Molecular Plant Advance Access originally published online on June 25, 2009
Molecular Plant 2009 2(5):1107-1122; doi:10.1093/mp/ssp042
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Global Analysis of Gene Expression Profiles in Brassica napus Developing Seeds Reveals a Conserved Lipid Metabolism Regulation with Arabidopsis thaliana
a National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Science (SIBS), Chinese Academy of Sciences, 300 Fenglin Road, 200032 Shanghai, China
b United Gene Holdings, Ltd. 3–5, 1111 ZhongshanBeier Road, 200092 Shanghai, China
c The Nature Products and Glycoconjugate Research Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 47 Zhongshan Road, 16023 Dalian, Liaoning, China
1 To whom correspondence should be addressed. E-mail hwxue{at}sibs.ac.cn, fax 00 86 21 54924060, tel. 0086 21 54924059.
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Abstract |
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In order to study Brassica napus fatty acid (FA) metabolism and relevant regulatory networks, a systematic identification of fatty acid (FA) biosynthesis-related genes was conducted. Following gene identification, gene expression profiles during B. napus seed development and FA metabolism were performed by cDNA chip hybridization (>8000 EST clones from seed). The results showed that FA biosynthesis and regulation, and carbon flux, were conserved between B. napus and Arabidopsis. However, a more critical role of starch metabolism was detected for B. napus seed FA metabolism and storage-component accumulation when compared with Arabidopsis. In addition, a crucial stage for the transition of seed-to-sink tissue was 17–21 d after flowering (DAF), whereas FA biosynthesis-related genes were highly expressed primarily at 21 DAF. Hormone (auxin and jasmonate) signaling is found to be important for FA metabolism. This study helps to reveal the global regulatory network of FA metabolism in developing B. napus seeds.
Key Words: Fatty acid • Brassica napus • seed development • starch • EST
Received for publication April 8, 2009. Accepted for publication May 28, 2009.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMethodsSUPPLEMENTARY DATAFUNDING |
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The seeds of Brassica napus (rapeseed) contain abundant fatty acids (FAs) such as oleic acid and linoleic acid, and this plant has served as one of the main sources of plant oil for humans. Numerous studies have focused on increasing the yield and improving the quality of rapeseed through genetic breeding (Meng et al., 1998; Zhang et al., 2004, 2006) and there are detailed studies on FA metabolism and its regulation; these provide important information for future breeding.
There are 
700 genes encoding lipid metabolism-related proteins in Arabidopsis (Beisson et al., 2003) and analyses of their expression patterns are expected to facilitate the understanding of lipid metabolism on a wide level. Currently, a common flux model indicates that during lipid synthesis in seeds, the sugars are first transported into the endosperm from the source tissues and then absorbed by the embryo. The sugars, including sucrose, glucose, and fructose, are used as carbon sources. The cleavage product of sucrose, by sucrose synthase, is utilized through both the cytosolic and the plastidic glycolytic pathway. The multiple transporters, including the glucose-6-phosphate (Glc-6-P) translocator, the triose phosphate translocator, and the phosphoenolpyruvate (PEP) translocator, which are localized to the plastid membrane, exchange the intermediates that are generated during the glycolytic process from the cytosol to the plastid (Fischer and Weber, 2002). A parallel plastidic glycolytic pathway is also present in the B. napus plastid, and is closely linked to starch metabolism and the oxidative pentose phosphate pathway (OPPP) in plastids. All of the PEP and pyruvate generated by the cytosolic glycolytic and plastidic glycolytic processes are utilized as a carbon source to participate in FA synthesis in plastids. The malonyl–CoA, produced from pyruvate, can serve as a two-carbon unit in FA synthesis. Continuous FA biosynthesis is catalyzed by a series of FA synthases.
Recent studies have revealed important roles for FA metabolism in plant morphology, growth, pollen and seed development, and stress responses (Mou et al., 2000; Kachroo et al., 2003, 2004; Zheng et al., 2005, Zhang et al., 2008, Dong et al., 2009). Arabidopsis acyl–ACP thioesterases (FATB) affect both plant growth and seed development by regulating cellular components (Bonaventure et al., 2003). A deficiency of FATB results in greatly reduced concentrations of palmitate and stearate in almost all tissues. Enoyl–CoA reductase (ECR), a key enzyme in very-long-chain FA (VLCFA) synthesis, has an essential role in endocytic membrane trafficking and cell expansion (Zheng et al., 2005). Deficiency of the stearoyl–acyl carrier protein desaturase (SSI2/FAB2) results in the reduction of the concentration of oleic acid and leads to the constitutive activation of the NPR1-dependent and -independent defense responses (Kachroo et al., 2003).
Lipid biosynthesis and FA accumulation are regulated by many factors, including plant hormones. WIN1, an Arabidopsis ethylene response factor (ERF)-type transcription factor, acts as a transcriptional activator during epidermal wax accumulation. Overexpression of WIN1 resulted in an 4.5-fold leaf epidermal wax accumulation and stimulated the expression of genes involved in wax FA metabolism, including CER1, KCS1, and CER2 (Broun et al., 2004). ABA induces lipid biosynthesis in the developing B. napus embryo (Zou et al., 1995), and ABA INSENSITIVE4 (ABI4) acts as a repressor of lipid breakdown in Arabidopsis seed germination (Penfield et al., 2006). Although numerous studies have focused on the FA biosynthetic pathway (Murphy and Cummins, 1989; Ohlrogge and Browse, 1995; Kang and Rawsthorne, 1996; Da Silva et al., 1997), systemic studies on FA metabolism, specifically its regulation, are still deficient.
