ORIGINAL RESEARCH
`published: 19 November 2015
`doi: 10.3389/fpls.2015.01015
`
`Wrinkled1 Accelerates Flowering and
`Regulates Lipid Homeostasis
`between Oil Accumulation and
`Membrane Lipid Anabolism in
`Brassica napus
`
`Qing Li, Jianhua Shao, Shaohua Tang, Qingwen Shen, Tiehu Wang, Wenling Chen and
`Yueyun Hong*
`
`National Key Laboratory of Crop Genetic Improvement, College of Life Science and Technology, Huazhong Agricultural
`University, Wuhan, China
`
`Wrinkled1 (WRI1) belongs to the APETALA2 transcription factor family; it is unique to
`plants and is a central regulator of oil synthesis in Arabidopsis. The effects of WRI1 on
`comprehensive lipid metabolism and plant development were unknown, especially in
`crop plants. This study found that BnWRI1 in Brassica napus accelerated flowering and
`enhanced oil accumulation in both seeds and leaves without leading to a visible growth
`inhibition. BnWRI1 decreased storage carbohydrates and increased soluble sugars to
`facilitate the carbon flux to lipid anabolism. BnWRI1 is localized to the nucleus and
`directly binds to the AW-box at proximal upstream regions of genes involved in fatty
`acid (FA) synthesis and lipid assembly. The overexpression (OE) of BnWRI1 resulted
`in the up-regulation of genes involved in glycolysis, FA synthesis, lipid assembly, and
`flowering. Lipid profiling revealed increased galactolipids monogalactosyldiacylglycerol
`(MGDG), digalactosyldiacylglycerol (DGDG), and phosphatidylcholine (PC) in the leaves
`of OE plants, whereas it exhibited a reduced level of the galactolipids DGDG and MGDG
`and increased levels of PC, phosphatidylethanolamide, and oil [triacylglycerol (TAG)] in
`the siliques of OE plants during the early seed development stage. These results suggest
`that BnWRI1 is important for homeostasis among TAG, membrane lipids and sugars,
`and thus facilitates flowering and oil accumulation in B. napus.
`
`Keywords: Wrinkled1 (WRI1), oil accumulation, flowering, lipid homeostasis, transcriptional regulation, Brassica
`napus
`
`INTRODUCTION
`
`Lipids not only serve as storage components of high-density energy, but they also function
`as essential components of cell membranes and regulators of various cellular processes during
`growth, development, and stress responses (Wang et al., 2006; Hong et al., 2008, 2009; Phillips
`et al., 2009; To et al., 2012). Fatty acid (FA) synthesis and lipid assembly involve multiple
`steps (Li-Beisson et al., 2010). The initial precursors of lipid biosynthesis include acetyl-CoA
`and glycerol-3-phosphate, which are initially derived from glycolysis and the Calvin–Benson
`cycle in plants (Kang and Rawsthorne, 1996; Alonso et al., 2007). The acetyl-CoA carboxylase
`(ACCase) complex is made of three subunits, namely biotin carboxyl carrier protein (BCCP),
`biotin carboxylase (BC), and carboxyltransferase (CT); this complex is encoded by separated
`
`Edited by:
`Chandrashekhar Pralhad Joshi,
`Michigan Technological University,
`USA
`Reviewed by:
`Biswapriya Biswavas Misra,
`University of Florida, USA
`Upinder S. Gill,
`The Samuel Roberts Noble
`Foundation, USA
`
`*Correspondence:
`Yueyun Hong
`hongyy@mail.hzau.edu.cn
`
`Specialty section:
`This article was submitted to
`Plant Biotechnology,
`a section of the journal
`Frontiers in Plant Science
`Received: 03 September 2015
`Accepted: 02 November 2015
`Published: 19 November 2015
`Citation:
`Li Q, Shao J, Tang S, Shen Q,
`Wang T, Chen W and Hong Y (2015)
`Wrinkled1 Accelerates Flowering
`and Regulates Lipid Homeostasis
`between Oil Accumulation
`and Membrane Lipid Anabolism
`in Brassica napus.
`Front. Plant Sci. 6:1015.
`doi: 10.3389/fpls.2015.01015
`
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`Li et al.
