Ectopic Expression of Perilla frutescens WRI1 Enhanced Storage Oil Accumulation in Nicotiana benthamiana Leaves

Vegetable oils are indispensable in human and animal diets and have been widely used for the production of detergents, lubricants, cosmetics, and biofuels. The seeds of an allotetraploid Perilla frutescens contain approximately 35 to 40% oils with high levels of polyunsaturated fatty acids (PUFAs). WRINKELD1 (WRI1) encoding an AP2/ERF-type transcription factor is known to upregulate the expression of genes involved in glycolysis and fatty acid biosynthesis and TAG assembly. In this study, two WRI1 isoforms, PfWRI1A, and PfWRI1B were isolated from Perilla and predominantly expressed in developing Perilla seeds. The fluorescent signals from PfWRI1A:eYFP and PfWRI1B:eYFP driven by the CaMV 35S promoter were detected in the nucleus of the Nicotiana benthamiana leaf epidermis. Ectopic expression of each of PfWRI1A and PfWRI1B increased the levels of TAG by approximately 2.9- and 2.7-fold in N. benthamiana leaves and particularly, the enhanced levels (mol%) of C18:2, and C18:3 in the TAGs were prominent with the concomitant reduction in the amounts of saturated fatty acids. The expression levels of NbPl-PKβ1, NbKAS1, and NbFATA, which were known to be target genes of WRI1, significantly increased in tobacco leaves overexpressing PfWRI1A or PfWRI1B. Therefore, newly characterized PfWRI1A and PfWRI1B can be potentially useful for the enhanced accumulation of storage oils with increased PUFAs in oilseed crops.


Introduction
Plants accumulate triacylglycerols (TAGs), carbon, and energy storage components during seed or fruit development [1]. Storage oils produced from oil palm trees (Elaeis guineesis), soybean (Glycine max), corn (Zea mays), and rapeseed (Brassica napus) have been used for the production of edible oils and as industrial raw materials for manufacturing paints, detergents, lubricants, and cosmetics [2,3]. Recently, the usage of sustainable vegetable oils has been expanded tremendously to dissolve global warming caused by rapid human population growth and the increased ratio of carbon dioxide emitted from the consumption of large-scale fossil fuels [4,5]. To meet the increased demand for vegetable oils, oilseed crops with enhanced storage oil contents have been developed using genetic engineering technologies [6][7][8][9][10]. Therefore, it is a critical step to isolate novel genetic resources to contribute to the enhancement of TAG levels in oilseed crops.
To isolate PfWRI1A and PfWRI1B cDNAs and further examine their subcellular localization, the generated cDNAs were amplified by PCR using SacI-PfWRI1_F1 and XmaI-PfWRI1_R1 primers (Supplementary Table S1). The amplified PfWRI1s PCR products were digested with SacI (5 terminus) and XmaI (3 terminus) and then cloned into the binary vector pPZP212. Two WRI1 isoforms, PfWRI1A, and PfWRI1B were identified after sequencing of the clones. PfWRI1s were translationally fused with eYFP in the pPZP212 vector and expressed under the control of the CaMV promoter. Subsequently, the binary constructs were transformed into Agrobacterium tumefaciens GV3101 using the freeze-thaw method [49]. The transformed Agrobacteria suspended in infiltration media (10 mM MES, pH5.7, 10 mM MgCl 2 , 200 µM acetosyringone) were infiltrated into N. benthamiana leaves. The infiltrated leaves were observed using a confocal laser scanning microscope (Leica TCS SPE, Weitzlar, Germany) 48 h after infiltration. The YFP fluorescence signal was observed at 488 nm excitation and 532 nm emission wavelengths. To visualize the nucleus in the infiltrated leaves, the leaves were stained with DAPI solution (5 µg/mL in PBS) for 5 min, washed 1-2 times with DW, and then the fluorescent signals were obtained at 359 nm excitation and 461 nm emission.

