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Applied Biological Chemistry
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Enhancing seed oil content and fatty acid composition in camelina through overexpression of castor RcWRI1A and RcMYB306

Applied Biological Chemistry volume 67, Article number: 74 (2024) Cite this article

Abstract

Seed triacylglycerol (TAG), a major component of vegetable oil, consists of a glycerol esterified with three fatty acids. Vegetable oil has industrial applications and is widely used as edible oil. The increasing demand for plant oils, owing to population growth, it is crucial to enhance the oil content in seeds. We found castor WRINKLED1A (RcWRI1A) and R2R3-type MYB domain protein 306 (RcMYB306) which have homology with Arabidopsis WRI1 (AtWRI1) and AtMYB96 which regulate genes involved in fatty acid and TAG synthesis, respectively. These castor genes were separately and jointly overexpressed using seed-specific promoters in an oil crop, camelina (Camelina sativa). Overexpression of RcWRI1A, RcMYB306, or RcWRI1A + RcMYB306 increased the total seed oil content in camelina. However, this increase was not significantly different from that observed during the overexpression of RcWRI1A or/and RcMYB306. RcWRI1A overexpression increased the fatty acid content, including 16:0, 18:2, 18:3. Contrastingly, RcMYB306 overexpression increased the 18:1, 18:2, 18:3, 20:0 and 20:1 fatty acid. In the RcWRI1A + RcMYB306 lines, changes in fatty acid composition demonstrated the combined effects of these transcription factors. These results suggest that RcWRI1A and RcMYB306 can be used to improve the productivity of oil crops.

Introduction

Vegetable oil is an edible oil and is also used as a raw material in the industrial production of plastics, cosmetics, and lubricants [1]. It contains triacylglycerol (TAG), which comprises three fatty acids esterified with one glycerol [2]. The fatty acid composition of TAG in seeds differs depending on the plant species [3]. Olive oil has a high oleic acid (18:1Δ9) content and is used for frying or salad dressing [4, 5]. Perilla, flaxseed, and walnut oil have health benefits and are high in α-linolenic acid (18:3Δ9,12,15), an omega-3 fatty acid [6]. Castor (Ricinus communis L.) oil contains 80–90% ricinoleic acid (18:1Δ9-OH) with a hydroxy group on 12th carbon. It is used as an industrial raw material in various manufacturing processes, such as lubricant and paint production [7]. The demand for vegetable oils is increasing worldwide [8]. However, crop productivity is decreasing because of climate change caused by global warming [9]. Therefore, technological advancements are required to increase the TAG content of seeds, subsequently increasing vegetable oil production per unit area. Camelina (Camelina sativa) is an oil crop belonging to the Brassicaceae family. It has a short life cycle, grows in barren areas, and is easily transformed through the floral dipping method, a genetic engineering platform for plant oil enhancement [10, 11].

The transcription factors and genes involved in fatty acid and TAG synthesis have been identified in Arabidopsis (Arabidopsis thaliana) [12]. Fatty acids synthesized in plastids are exported to the cytosol to form acyl-coenzyme A (CoA) pools [12]. In the Kennedy pathway, three acyl-CoA molecules are sequentially added to one molecule of glycerol-3-phosphate to synthesize TAG using glycerol-3-phosphate acyltransferase, lysophosphatidic acid acyltransferase, and diacylglycerol acyltransferase 1 (DGAT1) in the endoplasmic reticulum (ER) [13, 14]. DGAT is a rate-limiting enzyme that transfers acyl-CoA to the sn-3 position of diacylglycerol (DAG) to generate TAG [15, 16]. Unsaturated fatty acids from the sn-2 position of phosphatidylcholine (PC) are transferred to the sn-3 position of DAG by phospholipid: DAG acyltransferase (PDAT) to synthesize TAG [17].

Arabidopsis WRINKLED1 (AtWRI1) belongs to a family of APETALA2 (AP2) transcription factors that regulate genes involved in fatty acid synthesis [18]. Orthologs of AtWRI1 have also been found in Brassica, corn, camelina, soybean, and castor [19,20,21,22,23]. MYBs are the largest family of the transcription factor in plants. The MYB protein is divided into several classes, one of which is the R2R3-type MYB TF, divided into 23 subgroups [24]. R2R3-type MYB domain protein 96 (MYB96), which exhibits drought stress or disease resistance related to ABA signaling, regulates PDAT1 and DGAT1 expression [25]. Furthermore, seed-specific overexpression of AtMYB96 enhances the TAG content in Arabidopsis seeds [25]. In contrast, AtMYB89 is a negative regulator for oil accumulation [26].

Castor is grown in tropical or subtropical regions and contains 30–50% oil in seeds [7]. Even though castor has high oil content in seeds, not much research has been conducted on the transcription factor of castor. In this study, two transcription factors, RcWRI1A and RcMYB306, were isolated from castor. It remains to be revealed whether overexpression of RcWRI1A and RcMYB306 effectively increases oil content in oil crops. Therefore, seed-specific overexpression of RcWRI1A and RcMYB306 were conducted in camelina as well as co-expression of RcWRI1A and RcMYB306. We investigated whether heterologous expression of RcWRI1A or/and RcMYB306 enhances the metabolism of fatty acids and TAG in camelina seeds and ultimately improves oil content.

