Characterization of CiWRI1 from Carya illinoinensis in Seed Oil Biosynthesis

: Pecan ( Carya illinoinensis ) is a widely consumed edible woody oil species that is rich in unsaturated fatty acids (FAs) that are beneﬁcial to human health. However, the genes and mechanisms regulating seed oil biosynthesis in pecan are not well understood. Here, we analyzed the expression patterns of genes involved in seed oil biosynthesis in two di ﬀ erent varieties of pecan with distinct fruit maturation schedules and oil contents. We cloned the C. illinoinensis WRINKLED 1 ( CiWRI1 ) gene, a homolog of Arabidopsis WRINKLED1 ( AtWRI1 ), which plays a key role in FA synthesis. Overexpressing CiWRI1 restored lipid synthesis in the Arabidopsis wri1-1 mutant and rescued other phenotypic defects such as plant height, root length, and germination rate, suggesting that CiWRI1 is an ortholog of the AtWRI1 and is involved in the regulation of FA synthesis. To investigate the mechanism of CiWRI1 regulation, we cloned C. illinoinensis BIOTIN CARBOXYL CARRIER PROTEIN ISOFORM 2 ( CiBCCP2 ) and determined that the CiWRI1 protein directly binds to an ASML1 / WRI1 (AW)-box motif in the CiBCCP2 gene promoter and thereby activates its transcription. CiBCCP2 overexpression partly rescued the phenotypic defects of the wri1-1 mutant, indicating that it is directly regulated by CiWRI1. Thus, de novo FA biosynthesis in seed is conserved across plant species; moreover, CiWRI1 regulates oil synthesis by directly controlling CiBCCP2 expression. These ﬁndings present novel potential targets for molecular-marker-assisted breeding of this commercially important plant.


Introduction
Pecan (Carya illinoinensis) is a widely consumed nut and woody oil species with a healthy oil content of 70%, of which 90% are monounsaturated fatty acids (MUFAs) and polyunsaturated FAs (PUFAs), and only a small proportion are saturated FAs (SFAs) [1,2]. In fact, the PUFA/SFA ratio in pecan is similar to that in olive oil, which is considered beneficial to human health [3]. A high MUFA/SFA ratio can reduce cancer incidence [4], and dietary intake of linoleic acid has been shown to reduce the risk of hypertension, coronary heart disease, and type 2 diabetes [5]. In addition to the high content of the essential FA α-linolenic acid, pecans are a rich source of vegetable protein and vitamin E [3], which are important nutrients for nerve cell metabolism and overall brain function.
As modern society becomes more health-conscious, there is increasing demand for healthier natural oils. A major aim of crop breeding is to cultivate new varieties with higher oil content and healthier proportions of various FAs. The oil content in plant seed is largely affected by genotype and to a lesser extent by environmental conditions. Lipid biosynthesis in plants is controlled by a variety of metabolic pathways and involves co-expression of enzymes and their regulatory factors as well as the transport of compounds between plastid, endoplasmic reticulum (ER), cytoplasm, and other subcellular structures. WRINKLED 1 (WRI1) is a transcription factor that plays an important role in plant development [6] by contributing to oil production in maturing seeds of Arabidopsis thaliana through regulation of glycolysis and FA biosynthesis [7][8][9]. Mutation of AtWRI1 in Arabidopsis thaliana results in severe defects in glycolysis and reduces seed oil content by up to 80% [7]; overexpressing WRI1 in the mutant rescues this phenotype [9]. WRI1 homologs have been identified in rapeseed [10,11] and corn; their overexpression in Arabidopsis and low-oil maize was shown to significantly increase seed triacylglycerol (TAG) content [10,12]. WRI1 regulates the transcription of genes encoding the glycolysis enzymes phosphoglycerate mutase, plastidic pyruvate kinase B subunit 1, and pyruvate dehydrogenase along with enzymes involved in FA biosynthesis during seed maturation such as biotin carboxyl carrier protein isoform 2 (BCCP2) and keto-ACP synthase [13]. WRI1 binds to a conserved cis element motif ASML1/WRI1 (AW) -box [14] in the promoter region of these genes as well as that of the SUCROSE SYNTHASE 2 gene in Arabidopsis [14][15][16]. Most research to date on oil production in plants has focused on herbaceous and crop plants, e.g., A. thaliana [17], rapeseed [18], and maize [19] or algae [20], and the molecular mechanisms of FA biosynthesis in woody oil plants is not well understood. Moreover, previous studies in pecan have addressed agronomically important issues such as the optimal method of graft propagation and cultivation. Although the genes involved in pecan FA and lipid synthesis have been studied on the level of transcriptome [2,21], their functions leading to TAG production have not been reported.
To this end, the present study examined the molecular mechanisms of lipid synthesis in pecan by identifying FA biosynthesis gene homologs and analyzing their temporal and spatial expression patterns in two accessions, Pawnee and Mahan, which have different fruit maturation schedules and oil contents. We also analyzed the function of CiWRI1 in lipid biosynthesis and identified its transcriptional target CiBCCP2. Our findings provide novel insight into the molecular mechanisms of lipid synthesis in woody oil plants.