The genome of B. napus, an allotetraploid (AACC, 1200 Mb), is a combination of the genomes from B. rapa (AA, 700 Mb) and B. oleracea (CC, 500 Mb). The model plant species Arabidopsis thaliana (125 Mb, 25 489 annotated genes, TAGI, 2000) belongs to the same family and shares high co-linearity with B. napus. The development of high-throughput technologies, such as large-scale sequencing, serial analysis of gene expression (SAGE), and cDNA chips (Velculescu et al., 1995; Schena et al., 1995), makes it possible to study gene expression profiles during B. napus seed development and the regulation of FA metabolism. The relative abundance of expressed sequence tags (ESTs) will also help to study gene expression levels at specific developmental stages or in specific tissues. The ESTs obtained from developing Arabidopsis seeds are a good resource for the study of the conversion of carbohydrates to seed oil in higher plants (White et al., 2000). In addition, this technique facilitates studies of similar metabolic pathways in developing seeds from Sesamum indicum (Suh et al., 2003).
Girke et al. (2000) identified a set of seed-specific genes of Arabidopsis using cDNA microarrays. Dong et al. (2004) detected 104 and 63 differentially expressed B. napus ESTs at 15 and 25 DAF (days after flowering), respectively, when compared with 10 DAF; based on this, the authors concluded that cell proliferation was active during 10–20 DAF. Park et al. (1993) sequenced 237 ESTs from the B. napus roots, whereas Hajduch et al. (2006) obtained 794 proteins from a B. napus developing seed through a proteomics strategy, in order to provide a resource linking the transcriptional and post-transcriptional regulatory network of seed development. Recently, a Brassica cDNA array that contained 10 642 genes from seed cDNA library was reported (Xiang et al., 2008). However, systemic gene expression profiles during B. napus seed development, especially fatty acid metabolism, have not yet been reported.
RAPESEED was established as a useful database for studies of the Brassica species (Wu et al., 2008). In this work, we present a detailed analysis of FA metabolism and regulation in the B. napus seed. A comparative analysis with Arabidopsis seeds has revealed the differential profiles and regulatory roles of starch metabolism, photosynthesis, and other co-factors in FA metabolism of B. napus.
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RESULTS |
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Serial Analysis of Gene Expression (SAGE) Reveals Active Lipid Metabolism during B. napus Early Seed Development
Immature seeds of B. napus at 5 and 9 DAF were collected and used to construct the SAGE library. Based on the total and unique tags that were obtained, a double-reciprocal plot and linetype-regress analysis revealed the presence of
35 000 expressed transcripts during B. napus early seed development (http://rapeseed.plantsignal.cn, Wu et al., 2008). Further analysis of the FA biosynthesis-related genes in the plastids showed that the genes encoding 3-ketoacyl-ACP synthase, 3-oxyacyl-ACP reductase (KR), enoyl-ACP reductase (ENR), and stearoyl-ACP-desaturase have approximately two copies; and that encoding the acyl carrier protein 1 (ACP) has 16 copies, resulting in a ratio of KR:ENR:ACP = 1:1:8. This is similar to previous work, which found that the transcripts of genes encoding KR, ENR, and ACP were at the invariable ratio of KR:ENR:ACP = 1:1.2:6.9 in seeds at 20–32 DAF (O'Hara et al., 2002), indicating the presence of an active FA metabolism at 9 DAF.
Conserved Enzymes Involved in Lipid Metabolism
Rapeseed embryos at different developmental stages were used to construct two cDNA libraries, and a total of 8 462 unique ESTs were obtained after normalization and sequencing (Wu et al., 2008). Previously, Beisson et al. (2003) systematically analyzed the lipid metabolism-related genes in the public database of Arabidopsis. According to the same criteria, the identified B. napus ESTs involved in lipid metabolism were classified into four processes: (1) metabolic pathways converting the photosynthate into seed oil; (2) FA elongation and degradation; (3) lipid metabolism in the endoplasmic reticulum and mitochondria; (4) other proteins involved in lipid metabolism (Table 1). These four processes include the processes of sugar importation into the seeds and the following glycolytic pathway, the transportation of the subsequent products, the biosynthesis of lipids in plastids, the production of FA in the endoplasmic reticulum and mitochondria, and other relevant processes. The similar components of the FA synthetic process (Table 1) suggest that lipid metabolism is conserved between B. napus and Arabidopsis.
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Although components of the lipid metabolism is likely to be conserved, different numbers of enzyme isoforms were found. In most cases, there were more isoforms of B. napus enzymes than those of Arabidopsis. Interestingly, most of these enzymes were starch (carbohydrate) metabolism-related genes (Table 1, in bold), indicating that a more active starch metabolism exists in the early stages of seed development in B. napus than in Arabidopsis. Additionally, although the ESTs were obtained from the early stage of seeds (3–19 DAF), genes encoding proteins involved in FA biosynthesis, elongation, transport, and even degradation were expressed.