Materials and methods

Plant materials and transformation

Camelina sativa cultivar Suneson was used for transformation. Wild-type and transgenic plant seeds were germinated on moist filter paper in a culture chamber at 16 °C under a 16 h light/8 h dark photoperiod. Plants were grown at 20 °C in a growth chamber under a 16 h light/8 h dark photoperiod. The Agrobacterium strain GV3101 was used to transform the pBinGlyRed3 vector. Agrobacterium cells were inoculated into 500 mL LB medium and incubated at 28 °C. Cultured cells were centrifuged at 3000 × g for 10 min at 4 °C. They were subsequently dissolved in a solution containing 5% sucrose and 0.05% (v/v) Silwet-L77. Eighteen plants were transformed using the floral dipping method [10]. After floral dipping, the plants were wrapped in black plastic for a day to maintain the humidity. This process was performed three times at intervals of 5–7 d. The fluorescent seeds were obtained using a green flash and a red filter to identify transgenic plants.

Gene cloning and vector construction

Total RNA was extracted from developing castor seeds, and cDNA was synthesized using a cDNA synthesis kit (Takara, Kusatsu, Japan). We designed primers covering the full-length open reading frame (ORF) to amplify the coding sequence (CDS) of RcWRI1A and RcMYB306 (Table S1). The PCR products were eluted using a PCR purification kit (Cosmo Genetech, Seoul, Korea) and cloned into the pGEM-T vector (Promega, Madison, WI, USA). The final vector, pBinGlyRed3, included the glycine promoter for seed-specific expression and DsRed3 as a selection marker [27]. There is an EcoRI site on both sides of the CDS in the pGEM-T vector and a unique EcoRI site in pBinGlyRed3. Therefore, RcWRI1A and RcMYB306 were cloned using EcoRI. To construct a co-expression vector containing both genes, RcMYB306 was cloned into a pKMS2 vector containing an oleosin promoter using NotI enzymes on both sides of the pGEM-T vector. Then, the oleosin promoter:RcMYB306:terminator cassette was cut out using the AscI restriction enzyme. This cassette was inserted into the AscI site of the pBinGlyRed3-RcWRI1A vector to complete the cloning of the co-expression vector.

Phylogenetic tree and protein sequence alignment

All protein sequences used for phylogenetic tree and sequence alignment were obtained from the National Center for Biotechnology Information (NCBI) and The Arabidopsis Information Resource (TAIR). The phylogenetic tree was generated by the Neighbor-Joining method with 1000 bootstrap replications in the MEGA7 program. Protein sequence alignment was conducted in DNAMAN program using Clustal W method.

Fatty acid analysis

The fatty acid composition and total oil content were analyzed through gas chromatography (GC). Seven seeds were reacted with 500 µL toluene and 1 mL of 5% H2SO4 including pentadecanoic acid (15:0) standard (100 µg/mL) in an 85 °C water bath for 2 h. Next, 1 mL of 0.9% NaCl and 1 mL hexane were added. The solution was mixed and centrifuged at 330 × g for 2 min to extract fatty acid methyl esters (FAME). The addition of 1 mL hexane, followed by centrifugation, was repeated three times. Then, the solution was evaporated using nitrogen gas. The FAME was diluted with 200 µL hexane and transferred into a GC vial for analysis using a GC-2030 (Shimadzu, Kyoto, Japan) machine and a DB-23 column (30 m × 0.25 mm, 0.25 μm film; Agilent, Santa Clara, CA, USA). The oven temperature range increased from 190 °C to 230 °C by 5 °C per min.

RT-PCR and RT-qPCR analysis

Total RNA was isolated from developing seeds in camelina transgenic plants using the method described in reference [28]. 2 µg of cDNA was synthesized using the PrimeScript 1st Strand cDNA Synthesis Kit (Takara, Kusatsu, Japan). Developing seeds were obtained during the oil accumulation period (18–24 DAF) based on reference [29]. Reverse transcription real-time PCR (RT-qPCR) was performed using TB Green Premix Ex Taq™ II (Takara, Kusatsu, Japan) reagent in a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). By subtracting the Cт values of the target gene and the endogenous control, the Cт value was determined. ACTIN2 was used as a control. To measure the relative expression level, the two Cт values were subtracted and obtained the value of 2(-ΔΔCт). Primers used in this study are listed in Table S1.

Results

Isolation of castor WRI1A and MYB306 cDNA for increasing seed oil in camelina

AtWRI1 functions as a master regulator by regulating transcription level of fatty acid and TAG biosynthesis genes [30]. The heterologous expression of various WRI1 genes increases the TAG content of seeds in several plants [18, 19, 31, 32]. In addition, the overexpression of AtMYB96 using a seed-specific promoter increased the TAG content in Arabidopsis [25]. In this study, we expressed RcWRI1A and RcMYB306 individually or simultaneously to increase TAG levels in camellia seeds. We attempted to compare the efficiency of these transcription factors in increasing oil content. Furthermore, we investigated whether the co-expression of two transcription factors could increase oil content compared to a single transcription factor.

