Diacylglycerol Acyltransferase 3(DGAT3) Is Responsible for the Biosynthesis of Unsaturated Fatty Acids in Vegetative Organs of Paeonia rockii

‘Diacylglycerol acyltransferase (DGAT)’ acts as a key rate-limiting enzyme that catalyzes the final step of the de novo biosynthesis of triacylglycerol (TAG). The study was to characterize the function of the DGAT3 gene in Paeonia rockii, which is known for its accumulation of high levels of unsaturated fatty acids (UFAs). We identified a DGAT3 gene which encodes a soluble protein that is located within the chloroplasts of P. rockii. Functional complementarity experiments in yeast demonstrated that PrDGAT3 restored TAG synthesis. Linoleic acid (LA, C18:2) and α-linolenic acid (ALA, C18:3) are essential unsaturated fatty acids that cannot be synthesized by the human body. Through the yeast lipotoxicity test, we found that the yeast cell density was largely increased by adding exogenous LA and, especially, ALA to the yeast medium. Further ectopic transient overexpression in Nicotiana benthamiana leaf tissue and stable overexpression in Arabidopsis thaliana indicated that PrDGAT3 significantly enhanced the accumulation of the TAG and UFAs. In contrast, we observed a significant decrease in the total fatty acid content and in several major fatty acids in PrDGAT3-silenced tree peony leaves. Overall, PrDGAT3 is important in catalyzing TAG synthesis, with a substrate preference for UFAs, especially LA and ALA. These results suggest that PrDGAT3 may have practical applications in improving plant lipid nutrition and increasing oil production in plants.


Introduction
Paeonia rockii is a promising woody oil crop which belongs to the family Paeoniaceae [1]. In addition to high ornamental value, its oil showed antibacterial, anti-inflammatory, immunity-enhancing, and other pharmacological effects. As a "new resource food" [2], P. rockii seeds are rich in unsaturated fatty acids, including α-linolenic acid (ALA), linoleic acid (LA), and oleic acid (OA), which account for up to 90% [3,4]. ALA is a type of ω-3 fatty acid which is essential to health but cannot be synthesized by the human body. It has excellent functions in anti-oxidation, lowering blood lipids and preventing cardiovascular and cerebrovascular diseases. Thus, exploring the oil synthesis mechanism is important in facilitating the oil production of P. rockii through genetic improvement.
Triacylglycerol (TAG) is the main storage form of vegetable oils [5]. Eukaryotes synthesize TAG mainly through two pathways ( Figure S1). The first pathway is the Kennedy Pathway, which relies on acyl-CoA and involves three successive acylation reactions to the glycerol main chain. The reaction begins with the formation of lyso-phosphatidic acid from glycerol-3-phosphate catalyzed by glycerol-3-phosphate acyltransferase (GPAT), which is 2 of 18 further acylated to phosphatidic acid; then, the phosphatidic acid is converted to diacylglycerol under the action of phosphatidic acid phosphatase. Diacylglycerol acyltransferase (DGAT) acts as a key rate-limiting enzyme in plant storage lipid accumulation. In the last step of the acylation reaction, DGAT catalyzes the generation of TAG in the sn-3 position of diacylglycerol [6,7]. The second pathway is the acyl-coA independent synthesis pathway, which uses phospholipids as the lipoyl donor and diacylglycerol as the lipoyl receptor [8,9]. Phospholipids: diacylglycerol acyltransferase (PDAT) transfers the phospholipids on the sn-2 fatty acids to the acyl glycerin sn-3; the formation of TAG and lyso-phospholipids occurs mainly in yeast and vascular plants [10,11].
In general, DGAT and PDAT, the key rate-limiting enzymes in the last acylation reaction of the TAG synthesis, play a crucial role in guiding the generation of TAG from the carbon sources [12]. Current studies have shown that there are at least three different DGATs in eukaryotes: DGAT1, DGAT2, and DGAT3 [13][14][15]. DGAT1 and DGAT2 are located on the endoplasmic reticulum (ER) [16] and are integral membrane proteins. DGAT3 is a soluble protein located in the cytoplasm [17][18][19]. It is generally believed that DGAT affects seed oil content, TAG content, fatty acid composition, and seed weight during seed development [20]. Therefore, it is possible to improve the oil content and fatty acid composition of oil-bearing crops by exploring the genes encoding active protein with DGAT [8,[21][22][23]. At present, the genes encoding functional DGAT1 or DGAT2 are isolated from a variety of plants, such as Arabidopsis thaliana, tobacco (Nicotiana tabacum L), peanut (Arachis hypogaea), rape (Brassica napus L), and Sesamumindicum. Their role in plant TAG biosynthesis has been demonstrated through overexpression and mutation downregulation techniques. For example, the AtDGAT1 mutant AS11 displayed reduced seed oil content by 20-40%, while the overexpression of AtDGAT1 resulted in a 9-12% increase in seed oil content and grain weight [24]. UrDGAT2 from Umbelopsis ramanniana was expressed by seed-specific promoters in soybean (Glycine max), generating a new soybean variety with significantly higher oil content [25]. Previous studies showed that DGAT1, DGAT2, and DGAT3 share little sequence homology with each other, indicating that they probably have different origins in plants [19,26]. DGAT1, DGAT2, and DGAT3 also show different expression patterns across different plant organs and tissues. The DGAT3 gene remains as a single copy in plants, except for a few species [26]. The soluble protein DGAT3 was first reported to be purified from the cytoplasm of the developing cotyledons of peanut (Arachis hypogaea) [18]. In Arabidopsis thaliana, AtDGAT3 can recycle LA and ALA into TAGs when the process of TAG decomposition is blocked [14]. Moreover, AtDGAT3 has recently been shown to be a metalloprotein involved in TAG biosynthesis [27]. During cotton (Gossypium) seed development, the overexpression of the GhDGAT3D gene leads to the increased accumulation of the TAG and C18:1 content [28]. In Camellia (Camelina sativa), CsDGAT3-3 also showed a strong catalytic capacity for TAG synthesis and a substrate preference for eicosenoic acid (EA) [29]. Nevertheless, the functions of DGAT1 and DGAT2 have been explored in a variety of plants, whereas the DGAT3 gene has only been characterized in few plant species.
Our study was to explore the function of the DGAT3 family members in Paeonia rockii. We identified and functionally characterized the PrDGAT3 gene in P. rockii. Then, we performed a bioinformatics analysis of DGAT3 and revealed its expression profiles among different tissues of P. rockii by qRT-PCR. Meanwhile, we examined the function of PrDGAT3 with stable transformation in Arabidopsis thaliana; transient transformation in Nicotiana benthamiana leaves; overexpression in yeast (Saccharomyces cerevisiae) mutant H1246; and gene silencing in P. rockii leaves. Finally, we showed that PrDGAT3 is a contributor to the oil synthesis of P. rockii and possibly has a certain relationship with the high content of ALA. Our findings provide an insight into the role of the DGAT3 gene and a new perspective on the improvement of lipid nutrition in plants.

