The MYB96 Transcription Factor Regulates Triacylglycerol Accumulation by Activating DGAT1 and PDAT1 Expression in Arabidopsis Seeds

The MYB96 Transcription Factor Regulates Triacylglycerol Accumulation by Activating DGAT1 and... Abstract Maturing seeds stimulate fatty acid (FA) biosynthesis and triacylglycerol (TAG) accumulation to ensure carbon and energy reserves. Transcriptional reprogramming is a key regulatory scheme in seed oil accumulation. In particular, TAG assembly is mainly controlled by the transcriptional regulation of two key enzymes, acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) and phospholipid:diacylglycerol acyltransferase 1 (PDAT1), in Arabidopsis seeds. However, the transcriptional regulators of these enzymes are as yet unknown. Here, we report that the R2R3-type MYB96 transcription factor regulates seed oil accumulation by activating the genes encoding DGAT1 and PDAT1, the rate-limiting enzymes of the last step of TAG assembly. Total FA levels are significantly elevated in MYB96-overexpressing transgenic seeds, but reduced in MYB96-deficient mutant seeds. Notably, MYB96 regulation of TAG accumulation is independent of WRINKLED 1 (WRI1)-mediated FA biosynthesis. Taken together, our findings indicate that FA biosynthesis and TAG accumulation are under independent transcriptional control, and MYB96 is mainly responsible for TAG assembly in seeds. Introduction Seeds are the products of plant sexual reproduction and enable the search for favorable places for germination. For long-term survival, seeds accumulate storage reserves, such as proteins, carbohydrates and lipids, mainly in the cotyledons during the maturation stage (Mansfield and Briarty 1991, Baud et al. 2008). Notably, oil constitutes 35–40% of the fresh weight of mature Arabidopsis seeds (Graham 2008), and triacylglycerol (TAG) is the most important storage form of seed oil (Kelly et al. 2011). Seed oil accumulation requires the co-ordination of de novo fatty acid (FA) biosynthesis and TAG assembly. FAs are synthesized in plastids using plastidial acetyl-CoA as the building block (Feria Bourrellier et al. 2010, Andre et al. 2012). The main pathway for generating acetyl-CoA is the glycolytic process mediated by the plastidial pyruvate dehydrogenase complex (Ke et al. 2000, Lin and Oliver 2008), which catalyzes oxidative decarboxylation of pyruvate to produce acetyl-CoA, CO2 and NADH (Sawers and Clark 2004). Then, formation of malonyl-CoA from acetyl-CoA and bicarbonate requires the acetyl-CoA carboxylase (ACC) multisubunit heteromeric enzyme complex with two catalytic sites (Baud et al. 2003, Li-Beisson et al. 2013): the biotin carboxylase domain of ACC catalyzes transfer of CO2 from bicarbonate to a biotin prosthetic group bound to biotin carboxyl carrier protein (BCCP) (Thelen and Ohlrogge 2002a, Li et al. 2011); the carboxyltransferase domain of ACC transfers the carboxyl group of carboxy-biotin to acetyl-CoA and yields malonyl-CoA (Shorrosh et al. 1996, Sasaki et al. 1997, Thelen and Ohlrogge 2002a, Thelen and Ohlrogge 2002b). The malonyl group of malonyl-CoA is then transferred to an acyl carrier protein by a malonyl-CoA:acyl carrier protein malonyltransferase (Thelen and Ohlrogge 2002a, Thelen and Ohlrogge 2002b). The production of 16- or 18-carbon acyl chains is catalyzed by FA synthase. Acetyl-CoA is used as a starting unit, and malonyl-ACP provides the two-carbon units at each step of chain elongation (Dehesh et al. 2001, Shockey et al. 2002, Thelen and Ohlrogge 2002b). The initial condensation reaction is catalyzed by 3-ketoacyl-ACP synthetase III (KASIII) (Dehesh et al. 2001, Lai and Cronan 2003), and KASI is responsible for subsequent elongation from six to 16 carbons (Pidkowich et al. 2007, Yang et al. 2016). KASII finally extends 16:0-ACP to 18:0-ACP (Pidkowich et al. 2007). In addition, two reductases, 3-ketoacyl-ACP reductase and enoyl-ACP reductase (Massengo-Tiasse and Cronan 2009, Wu and Xue 2010), and one dehydrase, hydroxyacyl-ACP dehydratase, are additionally