Increasing seed oil content in Brassica species through breeding and biotechnology

182 August 2013, Vol. 25, No. 8 Lipid Technology

Feature

Increasing seed oil content in Brassica speciesthrough breeding and biotechnology

Habibur Rahman, John Harwood, Randall Weselake

H.R. and R.W. are Professors in the Department of Agricultural, Food and Nutritional Science at the University of Alberta, Edmonton,Alberta, Canada T6G 2P5 E-mail: [email protected]. is Professor and Deputy Director of the School of Bioscience, Cardiff University, Cardiff CF10 3AX, United Kingdom

Summary

Increasing the seed oil content of Brassica species and other major oilseed crops is of paramount importance in maintaining a future supplyof vegetable oil for a growing global population. Currently, commercially-available Brassica species with enhanced seed oil content haveall been developed through plant breeding. Many quantitative trait loci including gene interactions are involved in the control of seed oilcontent. Despite this complexity, manipulation of specific steps in storage lipid biosynthesis using genetic engineering has resulted intransgenic lines of Brassica napus with increased seed oil content. Recent studies suggest that engineering of seed oil content can be guidedusing methods in metabolic analysis.

Introduction

Seeds of Brassica oil crops such as canola (Brassica napus L.) con-tain about 45% oil, and the meal remaining after oil extractioncontains about 40% protein. Since the development of canolacultivars (low erucic acid [22:1cisD13] oil and low glucosinolatemeal) of Brassica napus, the demand for this oil in the world mar-ket has increased steadily for use as a food oil and for industrialapplications. Currently, this oil contributes about 15% to theworld’s total supply of vegetable oils.

In Brassica, the cotyledon, which is a part of the matureembryo, and seed coat constitute the major part of the seed.Most of the seed oil and protein are stored in the cotyledon,while the seed coat contains very little oil. Accumulation of themajor amount of seed oil occurs between the 3rd and 7th weeksafter pollination, while accumulation of protein commencestwo to three weeks after the start of oil accumulation. After thatperiod, accumulation of these two major seed components con-tinues concurrently, but at a lower rate until maturity. In themature seed, oil content is largely determined by the geneticarchitecture of the maternal (seed producing) plant; the effect ofpollen and cytoplasm are relatively small. The maternal effecton seed oil content is primarily dictated by photosynthetic activ-ity in the silique wall tissue; and oil content in the mature seedcorrelates linearly with the area of the cotyledon cells filled withoil bodies. Seed oil content is also highly influenced by cultiva-tion practices and environmental conditions. For example, highnitrogen fertilizer increases protein content while reducing oilcontent. High temperatures generally accelerate plant develop-ment but, after anthesis, high temperature can shorten theduration of the seed filling period resulting in reduced seed oilcontent.

In this Feature article, we begin by examining the role of plantbreeding in increasing seed oil content in Brassica species, inparticular B. napus. We then provide a brief overview of the bio-chemistry of the oil formation process before moving on to dis-cuss genetic engineering approaches in increasing seed oil con-tent in B. napus. Finally, we discuss how metabolic control anal-ysis as well as examination of cotyledon metabolism using an in

silico model have provided new tools for guided genetic engi-neering of seed oil content.

