus seeds revealed abundant expression of three cruciferin subunits at the later stages of seed development, showing higher expression at 35 DPA compared to 20 DPA (Sjödahl et al. 1993). In winter B. napus seeds, however, napin and cruciferin genes showed differential expressions in seeds at 35 DAP compared to seeds at 23 DAP (Obermeier et al. 2009).

Oleosins are the most abundant proteins associated with oil bodies and play a role in the synthesis, metabolism, and stability of these bodies (Frandsen et al. 2001; Huang 1992). It is interesting to note that a positive correlation between accumulation of oleosins and oils exists in B. napus seeds(Huang 1992).

6 Carotenoid Accumulation and Gene Expression

Carotenoids are the second-most abundant pigments in nature. The carotenoid family consists of over 700 members (Britton 1998). These compounds are well-known antioxidants in higher plants. They are involved in the assembly of the photosystems and are essential components of the photosynthetic machinery. They harvest light in a broader range of the blue spectrum than chlorophyll and transfer energy to chlorophyll. Carotenoids also serve as photoprotective compounds by quenching both the triplet chlorophyll and singlet oxygen derived from excess light energy thus limiting membrane damage. In addition to their potential therapeutic properties for humans, carotenoids are also used as colorants in the food and cosmetics industries, and as supplements in livestock and fish feed formulations (Botella-Pavía and Rodríguez-Concepción 2006; Fraser and Bramley 2004; Taylor and Ramsay 2005). Carotenoids accumulate in B. napus seeds throughout development with the highest levels detected at approximately 35–45 DPA (Yu et al. 2008). Carotenoid accumulation drops significantly at the later stages of seed development with b-carotene and lutein accounting for more than 90 % of the total carotenoid content (Yu et al. 2008). Findings indicate that carotenoids provide oxidative stability to B. napus oil (Frankel 2005).

Transcriptomics of developing B. napus seeds have identified a large number of genes involved in secondary metabolism having differential expressions between 20 DPA and 35 DPA (Yu et al. 2010). This study also showed that in developing B. napus seeds, expressions of carotenoid genes varied with the changes in development stages and enzymes responsible for various carotenoid biosynthetic pathways showed their expression at distinct stages of development. The same study also noted higher expressions of the upstream genes involved in isoprenoid biosynthesis in seeds at 20 DPA compared to 35 DPA (Table 10.1).

Table 10.1 Expression of B. napus Seed Genes Encoding Enzymes Involved in Carotenoid and Isoprenoid Biosynthesis at 20 DPA Relative to 35 DPA. 1Fold change expressed as 20 DPA/35 DPA. (Reproduced from Yu et al. (2010; Plant Sci 178: 381–389) with permission from Elsevier)
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7 Phenolics Accumulation

Phenolics are secondary metabolites with variable phenolic structures and play important roles in protection against biotic and abiotic stresses (Auger et al. 2010; Yu and Jez 2008). These include flavonoids, phenylpropanoids, pro anthocyanins (PAs), and others. Phenolics are well known for their health benefits in humans, including antioxidant and antitumor properties (Gullett et al. 2010). Anthocyanins and PAs are important plant pigments, sharing common biosynthesis steps and intermediates with flavonoids (Li et al. 2010). Phenolics of B. napus seed coat consist mainly of PAs and/or their precursors (Auger et al. 2010). Among different Brassica species, elevated phenolics content was reported in the seed coat of a yellow-seeded B. carinata line compared to a genetically related brown-seeded line (Li et al. 2010; Marles et al. 2003). Furthermore, Li et al. (2010) reported that accumulation of flavonoids, phenylpropanoids, and lignans occurred in yellow seed coats, whereas the brown seed coats accumulated only phenylpropanoids and lignans. Transcriptomics analysis showed stronger expression of a gene encoding a chalcone-flavanoneisomerase-related protein (Arabidopsis At1g53520) involved in flavonoid biosynthetic pathway in developing seeds of B. napus especially at the early stages of development (20 DPA relative to 35 DPA; Yu et al. 2010).