RT-PCR and RT-qPCR Analyses
To investigate the expression of PfWRI1s in various Perilla organs, total RNA was extracted from leaves, stems, roots, and open flowers of approximately 3-month-old Perilla plants using a total RNA isolation kit (Qiagen). RNA was reverse transcribed into cDNA in the same manner as described above, and then the generated cDNA was subjected to the RT-PCR and RT-qPCR analyses using gene-specific primers (Table S1). RT-PCR was performed in a volume of 20 µL with Prime Taq Premix (GENET BIO, Daejeon, Republic of Korea); 1 cycle at 94 • C for 5 min, 28 cycles at 94 • C for 30 s, 61 • C for 30 s, and 72 • C for 30 s, and 1 cycle at 72 • C for 7 min. RT-qPCR was performed in a volume of 20 µL with TOPreal TM SYBR Green qPCR PreMix (Enzynomics, Daejeon, Republic of Korea); 1 cycle at 95 • C for 12 min, 30 cycles at 95 • C for 20 s, 60 • C for 20 s, 72 • C for 20 s, and 1 cycle at 95 • C for 10 s, 65 • C for 60 s, 97 • C for 1 s (CFX96 real-time PCR system, Bio-Rad, Heracles, CA, USA).
To examine the expression of PfWRI1s and their target genes in N. benthamiana leaves expressing PfWRI1A or PfWRI1B, total RNA was isolated from the transformed leaves using a total RNA isolation kit (Qiagen) and gene-specific primers of NbACT, NbBCCP, NbKAS1, NbENR1, NbFATA, NbPI-PKβ1, and NbPDH-E1α genes were designed by BLAST searches of the N. benthamiana genome sequence database (Sol Genomics Network, https: //solgenomics.net/organism/Nicotiana_benthamiana/genome (accessed on 26 November 2022) [51]) using their Arabidopsis orthologs as queries and based on the previous report (Supplementary Table S1) [52]. RT-PCR and RT-qPCR were performed as described above with some modifications. N. benthamiana Actin 2 gene (Niben101Scf06087g02002) was used to determine quantity and quality of cDNAs.

Transient Expression of PfWRI1A and PfWRI1B in N. benthamiana Leaves and Nile Red Staining
Agrobacterium containing PfWRI1A:eYFP, PfWRI1B:eYFP, or empty vector (pPZP212) were cultured to OD 600 = 0.8. Each Agrobacterium was mixed with Agrobacterium cell containing p19 in the infiltration solution, and then infiltrated into 4-5 week-old N. benthamiana leaves. The transformed leaves were stained with Nile red solution (10 µg/mL in 0.1 M Tris-HCl, pH 8.0) at room temperature for 30 min in dark conditions. It was washed twice for 5 min with Tris-HCl buffer (pH 8.0), and oil body formation was observed at 560 nm excitation and 615 nm emission wavelength using a confocal laser scanning microscope (TCS SPE, Leica Microsystems, Weitzlar, Germany).

TLC and GC Analyses
Six days after Agrobacterium infiltration, N. benthamiana leaves were finely ground under liquid nitrogen and freeze-dried and then used for TLC analysis. The dry residues (~5 mg) were mixed with glyceryl triheptadecanoate (C17:0) internal standard (15 µg/mL) in 1.5 mL of chloroform: methanol (2:1 = v/v) by vigorous vortexing and then mixed with 500 µL of 0.1 M KCl. After centrifugation, the lower lipid phase was concentrated under nitrogen gas and dissolved in 100 µL of chloroform. The extracted lipids were loaded on a TLC plate (Kieselgel 60, MERC) and separated in hexane: diethyl ether: acetic acid (70:30:1, v/v/v). After the TLC plate was sprayed with 80% acetone containing 0.01% primuline, the bands corresponding to TAGs were visualized under UV light. The marked TAG bands were scraped off, mixed with toluene and 1 mL of 5% H 2 SO 4 in methanol, and then methyl-esterified at 90 • C for 90 min. After 1.5 mL of aqueous 0.9% NaCl (w/v) was added, fatty acid methyl esters (FAMEs) were extracted with 2 mL of hexane three times. The concentrated FAMEs were analyzed on a GC-2010 (Shimadzu, Kyoto, Japan) equipped with flame ionization detector (FID). DB-23 column (30 mm × 0.25 mm, 0.25 µm film thickness; J&W Scientific, Folsom. CA, USA) was used and GC conditions were set to increase at a rate of 2.5 • C per minute from 160 • C to 220 • C. For each FAMEs, retention time and peak areas of internal standards were compared and analyzed. The composition and amount of FAMEs were analyzed by comparison of their retention times and peak areas with those of internal and individual standards.

