Comparative and Functional GenomicsVolume 2012 (2012), Article ID 914843, 14 pagesdoi:10.1155/2012/914843

Research Article

In Silico Identification and Comparative Genomics of Candidate Genes Involved in Biosynthesis and Accumulation of Seed Oil in Plants

Arti Sharma and Rajinder Singh Chauhan

Department of Biotechnology & Bioinformatics, Jaypee University of Information Technology, Waknaghat, P.O. Dumehar Bani, Kandaghat, Solan 173 215, India

Received 5 August 2011; Revised 27 September 2011; Accepted 14 October 2011

Academic Editor: Jinfa Zhang

Copyright © 2012 Arti Sharma and Rajinder Singh Chauhan. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Genes involved in fatty acids biosynthesis, modification and oil body formation are expected to be conserved in structure and function in different plant species. However, significant differences in the composition of fatty acids and total oil contents in seeds have been observed in different plant species. Comparative genomics was performed on 261 genes involved in fatty acids biosynthesis, TAG synthesis, and oil bodies formation in Arabidopsis, Brassica rapa, castor bean and soybean. In silico expression analysis revealed that stearoyl desaturase, FatB, FAD2, oleosin and DGAT are highly abundant in seeds, thereby considered as ideal candidates for mining of favorable alleles in natural population. Gene structure analysis for major genes, ACCase, FatA, FatB, FAD2, FAD3 and DGAT, which are known to play crucial role in oil synthesis revealed that there are uncommon variations (SNPs and INDELs) which lead to varying content and composition of fatty acids in seed oil. The predicted variations can provide good targets for seed oil QTL identification, understanding the molecular mechanism of seed oil accumulation, and genetic modification to enhance seed oil yield in plants.

1. Introduction

A major challenge mankind is facing in this century is the gradual exhaustion of the fossil energy resources. The combustion of those fossil fuels used in transportation is one of the key factors responsible for global warming and environment pollution due to large-scale carbon dioxide emissions. Thus, alternative energy sources based on sustainable and ecologically friendly processes are urgently required. At present gasoline or diesel are being largely substituted by two biofuels, bioethanol and biodiesel, capturing ~90% of the market [1]. Biodiesel is made from renewable biomass mainly by alkali-catalysed transesterification of triacylglycerols (TAGs) from plant oils [2]. Manipulation of biosynthetic pathways offers a number of exciting opportunities for plant biologists to redesign plant metabolism toward production of specific TAGs.

The biosynthesis of fatty acids in plants begins with the formation of acetyl Co-A from pyruvate. The acetyl CoA produced in plastids is activated to malonyl CoA; the malonyl group is subsequently transferred to acyl carrier protein (ACP) giving rise to malonyl ACP, the primary substrate of the fatty acid synthase complex. The formation of malonyl CoA is the committed step in fatty acid synthesis and is catalyzed by the highly regulated plastidic acetyl CoA carboxylase complex [3]. De novo fatty acid synthesis in the plastids occurs through a repeated series of condensation, reduction, and dehydration reactions that add two carbon units derived from malonyl ACP to the elongating fatty acid chain. A series of condensation reactions proceed with acetyl-CoA and malonyl-ACP, then acyl-ACP acceptors. Three separate condensing enzymes, or 3-ketoacyl-ACP synthases (KAS I–III) are necessary for the production of an 18-carbon fatty acid. Three additional condensation reactions are required; each condensation step to obtain a saturated fatty acid that is two carbons longer than at the start of the cycle. These reactions are catalysed by 3-ketoacyl-ACP reductase (KAR), 3-hydroxyacyl-ACP dehydratase (HD), and enoyl-ACP reductase (ENR). The first desaturation step also occurs in the plastid; while the acyl chain is still conjugated to ACP, a Δ 9-desaturase converts stearoyl ACP to oleoyl ACP. Termination of fatty acid elongation is catalyzed by acyl ACP thioesterases, which are two main types in plants. The FatA class removes oleate from ACP, whereas FatB thioesterases are involved in saturated and unsaturated acyl ACPs, and, in some species, with shorter-chain-length acyl ACPs [4–6]. After release from ACP, the free fatty acids are exported from the plastid and converted to acyl CoAs. Nascent fatty acids can be incorporated into TAGs in developing seeds [4]. Oleic acid can be further desaturated to oleate acids by FAD2 [7] and FAD6 [8] in the cytosol and the plastid, respectively. Cytosolic and plastid ω-3 desaturations that result in the production of linolenic acids are catalyzed by FAD3 [9] and FAD7 [10], respectively. Fatty acids can be incorporated into TAGs in developing seeds in a number of ways. For example, a series of reactions known as the Kennedy pathway results in the esterification of two acyl chains from acyl CoA to glycerol-3-phosphate to form phosphatidic acid (PA) and, following phosphate removal, diacylglycerol (DAG). A diacylglycerol acyltransferase (DGAT), using acyl CoA as an acyl donor, converts DAG to TAG. Two classes of DGAT enzymes have been isolated [11, 12], and orthologs have been identified in numerous plant species. DAG and phosphatidylcholine (PC) are interchangeable via the action of cholinephosphotransferase, suggesting a route for the flux of fatty acids into and out of PC. Acyl chains from PC can be incorporated into TAG, either via conversion back to DAG or by the action of a phospholipid diacylglycerol acyltransferase (PDAT) that uses PC as an acyl donor to convert DAG to TAG. There are two predominant seed oil storage proteins in plants: caleosin and oleosin. TAG assembled in these storage proteins form oil bodies in seeds.

The fatty acid composition of seed oil varies considerably both between species and within species. The variation of fatty acids occurs both in chain length and degrees of desaturation. Consequently, the fuel properties of biodiesel derived from a mixture of fatty acids are dependent on the composition of fatty acids in seed oil. Altering the fatty acid profile can, therefore, improve fuel properties of biodiesel such as cold-temperature flow characteristics, oxidative stability, and NOx emissions [13].

