**4. Strategies for improving oil content by modifying triacylglycerol metabolism in oil seed crops**

Since plant oils are commercially important, improving oil seed crops has long been a focus of breeders. Such breeding efforts, which began in ancient times, have led to the improvement of oil contents in several crops. In addition, the oil contents of the seeds of modern cultivars are significantly higher than those of wild species [23, 24]. Currently, many plant breeders have undertaken the challenge of further increasing seed oil contents. However, recent studies using quantitative trait loci analyses revealed that seed oil contents are controlled by many genes with additive effects [25–28], suggesting that traditional breeding methods based on cross-fertilization may be inadequate for further increasing seed oil contents.

**Figure 4.** Seeds of *Arabidopsis thaliana*. (A) Microscopic image of Arabidopsis seeds. (B) Electron microscopic image of Arabidopsis seed cells. OB: oil body, an oil storage organelle in seeds. PB: protein body, an organelle that accumulates seed storage protein.

By contrast, genetic transformation may be an effective tool for increasing seed oil contents. The model plant *Arabidopsis thaliana*, a close relative of the major oil crop *Brassica napus*, produces typical oil seeds (**Figure 4A** and **B**). The use of Arabidopsis drastically simplifies both the processes of screening mutants with abnormal phenotypes and generating transformants, in which genes of interest are introduced. The use of Arabidopsis has accelerated the process of uncovering TAG metabolic pathways in plants. In fact, most genes encoding enzymes involved in TAG metabolism have been identified based on characterization of Arabidopsis mutants defective in TAG metabolism [29, 30]. Elucidating TAG metabolic pathways has revealed the limiting factors and key enzymes in this process and has led to the development of novel strategies for improving seed TAG contents. In this section, we review important strategies for improving TAG contents in seeds based on metabolic engineering approaches.

#### **4.1. Enhancement of TAG biosynthesis**

addition to the *de novo* synthesis pathway, TAG is produced via an alternative pathway through phosphatidylcholine (hereafter referred to as PC) [15, 16]. PC, one of the main components of membrane lipids, is present in pools in the ER [16]. PC pools affect *de novo* TAG synthesis and the acyl-CoA pool in the cytosol to supply DAG as a precursor for TAG [16].

142 New Challenges in Seed Biology - Basic and Translational Research Driving Seed Technology

**Figure 3.** Triacylglycerol formation in the ER: (1) acyl-CoA:G3P acyltransferase, (2) acyl-CoA:LPA acyltransferase, (3)

Synthesized TAGs accumulate in compartments in the ER (**Figure 1C**), which are converted to vesicles through budding. The vesicles then develop into oil-accumulating organelles, i.e., oil bodies [16–19]. Oil bodies are TAG storage organelles with a single layer membrane, whose major membrane protein is oleosin [18, 20, 21]. Oleosin blocks adhesion between neighboring oil bodies, which allows small oil bodies to be packed tightly together without adhesion in the

**4. Strategies for improving oil content by modifying triacylglycerol**

Since plant oils are commercially important, improving oil seed crops has long been a focus of breeders. Such breeding efforts, which began in ancient times, have led to the improvement of oil contents in several crops. In addition, the oil contents of the seeds of modern cultivars are significantly higher than those of wild species [23, 24]. Currently, many plant breeders have undertaken the challenge of further increasing seed oil contents. However, recent studies using quantitative trait loci analyses revealed that seed oil contents are controlled by many

PA phosphatase, and (4) acyl-CoA:DAG acyltransferase.

**3.4. Triacylglycerol accumulation**

**metabolism in oil seed crops**

cells of oil seeds [22].

The TAG biosynthetic pathway, which extends across plastids, the cytosol, and the ER, involves many enzymes [30]. Additionally, DAG biosynthetic pathways include the *de novo* synthetic pathway and the phosphatidylcholine-derived pathway (hereafter referred to as PCderived pathway), whose activities greatly differ among plant species [15, 16]. Thus, increasing the activity of enzymes in the *de novo* or PC-derived pathway does not always increase seed oil contents in every plant species. On the other hand, TAG production from DAG by acyl-CoA:DAG acyltransferase is a common pathway among plants. Overexpressing acyl-CoA:DAG acyltransferase significantly increases seed TAG contents in Arabidopsis [31] and in other plants species [32–37]. These findings indicate that the conversion of DAG to TAG is one of the rate-limiting steps in the production of TAG in seeds, and they suggest that the acyl-CoA:DAG acyltransferase gene would be a promising target gene for increasing TAG contents.

#### **4.2. Suppression of TAG degradation**

Synthesized TAG is stored in oil bodies and degraded during germinative growth [17, 38, 39]. The TAG degradation pathway has also been uncovered, and most genes encoding enzymes in this pathway have been identified [17, 39]. The expression of these genes is upregulated after seed imbibition, and TAG degradation activity rapidly increases in imbibed seeds [40– 42]. These genes are also expressed during seed development in several plants [43, 44]. In fact, TAG degradation occurs in developing seeds [45–47]. Therefore, the TAG degradation pathway is activated during seed development, and seeds lose some of the TAG synthesized during seed development. This finding suggests that suppressing TAG degradation would be a promising strategy for improving seed oil contents. Oil degradation begins with TAG hydrolysis via TAG lipase. TAG lipase was genetically identified as *SUGAR DEPENDENT 1* in Arabidopsis [48] and was subsequently identified in rapeseed and Jatropha [47, 49]. Suppressing *SUGAR DEPENDENT 1* expression significantly increases seed oil contents [47, 49]. These reports indicate that suppressing TAG degradation via suppressing *SUGAR DEPENDENT 1* expression may represent an effective strategy for increasing seed oil contents in oil seed crops.
