Characterization of Ciwri1 from Carya Illinoinensis in Seed Oil Biosynthesis

Article Characterization of CiWRI1 from Carya illinoinensis in Seed Oil Biosynthesis

Xiaofeng Zhou 1 , Yuqiu Dai 2, Haijun Wu 2 , Peiqiao Zhong 3, Linjie Luo 4, Yangjuan Shang 1, Pengpeng Tan 1, Fangren Peng 1,* and Zhaoxia Tian 2,* 1 Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Longpan Road 159, Nanjing 210037, China; [email protected] (X.Z.); [email protected] (Y.S.); [email protected] (P.T.) 2 School of Life Sciences, University of Science and Technology of China, Huangshan Road 443, Hefei 230027, China; [email protected] (Y.D.); [email protected] (H.W.) 3 College of Life Sciences, Zhaoqing University, Zhaoqing Road, Zhaoqing 526061, China; [email protected] 4 College of Life Sciences, Anhui Normal University, Wuhu 241000, Anhui, China; [email protected] * Correspondence: [email protected] (F.P.); [email protected] (Z.T.); Tel.: +86-025-85427995 (F.P.); +86-0551-63600640 (Z.T.) Received: 16 June 2020; Accepted: 23 July 2020; Published: 28 July 2020

Abstract: 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 different 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.

Keywords: pecan oil content; fatty acid synthesis; CiWRI1; CiBCCP2

1. 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.

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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 [7–9]. 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 [14–16]. 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.

2. Materials and Methods

2.1. 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). Forests 2020, 11, 818 3 of 16

2.2. 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.

2.3. 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.

2.4. 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.

2.5. 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 0 × × (cold) probes were used as specific competitors. The sequences of primers used in EMSA are listed in Table S1.

2.6. 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 MgCl2, 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 × Forests 2020, 11, 818 4 of 16 vector and p35 S: CiWRI1 were co-transformed into tobacco leaves. Three days after transformation, tobacco leaves were used for RNA extraction.

2.7. 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 manufacturer0s 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 (N2) flow rate: 3 mL/min; gas (H2) flow rate: 47 mL/min; fuel gas (air) flow rate: 400 mL/min; shunt ratio: 1:3; injection volume: 1.0 µL.

2.8. Statistical Analysis Diﬀerences between two groups were evaluated with the Student s t test. p 0.05 was considered 0 ≤ statistically signiﬁcant.

3. Results

3.1. 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 (Table1, Figure S1), respectively. The early maturing variety Pawnee had higher seed oil and starch contents than late-maturing variety Mahan (Table1), 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 (Table1). 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 (Figure1a). 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 (Figure1b). Forests 2020, 11, 818 5 of 16

Table 1. Characteristics of Pawnee and Mahan varieties of pecan.

Maturation Seed Oil Soluble Sugar Protein Content Starch Content Variety Name Forests 2020, 11, x FOR PEER REVIEWTime(DAF) Content (%) Content (%) (mg/g) (mg/g) 5 of 17 Early Pawnee 152 1.41 74.43 1.32 1.68 0.01 2.11 0.16 17.76 4.36 maturing ± ± ± ± ± theLate two maturing varieties Mahan were similar, 173 0.82 with *** UFAs 71.59 including0.79 * oleic 1.75 acid0.01 and linoleic 3.85 acid0.54 *accounting 7.09 2.18for **90% of the total FA content (Figure± 1b). ± ± ± ± * p < 0.05, ** p < 0.01, *** p < 0.001 (Student0s t test). DAF, days after ﬂowering.

Figure 1. Fruit oil content and fatty acid (FA)(FA) composition inin twotwo pecan varieties.varieties. (a)) Seed oil contents of early maturingmaturing PawneePawnee and late-maturinglate-maturing Mahan varieties from 98 days after ﬂoweringflowering (DAF) toto 152152 DAFDAF (Pawnee)(Pawnee) oror 173173 DAFDAF (Mahan).(Mahan). N == 3 for each variety and time point, and 5 g samplesample waswas

used forfor eacheach analysis.analysis. *** pp << 0.001,0.001, Student0′ss t--test;test; ns, no significantsignificant didifference.fference. (b)) PercentagesPercentages ofof didifferentfferent FAFA speciesspecies inin seedsseeds ofof PawneePawnee andand Mahan.Mahan.