Nucleotide and Amino Acid Sequences of P. frutescens WRI1 Isoforms and Phylogenetic Tree in WRI1 Orthologs from Various Plant Resources
To isolate WRI1 orthologs in P. frutescens, total RNAs were isolated from developing Perilla seeds and then the converted cDNAs were subjected to the RT-PCR using PfWRI1 gene-specific primers (Supplementary Table S1). When the PCR products were sequenced, two 1200-nucleotide-long and 1197-nucleotide-long WR1 isoforms were isolated and named PfWRI1A and PfWRI1B, respectively ( Figure S1). The nucleotide sequence similarity between PfWRI1A and PFWRI1B was very similar (about 97%), suggesting that the two isoforms are very well conserved ( Figure S2). Next, the deduced amino acid sequences of PfWRI1A and PfWRI1B were compared with those of WRI1 orthologs from various plant resources including an Arabidopsis WRI1, and their phylogenetic tree was constructed. As shown in an Arabidopsis WRI1, both PfWRI1A and PfWRI1B harbor two AP2/EREBP DNA binding motifs and an 'IYL' motif instead of 'VYL', which is known to be a transcriptional activation motif in AtWRI1 ( Figures 1A and S2) [32]. The 17th and 18th serine residues in PfWRI1B were deleted in PfWRI1A, whereas thee amino acid residues corresponding to proline, serine, and serine at 384th to 386th in PfWRI1A were not present in PfWRI1B ( Figure 1A). The proline and serine motifs (or PEST motifs), which were reported to be involved in the regulation of WRI1 [53] were searched by ePESTfind (https://emboss.bioinformatics.nl/cgi-bin/emboss/epestfind (accessed on 1 June 2000) and then the PEST motifs were detected in the N-and C-terminal regions of Arabidopsis and Camelina WRI1s, but in the middle region of Perilla WRI1A and WRI1B ( Figure 1A). The putative amino acid residues, which correspond to the phosphorylation sites in the PEST motifs of AtWRI1 [53] were also shown in PfWRIs and CsWRI1s ( Figure 1A), but their regulatory roles in PfWRIs and CsWRI1s remain to be further investigated. In the search of plant WRI1s, we found that one WRI1 isoform was detected from various crops examined, except Zea mays (ZmWRI1a and ZmWRI1b) and Camelina sativa (CsWRI1 A, B, and C). In the phylogenetic tree of plants, PfWRI1s and the clade including ZmWRI1, Avena sativa AsWRI1, Cyperus esculentus CeWRI1, EgWRI1, and Ostreococcus lucimarinus Ol3404 were divided after the clade containing Brassica WRI1s were divided from the ancestor ( Figure 1B). The deduced amino acid sequence similarity of WRIs orthologs from various plant species are shown in Supplementary Table S2.

Expression of PfWRI1A and PfWRI1B in Various Perilla Organs
To examine the transcript levels of PfWRI1A and PfWRI1B in various Perilla organs, total RNAs were isolated from rosette leaves, stems, roots, open flowers, and developing seeds of P. frutescens plants, and subjected to the RT-PCR and RT-quantitative PCR analyses. Both RT-PCR and RT-qPCR analyses revealed that PfWRI1s were predominantly ex-pressed in developing Perilla seeds (Figure 2A,B). Initially, we tried to design PfWRI1Aor PfWRI1Bspecific primers. The design of PfWRI1B-specific primers was successful, but it failed to design PfWRI1A-specific primers due to a high sequence homology between PfWRI1A and PfWRI1B ( Figure S2). Thus, the total transcript levels of PfWRI1A and PfWRI1B were shown in Figure 2B To obtain the binary vector constructs containing PfWRI1A:eYFP or PfWRI1B:eYFP, the coding regions of the isolated PfWRI1A and PfWRI1B were fused in-frame eYFP under the control of CaMV 35S promoter in the pPZP212 binary vector ( Figure 3A). The PfWRI1A:eYFP and PfWRI1B:eYFP constructs were transformed into Agrobacterium (GV3101 strain), and the transformed Agrobacteria were infiltrated into the N. benthamiana leaves. The leaves containing the Agrobacterium-infiltrated sites were stained with a DAPI solution 48 h after infiltration and then observed using a confocal laser microscope. The fluorescent signals from both PfWRI1A:eYFP or PfWRI1B:eYFP constructs were merged with the DAPI signals, with the DAPI signals, indicating that PfWRI1A and PfWRI1B are localized in the nucleus in N. benthamiana leaf epidermal cells ( Figure 3B) and might play a role as a transcription factor.