Seed oil content is controlled by manyquantitative trail loci

In B. napus, seed oil content is controlled by a large number ofnuclear gene loci (QTL, quantitative trait loci), mostly with anadditive effect. Recent reports, however, indicate that epistaticinteractions as well as QTL X environment interactions are alsoinvolved in the control of this trait. To date, QTL mapping con-ducted by various researchers has indicated that 17 of the 19 B.napus chromosomes carry loci involved in the control of seed oilcontent [1]. Depending on the parents involved in the cross andthe size of the mapping population used in genetic studies, thenumber of QTL detected in the studies varied from 3 to 27. Theamount of phenotypic variance explained by these QTL variedfrom about 2% to more than 10%, while the additive effect ofthese QTL alleles varied from about 0.2% to more than 1.0%.Given the fact that the three Brassica genomes (A, B and C)evolved from an ancestral prototype through genome duplica-tion, rearrangements, chromosome fusion, and/or fission eventsand that B. napus is an amphidiploid of the A and C genome spe-cies (B. rapa and B. oleracea), the occurrence of such a high num-ber of loci involved in oil synthesis and accumulation in theseed is not surprising. Some of the QTL stably express underdifferent environmental conditions; however, the majority ofthe QTL are sensitive to environmental changes which makephenotypic selection for accumulation of positive QTL alleles ina cultivar a difficult task. Some genomic regions carry both oiland protein QTL where the allele increasing oil content iscoupled with the allele decreasing protein content and viceversa. This results in a reciprocal correlation between these twoseed components. Therefore, increasing oil content throughaccumulating positive alleles generally decreases protein con-tent in the seed. Of the two main products of canola seed, oil andseed meal, the price of seed meal is only a quarter as compared

i 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.lipid-technology.com

DOI 10.1002/lite.201300291

Lipid Technology August 2013, Vol. 25, No. 8 183

to the price of its oil. Therefore, increasing seed oil content incanola will ultimately raise the value of this oilseed crop.

Breeding for increased seed oil content

Among the various Brassica species, the most extensive breedingresearch for increasing seed oil content has been conducted onB. napus. Germplasm with the highest oil content can also befound in this species. Oil content in B. napus germplasm variesfrom about 35% to 52%. Interestingly, a line with about 65% oilhas recently been developed in China through pyramiding ofhigh oil alleles [2]. Despite the wide genetic variation present inthe germplasm of this crop, a commercial canola cultivar withthis high level of oil content is only rarely found. A cultivardeveloped in a breeding program does not carry a single desir-able trait such as extremely high oil content; it is rather a pack-age of good agronomic and seed quality properties includinghigh seed yield. Most of the plant-traits, which are measured in aquantitative scale, are controlled by multiple gene loci and theseloci may possess more than two alleles. Interaction of allelesamong the loci contributing to phenotypic variation is also acommon phenomenon for many plant traits. Thus, the favour-able properties in a cultivar or in an elite breeding line are theresult not only of accumulation of a greater number of favour-able alleles, but also superior combinations of alleles amongmany of the loci. This is generally achieved through severalcycles of breeding with an incremental progress in cultivar per-formance. In the last decade, plant breeders have steadilyincreased seed oil content in canola cultivars in Europe andCanada (Figure 1). The increase is relatively less prominent inthe case of European winter canola when compared to Canadianspring canola. This may partly be due to the longer timerequired for each cycle of breeding in this crop. This increase inoil content along with the increase in seed yield indeedincreased oil yield per unit area. Thus, much greater oil yield incanola is still possible through improving both seed oil contentand yield.

In breeding for cultivar development, the choice of the parentsto be used in crossing is critical for mining a new breeding line or

a cultivar with superior agronomic and seed quality properties.In the case of a trait like seed oil content, which is controlled by alarge number of loci (with each locus exerting a small effect), it ispossible that two parental lines with similar seed oil content aregenetically distinct for the QTL alleles. Cross-breeding of theselines would result in transgressive segregants with higher seedoil content through accumulation of the positive alleles from theparents. Identification of such genetically distinct parents, how-ever, is a difficult and cumbersome task in traditional breeding.Molecular plant breeding can play an important role in this. Onthe other hand, the use of an exotic germplasm in cross-breedingwith a locally adapted cultivar or elite breeding line to develop anew cultivar with high seed oil content may require repeatedcycles of breeding. Often, exotic germplasm carry several undesir-able traits, and use of this type of germplasm in cross-breedingmay disrupt the favourable allele combinations in the existingcultivars or elite breeding lines which have been achievedthrough several cycles of breeding. Given that each trait is con-trolled by several gene loci, cross-breeding would result in tre-mendous variation in the progeny due to re-shuffling of thealleles of the parents. Identification of a superior high-oil eliteline possessing all other desired traits often becomes difficult inone cycle of breeding. The traditional backcross breeding tech-nique is suitable for introgression of a highly heritable trait, con-trolled by one or two gene loci, into an elite line or cultivar; how-ever, this is difficult for a quantitative trait like seed oil content.Furthermore, current regulation of cultivar registration mayalso impose limitations on increasing seed oil content. For exam-ple, in Canada, it is required that a certain quantity of proteinneeds to be present in a canola cultivar for registration and culti-vation at a commercial scale. This restricts increasing seed oilcontent in a cultivar to the highest possible level due to reciprocalcorrelation between these two traits.

