**5. Novel strategies focused on carbon flux in seeds**

In production systems that utilize microorganisms and cultured cells for metabolic engineer‐ ing, individual cells synthesize organic compounds, providing a limitless source of carbon in the medium. By contrast, the seed, representing only one of many plant organs, synthesizes storage reserves using a limited carbon source provided by photosynthesis. This limited carbon source is thought to represent another limiting factor to the use of seeds as production systems, in addition to the limited activities of metabolic enzymes. To utilize seeds for oil production, it is important to obtain a comprehensive understanding of the factors that regulate TAG production, including carbon flow, metabolite transport, TAG metabolism, compartmentation, competition between other reserves, and so on. Recent studies identifying other factors that limit oil production in seeds have opened up the possibility of developing novel strategies for improving seed oil contents.

#### **5.1. Effective utilization of "the window" during seed development**

Fertilized embryos grow into mature seeds through sequential development. Seed storage oils are produced only during a short period of seed development; high TAG biosynthesis activity only occurs for a short period of time. The master transcription factor of TAG biosynthesis, WRINKLED1 (hereafter referred to as WRI1), is expressed during this short period and induces the expression of genes related to fatty acid biosynthesis [50–54]. Although WRI1 positively regulates TAG biosynthesis, the overexpression of *WRI1* fails to increase TAG contents in seeds [51, 53]. These findings suggest that, in order to increase seed TAG contents, it is necessary to reconsider the timing and duration of *WRI1* expression. Arabidopsis seeds initially accumulate TAG, followed by proteins [1]. A detailed analysis of seed development revealed that there is a time lag between the termination of TAG biosynthesis and the initiation of protein biosyn‐ thesis, i.e., there is a "window" period in the middle phase of seed development during which the activities of the TAG and protein synthesis pathways are low (**Figure 5**) [54]. Additionally, strong expression of *WRI1* during this window extends the duration of TAG biosynthesis and increases TAG contents in seeds [54], indicating that *WRI1* expression during this window effectively induces the TAG biosynthesis pathway. This finding has important implications for developing novel strategies for increasing seed TAG contents. The timing and duration of TAG and seed storage protein synthesis differ greatly among plant species. Therefore, identifying the window phase and fine tuning of *WRI1* expression are essential for generating oil crops with increased TAG contents in seeds.

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.

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

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 production systems that utilize microorganisms and cultured cells for metabolic engineer‐ ing, individual cells synthesize organic compounds, providing a limitless source of carbon in the medium. By contrast, the seed, representing only one of many plant organs, synthesizes storage reserves using a limited carbon source provided by photosynthesis. This limited carbon source is thought to represent another limiting factor to the use of seeds as production systems, in addition to the limited activities of metabolic enzymes. To utilize seeds for oil production, it is important to obtain a comprehensive understanding of the factors that regulate TAG production, including carbon flow, metabolite transport, TAG metabolism, compartmentation, competition between other reserves, and so on. Recent studies identifying other factors that limit oil production in seeds have opened up the possibility of developing

Fertilized embryos grow into mature seeds through sequential development. Seed storage oils are produced only during a short period of seed development; high TAG biosynthesis activity only occurs for a short period of time. The master transcription factor of TAG biosynthesis, WRINKLED1 (hereafter referred to as WRI1), is expressed during this short period and induces the expression of genes related to fatty acid biosynthesis [50–54]. Although WRI1 positively regulates TAG biosynthesis, the overexpression of *WRI1* fails to increase TAG contents in seeds

**4.2. Suppression of TAG degradation**

**5. Novel strategies focused on carbon flux in seeds**

**5.1. Effective utilization of "the window" during seed development**

novel strategies for improving seed oil contents.

in oil seed crops.

**Figure 5.** Oil and protein biosynthesis during seed development. (A) Phases of oil and protein biosynthesis during seed development in *Arabidopsis thaliana*. (B) Schematic diagram of oil and protein biosynthesis during seed develop‐ ment in *Arabidopsis thaliana*.

