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

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 (18:1 Δ9 ) 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 (18:3 Δ9,12,15 ), an omega-3 fatty acid [6].Castor (Ricinus communis L.) oil contains 80-90% ricinoleic acid (18:1 Δ9 -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 coexpression 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.

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 pBinG-lyRed3-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 DNA-MAN 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% H 2 SO 4 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 StepOne-Plus 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.

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.m000440and 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.m000440and 30,138.m004082were 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.

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 coexpress RcWRI1A and RcMYB306, the two genes were cloned into a single vector containing glycinin and oleosin promoters (Fig. 2a).
Transgenic plants transformed with GlyP: RcWRI1A, GlyP: RcMYB306, or GlyP: RcWRI1A + OleP: RcMYB306 vectors were selected from T 1 seeds.T 1 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 T 1 plants in each transgenic plant and RNA was extracted from the developing seeds of the T 1 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 T 2 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 T 1 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 T 2 generation plants.The PCR was conducted using gDNA to confirm whether the transgene was inserted in the T 2 generation (Fig. S4).Furthermore, two independent lines were selected for the T 3 generation.
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).

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 T 4 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).
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.

Fig. 3
Fig. 3 Fatty acid analysis of T 3 transgenic plants.(a) Analysis of total FAME content (µg/mg) in T 4 seeds and (b) total FAME content (µg/seed) in T 4 seeds.(c) The FAME per seed in T 4 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)

Fig. 4
Fig. 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)