required after each condensation step to obtain a saturated FA. The synthesis of long-chain FAs is terminated with the release of the acyl group from ACP by acyl-ACP thioesterases (Jones et al. 1995, Tjellstrom et al. 2013). FAs synthesized in plastids undergo conversion into acyl-CoA, and these acyl-CoAs are incorporated into TAG in the endoplasmic reticulum (Chapman and Ohlrogge 2012, Bates et al. 2013). FA chains are transferred from acyl-CoA to the glycerol-3-phosphate backbone at the sn-1 and sn-2 positions by the acyltransferase reactions of glycerol-3-phosphate acyltransferase and lysophosphatidic acid acyltransferase, respectively (Kim et al. 2005, Shockey et al. 2016, Singer et al. 2016). Then, lysophosphatidic acid at the sn-3 position is dephosphorylated by phosphatidate phosphatase to form diacylglycerol (DAG) (Reddy et al. 2010). A third FA is transferred to the sn-3 of DAG by acyl-CoA:diacylglycerol acyltransferase (DGAT; Durrett et al. 2010, Liu et al. 2012). Although DGAT1 is a major enzyme for TAG assembly in Arabidopsis (Zhang et al. 2009, Vanhercke et al. 2013), other enzymes also compensate for TAG biosynthesis. Phospholipid:diacylglycerol acyltransferase 1 (PDAT1) catalyzes an acyl-CoA-independent reaction, by which the sn-2 acyl group of phospholipids such as phosphatidylcholine and phosphatidylethanolamine is transferred to the sn-3 position of DAG (Banas et al. 2000, Dahlqvist et al. 2000, Stahl et al. 2004). Notably, the final acylation step in TAG biosynthesis, which converts DAG to TAG, is considered the rate-limiting event (Katavic et al. 1995, Routaboul et al. 1999, Zhang et al. 2009). Consistently, disruption of both DGAT1 and PDAT1 genes results in a 70–80% decrease in seed oil content with abnormal seed development (Zhang et al. 2009, Chapman and Ohlrogge 2012). Transcriptional reprogramming is a key molecular scheme for both FA biosynthesis and TAG assembly during seed maturation (Bates et al. 2013, Collakova et al. 2013). The APETALA2 (AP2)/ethylene-responsive element-binding protein (EREBP)-type WRINKLED 1 (WRI1) transcription factor regulates FA biosynthesis at the onset of seed maturation by binding directly to the promoters of FA biosynthetic genes, such as PLASTIDIC PYRUVATE KINASE BETA SUBUNIT 1 (PKp-β1) and BCCP2 (Baud et al. 2007, Baud and Lepiniec 2009, To et al. 2012). Disruption of WRI1 results in an approximately 80% decrease in total FA levels in Arabidopsis seeds (Focks and Benning 1998, Chapman and Ohlrogge 2012). In addition, the B3 domain-containing transcription factor LEAFY COTYLEDON 2 (LEC2) also unequivocally regulates seed oil accumulation, together with other master regulators of seed development, including LEC1, LEAFY COTYLEDON1-LIKE (LIL), FUSCA3 (FUS3) and ABSCISIC ACID INSENSITIVE 3 (ABI3) (Stone et al. 2001, Kagaya et al. 2005, Kim et al. 2013, Chen et al. 2015). These proteins control FA biosynthetic and TAG metabolic genes during seed maturation to ensure the accumulation of storage reserves (Baud et al. 2008). In Arabidopsis, ectopic expression of LEC2 leads to considerable FA and TAG accumulation even in vegetative tissues, whereas LEC2-deficient seeds have approximately 30% less oil (Angeles-Nunez and Tiessen 2011, Kim et al. 2013, Kim et al. 2015). Despite the importance of TAG assembly in seed oil accumulation, the transcriptional regulator(s) responsible for the process remains to be fully unraveled. In this study, we report that MYB96 is a crucial regulator of TAG accumulation during seed maturation, which transcriptionally activates DGAT1 and PDAT1. The MYB96 gene is highly expressed in developing seeds and positively regulates TAG biosynthesis in seeds. MYB96-mediated TAG accumulation is largely independent of a WRI1-mediated FA biosynthesis pathway, highlighting its specific role in TAG assembly. Results Expression of core TAG biosynthetic genes is down-regulated in myb96-deficient seeds The MYB96 transcription factor is known to regulate diverse aspects of plant growth and development. In particular, lipid