#### **5.2. Concentrating the carbon source into triacylglycerol biosynthesis by reducing the levels of other organic materials**

Seeds accumulate various organic materials, including carbohydrates and proteins, in addition to TAG. Therefore, reducing carbohydrate and protein levels would help direct the carbon source into TAG biosynthesis.

#### *5.2.1. Reducing polysaccharide biosynthesis*

Seeds are generally covered with seed coats. Members of the Brassica family, including Arabidopsis, rapeseed, and so on, accumulate large amounts of mucilage consisting of pectin [55, 56]. Therefore, a considerable volume of the carbon source is consumed by mucilage production. Shen et al. reported that knockout of *GLABRA2*, which encodes a WRKY tran‐ scription factor controlling epidermal development, increases seed oil content in Arabidopsis [57]. Subsequent research demonstrated that increasing oil contents by suppressing *GLA‐ BRA2* expression reduces mucilage biosynthesis [58]. These findings indicate that reducing mucilage biosynthesis causes the carbon source to be directed into TAG biosynthesis. This transfer of the carbon source via reduction of polysaccharide levels has been verified in leaves. Starch comprises one of the main storage reserves in leaves. On the other hand, TAG biosyn‐ thesis activity is quite low in leaves. Therefore, to accumulate TAG in leaves, it is essential to both suppress starch biosynthesis and activate TAG biosynthesis. Sanjaya et al. found that overexpressing *WRI1* while suppressing the production of a small subunit of adenosine diphospho (ADP)-glucose pyrophosphorylase, a key enzyme for starch biosynthesis, signifi‐ cantly increases TAG contents in leaves [59]. These results indicate that reducing polysacchar‐ ide biosynthesis leads to the funneling of the carbon source into TAG biosynthesis, representing a possible novel strategy for increasing seed TAG contents in oil crops.

#### *5.2.2. Reducing seed storage protein biosynthesis*

As described above, oil seeds accumulate large amounts of seed storage proteins in addition to TAG. Crop breeders have reported a negative correlation between TAG and protein contents, especially in soybean [60, 61], suggesting that reducing the levels of seed storage proteins increases TAG contents in seeds. However, in Arabidopsis, knockout of genes encoding major seed storage proteins has little effect on TAG contents in seeds, although the protein content is reduced [54, 62]. These results may be due to the time lag between TAG and protein biosynthesis (**Figure 5**): little of the surplus carbon source derived from the suppression of protein biosynthesis is utilized for TAG biosynthesis because the TAG biosynthesis activity is quite low when protein synthesis is active in the late phase of seed development [54]. Therefore, simultaneously overexpressing *WRI1* and reducing protein synthesis greatly increases seed TAG contents due to effective utilization of the surplus carbon source [54]. This finding indicates that reducing protein synthesis indeed provides the surplus carbon source required for TAG production which, when combined with the simultaneous activation of TAG biosynthesis, leads to increased TAG production. This finding also demonstrates that the combination of these functional strategies has an additive effect on seed TAG content (**Figure 6**) [54, 63]. Breeding lines with reduced SSP content have been established in several crops [64, 65]. Introduction of *WRI1* into these breeding lines represents a potentially important strategy for high-oil seed production.

Designing Novel Breeding Strategies for Producing High-Oil Crops Based on a Molecular Understanding of Triacylglycerol Metabolism http://dx.doi.org/10.5772/64465 147

**Figure 6.** During the enlargement of seeds, the activation of TAG biosynthesis and the reduction of protein synthesis occur simultaneously. *FUS3pro:WRI1*; seeds from transgenic plants harboring *WRI1* under the control of the *FUS3* pro‐ moter in wild-type plants (Col-0): *WRI1* is expressed during the window phase. *FUS3pro:WRI1/12s1.4*; seeds from transgenic plants harboring *WRI1* under the control of the *FUS3* promoter in a double knockout mutant of *12S1* and *12S4*, encoding major seed storage proteins in *Arabidopsis thaliana*.