Gene Expression Profiles by cDNA Microarray Analysis Reveals a Conserved Carbon Flux and Reductant Supply Compared with Arabidopsis
Gene expression profiles during seed development were studied through cDNA microarray. cDNA inserts of the ESTs were PCR amplified and used to generate the glass-based cDNA chips, and RNAs from immature seeds at different developmental stages were used for hybridization. Because the latest samples used to construct the cDNA library is seeds at 19 DAF, which is relatively late during seed development, some high expression genes at late developmental stages such as cruciferin, oleosin, and napin were not included in the library or in the cDNA chip. FA biosynthesis can be divided into several different functional groups: the cytosolic glycolytic pathway, the transport of carbon sources into plastids, starch metabolism, the plastidic glycolytic pathway, the plastidic OPPP, and the FA synthesis pathway. Analysis of the expression patterns of the genes involved in these processes showed that most FA synthesis-related genes, and some of the OPPP-related genes, are highly expressed at the later stages of 21–31 DAF (Figure 1), when compared with sugar or starch-related genes. Further cluster analysis on the FA synthesis-related genes in the chip (400) revealed four types of expression patterns: (1) no clear changes before 21 DAF, but a continuous increase during 21–31 DAF (Type I, Figure 2A), including those encoding enoyl-ACP reductase, 3-oxoacyl-ACP synthase I, and stearoyl ACP desaturase, etc., similar to those in Arabidopsis developing seeds (Ruuska et al., 2002); (2) a continuous increase during 25–31 DAF (Type II, Figure 2B), including those encoding ACCase (biotin carboxylase, 
-carboxyltransferase), long-chain acyl–CoA synthetase 8, delta 9 desaturase, omega-6 FA desaturase, and ACP1; (3) a specific increase during 9–19 DAF (Type III, Figure 2C), mainly including starch metabolism-related genes; and (4) no obvious change during the entire development of the seed (Type IV, Figure 2D), including FatA, FatB, FAE1, ketoacyl–CoA synthase, and other isoforms of ACP.
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In fact, some Type IV genes have a high background level of expression (three to five times higher than those of Type I and II), as confirmed by the SAGE studies (Wu et al., 2008). As an example, the copy numbers of beta-ketoacyl–CoA synthase (GAGGCCAAGG) and ketoacyl–CoA synthase I (GGCTGGTTCG) are 9 and 6, respectively, while enoyl–ACP reductase (TTATGTTTCT) and stearoyl ACP desaturase (ACAGAGAAGT) have two copies each (Type I pattern). The regulation of these genes is primarily post-transcriptional.
Compared with Arabidopsis, a mainly constitutive role of carbon was detected for B. napus lipid biosynthesis. A systemic investigation by quantitative real-time RT–PCR (qRT–PCR) analysis showed that expression of the sucrose transporter (EL626999) gene at later stages (19 DAF later) was two-fold higher than in the early stages (3, 9 DAF), similar to that of Arabidopsis. However, expression of the sucrose synthase (SuSy, EL628106) gene was unaltered during the entire developmental process (Figure 3A).
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Imported sucrose can be degraded by both SuSy and invertase. Previous studies have focused more on SuSy and less on invertase, which is thought to be mainly involved in development. One neutral invertase was isolated and an expression pattern analysis, by both chip hybridization and qRT–PCR, showed the expression of which is significantly increased during seed development (Figure 3A), suggesting that invertase has a possible role in lipid metabolism. However, as the invertase was transcribed at a very low level compared with SuSy transcription (Figure 3B), and also considering the high level of SuSy expression throughout seed development, SuSy may in fact be the main enzyme that degrades sucrose, and this is conserved in Arabidopsis.
Ruuska et al. (2002) showed that PEP was the main carbon source imported into the plastid by the PEP translocator (PPT) during FA synthesis in Arabidopsis. qRT–PCR analysis showed that the B. napus PPT-encoding gene was expressed at a very high level at all stages of seed development, and stayed high at later stages (Figure 3C), which is similar to the expression detected in Arabidopsis. In Arabidopsis, glucose-6-phosphate (Glc-6-P) provides the main carbon source for the biosynthesis of starch at early stages and the OPPP carbon skeleton at later stages, of seed development. In addition, the Glc-6-P translocator (GPT1 and GPT2) was highly expressed at early stages. A similar pattern of B. napus GPT expression (EL623322, EL626977) was detected. In addition, the homolog of Arabidopsis GLT1 (EL628734), a Glc-6-P exporter that mediates the export of glucose from the plastid, maintains a high expression level during the entire seed development, and expression is even higher at later stages (Figure 3D).
Reductants are required for FA synthesis (NADPH is necessary for reactions that are catalyzed by 3-ketoacyl-ACP reductase) and thus the supply of reductants severely affects the accumulation of lipids. Forty-four percent of NADPH that is used for FA synthesis is supplied by the OPPP in B. napus (Schwender et al., 2003). Eight of the annotated Arabidopsis enzymes and their isoforms, possibly involved in the plastid OPPP, were identified (Kruger and Schaewen, 2003) and were encoded by 13 B. napus genes.
Transaldolase and transketolase function at the branching point of the OPPP and determine whether the carbon source flows to FA synthesis. The reactions catalyzed by transketolase and transaldolase are at the stage after NADPH production, and hence they control the flow of the carbon source. An expression pattern analysis revealed a continuous increase in the expression of genes encoding transaldolase and transketolase, at 21 or 25 DAF, and this is very similar to the expression of Type I or II FA synthesis-related genes, respectively (Figure 4A).