Fatty acid biosynthetic pathway is highly conserved in plants, but there are significant variations in fatty acid contents and composition in plants (Table 1). What determines differences in the contents and composition of fatty acids and subsequently the total oil yield in the seeds is not understood. The availability of whole genome sequences, ESTs, and individual gene sequences from different oil rich plant species provide an opportunity to investigate what differences in the structure and sequences of genes determine variation in contents and composition so as to identify distinguishing gene signatures to assist in genetic improvement of crop plants either through marker-assisted breeding or by metabolic engineering [32]. Tanhuanpää et al. [33] developed an allele-specific PCR marker for oleic acid by comparing the wild-type and high-oleic allele of the FAD 2 gene locus in spring turnip rape (Brassica rapa ssp. oleifera). The accumulation of ricinoleic acid in transgenic Arabidopsis seeds was doubled by expressing the castor FAH12 hydroxylase in a FAD 2/FAE1 mutant [34]. The FatA and FatB genes of castor bean were heterologously expressed in Escherichia coli for biochemical characterization after purification, resulting in high catalytic efficiency of RcFatA on oleoyl-ACP and palmitoleoyl-ACP and high efficiencies of RcFatB for oleoyl-ACP and palmitoyl-ACP. The expression profile of these genes displayed the highest levels in expanding tissues that typically are very active in lipid biosynthesis such as developing seed endosperm and young expanding leaves [35]. Arabidopsis thaliana gene diacylglycerol acyltransferase (DGAT) coding for a key enzyme in TAG biosynthesis was expressed in tobacco under the control of a strong ribulose-biphosphate carboxylase small subunit promoter. This modification led up to a 20-fold increase in TAG accumulation in tobacco leaves and translated into an overall twofold increase in extracted fatty acids up to 5.8% of dry biomass in Nicotiana tabacum [36]. Dimov and Mollers [37] tested genetic variation for saturated fatty acid content in two sets of modern winter oilseed rape cultivars (Brassica napus L.) in field experiments under typical German growing conditions. They observed highly significant genetic differences among the cultivars for total saturated fatty acid content, which ranged from 6.8% to 8.1%. Singh et al. [38] constructed genetic map using AFLP, RFLP, and SSR markers for oil palm. They detected quantitative trait loci (QTLs) controlling oil quality (measured in terms of iodine value and fatty acid composition) and identified significant QTLs associated with iodine value (IV), myristic acid (C14 : 0), palmitic acid (C16 : 0), palmitoleic acid (C16 : 1), stearic acid (C18 : 0), oleic acid (C18 : 1), and linoleic acid (C18 : 2) content. The Brassica napus mutant line DMS100 carrying a G-to-A base substitution at the 5′ splice site of intron 6 in FAD 3 had reduced C18 : 3 content in oil seeds [39]. These studies suggest that the comparative analysis of oil biosynthesis and accumulation genes is a suitable strategy to investigate the molecular basis of oil content and composition variation in seed oils of different plant species. Additionally, these variations can be used to develop functional markers for increasing selection efficiency by marker- assisted selection in plant breeding.

Table 1: Variations for fatty acids and TAG biosynthesis pathway genes associated with high oil content in different plant species.

Targeted Genes
Descriptions of variations
Gene regions harboring variations
Plant/organism
References

FAD 2, FAD 3
SNP for high oleic acid and low linolenic acid
Exon
Brassica
Hu et al., 2006 [14]

Stearoyl—ACP desaturase
SNP for high stearic acid
Exon
Soybean
Zhang et al., 2008 [15]

FAD 2
SNPs for high oleic acid
Exon
Peanut
López et al., 2000 [16]

FAD 3
SNP for low linolenic acid
Intron-Exon junction
Soybean
Bilyeu et al., 2005 [17]

KAS I
SNPs and Indel associated with oleic acid content
5′UTR, Exon, Intron
Soybean
Ha et al., 2010 [18]

KAS III, ACCase, Stearoyl—ACP desaturase, DGAT
Indels, SNPs and SSRs associated with variation in composition and concentration of oil
—
Maize
Yang et al., 2010 [19]

FAD 2
3 base pair variation leads to change in amino acid which contribute to high oleate content in oil
Exon
Peanut
Bruner et al., 2001 [20]

DGAT1
3 bp Insertion leads to high oleic acid content
Exon
Maize
Zheng et al., 2008 [21]

FAD 2
SSR linked to oleic acid content

Soybean
Bachlava et al., 2008 [22]

FAD 3
Deletion in soybean FAD 3 leads to reduced linolenate
Exon
Soybean
Anai et al., 2005 [23]

KAS III
SNP associated with high palmitic acid content
Exon
Soybean
Aghoram et al., 2006 [24]

Stearoyl—ACP desaturase
SSRs associated with high stearic acid
—
Soybean
Spencer et al., 2003 [25]

Stearoyl—ACP desaturase
SSRs and INDELs associated with high stearic acid
—
Sunflower
Pérez-Vich et al., 2006 [26]

FatB
Deletions associated with low palmitic acid content
Exons and Introns
Soybean
Cardinal et al., 2007 [27]

In the present study, four plant species, Arabidopsis, Brassica, soybeans, and castor bean were considered for comprehensive analysis of fatty acid biosynthesis genes due to the availability of their genome sequences and several ESTs collections. Moreover, soybeans and brassicas are the biggest source of plant oil in the world, whereas castor bean contains unusual fatty acid ricinoleate that have chemical properties useful for industrial applications. The total seed oil contents of Arabidopsis, castor bean Brassica, and soybean are 30–37%, 40–45%, 30–40%, and 15–20%, respectively (Table 2) [28–31]. Plant oils are mostly composed of five common fatty acids, namely, palmitate (16 : 0), stearate (18 : 0), oleate (18 : 1), linoleate (18 : 2) and linolenate (18 : 3), although, depending on the particular species, longer or shorter fatty acids may also be major constituents. These fatty acids differ from each other in terms of acyl chain length and number of double bonds, leading to different physical properties. Here we put forward the questions (1) whether there are common variations in genes, if any, which contribute to increased seed oil content in plants? (2) Which are the major genes responsible for the higher amounts of five fatty acids mentioned above in different plant species? For answering these questions the present study aimed at (1) the identification of candidate genes for fatty acid biosynthesis, TAG synthesis and oil body formation proteins in plant species under study, (2) the comparative structure analysis of these candidate genes, (3) the in silico identification of sequence variations in fatty acid biosynthesis genes, and (4) the in silico association of sequence variations in candidate genes for oil content and composition.

Table 2: Fatty acid composition of four plant species.

Fatty acid composition (%)
Arabidopsis [28]
Castor bean [29]
Brassica [30]
Soybean [31]

Palmitic acid
8.7
2.0
1.5
7–11

Stearic acid
3.6
1.0
0.4
2–6

Oleic acid
15.0
7.0
22.0
22–34

Linolenic acid
29.0
—
6.8
5–11

Linoleic acid
19.2
5.0
14.2
43–56

Ricinoleic acid
—
86–90
—
—

Others
24.5
—
47 (Erucic)
—

Total oil content
30–37
45–50
33–40
15–20

2. Materials and Methods

2.1. Retrieval of Sequences

Thirty-two genes involved in the biosynthesis and storage of fatty acids were retrieved from Arabidopsis database (http://lipids.plantbiology.msu.edu/) by referring to the comprehensive lipid gene catalog provided by Beisson et al. [40]. The selected genes covered all the major biochemical events in the biosynthesis and storage of fatty acids [41, 42]. The protein sequences of these genes were used as query against castor bean database in TIGR (http://blast.jcvi.org/er-blast/index.cgi?project=rca1) and soybean database in soybase (http://soybase.org/). Full-length coding sequences of Brassica were downloaded from GenBank (http://www.ncbi.nlm.nih.gov/genbank/GenbankSearch.html). Protein function domains were examined with “CDD” from NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml).