The castor genes with the highest amino acid sequence homology to AtWRI1 and AtMYB96 were selected from the castor genomic database (http://www.plantgdb.org) using the BLASTP program. The highest homology with AtWRI1 and AtMYB96 in the protein database translated using the entire castor transcript was 30,069.m000440 and 30,138.m004082, with values of 6.0e− 102 and 6.0e− 91, respectively. Analysis of BLAST and phylogenetic tree was conducted on the isolated transcript in the castor genomic database. As a result, 30,069.m000440 and 30,138.m004082 were estimated to be RcWRI1A and RcMYB306, respectively (Fig. 1). It was confirmed that castor WRI1 has a 14-3-3 binding motif, a conserved VYL motif, and two AP2 domains such as other WRI1 (Fig. S1). The amino acid sequence of RcWRI1A showed 61% identity with AtWRI1. RcWRI1A has the same sequence as a spliced transcript of castor WRI1 [23]. The phylogenetic tree between RcMYB306 and AtMYBs revealed that the RcMYB306 belongs to subgroup 1 and is close to AtMYB30 (Fig. S2). However, since AtMYB306 does not exist in Arabidopsis, the phylogenetic tree was generated between RcMYB306, several plant MYB306, and AtMYBs belonging to subgroup 1 (Fig. 1b). As a result, it was confirmed that RcMYB306 was closer to MYB306s than to AtMYB30. In addition, the R2 and R3 domains of RcMYB306 are conserved similar to other MYB306s (Fig. S3). According to the alignment result and phylogenetic tree, two genes were designated as RcWRI1A and RcMYB306 in this study.

Fig. 1
figure 1

Evolutionary relationships of (a) RcWRI1A and (b) RcMYB306 with various plants WRI1 and MYB306. The phylogenetic tree was generated by the Neighbor-Joining method with a bootstrap test (1000 replicates) in the MEGA7 program (https://www.megasoftware.net)

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Selection of camelina transformants introduced with RcWRI1A, RcMYB306, and RcWRI1A + RcMYB306 vectors

To investigate whether RcWRI1A and RcMYB306 enhance TAG biosynthesis in seeds, these two genes were cloned into seed-specific expression vectors. Two vectors were constructed to express RcWRI1A and RcMYB306 using a seed-specific glycinin promoter (Fig. 2a). To co-express RcWRI1A and RcMYB306, the two genes were cloned into a single vector containing glycinin and oleosin promoters (Fig. 2a).

Fig. 2
figure 2

T-DNA vector map and selection of transgenic plants. (a) A vector map for seed-specific expression of RcWRI1A or/and RcMYB306. GlyP, Glycinin promoter (701 bp); RcWRI1A, RcWRI1A cDNA (1365 bp); Gly-T, Glycinin terminator (458 bp); NOS-T, Nos terminator; LB, left border; RB, light border; RcMYB306, RcMYB306 cDNA (1035 bp); OleP, oleosin promoter (580 bp); Gly-T R, Glycinin terminator reverse primer; 35 S P, 35 S promoter. (b) Transgene PCR using genomic DNA of T1 plants. WT, wild type; W, RcWRI1A OE line; M, RcMYB306 OE line; WM, RcWRI1A + RcMYB306 OE line. (c) RT-PCR analysis of transgenes from RNA isolated from developing seeds of T1 plant. (d) Total FAME (µg/mg) of T2 seeds harvested from T1 transformants. Error bars are SD of the mean. Statistical significance is indicated by an asterisk and was evaluated using one-way ANOVA with Tukey’s multiple comparison test (**p < 0.01)

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Transgenic plants transformed with GlyP: RcWRI1A, GlyP: RcMYB306, or GlyP: RcWRI1A + OleP: RcMYB306 vectors were selected from T1 seeds. T1 plants overexpressing (OE) RcWRI1A, RcMYB306, or RcWRI1A + RcMYB306 were designated as W1–W10, M1–M10, and WM1–WM10, respectively. To detect only the transgene and not the endogenous genes, a forward primer was designed to target the rear of the transgene, and a reverse primer was designed to target the front of the terminator (Fig. 2a). PCR of the genomic DNA confirmed the expected 477 bp (RcWRI1A) and 456 bp (RcMYB306) PCR bands in transgenic plants but revealed no PCR bands in wild type (WT) (Fig. 2b).