Plant Materials and Growth Conditions
The two accessions of pecan used in the study, Pawnee and Mahan, were both cultivated at the same pecan base in Luzhou Pecan Technology Co. (Shanbei Village, Liuhe District, Nanjing, China). The sampling time was from August to October 2017, with monthly average temperature of 31 • C in August, 27 • C in September, and 21 • C in October, respectively. The annual precipitation of Liuhe District in 2017 was 1255.1 mm, with monthly precipitation 217.1 mm in August, 176.4 mm in September, and 81 mm in October respectively.
The fruit of pecan were frozen in liquid nitrogen and stored at −80 • C. A. thaliana lines were in the Columbia-0 (Col-0) background. The seeds of wri1-1 mutant plants were obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus, OH, USA; Salk: CS69538). Seeds were sterilized with 70% ethanol containing 0.5% Tween-20 for 10 min followed by two washes with 95% ethanol and then air-dried on a clean benchtop. Sterilized seeds were grown at 21 • C under long-day conditions (16:8-h light/dark).

Gene Cloning
To clone the coding sequences of CiWRI1 and CiBCCP2, we search for homologs of the Arabidopsis genes WRI1 and BCCP2 in the pecan RNA-Seq data from our laboratory [22], and primers were designed according to these pecan RNA-Seq data. Sequence data have been deposited through the BankIt portal of the National Center Bioinformatic Institute (NCBI) with accession numbers of MT263946 for CiWRI1 and MT263945 for CiBCCP2. To obtain the 1245 bp promoter sequence of the CiBCCP2, primers were designed according to the unpublished whole genome sequencing data of pecan provided by Professor Youjun Huang in Zhejiang Agriculture and Forestry University.

Plasmid Construction
To construct the p35 S: CiWRI1 and p35 S: CiBCCP2 plasmids, the CiWRI1 and CiBCCP2 coding sequences were amplified by polymerase chain reaction (PCR) using Pawnee cDNA as a template and inserted into the backbone vector downstream of the 35 S promoter. The constructs were used to transform wri1-1 mutant plants. To generate the pCiBCCP2: 3 ×VENUS-NLS vector, a 1245-bp sequence upstream of the ATG of CiBCCP2 was used as the promoter. For p35 S: CiWRI1-green fluorescent protein (GFP) and p35 S: CiBCCP2-GFP, full-length CiWRI1 and CiBCCP2 cDNA from the pENTR vector was used in the Gateway LR recombination reaction. The sequences of primers used in plasmid construction are listed in Table S1.

Total RNA Extraction and Quantitative Reverse Transcription (qRT)-PCR
The miniBEST Plant RNA Extraction Kit (Takara Bio, Otsu, Japan) was used to isolate total RNA from pecan plants. A. thaliana total RNA was isolated using TRIzol reagent (Sigma-Aldrich, St. Louis, MO, USA). The Transcriptor First Strand cDNA Synthesis Kit (Roche Molecular Systems, Pleasanton, CA, USA) was used for cDNA synthesis. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using GoTaq qPCR Master Mix (Promega, Madison, WI, USA) on a PIKO REAL96 Real Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA) under the following conditions: 95 • C for 5 min; 40 cycles of 95 • C for 10 s, 57 • C for 30 s, and 72 • C for 30 s; 72 • C for 10 min; and 20 • C for 10 s. The qRT-PCR primers were designed according to the coding sequences of CiWRI1 and CiBCCP2 obtained in this study. C. illinoinensis ACTIN and A. thaliana TUBULIN genes were used to normalize target transcript levels. Three independent biological replicates were used for qRT-PCR. Primers used for qRT-PCR are listed in Table S1.

Recombinant CiWRI1 Protein Expression and Electrophoretic Mobility Shift Assay (EMSA)
The 6 His-MBP-CiWRI1 vector was transformed into the Escherichia coli Rosetta strain for expression and purification of recombinant CiWRI1 protein. Electrophoretic mobility shift assay (EMSA) was performed using the LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific, USA) according to the manufacturer s instructions, with the probes labeled with biotin. The 10 × and 50 × unlabeled (cold) probes were used as specific competitors. The sequences of primers used in EMSA are listed in Table S1.

