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Fatty Acid Biosynthesis and Triacylglycerol Accumulation in the Biofuel Plant Jatropha curcas

  • Yan-Bin Tao
  • Xiao-Di Hu
  • Zeng-Fu XuEmail author
Chapter
  • 275 Downloads

Abstract

Jatropha curcas L. is recognized as one of the most promising biofuel plants because of the high oil content and the good oil composition in its seeds. Previous studies have established our understanding of the oil yield and quality of Jatropha in germplasm from around the world, which could be helpful for breeding programs. To obtain elite varieties of Jatropha with desirable traits, genetic manipulation technology can offer a feasible strategy of spatiotemporal regulation for expressing the genes involved in oil biosynthesis because Jatropha has the advantages of being a tree of a short generation time and an easy genetic transformation system. Based on recent studies of Jatropha genomics and transgenic analyses, we identified the genes involved in de novo fatty acid (FA) synthesis and triacylglycerol (TAG) assembly here, and we reviewed the gene expression profiles and transgenic manipulation of these genes in Jatropha. This information will be useful for Jatropha oil improvement via the combinatorial metabolic engineering approach.

Keywords

Biosynthesis Fatty acids Genetic engineering Oil content Oil quality Triacyl glycerols 

10.1 Oil Content and Fatty Acid (FA) Composition in Jatropha Seeds

Due to its high content of non-edible seed oil, Jatropha is one of the most promising energy plants for use as a biodiesel production feedstock. For conventional breeding purposes, many studies helped in identification of superior germplasm with desirable traits such as high oil yield with superior quality. Generally, there is a positive correlation between seed oil content and seed development, indicating that the oil content increases linearly as the seed weight increases. During the early stages of Jatropha seed development, before 21 days after pollination (DAP), the seeds grow gradually, their oil content is below 10% (Sinha et al. 2015), and no oil bodies can be found in endosperm cells at 14 days after flowering (DAF) (Gu et al. 2012). After 21 DAP, during the seed filling stage, the seeds grow rapidly (Tao et al. 2014) in association with a sharp increase in oil content (Sinha et al. 2015). At seed maturity time, the oil content reaches its maximum, and the oil bodies occupy most of the space in the endosperm cells (Gu et al. 2012).

Due to different environmental and genetic factors, the highest oil contents differ among Jatropha germplasm accessions. Jatropha is native to Central America and more particularly Mexico and Guatemala, and it was presumably taken to tropical areas of Asia and Africa by Portuguese seafarers. In Mexico, the seed oil content of 17 native populations was relatively high, varying from 42.4% to 55.4% (Martinez-Diaz et al. 2017). Six Jatropha accessions derived from Colombia were transplanted to Spain. Three of them had seed oil content higher than 40% and up to the maximum of 42.4%, whereas the others showed seed oil content lower than 30% (Alburquerque et al. 2017). In India, Singh et al. (2016) collected 1 genotype (JCN01) from Cape Verde and 14 genotypes (JCN02-14) from India to select for elite germplasm. Most of these genotypes contained between 30% and 37.5% (JCN14) seed oil, except for one (JCN01), which contained only 27.7% seed oil. The oil yield per plant of JCN14 (0.45 kg) was five times that of JCN01 (0.09 kg). High heritability was recorded for the oil yield per plant (98.6%) followed by the seed oil content (98.2%). Thus, parental candidates for selective breeding programs should be chosen from germplasm with the largest oil yield per plant possible.

In addition to the oil content, the FA composition is also a key factor that should be considered during the germplasm selection because it determines the quality of the biodiesel. Whether a biodiesel fuel is suitable or not can be estimated by its crystallization capacity, oxidative stability, and ignition quality (Alburquerque et al. 2017). These properties have a direct correlation with the FA composition. If the number of carbons and saturation in FA chains are high, then the crystallization capacity is high, which results in the solidification of biodiesel at high temperatures. However, if the unsaturation level is too high, then the biodiesel may have a poor oxidative stability. Several parameters such as the saponification number (SN), iodine value (IV), and cetane number (CN) of the oil are used to describe these properties. SN and IV indicate the FA chain length and unsaturation levels, respectively. IV is considered as a measure of the oil stability. The CN, which is calculated on the basis of the SN and IV, indicates the ignition quality. The higher the CN, the shorter is the ignition delay time.

Most plant oils are generally composed of saturated palmitic acid (16:0) and stearic acid (18:0), monounsaturated oleic acid (18:1), and polyunsaturated linoleic acid (18:2) and linolenic acid (18:3). The major FAs in Jatropha oil are palmitic acid, stearic acid, oleic acid, and linoleic acid (Akintayo 2004; Kywe and Oo 2009; Wassner et al. 2016). During Jatropha seed development, the FA composition changes considerably. Sinha et al. (2015) found that palmitic acid accounted for over 30% of all FAs at early developmental stages but was reduced to approximately 15% in mature seeds. This reduction may be due to the fact that palmitic acid is the precursor of other FAs. Consequently, along with the seed development, the stearic and oleic acids increased from 2.14% to 6.02% and 3.90% to 44.38%, respectively. The linoleic acid increased gradually from 33.38 at 6 DAP to 47.19% at 51 DAP, but it decreased to 33.02% at maturity. Linolenic acid, which is not a major component of Jatropha oil, accounted for up to 26.32% of all FAs at the early developmental stages before 27 DAP, but it decreased sharply to 0.52% at 36 DAP and remained at 0.65% in mature seeds. According to the variation in the FA composition from 6 DAP to maturity, the SN and IV decreased gradually from 206.37 to 201.18 and 136.0 to 101.5, respectively, which indicated that the oil stability turned better. On the other hand, the CN increased up to 50.59 at maturity. Compared to the yellow ripe stage, a higher CN and a lower IV were present at the black ripe stage, indicating the superior ignition quality and higher oxidative stability of the oil. Therefore, harvesting Jatropha seeds at the black ripe stage could offer a guarantee of the oil quality.

