**3. Understanding metabolic routes in oilseed crops**

Oilseed plants represent an important renewable source of fatty acids because they accumulate them in the form of triacylglycerol (TAG) as major storage components in seeds [9]. In plants, the reactions for de novo fatty acid synthesis begin in plastids [10] and then exported to the cytoplasm following two inter-related metabolic pathways: an acyl-CoA-dependent pathway and an acyl-CoA-independent pathway [11].

In the dependent pathway, commonly known as the Kennedy pathway, the priming and elongation of nascent acyl chains requires acetyl- and malonyl-CoA, respectively, as direct precursors up to eighteen carbons in length [12]. In this pathway, the glycerol-3-phosphate acyltransferase (G3PAT) is the first enzyme that catalyzes the transfer of a fatty acid to glycerol-3-phosphate (G3P) to form lysophosphatidic acid (LPA). Then, the LPA is acylated by the lysophosphatidic acid acyltransferase (LPAAT) to yield phosphatidic acid (PA). Next, PA is dephosphorylated by the phosphatidic acid phosphatase (PAP) to form diacylglycerol (DAG) and finally, a diacylglycerol acyltransferase (DGAT) catalyzes the acylation of DAG to the production of TAG [13]. In the acyl-CoA-independent pathway, an alternative enzyme is used for the final acylation reaction, termed phospholipid:diacylglycerol acyltransferase (PDAT). PDAT directly transfers an acyl group from phosphatidylcholine (PC) to DAG, producing TAG [14].

Desaturation steps for fatty acids are catalyzed by plastidial stearoyl-acyl carrier protein (ACP) desaturases. After termination, free fatty acids are activated to CoA esters, exported from the plastid, and assembled into glycerolipids at the endoplasmic reticulum (ER) [9]. In addition, further modifications (desaturation, hydroxylation, elongation, etc.) occur in the ER while acyl chains are esterified to glycerolipids or CoA [15]. The low polarity of TAG is believed to result in the accumulation of this lipid between bilayer leaflets leading to the budding of storage organelles termed oil bodies [9]. The accumulation of hydroxy fatty acids depends on many factors, including the performance of the desaturases and efficient channeling of hydroxy fatty acids into storage triacylglycerols [16]. Fatty acid dehydrogenase (FAD) catalyzes the desaturation reaction, leading to the formation of unsaturated FA. Interestingly, studies have revealed that some desaturase enzymes (such as the FAD2 and FAD3 genes) could be regulated at the transcriptional level or at the post-translation level in response to low-temperature induction in model plants [17, 18]. Other important enzyme is FAH12 which belongs to a large family of fatty acid modification enzymes that are related to the *Arabidopsis* oleate D12-desaturase (FAD2) protein, which is responsible for the synthesis of polyunsaturated fatty acids [19].

High-quality RNAseq data have allowed the identification and an accurate quantification of expression of transcription factors and key genes related with lipid metabolic pathways in soybean [20], *Jatropha curcas* [21], *Arabidopsis* [22], peanut [23] and castor bean [24]. In castor, comparison of expression between tissues allowed identification of candidate genes which may be important for triricinolein synthesis in seed in addition to the oleate-12 hydroxylase. Moreover, in purified endoplasmic reticulum from castor endosperm, the site of TAG synthesis, less than 10 genes were found being differentially expressed. Two of these genes, the DGAT2 and PDAT1A, were cloned in transgenic plants expressing the oleate-12 hydroxylase, increasing 18:1-OH incorporation into seed oils and also the expression of additional genes [24].

thus increasing raw materials available for oleochemistry. In this perspective, it has came growing research efforts of scientist around the world seeking to expand the knowledge barrier on oilseed crops. **Figure 2** shows continuous growth in the number of scientific publications in this field, records subtracted from literature databases: Scopus, Web of Science, and ScienceDirect.