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`BnWRI1 in Flowering and Lipid Homeostasis
`
`genes in plants and catalyzes acetyl-CoA and CO2 to produce
`malonyl-CoA, the first committed step in de novo FA synthesis
`(Slabas and Fawcett, 1992; Ohlrogge and Browse, 1995; Ohlrogge
`and Jaworski, 1997; Voelker and Kinney, 2001; Sasaki and
`Nagano, 2004). Malonyl-CoA was then transferred to ACP
`protein by malonyl-CoA: ACP transferase (MAT) to initiate FA
`synthesis, and malonyl-CoA provides a two-carbon unit for acyl
`chain elongation as catalyzed by an FA synthase (FAS) complex
`(Ohlrogge and Browse, 1995). The synthesized FAs are either
`retained in the chloroplast for galactolipid synthesis or exported
`to the endoplasmic reticulum (ER) for membrane phospholipid
`and storage lipid [triacylglycerol (TAG)] assembly. The final
`step of TAG assembly as catalyzed by DAG acyltransferase
`(DGAT), which occurs
`in the Kennedy pathway,
`is also
`regarded as a critical reaction for oil accumulation (Slabas
`and Fawcett, 1992; Ohlrogge and Browse, 1995; Zou et al.,
`1999; Voelker and Kinney, 2001). The loss of DGAT1 resulted
`in reduced seed oil content, whereas DGAT1 overexpression
`(OE) enhanced the seed oil content in Arabidopsis (Zou et al.,
`1999; Jako et al., 2001). The OE of maize high-oil DGAT1-2
`also promoted oil accumulation in maize seeds (Zheng et al.,
`2008). Alternatively, phosphatidylcholine (PC) also provides
`an acyl chain toward DAG for TAG synthesis, as catalyzed
`by phospholipid:diacylglycerol acyltransferase (PDAT; Dahlqvist
`et al., 2000; Tarczynski and Shen, 2008; Zhang et al., 2009).
`Given the complicated networks that make up the lipid
`anabolic process,
`it would be more efficient
`to boost oil
`accumulation by enhancing multiple routes in a coordinated
`fashion including carbon partitioning, FA synthesis, and lipid
`assembly. Therefore, the identification of the key enzymes or
`master regulators involved in multiple steps simultaneously
`becomes an attractive approach for improving oil production
`(Ohlrogge and Jaworski, 1997; Ruuska et al., 2002; Cahoon et al.,
`2007; Mu et al., 2008). Transcriptomic profiling revealed that
`the genes encoding enzymes involved in FA synthesis is co-
`regulated to the rate of acyl chain synthesis, suggesting that
`transcriptional regulation plays an important role in the lipid
`biosynthesis process (Ruuska et al., 2002; Baud and Lepiniec,
`2009; Barthole et al., 2012). Recent studies have identified several
`transcription factors that are capable of governing multiple oil
`accumulation steps (Cernac and Benning, 2004; Shen et al., 2010;
`To et al., 2012). Wrinkled1 (WRI1) belongs to the APETALA2
`(AP2)-ethylene-responsive element binding protein family of
`transcription factors, and it acts as a central regulator in seed
`oil accumulation by modulating numerous genes simultaneously
`during late glycolysis and FA biosynthesis. A deficiency mutant
`of Arabidopsis WRI1 (AtWRI1) leads to wrinkled seeds with 80%
`less seed oil content in Arabidopsis (Focks and Benning, 1998;
`Cernac and Benning, 2004). The loss of AtWRI1 also leads to
`impaired seed germination and seedling establishment, whereas
`AtWRI1 OE enhances oil accumulation in Arabidopsis, which
`is accompanied by aberrant seedling development (Stone et al.,
`2001; Kwong et al., 2003; Cernac and Benning, 2004; Cernac et al.,
`2006; Baud et al., 2007). AtWRI1 binds to the AW-box consensus
`[CnTnG](n)7[CG] in the proximal promoter of target genes that
`are involved in the glycolysis and FA synthesis of Arabidopsis
`(Maeo et al., 2009; To et al., 2012).