Thus, under the situations of multiple QTL involved in thecontrol of seed oil content and sensitivity of the QTL to environ-mental change and cultivation practices, marker assisted selec-tion would enhance breeding efficiency for increased oil contentthrough accumulating the positive QTL alleles. As some of theQTL alleles show significant interaction with the environment,specific alleles may need to be selected for a given cultivationregion. Epistatic interactions of the alleles also need to be takeninto account for a knowledge-based improvement of seed oilcontent. To date, however, very limited QTL have actually beenused in breeding. This is primarily due to the large number ofQTL involved in the control of this trait and the small effect ofeach locus on phenotypic variation. This is also due to the lack ofintegrated knowledge of the QTL detected by different research-ers and the understanding of the relative importance of the QTL.The extent of allelic variation present in each of the QTL is alsonot known. The presence of multiple alleles would further addcomplexity to breeding for high oil cultivars through traditionaland molecular approaches. Furthermore, in some cases, if theinterval between the molecular markers bracketing the QTLregion is too large, the markers may get lost due to recombina-tion between the gene and the linked marker during meiosis.Therefore, fine mapping of the QTL region and readily accessiblemolecular markers will benefit breeding as well as cloning ofcandidate genes involved in seed oil synthesis and accumula-tion. Identification of candidate genes and their allelic variationwould greatly increase the efficiency of breeding.

Many genes in the amphidiploid species B. napus exist in mul-tiple copies with similar structure; therefore, it is difficult to dis-


Figure 1. Seed oil content in Brassica napus canola cultivars inCanada and the United Kingdom (UK). In case of Canada, aver-age seed oil content (dry weight basis) is shown for springcanola cultivars tested in public coop trials prior to registration,and for the UK, average seed oil content (at 9% moisture) isshown for registered winter canola cultivars (Data source: Ray-mond Gadoua, Canola Council, Canada, and Simon Kightley,National Institute of Agricultural Botany, UK).

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tinguish the orthologous from the paralogous genes. With thesequencing of Arabidopsis genome, considerable progress hasbeen made in understanding the key genes involved in accumu-lation of seed storage oil. In this regard, knowledge of the Arabi-dopsis genome, which is a close relative of Brassica species, canbe used to provisionally identify the candidate genes involved inseed storage oil synthesis and accumulation in B. napus, and con-sequently for allele searches in Brassica germplasm and subse-quent utilization in breeding. Some of the Arabidopsis ortholo-gous genes involved in lipid biosynthesis map to some of theQTL associated with seed oil content in B. napus [3].

Brassica napus lines with higher seed oil content generally pos-sess a thinner seed coat which results in greater contribution ofthe cotyledon to the whole seed and eventually to the higher oilcontent of the seed. Seed size, in general, does not show strong acorrelation with oil content. Therefore, selection for higher oilcontent along with thinner seed coat and greater seed size canbe used for increasing oil yield in B. napus canola.