Molecular Characterization of DGAT3 from P. rockii
The length of the protein encoded by PrDGAT3 is 390 aa. The analysis of the fatty acid index and the hydrophilicity index showed that PrDGAT3 is a water-soluble protein, lacking any transmembrane structure. The signal peptide prediction analysis reported that PrDGAT3 most likely contains a chloroplast transport peptide at the N-terminus. As shown in Figure 1A, PCAMBIA2300-PrDGAT3-GFP was the target experimental group. The subcellular localization experiment confirmed that the PrDGAT3 protein is localized in the chloroplasts. The conserved motifs of PrDGAT3 were identified with the MEME program and were compared with motifs of the DGAT3 protein from other species. Most of the motifs are highly conserved in DGAT3, including phosphorylation sites, tyrosine kinase phosphorylation conserved sites, and putative DGAT catalytic motifs ( Figure S2). (C) Phylogenetic tree and conserved motifs of the PrDGAT3. Different letters indicate significant difference at p < 0.05, as confirmed by one-way ANOVA with Tukey's post hoc test.

Expression Profile of PrDGAT3
In order to explore the function of PrDGAT3 in the oil synthesis of P. rockii, the expression profile of PrDGAT3 in different tissues of Paeonia rockii was studied by qRT-PCR, including roots, stems, leaves, petals, and seeds. The 18S-26S internal transcribed spacer (18S-26S ITS) gene of P. rockii was used as an internal reference to calculate the relative gene expression in each tissue (Table S1). As shown in Figure 1B, the PrDGAT3 gene expression was significantly different among the organs. PrDGAT3 was highly expressed in the stems and leaves but was barely detected in the petals. These results suggested that PrDGAT3 was highly expressed in the green tissues, which may be involved in the lipid synthesis in chloroplasts.  The Conserved Domain Database (CDD) was employed to identify the conserved functional domains of PrDGAT3. The results showed that the PrDGAT3 protein contains the typical domains of the thioredoxin-like [2Fe-2S] ferredoxin (TRX-like Fd) family, which suggested that PrDGAT3 may be involved in the binding of the [2Fe-2S] cluster. This domain was identified as being conserved among the DGAT3 family proteins.
In order to understand the evolution of DGAT3, we constructed a phylogenetic tree based on the protein sequences of the PrDGAT3 and DGAT3 proteins of other plant species. As shown in Figure 1C, it is noteworthy that the PrDGAT3 of P. rockii was closed in relation to the DGAT3 proteins from the Vitis riparia and Vitis vinifera, forming a separate cluster, suggesting that PrDGAT3 may share a common evolutionary ancestor with other Vitis L. species. These sequence analysis results confirmed that the cloned PrDGAT3 from P. rockii was of full-length. Based on previous functional validation in other species, we predicted that PrDGAT3 might be involved in the soluble TAG synthesis pathway [26,27].

Expression Profile of PrDGAT3
In order to explore the function of PrDGAT3 in the oil synthesis of P. rockii, the expression profile of PrDGAT3 in different tissues of Paeonia rockii was studied by qRT-PCR, including roots, stems, leaves, petals, and seeds. The 18S-26S internal transcribed spacer (18S-26S ITS) gene of P. rockii was used as an internal reference to calculate the relative gene expression in each tissue (Table S1). As shown in Figure 1B, the PrDGAT3 gene expression was significantly different among the organs. PrDGAT3 was highly expressed in the stems and leaves but was barely detected in the petals. These results suggested that PrDGAT3 was highly expressed in the green tissues, which may be involved in the lipid synthesis in chloroplasts.

Overexpression of PrDGAT3 Functionally Recovers oil Biosynthesis in TAG-deficient S. cerevisiae H1246
The yeast mutant H1246 has four TAG biosynthesis genes knocked out, including DGA1 (encoding DGAT), LRO1 (encoding PDAT), and ARE1 (encoding acyl-CoA: Sterol transferase 1 (ASAT1), and ARE2 (encoding ASAT2) and therefore lacks the ability to synthesize neutral oil (TAG); as a result, this mutant strain is fit for verifying the function of enzymes such as DGAT or PDAT [30]. The yeast expression vector pYES2 containing PrDGAT3 was constructed first and was then transformed into the yeast mutant strain H1246. After 48 h of culture, lipid extraction was performed. The lipid separation and Nile red fluorescence determination were performed by TLC and with a confocal laser scanning microscope. The H1246 strain and the wild-type yeast (INVSc1) transformed with an empty pYES2 vector were used as the negative and positive controls, respectively.
As shown in Figure 2A, strong and dense fluorescence signals were observed in the positive control yeast cells and also in the H1246 yeast cells overexpressing PrDGAT3, demonstrating that a large number of oil bodies were formed in the transgenic yeast cells expressing the target gene. However, there was only a very weak fluorescence signal in the negative control cells and almost no oil body was generated. To further confirm this, TLC observations were performed ( Figure 2B), and no TAG spot was detected in the negative control H1246 yeast cells containing the empty pYES2 vector. In contrast, there was an obvious TAG spot in the H1246 yeast cells expressing PrDGAT3 and the positive control cells. This indicated that PrDGAT3 expression restored the ability of H1246 yeast cells to synthesize TAG. In conclusion, the expression of PrDGAT3 successfully restored TAG synthesis and accumulation in H1246 yeast cells, which fully suggested that PrDGAT3 has DGAT activity.
To further examine the substrate preference of PrDGAT3 encoded enzymes, the fatty acid content and composition of the TAG synthesized in these yeast cells were examined by GC analysis by scraping the TAG spots off the TLC plate. As shown in Figure 2C, the H1246 yeast cells overexpressing PrDGAT3 significantly increased the total fatty acid content compared with the negative control. The oil content of the transgenic H1246 yeast cells was about one-third of that in the positive control yeast cells.