3.2. Expression PatternsPatterns of LipidLipid SynthesisSynthesis GenesGenes in PecanPecan The ﬁrstfirst stepstep ofof dede novonovo FAFA biosynthesisbiosynthesis inin plantplant plastidsplastids isis catalyzedcatalyzed byby acetyl-coenzymeacetyl-coenzyme A (CoA)(CoA) carboxylase carboxylase (ACCase), (ACCase), which which generates generates the essential the essential substrate substrate malonyl-CoA malonyl from-CoA the precursor from the acetyl-CoAprecursor acetyl by ATP-dependent-CoA by ATP carboxylation-dependent carboxylation (Figure2a). Following (Figure 2a the). Following transfer of thethe3-carbon transfer malonylof the 3- groupcarbonfrom malonyl malonyl-CoA group from to the malonyl essential-CoA thiol to the of acyl essential carrier thiol protein of acyl (ACP) carrier by malonyl-CoA:ACP protein (ACP) by transacylase,malonyl-CoA:ACP saturated transacylase, long-chain saturated FAs (typically long-chain 16 carbons FAs (typically or 18 carbons) 16 carbons are synthesizedor 18 carbons) by are FA synthasesynthesized [25 ]by (Figure FA synthase2a). Although [25] (Figure FA desaturation 2a). Although is initiallyFA desaturation catalyzed is by initially acyl-ACP catalyzed desaturase by acyl in- plastids,ACP desaturase additional in desaturation plastids, additional and a series desaturation of reactions leadingand a series to TAG of biosynthesis reactions leading take place to in TAG the ERbiosynthesis (Figure2a). take place in the ER (Figure 2a).

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FigureFigure 2. Expression 2. Expression patterns patterns of genesof genes involved involved in in FAs FAs biosynthesis biosynthesis in in pecans. pecans. (a ()a Working) Working model model of of triacylglyceroltriacylglycerol (TAG)(TAG) biosynthesisbiosynthesis pathway in in plants. plants. OB, OB, oil oil body. body. (b,c ()b Expression,c) Expression levels levels of CiWRI1 of CiWRI1(b) and(b) and CiBCCP2CiBCCP2 (c)( c in) in different different pecan pecan varieties quantified quantified by by quantitative quantitative reverse reverse transcription transcription polymerasepolymerase chain chain reaction reaction (qRT-PCR) (qRT-PCR with) wi threeth three biological biological replicates. replicates The. TheCarya Carya illinoinensis illinoinensis ACTIN ACTIN genegene was was used used to normalizeto normalize target target mRNA mRNA levels. levels. Primers Primers used used for for qRT-PCR qRT-PCR are are listed listed in in Table Table S1. 1. * p * p << 0.05,0.05, **** pp << 0.01;0.01; ns, no significantsignificant didifference.fference.

GivenGiven the the more more starch starch contents contents in thein the Pawnee Pawnee variety variety and and higher higher oil accumulationoil accumulation in thein the early early seedseed development development from from the rangethe range of 122 of to122 132 to DAF132 DAF (Figure (Figure1a), we 1a analyzed), we analyzed the expression the expression profile profile of CiWRI1of CiWRI1, the C., the illinoinensis C. illinoinensishomolog homolog of Arabidopsis of Arabidopsis WRI1 WRI1that that is involved is involved in lipid in lipid and and carbohydrate carbohydrate metabolismmetabolism [8]. [8].CiWRI1 CiWRI1level level in Pawneein Pawnee increased increased dramatically dramatically and and remained remained high high during during the the early early periodperiod of fruit of fruit development development from from 124 124 DAF DAF to 138 to 138 DAF DAF (Figure (Figure2b), 2b), in accordance in accordance with with the fasterthe faster oil oil accumulationaccumulation observed observed at this at stagethis stage (Figure (Figure1a). In 1a contrast,). In contrast,CiWRI1 CiWRI1level increasedlevel increased more slowly more slowly in the in late-maturingthe late-maturing variety variety Mahan Mahan (Figure (Figure2b), consistent 2b), consistent with its with lower its rate lower of oil rate accumulation of oil accumulation (Figure 1(a).Figure BCCP21a). isBCCP2 a subunit is a ofsubunit ACCase of thatACCase catalyzes that thecatalyzes first committed the first committed step in FA biosynthesis. step in FA biosynthesis. We observed We that,observed like CiWRI1 that, ,CiBCCP2 like CiWRI1in the, earlyCiBCCP2 maturing in the variety early Pawnee maturing was markedly variety Pawnee upregulated was during markedly earlyupregulated fruit development during early stages fruit (Figure development2c). stages (Figure 2c). 3.3. Structural Features of C. Illinoinensis WRI1 3.3. Structural Features of C. Illinoinensis WRI1 Given that WRI1 plays key roles in glycolysis and FA biosynthesis in Arabidopsis and our 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 observation that CiWRI1 was higher expressed in the early maturing Pawnee variety of pecan (Figure (Figure2b), which was associated with high oil and starch contents in the seed (Figure1a), we focused 2b), which was associated with high oil and starch contents in the seed (Figure 1a), we focused our our investigation of the molecular mechanism underlying FA biosynthesis in pecan on WRI1. We first 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 cloned the pecan WRI1 homolog CiWRI1, which had an open reading frame of 1194 bp encoding a a protein with a predicted length of 397 amino acids, molecular weight of 44.45 kDa, and theoretical 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 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