metabolic processes, including very long chain fatty acid biosynthesis, FA elongation and modification, and lipid breakdown, are under the control of MYB96 (Seo et al. 2011, Lee et al. 2015a, Lee et al. 2015b, Lee et al. 2015c). Thus, we hypothesized that additional lipid metabolic processes might be affected by MYB96. To examine this possibility, we widely analyzed expression of lipid metabolic genes in MYB96-overexpressing activation-tagging myb96-ox, MYB96-deficient myb96-2 and wild-type seedlings. Quantitative real-time reverse transcription–PCR (RT–qPCR) analysis revealed that transcript accumulation of DGAT1, PDAT1 and FATTY ACID DESATURASE 3 (FAD3) was elevated in myb96-ox seedlings, whereas DGAT1 and PDAT1 were significantly down-regulated in myb96-2 mutant seedlings (Supplementary Fig. S1). Considering the expression patterns of myb96-ox and myb96-2 mutants, the DGAT1 and PDAT1 genes appeared likely to be key regulatory targets of MYB96, possibly involved in the control of TAG biosynthesis. Since TAG is a major carbon storage form in seeds (Baud et al. 2008, Graham 2008), we hypothesized that MYB96 may mainly be involved in TAG accumulation in the seeds. In support of this, the MYB96 gene is highly expressed in seeds and regulates a variety of seed developmental and metabolic processes, including seed dormancy, germination and FA modification (Seo et al. 2009, Seo et al. 2011, Lee et al. 2015a, Lee et al. 2015c). Our RT–qPCR analysis also provided support for MYB96 being highly expressed in developing siliques containing immature seeds, with an expression level equivalent to leaf tissues (Supplementary Fig. S2). DGAT1 and PDAT1 were also highly expressed in developing seeds and vegetative tissues (Supplementary Fig. S2). These observations were also supported by a web-based Arabidopsis gene expression database (eFP browser; http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi) (Supplementary Fig. S3). Hence, while we performed expression profiling of lipid metabolic genes in seedlings (Supplementary Fig. S1), it was necessary to test the transcriptional regulation of the TAG assembly genes by MYB96 in developing seeds. To this end, we analyzed expression of key genes influencing FA and TAG metabolism in developing siliques of wild-type, myb96-ox and myb96-2 genotypes (Fig. 1). We harvested silique tissues at mid-developmental stages (Supplementary Fig. S4), during which seed oil accumulation actively occurs (Kwong et al. 2003, Fatihi et al. 2013, Li et al. 2017). Consistent with the results in seedlings (Supplementary Fig. S1), the glycolytic and late FA biosynthetic genes, including PKp2, PDH-E1a, BCCP2, MAT, KASI, KASIII, ENR1 and FATA (To et al. 2012), were negligibly affected in MYB96-misexpressing seeds (Fig. 1). Expression of WRI1, a gene encoding a transcriptional regulator of FA metabolism, was also independent of MYB96 activity (Fig. 1). Moreover, glycerol-3-phosphate dehydrogenase genes (GPDH and GPDHc1), which are responsible for the glycerol backbone supply (Shen et al. 2006, To et al. 2012), were also uninfluenced (Fig. 1). Fig. 1 View largeDownload slide Expression of genes involved in lipid metabolism in developing siliques of the wild-type, myb96-ox and myb96-2 genotypes. Long green mature siliques were harvested for each genotype (Stage 2 in Supplementary Fig. S4). Transcript accumulation was analyzed by quantitative real-time reverse transcription–PCR (RT–qPCR). The EUKARYOTIC TRANSLATION INITIATION FACTOR 4A1 (eIF4a) gene (At3g13920) was used as an internal control. Biological triplicates were averaged. Statistically significant differences between the wild type and mutants are indicated by asterisks (*P < 0.05, Student’s t-test). Bars indicate the SEM. Fig. 1 View largeDownload slide Expression of genes involved in lipid metabolism in developing siliques of the wild-type, myb96-ox and myb96-2 genotypes. Long green mature siliques were harvested for each genotype (Stage 2 in Supplementary Fig. S4). Transcript accumulation was analyzed