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The effects of photosynthesis on embryo development are at the level of CO2 fixation and energy production (Asokanthan et al., 1997; King et al., 1998; Ruuska et al., 2004; Schwender et al., 2004). Although SAGE showed high transcript levels of the rubisco small subunit (TAATTTCTAT) (Wu et al., 2008), expression of both the rubisco small subunit and the ribulose–bisphosphate carboxylase had no evident change during seed development (Figure 4B). A lack of change may be due to: (1) a high background expression of the rubisco small subunit or (2) rubisco activity is regulated at the protein level rather than at the transcription level. The phosphoribulokinase is critical for Ribulose-1,5-bisphosphate production and the expression of gene encoding phosphoribulokinase continuously increases at 25–31 DAF, similar to type II FA synthesis genes (Figure 4C).
The cytosolic phosphoenolpyruvate carboxylase (PEPCase) also has the ability to fix CO2, and the gene that encodes PEPCase has a different expression pattern when compared with phosphoribulokinase, namely there are two peaks of expression during seed development (Figure 4C), and this may be due to the involvement of PEPCase in multiple metabolic pathways. It has been previously shown that photoreaction of photosynthesis provides NADPH and ATP for FA synthesis, and 20–30% of light penetrated the siliques to the seeds (Eastmond et al., 1996). Indeed, nine photoreaction-related genes, including the light-harvesting complex and the photosystem II oxygen-evolving complex protein, are detected in seeds and their expression patterns are very similar to the expression of type I or II FA synthesis-related genes (Figure 4D), indicating that photoreaction may provide reductants and energy for FA synthesis.
Active Starch Metabolism in the Early Stages of B. napus Seed Development
Starch metabolism is very active in early embryo development, and gene expression analysis has also revealed that there is a relatively higher transcription level of starch metabolism (synthesis and decomposition)-related genes (including type III FA synthesis-related genes). White et al. (2000) obtained 10 522 cDNAs from developing Arabidopsis seeds and studied the enzymes involved in carbohydrate and lipid metabolism. When compared with the copy numbers of the corresponding genes in Arabidopsis, the starch-related genes of rapeseed were highly expressed, indicating a more active starch metabolism in the rapeseed seed (Table 2). Genes encoding the Glc-6-P translocator, ADP–Glc pyrophosphorylase, the starch-branching enzyme, and the starch phosphorylase are highly expressed in B. napus seeds, with the exception of that encoding the starch synthase, which is expressed at a similar level when compared with Arabidopsis (Table 2). At this stage, Glc-6-P was the main carbon source imported from the cytoplasm to the plastid by GPT in Arabidopsis (Fischer and Weber, 2002), and starch was indeed synthesized from the imported Glc-6-P, but not by photosynthesis in B. napus (Da Silva et al., 1997).
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Expression of the Glc-6-P translocator was sustained at a high level at early stages and decreased rapidly at 21 DAF (Figure 5A). This expression indicates that Glc-6-P is the main carbon source during early embryo development. In addition, genes involved in starch metabolism (including Type III FA synthesis-related genes) have differential expression patterns when compared with those involved in lipid biosynthesis (Type I, II, and IV FA synthesis-related genes). The majority of the starch metabolism-related genes reached maximum expression at 17–19 DAF and then expression subsequently decreased; however, a few exceptions were detected. Maximum expression of the Glc-6-P translocator and beta-amylase was attained at 19 DAF (Figure 5A), and expression of the starch branching enzyme first peaked at 12 DAF and then at 21 DAF (Figure 5B), suggesting that transcriptional control of the genes encoding proteins involved in starch synthesis or decomposition might not be regulated by the same pathway.
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Additionally, the isoforms of the same enzymes showed a differential expression pattern. The isoforms of the starch phosphorylase and the sucrose transporter have at least two kinds of expression patterns; however, these two patterns still have some similarities. The starch phosphorylase isoforms, EL626129 and EL626671, reached maximum expression at 21 DAF (Figure 5C), which differs from the expression of the other isoforms, EL626950 and EL626832, which peaked at 19 DAF (Figure 2C). The sucrose transporter isoform, EL625304, had the highest expressions at 9, 17, and 21 DAF, with no obvious changes at other stages, whereas expression of the isoform EL626776 reached a maximal level at 19 DAF.
ADP–Glc pyrophosphorylase (AGPase), which catalyzes Glu-1-P to produce ADP-glucose, is the limiting enzyme during starch synthesis. Studies using transgenic approaches have indicated that AGPase strictly controls starch synthesis during early seed development, and participates in the establishment of a sink capacity at embryo initiation and lipid accumulation (Vigeolas et al., 2004). AGPase is composed of two large subunits, which have structure-remodeling capabilities, and two small subunits that have catalytic activities (Okita et al., 1990). Although the small subunits harbor the basic activities of the entire enzyme, various combinations of the large and small subunits enhance the catalytic activity. During B. napus seed development, the expression patterns of the AGPase large and small subunits are clearly different. For instance, expression of the large subunit was not constant (Figure 5D) and expression of the small subunit reached its maximal level at 17 DAF, and then decreased gradually (Figure 5E). These expression patterns are similar to those of the starch metabolism-related genes, indicating that, at the transcriptional level, AGPase may be regulated by the small subunits.