Forests 2020, 11, 818 7 of 16 typical APETALA2 (AP2) DNA-binding domains (Figure S2). Sequence alignment of CiWRI1 with WRI1Forests from 2020 di, ﬀ11erent, x FOR speciesPEER REVIEW including Arabidopsis showed a high degree of homology, especially7 of 17 in the two AP2 domains (Figure3a), suggesting that CiWRI1 is an AP2-type transcription factor 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 belonging to the AP2/ethylene-responsive element-binding protein family of proteins and might has functionsfunctions similar similar to other to other known known WRI1-like WRI1-like proteins.proteins. In In the the phylogenetic phylogenetic analysis, analysis, CiWRI1 CiWRI1 clustered clustered in a subcladein a subclade comprising comprising ﬁve five species species and and showed showed the highest highest homology homology with with the theWRI1 WRI1-like-like genes genes of of QuercusQuercus suber suber (Figure (Figure3b), 3b), whose whose seeds seeds are are enriched enriched inin starch and and oil. oil.

FigureFigure 3. Sequence 3. Sequence alignment alignment of of CiWRI1 CiWRI1 in in didifferentfferent plant plant species. species. (a) ( aAmino) Amino acid acid sequence sequence alignment alignment of CiWRI1of CiWRI1 and and WRI1-like WRI1-like proteins proteins from from di differentfferent species.species. (b (b) )Phylogenetic Phylogenetic analysis analysis of CiWRI1 of CiWRI1 and and WRI1-like proteins. Black box indicates the first AP2 DNA-binding domain and the second AP2 DNA- WRI1-like proteins. Black box indicates the first AP2 DNA-binding domain and the second AP2 binding domain is in red. DNA-binding domain is in red.

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We next examined the the subcellular subcellular localization localization of of CiWRI1 CiWRI1 in in transiently transiently transfected transfected tobacco tobacco epidermepidermalal cells. cells. N.N. benthamiana benthamiana abaxialabaxial leaves leaves were were infiltrated inﬁltrated with with A.A. tumefaciens tumefaciens transformedtransformed with with p3p355 S:S: CiWRI1-GFPCiWRI1-GFP andand thenthen examinedexamined byby confocalconfocal microscopy. microscopy. GFP GFP ﬂuorescence fluorescence was was observed observed in in cell cellnuclei nuclei (Figure (Figure S3), 3), consistent consistent with with the the potential potential function function of of CiWRI1 CiWRI1 as as a a transcription transcription factor. factor.

3.4. CiWRI1 CiWRI1 is is F Functionalunctional C Conservedonserved with with WRI1 WRI1 in in Arabidopsis Arabidopsis We obtained a wri1wri1-1-1 mutantmutant of of Arabidopsis Arabidopsis (Salk: (Salk: CS69538) CS69538) from from the the Arabidopsis Arabidopsis Biological Biological Resource Center (ABRC), (ABRC), which which harbors harbors a a point point mutation mutation at at the the intron intron–exon–exon border border of of the the first ﬁrst intron intron (G3197A) (Figure (Figure 44a).a). Seeds of the wri1wri1-1-1 mutantmutant had had a awrinkled wrinkled appearance appearance and and were were smaller smaller than than the large, large, round round seeds seeds of of wild wild-type-type plants plants (Figure (Figure 4b).4b). As As previously previously reported reported [7 [–79–],9 ],we we observed observed that that the amount of of oil oil stored stored in in seeds seeds was was reduced reduced remarkably remarkably in in the the wri1wri1-1-1 mutantmutant (Figure (Figure 4e).4e).