DGAT activity.
To further examine the substrate preference of PrDGAT3 encoded enzymes, the fatty acid content and composition of the TAG synthesized in these yeast cells were examined by GC analysis by scraping the TAG spots off the TLC plate. As shown in Figure 2C, the H1246 yeast cells overexpressing PrDGAT3 significantly increased the total fatty acid content compared with the negative control. The oil content of the transgenic H1246 yeast cells was about one-third of that in the positive control yeast cells.

PrDGAT3 Manifests High Substrate Preference for LA and ALA Revealed by Yeast Lipid Toxicity Test
The Nile red observations showed that although the TAG content of the H1246 yeast cells expressing PrDGAT3 was significantly increased, it was still lower than that of the wild-type yeast cells (Figure 2A). This result might be due to the lack of suitable substrate in the transgenic yeast cells. Consequently, we hypothesized that the tree peony seed oil was rich in fatty acids such as OA (18:1), LA (18:2), and ALA (18:3). It may be a suitable substrate for PrDGAT3. We tested this hypothesis using the principle of yeast lipotoxicity. Subsequently, these exogenous fatty acids were separately added to the culture medium to grow yeast cells.
We observed the growth curve of the yeast for 48 h (Figure 3). We detected no significant difference in the OD between the control and the H1246 yeast expressing PrDGAT3, when cultured in medium without any exogenous UFAs. As expected, after the exogenous addition of UFAs, the H1246 yeast cells containing an empty vector grew slowly at the early stage, and the growth tended to stop after 22 h. However, the growth rate of H1246 transgenic yeast cells expressing PrDGAT3 increased sharply after 35 h of culture, especially when the exogenous LA and ALA were added, suggesting that PrDGAT3 can overcome the lipotoxicity of yeast by catalyzing the biosynthesis of TAG with exogenous UFAs and may be selective for substrates containing LA and ALA, especially ALA.
Subsequently, these exogenous fatty acids were separately added to the culture medium to grow yeast cells.
We observed the growth curve of the yeast for 48 h (Figure 3). We detected no significant difference in the OD between the control and the H1246 yeast expressing PrDGAT3, when cultured in medium without any exogenous UFAs. As expected, after the exogenous addition of UFAs, the H1246 yeast cells containing an empty vector grew slowly at the early stage, and the growth tended to stop after 22 h. However, the growth rate of H1246 transgenic yeast cells expressing PrDGAT3 increased sharply after 35 h of culture, especially when the exogenous LA and ALA were added, suggesting that PrDGAT3 can overcome the lipotoxicity of yeast by catalyzing the biosynthesis of TAG with exogenous UFAs and may be selective for substrates containing LA and ALA, especially ALA. Values are mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Transient Expression of PrDGAT3 Enhances oil and ALA Content in Nicotiana Benthamiana Leaves
To further examine the role of PrDGAT3 in lipid synthesis and the selective collection of UFAs, we chose N. benthamiana as a transient overexpression system. Previously, it was reported that the Agrobacterium-mediated transient transformation had a higher time efficiency than the stable transformation [31]. In addition, the N. benthamiana leaf tissue was suitable for the transient expression of the genes involved in fatty acid synthesis and TAG accumulation [32]. Here, we co-transformed the coding sequences of PrDGAT3 and viral protein P19, an inhibitor of ectopic gene silencing, into N. benthamiana leaves by Agrobacterium-mediated osmosis. The expression of PrDGAT3 in N. benthamiana was detected by semi-quantitative RT-PCR with the NbActin gene of N. benthamiana as an internal reference. As shown in Figure 4A, PrDGAT3 was expressed in N. benthamiana leaves at 6 days after Agrobacterium infection. Overexpression of PrDGAT3 significantly increased the number of lipid droplets (LDs) relative to the control leaves transformed with P19 alone (Figure 4B,C). It is worth noting that there was a considerable increase in the numbers of small-, medium-, and large-sized LDs of the N. benthamiana leaves expressing PrDGAT3, compared with the P19 control, by 1.8-, 2.1-, and 2.2-fold, respectively ( Figure 4D).

Reduced Expression of PrDGAT3 in Paeonia rockii Represses the Oil Accumulation and Affects Fatty Acids Composition
To further verify the function of PrDGAT3 in oil accumulation, we infected the leaves of Paeonia rockii seedlings and inhibited the endogenous expression of PrDGAT3, utilizing the virus-induced gene silencing (VIGS) technique. One week after infection, we observed many green fluorescence spots within the leaves inoculated with TRV2-GFP and TRV2-PrDGAT3-GFP but not in the MOCK leaves under the excitation by blue light ( Figure 5A). Furthermore, the expression of TRV1 and TRV2 RNA was identified in the leaves of the Then, we lyophilized the leaves and extracted the lipids for methyl esterification. GC analysis of the fatty acids ( Figure 4E) showed that the expression of the PrDGAT3 gene in the tobacco leaf tissue significantly promoted the total oil content of the leaves, which was about twice that of the control leaves transformed with P19 alone. The expression of PrDGAT3 also resulted in significant changes in the fatty acid composition ( Figure 4F). The polyunsaturated fatty acids LA and ALA were significantly increased, with ALA increased by 10% when compared with the control leaves. These results suggested that PrDGAT3 most likely prefers UFAs as substrates for TAG synthesis.
In conclusion, our transient leaf expression assays showed that heterologous PrDGAT3 expression in tobacco can significantly increase oil content in leaves and selectively transfer endogenous UFAs to TAG.