`
`The biological significance of WRI1 has been extensively
`studied in relation to oil accumulation in Arabidopsis. The role of
`WRI1 in comprehensive lipid regulation in other plant species,
`particularly in crop plants, remains to be elucidated. A recent
`study showed that the WRI1 homolog from maize is able to
`compensate for the impaired oil accumulation and seedling
`establishment of the Atwri1 mutant in Arabidopsis (Cernac
`et al., 2006; Pouvreau et al., 2011). The OE of ZmWRI1 in
`maize increased the levels of FAs and some amino acid residues
`(Pouvreau et al., 2011), suggesting that the role of WRI1 in
`oil accumulation is highly conserved between monocot and
`dicot plants. Arabidopsis that overexpresses AtWRI1 exhibits
`undesirable agronomic traits, with retarded growth and reduced
`biomass (Lotan et al., 1998; Stone et al., 2001; Cernac and
`Benning, 2004; Wang et al., 2007; Mu et al., 2008), whereas
`ZmWRI1 OE in maize promotes oil accumulation without visible
`side effects on growth and development (Shen et al., 2010). The
`results suggest that the role of WRI1 in oil synthesis is conserved
`but distinguishable in different plant species. The WRI1 in
`different plant species may exhibit a unique role in addition to
`its effect on oil accumulation. Furthermore, most WRI1 studies
`have been focused on oil accumulation, and the effects of WRI1
`on membrane phospholipids and galactolipids have remained
`unknown. In the present study, we characterized BnWRI1
`(BnaA09g34250D) from Brassica napus (B. napus). BnWRI1 OE
`in B. napus resulted in enhanced lipid anabolism by binding
`to the cis-element CnTnG (n)7CG in the promoter regions of
`genes involved in FA synthesis and lipid assembly to up-regulate
`these target genes. BnWRI1 promotes oil accumulation and
`thylakoid membrane monogalactosyldiacylglycerol
`(MGDG),
`digalactosyldiacylglycerol
`(DGDG),
`and PC biosynthesis
`to regulate homeostasis among membrane lipids, oils, and
`carbohydrates. Therefore, BnWRI1 OE facilitates flowering,
`reproduction, and oil production without visible side effects on
`the growth of B. napus.
`
`MATERIALS AND METHODS
`
`Plant Materials and Growth Conditions
`The Westar cultivar of canola (B. napus L.) was used in this
`study. Seeds were germinated in either Murashige and Skoog
`(MS) plates or soil in pots. Three-week-old seedlings were then
`transferred to pots containing soil. The plants were raised in
`a growth room under 16 h light (25◦C)/8 h dark (20◦C), a
`photosynthetic photon flux density of 200–300 mmol m−2 s−1,
`and 60% relative humidity, or natural conditions during winter-
`spring seasons in Wuhan, China. For the field growth test,
`3-week-old seedlings were transferred to the field at suitable
`spacing (33 cm × 50 cm) that were arranged in a one-way
`randomized block design with 30 plants/lines per block, and three
`replications.
`
`Gene Cloning, Vector Construction, and
`B. napus Plant Transformation
`To obtain the full-length BnWRI1 cDNA,
`total RNA was
`extracted from the leaves of 4-week-old B. napus plants, and it
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`Li et al.
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`BnWRI1 in Flowering and Lipid Homeostasis
`
`was subjected to reverse transcription to obtain first strand
`cDNA according to the manufacturer’s instructions (Trans-
`full-length BnWRI1
`Gene Biotech, Beijing, China). The
`cDNA was amplified by PCR with primers BnWRI1F 5(cid:3)-
`GGATCCATGAAGAGACCCTTAACCACT-3(cid:3) and BnWRI1R
`5(cid:3)-GAGCTCTCAGACAGAATAGTTCCAAGAA-3(cid:3), and then it
`was ligated into binary vector pBI121, which had been digested by
`SacI and BamHI. The resulting construct was transformed into
`B. napus by Agrobacterium GV3101 mediation with hypocotyls
`used as explants for regeneration. The transgenic shoots were
`first selected on kanamycin (50 μg/ml), and then the kanamycin
`resistant shoots were transferred to MS medium containing
`1-naphthaleneacetic acid for rooting. The transgenic plants were
`further confirmed by PCR with a pBI121 vector and BnWRI1
`sequence specific primers (Supplemental Table S1).
`
`Subcellular Localization
`The full-length cDNA of BnWRI1 was ligated into pCAMB-
`IA1301 vector that had been digested by the restriction enzymes
`SacI and BamHI. The construct containing BnWRI1-GFP was
`introduced into Agrobacterium GV3101 and infiltrated into
`tobacco leaves for 24 h to obtain transient protein expression
`under the control of the 35S promoter. Subcellular localization
`was visualized under a confocal
`laser scanning microscope
`(Leica, Biberach, Germany) with the exciter filter HFT488 and
`the transmitting optical filter BP505–530 to observe the green
`fluorescence. The nuclei were labeled with 4(cid:3),6-diamidine-2-
`phenylindole dihydrochloride (DAPI) staining.
`
`RNA Extraction and Quantitative
`Real-time PCR
`Total RNA was extracted from various tissues at different stages
`using TransZol reagent (TransGen Biotech, Beijing, China), and
`it was then treated with RNase-free DNaseI (NEW ENGLAND
`Biolabs, Ipswitch, MA, USA) to remove any contaminating DNA.