Figure 4. 4. ComplementationComplementation of ofCiWRI1CiWRI1 in inwri1wri1-1-1 mutants.mutants. (a) i (dentificationa) identiﬁcation of the of wri1 the -wri1-11 mutantmutant of Arabidopsisof Arabidopsis. (b.() Seedb) Seed phenotypes phenotypes of ofwild wild-type-type and and wri1wri1-1-1 mutantmutant plants. plants. (c) (cSchematic) Schematic of of vector vector construction for CiWRI1 overexpression in the wri1-1wri1-1 mutantmutant ofof ArabidopsisArabidopsis.(. (d) Expression of CiWRI1 in inthree three individual individualp35 S:p3 CiWRI15 S: CiWRI1/wri1-1 transgenic/wri1-1 transgenic plants in the plants T1 generation in the T1 detected generation by semiquantitative detected by semiquantitativeRT-PCR. (e) Oil content RT-PCR. of seedse Oil from content wild-type, of seedswri1-1 frommutant, wild-type, and p35 wri1 S:- CiWRI11 mutant,/wri1-1 andtransgenic p35 S: CiWRI1plants./wri1 Seeds-1 fromtransgenic three plants.independent Seeds p35 from S: three CiWRI1 independent/wri1-1 transgenic p35 S: CiWRI1 plants/ werewri1-1 used transgenic for lipid plantsanalysis. wereN used= 3 for for each lipid genotype analysis. N with = 3 aboutfor each 2.4 genotype g seeds forwith each about analysis. 2.4 g seeds *** p for< 0.001each analysis. (Student’s ***t test). p < 0.001 Scale (Student bar in, Bars′s t test).= 100 Scaleµm (barb). in, Bars = 100 μm (b).

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To determine whether CiWRI1 is involved in FA biosynthesis like Arabidopsis WRI1, we transformedTo determine the wri1 whether-1 mutant CiWRI1 with is involved the p35 inS:FA CiWRI1 biosynthesis vector, like whichArabidopsis yieldedWRI1, three weindividual transformed T1 thetransgenicwri1-1 mutant plants with(Figure the 4cp35). RNA S: CiWRI1 was extractedvector, which from yielded the leaves three for individual semi-qRT T1-PCR transgenic analysis plants. We (Figuredetected4c). CiWRI1 RNA wasexpression extracted in the from wri1 the-1 leavesmutant for background semi-qRT-PCR in T1 analysis.transformants We detected but not inCiWRI1 Col-0 expressionwild-type inand the wri1wri1-1-1 mutantmutant of background Arabidopsis inplants T1 transformants (Figure 4d). butThe notseed in oil Col-0 content wild-type was markedly and wri1-1 mutantreduced of inArabidopsis the wri1-1 plantsmutant (Figure compared4d). to The Col seed-0 wild oil-type content plants was (13 markedly% vs. 40%) reduced (Figure in 4e). the Despitewri1-1 mutantthe 35S comparedpromoter is to known Col-0 wild-typeto be constitutive plants but (13% less vs. effective 40%) (Figure in seeds4e). [26 Despite–29], however, the 35S the promoter decrease is knownof seed to oil be content constitutive in the but wri1 less-1 mutant eﬀective was in seedsrescued [26 by–29 overexpressing], however, the decreaseCiWRI1, as of evidenced seed oil content by the in thetotalwri1-1 oil contentmutant of was 26% rescued in the seeds by overexpressing of p35 S: CiWRI1/wrCiWRI1i1-,1 as transgenic evidenced plants by the which total was oil content almost oftwice 26% inas the much seeds as of thep35 oil S: CiWRI1 content/ wri1-1 in thetransgenic wri1-1 seeds plants (Figure which 4e). was These almost results twice as suggest much asthat the CiWRI1 oil content is ininvolved the wri1-1 in FAseeds biosynthesis. (Figure4e). These results suggest that CiWRI1 is involved in FA biosynthesis. WRI1WRI1 functionsfunctions inin multiplemultiple developmentaldevelopmental processes in Arabidopsis [6][6].. To To determine determine whether whether thethe samesame isis truetrue forfor CiWRI1,CiWRI1, wewe comparedcompared other phenotypic features features of of Col Col-0,-0, wri1wri1-1-1, ,and and p3p355 S S:: CiWRI1CiWRI1/wri1/wri1-1-1transgenic transgenic plants. plants. PlantPlant heightheight waswas signiﬁcantlysignificantly reduced in in the the wri1wri1-1-1 mutant,mutant, but but was was normalnormal inin allall threethreep35 p35 S:S: CiWRI1CiWRI1/wri1/wri1-1-1 transgenictransgenic plantsplants (Figure 55a,b).a,b). Root growth defects in in the the mutantsmutants were were also also largely largely rescued rescued by byCiWRI1 CiWRI1overexpression overexpression (Figure (Figure5c,d). 5c,d). Seed Seed germination germination rate rate was dramaticallywas dramatically decreased decreased in the inwri1-1 the wri1mutant-1 mutant compared compared to wild-type to wild- plantstype plants (Figure (Figure5e), but 5e), was but partly was rescuedpartly rescued in p35 S: in CiWRI1 p35 S: CiWRI1transgenic transgenic plants plants (Figure (Figure5e). These 5e). These data suggest data suggest that CiWRI1 that CiWRI1has the has same the functionalitysame functionality that AtWRI1 that AtWRI1. .