2.2. Prediction of Gene Structures

Gene models for castor bean and soybean genomes were downloaded from Phytozome (http://www.phytozome.net/). The Arabidopsis gene models were downloaded from TAIR (http://www.arabidopsis.org/). Arabidopsis, castor bean, Brassica. Rapa, and soybean sequences were further annotated for gene models (open reading frames, including the 5′UTRs and 3′UTRs) using gene prediction algorithms of FGenesH (http://linux1.softberry.com/berry.phtml?topic=fgenesh&group=programs&subgroup=gfind) [43–45] (see Table 1 of the Supplementary Material available online at doi:10.1155/2012/914843). Sequence identity among Brassica rapa, castor bean, soybean, and Arabidopsis genes was confirmed using ClustalW in MegAling in DNASTAR (DNASTAR Inc., Madison, WI, USA). The in silico expression status of candidate genes belonging to different families was searched with an e-value cutoff 0.0 in the ESTdb of NCBI (National Centre for Biotechnology Information) at http://www.ncbi.nlm.nih.gov/BLAST/ and TIGR (The Institute of Genomic Research) at http://blast.jcvi.org/er-blast/index.cgi?project=rca1.

3. Results

3.1. Comparative Genomics of Fatty Acid Biosynthesis Genes in Major Oil Seed Plant Species

The fatty acid biosynthesis pathway includes 32 gene families involved in the conversion of acetyl Co-A into different fatty acids and their storage in oil bodies. A total of 68 protein sequences were retrieved for 32 gene families from the comprehensive lipid gene catalog of Arabidopsis [40] and functional domains were identified for each gene family. The 68 protein sequences from Arabidopsis were queried for fatty acid biosynthesis genes in Brassica rapa, soybean, and castor bean databases. A total of 261 genes belonging to 32 gene families were identified and retrieved from four plant species, out of which, 68 were from Arabidopsis, 62 from Brassica rapa, 55 from castor bean, and 76 from soybean (Table 3). Detailed gene structures, exon- intron coordinates of each gene are given in Supplementary Table  1.

Table 3: Oil synthesis and accumulation genes in Arabidopsis, Brassica rapa, castor bean, and soybean.

Category
Gene name
Accession number
Coding DNA sequence length (bp)

Arabidopsis
Brassica rapa
Castor bean
Soybean
Arabid-opsis
Brassica rapa
Castor bean
Soybean

ACCase
ACCase
At1g36180
X77382
29908.m005991
Glyma04g11550
5997
2193
6723
6834

Alpha-carboxyl transferase
At2g38040
AY538675
27798.m000585
Glyma18g42280
2346
2295
2355
2130

Beta-carboxyl transferase
ATCG00500
Z50868
28890.m000006
—
636
984
528
—

Biotin carboxylase
At5g35360
AY034410
30185.m000954
Glyma05g36450
1683
1608
1935
1731

Biotin carrier
At5g15530
AY538674
29630.m00080929929.m004560
Glyma08g03120
726
783
780924
1611

Malonyl Co-A transacylase
At2g30200
AJ007046
30113.m001448
Glyma18g06500
1104
993
987
1113

Beta-Ketoacyl ACP synthase I
At5g46290
AF244519
29693.m002034
Glyma10g04680
1422
1380
1437
1452

Beta-Ketoacyl ACP synthase II
At1g74960
AF244520
29739.m003711
Glyma13g19010
1770
1302
2013
1305

Beta-Ketoacyl ACP synthase III
At1g62640
AF179854
28455.m000368
Glyma09g41380Glyma15g00550Glyma18g44350
1215
186
1233
11948311254

Elongase
3-Ketoacyl- acp- dehydrase
At1g62610At3g46170At3g55290At3g55310
AF382146
30147.m013777
Glyma08g10760Glyma18g01280
924867822822

735
924963

3-Ketoacyl- Co-A reductase (KAR)
At1g24360
AY196197
29929.m004732
Glyma11g37320
927
960
987
963

Enoyl-ACP reductase (ENR)
At2g05990
AJ243087AJ243088AJ243089AJ243090x95462
27843.m00016029650.m000277
Glyma11g10770Glyma12g03060Glyma18g31780
11155
11581161116111641158
10831083
117612031533

Hydroxyacyl ACP Dehydrase (HD)
At2g22230
AF382146
30200.m000354
Glyma05g24650Glyma08g07870Glyma08g19200Glyma15g05800
663
672
534
417513219822

Plastidial 1 acylglycerol phosphate acyltransferase
At4g30580
—
29687.m000572
Glyma06g28540Glyma12g28470
1071
—
987
24061776

Plastidial Glycerol phosphate acyltransferase
At1g32200
—
30068.m002660
Glyma09g34110
1380
—
1236
1413

Monogalactosylacylglycerol desaturase (FAD 5)
At3g15850

29841.m002863
Glyma07g03370Glyma08g22730
1116

1161
11011173

Stearoyl-ACP desaturase
At1g43800At2g43710At3g02610At3g02630At3g02620At5g16230At5g16240
X63364X74782AY642537
27985.m00087728470.m00042829929.m00451530020.m000203
Glyma02g15600Glyma07g32850Glyma13g08970Glyma14g27990
11761206984119198412061185
120012061200
11763369601191
1176117611851014

Oleate desaturase (FAD 6)
At4g30950
AY642535AY642540
29696.m000105
Glyma02g36460
1347
13321293
825
1287

Desaturase
Linoleate desaturase (FAD 7)
At3g11170
AY592974AY599884FJ985689FJ985690FJ985691L01418L22962
28176.m00027329681.m00136029814.m000719
Glyma01g29630Glyma03g07570Glyma07g18350
1467
1320115211341299133511521134
135911311383
135913621362

Linoleate desaturase (FAD 8)
At5g05580
AY592974AY599884FJ985691
28176.m00027329681.m00136029814.m000719
Glyma01g29630Glyma03g07570Glyma07g18350
13081152
132011521335
135911311383
135913621362

ER-Oleate desaturase (FAD 2)
At3g12120
AY577313DQ518276DQ518277DQ518278FJ907397FJ907398FJ907399FJ907400FJ907401FJ952144
28035.m000362
Glyma03g30070Glyma09g17170Glyma10g42470Glyma19g32940Glyma20g24530
1152
1155780780780115511551155115511551155
1164
11521161114011521140

ER-Linoleate desaturase (FAD 3)
At2g29980
AY592974AY599884FJ985689L01418L22962
29681.m001360
Glyma01g29630Glyma07g18350
1161
13201152113411521134
1131
13591362

Thioesterase
Acyl-ACP thioesterase (FatA)
At3g25110At4g13050
X87842
30217.m00026229842.m003515
Glyma08g46360Glyma18g36130
10891104
1176
1269753
11911125

Palmitoyl-ACP thioesterase (FatB)
At1g08510
DQ847275Fj715952
29848.m004677
Glyma0421910Glyma05g08060Glyma06g23560Glyma17g12940
1239
12451239
1260
1332125114221251