The FA compositions were analyzed in six Jatropha accessions grown in Spain (Alburquerque et al. 2017). It was found that the oil quality varied among Jatropha accessions even when all the seeds are collected at the black ripe stage. These accessions showed the same FA composition profile but different FA amounts. The palmitic acid varied from 11.64% to 15.45%, the stearic acid varied from 3.52% to 8.18%, the oleic acid varied from 25.92% to 44.08%, and the linoleic acid varied from 33.79% to 52.20%. The higher oleic acid amounts were recorded from three accessions (4-5, 6-3, and 8-8), while their linoleic acid amounts were lower. By contrast, lower oleic acid amounts and higher linoleic acid amounts were recorded in another three accessions. A similar pattern was shown in Jatropha planted in India, but the variation was not as obvious (Singh et al. 2016). Biodiesel derived from oil with a high oleic acid level has excellent properties in terms of ignition quality, nitrogen oxide (NOx) emissions, and fuel stability (Graef et al. 2009). Accordingly, higher CN and lower IV were associated with seed oil of Jatropha accessions 4-5, 6-3, and 8-8. Additionally, the saturated FA amounts in these three accessions were higher than those in the others. Therefore, the biodiesel obtained from these accessions are also good candidates for environmentally friendly oils with lower NOx emissions because reduced NOx emissions are correlated with increasing CN values and saturation and decreasing IV. Besides the genetic component, Wassner et al. (2016) found that the temperature during the seed filling period could affect the FA composition significantly. The mature seeds harvested at cool temperatures (17.0–19.0 °C) produced oil with the largest linoleic acid content (47.5–45.4%) and the lowest oleic acid content (31.7–33.4%). However, under warm temperatures (27.9 and 28.7 °C), the opposite response pattern with the lowest linoleic acid contents (28.8% and 31.8%) and the highest oleic acid contents (47.1% and 45.4%) occurred. Consequently, a higher CN and lower IV were associated to seed oil obtained under warm conditions. This finding indicates that harvesting seeds matured under warmer temperatures helps in obtaining good-quality oil. However, the oil content was not correlated with temperature changes.

Taken together, the oil content and quality of Jatropha accessions vary according to genetic and local environmental factors. As a result, to select breeding candidates, a good approach is to choose the best performing plants in the location of interest. In addition to the conventional breeding approach, genetic manipulation technology can offer a feasible strategy for breeding Jatropha with a high oil yield and quality, especially because several efficient transformation systems have been established (Fu et al. 2015). Thus, investigating the molecular basis of oil biosynthesis during seed development becomes a prerequisite because it is essential for understanding the genetic factors that regulate the lipid biosynthesis and accumulation in Jatropha seeds.

10.2 Triacylglycerol (TAG) Biosynthesis

Plant oil (TAG) biosynthesis generally involves the following two pathways: de novo FA synthesis occurring in plastids (Fig. 10.1) and TAG assembly (Fig. 10.2) occurring in the endoplasmic reticulum (ER) (Ohlrogge and Chapman 2011). Plant TAGs, which are energy-dense lipids that accumulate in oilseed plants, serve as an energy reservoir for seed germination and the early growth of young seedlings. It is the major component of the plant oil. Three FA chains (usually 16 or 18 carbons long) esterified to a glycerol backbone constitute the TAGs (Durrett et al. 2008).
Fig. 10.1

De novo fatty acid (FA) biosynthetic pathway in Jatropha curcas. ACC acetyl-CoA carboxylase (ACCase), ACP acyl carrier protein, CoA Coenzyme A, EAR enoyl-ACP reductase, FAD2, 3, 6, 8, fatty acid desaturase 2, 3, 6, 8, FATA acyl-ACP thioesterase A, FATB acyl-ACP thioesterase B, HAD β-hydroxyacyl-ACP dehydratase, KAR β-ketoacyl-ACP reductase, KAS I, II, III, β-ketoacyl-ACP synthase I, II, III, LCACS long-chain acyl-CoA synthase, LPCAT acyl-CoA:lysophosphatidylcholine acyltransferase, MCAT malonyl-CoA:ACP transacylase, SAD stearoyl-ACP desaturase

Fig. 10.2

Triacylglycerol (TAG) assembly pathway in Jatropha curcas. DGAT diacylglycerol acyltransferase, GPAT glycerol-3-phosphate acyltransferase, LPAAT lysophosphatidic acid acyltransferase, PAP phosphatidic acid phosphatase, PC phosphatidylcholine, PDAT phospholipid:diacylglycerol acyltransferase, and PDCT phosphatidylcholine:diacylglycerol cholinephosphotransferase