Oil Body Formation and TAG Measurement in N. benthamiana Leaves Expressing PfWRI1A or PfWRI1B
To investigate the functional activities of PfWIR1A and PfWRI1B, Agrobacteria containing PfWRI1A:eYFP or PfWRI1B:eYFP driven by the CaMV 35S promoter were infiltrated in N. benthamiana leaves. The leaves containing the Agrobacterium-infiltrated sites were stained with Nile red solution 6 d after infiltration and then oil bodies were observed under confocal microscopy. Ectopic expression of PfWIR1A or PfWRI1B caused the formation of oil bodies in N. benthamiana leaves, but no signals corresponding to oil bodies were observed in N. benthamiana leaves infiltrated with the pPZP212 binary vector without PfWRI1 ( Figure 4A). The number of oil bodies significantly increased in the leaves expressing PfWIR1A or PfWRI1B relative to the control, indicating that the PfWRI1A and PfWRI1B may contribute to the accumulation of storage oils in N. benthamiana leaves (Figure 4B).
Subsequently, the amount of TAGs accumulated in the oil bodies in N. benthamiana leaves expressing PfWRI1A or PfWRI1B was measured by TLC and GC with a flame ionization detector. Briefly, the N. benthamiana leaves expressing PfWRI1A or PfWRI1B were lyophilized and then subjected to the extraction of total lipids. After the chloroform-extracted lipids were separated by TLC and visualized with 0.01% primuline under UV light (Supplementary Figure S3), the bands corresponding to the TAGs were eluted, and then further analyzed by GC. The levels of total FAMEs in N. benthamiana leaves expressing PfWRI1A and PfWRI1B increased approximately 2.9-and 2.7-fold relative to the control transformed with the pPZP212 binary vector without PfWRI1, respectively ( Figure 4C). In

Oil Body Formation and TAG Measurement in N. benthamiana Leaves Expressing PfWRI1A or PfWRI1B
To investigate the functional activities of PfWIR1A and PfWRI1B, Agrobacteria containing PfWRI1A:eYFP or PfWRI1B:eYFP driven by the CaMV 35S promoter were infiltrated in N. benthamiana leaves. The leaves containing the Agrobacterium-infiltrated sites were stained with Nile red solution 6 d after infiltration and then oil bodies were observed under confocal microscopy. Ectopic expression of PfWIR1A or PfWRI1B caused the formation of oil bodies in N. benthamiana leaves, but no signals corresponding to oil bodies were observed in N. benthamiana leaves infiltrated with the pPZP212 binary vector without PfWRI1 ( Figure 4A). The number of oil bodies significantly increased in the leaves expressing PfWIR1A or PfWRI1B relative to the control, indicating that the PfWRI1A and PfWRI1B may contribute to the accumulation of storage oils in N. benthamiana leaves ( Figure 4B).
Subsequently, the amount of TAGs accumulated in the oil bodies in N. benthamiana leaves expressing PfWRI1A or PfWRI1B was measured by TLC and GC with a flame ionization detector. Briefly, the N. benthamiana leaves expressing PfWRI1A or PfWRI1B were lyophilized and then subjected to the extraction of total lipids. After the chloroformextracted lipids were separated by TLC and visualized with 0.01% primuline under UV light (Supplementary Figure S3), the bands corresponding to the TAGs were eluted, and then further analyzed by GC. The levels of total FAMEs in N. benthamiana leaves expressing PfWRI1A and PfWRI1B increased approximately 2.9-and 2.7-fold relative to the control transformed with the pPZP212 binary vector without PfWRI1, respectively ( Figure 4C). In fatty acid composition analysis, the levels of all fatty acid components except C16:1 significantly increased in the leaves expressing PfWRI1A:eYFP or PfWRI1B:eYFP compared with the control expressing eYFP ( Figure 4D). The ratio of polyunsaturated fatty acids, fatty acid composition analysis, the levels of all fatty acid components except C16:1 significantly increased in the leaves expressing PfWRI1A:eYFP or PfWRI1B:eYFP compared with the control expressing eYFP ( Figure 4D). The ratio of polyunsaturated fatty acids, C18:2 and C18:3 were elevated by approximately 2-and 3-fold, but the proportions of saturated fatty acids, C16:0, C18:0, and C24:0 decreased by approximately 22%, 34%, and 50% in N. benthamiana leaves expressing PfWRI1A and PfWRI1B compared with the control, respectively ( Figure 4E). Taken together, ectopic expression of each of PfWRI1A and PfWRI1B enhanced the levels of TAG with unsaturated fatty acids (C18:2, and C18:3) in N. benthamiana leaves.