Four independent plants were randomly selected among the 10 T1 plants in each transgenic plant and RNA was extracted from the developing seeds of the T1 transgenic plants. Then, RT-PCR was performed to assess RcWRI1A and RcMYB306 expression. This analysis confirmed that the two genes were stably expressed in each of the four transformed lines (Fig. 2c). The total FAME content in the seeds was analyzed in transgenic plants. The total FAME per mg in T2 seeds was increased in the three kinds of transgenic plants compared to the WT but was not significantly increased in some of the overexpressed lines (Fig. 2d). Because the T1 generation was not a homozygous plant, W5, W8, M5, M7, WM7, and WM10 lines, which had high transgene expression in developing seeds, were selected and transmitted to the T2 generation plants. The PCR was conducted using gDNA to confirm whether the transgene was inserted in the T2 generation (Fig. S4). Furthermore, two independent lines were selected for the T3 generation.

Fatty acid analysis of RcWRI1A, RcMYB306, and RcWRI1A + RcMYB306 transgenic plants

Fatty acid analysis was performed on T4 seeds derived from T3 plants to investigate whether the RcWRI1A- and RcMYB306-induced oil content increase was maintained in the T3 generation (Fig. 3a, b). Except for RcWRI1A OE #8-2-3 and #8-2-4 lines, the total FAME per mg increased by 14% in the RcWRI1A OE #5-5-1 and #5-5-6 lines compared to that in the WT (Fig. 3a). All transgenic plants in RcMYB306 and RcWRI1A + RcMYB306 OE lines had higher total FAME per mg of seeds (Fig. 3a). RcMYB306 OE lines increased the total FAME per mg by 7–10% compared to the WT (Fig. 3a). In addition, the co-expression of RcWRI1A and RcMYB306 caused 13–17% increase in total FAME per mg in seeds (Fig. 3a). The RcWRI1A, RcMYB306, and RcWRI1A + RcMYB306 OE lines increased the total FAME per seed by 14–21%, 13–19%, and 15–26%, respectively (Fig. 3b).

Fig. 3
figure 3

Fatty acid analysis of T3 transgenic plants. (a) Analysis of total FAME content (µg/mg) in T4 seeds and (b) total FAME content (µg/seed) in T4 seeds. (c) The FAME per seed in T4 seeds compared to the WT. Error bars are SD of the mean. Statistical significance is indicated by an asterisk and was evaluated using one-way ANOVA with Tukey’s multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001)

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Since the total fatty acid content of the three types of overexpression plants increased, the change in fatty acid content per seed was investigated compared to WT (Fig. 3c). The RcWRI1A OE lines significantly increased the 16:0, 18:2, and 18:3 contents in seed oil. In the RcMYB306 OE lines, 18:1, 18:3 contents significantly increased, and 18:2 content slightly increased. In addition, 20:0 and 20:1, which did not change in RcWRI1A OE lines, increased in RcMYB306 OE lines. The RcWRI1A + RcMYB306 OE lines showed changes in fatty acid increase that were somewhat different from those of the RcWRI1A and RcMYB306 OE lines, respectively. The increases in 16:0, 18:2, and 18:3 were similar to RcWRI1A OE lines, but the increases in 18:1 which showed increases such as RcMYB306 OE lines (Fig. 3c). In addition, 20:0 and 20:1 was decreased in RcWRI1A + RcMYB306 OE lines compared that of RcMYB306 OE lines (Fig. 3c). This result showed that seed fatty acid content was increased in the RcWRI1A and RcMYB306 OE lines. There was no significant difference in total FAME but composition of FAME per seed in the RcWRI1A + RcMYB306 OE lines was different compared to the RcWRI1A or RcMYB306 OE lines (Fig. 3). Even though total FAME was increased in RcWRI1A, RcMYB306, and RcWRI1A + RcMYB306 OE lines, seed size was not affected in all lines (Fig. S5).

Regulation of fatty acid and target gene expression by RcWRI1A and RcMYB306 in camelina

The expression of fatty acid biosynthesis genes targeted by each of transcription factors was analyzed between RcWRI1A, RcMYB306, and RcWRI1A + RcMYB306 OE lines (Fig. 4). A comparative analysis was performed on one line of each OE lines. The transgenes (RcWRI1A and RcMYB306) were overexpressed in a seed-specific manner for each OE line (Fig. 4a). In the developing seeds of the RcWRI1A OE line, the expression of camelina BCCP2, KASI, KASII, MAT, PDH and PKP2, which were reported as targets of AtWRI1, was upregulated as expected. In addition, expression of FAD2 which is a desaturase enzyme, was also increased. However, the expression of FAE1, DGAT1, and PDAT1 was downregulated (Fig. 4b, c). In the RcMYB306 OE line, expression of KASI, KASII, MAT, PDH and PKP2 were upregulated similarly to the RcWRI1A OE line, and expression of FATA and FAD3, which were unchanged in the RcWRI1A OE line, were increased. Expression of FAE1 and DGAT1 was also upregulated (Fig. 4b, c). The RcWRI1A + RcMYB306 OE lines showed the expression pattern of mixed genes of RcWRI1A and RcMYB306 OE lines. MAT, PKP2, PDH, and PDAT1 genes showed a greater increase than in the single OE line. However, the expression of KASI, FAD2, and FAE1 was reduced more than that of overexpression of single gene (Fig. 4b, c).