FA biosynthesis is initiated by the conversion of acetyl-CoA to malonyl-CoA, which is catalyzed by heteromeric acetyl CoA carboxylase (ACCase). This step is generally considered to be rate limiting. The second step is the formation of malonyl-ACP by transferring a malonyl group from malonyl-CoA to an acyl carrier protein (ACP), which is catalyzed by malonyl-CoA:ACP transacylase (MCAT). The third step is the condensation of malonyl-ACP and acyl-CoA to form acetoacetyl-ACP (4:0-ACP), which is catalyzed by β-ketoacyl-ACP synthase III (KAS III). The fourth step is the reduction of acetoacetyl-ACP to form β-hydroxybutyryl-ACP, which is catalyzed by β-ketoacyl-ACP reductase (KAR). The fifth step is dehydrating β-hydroxybutyryl-ACP with β-hydroxyacyl-ACP dehydratase (HAD) to form β-trans-butenoyl-ACP. The next reaction is the reduction of double bond in β-trans-butenoyl-ACP to form butyryl-ACP, which is catalyzed by enoyl-ACP reductase (EAR). Further chain elongation up to palmitoyl-ACP (16:0-ACP) is catalyzed by β-ketoacyl-ACP synthase I (KAS I) in six consecutive reactions. Next, the palmitoyl-ACP is converted to stearoyl-ACP (18:0-ACP), which is catalyzed by β-ketoacyl-ACP synthase II (KAS II). Stearoyl-ACP desaturase (SAD) catalyzes the first desaturation step in FA biosynthesis, converting stearoyl-ACP to oleoyl-ACP (18:1Δ9-ACP). This step is also considered rate-limiting because it converts saturated FAs to unsaturated FAs. Finally, the chain elongation is terminated by acyl-ACP thioesterases that hydrolyze the thioester bond, releasing free FAs. Thus, this step is regarded as the primary determinant of the chain length and level of saturated FAs. FATA and FATB, which are encoded by FatA and FatB, respectively, are two distinct acyl-ACP thioesterases. FATA acts on unsaturated acyl-ACPs, and FATB prefers saturated acyl-ACPs. These free FAs are then converted to acyl-CoAs by long-chain acyl-CoA synthase (LCACS) to be bound by acyl-CoA-binding proteins (ACBPs), which are able to protect them from acyl-CoA hydrolases and transport them to the ER. Chain elongation occurs in the acyl-CoA pool, whereas desaturation reactions occur in the acyl-phosphatidylcholine (PC) pool (Singh et al. 2005). Modifications that occur in the ER recruit oleoyl-CoA as the substrate that can be desaturated to polyunsaturated FAs (PUFAs). Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) catalyzes the formation of PC (C18:1Δ9-PC) from acyl-CoA (C18:1Δ9-CoA) by mediating acyl-exchange between acyl moieties at the sn-2 position of PC and the acyl-CoA pool. Fatty acid desaturase 2 or 6 (FAD2/6) desaturates the oleic acid (18:1Δ9) to form linoleic acid (18:2Δ9,12) in plants. The linoleic acid can be further desaturated by FAD3/8 to produce α-linolenic acid (18:3Δ9,12,15). These desaturases and their divergent forms can also edit the acyl chains to be esterified to PC.

In the ER, TAG assembly sequentially consumes the acyl-CoA using glycerol-3-phosphate (G3P) as a substrate through the Kennedy pathway, which involves four steps. G3P, the precursor of the glycerol backbone, is catalyzed by glycerol-3-phosphate acyltransferase (GPAT) to form lysophosphatidic acid (LPA), which is further catalyzed by lysophosphatidic acid acyltransferase (LPAAT) to produce phosphatidic acid (PA). The next step converts the PA to diacylglycerol (DAG), and it is catalyzed by phosphatidic acid phosphatase (PAP). Finally, diacylglycerol acyltransferase (DGAT) converts DAG to TAG by using an acyl-CoA as acyl donor. To transfer PUFAs-PC to form TAG, two routes are available. The PC substrate for this process is provided by the DAG produced in the Kennedy pathway, which can be converted to PC by the catalyzation of CDP-choline:DAG cholinephosphotransferase (CPT). PC backbones carrying PUFAs can be converted into DAG through the removal of the phosphocholine headgroup through the action of phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) (Ichihara and Suda 2003; Singh et al. 2005). This process allows PUFAs to form TAG through the Kennedy pathway. The second route involves the phospholipid:diacylglycerol acyltransferase (PDAT)-mediated transacylation of PUFAs from PC to the DAG to form TAG (Dahlqvist et al. 2000). Various TAGs are then assembled in oleosins to form oil bodies in the seeds.

10.3 Expression Analysis of FA Biosynthetic Pathway Genes During Seed Development

Using the Jatropha genome sequences (Wu et al. 2015) uploaded in NCBI (https://www.ncbi.nlm.nih.gov/), we first retrieved all the genes and proteins that are related to oil synthesis in Arabidopsis thaliana from the TAIR database (http://www.arabidopsis.org/) based on previous reports. The cDNA and protein sequences of Jatropha were aligned against known A. thaliana FA genes and proteins using BLASTN and BLASTP with E-values of 1e−10 and 1e−5, respectively (Altschul et al. 1990). All predicted proteins were then subjected to PFAM analysis, and the candidate proteins containing certain domains of the respective gene families were checked manually and verified with the results of the BLASTP. The genes encoding the key enzymes involved in Jatropha FA biosynthesis are listed in Table 10.1.
Table 10.1