FigureFigure 5. 5.Rescue Rescue ofof developmentaldevelopmental defects in wri1-1wri1-1 mutant by CiWRI1..( (aa)) Plant Plant height height in in 35 35-day-old-day-old wildwild type, type,wri1-1 wri1mutant,-1 mutant, and three and individual three individualp35 S: CiWRI1 p35 S/wri1-1: CiWRI1transgenic/wri1-1 plants.transgenic (b) Quantiﬁcation plants. (b) ofQuantification plant heights of in plant (a). (heightsc) Root in length (a). (c) in Root 12-day-old length in wild-type, 12-day-oldwri1-1 wild-type,mutant, wri1 and-1 mutant, three individualand three p35individual S: CiWRI1 p3/wri1-15 S: CiWRI1transgenic/wri1 plants.-1 transgenic (d) Quantiﬁcation plants. (d) ofQuantification root lengths in of (c roo). (te ) lengths Germination in (c). rates(e) ofGermination wild-type ( nrates= 5 of for wild each-type time (n point, = 5 for100 each seeds time forpoint, each 100 biological seeds for replicate),each biologicalwri1-1 replicate),mutant wri1 (n =-3 for1 mutant each time (n = point, 3 for 100each seeds time forpoint, each 100 biological seeds for replicate), each biological and three replicate), individual and threep35 S: individual CiWRI1/wri1-1 p35 transgenicS: CiWRI1/ plantswri1-1 (Linetransgenic 1, n = plants3 for each(Line time 1, n = point; 3 for Lineeach 2,timen = point;3 for eachLine 2, time n = point;3 for each Line time 3, n point;= 4 for eachLine time 3, n = point; 4 for 100each seeds time forpoint; each 100 biological seeds for replicate). each biological *** p < replicate).0.001, Student’s *** p < t0.001 test., BarsStudent’s=1 cm t test. (a,c). Bars =1 cm (a,c).