Opposite Expression Patterns of Genes during 17–21 DAF
A cluster analysis of the 4000 differentially expressed genes revealed two different groups of genes (854 genes in total) with a completely opposite pattern of expression during 17–21 DAF; 376 genes were repressed (upper panel), while the other 478 genes were stimulated (bottom panel) (Figure 6A). The transition stage from heart-shaped to torpedo embryos and the initiation of FA synthesis occur 17–21 DAF. A detailed analysis revealed that 102 of the repressed genes encode the proteins that are involved in replication, transcription, and translation (Figure 6B and Supplemental Table 1). In addition, 46 ribosomal proteins were repressed (only one was stimulated), indicating that protein translation was heavily inhibited at this stage. Six photosynthetic genes, including those that encode the light-harvesting chlorophyll a/b binding protein, the photosystem II protein, psbW, and cp29, were repressed and only two genes encoding thylakoid lumenal 25.6 kDa protein and thylakoid-bound ascorbate peroxidase were specifically up-regulated. In addition, 5- or 2-electron flow- and ATP-related genes were repressed and up-regulated, respectively.
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Possible Regulators of FA Synthesis during Seed Filling
Our studies indicate that FA synthesis is a rigorous process controlled largely at the transcriptional level; however, few transcription factors have been reported to regulate the FA metabolism related to intracellular signaling and storage. Thus, the genes encoding proteins that are related to transcription (transcription factors), signal transduction, and hormone function, with similar patterns of expressions of FA synthesis, were identified and analyzed.
Although approximately 320 transcription factors are expressed during seed development, only seven had an expression pattern similar to FA synthesis-related genes (Type I or II) including AP2, MADS, bHLH, etc. (Figure 7A and Table 3). The majority of these genes have not been previously reported, except for a MADS-box protein, FLOWERING LOCUS F, which is a repressor of flowering in Arabidopsis and may be involved in GA signaling. Six signal transduction-related genes have similar FA synthesis-related gene expression patterns (type I or II), including MAP kinase, the phosphatidylinositol 3- and 4-kinase families, and phospholipase C (Figure 7B and Table 3). These genes primarily belong to two functional groups, either cell division or the phosphatidylinositol-signaling pathway.
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Plant hormones have important roles in organ patterning, plant tropism, and root/shoot development. It was previously demonstrated that ABA is closely related to FA biosynthesis and mobilization (Zou et al., 1995). However, no ABA-related genes were detected with expression patterns similar to that of FA synthesis-related genes. Five auxin (IAA) de novo synthesis-related genes were identified, and four of them show similar expression patterns to FA synthesis-related genes, including nitrilase, nitrilase 2, and two isoforms of the tryptophan synthase beta-subunit (Table 3). The similar expression patterns indicate an important role for auxin in FA metabolism and in B. napus embryogenesis. Additionally, one gene encoding a jasmonate-inducible protein was shown to have a similar pattern of expression to that of the FA synthesis-related genes (Type II) (Figure 7C and Table 3).
These results indicated that auxin and jasmonates (JA) might play important parts in FA metabolism during seed development. Thus, the seed FA components in the auxin signaling mutant axr1-3, tir1-1, and the JA signaling mutant coi1 were analyzed. The results show changes of FA components in the axr1-3 mutant, including a decrease in palmitic acid (FA16:0) and linoleic acid (FA18:2), and an increase in stearic acid (FA18:0), oleic acid (FA18:1), gondoic acid (FA20:1), and erucic acid (FA22:1) (Figure 8A and 8B and Supplemental Table 2). Additionally, the total FA content of the axr1-3 seed increased when compared with that of wild-type (WT) (Figure 8C and Supplemental Table 3). FA components of the coi1 seed also change dramatically, with a decrease in palmitic acid, palmitoleic acid (FA16:1), linoleic acid, and eicosadienoic acid (FA20:2), and an increase in oleic acid and gondoic acid (Figure 8A and 8B and Supplemental Table 2). The total FA content of the coi1 seed also increased dramatically (38% compared with WT) (Figure 8C and Supplemental Table 3).
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Neither the FA components nor the content of the tir1-1 seed underwent an obvious change (Figure 8A–8C and Supplemental Tables 2 and 3). Interestingly, the variability of axr1-3 and coil was very similar (a decrease in palmitic acid (FA16:0) and linoleic acid (FA18:2), and an increase in oleic acid (FA18:1), gondoic acid (FA20:1), and the total FA content in the seed).
In addition, more than 100 genes were detected that had an expression pattern similar to that of the FA synthesis-genes (Supplemental Table 4), including those related to secondary metabolism and the stress response.
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DISCUSSION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMethodsSUPPLEMENTARY DATAFUNDING |
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Studies on the regulation of B. napus lipid metabolism have important fundamental and useful applications. Here, we provide a systemic analysis of the gene expression profiles of B. napus, followed by a comparative analysis of FA metabolism gene expression to Arabidopsis. This work has revealed global insight for understanding the regulatory network of FA metabolism of the B. napus seed, and will thus be helpful for oil-crop engineering.