Nicotiana Benthamiana Infiltration
To examine the subcellular localization of CiWRI1 and CiBCCP2 proteins, Agrobacterium tumefaciens strains harboring p35 S: CiWRI1-GFP and p35 S: CiBCCP2-GFP were cultured at 28 • C for two days. The cells were harvested by centrifugation at 3500 rpm for 10 min and resuspended in infiltration buffer composed of 10 mM 2-(N-morpholino) ethanesulfonic acid, 10 mM MgCl 2 , and 150 mM acetosyringone (pH 5.8), followed by incubation for 2 h at room temperature. The surface of abaxial leaves of four-week-old N. benthamiana plants was infiltrated with the cell suspension using an injector. The leaves were imaged with a confocal microscope (LSM710; Zeiss, Oberkochen, Germany) after four days. To verify whether CiWRI1 activates CiBCCP2 expression in vivo, the pCiBCCP2: 3 ×VENUS-NLS vector and p35 S: CiWRI1 were co-transformed into tobacco leaves. Three days after transformation, tobacco leaves were used for RNA extraction.

Lipid Analysis
To determine the oil content in seeds of Arabidopsis wild-type, wri1-1 mutants and p35 S: CiWRI1/wri1-1 transgenic plants, fresh seeds were dried 48 h at 65 • C with less than 5% moisture content. Oil content determination was performed with TD-NMR (time-domain nuclear magnetic resonance) using BRUKER minispec-mqone Seed Analyzer (Bruker BioSpin GmbH, Ettlingen, Germany) according to the manufacturer s instructions. Standard curves were obtained from a reference sample of lipids extracted from mature seeds of Arabidopsis thaliana by Soxhlet extraction [23]. Seeds from three individual p35 S: CiWRI1/wri1-1 transgenic plants were used for lipid analysis. The oil of pecan was extracted by Soxhlet extraction and the content was determined as previously described [23].
The oil components were analyzed by gas chromatography [24]. The light yellow oils were obtained by the Soxhlet extraction after evaporation of the organic solvent. We added 3 mL of 0.5 moL/L KOH-methanol solution to a round-bottomed flask, mixed and then connected to a reflux device, heated in a water bath at 100 • C, and the receiver flask was shaken every 30-60 s. After 5 min, 2 mL of 14% boron trifluoride methanol solution was added from the top of the condensate for 5 min, and finally 2 mL of hexane reflux extraction was added for 2 min, cooled to room temperature and transferred to a 20 mL plugged test tube. The samples were analyzed by Shimadzu GC-2010 (Shimadzu Corporation, Kyoto, Japan) gas chromatography with the following conditions: column: DB-WAX 30 M I.D.; inlet temperature: 250 • C; detector temperature: 250 • C; program: 100 • C (3 min), 10 • C/min to 180 • C (1 min), 3 • C/min to 240 • C (9 min); carrier gas (N 2 ) flow rate: 3 mL/min; gas (H 2 ) flow rate: 47 mL/min; fuel gas (air) flow rate: 400 mL/min; shunt ratio: 1:3; injection volume: 1.0 µL.

Statistical Analysis
Differences between two groups were evaluated with the Student s t test. p ≤ 0.05 was considered statistically significant.

Energy Metabolism in Two Varieties of Pecan with Different Mature Periods
Pawnee and Mahan are two popular varieties of pecan differing in terms of fruit maturation schedule and oil content. Under the growth conditions in this study, the fruit maturation time of Pawnee and Mahan was about 152 days after flowering (DAF) and 173 DAF (Table 1, Figure S1), respectively. The early maturing variety Pawnee had higher seed oil and starch contents than late-maturing variety Mahan ( Table 1), suggesting that energy metabolism-including of carbohydrates and lipids-was more active in the early maturing variety Pawnee at the weather conditions we tested in 2017. However, there was a negative correlation between oil and protein contents in pecans, with higher protein levels detected in Mahan than in Pawnee (Table 1). In the critical period of fruit development from early August to mid-September, during which various substances accumulate and undergo transformation in the pecan embryo, there was a rapid increase in oil content from 122 DAF to 132 DAF in Pawnee and from 132 DAF to 152 DAF in Mahan (Figure 1a). The FA compositions in the two varieties were similar, with UFAs including oleic acid and linoleic acid accounting for 90% of the total FA content ( Figure 1b). the two varieties were similar, with UFAs including oleic acid and linoleic acid accounting for 90% of the total FA content ( Figure 1b).