Fig. 4
figure 4

RT-qPCR analysis in developing seeds of transgenic plants compared to the wild type. (a) Expression pattern of RcWRI1A and RcMYB306 in transgenic plants. (b) Expression pattern of fatty acid biosynthesis genes and acyltransferase genes. (c) Upregulated and downregulated genes are indicated by red and blue color in RcWRI1A OE, RcMYB306 OE lines, and RcWRI1A + RcMYB306 lines. Error bars are SD of the mean. Statistical significance is indicated by different letters and was evaluated using one-way ANOVA with Tukey’s multiple comparison test (p < 0.05)

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Discussion

Much research has been performed to improve the oil content in oil crops [33, 34]. One of the strategies for increasing TAG in leaves promote the increase of fatty acid content by transcription factors (Push), transfer fatty acids to TAG through acyltransferases (Pull), and maintain the oil body structure (Protect); this is referred to as the 3P strategy [35, 36]. In this study, push and pull were induced by seed-specific overexpression of RcWRI1A and RcMYB306 in camelina seeds (Fig. 2). Seed-specific overexpression of RcWRI1A and RcMYB306 increased the fatty acid content by 14% and by 7–10% respectively in camelina T4 seeds, with positive effects observed for RcWRI1A and RcMYB306 individual overexpression (Fig. 3). The simultaneous expression of RcWRI1A and RcMYB306 did not more increase seed oil content but resulted in different fatty acid composition compared with that of RcWRI1A or RcMYB306 OE lines (Fig. 3).

Gene expression analysis explains the differences between fatty acid content and fatty acid composition in the RcWRI1A, RcMYB306, and RcWRI1A + RcMYB306 OE lines (Figs. 3 and 4). Expression of PKp2, MAT, KASI, KASIII, ENR, and FATA was significantly downregulated in AtWRI1 mutant [18]. In addition, PKP-β1, BCCP2, ACP1, and KASI are directly regulated by AtWRI1 [37, 38]. In previous report, transient expression of RcWRI1A in Nicotiana benthamiana leave increases the expression of ACP1, PDH-E1α, KASI, BCCP2, PKP-α, PKP-β1 [23]. In our study, RcWRI1A overexpression of camelina led to upregulation expression of PKP2, BCCP2, KASI, which are direct targets of AtWRI1, in addition to increasing the expression of KASII, MAT, PDH (Fig. 4b). When expression of RcWRI1A in N. benthamiana, increase the 18:1, 18:3, 22:0 fatty acids and decrease 18:0 and 20:0 fatty acids in leaves [23]. It showed a different pattern in the seed fatty acid composition of the RcWRI1A OE lines, which has no decrease in 18:0 and 20:0 fatty acids and an increase in 16:0, 18:2, and 18:3 content (Fig. 3c). The 18:2 content increase in RcWRI1A OE lines may be due to the increased expression of FAD2 and the increase in 16:0 is expected to be due to the higher expression of KASI. Unexpectedly, the expression of FAD3 was not upregulated but the 18:3 content was increased (Fig. 4b).

The increase of 20:1 content in RcMYB306 OE lines is presumed to be an increase in FAE1 expression. This result is consistent with the increased expression of FAE1 and 20:1 content in the AtMYB96 overexpression line [39]. In addition, AtMYB96 has been shown to upregulate the expression of DGAT1 and PDAT1 [25]. Expression of DGAT1 was also elevated in RcMYB306 OE lines, but PDAT1 expression was not significantly different from WT (Fig. 4b). When the seed fatty acid composition was examined in AtMYB96 OE lines, the 16:0, 18:0, 18:1, 18:2 and 18:3 contents all increased [25]. This is consistent with the increase in most fatty acid levels in RcMYB306 OE lines (Fig. 3c). In particular, the 18:1 content did not increase in the RcWRI1A OE line but increased in the RcMYB306 OE line. The reason is the increase of DGAT1 expression in the RcMYB306 OE line, which results in an increase of 18:1 content, which is a DGAT1 substrate [40]. In addition, the increase in 18:3 content may be due to increased expression of FAD3. However, the RcMYB306 OE line also showed increased expression of KASI, KASII, MAT, PKP2, PDH, and FATA unlike AtMYB96 OE lines (Fig. 4b).

MYB306 research was conducted in several plants [41,42,43]. Paeonia suffruticosa MYB306 (PsMYB306) negatively regulate bud dormancy by directly binding to the promoter of PsNCED3 and activating it [41]. NtMYB306a is highly expressed in trichomes and is involved in wax alkane biosynthesis in leaves [42]. Apple MdMYB306-like interacts with MdMYB17 and MdbHLH33 and inhibits anthocyanin synthesis [43]. Since MYB306 shows various functions in various crops, additional research in other fields as well as controlling the oil content of RcMYB306 appears to be necessary.

