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. Forests 2020, 11, 818; doi:10.3390/f11080818 www.mdpi.com/journal/forests Forests 2020, 11, 818 2 of 16 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.

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