Expression Patterns of Lipid Synthesis Genes in Pecan
The first step of de novo FA biosynthesis in plant plastids is catalyzed by acetyl-coenzyme A (CoA) carboxylase (ACCase), which generates the essential substrate malonyl-CoA from the precursor acetyl-CoA by ATP-dependent carboxylation ( Figure 2a). Following the transfer of the 3carbon malonyl group from malonyl-CoA to the essential thiol of acyl carrier protein (ACP) by malonyl-CoA:ACP transacylase, saturated long-chain FAs (typically 16 carbons or 18 carbons) are synthesized by FA synthase [25] (Figure 2a). Although FA desaturation is initially catalyzed by acyl-ACP desaturase in plastids, additional desaturation and a series of reactions leading to TAG biosynthesis take place in the ER (Figure 2a).

Expression Patterns of Lipid Synthesis Genes in Pecan
The first step of de novo FA biosynthesis in plant plastids is catalyzed by acetyl-coenzyme A (CoA) carboxylase (ACCase), which generates the essential substrate malonyl-CoA from the precursor acetyl-CoA by ATP-dependent carboxylation ( Figure 2a). Following the transfer of the 3-carbon malonyl group from malonyl-CoA to the essential thiol of acyl carrier protein (ACP) by malonyl-CoA:ACP transacylase, saturated long-chain FAs (typically 16 carbons or 18 carbons) are synthesized by FA synthase [25] (Figure 2a). Although FA desaturation is initially catalyzed by acyl-ACP desaturase in plastids, additional desaturation and a series of reactions leading to TAG biosynthesis take place in the ER ( Figure 2a).  Table 1. * p < 0.05, ** p < 0.01; ns, no significant difference.
Given the more starch contents in the Pawnee variety and higher oil accumulation in the early seed development from the range of 122 to 132 DAF (Figure 1a), we analyzed the expression profile of CiWRI1, the C. illinoinensis homolog of Arabidopsis WRI1 that is involved in lipid and carbohydrate metabolism [8]. CiWRI1 level in Pawnee increased dramatically and remained high during the early period of fruit development from 124 DAF to 138 DAF (Figure 2b), in accordance with the faster oil accumulation observed at this stage ( Figure 1a). In contrast, CiWRI1 level increased more slowly in the late-maturing variety Mahan (Figure 2b), consistent with its lower rate of oil accumulation ( Figure   1a). BCCP2 is a subunit of ACCase that catalyzes the first committed step in FA biosynthesis. We observed that, like CiWRI1, CiBCCP2 in the early maturing variety Pawnee was markedly upregulated during early fruit development stages (Figure 2c).

Structural Features of C. Illinoinensis WRI1
Given that WRI1 plays key roles in glycolysis and FA biosynthesis in Arabidopsis and our observation that CiWRI1 was higher expressed in the early maturing Pawnee variety of pecan ( Figure  2b), which was associated with high oil and starch contents in the seed (Figure 1a), we focused our investigation of the molecular mechanism underlying FA biosynthesis in pecan on WRI1. We first cloned the pecan WRI1 homolog CiWRI1, which had an open reading frame of 1194 bp encoding a protein with a predicted length of 397 amino acids, molecular weight of 44.45 kDa, and theoretical isoelectric point of 6.24. An analysis of the deduced amino acid sequence of CiWRI1 revealed two typical APETALA2 (AP2) DNA-binding domains ( Figure 2). Sequence alignment of CiWRI1 with WRI1 from different species including Arabidopsis showed a high degree of homology, especially in  Table S1. * p < 0.05, ** p < 0.01; ns, no significant difference.
Given the more starch contents in the Pawnee variety and higher oil accumulation in the early seed development from the range of 122 to 132 DAF (Figure 1a), we analyzed the expression profile of CiWRI1, the C. illinoinensis homolog of Arabidopsis WRI1 that is involved in lipid and carbohydrate metabolism [8]. CiWRI1 level in Pawnee increased dramatically and remained high during the early period of fruit development from 124 DAF to 138 DAF (Figure 2b), in accordance with the faster oil accumulation observed at this stage ( Figure 1a). In contrast, CiWRI1 level increased more slowly in the late-maturing variety Mahan (Figure 2b), consistent with its lower rate of oil accumulation (Figure 1a). BCCP2 is a subunit of ACCase that catalyzes the first committed step in FA biosynthesis. We observed that, like CiWRI1, CiBCCP2 in the early maturing variety Pawnee was markedly upregulated during early fruit development stages (Figure 2c).