TAG synthesis
Diacylglycerol Acyltranferase (DGAT 1)
At2g19450
AF164434
29912.m005373
Glyma13g16560
1593
1512
1830
1347

Diacylglycerol Acyltranferase (DGAT 2)
At3g51520
AF155224
29682.m000581
Glyma09g32790
945
1056
768
987

Lysophosphosphatidic acid acyltransferase (LPAAT)
At1g01610At1g51260At1g78690At1g80950At2g27090At2g38110At3g05510At3g11430At3g18850At3g57650At4g00400At5g06090
AF111161Gu045434GU04535Gu045436Z49860
27810.m00064629851.m00244830169.m00643330170.m01399029736.m00207029822.m00344129969.m00026730174.m008615
Glyma01g27900Glyma03g01070Glyma03g14180Glyma07g07580Glyma07g17720Glyma10g23560Glyma18g42580
15121119873114022321506134715091146117015031503
10351173117611739361188
11881050852738151515395941506
1536678858675149114281620

Diacylglycerol cholinephosphotransferase
At3g25585
AY179560
30138.m003845
Glyma12g08720Glyma0214210
1170
1449
1449
7711188

Digalactosyldiacyglycerol synthase (DGD1)
At3g11670
—
28726.m000069
Glyma03g36050Glyma19g38720
2388
—
2538
23522361

ER Phosphatidate Phosphatase
At1g15080
—
29586.m00062029660.m00076029660.m00075929660.m000759
Glyma09g18450Glyma10g41580Glyma20g25650
813
—
954564930945
957969909

Oil body protein
Caleosin
At1g23240At1g23250At1g70670At1g70680At2g33380At4g26740At5g55240
AY966447DQ140380
29673.m00093230008.m000820
Glyma3g41030Glyma09g22310Glyma09g22330Glyma09g22580Glyma09g25350Glyma10g33350Glyma19g43680Glyma20g34300
669663588552711738732
717705
597702
723615606384384570723402

Oleosin
At1g48990At2g25890At3g01570At3g18570At3g27660At4g25140At5g40420At5g51210
DQ328612S37032
29794.m00337230147.m01389130147.m014333
Glyma05g07880Glyma14g15020Glyma17g13120
510450552501576522600426
699426
564489495
492492471

3.2. Expression Status of Fatty Acid Biosynthesis Genes

In silico expression analysis revealed that for 32 gene families, ESTs were detected for 68 genes in Arabidopsis, 62 genes in Brassica, 49 genes in castor bean, and 76 genes in soybean (Figure 1). Thirteen genes of Arabidopsis, 15 from castor bean, 8 from soybean, and 2 from Brassica showed tissue preferential expression patterns as per their identities to ESTs from tissue-specific libraries. Twenty-two genes from four plant species were expressed in seeds, 4 in leaves, 3 in flower, and 1 in roots (Table 4). FAD 2 and one homolog of Stearoyl desaturase gene had maximum seed ESTs in castor bean.

Table 4: In silico expression status of fatty acids biosynthesis and accumulation genes.

Tissue
Gene
Accession no.

Arabidopsis
B. rapa
Soybean
Castor bean

Seeds
Alpha carboxyltransferase

27798.m000585

Enoyl ACP reductase

27843.m000160

Stearoyl desaturase

X74782
Glyma13g08970
27985.m000877

FAD-2

Glyma10g42470
28035.m000362

ER Phosphatidate Phosphatase

29660.m000760

DGAT 2

Glyma17g06120
29682.m000581

FatB

Glyma17g12940
29842.m003515

Oleosin

S37032
Glyma14g15020
30147.m014333

Oleosin
At5g40420

Glyma17g13120
30147.m013891

Oleosin

29794.m003372

Hydroxyacyl ACP dehydrase

30200.m000354

Caleosin
At5g55240

Glyma20g34300

Leaves
FatB

29848.m004677

LPAAT

Glyma07g07580

3-Ketoacyl- acp- dehydrase
At3g55290

At3g55310

Flowers
DGD1

28726.m000069

Beta- carboxyl transferase

28890.m000006

ACCase

29908.m005991

Roots
Stearoyl desaturase
At3g02620

Roots + flowers
Stearoyl desaturase
At3g02610

Seed + flowers
Oleosin
At1g48990

Leaves + flowers
FAD 7
At3g11170

Oleosin
At2g25890

At3g18570

Caleosin
At1g23240

At1g23250

At4g26740

Figure 1: In silico transcript abundance (based on matching ESTs available in the database) of oil biosynthesis and accumulation genes in different tissues.

3.3. Comparative Analysis of Gene Structures in Different Plant Species

Comparative genomics of fatty acid biosynthesis genes was done to understand as what determines differences, if any, for variations in contents and compositions of fatty acids in different plant species. The gene structure analysis revealed that the exon-intron structure of fatty acid biosynthesis genes in castor bean and soybean gene homologs shared more structure similarity in comparison to Arabidopsis fatty acid biosynthesis genes. However, insertion, deletion, and intron size variations were found in castor bean and soybean genes with reference to Arabidopsis. Fatty acid biosynthesis genes of Brassica rapa were not analyzed for gene structure because for most of the Brassica genes only coding DNA sequences were available in the GeneBank.

Conversion of acetyl Co-A to malonyl Co-A by acetyl carboxylase (ACCase) is the most committed step in fatty acid biosynthesis. Exon/intron number and CDS length for ACCase gene was almost same between castor bean (31 exons) and soybean (33 exons), whereas slightly less in Arabidopsis (26 exons). Comparative structural analysis revealed that homomeric ACCase gene from Arabidopsis (1–26 exons) showed microsynteny with castor bean (6–31 exons) and soybean (6–33 exons), with a 3 bp deletion in 8th and 26th exons of castor bean, 3 bp deletion and 3 bp insertion in 29th and 31st exons of soybean, and a 12 bp insertion in 24th and 26th exons of castor bean and soybean, respectively. First five exons of homomeric ACCase in castor bean and soybean (missing in Arabidopsis) showed colinearity for exon size, with the exception of a 3 bp insertion in the first exon of castor bean gene. Sixteenth exon of ACCase in castor bean showed sequence identity to 3 exons (16th, 17th, and 18th) of soybean (Figure 2).

Figure 2: Structure of ACCase gene in Arabidopsis (26 exons), castor bean (31 exons), and soybean (33 exons); thick arrows and thin lines represented exons and introns, respectively. Arabidopsis 1–26 exons showed identity to 6 to 31st and 6 to 33rd exons of castor bean and soybean, respectively; 16th exon of castor bean showed identity to three exons of soybean (16th, 17th, and 18th). A 3 bp deletion (del) in the 8th and 26th exons of castor bean, 3 bp deletion and 3 bp insertion (in) in the 31st and 29th exons of soybean, and a 12 bp insertion in the 24th and 26th exons of castor bean and soybean, respectively. At1g36180: Arabidopsis ACCase gene; 29908.m005991: Castor bean ACCase gene; Glyma04g11550: Soybean ACCase gene.