Induced Expzression of WRI1 s Target Genes in N. benthamiana Leaves Expressing PfWRI1A or PfWRI1B
To investigate whether or not the ectopic expression of PfWRI1A and PfWRI1B upregulates the expression of WRI1's target genes, total RNAs were extracted from N. benthamiana leaves expressing PfWRI1A or PfWRI1B 2 d after Agrobacterium infiltration and converted to the cDNAs. The synthesized cDNAs were subjected to RT-PCR and RT-qPCR analyses using the gene-specific primers, which were searched from the N. benthamiana transcriptome database (https://solgenomics.net/organism/Nicotiana_benthamiana/genome (accessed on 26 November 2014). N. benthamiana leaves transformed with the pPZP212 binary vector without PfWRI1 were used as a control. In RT-PCR analysis, we observed the induced expression of NbBCCP2, NbKAS1, and NbPl-PKβ1 in N. benthamiana leaves expressing PfWRI1A or PfWRI1B relative to the control, but no significant differences in the levels of NbENR, NbFATA, and NbPDH-E1α transcripts were observed between N. benthamiana leaves expressing PfWRI1A or PfWRI1B and the control leaves ( Figure 5A). We further measured the levels of PfWRI1, NbBCCP2, NbKAS1, and NbPl-PKβ1 transcripts by RT-qPCR. The NbACT was used for the determination of the quantity and quality of cDNAs. As shown in Figure 5B, the expression of NbPl-PKβ1 transcripts was upregulated by approximately 15-and 16-fold in N. benthamiana leaves expressing PfWRI1A and PfWRI1B relative to the control, respectively. The expression of NbBCCP2 and NbKAS1 was also elevated by approximately two-to three-fold in N. benthamiana leaves expressing PfWRI1A or PfWRI1B compared with the control. The results revealed that PfWRI1A and PfWRI1B are able to accumulate storage oils in N. benthamiana leaves by the upregulation of their target genes such as NbBCCP2, NbKAS1, and NbPl-PKβ1.
PfWRI1A:eYFP, PfWRI1B:eYFP, or empty vector. Total lipids were extracted from the transformed N. benthamiana leaf disks and fractionated by thin-layer chromatography. The TAG fractions were eluted, transmethylated, and then the fatty acid methyl esters were analyzed by gas chromatography. Values are averages and SD of four individual experiments. Data were statistically analyzed using ANOVA test (p < 0.05).