The RcWRI1A + RcMYB306 OE line did not differ significantly from the increase in total fatty acid content of RcWRI1A and RcMYB306 OE lines (Fig. 3). This means that among the fatty acid biosynthesis genes targeted by each transcription factor, the expression of some genes increased more than that of the single OE lines, on the other hand, the expression of some genes decreased more than that of the single OE lines (Fig. 4). This suggests that co-overexpression of the exogenous transcription factors RcWRI1A and RcMYB306 causes the interaction between them, leading to upregulation of some target genes and downregulation of some genes in fatty acid metabolism in camelina (Fig. 4).

In conclusion, we demonstrated the potential of RcWRI1A and RcMYB306 to enhance fatty acid content in camelina seeds when expressed individually. The simultaneous expression of both transcription factors changes the fatty acid composition but not lead to an increase in seed oil content compared to that of RcWRI1A or RcMYB306 OE lines. Complex genetic interactions, metabolic imbalances, and regulatory interference may have contributed to this outcome. Therefore, further investigations are required to address these challenges and optimize strategies for increasing camelina seed oil production.

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

References

  1. Kumar A, Sharma A, Upadhyaya KC (2016) Vegetable Oil: Nutritional and Industrial Perspective. Curr Genomics 17:230–240

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Coleman RA, Lee DP (2004) Enzymes of triacylglycerol synthesis and their regulation. Prog Lipid Res 43:134–176

    Article  CAS  PubMed  Google Scholar 

  3. Ohlrogge JB (1994) Design of New Plant products: Engineering of fatty acid metabolism. Plant Physiol 104:821–826

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Casal S, Malheiro R, Sendas A, Oliveira BP, Pereira JA (2010) Olive oil stability under deep-frying conditions. Food Chem Toxicol 48:2972–2979

    Article  CAS  PubMed  Google Scholar 

  5. Varela G, Ruiz-Roso B (2000) Some Nutritional Aspects of Olive Oil in Handbook of Olive Oil: Analysis and Properties (Harwood, J. & Aparicio, R., eds) 565–582

  6. Asif M (2011) Health effects of omega-3,6,9 fatty acids: Perilla frutescens is a good example of plant oils. Orient Pharm Exp Med 11:51–59

    Article  PubMed  PubMed Central  Google Scholar 

  7. Patel VR, Dumancas GG, Kasi Viswanath LC, Maples R, Subong BJ (2016) Castor Oil: Properties, uses, and optimization of Processing parameters in Commercial Production. Lipid Insights 9:1–12

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Lu C, Napier JA, Clemente TE, Cahoon EB (2011) New frontiers in oilseed biotechnology: meeting the global demand for vegetable oils for food, feed, biofuel, and industrial applications. Curr Opin Biotechnol 22:252–259

    Article  CAS  PubMed  Google Scholar 

  9. Wheeler T, von Braun J (2013) Climate change impacts on global food security. Science 341:508–513

    Article  CAS  PubMed  Google Scholar 

  10. Lu C, Kang J (2008) Generation of transgenic plants of a potential oilseed crop Camelina sativa by Agrobacterium-mediated transformation. Plant Cell Rep 27:273–278

    Article  CAS  PubMed  Google Scholar 

  11. Vollmann J, Eynck C (2015) Camelina as a sustainable oilseed crop: contributions of plant breeding and genetic engineering. Biotechnol J 10:525–535

    Article  CAS  PubMed  Google Scholar 

  12. Li-Beisson Y, Shorrosh B, Beisson F, Andersson MX, Arondel V, Bates PD, Baud S, Bird D, Debono A, Durrett TP, Franke RB, Graham IA, Katayama K, Kelly AA, Larson T, Markham JE, Miquel M, Molina I, Nishida I, Rowland O, Samuels L, Schmid KM, Wada H, Welti R, Xu C, Zallot R, Ohlrogge J (2013) Acyl-lipid metabolism. Arabidopsis Book 11:e0161

    Article  PubMed  PubMed Central  Google Scholar 

  13. Weiss SB, Kennedy EP, Kiyasu JY (1960) The enzymatic synthesis of triglycerides. J Biol Chem 235:40–44

    Article  CAS  PubMed  Google Scholar 

  14. Bates PD (2016) Understanding the control of acyl flux through the lipid metabolic network of plant oil biosynthesis. Biochim Biophys Acta 1861:1214–1225

    Article  CAS  PubMed  Google Scholar 

  15. Ichihara K, Takahashi T, Fujii S (1988) Diacylglycerol acyltransferase in maturing safflower seeds: its influences on the fatty acid composition of triacylglycerol and on the rate of triacylglycerol synthesis. Biochim Biophys Acta 958:125–129

    Article  CAS  PubMed  Google Scholar 

  16. Zou J, Wei Y, Jako C, Kumar A, Selvaraj G, Taylor DC (1999) The Arabidopsis thaliana TAG1 mutant has a mutation in a diacylglycerol acyltransferase gene. Plant J 19:645–653

