As mentioned above, FATB is a key enzyme in FA biosynthesis that catalyzes the removal of ACP from 16:0-ACP and 18:1-ACP to generate NEFAs. The knockdown of
GmFATB1 expression decreases UFA content in soybean seeds
[27]. Similarly, the expression of
BnFATB in rapeseed appears to be highly and positively correlated with seed oil content (SOC), as evidenced by an analysis integrating quantitative trait loci (QTLs) from nine different populations
[28].
LPAT is one of the key enzymes in the Kennedy pathway that converts acyl-CoA to PA. The heterologous expression of a yeast (
Saccharomyces cerevisiae) sn-2 acyltransferase gene in rapeseed resulted in increased FA content by 8–22%
[29]. Cloning and spatiotemporal expression of
AhLPAT2 from peanut (
Arachis hypogaea) showed that increased expression of this gene was closely related to seed oil content; moreover, heterologous expression of
AhLPAT2 in Arabidopsis from a seed-specific promoter significantly increased SOC
[30].
DGAT is a key enzyme that acylates DAG at the sn-3 position. Seed-specific overexpression of Arabidopsis
DGAT in wild-type plants resulted in an increase in SOC and seed weight from 29% to 35%, along with an increase in the average thousand-seed weight from 19 mg in non-transgenic plants to 23 mg in the overexpression lines
[31], which was consistent with the results of heterologous expression of
DGAT1 from sesame (
Sesamum indicum) in Arabidopsis
[32].
GDSL esterase is a type of hydrolase that is widely involved in various physiological activities in plants and is important in oil metabolism. The name of the enzyme derives from its conserved domain (GDSL), where G, D, and S represent glycine (Gly), aspartate (Asp), and serine (Ser), respectively
[33]. Arabidopsis has five GDSL-like lipase genes (also called
SEED FATTY ACID REDUCER [
SFAR] genes) that decrease SOC by acting downstream of the gibberellin signaling (GA) pathway since GA may affect the seed FA content via SFARs
[34]. The overexpression or knockout of each
SFAR gene significantly lowered or increased SOC, respectively, and altered FA composition. The heterologous expression of a
GDSL gene from oil palm (
Elaeis guineensis) in Arabidopsis resulted in a 9.5% increase in total FA content compared to the wild type.
In addition to these important pathway enzymes and their encoding genes directly involved in de novo FA biosynthesis and TAG assembly, recent studies have identified several genes annotated as participating in carbon source provisioning and photosynthetic pathways that may also be involved in regulating SOC
[35]. For instance, trehalose-6-phosphate (T6P), a metabolic precursor of sucrose and a key signaling molecule for plants to respond to carbon availability and regulate growth and development, also regulates FA biosynthesis by inhibiting sucrose non-fermenting 1-related kinase 1 (SnRK1). Indeed, incubation of rapeseed suspension cells in T6P-containing medium or heterologous expression of
T6P synthase from
Escherichia coli in
Nicotiana benthamiana significantly increased FA biosynthesis rates
[36]. CALCINEURIN B-LIKE PROTEIN-INTERACTING PROTEIN KINASEs (CIPKs) are a family of energy-signaling protein kinases in plants. Relative to non-transgenic plants, the overexpression of
BnCIPK9 during seed development in transgenic rapeseed lowered oil biosynthesis
[37].
2.2. Transcription Factors Involved in Regulation
Looking for transcription factors (TFs) that regulate the expression of multiple genes is also an effective method to increase SOC
[38]. Currently, well-studied TFs in plants mainly include WRINKLED1 (WRI1), LEAFY COTYLEDON1 (LEC1), LEC2, LEC1-LIKE (LIL), FUSCA3 (FUS3), and ABSCISIC ACID INSENSITIVE3 (ABI3).