Two distinct classes of thioesterases, FatA and FatB, are responsible for release of fatty acids from ACP by thioesterases. FatA gene structure was diverse with respect to exons number (varying from 5 to 11) among four plant species. Two homologs of FatA gene were present in Arabidopsis, castor bean, and soybean, whereas FatB gene had 4 homologs in soybean. The first exon of FatB gene had an insertion of 3 bp in castor bean and 27 bp insertion in one of soybean homologs (Glyma0421910) and other three homologs of soybean (Glyma05g08060, Glyma17g12940, and Glyma06g23560) had 6 bp deletion compared to Arabidopsis (Figure 3). An 69 bp insertion of one exon was present in FatB genes of castor bean and soybean but was absent in Arabidopsis. The last exon of FatB (5th exon) in Arabidopsis showed homology to the last exon (6th exon) of one of the homologs of soybean (Glyma04g21910) and last two exons (6th and 7th) of another homolog of soybean (Glyma06g23560), whereas last exon of castor bean showed homology to the last exon of other two homologs of soybean (Glyma05g08060 and Glyma17g12940).

Figure 3: Structure of FatB (palmitoyl thioesterase) gene in Arabidopsis (At1g08510), castor bean (29848.m004677), and four soybean homologs (Glyma0421910, Glyma05g08060, Glyma17g12940, and Glyma06g23560). The 5th exon of FatB in Arabidopsis showed homology to the 6th exon of one of the homologs of soybean (Glyma04g21910) and last two exons (6th and 7th) of another homolog of soybean (Glyma06g23560), whereas 6th exon of castor bean showed homology to the 6th exons of other two homologs of soybean (Glyma05g08060 and Glyma17g12940).

Stearoyl ACP desaturase gene had maximum number of homologs (6 in Arabidopsis, 3 in Brassica, 4 in soybean, and 4 in castor bean) in fatty acid desaturase category of enzymes. Oleoyl desturase (Fad2) and Linoleate desaturase (Fad3) genes showed more relatedness in relation to number and sizes of exons and introns in each homolog among four plant species. Oleoyl desaturase (FAD 2) had only one exon in Arabidopsis, castor bean, and soybean with an insertion of 12 bp in the exon of castor bean and 9 bp insertion in the exon of one homolog of soybean (Glyma09g17170). FAD 3 gene structure was conserved with respect to exon-intron number and size between Arabidopsis, castor bean, and soybean except for first and last exons. A 21 bp deletion in the first exon of castor bean (29681.m001360) and an insertion of 210 and 213 bp was observed in two homologs of soybean (Glyma01g29630 and Glyma07g18350), respectively. Two deletions of 3 and 12 bp were observed in the last exon (8th exon) of castor bean and soybean, respectively. A deletion of 6 bp was observed in the 3rd exon of FAD 3 of castor bean. An SNP (G→A) was also identified at the exon-intron junction of FAD 3 gene in the 3rd exon of one homolog of soybean (Glyma01g29630) with respect to castor bean, Arabidopsis, and other homologs of soybean (Figure 4).

Figure 4: Structure of FAD 3 (linoleoyl desaturase) gene in Arabidopsis (At2g29980), castor bean (29681.m001360), and two soybean homologs (Glyma01g29630, Glyma07g18350). Exon/intron numbers are conserved in FAD 3 while variation in sizes was observed in the first and last exons. SNP identified in the 6th exon of soybean homolog (Glyma01g29630) was reported to be associated with low linolenic acid content [17].

The DGAT gene involved in TAG (Tri-acyl Glyceride) synthesis has two isoforms, DGAT-1 and DGAT-2. These two genes showed variation in number and sizes of exons and introns. DGAT-1 gene had 15 exons in Arabidopsis, 13 exons in castor bean, and 16 exons in soybean. DGAT-2 had 8 exons in Arabidopsis and castor bean and 7 exons in soybean. The detailed comparative genomics of fatty acid biosynthesis genes in 4 oil seed plant species provided insights to undertake identification and utilization of castor bean fatty acid biosynthesis genes and sequence variations for the development of candidate gene markers in Jatropha.

Fatty acid biosynthesis genes showed evolutionary relatedness but there is no synteny in gene order and position of genes on the chromosomes. Location of genes on chromosomes in Arabidopsis and soybean is given in Supplementary Table 2.

4. Discussion

In general, plant oil biosynthesis mostly follows the common biosynthetic pathways for fatty acids in the plastid as well as TAG in the endoplasmic reticulum (ER) and the oil further accumulates in oil bodies. However, there are significant differences for content and composition of seed oil in different plant species. Using comparative genomics, we tried to infer the effect of change in gene structure differences on oil content in different plant species. In this study, 261 genes involved in biosynthesis and accumulation of seed oil were identified in four oil seed plant species, Arabidopsis, Brassica, castor bean, and soybean. The genes corresponded to six different categories (ACCase, desturase, elongase, thioesterase, TAG synthesis and oil body proteins). Gene families corresponding to these six categories of enzymes had multiple copies in plant species with the exception of homomeric ACCase.

In higher plants, many proteins and enzymes are encoded by gene families, and in Arabidopsis, it has been estimated that 20% of genes are members of gene families [46]. The existence of gene families can sometimes reflect additional levels of genetic control or isoforms of proteins with specific functions. Therefore, it is of interest to detect potential gene families involved in the fatty acid biosynthesis pathway. There is a possibility that different copies of fatty acid biosynthesis genes are present in low oil content genotypes which gives leaky phenotypes as in the case of starch biosynthesis pathway where different copies of genes were responsible for low, medium, and high amylase contents in rice [47].

The oil biosynthesis may be limited by the production of fatty acids [48], which is regulated by acetyl CoA carboxylase (ACCase). Reduction of ACCase activity lowered (1.5–16%) the fatty acid content in transgenic seeds [49]. Conversion of acetyl Co-A to malonyl Co-A by acetyl carboxylase (ACCase) is the most committed step in fatty acid biosynthesis. ACCase of castor bean and soybean showed microsynteny to Arabidopsis, with a 3 bp deletion in 8th and 26th exons in castor bean, 3 bp deletion and 3 bp insertion in 29th and 31st exons in soybean and a 12 bp insertion in 24th, and 26th exons of castor bean and soybean, respectively with respect to Arabidopsis. These sequence variations in ACCase genes may be possibly influencing the variations in fatty acid composition and content in seed oil among Arabidopsis, castor bean, and soybean, as fatty acid content and composition was altered in many plant species with the variations in sequences or expression of ACCase gene [19, 50]. Yang et al. [19] identified two SNPs (T→G, G→A) in ACCase gene which lead to increase (1.3%) in oleic acid, lenolenic acid, and lenoleic acid content in maize. Addition of a plastid transit sequence targeted the introduced ACCase protein to chloroplasts, ultimately resulting in a 5% increase in seed oil of rapeseed [50]. The insertion or deletion identified in our analysis between Arabidopsis, castor bean, and soybean might be responsible for reduction or enhancement of ACCase activity, which is associated with the variations in total fatty acid composition in seed oil among these plant species.