Structural Features of C. Illinoinensis WRI1
Given that WRI1 plays key roles in glycolysis and FA biosynthesis in Arabidopsis and our observation that CiWRI1 was higher expressed in the early maturing Pawnee variety of pecan (Figure 2b), which was associated with high oil and starch contents in the seed (Figure 1a), we focused our investigation of the molecular mechanism underlying FA biosynthesis in pecan on WRI1. We first cloned the pecan WRI1 homolog CiWRI1, which had an open reading frame of 1194 bp encoding a protein with a predicted length of 397 amino acids, molecular weight of 44.45 kDa, and theoretical isoelectric point of 6.24. An analysis of the deduced amino acid sequence of CiWRI1 revealed two typical APETALA2 (AP2) DNA-binding domains ( Figure S2). Sequence alignment of CiWRI1 with WRI1 from different species including Arabidopsis showed a high degree of homology, especially in the two AP2 domains (Figure 3a), suggesting that CiWRI1 is an AP2-type transcription factor belonging to the AP2/ethylene-responsive element-binding protein family of proteins and might has functions similar to other known WRI1-like proteins. In the phylogenetic analysis, CiWRI1 clustered in a subclade comprising five species and showed the highest homology with the WRI1-like genes of Quercus suber (Figure 3b), whose seeds are enriched in starch and oil.
Forests 2020, 11, x FOR PEER REVIEW 7 of 17 the two AP2 domains (Figure 3a), suggesting that CiWRI1 is an AP2-type transcription factor belonging to the AP2/ethylene-responsive element-binding protein family of proteins and might has functions similar to other known WRI1-like proteins. In the phylogenetic analysis, CiWRI1 clustered in a subclade comprising five species and showed the highest homology with the WRI1-like genes of Quercus suber (Figure 3b), whose seeds are enriched in starch and oil.  We next examined the subcellular localization of CiWRI1 in transiently transfected tobacco epidermal cells. N. benthamiana abaxial leaves were infiltrated with A. tumefaciens transformed with p35 S: CiWRI1-GFP and then examined by confocal microscopy. GFP fluorescence was observed in cell nuclei ( Figure S3), consistent with the potential function of CiWRI1 as a transcription factor.

CiWRI1 is Functional Conserved with WRI1 in Arabidopsis
We obtained a wri1-1 mutant of Arabidopsis (Salk: CS69538) from the Arabidopsis Biological Resource Center (ABRC), which harbors a point mutation at the intron-exon border of the first intron (G3197A) (Figure 4a). Seeds of the wri1-1 mutant had a wrinkled appearance and were smaller than the large, round seeds of wild-type plants ( Figure 4b). As previously reported [7][8][9], we observed that the amount of oil stored in seeds was reduced remarkably in the wri1-1 mutant (Figure 4e). We next examined the subcellular localization of CiWRI1 in transiently transfected tobacco epidermal cells. N. benthamiana abaxial leaves were infiltrated with A. tumefaciens transformed with p35 S: CiWRI1-GFP and then examined by confocal microscopy. GFP fluorescence was observed in cell nuclei (Figure 3), consistent with the potential function of CiWRI1 as a transcription factor.