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Forests 2020, 11, x FOR PEER REVIEW 10 of 17 3.5. Cloning of the ACCase Subunit BCCP2 3.5. Cloning of the ACCase Subunit BCCP2 GivenGiven that that the firstthe first step step of FA of synthesisFA synthesis in Arabidopsis in Arabidopsisis catalyzedis catalyzed by by ACCase, ACCase, we we cloned cloned the the pecan homologpecan of homolog the BCCP2 of thegene BCCP2 encoding gene encoding an ACCase an ACCase subunit. subunit. CiBCCP2 CiBCCP2 was was predicted predicted to to have have anan open readingopen frame reading of 852frame bp of and 852 encode bp and aencode 283-amino a 283- acidamino protein acid protein with awith molecular a molecular weight weight of 30 of kDa 30 and theoreticalkDa and isoelectric theoretical point isoelectric of 8.50. point Analysis of 8.50. of the Analysis amino of acid the sequence amino acid revealed sequence an AMKLMN revealed an motif (FigureAMKLMN S4) that motif is a conserved(Figure 4) that biotinylation is a conserved site biotinylation and functional site and domain functional of domain the BCCP2 of the subunit BCCP2 [30]. Sequencesubunit alignment [30]. Sequence showed alignment that CiBCCP2 showed that is highly CiBCCP2 homologous is highly homologous to BCCP2 to in BCCP2 other plantin other species includingplant Arabidopsis species including, especially Arabidopsis the C-terminal, especially AMKLMN the C-terminal sequence AMKLMN (Figure sequence6a), implying (Figure functional 6a), implying functional conservation with previously identified BCCP2-like proteins in different species. conservation with previously identified BCCP2-like proteins in different species. In the phylogenetic In the phylogenetic analysis, CiBCCP2 clustered in a subclade comprising five species and showed analysis,the highest CiBCCP2 homology clustered to in the a subclade BCCP2-like comprising gene in Q. five suber species (Figure and 6b) showed as observed the highest for the homology pecan to the BCCP2-likehomolog of gene WRI1 in gene,Q. suber providing(Figure further6b) as observed evidence for for the a conserve pecan homologd mechanism of WRI1 of FAgene, synthesis providing furtherbetween evidence these for two a species. conserved mechanism of FA synthesis between these two species.

FigureFigure 6. Sequence 6. Sequence alignment alignment of CiBCCP2 of CiBCCP2 in di ﬀ inerent different plant plantspecies. species. (a) Amino (a) Amino acid sequence acid sequence alignment of CiBCCP2alignment and of BCCP2-likeCiBCCP2 and proteins BCCP2- inlike indi proteinsﬀerent in species. indifferent (b) species Phylogenetic. (b) Phylogenetic analysis of analysis CiBCCP2 of and BCCP2-likeCiBCCP2 proteins. and BCCP2 Black-like box proteins. indicates Black the box AMKLMN indicates the biotinylation AMKLMN biotinylation domains. domains.

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). Forests 2020, 11, 818 11 of 16

3.6. 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 (Figure7a). Each binding motif of (CnTnG)(n)7(CG) contained conserved CnTnG and CG sequences separated by 7 bp of random nucleotides (Figure7a). 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 (Figure7b), 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 (Figure7c). 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 (Figure7d), as previously reported.Forests However, 2020, 11, x thisFOR PEER was REVIEW reversed in plants by overexpressing CiWRI1 of pecan (Figure12 of 17 7d).

Figure 7.FigurePositive 7. Positive regulation regulation of CiBCCP2of CiBCCP2 byby CiWRI1.CiWRI1. (a) (Tawo) Two predicted predicted ASML1 ASML1/WRI1 (AW/WRI1)-boxes (AW)-boxes (shown in boxes) in the CiBCCP2 promoter; the first (P1) is located in the region from 271 bp to 284 (shown in boxes) in the CiBCCP2 promoter; the first (P1) is located in the region from 271 bp to 284 bp bp and the second (P2) from 165 bp to 178 bp upstream of ATG (red). (b) EMSA showing the binding and the secondof CiWRI1 (P2) to fromthe CiBCCP2 165 bp promoter. to 178 bpThe upstream fragment containing of ATG (red).the first ( b(P1)) EMSA AX-box showing sequence was the binding of CiWRI1 toused the asCiBCCP2 a probe. Thepromoter. arrow shows The specific fragment interactions. containing (c) Upregulation the first of CiBCCP2 (P1) AX-box by co-expression sequence was used as a probe.of p3 The5 S: arrow CiWRI1 shows vector specific in tobacco. interactions. Results are representative (c) Upregulation of three of independentCiBCCP2 by biological co-expression of p35 S: CiWRI1replicates.vector (d) BCCP2 in tobacco. expression Results in wild are-type representative Col-0, wri1-1 of mutant, three independent and p35 S: CiWRI1 biological/wri1-1 replicates. transgenic Arabidopsis. Three individual p35 S: CiWRI1/wri1-1 transgenic plants were used for qRT- (d) BCCP2PCR.expression * p < 0.05, in*** wild-typep < 0.001 (Student’s Col-0, twri1-1 test). mutant, and p35 S: CiWRI1/wri1-1 transgenic Arabidopsis. Three individual p35 S: CiWRI1/wri1-1 transgenic plants were used for qRT-PCR. * p < 0.05, *** p < 0.001 (Student’sTot examinetest). 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.