Studies in transgenic plants have demonstrated that thioesterases contribute to the regulation of fatty acid chain length [51]. Typically, FatB accepts saturated acyl-ACP substrates of varying length, while FatA is specific to unsaturated fatty acids and acts on C18:1, oleic, acyl-ACPs [51]. In Brassica napus and Arabidopsis, genetic engineering of Acyl-ACP thioesterase (FatB) resulted in maximum increase of 58% in palmitic acid content [52, 53]. Preventing the release of saturated fatty acids from ACP by downregulating FatB, which encodes a palmitoyl ACP thioesterase, lowered the levels of saturated fatty acids [54]. Variations in palmitate content in seed oil in plant species can be related to the variations in FatB gene [27, 52, 53]. Cardinal et al. [27] identified deletion in exon-inrton junction in one homolg of FatB gene which was associated with low palmitic acid content in soybean cultivar Century (N79-2077 and N93-2008). Palmitate content was ~8% in Arabidopsis [55], ~2% in castor bean [56] and 7–11% in soybean [57]. Variations in the amount of palmitic acid in the seeds of Arabidopsis, castor bean, and soybean might be due to deletions in first exon of FatB gene, which can be further utilized for identification of markers associated with high level of palmitate (saturated fatty acid) in total seed oil in plant species desired for biodiesel purpose.

Soybean lines with high levels of oleic acid (85%) and low levels of saturated fatty acids (6%) have been developed using a transgenic strategy that results in downregulation of two genes, FAD 2, and FatB involved in fatty acid synthesis. Downregulation of the FAD 2 gene, encoding a Δ12 fatty acid desaturase, prevented the conversion of oleic acid to polyunsaturated fatty acids, resulting in increased levels of oleic acid. Additionally, preventing the release of saturated fatty acids from acyl carrier protein (ACP) by downregulating FatB gene, which encodes a palmitoyl ACP (acyl carrier protein) thioesterase, lowered the levels of saturated fatty acids [54]. Hu et al. [14] sequenced the FAD 2 gene fragment from the mutant line DMS100 and wild-type line Quantum of Brassica napus, and identified a single nucleotide mutation (C→T) in the FAD 2 gene. This particular mutation created a stop codon (TAG) leading to premature termination of the peptide chain during translation which leads to high oleic acid content in mutant line DMS100. B. napus mutant line DMS100 carrying a G-to-A substitution at the 5′ splice site of intron 6 in FAD 3 had reduced lenolenic acid content in seed oil [39]. In our analysis insertions or deletions in FAD 2 and FAD 3 genes of soybean might be the possible causes of higher oleate and linoleate content in high oil yielding soybean genotypes. Higher amount of ricinoleic acid in castor bean can be due to an insertion in the FAD 2 gene resulting in higher level of oleic acid because oleic acid is further utilized as a substrate by fatty acid hydroxylase (FAH) to convert oleate to ricinoleate. Low level of linoleate in castor bean oil may be due to a deletion in the 3rd exon of FAD 3 gene because each copy of FAD 3 in Arabidopsis and soybean is conserved.

In our analysis, the acyl-CoA:diacylglycerol acyltransferases (DGAT) gene was highly diverse, which might be involved in the overall variation in triacylglycerols in the oil among the plant species as it is a key enzyme in determining the levels of triacylglycerols in seed oils [58, 59]. Burgal et al. [58] demonstrated that coexpressing the castor bean DGAT2 gene with the castor FA 12 hydroxylase resulted in almost double the levels of hydroxylated fatty acids in neutral lipids (up to 30% of total, compared with 17% in the absence of DGAT2). In our study, most of the variations observed in the coding regions are either insertion or deletion of 3 bp or multiple of three that represent codon usage which either leads to shift in reading frame or functional mutation that are expected to be related to oil content. Thus, the sequence variations identified in fatty acid biosynthesis genes in this study can be tested for their functional role in altering content and composition of seed oil in Jatropha.

5. Conclusion

Comparative genomics, for gene structures and coding sequence variations, was performed on 261 genes involved in fatty acids biosynthesis, TAG synthesis, and oil bodies formation in four oil seed plant species, Arabidopsis, Brassica rapa, castor bean, and soybean to understand whether differences in gene structures or coding sequence determine preferential biosynthesis of higher amounts of particular fatty acids and their contents in the seeds of different plant species. Overall comparative gene structure of fatty acid biosynthesis related genes provided an insight to improve oil quality for biodiesel by exploiting the variations for engineering FAD5, FAD6, and FatB genes to enhance the content of saturated fatty acids. The variations in FAD2, FAD3, Stearoyl desaturase, DGAT-1, and DGAT-2 will be helpful to enhance the oil content in plants. The close relationship between genes under study would be helpful for comparative genomics to study these genes in related species for oil content modification.

References

D. Antoni, V. V. Zverlov, and W. H. Schwarz, “Biofuels from microbes,” Applied Microbiology and Biotechnology, vol. 77, no. 1, pp. 23–35, 2007.

F. Ma and M. A. Hanna, “Biodiesel production: a review,” Bioresource Technology, vol. 70, no. 1, pp. 1–15, 1999.

B. J. Nikolau, J. B. Ohlrogge, and E. S. Wurtele, “Plant biotin-containing carboxylases,” Archives of Biochemistry and Biophysics, vol. 414, no. 2, pp. 211–222, 2003.

K. M. Mayer and J. Shanklin, “Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approach,” BMC Plant Biology, vol. 7, article 1, 2007.

M. R. Pollard, L. Anderson, C. Fan, D. J. Hawkins, and H. M. Davies, “A specific acyl-ACP thioesterase implicated in medium-chain fatty acid production in immature cotyledons of Umbellularia californica,” Archives of Biochemistry and Biophysics, vol. 284, no. 2, pp. 306–312, 1991.

J. J. Salas and J. B. Ohlrogge, “Characterization of substrate specificity of plant FatA and FatB acyl-ACP thioesterases,” Archives of Biochemistry and Biophysics, vol. 403, no. 1, pp. 25–34, 2002.

J. Okuley, J. Lightner, K. Feldmann, N. Yadav, E. Lark, and J. Browse, “Arabidopsis FAD2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis,” Plant Cell, vol. 6, no. 1, pp. 147–158, 1994.

W. D. Hitz, T. J. Carlson, J. R. Booth, A. J. Kinney, K. L. Stecca, and N. S. Yadav, “Cloning of a higher-plant plastid omega-6 fatty acid desaturase cDNA and its expression in a cyanobacterium,” Plant Physiology, vol. 105, no. 2, pp. 635–641, 1994.