CiWRI1 is Functional Conserved with WRI1 in Arabidopsis
We obtained a wri1-1 mutant of Arabidopsis (Salk: CS69538) from the Arabidopsis Biological Resource Center (ABRC), which harbors a point mutation at the intron-exon border of the first intron (G3197A) (Figure 4a). Seeds of the wri1-1 mutant had a wrinkled appearance and were smaller than the large, round seeds of wild-type plants ( Figure 4b). As previously reported [7][8][9], we observed that the amount of oil stored in seeds was reduced remarkably in the wri1-1 mutant (Figure 4e).  To determine whether CiWRI1 is involved in FA biosynthesis like Arabidopsis WRI1, we transformed the wri1-1 mutant with the p35 S: CiWRI1 vector, which yielded three individual T1 transgenic plants (Figure 4c). RNA was extracted from the leaves for semi-qRT-PCR analysis. We detected CiWRI1 expression in the wri1-1 mutant background in T1 transformants but not in Col-0 wild-type and wri1-1 mutant of Arabidopsis plants (Figure 4d). The seed oil content was markedly reduced in the wri1-1 mutant compared to Col-0 wild-type plants (13% vs. 40%) (Figure 4e). Despite the 35S promoter is known to be constitutive but less effective in seeds [26][27][28][29], however, the decrease of seed oil content in the wri1-1 mutant was rescued by overexpressing CiWRI1, as evidenced by the total oil content of 26% in the seeds of p35 S: CiWRI1/wri1-1 transgenic plants which was almost twice as much as the oil content in the wri1-1 seeds (Figure 4e). These results suggest that CiWRI1 is involved in FA biosynthesis.
WRI1 functions in multiple developmental processes in Arabidopsis [6]. To determine whether the same is true for CiWRI1, we compared other phenotypic features of Col-0, wri1-1, and p35 S: CiWRI1/wri1-1 transgenic plants. Plant height was significantly reduced in the wri1-1 mutant, but was normal in all three p35 S: CiWRI1/wri1-1 transgenic plants (Figure 5a,b). Root growth defects in the mutants were also largely rescued by CiWRI1 overexpression (Figure 5c,d). Seed germination rate was dramatically decreased in the wri1-1 mutant compared to wild-type plants (Figure 5e), but was partly rescued in p35 S: CiWRI1 transgenic plants (Figure 5e). These data suggest that CiWRI1 has the same functionality that AtWRI1. To determine whether CiWRI1 is involved in FA biosynthesis like Arabidopsis WRI1, we transformed the wri1-1 mutant with the p35 S: CiWRI1 vector, which yielded three individual T1 transgenic plants (Figure 4c). RNA was extracted from the leaves for semi-qRT-PCR analysis. We detected CiWRI1 expression in the wri1-1 mutant background in T1 transformants but not in Col-0 wild-type and wri1-1 mutant of Arabidopsis plants (Figure 4d). The seed oil content was markedly reduced in the wri1-1 mutant compared to Col-0 wild-type plants (13% vs. 40%) (Figure 4e). Despite the 35S promoter is known to be constitutive but less effective in seeds [26][27][28][29], however, the decrease of seed oil content in the wri1-1 mutant was rescued by overexpressing CiWRI1, as evidenced by the total oil content of 26% in the seeds of p35 S: CiWRI1/wri1-1 transgenic plants which was almost twice as much as the oil content in the wri1-1 seeds (Figure 4e). These results suggest that CiWRI1 is involved in FA biosynthesis.
WRI1 functions in multiple developmental processes in Arabidopsis [6]. To determine whether the same is true for CiWRI1, we compared other phenotypic features of Col-0, wri1-1, and p35 S: CiWRI1/wri1-1 transgenic plants. Plant height was significantly reduced in the wri1-1 mutant, but was normal in all three p35 S: CiWRI1/wri1-1 transgenic plants (Figure 5a,b). Root growth defects in the mutants were also largely rescued by CiWRI1 overexpression (Figure 5c,d). Seed germination rate was dramatically decreased in the wri1-1 mutant compared to wild-type plants (Figure 5e), but was partly rescued in p35 S: CiWRI1 transgenic plants (Figure 5e). These data suggest that CiWRI1 has the same functionality that AtWRI1.

Cloning of the ACCase Subunit BCCP2
Given that the first step of FA synthesis in Arabidopsis is catalyzed by ACCase, we cloned the pecan homolog of the BCCP2 gene encoding an ACCase subunit. CiBCCP2 was predicted to have an open reading frame of 852 bp and encode a 283-amino acid protein with a molecular weight of 30 kDa and theoretical isoelectric point of 8.50. Analysis of the amino acid sequence revealed an AMKLMN motif ( Figure S4) that is a conserved biotinylation site and functional domain of the BCCP2 subunit [30]. Sequence alignment showed that CiBCCP2 is highly homologous to BCCP2 in other plant species including Arabidopsis, especially the C-terminal AMKLMN sequence (Figure 6a), implying functional conservation with previously identified BCCP2-like proteins in different species. In the phylogenetic analysis, CiBCCP2 clustered in a subclade comprising five species and showed the highest homology to the BCCP2-like gene in Q. suber (Figure 6b) as observed for the pecan homolog of WRI1 gene, providing further evidence for a conserved mechanism of FA synthesis between these two species.