Induced Expzression of WRI1′s Target Genes in N. benthamiana Leaves Expressing PfWRI1A or PfWRI1B
To investigate whether or not the ectopic expression of PfWRI1A and PfWRI1B upregulates the expression of WRI1's target genes, total RNAs were extracted from N. benthamiana leaves expressing PfWRI1A or PfWRI1B 2 d after Agrobacterium infiltration and converted to the cDNAs. The synthesized cDNAs were subjected to RT-PCR and RT-qPCR analyses using the gene-specific primers, which were searched from the N. benthamiana transcriptome database (https://solgenomics.net/organism/Nicotiana_benthamiana/genome (accessed on 26 November 2014). N. benthamiana leaves transformed with the pPZP212 binary vector without PfWRI1 were used as a control. In RT-PCR analysis, we observed the induced expression of NbBCCP2, NbKAS1, and NbPl-PKβ1 in N. benthamiana leaves expressing PfWRI1A or PfWRI1B relative to the control, but no significant differences in the levels of NbENR, NbFATA, and NbPDH-E1 transcripts were observed between N. benthamiana leaves expressing PfWRI1A or PfWRI1B and the control leaves (Figure 5A). We further measured the levels of PfWRI1, NbBCCP2, NbKAS1, and NbPl-PKβ1 transcripts by RT-qPCR. The NbACT was used for the determination of the quantity and quality of cDNAs. As shown in Figure 5B, the expression of NbPl-PKβ1 transcripts was upregulated by approximately 15-and 16-fold in N. benthamiana leaves expressing PfWRI1A and PfWRI1B relative to the control, respectively. The expression of NbBCCP2 and NbKAS1 was also elevated by approximately two-to three-fold in N. benthamiana leaves expressing PfWRI1A or PfWRI1B compared with the control. The results revealed that PfWRI1A and PfWRI1B are able to accumulate storage oils in N. benthamiana leaves by the upregulation of their target genes such as NbBCCP2, NbKAS1, and NbPl-PKβ1.  Total RNA was isolated from N. benthamiana leaves expressing PfWRI1A, PfWRI1B, or empty vector control and converted to the cDNA, which was subjected to the RT-PCR analysis. N. benthamiana Actin2 was used to determine cDNA quantity and quality. (B) RT-qPCR analysis of its target genes in N. benthamiana leaves expressing PfWRI1A or PfWRI1B. NbActin2 was used to normalize the levels of PfWRI1s and their target genes' transcripts. Each value is the mean of three independent measurements ± standard error. Data were statistically analyzed using ANOVA test (p < 0.01). * ND indicates non-detected.