Starch Metabolism Is More Active in B. napus Lipid Synthesis
The amounts of lipids in the B. napus seeds reached approximately 45% after maturation, and there was a short stage of starch accumulation during early seed development. There was little or no starch in the mature B. napus seeds, while starch synthesis and decomposition were active at the early stages of embryo development and reached maximal levels at the early-mid stage of development. Compared with that of Arabidopsis (White et al., 2000), expression of B. napus starch metabolism-related genes was higher (Table 2), indicating a more active starch metabolism in the B. napus seeds. Indeed, between Arabidopsis and B. napus, nearly 30 starch metabolism and OPPP-related genes had differential expression patterns, and this is consistent with an active starch metabolism at the early stages of B. napus seed development before the initiation of FA biosynthesis.
In B. napus, plants with an embryo-specific deficiency of AGPase, the activity of which decreases to 50% at the early developmental stage, as well as decreased starch content and rate of starch synthesis (Vigeolas et al., 2004), showed a 50% decrease in the lipid content of the seed. However, this difference was reduced, along with the embryo development, and there was no remarkable difference between mature antisense and WT seeds. This suggests that the suppression of starch synthesis will delay FA synthesis, while starch metabolism at the early stages does not have much of an affect. However, phosphoglucomutase (PGM, EC 2.7.5.1 [EC] ) catalyzes the readily reversible interconversion of Glc-1-P and Glc-6-P, and the seed oil content of the Arabidopsis plastidic pgm mutant was reduced to 40% when compared with WT (Periappuram et al., 2000). In the pgm mutant, the total enzyme activity was defective during all of the developmental stages, which was different from that of the AGPase antisense plants (the AGPase activity was nearly equal to that of the WT plants at later stages of FA synthesis), indicating that the starch synthesis at the early stage of embryo development is indispensable and crucial for the later lipid synthesis.
The accumulated starches in the seeds provide only a few carbon sources during the later developmental stage, and imported sugars are the main carbon source. Starch synthesis at the early stage is helpful for the embryo to become a sink organ before FA synthesis (Da Silva et al., 1997). When starch synthesis was blocked in the embryo, the importation of the carbon source into the plastid was dramatically reduced and the formation of the embryo as a sink tissue was impaired (Vigeolas et al., 2004). High expression of GLT1 during seed development suggests that the substrates may exchange between the cytosol and the plastid (Weber et al., 2000). This indicates that active starch metabolism has important roles in the B. napus embryo development, in the storage of accumulated lipids, and thus significantly contributes to the high lipid content when compared with Arabidopsis.
17–21 Days after Flowering: An Important Stage for Developing B. napus Seeds
A cluster analysis revealed two groups of genes with primarily opposite expression patterns at 17–21 DAF, and protein translation was heavily inhibited at this stage. Previous studies indicated that initiation of the synthesis of storage substances would result in decreased cell division, which is consistent with the observation of a reduction in transcripts of ribosomal, DNA replication, and RNA transcription-related proteins. At this stage, cell division was restricted and accumulation of the storage protein was not initiated. With regard to the relevant biochemical characteristics, we hypothesize that the suppression of protein translation occurs in order to accumulate energy and substances for the initiation of FA synthesis that occurs near 21 DAF. In addition to FA synthesis, these proteins, which are again up-regulated, are involved in the synthesis of storage proteins. Together with the previous theory, a temporary accumulation of starch is helpful as a sink organ and this suggests that starch synthesis first forms sink capacities, and then further enhances this capability through the accumulation of energy and storages.
As a critical plant hormone, auxin is involved in and regulates multiple developmental processes (Woodward and Bartel, 2005). Several auxin-induced genes, and several genes implicated in de novo auxin (IAA) biosynthesis or active IAA release, including nitrilase (NIT), tryptophan synthase beta-subunit, and IAA-Ala hydrolase, are highly expressed during 17–21 DAF. This indicates that auxin may have possible roles in these processes (Table 3).
Multiple Factors Are Involved in FA Synthesis Regulation and Interlacing of Transcriptional and Post-Transcriptional Regulation for B. napus Seed–FA Synthesis
Both transcriptional and post-transcriptional regulators are involved in B. napus seed development. It has been previously shown that Napin and cruciferin are two major storage proteins of the B. napus seeds. In particular, cruciferin has many isoforms and very abundant protein levels, specifically at later stages of seed development (4, 5, and 6 weeks after flowering, WAK) (Hajduch et al., 2006).
An expression profile analysis showed that Type I and II FA synthesis-related genes started to increase at 21 or 25 DAF, and reached a peak of expression at 31 DAF. This pattern of expression is consistent with proteomic studies that showed that FA synthesis-related proteins have a bell-shaped pattern and are highly expressed at 4 or 5 WAK (then decrease at 6 WAK, Hajduch et al., 2006). Interestingly, the majority of the previously identified proteins were represented by multiple isoelectric forms (Hajduch et al., 2006), which suggested the presence of a post-transcriptional modification. The gene encoding Rubisco has a pattern of expression like that of the Type IV FA synthesis-related genes and contributes to CO2 recycling. Expression of the Rubisco small subunit-encoding gene was not changed during seed development, albeit the intensity was at a high level. In addition, the large subunit of Rubisco was expressed at a high level during the entire seed development, as revealed by proteomic studies (Hajduch et al., 2006).