Summary of fatty acid (FA) biosynthetic genes in Jatropha curcas

Gene

Gene ID

Protein ID

Annotation

ACC1-LIKE

105638840

NP_001295714.1

Acetyl-CoA carboxylase 1-like, cytosolic ACCase

accA

105649954

XP_012092191.1

ACC- CTα methylcrotonoyl-CoA carboxylase subunit alpha, mitochondrial

accB-1

105644890

XP_012085781.1

ACC-BCCP1 X1 biotin carboxyl carrier protein of acetyl-CoA carboxylase 1, chloroplastic isoform X1

XP_012085782.1

ACC-BCCP1 X2 biotin carboxyl carrier protein of acetyl-CoA carboxylase 1, chloroplastic isoform X2

accB-2

105644147

NP_001295674.1

ACC-BCCP2 biotin carboxyl carrier protein of acetyl-CoA carboxylase 2, chloroplastic

accC

105643965

NP_001292959.1

ACC-BC biotin carboxylase 1, chloroplastic

accD

7564871

YP_002720121.1

ACC- CTβ, chloroplastic

MCAT

105639234

NP_001306855.1

Malonyl-CoA-acyl carrier protein transacylase, mitochondrial

KAS III

105645991

NP_001292956.1

Beta-ketoacyl-ACP synthase 3, chloroplastic

KAR

105644771

XP_012085628.1

Ketoacyl ACP reductase

KAR-LIKE

105640730

XP_012080517.1

Low-quality protein: beta-keto acyl-carrier protein reductase, chloroplastic-like