Discussion
Since the usage of vegetable oils has increased tremendously as a sustainable and alternative energy resource, it is known to be the critical step in elevating oil content in oilseed crops [2][3][4]. Vanhercke et al. [54] reported that the "Push-Pull-Protect" module was suggested to increase storage oil deposition in genetically engineered seeds or leaves by an increase in the production of fatty acids in the plastids (Push), an increase in TAG assembly and accumulation (Pull), and a decrease in TAG hydrolysis or catabolism (Protect). WRINKLED1 (WRI1) is well known as a "Push" factor to activate fatty acid biosynthesis by the upregulation of genes involved in glycolysis and fatty acid biosynthesis and thereby enhances TAG accumulation [28][29][30]. In this study, we identified a noble genetic resource, P. frutescens WRI1 genes, which contribute to the increased accumulation of storage oils in N. benthamiana leaves.
Since the WRI1 was first reported from Arabidopsis (Cernac and Benning, 2004), several WRI1 orthologs have been characterized from various monocot and dicot plants including Brassica napus [55], Camelina sativa [37], Zea mays [35,56], Jatropha curcas [38], and Oryza sativa [57]. In the previous report [42], the WRI1 orthologs from Arabidopsis (AtWRI1), potato (StWRI1), oat (AsWRI1), poplar (PtWRI1), and nutsedge (CeWRI1) were transiently expressed in tobacco leaves and then a significant increase in TAG accumulation was observed in all transformed leaves relative to the control. Ectopic expression of C. sativa WRI1A, B, or C induced the formation of oil bodies in N. benthamiana leaves and eventually increased TAG levels by approximately 2.5-to 4.0-fold in the leaves compared to the control [37]. The formation of oil bodies was also observed in N. benthamiana leaves expressing PfWRI1A or PfWRI1B by the Nile red staining ( Figure 4A). In the tobacco leaves expressing PfWRI1A and PfWRI1B, the levels of TAGs were significantly elevated relative to the control ( Figure 4B), indicating that the PfWRI1A and PfWRI1B enhance storage oil deposition in leaves, which have been used for the production of vegetable oils as an alternative non-seed organ [48].
In the fatty acid composition in the TAG fractions accumulated in N. benthamiana leaves expressing AtWRI1, StWRI1, AsWRI1, PtWRI1, or CeWRI1, interestingly the ratio of C18:3 were increased, but the proportions of C18:0 were remarkably decreased in all transgenic leaves compared to transformed control [42]. An et al. [37] reported that the levels (mol%) of C18:1 were increased, but the amounts of C18:0 were decreased in the TAG fractions isolated from leaves expressing CsWRI1A, B, or C. Interestingly, we observed an increase and a decrease in the levels (mol%) of polyunsaturated fatty acid (C18:2 and C18:3) and saturated fatty acids (C16:0, C18:0, and C24:0), respectively, in the TAG fractions induced by the overexpression of PfWRI1A or PfWRI1B compared to the transformed control ( Figure 4C). A similar observation was also reported in N. benthamiana leaves expressing RcWRIA or RcWRIB [52]. Therefore, the overexpression of plant WRI1s can alter the ratio of saturated and unsaturated fatty acids in the TAGs accumulated in leaves and they are possibly useful for the production of storage oils with high unsaturated fatty acids in vegetative organs.
Several WRI1 orthologs including PfWRI1 showed their predominant expression in developing seeds or oil-rich non-seed tissues (Figure 2) [58], indicating that they play a crucial role in the deposition of seed storage materials. WRI1s were known to be a key transcriptional regulator, which activates the expression of several genes involved in late steps of glycolysis, fatty acid biosynthesis, and TAG assembly such as BCCP2, Pl-PKβ1, PDHE1α, enoyl-ACP reductase (EAR), ACP1, KAS1, and DGAT1 [29][30][31]38]. The expression of CsWRIs caused the upregulation of BCCP2 and Pl-PKβ1 in developing seeds of Arabidopsis wri1-3 [37]. Transient expression of RcWRI1-A or RcWRI1-B in N. benthamiana leaves significantly activated the expression of ACP1, PDHE1α, KAS1, BCCP2, Pl-PKβ1, and PlPKα [52]. We also observed that the expression of Pl-PKβ1, BCCP2, and KAS1 was significantly enhanced in N. benthamiana leaves by the expression of PfWRI1s ( Figure 5).
Arabidopsis WRI1 and its orthologs were reported to be localized in the nucleus [36][37][38]40,59,60], and the fluorescent signals from PfWRI1A:eYFP and PfWRI1B:eYFP were also observed in the nucleus (Figure 3). When the nuclear localization signal (NLS) sequences were searched on the website (https://www.novoprolabs.com/tools/nls-signalprediction)), the "PRPKRAKRA" motif was predicted in AtWRI1 protein. In the case of PfWRI1A and PfWRI1B, the "VKPKPKRVRAK" motif might be important to be localized in the nucleus. In addition, mutations in any of the three "VYL" residues, which are present in the first AP2 domain of AtWRI1 partially rescued the low seed oil phenotype of wri1-1 to the wild type and mutations in all three residues failed to restore the fatty acid content of wri1-1, indicating that the motif is essential for the function of AtWRI1 [36]. However, Ji et al. [52] reported that both RcWRI1A with the VYL motif and RcWRI1B without the VYL motif were functionally active and restored the wrinkled seed phenotype of wri1-1. Although PfWRI1s also contain the "IYL" motif instead of "VYL" (Figure 1A), the role of the "IYL" motif remains to be further investigated in the transcriptional regulation.

Conclusions
Transcriptome and genetic resources from Perilla [46,47] enabled us to isolate two WRI1 isoforms, PfWRI1A and PfWRI1B genes from developing Perilla seeds. Ectopic expression upregulated the expression of their target genes in N. benthamiana leaves and stimulated storage oil accumulation. These findings can be applied to the production of sustainable and renewable storage oils in leafy biomass to meet the increasing demand for their usage.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/plants12051081/s1, Table S1: Primers used in this study. Table S2: The deduced amino acid sequence similarity of WRIs orthologs from various plant species. Figure S1: Nucleotide and deduced amino acid sequences of P. frutescens WRI1A and WRI1B isoforms. Figure S2: Design of PfWRI1Aor PfWRI1B-specific primers. Figure   Data Availability Statement: All data supporting the finding of this study are available within the paper and its Supplementary Materials published online.