Oilseed plants represent an important renewable source of fatty acids because they accumulate them in the form of triacylglycerol (TAG) as major storage components in seeds [9]. In plants, the reactions for de novo fatty acid synthesis begin in plastids [10] and then exported to the cytoplasm following two inter-related metabolic pathways: an acyl-CoA-dependent

In the dependent pathway, commonly known as the Kennedy pathway, the priming and elongation of nascent acyl chains requires acetyl- and malonyl-CoA, respectively, as direct precursors up to eighteen carbons in length [12]. In this pathway, the glycerol-3-phosphate acyltransferase (G3PAT) is the first enzyme that catalyzes the transfer of a fatty acid to glycerol-3-phosphate (G3P) to form lysophosphatidic acid (LPA). Then, the LPA is acylated by the lysophosphatidic acid acyltransferase (LPAAT) to yield phosphatidic acid (PA). Next, PA is dephosphorylated by the phosphatidic acid phosphatase (PAP) to form diacylglycerol (DAG) and finally, a diacylglycerol acyltransferase (DGAT) catalyzes the acylation of DAG to the production of TAG [13]. In the acyl-CoA-independent pathway, an alternative enzyme is used for the final acylation reaction, termed phospholipid:diacylglycerol acyltransferase (PDAT). PDAT directly transfers an acyl group from phosphatidylcholine (PC) to DAG, producing TAG [14]. Desaturation steps for fatty acids are catalyzed by plastidial stearoyl-acyl carrier protein (ACP) desaturases. After termination, free fatty acids are activated to CoA esters, exported from the plastid, and assembled into glycerolipids at the endoplasmic reticulum (ER) [9]. In addition, further modifications (desaturation, hydroxylation, elongation, etc.) occur in the ER while acyl chains are esterified to glycerolipids or CoA [15]. The low polarity of TAG is believed to result in the accumulation of this lipid between bilayer leaflets leading to the budding of storage organelles termed oil bodies [9]. The accumulation of hydroxy fatty acids depends on many factors, including the performance of the desaturases and efficient channeling of hydroxy fatty acids into storage triacylglycerols [16]. Fatty acid dehydrogenase (FAD) catalyzes the desaturation reaction, leading to the formation of unsaturated FA. Interestingly, studies have revealed that some desaturase enzymes (such as the FAD2 and FAD3 genes) could be regulated at the transcriptional level or at the post-translation level in response to low-temperature induction in model plants [17, 18]. Other important enzyme is FAH12 which belongs to a large family of fatty acid modification enzymes that are related to the *Arabidopsis* oleate D12-desaturase (FAD2) protein, which is responsible for the synthesis of polyunsaturated fatty acids [19].

High-quality RNAseq data have allowed the identification and an accurate quantification of expression of transcription factors and key genes related with lipid metabolic pathways in soybean [20], *Jatropha curcas* [21], *Arabidopsis* [22], peanut [23] and castor bean [24]. In castor,

**3. Understanding metabolic routes in oilseed crops**

pathway and an acyl-CoA-independent pathway [11].

298 Advances in Seed Biology

Gathering the RNAseq information and advances in plant transformation technology is possible now engineering plants for the production of oilseed fatty acids. In a remarkable research, Arabidopsis plant was modified introducing a fatty acid hydroxylase from castor plant, it leading to produce some ricinoleic acid and an unusual fatty acid in the seed [25]. Reactions in triacylglycerol biosynthesis have also been manipulated to increase seed oil content. Studies have suggested that the level of DGAT activity during seed development may have a substantial effect on the flow of carbon into seed oil. Thus, overexpression of cDNAs encoding either *Arabidopsis* DGAT1 or a variant of *B. napus* DGAT1 during seed development in *B. napus* resulted in increased seed oil content under both greenhouse and field conditions [26].

However, in the last decade, the scientists have realized that the manipulation of single genes only contribute with limited value to change the metabolic pathways. Nowadays, there are strategies focused on more complex approaches involving simultaneous overexpression or suppression of multiple genes to achieve optimal metabolic flux [27]. Understanding a metabolic network would facilitate the production of natural products and the synthesis of novel molecules in a predictable and useful manner [16]. For this reason, the metabolic engineering in oilseed plants has attracted industrial and academic researchers in the last decade.