HAD

105645386

XP_012086368.1

Hydroxyacyl-ACP dehydratase

EAR1

105646511

XP_012087759.1

Enoyl-[acyl-carrier-protein] reductase 1[NADH], chloroplastic

EAR2

105634472

XP_012072726.1

Enoyl-[acyl-carrier-protein] reductase 2[NADH], chloroplastic isoform X1

XP_020535082.1

Enoyl-[acyl-carrier-protein] reductase 2[NADH], chloroplastic isoform X2

KAS I-1

105633975

NP_001292958.1

Beta-ketoacyl-ACP synthase I, chloroplastic

KAS I-2

105645646

XP_012086687.1

Beta-ketoacyl-ACP synthase I, chloroplastic

KAS II

105638018

NP_001292950.1

Beta-ketoacyl-ACP synthase II, chloroplastic precursor

mtKAS

105640881

XP_012080672.1

Beta-ketoacyl-ACP synthase, mitochondrial

FATA

105644367

NP_001292940.1

FATA, oleoyl-acyl carrier protein thioesterase, chloroplastic

FATB1

105639285

XP_012078682.1

Palmitoyl or stearoyl acyl-ACP thioesterase, chloroplastic

FATB-Like1

105636910

NP_001292931.1

Palmitoyl acyl-ACP thioesterase, chloroplastic-like

FATB-Like2

105634942

XP_020535202.1

Palmitoyl acyl-ACP thioesterase, chloroplastic-like

FATB-Like3

105634943

NP_001292949.1

Palmitoyl acyl-ACP thioesterase, chloroplastic-like

SAD5a

105644924

XP_012085823.1

Stearoyl-[acyl-carrier-protein] 9-desaturase 5, chloroplastic

SAD5b

105644925

NP_001295684.1

Acyl-[acyl-carrier-protein] desaturase, chloroplastic-like

SAD6

105629158

XP_012066083.1

Stearoyl-[acyl-carrier-protein] 9-desaturase 6, chloroplastic

SAD7

105628257

NP_001292942.1

Acyl-[acyl-carrier-protein] desaturase 7, chloroplastic

FAD2–1

105646514

NP_001295707.1

Omega-6 fatty acid desaturase, endoplasmic reticulum isozyme 2

FAD2–2

105650891

XP_012093240.1

Bifunctional desaturase/conjugase FADX

FAD3

105630235

NP_001292929.1

Omega-3 fatty acid desaturase, endoplasmic reticulum-like

FAD4

105632931

XP_012070789.1

Fatty acid desaturase 4, chloroplastic

FAD6

105633504

NP_001292968.1

Omega-6 fatty acid desaturase, chloroplastic

FAD8a

105646096

NP_001292928.1

Omega-3 fatty acid desaturase, chloroplastic

FAD8b

105646580

NP_001295746.1

Omega-3 fatty acid desaturase, chloroplastic-like

FAD8c

105631214

XP_012068645.1

Omega-3 fatty acid desaturase, endoplasmic reticulum

LACS1

105642647

XP_012082936.1

Long-chain acyl-CoA synthetase 1

LACS2

105631770

XP_012069337.1

Long-chain acyl-CoA synthetase 2

LACS4a

105645675

XP_012086723.1

Long-chain acyl-CoA synthetase 4

LACS4b

105645676

XP_012086726.1

Long-chain acyl-CoA synthetase 4

LACS6

105632205

XP_012069918.1

Long-chain acyl-CoA synthetase 6, peroxisomal

LACS7

105647541

XP_012089058.1

Long-chain acyl-CoA synthetase 7, peroxisomal

LACS8

105641541

XP_012081513.1

Long-chain acyl-CoA synthetase 8

LACS9

105638933

XP_012078234.1

Long-chain acyl-CoA synthetase 9, chloroplastic

ACCase catalyzes the first step of FA biosynthesis, which comprises the following four subunits: a carboxyltransferase α-subunit (CTα) encoded by accA, a biotin carboxyl carrier protein (BCCP) encoded by accB, a biotin carboxylase (BC) encoded by accC, and a carboxyltransferase β-subunit (CTβ) encoded by accD (Gu et al. 2011). In Jatropha, single copies of ACC1-LIKE, accA, accC, and accD were each found. The ACC1-LIKE encodes a cytosolic ACCase. There are two accB genes, which are designated as accB1 and accB2, with accB1 encoding ACC-BCCP 1 and accB2 encoding ACC-BCCP 2. ACC-BCCP 1 has two isoforms, which are formed by mRNA alternative splicing. So, there are three ACC-BCCP proteins in Jatropha. During seed development, ACC1-LIKE was highly expressed during late stages (40 and 50 DAP), while its expression levels were low at early stages (Xu et al. 2011). Consistently, accA, accB, and accD were predominantly expressed at 42 DAP, while accC was expressed at low levels during whole seed development. In addition, accD was also highly expressed in the leaves (Gu et al. 2011). In the second reaction, a single MCAT was found in Jatropha. MCAT began to be expressed at 14 DAP, and its expression levels gradually reduced down to the minimum at 42 DAP, but it increased sharply in the mature seeds (56 DAP) (Gu et al. 2012).

KAR, HAD, and EAR are involved in the formation of common intermediates in FA biosynthesis. KAR and KAR-LIKE were found in Jatropha. KAR was significantly expressed during seed development except in mature seeds (Gu et al. 2012). A single HAD started being expressed at 14 DAP and reached its maximum at 28 DAP, and its levels decreased gradually in the later stages (Gu et al. 2012). Two EAR genes (EAR1 and EAR2) are present in Jatropha. EAR2 encoded by EAR2 has two isoforms, which are formed by mRNA alternative splicing. The EAR1 was primarily expressed during the mid to late stages (28–42 DAP) (Gu et al. 2012).

The KAS gene family includes KAS I, KAS II, and KAS III. Two KAS I genes (KAS I-1 and KAS I-2) and single copies of KAS II, KAS III, and mtKAS were found. KAS I and KAS II are responsible for the synthesis of palmitic acid (16:0) and stearic acid (18:0), respectively. KAS III is involved in controlling the initial reaction to form C4:0-ACP. The strongest expression of KAS I was detected at mid stage (28 DAP) (Gu et al. 2012), which may lead to a high content of palmitic acid before 36 DAP (Sinha et al. 2015). KAS II was predominantly expressed at 42 DAP (Gu et al. 2012), resulting in the highest content of stearic acid exhibited at that stage (Sinha et al. 2015). By contrast, KAS III was constitutively expressed in Jatropha (Li et al. 2008), and its expression levels during seed development were the strongest at the mid-stage (Gu et al. 2012).

SAD is responsible for the synthesis of oleic acid (18:1Δ9), which is the first desaturation step in FA biosynthesis. We found four SAD genes, which were designated as SAD5a, SAD5b, SAD6, and SAD7 according to their homologs in A. thaliana. SAD was predominantly expressed in mature seeds (56 DAP) (Gu et al. 2012), which is consistent with the high amount of oleic acid noted at the same stage (Sinha et al. 2015). The next desaturations rely on fatty acid desaturase (FAD). Oleic acid (18:1Δ9) as a substrate is catalyzed by FAD2 or FAD6 to form linoleic acid (18:2Δ9,12), which is further desaturated by FAD3 or FAD8 to produce linolenic acid (18:2Δ9,12,15). Only two FAD2-1 and FAD2-2 and a single FAD6 genes were identified for FAD2 and FAD6, respectively. The expression levels of these genes were detected at early stages up to the maximum at the mid stages and then subsequently reduced (Gu et al. 2012). A single FAD4 was identified, and its substrate remains unknown. There are four FAD genes, FAD3, FAD8a, FAD8b, and FAD8c, which belong to the omega-3 fatty acid desaturase family.

FATA and FATB encode two distinct acyl-ACP thioesterases that terminate FA chain elongation. A single FATA gene encodes the FATA enzyme, which catalyzes oleoyl-ACP, releasing free oleic acid. The expression levels of FATA increased gradually during seed development up to the maximum at 42 DAP, but they were reduced sharply in mature seeds. FATB encodes FATB, which prefers the saturated palmitoyl-ACP (16:0) and stearoyl-ACP as substrates. The four FATB genes FATB1, FATB-Like1, FATB-Like2, and FATB-Like3 were found. The FATB1 expression levels increased during later developmental stages (Wu et al. 2009). The free FAs released from acyl-ACPs are converted to acyl-CoAs by LCACS. Here, we identified the following eight LCACS genes: LCACS1, LCACS2, LCACS4a, LCACS4b, LCACS6, LCACS7, LCACS8, and LCACS9. The expression pattern of LCACS is almost the same as that of HAD (Gu et al. 2012).