One of the most abundant phenolics in B. napus seed is sinapine, also known as sinpoyl choline. Sinapine is unique to cruciferous oilseeds with its levels in B. napus seeds ranging from 0.7 to 4 % (Blair and Reichert 1984); most of which (approximately 90 %) is located in the embryo (non-hull) fraction (Wang et al. 1998). B. napus accumulates a mixture of sinapate esters, including glucose, gentiobiose, and kaempferol glycoside esters as well as sinapine (sinapoylcholine), sinapoylmalate, and an unusual cyclic spermidine amide (Baumert et al. 2005). The expression of two genes, involved in the final two steps of sinapine biosynthesis, SGT (UDP-glucose:sinapate glucosyl transferase) and SCT (sinapoylglucose:choline sinapoyl transferase), have been studied (Milkowski et al. 2004). The activities of SGT and SCT were regulated at the transcriptional level during seed and seedling development of B. napus. The expression of SCT was specific to developing seeds, whereas SGT expression increased during early stages of seed development until the early cotyledonary stage, and then maintained that level until later stages. The SGT expression reached its highest level in two-day-old seedlings, and then decreased significantly.

Because of the antinutritional nature of sinapine, repeated attempts have been made to reduce its levels in Brassica seeds (Baumert et al. 2005; Hüsken et al. 2005; Nair et al. 2000; Velasco and Möllers 1998), but the highest reduction of ∼ 90 % was obtained by concomitantly silencing of both ferulic acid 5-hydroxylase (FAH) and SCT genes in the phenylpropanoid pathway (Bhinu et al. 2009). The sinapine reduction trait was stable over several generations and also under normal field conditions. However, silencing of FAH and SCT genes in Arabidopsis had pleiotropic effects beyond sinapine biosynthesis (Huang et al. 2009a). Transcriptomics of Arabidopsis fah and sct mutants documented more than 4,500 different transcripts with greater than four-fold difference in expression in developing siliques of the mutants compared to wild type control. Most of the genes involved in a variety of metabolic processes showed up to 20 % difference in their expression levels compared to wild type. Many genes of the phenylpropanoid pathway also showed changes in their expression levels (Fig. 10.1; Huang et al. 2009a).

Fig. 10.1
figure 1

Microarray-based identification of a large number of genes with prominent expression changes in developing siliques of a f5h and b sct plants harboring a single T-DNA insertion. The color-coded legends represent different functional groups of genes. (With kind permission from Springer Science + Business Media: Huang et al. 2009a. (Fig. 1))

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8 Regulatory and Signaling Genes Related to Seed Development

Embryonic genes in B. napus are repressed in vegetative organs, and their derepression during seed development is regulated by a complex network of transcriptional and post transcriptional regulatory factors (Vicente-Carbajosa and Carbonero 2005)reference added in the reference section. Changes in seed physiology and composition during seed development are regulated by regulatory networks, involving TFs and protein modifying enzymes. Such factors would play major roles in the timing of expression of storage compounds. In Arabidopsis, as many as 2,000 TF genes have been reported, representing more than 6 % of all total genes (Riechmann and Ratcliffe 2000; Riechmann et al. 2000). In B. napus, 320 TFs were reported to be expressed during seed development, and seven of them had expression patterns similar to those of genes related to FA biosynthesis (Niu et al. 2009). A number of TFs were found to be differentially expressed in seeds at 20 DPA and 35 DPA (Yu et al. 2010). A B3 family protein, similar to Arabidopsis At3g18960, is a good example as its expression was strongly upregulated in seeds at 20 DPA. The B3 domain-containing TFs were previously reported to regulate several aspects of seed development and accumulation of seed storage compounds in Arabidopsis (Meinke et al. 1994; Santos-Mendoza et al. 2005; Stone et al. 2001). Two TFs, belonging to the AP2/ERF family, which play important roles in determining seed size, weight, and regulation of storage compound biosynthesis, were reported to be expressed at a higher level at 35 DPA relative to 20 DPA (Yu et al. 2010). Of these, the AtRAP2.2 TF was shown to regulate phytoene synthase and phytoenedesaturase genes, which catalyze important steps in the carotenoid biosynthesis pathway.

Yu et al. (2010) reported a number of bZIP TFs to be differentially expressed over the course of B. napus DH12075 seed development. They reported that increased expression of ABI5, a bZIP TF involved in ABA signaling (Finkelstein and Lynch 2000), was accompanied by stimulation of 13 LEA genes. Abiotic stress-induced TF, AZF2, was also upregulated in B. napus seeds at 35 DPA compared to 20 DPA (Yu et al. 2010).