Reduced Expression of PrDGAT3 in Paeonia rockii Represses the Oil Accumulation and Affects Fatty Acids Composition
To further verify the function of PrDGAT3 in oil accumulation, we infected the leaves of Paeonia rockii seedlings and inhibited the endogenous expression of PrDGAT3, utilizing the virus-induced gene silencing (VIGS) technique. One week after infection, we observed many green fluorescence spots within the leaves inoculated with TRV2-GFP and TRV2-PrDGAT3-GFP but not in the MOCK leaves under the excitation by blue light ( Figure 5A). Furthermore, the expression of TRV1 and TRV2 RNA was identified in the leaves of the inoculated line through RT-PCR ( Figure 5C). In addition, the qRT PCR exhibited that the transcription level of PrDGAT3 in the leaves inoculated with TRV2-PrDGAT3-GFP was significantly lower than that of the MOCK and TRV2-GFP lines ( Figure 5B). Meanwhile, we detected remarkably decreased expressions of fatty acid biosynthesis genes, including MOD1, FAD2, LPAAT1, NFYC, DGAT1, DGAT2, and PDAT2, in the PrDGAT3-silenced leaves (Supplementary Table S2). It is worth noting that the expressions of DGAT1, PDAT2, FAD2, and MOD1 significantly decreased with the reduction in the endogenous PrDGAT3 transcription level ( Figure 5B). These results revealed that PrDGAT3 may interact with other fatty acid biosynthetic genes in the process of fatty acid and oil accumulation. inoculated line through RT-PCR ( Figure 5C). In addition, the qRT PCR exhibited that the transcription level of PrDGAT3 in the leaves inoculated with TRV2-PrDGAT3-GFP was significantly lower than that of the MOCK and TRV2-GFP lines ( Figure 5B). Meanwhile, we detected remarkably decreased expressions of fatty acid biosynthesis genes, including MOD1, FAD2, LPAAT1, NFYC, DGAT1, DGAT2, and PDAT2, in the PrDGAT3-silenced leaves (Supplementary Table S2). It is worth noting that the expressions of DGAT1, PDAT2, FAD2, and MOD1 significantly decreased with the reduction in the endogenous PrDGAT3 transcription level ( Figure 5B). These results revealed that PrDGAT3 may interact with other fatty acid biosynthetic genes in the process of fatty acid and oil accumulation.
In addition, GC analysis was performed on the PrDGAT3-silenced leaves. Compared with the TRV2-GFP control and the blank control, the total oil content in the PrDGAT3silenced leaves decreased significantly to 15.60 mg/g compared with 31.79 mg/g and 29.52 mg/g ( Figure 5D). Notably, compared with the other two control groups, the proportion of the UFAs LA and ALA in the PrDGAT3-silenced leaves was significantly reduced. The proportion of LA was 16.16% after PrDGAT3 silencing, compared with 21.11% and 21.18% in the TRV2-GFP control and MOCK, respectively. The proportion of ALA was 48.07% after PrDGAT3 silencing, compared with 52.39% and 53.42% in the TRV2-GFP control and MOCK, respectively ( Figure 5E). These results further demonstrated that the expression level of PrDGAT3 can affect the lipid content and fatty acid composition.  In addition, GC analysis was performed on the PrDGAT3-silenced leaves. Compared with the TRV2-GFP control and the blank control, the total oil content in the PrDGAT3silenced leaves decreased significantly to 15.60 mg/g compared with 31.79 mg/g and 29.52 mg/g ( Figure 5D). Notably, compared with the other two control groups, the proportion of the UFAs LA and ALA in the PrDGAT3-silenced leaves was significantly reduced. The proportion of LA was 16.16% after PrDGAT3 silencing, compared with 21.11% and 21.18% in the TRV2-GFP control and MOCK, respectively. The proportion of ALA was 48.07% after PrDGAT3 silencing, compared with 52.39% and 53.42% in the TRV2-GFP control and MOCK, respectively ( Figure 5E). These results further demonstrated that the expression level of PrDGAT3 can affect the lipid content and fatty acid composition.

Stable Expression of PrDGAT3 Increases Oil, Especially LA and ALA Accumulation, in Transgentic Arabidopsis Rosette Leaves
Transgenetic Arabidopsis lines expressing pCAMBIA1300-PrDGAT3 were identified and selected for phenotype analysis ( Figure S3). Rosette leaves of three independent lines were collected from OE-4, -8, and -10, with relatively high PrDGAT3 expression ( Figure 6A).