The OPPP provides components of the reductants for FA synthesis and several genes involved in OPPP are highly expressed at later stages of seed development (after 21 DAF, four transketolase, two transaldolase, and one ribulose-5-P epimerase). Similarly, analysis by 2-DE gel electrophoresis showed that five isoforms of 6-phosphogluconate dehydrogenase and one transaldolase, and three isoforms of 6-phosphogluconate dehydrogenase and the transaldolase maintained high levels of protein during 3–5 WAK (Hajduch et al., 2006), similar to the levels of transcription.
Jasmonate Signaling Regulates FA Metabolism
COI1 mediates the JA signaling through the SCFC°I1 ubiquitin–ligase complex (similar to auxin–TIR1–AUX/IAA in auxin signaling) (Xu et al., 2002). This complex is very similar to the auxin signaling SCFTIR1 ubiquitin–ligase complex, except the F-box protein, TIR1, is replaced with an alternate F-box protein, COI1 (Gray et al., 1999, 2001). AXR1 forms a heterodimer with the ECR1 protein and activates the conjugation of the ubiquitin-related protein, RUB1/NEDD8, to a member of the SCF complex, AtCUL1. The RUB1-modified AtCUL1 is the functional form and participates in the SCFC°I1 and SCFTIR1 complex in the JA and auxin signaling pathways, respectively (del Pozo et al., 1998, 1999, 2002). The tendency of FA changes in the axr1-3 and coi1 mutant seed are consistent. However, the FAs of tir1-1 seed do not have obvious changes (Figure 8A–8C and Supplemental Tables 3 and 4), and thus it is concluded that the FA changes of axr1-3 are derived from the defect in JA signaling. Indeed, the FA changes are extremely severe in the coi1 seed because of the severe defect in JA signaling. The ssi2/fab2 mutant, which results in reduction of oleic acid (FA18:1), is compromised in JA-related signaling (Nandi et al., 2003; Kachroo et al., 2003, 2004), indicating that FA signaling affects JA signaling. Our results show that JA signaling may modulate FA metabolism through a feedback regulation.
These results indicate that proper repression of JA signaling will change the FA composition and increase the seed production. This provides new insight for the engineering of oil-producing plants (including B. napus). The similar expression profiles of several of the auxin biosynthesis-related genes with Type I or II FA synthesis-related genes indicate the important roles of auxin in FA biosynthesis. The reason for the unchanged FAs in the tir1-1 mutant may be because of redundancy of the F-box proteins in Arabidopsis. Another auxin signaling mutant, axr2-1, which is a gain-of-function AXR2/IAA7 mutant, was then analyzed (Supplemental Figure 1). Both of the components and the content of the FAs are changed. However, the altered pattern of FA components was not similar to that of axr1-3 or coi1, indicating that different regulation of auxin and JA in FA biosynthesis occurs.
In total, this study provides information to allow for a further understanding of the FA metabolism of B. napus. In addition, this work demonstrates the critical role of starch metabolism and the relevant regulatory networks, which will be very helpful for molecular breeding of oil crops in the future.
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Plant Materials
Brassica napus plants (Huyou15) were planted in the field in late September (last 10 d) under natural conditions. These plants flowered in late March (last 10 d) of the second year. The temperature in Shanghai is mainly lowest 10°C at night and highest 20°C in the daytime in late March (last 10 d); lowest 15°C at night and highest 26°C in the daytime in late April (last 10 d). Siliques at 3–21 (every 2 d), 25, or 31 DAF of Brassica napus were collected. Each flower was labeled after flowering and harvested accurately. Immature seeds were separated from silique coats and used for RNA extraction, construction of cDNA libraries (library I and library II), and microarray hybridization.
Construction of cDNA Libraries and cDNA Sequencing, cDNA Array Generation, Hybridization, and Data Analysis
Briefly, total RNAs were extracted from the immature seeds with TRIzol reagent according to the manufacturer's introductions (GIBCO/BRL, California, USA), and mixtures of equal amounts of materials of 3, 5, 7, and 9 DAF or 11, 13, 15, 17, and 19 DAF were used to construct two cDNA libraries. First-strand cDNA was synthesized from poly(A)+ RNA (QIAGEN, Hilden, Germany) using PowerScript Reverse Transcriptase. After synthesis of second-strand cDNA, protease K-Sfi-digested cDNA fragments were ligated into pBSF vector. The insertions were sequenced from the 5'-end. Chromatograms were processed in bulk using PHRED. The 5'- and 3'-ambiguous sequences were trimmed and vector sequences were removed. Sequences of less than 150 bp or with more than five ambiguous bases in the first 350 bases were not processed. If more than 100 bp had more than 90% identity between two sequences, the one nearer to the 5'-end was selected.
Further, inserts of all independent cDNA clones were amplified by PCR using primers T3 and T7 flanking both sides of cDNA inserts, and spotted on the glass-based chips. In total, there are 8192 spots, including 8095 B. napus genes, 81 blanks, and 16 negative controls, on the chip, which is divided into 32 sub-matrices. Every sub-matrix contains 16 x 16 spots, and the distance between two spots is 0.281 mm. Total RNAs extracted from immature seeds at different developmental stages (7, 9, 12, 17, 19, 21, 25, and 31 DAF, respectively) were used for hybridization. Probes were marked with Cy5-dCTP (test) or Cy3-dCTP (reference), respectively.