10.4 Expression Analysis of TAG Biosynthetic Pathway Genes During Seed Development

Using the same approach, we found genes encoding the key enzymes involved in Jatropha TAG assembly, and they are listed in Table 10.2.
Table 10.2

Summary of triacylglycerol (TAG) biosynthetic genes in J. curcas

Gene

Gene ID

Protein ID

Annotation

GPAT1

105629551

XP_012066550.1

Glycerol-3-phosphate acyltransferase 1

GPAT2a

105638856

XP_012078122.1

Probable glycerol-3-phosphate acyltransferase 2

GPAT2b

105638854

XP_012078119.1

Probable glycerol-3-phosphate acyltransferase 2

GPAT3a

105642646

XP_012082935.1

Probable glycerol-3-phosphate acyltransferase 3

GPAT3b

105647808

XP_012089426.1

Probable glycerol-3-phosphate acyltransferase 3 isoform X1

XP_020540395.1

Probable glycerol-3-phosphate acyltransferase 3 isoform X2

GPAT5

105647393

XP_012088851.1

Glycerol-3-phosphate acyltransferase 5

GPAT6a

105646620

XP_012087892.1

Glycerol-3-phosphate 2-O-acyltransferase 6

GPAT6b

105642423

XP_012082634.1

Glycerol-3-phosphate 2-O-acyltransferase 6

GPAT6c

105649803

XP_012091984.1

Glycerol-3-phosphate 2-O-acyltransferase 6

GPAT8

105642972

XP_012083373.1

Probable glycerol-3-phosphate acyltransferase 8

GPAT9

105630072

NP_001295680.1

Glycerol-3-phosphate acyltransferase 9

GPAT-LIKE

105640970

XP_012080786.1

Glycerol-3-phosphate acyltransferase, chloroplastic-like

LPAAT1

105633522

XP_012071517.1

Lysophosphatidic acid acyltransferase 1, chloroplastic

LPAAT2a

105634720

NP_001295696.1

Lysophosphatidic acid acyltransferase 2

LPAAT2b

105648809

XP_012090707.1

Lysophosphatidic acid acyltransferase PLS1

LPAAT4

105633742

XP_012071774.1

Probable lysophosphatidic acid acyltransferase 4

LPAAT5

105631921

XP_012069535.1

Probable lysophosphatidic acid acyltransferase 5

LPAAT-LIKE

105632950

XP_012070810.1

Lysophosphatidic acid acyltransferase isoform X1

XP_012070815.1

Lysophosphatidic acid acyltransferase isoform X2

PAP2a

105636922

NP_001295641.1

Phosphatidic acid phosphatase 2

PAP2b

105638770

XP_012078018.1

Phosphatidic acid phosphatase 2 isoform X1

XP_020536804.1

Phosphatidic acid phosphatase 2 isoform X2

XP_012078019.1

Putative phosphatidic acid phosphatase 2, chloroplastic isoform X3

PAP2c

105636725

XP_012075457.1

Phosphatidic acid phosphatase 2

PAP3

105636923

XP_012075719.1

Phosphatidic acid phosphatase 3 isoform X1

XP_012075720.1

Putative phosphatidic acid phosphatase 3, chloroplastic isoform X2

DGAT-1

105637897

NP_001292926.1

Diacylglycerol O-acyltransferase 1

DGAT-2

105646335

NP_001292973.1

Diacylglycerol O-acyltransferase 2

DGAT-3

105642702

XP_012083005.1

Diacylglycerol O-acyltransferase 3, cytosolic

PDAT1

105631411

XP_012068907.1

Phospholipid:diacylglycerol acyltransferase 1

PDAT1-LIKE

105637933

XP_020536294.1

Phospholipid:diacylglycerol acyltransferase 1-like isoform X1

XP_020536295.1

Phospholipid:diacylglycerol acyltransferase 1-like isoform X2

PDAT2

105637340

XP_012076169.2

Putative phospholipid:diacylglycerol acyltransferase 2

PDCT

105634846

XP_012073167.1

Phosphatidylcholine:diacylglycerol cholinephosphotransferase

At the beginning of TAG assembly, GPAT catalyzes glycerol-3-phosphate, the precursor of the glycerol backbone, to LPA. There are 12 GPAT genes in Jatropha, namely, GPAT1, GPAT2a, GPAT2b, GPAT3a, GPAT3b, GPAT5, GPAT6a, GPAT6b, GPAT6c, GPAT8, GPAT9, and GPAT-LIKE. The predominant expression levels of GPAT were detected at the early to mid stages (14–28 DAP) (Gu et al. 2012) because this enzyme is involved in the first step of TAG assembly. LPAAT and PAP are involved in the formation of intermediates. The six LPAAT genes LPAAT1, LPAAT2a, LPAAT2b, LPAAT4, LPAAT5, and LPAAT-LIKE were identified. The expression patterns of LPAAT1and LPAAT2 are similar. They were expressed during seed development, with the highest levels at mid stage. LPAAT5 was also highly expressed at the mid stage, but the levels at other stages were very low. By contrast, the expression of LPAAT4 was increased gradually from the early stages to maturity. Four PAP genes, PAP2a, PAP2b, PAP2c, and PAP3, were identified. PAP started being expressed at the early stage and reached its maximum level at 28 DAP and then subsequently decreased considerably in the later stages (Gu et al. 2012).