`The resulting RNA was used for first strand synthesis by reverse
`transcriptase with an oligo-d (T) 18 primer (TransGen Biotech,
`Beijing, China) to obtain cDNA according the manufacturer’s
`protocol. Quantitative real-time PCR was performed with SYBR
`Green PCR Master Mix (TransGen Biotech, Beijing, China)
`on a single-color Real-time PCR Detection System (Bio-Rad,
`Hercules, CA, USA). A BnActin gene was used as the standard
`control. The quantitative real-time PCR conditions were as
`follows: 95◦C for 1 min; 40 cycles of 95◦C for 30 s, 55◦C for
`30 s, 72◦C for 30 s; and 72◦C for 10 min for the final extension.
`The primers used for real-time PCR are listed in Supplemental
`Table S2.
`
`Lipid Extraction and Analyses
`Lipids were extracted from the leaves, developing siliques,
`and mature seeds. The lipids were separated on a thin layer
`chromatography (TLC) plate with developing solvent consisting
`of petroleum ether, ethyl ether, and acetic acid (80:20:1, v/v).
`The separated lipids were visualized with iodine vapor and the
`spots were scraped for measurement by GC analysis (Agilent
`7890A, Santa Clara, CA, USA) after a methyl ester reaction with
`
`methanol and toluene containing 5% H2SO4 at 80◦C for 3–4 h.
`To measure the seed oil contents, oil was extracted from the
`seeds and tested by GC analysis after the methyl ester reaction as
`detailed above. The GC running conditions were as follows: the
`injection port temperature was 180◦C, and the oven temperature
`was set at 180◦C for 2 min and was increased by 10◦C/min up to
`220◦C for 5 min. The temperature of the flame ionization detector
`was 280◦C with flow rates of 30, 300, and 25 ml/min for hydrogen,
`air, and helium, respectively.
`
`Electrophoretic Mobility-shift Assay
`The full-length cDNA of Bn WRI1 was amplified by using the
`forward primer 5(cid:3)-CCCGGGTATGAAGAGACCCTTAACCAC-
`3(cid:3) coupled with the reverse primer 5(cid:3)-GGATCCCGACAG
`AATAGTTCCAAGAA-3(cid:3), and then it was ligated to pET28a
`vectors that were digested by BamHI and SacI. This construct
`was transformed into Escherichia coli strain Rosetta (DE3), and
`the BnWRI1 protein was expressed by induction with 0.6 mM
`isopropyl-β-D-thiogalactopyranoside (IPTG) while the strain
`was grown in Luria Bertani (LB) medium overnight at 20◦C.
`The cells were harvested and lysed by sonication in a buffer
`(300 mM NaCl, 20 mM Tris-HCl, pH 8.0, 10 mM imidazole, 5%
`glycerol, and 50 mM NaH2PO4). The cell lysate was centrifuged
`at 12,000 r/min for 20 min. The supernatant was incubated
`with Ni-NTA resin (Shanghai Sangon, http://www.sangon.com)
`for 3 h at 4◦C. The BnWRI1 protein was eluted from the
`resin after three washes with wash buffer (50 mM NaH2PO4,
`300 mM NaCl, and 20 mM imidazole, pH 8). Protein from
`E. coli cells containing only the pET28a vector was used as
`a negative control. The DNA sequences that were 300 and
`250 bp upstream from the start codon of KASI and GPAT9,
`respectively, were amplified from Arabidopsis with the KASI
`forward primer 5(cid:3)-GAATTCTGTTGAGTTACGAATTGGAG-
`3(cid:3), coupled with the KASI reverse primer 5(cid:3)-GAGCTCATTGAG
`AGAGGTATTGAGAG-3(cid:3), and the GPAT9 forward primer
`5(cid:3)-GAATTCACATAATATGTCCAAGATCATT-3(cid:3) coupled with
`the GPAT9 reverse primer 5(cid:3)-GAGCTCCTATTATACTTATA
`CCACAT-3(cid:3). The substitutive nucleotide (C→T, T→C, G→A)
`mutant at the AW-box [CnTnG](n)7[CG] was amplified by using
`a similar approach. The amplified DNA fragments containing
`native or mutant AW-box were incubated with purified BnWRI1
`protein in binding buffer (20 mM Tris-HCl, pH 8.0, 250 mM
`NaCl, 2 mM MgCl2, 1% glycerol, 1 mg/ml BSA, 1 mM DTT) for
`1 h at 4◦C. The resulting mixture was separated on native PAGE
`(6%) by electrophoresis and was visualized under UV light. The
`binding activity of BnWRI1 to the AW-box was also determined
`with biotin labeled DNA probes using a chemiluminescent
`electrophoretic mobility-shift assays (EMSA) kit (Beyotime,
`China) according to the manufacturer’s instructions.