D. W. Reed, U. A. Schafer, and P. S. Covello, “Characterization of the Brassica napus extraplastidial linoleate desaturase by expression in Saccharomyces cerevisiae,” Plant Physiology, vol. 122, no. 3, pp. 715–720, 2000.

K. Iba, S. Gibson, T. Nishiuchi et al., “A gene encoding a chloroplast ω-3 fatty acid desaturase complements alterations in fatty acid desaturation and chloroplast copy number of the fad7 mutant of Arabidopsis thaliana,” Journal of Biological Chemistry, vol. 268, no. 32, pp. 24099–24105, 1993.

S. Cases, S. J. Smith, Y. W. Zheng et al., “Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis,” Proceedings of the National Academy of Sciences of the United States of America, vol. 95, no. 22, pp. 13018–13023, 1998.

K. D. Lardizabal, J. T. Mai, N. W. Wagner, A. Wyrick, T. Voelker, and D. J. Hawkins, “DGAT2 is a new diacylglycerol acyltransferase gene family. Purification, cloning, and expression in insect cells of two polypeptides from Mortierella ramanniana with diacylglycerol acyltransferase activity,” Journal of Biological Chemistry, vol. 276, no. 42, pp. 38862–38869, 2001.

A. Serdari, E. Lois, and S. Stournas, “Impact of esters of mono- and dicarboxylic acids on diesel fuel quality,” Industrial and Engineering Chemistry Research, vol. 38, no. 9, pp. 3543–3548, 1999.

X. Hu, M. S. Gilbert, M. Gupta, and S. A. Thompson, “Mapping of the loci controlling oleic and linolenic acid contents and development of fad2 and fad3 allele-specific markers in canola (Brassica napus L.),” Theoretical and Applied Genetics, vol. 113, no. 3, pp. 497–507, 2006.

P. Zhang, J. W. Burton, R. G. Upchurch, E. Whittle, J. Shanklin, and R. E. Dewey, “Mutations in a ⁹-stearoyl-ACP-desaturase gene are associated with enhanced stearic acid levels in soybean seeds,” Crop Science, vol. 48, no. 6, pp. 2305–2313, 2008.

Y. López, H. L. Nadaf, O. D. Smith, J. P. Connell, A. S. Reddy, and A. K. Fritz, “Isolation and characterization of the Δ12-fatty acid desaturase in peanut (Arachis hypogaea L.) and search for polymorphisms for the high oleate trait in Spanish market-type lines,” Theoretical and Applied Genetics, vol. 101, no. 7, pp. 1131–1138, 2000.

K. D. Bilyeu, L. Palavalli, D. A. Sleper, and P. R. Beuselinck, “Mutations in soybean microsomal omega-3 fatty acid desaturase genes reduce linolenic acid concentration in soybean seeds,” Crop Science, vol. 45, no. 5, pp. 1830–1836, 2005.

B. K. Ha, M. J. Monteros, and H. R. Boerma, “Development of SNP assays associated with oleic acid QTLs in N00-3350 soybean,” Euphytica, vol. 176, no. 3, pp. 403–415, 2010.

X. Yang, Y. Guo, J. Yan et al., “Major and minor QTL and epistasis contribute to fatty acid compositions and oil concentration in high-oil maize,” Theoretical and Applied Genetics, vol. 120, no. 3, pp. 665–678, 2010.

A. C. Bruner, S. Jung, A. G. Abbott, and G. L. Powell, “The naturally occurring high oleate oil character in some peanut varieties results from reduced oleoyl-PC desaturase activity from mutation of aspartate 150 to asparagine,” Crop Science, vol. 41, no. 2, pp. 522–526, 2001.

P. Zheng, W. B. Allen, K. Roesler et al., “A phenylalanine in DGAT is a key determinant of oil content and composition in maize,” Nature Genetics, vol. 40, no. 3, pp. 367–372, 2008.

E. Bachlava, R. E. Dewey, J. Auclair, S. Wang, J. W. Burton, and A. J. Cardinal, “Mapping genes encoding microsomal ω-6 desaturase enzymes and their cosegregation with QTL affecting oleate content in soybean,” Crop Science, vol. 48, no. 2, pp. 640–650, 2008.

T. Anai, T. Yamada, T. Kinoshita, S. M. Rahman, and Y. Takagi, “Identification of corresponding genes for three low-α-linolenic acid mutants and elucidation of their contribution to fatty acid biosynthesis in soybean seed,” Plant Science, vol. 168, no. 6, pp. 1615–1623, 2005.

K. Aghoram, R. F. Wilson, J. W. Burton, and R. E. Dewey, “A mutation in a 3-keto-acyl-ACP synthase II gene is associated with elevated palmitic acid levels in soybean seeds,” Crop Science, vol. 46, no. 6, pp. 2453–2459, 2006.

M. M. Spencer, V. R. Pantalone, E. J. Meyer, D. Landau-Ellis, and D. L. Hyten Jr., “Mapping the Fas locus controlling stearic acid content in soybean,” Theoretical and Applied Genetics, vol. 106, no. 4, pp. 615–619, 2003.

B. Pérez-Vich, A. J. Leon, M. Grondona, L. Velasco, and J. M. Fernández-Martínez, “Molecular analysis of the high stearic acid content in sunflower mutant CAS-14,” Theoretical and Applied Genetics, vol. 112, no. 5, pp. 867–875, 2006.

A. J. Cardinal, J. W. Burton, A. M. Camacho-Roger, J. H. Yang, R. F. Wilson, and R. E. Dewey, “Molecular analysis of soybean lines with low palmitic acid content in the seed oil,” Crop Science, vol. 47, no. 1, pp. 304–310, 2007.

Y. Li, F. Beisson, M. Pollard, and J. Ohlrogge, “Oil content of Arabidopsis seeds: the influence of seed anatomy, light and plant-to-plant variation,” Phytochemistry, vol. 67, no. 9, pp. 904–915, 2006.

L. Velasco and H. C. Becker, “Estimating the fatty acid composition of the oil in intact-seed rapeseed (Brassica napus L.) by near-infrared reflectance spectroscopy,” Euphytica, vol. 101, no. 2, pp. 221–230, 1998.

L. C. D. S. Ramos, J. S. Tango, A. Savi, and N. R. Leal, “Variability for oil and fatty acid composition in castorbean varieties,” Journal of the American Oil Chemists' Society, vol. 61, no. 12, pp. 1841–1843, 1984.

N. K. Sangwan, K. Gupta, and K. S. Dhindsa, “Fatty acid composition of developing soybeans,” Journal of Agricultural and Food Chemistry, vol. 34, no. 3, pp. 415–417, 1986.

G. M. Rubin, M. D. Yandell, J. R. Wortman et al., “Comparative genomics of the eukaryotes,” Science, vol. 287, no. 5461, pp. 2204–2215, 2000.