Seed oil formation

Before discussing biotechnological approaches for increasingseed oil content in B. napus, it is useful to describe briefly the pro-cess of seed oil formation. Seed oil is predominantly composedof triacylglycerol (TAG), which contains three fatty acids esteri-fied to a glycerol backbone. The process of TAG formation can bethought of as occurring in two major blocks where block A refersto fatty acids synthesis and block B refers to glycerolipid assem-bly and acyl-editing (Figure 2) [4, 5]. Fatty acids are synthesizedin the plastids of oil-forming cotyledons. Formation of monoun-saturated fatty acids also occurs in the plastid, important for thehigh proportion of oleic acid (18:1cisD9) in canola TAG. After beingreleased from the fatty acid synthesizing machinery, the fattyacids are exported to the cytoplasm where they become esteri-fied to coenzyme A (CoA) to form the acyl-CoA pool, which canbe thought of as an intermediate connecting block A to block B.In some cases, the acyl-CoA can undergo elongation to formlonger hydrocarbon chains. The elongation of oleoyl-CoA to eru-coyl (22:1cisD13)-CoA is largely absent in modern day canola due togenetic mutations inactivating the elongation reactions. sn-Gly-cerol 3-phosphate (G3P) serves as the starting point for acyl-CoA-dependent assembly of fatty acids on the glycerol backbonewhich is catalyzed mainly by the membrane-bound acyltrans-ferases of the linear G3P (or Kennedy) pathway leading to TAG.The phosphate group is removed from the glycerol backboneprior to the final acylation which is catalyzed by diacylglycerolacyltransferase (DGAT). The process of TAG production in plantswith TAG containing polyunsaturated fatty acids (PUFA), such asB. napus, however, is not simply a linear pathway but involves acomplex interaction with the membranes of the endoplasmicreticulum membrane (ER). The diacylglycerol (DAG) skeletongenerated in the Kennedy pathway can be converted to phospha-tidylcholine of the ER which is the site of formation of the PUFA,linoleic (18:2cisD9,12) and �-linolenic (18:3cisD9,12,15) acids, catalyzedby membrane-bound desaturases. Various acyl-editing mechan-isms can then return PUFA-enriched DAG to the Kennedy path-way and also enrich the acyl-CoA pool in PUFA-CoA. PUFA canalso be transferred from the sn-2 position of phosphatidylcho-line directly to DAG to form TAG via the catalytic action of phos-pholipid:diacylglycerol acyltransferase (PDAT), which is an acyl-CoA-independent process. TAG generated via the catalytic action

of DGAT and PDAT accumulates between the outer leaflets of theER and eventually pinches off the ER in the form of oil bodieswhich range from 0.2 to 2 microns in diameter.

Genetic engineering approaches for increasingseed oil content

Genetic engineering to increase seed oil content in Brassica spe-cies is essentially at the experimental or pre-commercializationstage. Despite the fact that seed oil content in Brassica species iscontrolled by many QTL, there have been many reported suc-cesses of increasing seed oil content in B. napus by manipulatingspecific steps in fatty acids synthesis, glycerolipid assembly orother pathways in carbon metabolism [5]. Over-expression ofgenes encoding transcription factors, which are known to up-regulate many reactions in fatty acid synthesis and glycolysis,has also resulted in increased seed oil content, but most of thesestudies have been conducted with the model plant, Arabidopsis[5].

Over-expression of a gene encoding Arabidopsis cytoplasmicacetyl-CoA carboxylase (ACCase) in the plastid during seed devel-opment in B. napus represents an early success in boosting seedoil content in this oleaginous crop [5]. ACCase catalyzes the for-mation of malonyl-CoA, from acetyl-CoA, which serves as a two-carbon donor in fatty acid synthesis and is the first committedstep in the process. It was hypothesized that the cytoplasmicform of ACCase, which is involved in providing malonyl-CoA forfatty acid elongation in the ER, was not subject to the same regu-lation as the plastidial form of the enzyme. Therefore, cytoplas-mic ACCase may be more effective in driving overall fatty acidsynthesis than the plastidial form of the enzyme. In a later study,a gene encoding a yeast lysophosphatidate sn-2 acyltransferasewas heterologously over-expressed in B. napus resulting inincreased seed oil content [5]. Over-expression of type-1 DGAT toincrease seed oil content in B. napus represents another success[4]. Previous metabolic studies have demonstrated that DAG wasthe next most abundant Kennedy pathway compound after TAGduring seed development in B. napus suggesting that the DGAT-catalyzed reaction represented a “bottleneck” in the process ofTAG formation [5].