`
`Measurements of Protein, Starch, and
`Soluble Sugar
`Proteins were extracted from leaves and seeds by homogenizing
`in buffer containing 50 mM Tris-HCl, pH 8.0, 250 mM NaCl,
`1 mM EDTA, and 1% (w/v) SDS, and incubating the mixture
`for 2 h at 25◦C. The homogenate was centrifuged at 16,000 g for
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`
`BnWRI1 in Flowering and Lipid Homeostasis
`
`FIGURE 1 | BnWRI1 expression pattern and overexpression (OE) of
`BnWRI1 in Brassica napus. (A) Transcript level of BnWRI1 in different
`B. napus tissues at seedling, bolting, flowering and seed developing stages.
`The expression level was quantified by real-time PCR normalized to the
`expression of BnActin. Values are mean ± SD (n = 3). R, root; St, stem; L,
`leaf; Bu, flower bud; F, flower; and Si15, Si30, and Si45, siliques at 15, 30,
`and 45 days, respectively, after anthesis. (B) The construct containing
`BnWRI1 in the binary vector pBI121. (C,D) The transcript level of BnWRI1 in
`BnWRI1-OE plants according to semi-quantitative RT-PCR (C) and
`quantitative real-time PCR (D). Total RNA was extracted from the leaves of
`6-week-old plants, and the expression level was detected by using
`BnWRI1-specific primers. The expression levels were normalized to BnActin.
`OE2, OE16, and OE16 represent BnWRI1-OE lines. Values are mean ± SD
`(n = 3). ∗∗Indicates significant difference at P < 0.01 compared with the WT
`based on Student’s t-test.
`
`10 min, the supernatant was diluted 200 times and the protein
`concentration was measured by Lowry D protein assay (Bio-Rad).
`Soluble sugars were measured using phenol-sulfuric acid method
`(DuBois et al., 1956; Chow and Landhausser, 2004). In brief, leaf
`samples (1 g fresh weight) were homogenized with deionized
`water and filtered. The extract (50 μl) was mixed with 450 μl of
`sulfuric acid containing anthrone (2 mg/ml) at 95◦C, and then the
`absorbance at 625 nm was monitored by spectrometer (Infinite
`M200 PRD, Untersbergstr, Austria). For starch extraction, the
`remaining sediment was suspended in a solution containing 0.2 N
`KOH and incubated at 95◦C for 1 h, followed by the addition of
`1 N acetic acid and incubation for 15 min. After centrifugation at
`16,000 g for 5 min, the starch in the supernatant was measured by
`using a method similar to that of soluble sugars.
`
`FIGURE 2 | Accelerated flowering in BnWRI1-OE plants. (A) BnWRI1-OE
`and WT plants at the vegetative growth stage. The pictures were taken of
`plants that were grown in pots at 30 days after germination. (B) Accelerated
`flowering in BnWRI1-OE plants. The picture was taken of 145-day-old plants
`grown in the field. (C) Days to bolting based on a flowering rate of 50%
`(n = 20, r = 3). (D) The flowering rate of OE and WT plants grown under the
`same conditions. Values are mean ± SD (n = 20, r = 3). (E) Inflorescent
`branch number of BnWRI1-OE and WT. The data were collected from mature
`plants (175-day-old) grown in the field (n = 12, r = 3). (F) The fresh weights of
`aerial part from 175-day-old plants grown in the field. Values are mean ± SD
`(n = 12, r = 3). ∗,∗∗Indicate significant differences at P < 0.05 and P < 0.01,
`respectively, compared with the WT based on Student’s t-test.
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`Li et al.
`
`BnWRI1 in Flowering and Lipid Homeostasis
`
`FIGURE 3 | Enhanced seed oil accumulation in BnWRI1-OE plants. Total TAG content (A) and fatty acid (FA) composition (B) in the seeds of BnWRI1-OE and
`WT. Total TAG content (C) and FA composition (D) in the leaves of BnWRI1-OE and WT. Lipids were extracted from the leaves of 8-week-old plants and were
`separated by using a thin layer chromatography (TLC) plate. TAG spots were scraped and extracted for methylation and GC measurement. Values are mean ± SD
`(n = 4); ∗∗P < 0.01.