Discussion
Oil content and fatty acid composition are the most important agronomic traits of oil crops. The transcriptional regulation of biosynthetic genes is the major factor affecting the TAG biosynthesis. In previous studies, more and more key genes related to lipid biosynthesis and accumulation have been revealed. DGAT1 was highly expressed in the developing embryos of oil crops, and its expression level was related to oil accumulation during The results showed that the overexpression of PrDGAT3 displayed significantly increased total fatty acid content and different fatty acid composition. Compared with the wild-type Arabidopsis, the total fatty acid content in the rosettes of the OE-4, -8 and -10 trans-genic lines increased to 26.6 mg/g, 21.1 mg/g, and 30.8 mg/g, respectively ( Figure 6B). In addition, the contents of LA in the OE-4, OE-8, and OE-10 transgenic lines were 20.77%, 20.23%, and 22.82%, respectively, which is significantly higher than the 13.75% in the wild type. Similarly, the contents of ALA were 43.44%, 40.29%, and 48.44% in the OE-4, -8, and -10 transgenic lines, respectively, compared with that of 31.0% in the wild type ( Figure 6C). Interestingly, the OE seedlings did not show obvious phenotypic or developmental differences compared with the wild type ( Figure S3), suggesting that PrDGAT3 may play a minor role in other physiological processes. Collectively, these results demonstrated that PrDGAT3 was involved in the regulation of lipid biosynthesis and fatty acid composition.
To further explore the effect of the overexpression of PrDGAT3 on lipid synthesis in Arabidopsis Thaliana, we examined the expression of various fatty acid and TAG biosynthesis genes in rosette leaves with qRT-PCR (Supplementary Table S2). Firstly, we analyzed the expression profile of seven key TAG synthesis genes, including AtDGAT1, AtDGAT2, AtDGAT3, AtGPAT6, AtPDAT1, AtPDAT2, and AtOLEOSIN (AtOLEO3) ( Figure 6D). Next, we analyzed the expression levels of AtLEC1 and AtLEC2, two positive regulators in the process of lipid synthesis. We detected about 7-to 13-fold higher transcription levels of LEC1 and 1.5-to 5-fold higher transcription levels of LEC2 in the OE-4, OE-8, and OE-10 transgenic lines compared with that of the wild type. Finally, we quantified the expression levels of four key fatty acid biosynthetic genes AtSUS2, AtKASI, AtMOD, and AtFAD2. Compared with the wild type, the expression levels of most genes were slightly different from those of the wild type, but interestingly, the expression level of AtSUS2 in the rosette leaves of the transgenic lines was significantly increased. In addition, the expression level of AtDGAT2 in the rosette leaves of the transgenic lines was significantly decreased. However, the expressions of AtDGAT1 and AtPDAT2 were increased significantly. More notably, the expression of AtOLEOSIN (AtOLEO3) increased 10-24 times in the rosette leaves of the transgenic lines. All together, these findings corroborated that PrDGAT3 may potentially interact with other genes in lipid synthesis and metabolism during plant development.

Discussion
Oil content and fatty acid composition are the most important agronomic traits of oil crops. The transcriptional regulation of biosynthetic genes is the major factor affecting the TAG biosynthesis. In previous studies, more and more key genes related to lipid biosynthesis and accumulation have been revealed. DGAT1 was highly expressed in the developing embryos of oil crops, and its expression level was related to oil accumulation during seed development [12,33]. The inactivation of DGAT1 resulted in reduced seed oil content in Arabidopsis mutant AS11 [34]. The co-transformation of VfFAD2 and VfDGAT2 genes in the oilseed of Vernicia fordii resulted in a 17.10% increase in Eicosatenoic Acid (C20:3) in Arabidopsis [35]. Past studies have shown that DGATs are the ultimate rate-limiting enzymes for TAG biosynthesis in eukaryotes [36,37]. Compared with the commonly studied DGAT1 and DGAT2 gene family members, DGAT3 gene has only been identified in a few plant species, and its function is still unknown [18,[27][28][29].
In this study, we identified and cloned PrDGAT3 from Paeonia rockii. One or two DGAT3 genes have been reported in the genomes of various plants [26], ranging from lower prokaryotes such as Chlamydomonas reinhardtii and Phaeodactylum tricornutum to important cash crops such as soybean, corn, and camelina. Two isoforms of the DGAT3 family were identified from the genome of oil palm (Elaeis guineensis) [38], and three isoforms were identified from peanut [17]. The structural analysis of DGAT3 proteins showed that most plant DGAT3 proteins were located in the cytoplasm and were water-soluble proteins. In agreement with this finding, our study revealed that PrDGAT3 of P. rockii was a watersoluble protein without a transmembrane structure. Phylogenetic analysis ( Figure 1C) showed that the PrDGAT3 protein of P. rockii had the highest homology with the DGAT3 protein of the genus Vitis, suggesting that the two species may have shared a common ancestor during evolution. Similarly to the other DGAT3s, PrDGAT3 also contained a TRX-like Fd domain, which was a conserved thioredoxin-like ferredoxin domain located in the C-terminal region of the DGAT3s, and it contained four conserved cysteines involved in [2Fe-2S] cluster binding. At the same time, this domain indicated that DGAT3 was a metalloprotein, and its function may be related to iron utilization. Consistently with this, the expression of AtDGAT3 was up-regulated in the leaves and roots of Arabidopsis under iron stress [39], while the over-accumulation of iron led to the down-regulation of the AtDGAT3 expression in flowers [40]. On the other hand, it is speculated that DGAT3 had a dual activity in which electron transfer was coupled to the acylation reaction [41]. Furthermore, the DGAT3 conserved motif was mostly located in the N-terminal region, indicating that it was the most conserved region. The presence of multiple functional motifs in the PrDGAT3 protein may indicate that their function was influenced or regulated by other factors. Therefore, further experiments are needed to elucidate the exact role of this domain in the DGAT3 protein.
Interestingly, the transiently expressed PrDGAT3 protein was localized in the chloroplasts of tobacco leaf cells ( Figure 1A), which was consistent with the prediction of the chloroplast transport peptide in PrDGAT3 protein. Notably, the DGAT3 activity has also been detected in the cytosolic and plastid fractions [18], predicting an N-terminal chloroplast transport peptide in AtDGAT3 [27]. In higher plants, TAG accumulates as lipid droplets in the cytoplasm and in plastids [42,43], which include chloroplasts, chromatids, and alba bodies [44][45][46]. An in vitro assay has shown that even a small increase in ALA can cause severe damage to membrane structure and function within the chloroplasts; so, chloroplasts are highly sensitive to free fatty acids [47][48][49]. One of the primary functions of LDs is to buffer the levels of cytotoxic lipids and release stored neutral lipids in response to membrane homeostasis and cell growth requirements [50]. Therefore, in contrast to the TAG in the cytoplasm, the main function of the TAG in chloroplasts is to reduce the damage caused by lipotoxicity, thereby reducing the direct damage caused by lipids to the organism. In addition, DGAT1, a key rate-limiting enzyme involved in TAG synthesis in the endoplasmic reticulum, has been shown to be responsible for the chloroplast envelope [51,52], and the activity of Arabidopsis PDAT1 has also been shown to be associated with the microsomal membrane [11]. However, to date, the relationship between the cytoplasm and the chloroplast TAG metabolism is still unclear. It is crucial to explore the metabolism of TAG in the chloroplasts and the enzymes that catalyze the final step of the TAG assembly [53]. Collectively, these data showed that DGAT3 subcellular localization is different from that of higher plants; hence, future experimentations are required to illustrate the exact role of PrDGAT3 in chloroplast TAG synthesis and its association with cytoplasmic TAG metabolism.
Previous studies have shown that DGAT3 has different substrate selection for fatty acids in various plant species. For example, the transient expression of AtDGAT3 in tobacco leaves resulted in increased TAG accumulation compared with the AtDGAT1-expressing control, which significantly enhanced the LA and ALA accumulation [14], and this activity was also verified by the in vitro assay of bacterially expressed AtDGAT3 [27]. CsDGAT3-3 in camellia seeds was functionally verified by the Saccharomyces cerevisiae system, in the in vitro and tobacco experiments. The results showed that CsDGAT3-3 had high DGAT activity against UFAs, especially against 20:1n-9. The RgDGAT3 gene from the Rhodotorula glutinis [54] preferentially selected for C18 UFAs in restoring TAG synthesis in the Saccharomyces cerevisiae mutant H1246. In our study, the functional complementation experiment in the yeast strain H1246 and the yeast lipotoxicity assay, as well as the heterogeneously transient expression in tobacco leaves and the stable expression in Arabidopsis Thaliana and PrDGAT3 gene silencing, demonstrated that PrDGAT3 has a high DGAT activity and substrate preference for UFAs, particularly LA and ALA.
Both the transient and the stable overexpression of PrDGAT3 induced a significant increase in the total oil content of the plant and altered its fatty acid composition ( Figures 4A,C and 6B,C). In addition, the silencing of PrDGAT3 resulted in lower total plant oil content and the composition of several major fatty acids ( Figure 5B,C). The expression of genes involved in the regulation of lipid biosynthesis is critical for TAG accumulation. With qRT-PCR, we found that overexpression or silencing of PrDGAT3 affected the expression of several key genes involved in TAG biosynthesis. For example, AtOLEO3 is an oil body protein, which has important effects on fatty acid biosynthesis. The expression level of AtOLEO3 was significantly increased in the PrDGAT3 overexpression lines. As key rate-limiting enzymes in the last step of TAG biosynthesis, the expression levels of AtDGAT1 and AtPDAT2 increased significantly in the PrDGAT3 overexpressing lines. Similarly, the expression levels of PrDGAT3 decreased when PrDGAT3 was silenced, indicating that DGAT3 may have a potential relationship with DGAT1 and PDAT2 in plants. LEC1 and LEC2, are important in regulating seed maturation and lipid accumulation in Arabidopsis [55]. More interestingly, we found to our surprise that the expression levels of LEC1 and LEC2 increased in the PrDGAT3 overexpression lines. Further studies are needed to determine whether DGAT3 is related to LEC1 and LEC2.