DGAT catalyzes the final step of TAG biosynthesis, and its level is given with respect to TAG accumulation. We identified three DGAT genes, DGAT1, DGAT2, and DGAT3, in Jatropha. DGAT1 and DGAT2 are generally known (Hobbs et al. 1999; Oelkers et al. 2000), whereas DGAT3 (cytoDGAT) has been identified recently and is involved in the cytosolic TAG biosynthetic pathway (Saha et al. 2006). DGAT1 incorporates the usual FAs into TAG, whereas DGAT2 prefers unusual FAs such as ricinoleate and vernolic acid. DGAT1 exhibited high expression levels at 28 and 42 DAP (Gu et al. 2012). The expression of DGAT2 also detected in leaves was much higher than in seeds (Xu et al. 2011). There have been no reports about the expression pattern of cytoDGAT in Jatropha thus far. In peanuts, AhDGAT (cytoDGAT), which prefers oleoyl-CoA as the acyl donor, was detected only in immature seeds (Saha et al. 2006).

To transfer PUFAs-PC into TAG, PDAT and PDCT mediated the process in two distinct ways. The three PDAT genes (PDAT1, PDAT1-LIKE, and PDAT2) and a single PDCT were found. High expression levels of PDAT were found in leaves and in the late developmental stages of the seeds (Xu et al. 2011). Studies on Jatropha PDCT are lacking. In flax, two PDCT genes, LuPDCT1 and LuPDCT2, displayed similar expression profiles, which were expressed at very low levels in vegetative and floral tissues but at high levels in the mid to late stages of embryo development (Wickramarathna et al. 2015). The seed-specific expression of LuPDCT1 and LuPDCT2 in A. thaliana resulted in increases in C18-PUFAs, with an attendant decrease in the oleic acid content. Therefore, to increase the proportion of oleic acid in Jatropha, which helps to improve the biodiesel quality, a reduction in the PDCT expression would be a suitable approach.

Altogether, the FA and TAG biosynthetic genes that we identified here can facilitate an understanding of the oil biosynthesis mechanisms in Jatropha.

10.5 Genetic Engineering of FA Composition and TAG Accumulation in Jatropha

Despite the high potential of Jatropha, an available elite germplasm with desirable traits for this plant is still under selective breeding at present, and thus, unreliable oil yields and quality limit its use in the biodiesel industry. Therefore, genetic improvement has become an imperative for Jatropha breeding, especially Sood and Chauhan (2015) found that genes from FA and TAG biosynthetic pathway were expressed at a higher level in accessions with large oil contents than in accessions with low oil contents. The understanding of spatiotemporal regulation of the genes involved in the process of oil biosynthesis in Jatropha is essential for the improvement of oil production from Jatropha.

As we mentioned above, biodiesel derived from oil with a high monounsaturated FA content has excellent properties with respect to the ignition quality, NOx emissions, and oxidative stability. However, Jatropha oil contains 30–52% PUFAs (primarily linoleic acid), which negatively impacts its biodiesel quality, although FA unsaturation relieves the solidification of fuel at a cold temperature. One available approach is to reduce the conversion of oleic acid to linoleic acid. FAD2 is the key enzyme responsible for linoleic acid production in plants. Qu et al. (2012) isolated two FAD2 genes from Jatropha and downregulated the expression of JcFAD2-1 in a seed-specific manner through RNA interference (RNAi). The resulting transgenic plants exhibited a significant increase in oleic acid (>78%) and a corresponding decrease in PUFAs (<3%) in the seed oil, while the control Jatropha contained approximately 37% oleic acid and 41% PUFAs. As a result, the oil of the transgenic Jatropha produced a cetane number as high as 60.2, which is similar to the required cetane number (CN) for conventional diesel fuels (60) in Europe. It is well-known that the CN value is perhaps the most important factor for biodiesel use because it indicates the ignition quality of diesel fuels and also negatively correlates with the NOx emissions (McCormick et al. 2001). The higher the CN, the shorter the ignition delay time and the lower are the NOx emissions. The CN value can be increased by increasing the chain length and the saturation level of FA (Vaknin et al. 2011). KAS II in FA biosynthesis catalyzes the conversion of palmitoyl-ACP (16:0-ACP) to stearoyl-ACP (18:0-ACP). The overexpression of the Jatropha KAS II (JcKASII) gene under the CaMV35S promoter in A. thaliana resulted in decreases in the C16:0 and increases in the C18:0 in transgenic plants (Wei et al. 2012). This result indicates that JcKASII could promote the conversion of C16:0 into C18:0 FA and increase its accumulation in Jatropha seed oil. As outlined above, a FATB gene designated as JcFATB1 was isolated from Jatropha. When the seed-specific expression of JcFATB1 was induced in A. thaliana by transgenesis, it led to a three- to fourfold increase in accumulated palmitic acid and moderate increases in other saturated FAs, along with reductions in the unsaturated FAs (Wu et al. 2009). This result confirms that JcFATB1 has a higher affinity for the catalysis of saturated acyl-ACPs, especially palmitoyl-ACP. These manipulations can be performed in Jatropha to increase the chain length and saturation of FAs to improve the ignition quality and reduce NOx emissions.