`
`RESULTS
`
`Expression Pattern and the Effect of
`BnWRI1 on Flowering in B. napus
`To investigate the temporal and spatial distribution of BnWRI1
`mRNA in B. napus, total RNA was extracted from various tissues
`at different stages and used for analysis by quantitative real-
`time PCR. During the seedling and bolting stages, the BnWRI1
`transcript level was higher in leaves and flower buds than it was
`in roots and stems (Figure 1A). During the flowering stage, the
`BnWRI1 expression was higher in flowers than in leaves and
`stems. The transcript level was rapidly up-regulated in siliques
`and was highest at 30 days after anthesis (Figure 1A).
`To explore the biological function of BnWRI1 in B. napus, full-
`length BnWRI1 cDNA was cloned by reverse transcription PCR
`by using mRNA that was extracted from leaves as a template,
`and the cDNA was ligated into binary vector pBI121. The
`resulting construct containing BnWRI1 was transformed into
`B. napus under the control of the 35S promoter (Figure 1B).
`More than 30 independent transgenic lines were obtained, and
`the BnWRI1 transcript level in transgenic plants was much
`higher than that of the wild-type (WT; Figures 1C,D). Three
`
`representative, independent BnWRI1 OE lines, OE2, OE16, and
`OE17, were selected randomly from 30 transgenic lines, and
`they were used for further characterization. These plants were
`grown under natural conditions either in the field or in pots,
`and no visual growth change was observed between OE and WT
`plants, which showed a similar leaf size, leaf number, and growth
`rate during the vegetative growth stage (Figure 2A). However,
`BnWRI1 accelerated flowering; OE plants bolted and flowered
`4 to 6 days earlier than WT plants (Figures 2B,C). At 136 days
`after germination, 60% of the OE plants were flowering, whereas
`only 23% of the WT plants were flowering (Figure 2D). The
`earlier flowering in OE plants did not cause changes in the total
`number of inflorescent branches and biomass at the mature stage
`compared with the results for the WT plants (Figures 2E,F).
`Overexpression of BnWRI1 Enhances Oil
`Accumulation in Seeds and Leaves
`without Undesirable Agronomic Traits
`To investigate the role of BnWRI1 in oil (TAG) synthesis, the oil
`content was measured in both the seeds and leaves of OE and
`WT plants. The oil content of BnWRI1-OE seeds was significantly
`higher than that of the WT, and it was increased by 31, 38,
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`Li et al.
`
`BnWRI1 in Flowering and Lipid Homeostasis
`
`FIGURE 4 | The effect of BnWRI1 on partitioning among lipids, sugars, and proteins in B. napus. (A) Total FA contents in leaves from BnWRI1-OE and WT
`plants. (B,C) The soluble sugars, starch, total sugars, and proteins in the leaves of 2-month-old plants. (D,E) The soluble sugar, starch, total sugar, and protein
`contents in mature seeds. Values are mean ± SD (n = 3). DW, dry weight; ∗P < 0.05, ∗∗P < 0.01.
`
`and 18% in OE2, OE16, and OE17, respectively (Figure 3A).
`FA profiling revealed that oleic acid (18:1) contributed mostly
`to oil accumulation in OE plants. Other FA species such as 16:0
`and 18:2 also increased in OE seeds (Figure 3B). The OE of
`BnWRI1 also increased the oil contents of vegetative tissues. The
`oils in the leaves were primarily composed of 16:0, 18:1, and
`18:0 FA species, which account for ∼90% of the total FA species.
`The total TAG contents of the leaves from OE2, OE16, and
`OE17 increased by 28, 67, and 63%, respectively, in comparison
`with the WT plants (Figure 3C). The enhanced TAG in the
`OE leaves resulted from the increase of 16:0, 18:0, 18:1, and
`18:2 FA species (Figure 3D). The relative increase in leaf oil
`content from BnWRI1 OE was greater than that of the seed oil
`content.