P. Tanhuanpää, J. Vilkki, and M. Vihinen, “Mapping and cloning of FAD2 gene to develop allele-specific PCR for oleic acid in spring turnip rape (Brassica rapa ssp. oleifera),” Molecular Breeding, vol. 4, no. 6, pp. 543–550, 1998.

M. A. Smith, H. Moon, G. Chowrira, and L. Kunst, “Heterologous expression of a fatty acid hydroxylase gene in developing seeds of Arabidopsis thaliana,” Planta, vol. 217, no. 3, pp. 507–516, 2003.

A. Sánchez-García, A. J. Moreno-Pérez, A. M. Muro-Pastor, J. J. Salas, R. Garcés, and E. Martínez-Force, “Acyl-ACP thioesterases from castor (Ricinus communis L.): an enzymatic system appropriate for high rates of oil synthesis and accumulation,” Phytochemistry, vol. 71, no. 8-9, pp. 860–869, 2010.

V. Andrianov, N. Borisjuk, N. Pogrebnyak et al., “Tobacco as a production platform for biofuel: overexpression of arabidopsis DGAT and LEC2 genes increases accumulation and shifts the composition of lipids in green biomass,” Plant Biotechnology Journal, vol. 8, no. 3, pp. 277–287, 2010.

Z. Dimov and C. Mollers, “Genetic variation for saturated fatty acid content in a collection of European winter oilseed rape material (Brassica napus),” Plant Breeding, vol. 129, no. 1, pp. 82–86, 2010.

R. Singh, S. G. Tan, J. M. Panandam et al., “Mapping quantitative trait loci (QTLs) for fatty acid composition in an interspecific cross of oil palm,” BMC Plant Biology, vol. 9, pp. 114–133, 2009.

X. Hu, M. Sullivan-Gilbert, M. Gupta, and S. A. Thompson, “G-to-A mutation at a 5_splice site of fad3c caused impaired splicing in a low linolenic mutant of canola (Brassica napus L.),” Plant Biotechnology, vol. 24, no. 4, pp. 397–400, 2007.

F. Beisson, A. Koo, S. Ruuska et al., “Arabidopsis genes involved in acyl lipid metabolism. A 2003 census of the candidates, a study of the distribution of expressed sequence tags in organs, and a web-based database,” Plant Physiology, vol. 132, no. 2, pp. 681–697, 2003.

S. Mekhedov, I. O. De de, and J. Ohlrogge, “Toward a functional catalog of the plant genome. A survey of genes for lipid biosynthesis,” Plant Physiology, vol. 122, no. 2, pp. 389–401, 2000.

J. Ohlrogge and J. Browse, “Lipid biosynthesis,” Plant Cell, vol. 7, no. 7, pp. 957–970, 1995.

J. Yu, S. Hu, J. Wang et al., “A draft sequence of the rice genome (Oryza sativa L. ssp. indica),” Science, vol. 296, no. 5565, pp. 79–92, 2002.

S. A. Goff, D. Ricke, T. H. Lan et al., “A draft sequence of the rice genome (Oryza sativa L. ssp. japonica),” Science, vol. 296, no. 5565, pp. 92–100, 2002.

A. M. Krill, M. Kirst, L. V. Kochian, E. S. Buckler, and O. A. Hoekenga, “Association and linkage analysis of aluminum tolerance genes in maize,” Plos One, vol. 5, no. 4, Article ID e9958, 2010.

M. Bevan, I. Bancroft, E. Bent et al., “Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana,” Nature, vol. 391, no. 6666, pp. 485–488, 1998.

Z. Tian, Q. Qian, Q. Liu et al., “Allelic diversities in rice starch biosynthesis lead to a diverse array of rice eating and cooking qualities,” Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 51, pp. 21760–21765, 2009.

X. M. Bao and J. Ohlrogge, “Supply of fatty acid is one limiting factor in the accumulation of triacylglycerol in developing embryos,” Plant Physiology, vol. 120, no. 4, pp. 1057–1062, 1999.

J. J. Thelen and J. B. Ohlrogge, “Metabolic engineering of fatty acid biosynthesis in plants,” Metabolic Engineering, vol. 4, no. 1, pp. 12–21, 2002.

K. Roesler, D. Shintani, L. Savage, S. Boddupalli, and J. Ohlrogge, “Targeting of the Arabidopsis homomeric acetyl-coenzyme A carboxylase to plastids of rapeseeds,” Plant Physiology, vol. 113, no. 1, pp. 75–81, 1997.

A. Jones, H. M. Davies, and T. A. Voelker, “Palmitoyl-acyl carrier protein (ACP) thioesterase and the evolutionary origin of plant acyl-ACP thioesterases,” The Plant Cell, vol. 7, no. 3, pp. 359–371, 1995.

T. A. Voelker, T. R. Hayes, A. M. Cranmer, J. C. Turner, and H. M. Davies, “Genetic engineering of a quantitative trait: metabolic and genetic parameters influencing the accumulation of laurate in rapeseed,” Plant Journal, vol. 9, no. 2, pp. 229–241, 1996.

P. Dormann, T. A. Voelker, and J. B. Ohlrogge, “Accumulation of palmitate in Arabidopsis mediated by the acyl-acyl carrier protein thioesterase FATB1,” Plant Physiology, vol. 123, no. 2, pp. 637–643, 2000.

T. Buhr, S. Sato, F. Ebrahim et al., “Ribozyme termination of RNA transcripts down-regulate seed fatty acid genes in transgenic soybean,” Plant Journal, vol. 30, no. 2, pp. 155–163, 2002.

G. Bonaventure, J. J. Salas, M. R. Pollard, and J. B. Ohlrogge, “Disruption of the FATB gene in Arabidopsis demonstrates an essential role of saturated fatty acids in plant growth,” Plant Cell, vol. 15, no. 4, pp. 1020–1033, 2003.

E. Akbar, Z. Yaakob, S. K. Kamarudin, M. Ismail, and J. Salimon, “Characteristic and composition of Jatropha curcas oil seed from Malaysia and its potential as biodiesel feedstock feedstock,” European Journal of Scientific Research, vol. 29, no. 3, pp. 396–403, 2009.

A. J. Kinney, “Genetic engineering of oilseeds for desired traits,” in Genetic Engineering, J. K. Setlow, Ed., vol. 19, pp. 149–166, Plenum Press, New York, NY, USA, 1997.

J. Burgal, J. Shockey, C. Lu et al., “Metabolic engineering of hydroxy fatty acid production in plants: RcDGAT2 drives dramatic increases in ricinoleate levels in seed oil,” Plant Biotechnology Journal, vol. 6, no. 8, pp. 819–831, 2008.

M. Venegas-Calerón, O. Sayanova, and J. A. Napier, “An alternative to fish oils: metabolic engineering of oil-seed crops to produce omega-3 long chain polyunsaturated fatty acids,” Progress in Lipid Research, vol. 49, no. 2, pp. 108–119, 2010.