GmMYB73 gene was identified to have variable expression levels during soybean seed development and to promote lipid accumulation in transgenic Arabidopsis. The effect of GmMYB73 on lipid accumulation may be achieved through reduction of GL2 expression, a negative regulator of oil accumulation, and then release of GL2-inhibited PLDα1 expression.
Overexpression of GmMYB73 enhanced seed size and thousand-seed-weight of Arabidopsis transgenic plants (Figure 3e). The GmMYB73 also rescued the seed phenotype and thousand-seed weight in try cpc double mutant (Figure 3). These results indicate that GmMYB73 or its homologues in Arabidopsis regulates seed development in addition to control of trichome formation. Several other genes involved in regulation of trichome formation also affect seed development. These include TRANSPARENT TESTA GLABRA2 and TRANSPARENT TESTA GLABRA1 in seed coat differentiation; gl2 mutants do not produce releasable mucilage ; GL3, EGL3, and TTG1 regulate seed coat proanthocyanidin biosynthesis [55, 56]. Some of these genes also play roles in seed size control and thousand-seed weight regulation . It should be mentioned that gl2-1 seeds (Ler ecotype) show no change in thousand-seed weight when compared to WT . In contrast, the present gl2-2 (Col ecotype) exhibited slight but significant increase in thousand-seed weight when compared to control (Figure 3e). This discrepancy may be derived from different ecotypes used and/or different storage time for seeds. There are other genes that regulate both seed development and aspects of plant growth and development. Inhibition of ethylene receptor gene OsETR2 expression in rice leads to increased thousand-grain weight and early flowering . Mutation of MHZ7/OsEIN2 affects grain shape and senescence . Ethylene and salt-induced NIMA-related kinase NEK6 enhances plant growth and seed yield in Arabidopsis; however, thousand-seed weight and seed width are reduced . Recently, two DOF genes DOF4.2 and DOF4.4 have been found to promote shoot branching but affect seed/silique development in Arabidopsis . Other genes involved in regulating seed size and/or development are reviewed in .
GmMYB73 overexpression promotes fatty acid accumulation in transgenic Arabidopsis and transgenic Lotus plants (Figures 4 and 5). The gene also fully rescued the total lipid level and partially rescued the total fatty acid level in try cpc double mutant (Figure 4), indicating that the GmMYB73 is involved in upregulation of fatty acid accumulation. It should be noted that the increase of total fatty acid levels in seeds of the GmMYB73-transgenic Arabidopsis plants was likely not due to the increase in any specific fatty acid but rather due to the overall increase in each fatty acid level (Figure 4b, c). However, in leaves of the transgenic plants, the increase of the total fatty acids was due to an increase in linolenic acid (18:3) (Figure 4d, e). The difference in the accumulation patterns of fatty acids in seed and leaf tissues may be due the physiological and metabolic difference of these organs, where seed is a sink organ, while the leaf is a source organ. In GmMYB73-transgenic Lotus plants, the increase of total fatty acids in leaves is mainly due to increase of linolenic acid (18:3) (Figure 5f), similar to the case in leaves of Arabidopsis transgenic plants (Figure 4e). However, the seeds of transgenic Lotus plants showed a different change in fatty acid compositions compared to seeds of transgenic Arabidopsis, and the increase of total fatty acids was attributed to an increase in linolic acid (18:2) and/or linolenic acid (18:3) (Figure 5). The different accumulation patterns of fatty acid compositions in seeds of transgenic Arabidopsis and Lotus plants suggest that some different mechanisms may be affected in lipid accumulation in these plant species.
Due to the difficulty of soybean plant transformation, we adopted a method for generating transgenic hairy roots in soybean. GmMYB73 overexpression in soybean transgenic hairy roots increased the total fatty acid levels and this elevation was likely due to the increase in linolic acid (18:2) and linolenic acid (18:3) levels (Figure 5h, i). It should be mentioned that the changes in fatty acid composition in soybean transgenic hairy roots were very similar to those in leaves of transgenic Arabidopsis plants (Figures 4e and 5i), suggesting that similar mechanisms may have been affected in vegetative organs of Arabidopsis and soybean. It is possible that GmMYB73 would promote lipid accumulation in soybean seeds as it did in Arabidopsis seeds although further studies are needed to confirm this.