In addition to modifying the FA composition, the efforts toward increasing the oil content in seeds are considerable. GPAT catalyzes the first step in TAG assembly. Two GPAT genes were isolated from Jatropha and were then characterized in A. thaliana (Misra et al. 2017). The expression levels of JcGPAT2 were higher than the JcGPAT1 levels during Jatropha seed development. The oil contents in transgenic A. thaliana overexpressing JcGPAT1 under the CaMV35S promoter or the seed-specific promoter increased by 13% and 20% more than the control, respectively. On the other hand, the oil contents in transgenic A. thaliana overexpressing JcGPAT2 under the same two promoters increased by 42% and 60%, respectively, more than the control. It appears that JcGPAT2 plays a more important role than JcGPAT1 in TAG biosynthesis. These results were consistent with the fact that the oil content in seeds of antisense transgenic lines of JcGPAT1 and JcGPAT2 under the control of the CaMV35S promoter was lower than in the control. This result indicates that increasing the intermediates in the TAG biosynthesis pathway can regulate oil accumulation. In the final step of TAG biosynthesis, the overexpression of JcDGAT1and JcDGAT2 in transgenic tobacco resulted in seed oil content increases of 32.8% and 31.8%, respectively (Xu et al. 2014). The FA composition was also different in transgenic plants modified for JcDGAT2. The proportion of linoleic acid in the JcDGAT2 transgenic lines increased significantly compared with that in the JcDGAT1 transgenic lines and the control, and correspondingly, the oleic acid significantly decreased. These results suggest that JcDGAT1 is a better choice for the improvement of oil content compared to JcDGAT2, since JcDGAT2 could entail a decrease in the oil quality. Recently, the A. thaliana DGAT1 gene AtDGAT1 was used to improve oil accumulation in Jatropha. This gene successfully increased the oil content by 20–30% in seeds and 1.5- to 2.0-fold in the leaves of Jatropha through genetic engineering and entailed a correlated increase in oleic acid and a decrease in PUFAs (Maravi et al. 2016).

Another way to accumulate oil content is by preventing the degradation of TAG by TAG lipases. During seed germination, TAG lipases hydrolyze TAG into glycerol and free fatty acids (FFAs) (Ma et al. 2017). Jatropha oil contains a high FFA level up to 27% (Kywe and Oo 2009; Kim et al. 2014). However, the FFA content must be as low as possible to avoid acidic transesterification and improve the biodiesel storage stability. The sugar-dependent 1(SDP1) gene encodes SDP1, which is specifically responsible for the first step in TAG degradation. The downregulation of JcSDP1 expression in Jatropha was achieved by RNAi technology, and the JcSDP1-RNAi transgenic plants accumulated 13–30% higher total lipids, along with a 7% compensatory decrease in the protein content, than the control (Kim et al. 2014). Moreover, the FFA content in the seed oil decreased from 27% in the control plants to 8.5% in the transgenic plants. The considerable reduction in FFA resulting from this approach will be of great value when dealing with the high FFA problem of oil from Jatropha for biodiesel production.

In addition to genes that encode the key enzymes involved in oil biosynthesis, transcription factors (TFs) are also involved in regulating lipid biosynthesis. However, in contrast to the many functional data on the oil biosynthetic pathway in Jatropha, TFs have been rarely functionally characterized. At present, only a JcWRI1 encoding WRINKLED1 (WRI1), which is a member of the large plant-specific APETALA2 (AP2) family of TFs involved in controlling seed oil biosynthesis, was characterized in Jatropha. The overexpression of JcWRI1 increased the seed oil contents in both transgenic A. thaliana and Jatropha (Ye et al. 2018). In contrast to oil biosynthesis genes, increase in seed oil synthesis induced by the JcWRI1 in transgenic lines did not appear to be correlated with a modified FA composition; however, this functional difference may depend on TF and target genes that are considered.

10.6 Conclusions and Future Perspective

Recently, increasing the oil content in non-seed tissues has been proposed as a novel platform for meeting global vegetable oil production needs. The TAG accumulation in vegetative tissues has the potential to outyield current oilseed crops, especially when engineered in high-biomass crops (Ohlrogge and Chapman 2011; Weselake 2016). In Jatropha, excessive vegetative growth is one reason for low oil yields. Increasing the oil content in leaves is one way to improve the oil yield. Maravi et al. (2016) reported that the overexpression of AtDGAT1 effectively increased the oil content by 1.5- to 2.0-fold in the leaves of Jatropha. In addition, the thick pericarps could be another candidate platform to test by directing oil biosynthesis genes using pericarp-specific promoters. However, the increased levels of TAG in non-seed tissues are still lower than they are in seeds. This pattern can largely be attributed to the single-gene strategy of most studies. To address this problem, a variety of combinatorial metabolic engineering approaches have been established to increase the storage of lipids through the de novo fatty acid (“Push”) and TAG assembly (“Pull”) pathways while stabilizing the cytosolic lipid droplets (“Package”) and minimizing lipid turnover (“Protect”). With this strategy, transgenic tobacco accumulated until 30–33% TAG in the leaves (Vanhercke et al. 2017), and there was a greater than 100-fold increase in TAG accumulation, to levels up to 3.3% of the tuber dry weight of transgenic potatoes (Liu et al. 2017). Because a great deal of functional information about oil biosynthesis in Jatropha has been revealed, the combinatorial metabolic engineering approach would be optimal for Jatropha oil improvement.

Notes

Acknowledgments

This work was supported by funding from the Natural Science Foundation of Yunnan Province (2016FB048), the West Light Foundation and the Plant Germplasm Innovation Program of the Chinese Academy of Sciences (CAS, kfj-brsn-2018-6-008), and the CAS 135 program (2017XTBG-T02). The authors gratefully acknowledge the Central Laboratory of the Xishuangbanna Tropical Botanical Garden for providing research facilities.

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© Springer Nature Singapore Pte Ltd. 2019

Authors and Affiliations

  1. 1.CAS Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical GardenChinese Academy of SciencesMenglaChina

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