Metabolic analysis for informed geneticengineering of seed oil content

Various types of metabolic analyses, based on the use of feedingB. napus embryos with precursor compounds labeled with iso-


Figure 2. Overview of fatty acid synthesis and glycerolipid meta-bolism leading to production triacylglycerol (TAG) in developingembryos of Brassica napus. Image designed based on Wese-lake et al. [4, 5]. ER, endoplasmic reticulum; G3P, sn-glycerol 3-phosphate.

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topes (e.g. 14C and 13C) or in silico computational modeling ofmetabolism in embryos, have been useful in identifying reac-tions or pathways that could be potentially manipulated toincrease storage product accumulation [4–6]. Harwood and col-leagues have used an approach known as top-down metaboliccontrol analysis to examine entire pathways [4]. In this regard,block A and block B shown in Figure 2 can be viewed in terms oftheir individual contributions to the control of TAG production.When applied to developing embryos of B. napus, this approachindicated that 70% of the control associated with TAG produc-tion was due to block B, where glycerolipid assembly occurred[4]. In transgenic lines over-expressing type-1 DGAT in anembryos-specific fashion, control in block B decreased to 50%indicating that the increased abundance of DGAT enzyme wasopening up the “bottleneck” thereby allowing more DAG to beconverted to TAG.

The process of storage lipid synthesis, however, sits in the con-text of other metabolic pathways. For example, G3P, which pro-vides the glycerol backbone for TAG production, is derived fromdihydroxyacetone phosphate produced through glycolysis.Indeed, one of the early successes in increasing seed oil contentin B. napus was based on increasing the availability of G3P forTAG assembly [5]. Schwender & Hay [6] have developed a predic-tive biochemical systems-level approach based on in silico-simu-lated associations between metabolic carbon partitioning andshifts in TAG versus protein content. A large-scale stoichiometricmodel (bna572) of B. napus seed storage metabolism was used forthis purpose. Interestingly, among the highest TAG-responsivereactions were those involved in sugar catabolism. Subse-quently, the investigators suggested that it might be worthwhilemanipulating sucrose catabolism by glycolysis to boost seed oilcontent in B. napus.

Conclusions

It will be clear from the foregoing that there are several opportu-nities for enhancing seed oil levels in B. napus. While breedinghas had a spectacular initial success in developing cultivars withincreased seed oil content, there is still much to learn about thegenes regulating seed oil content and their interactions. In addi-tion, use of metabolic control analysis to inform genetic manip-ulation offers new possibilities for increasing seed oil content. Ithas already demonstrated promise in increasing seed oil contentby around 10% through single gene manipulations. With thecontinued demand for commodity oils and limited agriculturalland, metabolic engineering to increase seed oil content offersmuch promise for the future.

RW and HR were supported by the Natural Sciences and EngineeringResearch Council of Canada, Alberta Innovatives Bio Solutions, theAlberta Canada Producers Commission and the Alberta Crop IndustryDevelopment Fund. Work in JH’s lab was supported by the MalaysianPalm Oil Board, DuPont and the BBSRC (UK).

References

[1] Delourme, R. et al., Theor. Appl. Genet. 2006, 113, 1331–1345.

[2] Hu, Z.-Y. et al., PLOS ONE 2013, 8, e62099.

[3] Zhao, J. et al., Theor. Appl. Genet. 2012, 124, 407 –421.

[4] Weselake , R.J. et al., J. Exp. Bot. 2008, 59, 3543 –3549.

[5] Weselake, R.J. et al., Biotechnol. Adv. 2009, 27, 866–878.

[6] Schwender, J., Hay, J.O., Plant Physiol. 2012, 160, 1218–1236.


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Increasing seed oil content in Brassica species through breeding and biotechnology