Cloning of the ACCase Subunit BCCP2
Given that the first step of FA synthesis in Arabidopsis is catalyzed by ACCase, we cloned the pecan homolog of the BCCP2 gene encoding an ACCase subunit. CiBCCP2 was predicted to have an open reading frame of 852 bp and encode a 283-amino acid protein with a molecular weight of 30 kDa and theoretical isoelectric point of 8.50. Analysis of the amino acid sequence revealed an AMKLMN motif (Figure 4) that is a conserved biotinylation site and functional domain of the BCCP2 subunit [30]. Sequence alignment showed that CiBCCP2 is highly homologous to BCCP2 in other plant species including Arabidopsis, especially the C-terminal AMKLMN sequence (Figure 6a), implying functional conservation with previously identified BCCP2-like proteins in different species. In the phylogenetic analysis, CiBCCP2 clustered in a subclade comprising five species and showed the highest homology to the BCCP2-like gene in Q. suber (Figure 6b) as observed for the pecan homolog of WRI1 gene, providing further evidence for a conserved mechanism of FA synthesis between these two species.  We examined the subcellular localization of CiBCCP2 in plant cells by infiltration of N. benthamiana abaxial leaves with A. tumefaciens transformed with p35 S: CiBCCP2-GFP. GFP fluorescence of CiBCCP2-GFP was detected in the cytoplasm and colocalized with plastids ( Figure S5).

Regulation of CiBCCP2 Expression by CiWRI1
BCCP2 expression in Arabidopsis has been shown to be regulated by the WRI1 [15]. An analysis of the CiBCCP2 promoter revealed two AW-boxes-i.e., from 271 bp to 284 bp (P1) and from 165 bp to 178 bp (P2) upstream of ATG-that were predicted to bind WRI1 (Figure 7a). Each binding motif of (CnTnG)(n)7(CG) contained conserved CnTnG and CG sequences separated by 7 bp of random nucleotides (Figure 7a). To determine whether CiWRI1 directly regulates CiBCCP2 expression, we expressed CiWRI1 in bacteria and performed EMSA with biotin-labeled probes. CiWRI1 strongly bound to one of the AW-boxes in P1 of the CiBCCP2 promoter and was competed by non-biotinylated competitive probes (Figure 7b), indicating that CiWRI1 directly associates with the CiBCCP2 promoter. We further examined whether CiWRI1 regulates CiBCCP2 transcription by co-expressing the pCiBCCP2: 3 × VENUS and p35 S: CiWRI1 constructs in tobacco epidermal cells. CiBCCP2 expression was markedly enhanced by co-transfection of pCiBCCP2: 3 × VENUS and p35 S: CiWRI1 (Figure 7c). We then evaluated BCCP2 expression in Col-0 wild-type, wri1-1 mutant, and p35 S: CiWRI1/wri1-1 transgenic Arabidopsis plants and found that BCCP2 was downregulated in the mutant (Figure 7d), as previously reported. However, this was reversed in plants by overexpressing CiWRI1 of pecan (Figure 7d). To examine the interaction between CiWRI1 and CiBCCP2 genetically, we generated a construct in which CiBCCP2 was expressed under the control of the 35 S promoter (Figure 8a) and used it to transform wri1-1 mutant of Arabidopsis plants. We obtained three individual T1 transgenic plants and extracted RNA from the leaves for analysis (Figure 8b). CiBCCP2 was highly expressed in T1 transformants but not in Col-0 wild-type and wri1-1 mutant of Arabidopsis (Figure 8c). The decrease in plant height (Figure 8b,d), root defects (Figure 8f,g), and lower germination rate (Figure 8e) in the mutant were partly rescued by overexpressing CiBCCP2. Thus, our data suggest that CiWRI1 directly regulates CiBCCP2 transcription during plant development. To examine the interaction between CiWRI1 and CiBCCP2 genetically, we generated a construct in which CiBCCP2 was expressed under the control of the 35 S promoter (Figure 8a) and used it to transform wri1-1 mutant of Arabidopsis plants. We obtained three individual T1 transgenic plants and extracted RNA from the leaves for analysis (Figure 8b). CiBCCP2 was highly expressed in T1 transformants but not in Col-0 wild-type and wri1-1 mutant of Arabidopsis (Figure 8c). The decrease in plant height (Figure 8b,d), root defects (Figure 8f,g), and lower germination rate (Figure 8e) in the mutant were partly rescued by overexpressing CiBCCP2. Thus, our data suggest that CiWRI1 directly regulates CiBCCP2 transcription during plant development.