Analysis of differential gene expression between two cultivars of B. napus showed that regulation of TFs during seed development can also be cultivar specific (Fei et al. 2007). Two genes encoding TFs (At1g50600 and At5g10280) were specifically downregulated in seeds of AC Excel cultivar of B. napus compared to DH12075. The gene At1g50600 coded for a scarecrow-like TF has been shown to be associated with signaling and development in Arabidopsis (Pysh et al. 1999). The TFs, MYB66 and the gene encoding a MYB family member (homolog to Arabidopsis At5g14750) was upregulated in DH12075. The MYB family of TFs is known to be involved in the regulation of secondary metabolism, control of cell shape, disease resistance, hormone, and stress response (Martin and Paz-Ares 1997). Furthermore, a gene encoding ethylene responsive MBF1 family protein, a transcriptional coactivator, was upregulated specifically in DH12075 (Matilla 2000; Yu et al. 2010).

Seed development is accompanied by large-scale chromatin remodeling and histone modifications, with chromatin modifications starting as early as the decondensation of chromosomes from pollen grains immediately after fertilization (reviewed by Baroux et al. 2007). Histones are a component of chromatin, the protein-DNA complex involved in DNA packaging and transcriptional regulation (Okada et al. 2005). Covalent histone modifications are important for chromatin remodeling and transcriptional regulation of gene expression (Jenuwein and Allis 2001). Four histone H3 genes (Arabidopsis At1g09200, At5g10400, At5g65350, and At5g65360) and four H2B genes (Arabidopsis At1g07790, At2g37470, At3g53650, and At5g22880) had higher expression in B. napus seeds at 20 DPA relative to 35 DPA (Yu et al. 2010).

Signal transduction through phosphorylation processes regulates many cellular events and metabolic pathways (Chevalier and Walker 2005). Six signal transduction-related genes were found to have similar expression patterns as genes involved in FA synthesis in developing B. napus seeds (Niu et al. 2009). These genes include MAP kinase, the phosphatidylinositol 3- and 4-kinase families, and phospholipase. Yu et al. (2010) also observed higher expression of CDPK6 (Arabidopsis At4g23650) and CDPK9 (Arabidopsis At5g23580) at 20 DPA, while CDPK19 (Arabidopsis At5g19450) had a weaker expression in seeds at 20 DPA relative to 35 DPA. Previous reports showed that rice CDPK (SPK) played a crucial role in the accumulation of storage products during seed development (Asano et al. 2002). Other kinases differentially expressed during seed development were phosphatidylinositol-4-monophosphate 5-kinase (PIP5K9; Arabidopsis At3g09920) and pyruvate orthophosphate dikinase (Arabidopsis At4g15530), which were downregulated in seeds at 20 DPA compared to 35 DPA (Yu et al. 2010). In addition, cultivar-specific differential expression of protein kinases was also reported (Fei et al. 2007). Four upregulated genes encoding protein kinases (Arabidopsis At3g09830, At1g28440, At1g75970, and At2g33830) were specifically identified in seeds of cultivar DH 12075 compared to AC Excel.

Ubiquitination plays an essential role in a large number of eukaryotic cellular processes by targeting proteins for proteasome-mediated degradation (reviewed in Hershko and Ciechanover 1998). Polyubiquitin genes are transcribed and translated into polyproteins that are cleaved into monomers by specific proteases (Baker et al. 1992). Four such genes (Arabidopsis At2g01340, At4g05050, At5g03240, and At5g19990) were found to be upregulated specifically in seeds of cultivar AC Excel in comparison to DH12075 (Fei et al. 2007). These genes are related to protein metabolism, including polyubiquitins. These results reinforce the fact that differential regulation of protein metabolism and signal transduction exists between different cultivars of B. napus.

9 Concluding Remarks

Numerous efforts were concentrated on better understanding the underlying mechanisms regulating seed development in both the model plant Arabidopsis and the economically important oilseed crop rapeseed. Attempts so far have included conventional approaches like studying the seed composition at various stages of development, and novel approaches involving global gene expression profiling directly from the target seed compartments. These studies have contributed greatly to our understanding of the whole processes and complexities of different pathways operating simultaneously in the developing seeds. Yet, biology behind seed development remains largely a mystery and the underlying mechanisms a paradox. Recent advances in technologies, such as laser capture microdissection, will help deep profiling of gene expression from different cell types and seed compartments. Completion of the B. napus genome sequencing and advances in NGS will further our understanding of developmental mechanisms in seeds. Transcriptomics and proteomics approaches combined with a sequence database could complement sequence analysis and contribute to deciphering the seed development blackbox in B. napus and other oilseed plants.