Plant Materials and Regents
The Paeonia rockii used in the experiment was grown in the tree peony Germplasm Resource nursery of Northwest A&F University in Shaanxi Province, China. The P. rockii was managed from seedling planting to seed harvest. The roots, stems, leaves, petals, and seeds of P. rockii were collected in liquid nitrogen and then stored at −80 • C. Two-yearold seedlings were used for virus-induced gene silencing (VIGS). Escherichia coli DH5α, Agrobacterium tumefaciens GV3101, and yeast (Saccharomyces cerevisiae) wild-type strain (INVSc1) were purchased from Shanghai Viland Biotechnology Company. The yeast TAGdeficient strain H1246 was generously donated by professors of Hainan University. The yeast expression vector pYES2 was stored in the Oil Tree Peony Engineering Center of Northwest A&F University. The yeast transformation kit and yeast plasmid extraction kit were purchased from Beijing Coolaber Technology Company. The plant expression vectors pCAMBIA1300 and pCAMBIA2300-GFP and the gene silencing vector TRV2-GFP were preserved by the Oil Tree Peony Engineering Center of Northwest A&F University. The toxicity test of the yeast fatty acids was carried out by adding a single fatty acid with a concentration of 1 mM into the culture medium. The long-chain fatty acids used included oleic acid (OA, C18:1, 99% purity), linoleic acid (LA, C18:2, 99% purity), and α-linolenic acid (ALA, C18:3, 98% purity), purchased from Beijing Solebo Technology Company.

The Cloning of PrDGAT3 and Sequence Analysis
We found the DGAT3 gene fragment in the previous transcriptome data and cloned the full-length gene by the chromosome step. The full-length CDS of PrDGAT3 was cloned from P. rockii ( Figure S4). Based on the protein sequence, we constructed an unrooted phylogenetic tree of the PrDGAT3 and 18 DGAT3 protein sequences from other plant species with MEGA6.0, using the neighbor-joining (NJ) method (bootstrap replicates:1000); the GenBank accession numbers of the sequences are listed in Table S3. The basic physical and chemical properties of the PrDGAT3 protein were analyzed. The hydrophilicity, molecular weight, and isoelectric point of the PrDGAT3 homologous proteins were predicted using the online analysis website (http://www.expasy.org, accessed on 8 March 2022). The transmembrane (TM) structure of the PrDGAT3 proteins was predicted using the online software TMHMM Server v.2.0 (http://www.cbs.dtu.dk/services/TMHMM/, accessed on 8 March 2022). The amino acid sequence of PrDGAT3 was submitted to the MEME program to identify conserved protein motifs [56]. The amino acid sequence of PrDGAT3 was analyzed by the PRALINE program (http://www.ibi.vu.nl/programs/Pralinewww/, accessed on 8 March 2022).