Discussion
In the oil biosynthesis among the woody oil plants, so far, only a few species like Jatropha [31][32][33], has been widely studied. However, the study of oil synthesis in other woody plants, especially

Discussion
In the oil biosynthesis among the woody oil plants, so far, only a few species like Jatropha [31][32][33], has been widely studied. However, the study of oil synthesis in other woody plants, especially in edible woody oil plants such as pecan (Carya cathayensis), is quite limited. Although the transcriptome analysis of genes involved in lipid biosynthesis of the pecan has been studied [2,21], the key regulatory genes and their functions involved in fatty acids and oil biosynthesis, and whether it shares a conserved mechanism with other species like in Arabidopsis, are far from clear.
Pecan nut is a kind of high-grade nut that is rich in various microelements, amino acids, unsaturated fatty acids and so on, which is a highly efficient and eco-economic woody oil-bearing tree species with great market competitiveness. In recent years, with the increasing demand of woody oil-bearing tree in the world, the planting area of pecan has been continuously expanded, which need to cultivate excellent varieties to meet the growing market demand. One of the most critical limiting factors is to understand the molecular basis of oil synthesis in pecan and apply the knowledge in molecular assisted breeding.
De novo FAs biosynthesis in plants is catalyzed by a complex network of catalytic enzymes whose expression and activities are tightly controlled. The transcription factor WRI1 plays an important role in lipid biosynthesis by regulating the transcription of BCCP2, which encodes a subunit of ACCase, the enzyme catalyzing the first step of FA biosynthesis [15]. We observed that, in early fruit development, the pecan homologs of WRI1 and BCCP2 were rapidly upregulated and continued to be highly expressed in the early maturing variety of pecan, which was associated with a high oil content (Figure 1a,b). By cloning the full-length open reading frames of CiWRI1 and CiBCCP2 we observed a high degree of homology to WRI1 and BCCP2 in other species (Figures 3 and 6). In particular, the pecan protein of CiWRI1 and CiBCCP2 showed the highest similarity to those in Q. suber, which also belongs to the plant order Fagales.
The critical role of WRI1 in FA biosynthesis was evidenced by the defect in oil storage in seeds of the Arabidopsis wri1-1 mutant, which was rescued by CiWRI1 overexpression (Figure 4e). Over-expressions of Arabidopsis WRI1 have been shown to increase oil accumulating in the oil palm [34] and castor bean [35]. While the WRI1-like genes from Brassica napus [10,36] and soybean [37] were observed positively regulates seed oil accumulation. These data suggested that WRI1-mediated FA biosynthesis is conservative during plant evolution.
WRI1 deficiency was associated with a range of other phenotypes [6] including reduced plant height and root length [38] and low germination rate [39]. These developmental defects were also rescued by CiWRI1 overexpression (Figure 5), demonstrating the functional conservation of CiWRI1 in the regulation of FA biosynthesis and plant development. However, it remains unclear whether the developmental defects in the wri1-1 mutant were caused by impaired FA metabolism. One possibility is that decreased FA biosynthesis and deposition resulted in an inadequate supply of TAG or other metabolic intermediates required for plant growth and development. For instance, seed oil serves as a carbon and energy source that can be converted to glucose via gluconeogenesis for germination and seedling establishment [9]. Alternatively, WRI1 may have additional targets that regulate different processes in plant development. To distinguish between these two scenarios, we overexpressed CiBCCP2 in the wri1-1 mutant background and found that the developmental defects were rescued (Figure 8), suggesting that wri1-1 defects in the development were caused in part by impaired FA metabolism.

Conclusions
In conclusion, our data indicate that de novo FAs biosynthesis in pecan is a conserved process, with similarities to Arabidopsis and other plant species. CiWRI1 contributes to FA and oil synthesis and directly regulates CiBCCP2 transcription. Additionally, we showed that the expression levels of CiWRI1 and CiBCCP2 genes in TAG biosynthesis are higher expressed in the early maturing variety of pecan (Figure 2b,c) with high oil content in the seed (Figure 1a). Thus, lipid biosynthesis genes can serve as targets in molecular marker-assisted breeding strategies to generate improved varieties of pecan.
Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4907/11/8/818/s1, Figure S1 Fruit development in Pawnee and Mahan pecans at different periods. Figure S2 Open reading frame (ORF) and deduced amino acid sequence of CiWRI1. Figure S3 Subcellular localization of CiWRI1. Figure S4 Open reading frame (ORF) and deduced amino acid sequence of CiBCCP2. Figure S5 Subcellular localization of CiBCCP2. Table S1: Primer sequences used this study.
Author Contributions: Z.T., F.P., and X.Z. designed the experiments, analyzed the data and wrote the paper. Y.D. and L.L. performed the electrophoretic mobility shift assays. P.Z. and H.W. carried out the Nicotiana benthamiana infiltration. Y.S. and P.T. assisted with pecan sample collection. X.Z. performed all other experiments. All authors have read and agreed to the published version of the manuscript.