    Article  CAS  PubMed  Google Scholar 

  17. Dahlqvist A, Stahl U, Lenman M, Banas A, Lee M, Sandager L, Ronne H, Stymne H (2000) Phospholipid: diacylglycerol acyltransferase: an enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants. Proc Natl Acad Sci U S A 97:6487–6492

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. To A, Joubes J, Barthole G, Lecureuil A, Scagnelli A, Jasinski S, Lepiniec L, Baud S (2012) WRINKLED transcription factors orchestrate tissue-specific regulation of fatty acid biosynthesis in Arabidopsis. Plant Cell 24:5007–5023

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Liu J, Hua W, Zhan G, Wei F, Wang X, Liu G, Wang H (2010) Increasing seed mass and oil content in transgenic Arabidopsis by the overexpression of wri1-like gene from Brassica napus. Plant Physiol Biochem 48:9–15

    Article  CAS  PubMed  Google Scholar 

  20. Shen B, Allen WB, Zheng P, Li C, Glassman K, Ranch J, Nubel D, Tarczynski MC (2010) Expression of ZmLEC1 and ZmWRI1 increases seed oil production in maize. Plant Physiol 153:980–987

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. An D, Kim H, Ju S, Go YS, Kim HU, Suh MC (2017) Expression of Camelina WRINKLED1 isoforms rescue the seed phenotype of the Arabidopsis wri1 mutant and increase the Triacylglycerol Content in Tobacco leaves. Front Plant Sci 8:34

    Article  PubMed  PubMed Central  Google Scholar 

  22. Zhang D, Zhao M, Li S, Sun L, Wang W, Cai C, Dierking EC, Ma J (2017) Plasticity and innovation of regulatory mechanisms underlying seed oil content mediated by duplicated genes in the palaeopolyploid soybean. Plant J 90:1120–1133

    Article  CAS  PubMed  Google Scholar 

  23. Ji XJ, Mao X, Hao QT, Liu BL, Xue JA, Li RZ (2018) Splice variants of the Castor WRI1 gene upregulate fatty acid and oil biosynthesis when expressed in Tobacco leaves. Int J Mol Sci 19

  24. Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L (2010) MYB transcription factors in Arabidopsis. Trends Plant Sci 15:573–581

    Article  CAS  PubMed  Google Scholar 

  25. Lee HG, Kim H, Suh MC, Kim HU, Seo PJ (2018) The MYB96 transcription factor regulates Triacylglycerol Accumulation by activating DGAT1 and PDAT1 expression in Arabidopsis seeds. Plant Cell Physiol 59:1432–1442

    CAS  PubMed  Google Scholar 

  26. Li D, Jin C, Duan S, Zhu Y, Qi S, Liu K, Gao C, Ma H, Zhang M, Liao Y, Chen M (2017) MYB89 transcription factor represses seed oil Accumulation. Plant Physiol 173:1211–1225

    Article  CAS  PubMed  Google Scholar 

  27. Kim HJ, Silva JE, Vu HS, Mockaitis K, Nam JW, Cahoon EB (2015) Toward production of jet fuel functionality in oilseeds: identification of FatB acyl-acyl carrier protein thioesterases and evaluation of combinatorial expression strategies in Camelina seeds. J Exp Bot 66:4251–4265

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Onate-Sanchez L, Vicente-Carbajosa J (2008) DNA-free RNA isolation protocols for Arabidopsis thaliana, including seeds and siliques. BMC Res Notes 1:93

    Article  PubMed  PubMed Central  Google Scholar 

  29. Rodríguez-Rodríguez MF, Sánchez-García A, Salas JJ, Garcés R, Martínez-Force E (2013) Characterization of the morphological changes and fatty acid profile of developing Camelina sativa seeds. Ind Crop Prod 50:673–679

    Article  Google Scholar 

  30. Kong Q, Yuan L, Ma W (2019) WRINKLED1, a Master Regulator in Transcriptional Control of Plant Oil Biosynthesis. Plants (Basel) 8

  31. Li W, Wang L, Qi Y, Xie Y, Zhao W, Dang Z, Zhang J (2022) Overexpression of WRINKLED1 improves the weight and oil content in seeds of flax (Linum usitatissimum L). Front Plant Sci 13:1003758

    Article  PubMed  PubMed Central  Google Scholar 

  32. Kang NK, Kim EK, Kim YU, Lee B, Jeong WJ, Jeong BR, Chang YK (2017) Increased lipid production by heterologous expression of AtWRI1 transcription factor in Nannochloropsis salina. Biotechnol Biofuels 10:231

    Article  PubMed  PubMed Central  Google Scholar 

  33. Zafar S, Li YL, Li NN, Zhu KM, Tan XL (2019) Recent advances in enhancement of oil content in oilseed crops. J Biotechnol 301:35–44

    Article  CAS  PubMed  Google Scholar 

  34. Savadi S, Lambani N, Kashyap PL, Bisht DS (2017) Genetic engineering approaches to enhance oil content in oilseed crops. Plant Growth Regul 83:207–222