`
`The Effect of BnWRI1 on Total Lipids,
`Sugar, and Protein Accumulation
`tissues,
`green
`Carbohydrates
`are
`photosynthesized
`in
`predominantly in leaves, which are the major source tissue,
`providing precursors for lipid and protein synthesis. Carbon
`partitioning among these metabolites is a major factor that
`influences lipid accumulation. To determine whether enhanced
`oil accumulation resulted from alterations in carbon partitioning,
`the contents of the total
`lipids, soluble sugars, starch, and
`proteins were measured in the leaves of 3-month-old plants
`at the flowering stage. The total
`lipid content of OE leaves
`was significantly higher than that of WT, and it was increased
`
`by 24, 47, and 25% for OE2, OE16, and OE17, respectively
`(Figure 4A). BnWRI1-OE leaves exhibited increased soluble
`sugars with reduced starch contents compared with WT plants
`(Figures 4B,C). However, the total sugars and total protein
`content in OE leaves were not substantially different from those
`of WT plants (Figures 4B,C). In comparison with those in the
`leaves, the contents of soluble sugars, starch, total sugars, and
`proteins in mature seeds were less altered between the OE and
`WT (Figures 4D,E). The starch contents of OE2 and OE16 seeds
`were lower than that of the WT, whereas the soluble sugar, total
`sugar, and protein contents in OE seeds were not significantly
`different from that of the WT (Figures 4D,E). These results
`indicate that enhancing the lipid content by overexpressing
`BnWRI1 without reducing sugars and proteins, but enhanced
`leaf sugar moves from storage starch to soluble sugars for lipid
`accumulation.
`
`BnWRI1 Binds to the Proximal Upstream
`Regions of Genes Involved in Lipid
`Anabolism
`into the molecular mechanism of BnWRI1
`To get
`insight
`in lipid metabolism and carbon partitioning, the subcellular
`localization of BnWRI1 and its putative target genes involved
`in lipid metabolism were investigated. The full-length cDNA
`of BnWRI1 was fused with GFP at the C-terminus and then
`transiently expressed in the epidermal cells of tobacco leaves by
`Agrobacterium infiltration. Green fluorescent BnWRI1-GFP was
`
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`Li et al.
`
`BnWRI1 in Flowering and Lipid Homeostasis
`
`FIGURE 5 | Nuclear localization of BnWRI1. (A) Green fluorescence of BnWRI1-GFP. (B) Nucleus stained with DAPI. (C) Bright image. (D) Merged image of the
`same cells observed in (A). BnWRI1-GFP was transiently expressed in Nicotiana benthamiana epidermal cells and visualized under by confocal laser scanning
`microscopy. The nuclear localization of BnWRI1 was indicated by green fluorescence that was overlaid with the nucleus indicated by DAPI staining.
`
`visualized by confocal laser scanning microscopy (Figure 5A),
`and the resulting image was overlaid with a nucleus marked by
`4(cid:3),6-diamidine-2-phenylindole dihydrochloride (DAPI) staining,
`confirming that the introduced BnWRI1 is localized to the
`nucleus (Figures 5B–D). The proximal upstream regions of
`Arabidopsis genes such as pyruvate kinase (PKp), BCCP2,
`KASI, LPAT2, and GPAT9 that are involved in glycolysis, FA
`synthesis, and lipid assembly contain the AW-box featured
`with [CnTnG](n)7[CG] (Figure 6A). To test whether BnWRI1
`binds to the target DNA fragments containing the AW-box,
`the BnWRI1 protein was expressed in E. coli and purified for
`binding assays (Figure 6C). The DNA fragments of 250–300 bp
`that contained the AW-box [CnTnG](n)7[CG] in the promoter
`region of two representative genes known as KASI and GPAT9
`were amplified and used for EMSA. When BnWRI1 protein
`was incubated with the DNA fragment amplified from the KASI
`promoter containing the AW-box [CnTnG](n)7[CG], the target
`DNA was bound to BnWRI1, as indicated by the shifted bands at
`the top of gel when it was visualized under UV light (Figure 6D).
`However, when the DNA fragment with a mutant AW box
`[TnCnA](n)7[CG] was explored,
`the binding between the
`
`BnWRI1 and the DNA fragment was abolished, as shown by the
`observation that the DNA band position remaining unchanged
`in the gel (Figures 6B,D). A similar gel shift was observed
`when BnWRI1 protein was incubated with the DNA fragment
`containing an AW box [CnTnG](n)7[CG] from the promoter
`region of GAPT9 (Figure 6D). The binding was diminished when
`the consensus was mutated to [CnTnG](n)7[TA] for the GPAT9
`promoter (Figures 6B,D). A similar result was found when the
`DNA probes were labeled with biotin (Figure 6E). These results
`suggest that BnWRI1 specifically binds to the promoter region
`containing the AW-box, which is conserved with Arabidopsis
`WRI1, and the AW-box is essential for the interaction between
`BnWRI1 and the target gene promoters.