GmMYB73 reduced GL2 expression (Figure 2), and GL2 has been found to be involved in oil accumulation in seeds. Shen et al.  reported that mutation in GL2 gene led to increase in seed oil content compared to wild type levels. Overexpression of BnaC.GL2.b in Arabidopsis affected the seed oil accumulation . More recently, Shi et al.  found that the PLDZ1/2 genes, targets of GL2 , are not involved in seed oil accumulation. However, blocking MUM4, another downstream target of GL2, was found to reduce mucilage biosynthesis, which may be linked to an increase in seed oil production . Presently, we found that GmMYB73-overexpressing plants and Arabidopsis gl2-2 mutant had higher levels of PLDα1, due to inhibition of GL2 expression by GmMYB73 (Figures 2 and 6). GL2 has been shown to bind to PLDα1 promoter and inhibit PLDα1 expression (Figure 7). Therefore, GmMYB73 may promote lipid accumulation by suppressing GL2 expression, and thus enhancing PLDα1 expression.
PLD hydrolyses the P-O bond of phosphatidylcholine (PC) to produce phosphatidic acid (PA) and choline. The PA is converted to 1,2-sn-diacylglycerol (DAG)  by the action of PA phosphatase and then DAG can be acylated to produce triacylglycerol (TAG). In developing soybean seeds, the major pathway for TAG formation is through conversion of PC to DAG and acylation of DAG to produce TAG . Recently, Lee et al. [51, 52] reported that the lipid profile was changed by suppression of PLDα in the soybean seeds and the total lipids and TAG signals tended to decrease in fresh seeds of PLDα-knockdown soybean. Presently, we find that PLDα1 mutation in Arabidopsis substantially reduced the TAG levels in both seeds and leaves (Tables 1 and 2). In contrast, GmMYB73-transgenic Arabidopsis plants substantially had more TAG in seeds and leaves compared to Col-0 (Tables 1 and 2). It is noted that in seeds, most of the lipid are TAG, whereas in leaves, the major lipid species are PC and PA (Tables 1 and 2). The roughly negative correlation between PA and PC contents in leaves of both GmMYB73-transgenic plants and pldα1 mutant (Tables 1 and 2) suggests that GmMYB73 may finally promote PA and possible DAG and TAG accumulation through PC conversion by PLDα1 function. Similar case may happen in GmMYB73-transgenic seeds although PA levels were not high, possibly due to an efficient conversion to DAG and TAG. All these data suggest that GmMYB73 may enhance lipid accumulation at least partially through repression of GL2 and promotion of PLDα1. Other downstream genes (e.g., MUM4) may also be involved in this process . It should be mentioned that the GmMYB73-overexpresing Arabidopsis plants have no or very little trichomes. Considering that trichome contained a multitude of wax components and can secrete lipids , the lack of trichome may save total energy and resources for lipid biosynthesis in other organs such as seeds and leaves of transgenic plants. Recently, PLDα1 overexpression has been found to improve drought tolerance and increase seed yield , lending support to our present study.
It is interesting to note that, although GmMYB73 enhances lipid contents in transgenic seeds, its expression during soybean seed development is not consistent with the fatty acid accumulation pattern at these stages (Figure 1). The discrepancy may be due to GmMYB73 being an upstream regulator and hence being expressed early in seed development in order to regulate downstream genes including GL2, PLD and possible other genes. At later stage, the GmMYB73 is reduced and the downstream factors and lipid biosynthesis genes are activated for lipid accumulation. Another gene GmbZIP123, with an increase in expression during soybean seed development, increases lipid contents in seeds of transgenic Arabidopsis plants through promotion of sugar translocation by activation of sucrose transporter genes and cell-wall invertase genes .