Every sample was biologically repeated (two times) and the arrays were scanned with ScanArray4000 Standard Biochip Scanning System (Axon Instruments, California, USA). The mean fluorescence intensities for each cDNA clone were determined using GenePix Pro (Ver 3.0). Cy5 or Cy3 signal intensity of each gene was the feature pixel intensity minus the background pixel intensity. In order to avoid the weak signal interference of Cy5, signals less than 200 are replaced with 200.
Genes used for normalization followed two principles: (1) both Cy3 and Cy5 intensities were >200 or one of them >800; (2) Cy5/Cy3 value of gene was between 0.1 and 10. Gene expression at each developmental stage was compared to that at 3 DAF, and ratios were rescaled by setting that at 3 DAF as 1. The correlation for gene expression levels was analyzed using Microsoft Excel with the function of bivariate Pearson correlations. The t-test fellow by False Discovery Rate (FDR) was performed using Maanova base on R 2.4.1 of significance analysis to discover the genes with significant changes. The chip with fewer error probe signals of two replicates was chosen for further analysis. Then, ratios <0.5 or >2 were considered as down- or up-regulated, respectively. GeneCluster 2.0 was used for the cluster analysis. Mev4.0 was used for the hierarchical analysis (Saeed et al., 2003). The fold change of given stages compared to 3 DAF was Log2 transformed in hierarchical analysis. The genes at different developmental stages were median centered.
Quantitative Real-Time Reverse Transcription PCR Analysis
Quantitative real-time RT–PCR (qRT–PCR) was performed to study the gene expressions. Seed at 3, 9, 19, 25, and 31 DAF were collected for RNA extraction using RNeasy Plant Mini Kit (QIAGEN, Cat. No. 74904). qRT–PCR analysis was performed with the RotorGene 3000 system (Corbett Research) through the SYBR green detection protocol (TOYOBO, Cat. No. QPK-201). B. napus Actin (AF111812
[GenBank]
) mRNA was used as an internal control and relative amounts of tested mRNA were calculated using the comparative threshold cycle method (Rotor-Gene software, version 6.0.19; Corbett Research). Primers used were as follows: Actin (sense, 5'-TGAAGATCAAGGTGGTCGCA-3', and reverse, 5'-AGAAGGCAGAAACACTTAGAAG-3’), sucrose transporter (EL626999, sense, 5'-TATGCTTGTTCAGCCCATCGT-3', and reverse, 5'-GCGTTCTCGGCGGTTTGTC-3’), SuSy (EL628106, sense, 5'-GAGACAGAGATGCTCCAACG-3', and reverse, 5'-TCCATTTGCGAACAATACCCT-3’), invertase (EL625354, sense, 5'-AATGGTGGAACAGATAGTGAAG-3', and reverse, 5'-GAGATGGCATGAAGTCGAAAAC-3’), PPT (DQ985809
[GenBank]
, sense, 5'-GTTGATGTCTCTCTTTCTGATG-3', and reverse, 5'-CCTGCTGGTATGCGTGGAAG-3’), GLT1 (EL628734, sense, 5'-CTCCTCCGTCAAAGCTCGAT-3', and reverse, 5'-CCAAGGTGATAGCCAAACAAT-3’).
Fatty Acid Analysis through GC/MS
To determine fatty acid compositions, 30 mg Arabidopsis seeds were harvested and FA methyl esters were prepared according to Browse et al. (1986) with few modifications. 50 µL of nonadecanoic acid methyl ester (Fluka, 74208) stock solution (2 mg mL–1 in hexone) was added as internal references. Before analysis, 10 µl each of pyridine and N-methyl-N-trimethylsilyl-trifluoroacetamide (Fluka, 69479) were added and incubated at 37°C for 30 min. GC/MS was performed using the Agilent 5975 inert GC/MS system (Agilent Technology, California, USA) with the HP-INNOWax column (Agilent, 19091N-133).
Notes
All the described ESTs have been deposited in GenBank (www.ncbi.nlm.nih.gov/Genbank/) under accession numbers EL622496–EL630838 and EL680848–EL680966. SAGE and microarray data were deposited in GEO (Gene Expression Omnibus, www.ncbi.nlm.nih.gov/geo/) with the Series Number GSE7204
[NCBI GEO]
. Data including comparison with Arabidopsis thaliana, Brassica oleracea, and other species of genus Brassica, and results of SAGE and cDNA chip hybridization, are available at http://rapeseed.plantsignal.cn.
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMethodsSUPPLEMENTARY DATAFUNDING |
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Supplementary Data are available at Molecular Plant Online.
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMethodsSUPPLEMENTARY DATAFUNDING |
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This study was supported by the State Key Project of Basic Research (973, 2006CB101603), the Chinese Academy of Sciences (No. KSCX2-SW-328), and the Hi-Tec program (863, No. 2003AA222101).
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Acknowledgements |
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We thank Dr Dao-Xin Xie (Tsinghua University) for providing the seeds of coi1, and Dr Hong-Quan Yang (Shanghai Institute of Plant Physiology and Ecology) for providing the seeds of tir1-1, axr1-3, and axr2-1. No conflict of interest declared.
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2 These authors contributed equally to the studies.
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