Expression Analysis of PrDGAT3
The qRT-PCR primers were designed by Oligo6.0, and P. rockii's 18S-26S internal transcribed spacer (18S-26S ITS) gene [57] was used as the internal reference gene, as shown in Table S1. RNA from the seeds and other organs was extracted using the RNAprep Pure Plant kit (Tiangen Beijing, China); subsequently, approximately 1 µg of total RNA was used for reverse transcription, using the PrimeScript TM RT reagent Kit (TaKaRa Dalian, China). Three copies of each sample were analyzed. The relative expression analyses were performed by the 2−∆∆CT values [58]. The reverse transcription of the P. rockii cDNA was used as a template for PCR amplification. The PCR products were purified by the DNA Gel Extraction Kit (Sangon), and then, the amplified PCR fragments were inserted into the pMD19-T vector. After that, positive clones were screened for gene sequencing. The correctly sequenced plasmids were stored in the refrigerator at −20 • C for later usage.

Construction of PrDGAT3 Expression Vectors
The plasmid pCAMBIA1300-PrDGAT3 was constructed by inserting the CDS of PrDGAT3 into pCAMBIA1300. The vector pCAMBIA2300-PrDGAT3-GFP was generated for subcellular localization by inserting the CDS of PrDGAT3 without the stop codon into the pCAMBIA2300-GFP vector. The gene fragment was amplified and linked to TRV2 to construct TRV2-PrDGAT3-GFP vectors for VIGS analysis. To construct the yeast overexpression vector, we inserted the CDS of PrDGAT3 into the yeast expression vector pYES2. The primers used for gene cloning and vector construction in this study were designed by Oligo 6.0 software and are shown in Supplementary Table S1.

Subcellular Location
To identify the subcellular location of PrDGAT3, the pCAMBIA2300-PrDGAT3-GFP vector was transiently transformed into tobacco leaf cells. The recombinant plasmid pCAMBIA2300-PrDGAT3-GFP was first transferred into the receptor cells of Agrobacterium tumefaciens GV3101 by the freeze-thaw method [32]. The Agrobacteria were cultured until OD600, reaching 0.4 in the YEP medium containing kanamycin (50 µg/mL) and rifampicin (50 µg/mL), suspended with equal volume buffer (10 mM MES, 200 uM Acetosyringone, and 10 mM MgCl2), and then injected into 4-week-old tobacco leaves through the lower epidermis. After 4-6 days, the injected tobacco leaves were scanned for GFP signals under a confocal laser scanning microscope (LEICA TCS SP8, Germany Heidelberg) with an excitation wavelength of 488 nm and a spontaneous light detection wavelength of 640 nm.

Heterogeous Expression in the S. cerevisiae Mutant H1246 Strain
The recombinant plasmid (pYES2-PrDGAT3) was transformed into yeast strain H1246 [59] by the LiAc method. The empty pYES2 vector was transformed into wild-type yeast strain INVSc1 and yeast strain H1246, as the positive control and negative control, respectively. The positive colonies were screened on the lowest medium plate lacking uracil (SC-U), and the positive colonies were screened by PCR (primers are in Supplementary  Table S1). Firstly, the recombinant yeast cells were grown in 10 mL SC-URA inhibitory medium with 2% (w/v) glucose overnight at 200 r/min at 30 • C and then inoculated at a starting point of OD600 of 0.1 in a minimum medium containing 2% (w/v) galactose and 1% (w/v) raffinose (referred to as the induction medium) to induce gene expression (200 r/min; 30 • C for 36 h). The cells were collected by centrifugation (5000× g, 8 min) and washed with double-distilled water three times. The cells used for the subsequent lipid analysis were dried in a freeze dryer and stored at room temperature.

Yeast Fatty Acid Toxicity Test for acyl-CoA Substrate Specificity Assay
A yeast fatty acid toxicity test was employed to determine the amount of free fatty acids added to a culture medium that can produce lipid toxicity for TAG-deficient yeast. Nevertheless, if the transgenic yeast can produce acyltransferase activity that can selectively convert free fatty acids and restore TAG accumulation, it can overcome lipid toxicity and grow normally [60]. We evaluated the effects of exogenous fatty acids on total TAG content and fatty acid composition in yeast expressing PrDGAT3; at the same time, we identified the substrate preference of PrDGAT3. To scatter the fatty acids into the medium, a solution of 1 mM fatty acid solution was first dissolved in 0.5 M ethanol and then diluted in a warm medium containing 0.06% (v/v) tyloxapol (Sigma) before mixing with the induction medium. Approximately 30 mg of dry yeast was used for culturing in an induction medium supplemented with different fatty acids (1 mM) OA, LA, and ALA. The yeast cultured in an induction medium without exogenous fatty acid was used as the control group. Then, the yeast was placed in a 28 • C shaking box at 200 r/min for 48 h. During this time, the cell density was measured by UV spectrophotometer at different time points to make the yeast growth curve (A600 nm).

Lipid Extraction, Fatty Acid Analysis, and Visualization of Lipid Droplets
Lyophilized yeast was ground into powder, and the total lipid was extracted using the n-hexane: isopropanol (3:2) method [61]. A solvent mixture of n-hexane: diethyl ether: acetic acid (80:20:2, v/v/v) was used to separate the labels from the total lipids by thin layer chromatography (TLC). The separated TAG bands were scraped off the silica gel plate and eluted with 2 mL n-hexane for 2 h. The supernatant passed through a 0.22 µm organic filter and was placed in a new centrifuge tube. In accordance with Zhang [62], the fatty acid composition analysis of fatty acid methyl ester was carried out with C17:0 as an internal standard. Three copies of each sample were analyzed [63]. Fatty acid methyl ester (FAME) was analyzed by the Agilent 8890 gas chromatography system (Agilent, Santa Clara, CA, USA), equipped with an HP-Innowax (60 m × 0.25 mm, 0.25 µm) elastic quartz capillary column with nitrogen as a carrier gas at a 250 • C inlet temperature and a 130 • C chamber start-up temperature.
The Nile red fluorescence assay was applied to observe intracellular lipid droplets using the confocal laser scanning microscope (LEICA TCS SP8, Germany Heidelberg). A 1 mL yeast cell culture was obtained by centrifugation (5000× g for 10 min) for 36 h of growth and was re-suspended with double-distilled water to OD600 = 0.5. Then, 200 µL yeast cells was stained with 10 µL Nile red (5 mg/mL methanol), 50 µL dimethyl sulfoxide, and 750 µL double-distilled water in the dark for 15 s [29]. Nile red was excited at 488 nm with emission wavelengths of 560-620 nm and then observed by confocal microscopy.