    Article  CAS  Google Scholar 

  35. Park K, Sanjaya SA, Quach T, Cahoon EB (2021) Toward sustainable production of value-added bioenergy and industrial oils in oilseed and biomass feedstocks. Gcb Bioenergy 13:1610–1623

    Article  CAS  Google Scholar 

  36. Vanhercke T, El Tahchy A, Liu Q, Zhou XR, Shrestha P, Divi UK, Ral JP, Mansour MP, Nichols PD, James CN, Horn PJ, Chapman KD, Beaudoin F, Ruiz-Lopez N, Larkin PJ, de Feyter RC, Singh SP, Petrie JR (2014) Metabolic engineering of biomass for high energy density: oilseed-like triacylglycerol yields from plant leaves. Plant Biotechnol J 12:231–239

    Article  CAS  PubMed  Google Scholar 

  37. Maeo K, Tokuda T, Ayame A, Mitsui N, Kawai T, Tsukagoshi H, Ishiguro S, Nakamura K (2009) An AP2-type transcription factor, WRINKLED1, of Arabidopsis thaliana binds to the AW-box sequence conserved among proximal upstream regions of genes involved in fatty acid synthesis. Plant J 60:476–487

    Article  CAS  PubMed  Google Scholar 

  38. Yang Y, Kong Q, Lim ARQ, Lu S, Zhao H, Guo L, Yuan L, Ma W (2022) Transcriptional regulation of oil biosynthesis in seed plants: current understanding, applications, and perspectives. Plant Commun 3:100328

    Article  PubMed  PubMed Central  Google Scholar 

  39. Lee HG, Park BY, Kim HU, Seo PJ (2015) MYB96 stimulates C18 fatty acid elongation in Arabidopsis seeds. Plant Biotechnol Rep 9:161–166

    Article  Google Scholar 

  40. Cases S, Stone SJ, Zhou P, Yen E, Tow B, Lardizabal KD, Voelker T, Farese RV Jr (2001) Cloning of DGAT2, a second mammalian diacylglycerol acyltransferase, and related family members. J Biol Chem 276:38870–38876

    Article  CAS  PubMed  Google Scholar 

  41. Yuan Y, Zeng L, Kong D, Mao Y, Xu Y, Wang M, Zhao Y, Jiang CZ, Zhang Y, Sun D (2024) Abscisic acid-induced transcription factor PsMYB306 negatively regulates tree peony bud dormancy release. Plant Physiol 194:2449–2471

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Yu J, Lei B, Zhao H, Wang B, Kakar KU, Guo Y, Zhang X, Jia M, Yang H, Zhao D (2022) Cloning, characterization and functional analysis of NtMYB306a gene reveals its role in wax alkane biosynthesis of tobacco trichomes and stress tolerance. Front Plant Sci 13:1005811

    Article  PubMed  PubMed Central  Google Scholar 

  43. Wang S, Zhang Z, Li LX, Wang HB, Zhou H, Chen XS, Feng SQ (2022) Apple MdMYB306-like inhibits anthocyanin synthesis by directly interacting with MdMYB17 and MdbHLH33. Plant J 110:1021–1034

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

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Funding

This work was supported by the Mid-Career Researcher Program (NRF-2020R1A2C2008175), the Basic Research Laboratory Program (RS-2024-00410854) of the National Research Foundation (NRF), and the New Breeding Technologies Development Program (RS-2024-00322277) of the Rural Development Administration (RDA) grants funded by the Korea government (MIST), and the faculty research fund of Sejong University in 2023.

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Authors and Affiliations

  1. Department of Molecular Biology, Sejong University, Seoul, 05006, Republic of Korea

    Mid-Eum Park, Inyoung Kim & Hyun Uk Kim

  2. Department of Bioindustry and Bioresource Engineering, Sejong University, Seoul, 05006, Republic of Korea

    Hye Ji Lee & Hyun Uk Kim

  3. Department of Life Sciences, Sogang University, Seoul, 04107, Republic of Korea

    Mi Chung Suh

  4. Department of Agricultural Biotechnology, National Institute of Agricultural Sciences, Rural Development Administration, Jeonju, 54875, Republic of Korea

    Kyeong-Ryeol Lee

  5. Plant Engineering Research Institute, Sejong University, Seoul, 05006, Republic of Korea

    Hyun Uk Kim

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Contributions

H.U.K supervised the project. H.U.K and M.-E.P designed the research. M.-E.P performed fatty acid analysis, vector construction, camelina transformation. M.-E.P, I.K, H.J.L performed RT-qPCR analysis. H.U.K and M.-E.P wrote paper. H.U.K, M.-E.P, M.C.S, K.-R.L analyzed the data. All authors read and approved the final manuscript.

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Correspondence to Hyun Uk Kim.

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Park, ME., Kim, I., Lee, H.J. et al. Enhancing seed oil content and fatty acid composition in camelina through overexpression of castor RcWRI1A and RcMYB306. Appl Biol Chem 67, 74 (2024). https://doi.org/10.1186/s13765-024-00927-1

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