`
`Overexpression of BnWRI1 Up-regulates
`the Transcript Level of Genes in
`Glycolysis, Fatty Acid Synthesis, Lipid
`Assembly, and Flowering
`To investigate whether BnWRI1 OE up-regulated the transcript
`level of genes involved in the lipid anabolism process, RNA
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`November 2015 | Volume 6 | Article 1015
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`Li et al.
`
`BnWRI1 in Flowering and Lipid Homeostasis
`
`FIGURE 6 | BnWRI1 binding to proximal upstream regions of genes involved in lipid anabolism. (A) The AW-box [CnTnG](n)7[CG] in the proximal upstream
`regions of genes involved in glycolysis, FA synthesis, and lipid assembly. (B) Consensus [CnTnG](n)7[CG] of the AW-box and its mutant AW-box [TnCnA](n)7[CG] of
`the KASI promoter or [CnTnG](n)7[TA] of GPAT9 promoter. (C) Recombinant BnWRI1 protein (50 kD) expressed in E. coli cells. (D) BnWRI1 specifically binds to the
`promoter region of KASI and GPAT9 containing the AW-box as assayed by EMSA. The binding was abolished when the conserved sequence was mutated into the
`[TnCnA](n)7[CG] of the KASI promoter or the [CnTnG](n)7[TA] of the GPAT9 promoter. The purified BnWRI1 protein (0.4 μg) was incubated with the target DNA
`fragment for 1 h. The resulting complex was visualized under UV light. The lanes from left to right: M, DNA ladder; KASI promoter only; BnWRI1 (0.4 μg) + KASI
`promoter; KASI mutant promoter only; BnWRI1 (0.4 μg) + KASI mutant promoter; GPAT9 promoter only; BnWRI1 (0.4 μg) + GPAT9 promoter; GPAT9 mutant
`promoter only; BnWRI1 (0.4 μg) + GPAT9 mutant promoter; BnWRI1 only. (E) The binding activity of BnWRI1 to the AW-box or its mutant AW-box (mAW) in the
`promoter regions of KASI and GPAT9 by EMSA using the DNA probes labeled with biotin. The shifted band is indicated by the arrow.
`
`was extracted from the leaves and analyzed by quantitative real-
`time PCR. These genes include PKp2 in glycolysis, BCCP2,
`MAT, KASI, ENR1, and acyl-ACP thioesterase (FATA) in the
`FA biosynthetic process, and GPAT9, LPAT2, and DGAT1 in
`lipid assembly. The transcript levels of the tested genes were all
`significantly higher in OE plants than in the WT. Despite the
`significant elevation of genes in multiple pathways by BnWRI1,
`the regulation of BnWRI1 in specific routes differs to variable
`extents. The transcript level of genes involved in glycolysis and
`FA synthesis including PKp2, MAT, KASI, ENR1, and FATA was
`up-regulated the most, and the expression levels in OE plants
`were more than twofold that of the WT (Figures 7A,B). The
`BnWRI1 enhanced expression of genes involved in FA synthesis
`was most prominent among the tested genes (Figure 7B).
`Moreover, RNA accumulation for the genes involved in lipid
`assembly was also strongly induced in OE plants. The mRNA
`level of GPAT9 in OE plants accumulated more than twofold
`that of the WT (Figure 7C). The LPAT2 and DGAT1 transcript
`levels were also substantially higher in OE than in WT plants
`(Figures 7C,D). In addition,
`the FLOWERING LOCUS T
`(FT) is a key regulator in the control of flowering time in
`several plant species, and the FT expression level in OE plants
`was threefold higher than that of the WT (Figure 7E). The
`results suggest that BnWRI1 synchronously promotes multiple
`pathways in transcriptional regulation to enhance the plant
`reproductive process, seed development, and oil accumulation in
`B. napus.
`
`The Effect of BnWRI1 on the Membrane
`Lipid Composition
`Most studies have focused on the effect of WRI1 on oil
`accumulation, but
`the effect of WRI1 on the membrane
`lipid composition remains elusive. The OE of BnWRI1 in
`B. napus leads to the involvement of numerous genes in
`glycolysis, FA biosynthesis, and the lipid assembly process,
`indicating its significance in lipid anabolism. To investigate
`the effect of BnWRI1 on various lipid metabolisms further,
`phospholipids and galactolipids from leaves or siliques during
`flowering stages were analyzed. The phosphatidylethanol (PE)
`and phosphatidylglycerol (PG) in leaves remained comparable
`between OE and WT plants (Figure 8A). However, BnWRI1
`OE resulted in a significant increase in MGDG, DGDG, and
`PC in leaves relative to their levels in WT plants (Figure 8A).
`The incre