WRI1 is an APETALA2 (AP2)/ETHYLENE-RESPONSIVE ELEMENT BINDING PROTEIN (EREBP) that regulates genes involved in glycolysis and sucrose entry into TAG
[39]. WRI1 was discovered in Arabidopsis in 1998 as a mutant affecting seed storage accumulation. Compared with the wild type, the mutant was unable to convert glucose and sucrose into precursors for fatty acid synthesis during seed development and reduced the activity of several glycolytic enzymes such as hexokinase and phosphofructokinase, resulting in an 80% reduction in SOC
[40]. The oil content of
N. benthamiana leaves expressing
WRI1 from castor bean showed a 4.3–4.8 times increase compared to the control group. The Arabidopsis
wri1 loss-of-function mutant was almost completely rescued by the strong expression of castor bean
WRI1 from a seed-specific promoter, resulting in a total FA content close to that of non-transgenic seeds
[41]. LEC1 is a member of the nuclear factor-YB (NF-YB) family of TFs, and the individual overexpression of Arabidopsis
LEC1 or
L1L, or the overexpression of their homologous genes
BnLEC1 or
BnL1L from rapeseed, resulted in significantly increased FA levels in transgenic Arabidopsis
[42][43].
FUS3, another member of the plant-specific B3 domain family, plays an important role in recognizing and binding to the RY element CATGCA, which is found in the promoters of many genes.
fus3 mutants in rapeseed showed a lower total SOC than the wild type
[44]. Inducible expression of Arabidopsis
FUS3 increased the oil content of Arabidopsis seedlings to 6% of dry weight, which was more than 50-fold higher than that of non-transgenic seeds. Similarly, the inducible expression of
FUS3 in
Nicotiana tabacum L. cv Bright Yellow2 (BY2) cells increased TAG accumulation, and the co-expression of
FUS3 and
DGAT1 further increased the TAG levels to 4% of the dry weight.
ABI3 is also a member of the B3 domain TF family. The heterologous expression of
GmABI3 from soybean in Arabidopsis significantly increased TAG content and altered the FA composition of seeds. In addition,
GmABI3 expression successfully complemented the phenotype and oil content of Arabidopsis
abi3 mutant seeds
[45]. In another study, tobacco leaf protoplasts were prepared for transfection with effector constructs overexpressing
WRI1 and/or
DGAT1 alone or together with one of the master regulators
ABI3,
FUS3,
LEC1, or
LEC2. The co-expression of
FUS3 and
ABI3 with
WRI1 and
DGAT1 significantly increased the content of total non-polar lipids of the protoplasts. Notably, the expression of
ABI3 overexpression alone resulted in the highest accumulation of total non-polar lipid in protoplasts. Furthermore, in contrast to
LEC2, the co-expression of
ABI3 further increased the lipid content of protoplasts transiently expressing
WRI1 and
DGAT1 [46].
MYB TFs comprise one of the largest gene families in plants and are associated with the regulation of plant growth and development, metabolism, morphology, and cellular patterning
[47]. MYB92 can directly bind to the promoter of and activate the transcription of
Biotin carboxyl carrier protein 2 (
BCCP2), which encodes a component of the FA biosynthetic pathway. The overexpression of Arabidopsis
MYB92 in
N. benthamiana induced the expression of FA biosynthesis genes, resulting in the accumulation of various types of lipids
[48]. DECREASE WAX BIOSYNTHESIS 2 (DEWAX2) is a member of the AP2-EREBP TF family in Arabidopsis and negatively regulates epidermal wax deposition by directly binding to the promoters of
LACS1,
LACS2,
KCSII, and
ECERIFERUM1 (
CER1) to inhibit their expression
[4].
Growth-regulating factor 2a (
BnRGF2a) was identified in rapeseed based on its differential expression between two rapeseed lines with differing seed oil production. Arabidopsis lines overexpressing
BnGRF2 showed improved seed quality and oil content. In addition, transcriptome analysis revealed that some genes related to cell proliferation, photosynthesis, and oil biosynthesis were upregulated in these overexpression lines, suggesting that cell number and photosynthesis are linked to the observed increase in seed weight and oil content
[49].
2.3. Advances in Multi-Omics Studies of Oilseed Crops
With the abundance of high-throughput sequencing information, the mining of large amounts of biological data is becoming increasingly popular and feasible. The availability of genomic, transcriptomic, and other multi-omics data provides a powerful avenue to unravel the detailed regulation of lipid metabolism
[50][51]. From an omics perspective, the following aspects of lipid metabolism in oilseed crops are currently of interest (
Figure 3).