Transient Overexpression in Nicotiana Benthamiana Leaves
The recombinant plasmid pCAMBIA1300-PrDGAT3 was transferred into the competent cells of Agrobacterium tumefaciens GV3101 by the freeze-thaw method. The positive colonies were selected by COLONY PCR after being cultured for 48 h on a solid YEP medium containing kanamycin (50 µg/mL) and rifampicin (50 µg/mL) under dark conditions at 28 • C. The positive colonies were then sub-cultured in liquid medium containing the same antibiotic of YEP and continued to be shaken overnight at 28 • C. The Agrobacteria cells were then collected by centrifugation at 8000 r/min for 10 min and suspended in osmotic buffer (300 µM acetosyringone, 10 mM MES, 10 mM MgCl2, pH 5.7) until the final OD600 was 0.4-0.6. The culture was kept in the dark at 28 • C for 3 h. In order to improve the expression of PrDGAT3 in the tobacco leaves, agrobacterium suspensions containing P19 silencing inhibitors and pCAMBIA1300-PrDGAT3 suspensions were mixed in a 1:1 ratio and injected into the reverse side of 4-week-old tobacco leaves with sterile syringes. The leaves injected with the Agrobacterium tumefaciens P19 were used as the control. The tobacco plants injected with the Agrobacteria solution were placed in the culture chamber. On the fourth day, some tobacco leaves were dyed with Nile red for the lipid droplet visualization experiment. The lipid droplets were stained with 2µg/mL Nile red (raw solution) in 0.01 mM phosphate buffer (pH 7.2). A confocal laser scanning microscope (LEICA TCS SP8, Germany) was used to obtain the confocal images according to the method described above. On the fifth day, approximately 0.1 g of tobacco leaves per replicate was weighed out after stabilizing the leaf moisture content in a freeze drier for 72 h; three biological replicates were prepared for each sample. Then, the leaves were placed in a 10 mL glass test tube and a 4 mL 2.5% methanol sulfate solution was added first; then, they were put into a water bath at 80 • C for 2 h in the dark. After dropping to room temperature, 1 mL 0.9% sodium chloride solution and 1 mL n-hexane were added sequentially. After shaking and full extraction and 250× g centrifuge for 5 min, the supernatant was isolated with 0.22 µm organic filter membrane placed into the sample bottle for GC (gas chromatography) detection. The gas chromatographic conditions were the same as those described above.

Virus-Induced Gene Silencing of PrDGAT3
The seedlings of Paeonia rockii were dyed according to the method of Xie [64]. TRV2-PrDGAT3-GFP, TRV2-GFP, and TRV1 were transformed into Agrobacterium GV3101, respectively. The transformed GV3101 were grown in YEP medium containing kanamycin (50 µg/mL), rifampicin (50 µg/mL), and gentamicin (50 µg/mL) at 28 • C with constant shaking overnight. After the supernatant was dumped, the cells were treated with Acetosyringone containing 10 mM morpholinoethulfonic Acid (MES) and 100 µM Acetosyringone (AS) and 10 mM MgCl2 immersion buffer to re-suspend the Agrobacteria and adjust the OD600 to 1.2~1.5. The Agrobacteria solution containing TRV1 was mixed with the Agrobacteria solution containing TRV2-GFP and TRV2-PrDGAT3-GFP in equal volumes, and then, the mixed bacterial solution was placed at 28 • C, 90 r/min and sheltered from light for 3 h. The leaves of the P. rockii seedlings infected with TRV2-GFP were used as a control. After 6 days of infection, the leaves were used for GFP expression analysis. After 14 days of infection, the leaf samples of the GFP-expressed plants were collected for subsequent fatty acid content determination and gene expression evaluation with real-time quantitative PCR. The specific methods were described above.

Overexpression of PrDGAT3 in Arabidopsis Thaliana
The recombinant plasmid pCAMBIA1300-PrDGAT3 was transformed into Agrobacterium tumefaciens strain GV3101. Subsequently, the Agrobacterium strains were used to transform the wild-type Arabidopsis (Columbia) by the floral dipping method [65]. T0 seeds were germinated on 1/2 MS medium containing hygromycin (20 mg/l) to screen the positive transformed Arabidopsis lines. The seedlings with normal root growth were cultured in the substrate for two to three weeks before the collection of the young leaves. Semi-quantitative RT-PCR was used to identify the seedlings transformed with PrDGAT3 (Supplementary Table S1). These transgenic lines were grown into a T2 generation to obtain homozygous lines. Mature Arabidopsis leaves of the T2 transgenic lines were collected and randomly selected for different lines; the fatty acids were extracted for analysis. The specific methods were described above.

Conclusions
In this study, we systematically dissected the phylogeny, subcellular localization, and functional roles of PrDGAT3 in Paeonia rockii, a promising woody oil crop. PrDGAT3 is mainly expressed in the vegetative organs, such as stems and leaves. With protein motif analysis, we found that PrDGAT3 protein shares a conserved domain with the same family members of other species. By integrating functional assays in yeast mutant H1246 with and without exogenous fatty acid feeding, as well as PrDGAT3 gene silencing, the transient expression in tobacco leaves and the stable expression in Arabidopsis Thaliana, we detected that PrDGAT3 has a high DGAT activity and a substrate selection for UFAs, particularly linoleic acid (LA, C18:2) and α-linolenic acid (ALA, C18:3). Our findings provide new knowledge to advance our understanding of the regulation of TAG biosynthesis and metabolism in plants, and in particular, they provide a new insight into the genetic improvement of oil yield and quality through biotechnology.