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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 (Figure8a) 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 (Figure8b). CiBCCP2 was highly expressed in T1 transformants but not in Col-0 wild-type and wri1-1 mutant of Arabidopsis (Figure8c). The decrease in plant height (Figure8b,d), root defects (Figure8f,g), and lower germination rate (Figure8e) in the mutant were partly rescued by overexpressing CiBCCP2. Thus, our data suggest that CiWRI1 directly regulatesForests 2020, CiBCCP2 11, x FOR PEER transcription REVIEW during plant development. 13 of 17

FigureFigure 8. 8. CiBCCP2 rescues developmental defects in the the wri1-1wri1-1 mutant.mutant. ( a) Schematic of vector vector constructionconstruction for CiBCCP2 overexpressionoverexpression in in the the wri1wri1-1-1 mutantmutant of of ArabidopsisArabidopsis. (.(b) bPlant) Plant height height in 33 in- 33-day-oldday-old wild wild-type,-type, wri1wri1-1-1 mutant,mutant, and and three three independent independent p35p35 S: CiBCCP2 S: CiBCCP2/wri1/wri1-1-1 transgenictransgenic plants. plants. (c) (CiBCCP2c) CiBCCP2 expressionexpression in three three independent independentp35 p3 S:5 CiBCCP2 S: CiBCCP2/wri1-1/wri1transgenic-1 transgenic plants in plants the T1 generation in the T1 detectedgeneration by semiquantitativedetected by semiquantitative RT-PCR. (d) Quantiﬁcation RT-PCR. (d) ofQuantification plant heights inof ( plantb). (e ) heights Germination in (b rates). (e) inGermination wild-type ( nrates= 5 in for wild each-type time ( point,n = 5 for 100 each seeds time for point, each biological100 seeds for replicate), each biologicalwri1-1 mutant replicate), (n = wri13 for- each1 mutant time ( point,n = 3 for 100 each seeds time for point, each biological100 seeds replicate),for each biological and three replicate), independent and threep35 S: independent CiBCCP2/wri1-1 p35 transgenicS: CiBCCP2 plants/wri1-1 (Line transgenic 6, n = 4plants for each (Line time 6, n point; = 4 for Line each 8, timen = 3point; for each Line time 8, n point;= 3 for Lineeach 9,timen = point;4 for eachLine time9, n = point; 4 for each 100 seeds time forpoint; each 100 biological seeds for replicate). each biological (f) Root replicate length in). ( 12-day-oldf) Root length wild-type, in 12-daywri1-1-old mutant,wild-type, and wri1 three-1 independentmutant, and p35 three S: CiBCCP2 independent/wri1-1 p3transgenic5 S: CiBCCP2 plants./wri1 (-g1) Quantiﬁcationtransgenic plants. of root (g) lengthsQuantification in (f). * pof< root0.05, lengths ** p < 0.01,in (f). *** * p << 0.0010.05, ** (Student’s p < 0.01, t***test). p < Bars0.001= (Student’s1 cm (b,f). t test). Bars =1 cm (b,f).

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4. Discussion In the oil biosynthesis among the woody oil plants, so far, only a few species like Jatropha [31–33], 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 (Figure1a,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 (Figures3 and6). 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 (Figure4e). 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 (Figure5), 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 (Figure8), suggesting that wri1-1 defects in the development were caused in part by impaired FA metabolism.

5. 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 (Figure2b,c) with high oil content in the seed (Figure1a). Thus, lipid biosynthesis genes Forests 2020, 11, 818 14 of 16 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. Funding: F.P. was supported by a grant from the National Key R & D Program of China (nos. 2018YFD1000600 and 2018YFD1000604). Z.T. was supported by a grant from the National Natural Science Foundation of China (31300248). Acknowledgments: We thank Zengfu Xu and Mingyong Tang for assistance with the lipid analysis, and Youjun Huang shares the unpublished data. Conflicts of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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