by quantitative real-time reverse transcription–PCR (RT–qPCR). The EUKARYOTIC TRANSLATION INITIATION FACTOR 4A1 (eIF4a) gene (At3g13920) was used as an internal control. Biological triplicates were averaged. Statistically significant differences between the wild type and mutants are indicated by asterisks (*P < 0.05, Student’s t-test). Bars indicate the SEM. However, the genes encoding DGAT1 and PDAT1, the rate-limiting enzymes for TAG assembly, were significantly repressed in myb96-2, but up-regulated in myb96-ox, especially at mid-stages of silique development (Fig. 1; Supplementary Fig. S4). In contrast, the two genes were not differentially expressed in dry seeds of myb96-ox and myb96-2 plants (Supplementary Fig. S5), possibly because TAG metabolic processes have been completed in dry seeds. These results suggest that MYB96 primarily regulates TAG accumulation in immature seeds, possibly by activating metabolic enzymes involved in the final acylation step of TAG assembly, rather than FA biosynthesis. TAG levels are reduced in myb96-deficient mutant seeds DGAT1 and PDAT1 are key rate-limiting enzymes in TAG biosynthesis (Katavic et al. 1995, Routaboul et al. 1999, Zhang et al. 2009). To assess the connection between MYB96 and TAG biosynthesis, we employed wild-type and MYB96-deficient myb96-2 mutant seeds (Lee et al. 2015b, Lee et al. 2016) and measured total FA contents. Total FAs were extracted from dry seeds of all genotypes and analyzed by gas chromatography (GC). Quantitative analysis of total FAs revealed that myb96-2 seeds had approximately 20% lower contents of total FAs compared with wild-type seeds (Fig. 2A;Supplementary Fig. S6), despite the dry weight of myb96-2 mutant seeds being comparable with that of wild-type seeds (Supplementary Fig. S7). FA composition was further analyzed, and the levels of most FA species significantly decreased in myb96-2 relative to wild-type seeds (Fig. 2B). To support this result, we also tested the additional myb96-1 mutant, which is a weak mutant allele possibly due to the T-DNA insertion in the intron (Fig. 2C;Seo et al. 2009). However, unexpectedly, although expression of DGAT1 and PDAT1 was reduced in myb96-1 seeds (Fig. 2C), total FA contents were negligibly influenced (Supplementary Fig. S8), which may be due to insufficient reduction of DGAT1 and PDAT1 in myb96-1 to influence TAG accumulation. Fig. 2 View largeDownload slide Quantification of total fatty acid levels in wild-type and myb96-2 mutant seeds. In (A) and (B), the abundance of total fatty acids in dry seeds was determined by gas chromatography (GC). At least 300 seeds per independent line were measured in each replicate. Triplicate experiments were averaged. Statistically significant differences between the wild type and the myb96-2 mutant are indicated by asterisks (Student’s t-test, *P < 0.05, **P < 0.01). Bars indicate the SEM. (A) Total fatty acid contents. (B) Fatty acid profiles. (C) Expression of MYB96, DGAT1 and PDAT1 in immature seeds of myb96 mutants. Developing siliques were harvested for total RNA isolation. Transcript accumulation was analyzed by RT–qPCR. The eIF4a gene was used as an internal control. Biological triplicates were averaged. Different letters represent a significant difference at P < 0.05 (one-way ANOVA with Fisher’s post-hoc test). Bars indicate the SEM. Fig. 2 View largeDownload slide Quantification of total fatty acid levels in wild-type and myb96-2 mutant seeds. In (A) and (B), the abundance of total fatty acids in dry seeds was determined by gas chromatography (GC). At least 300 seeds per independent line were measured in each replicate. Triplicate experiments were averaged. Statistically significant differences between the wild type and the myb96-2 mutant are indicated by asterisks (Student’s t-test, *P < 0.05, **P < 0.01). Bars indicate the SEM. (A) Total fatty acid contents. (B) Fatty acid profiles. (C) Expression of MYB96, DGAT1 and PDAT1 in immature seeds of myb96 mutants. Developing siliques were harvested for total RNA isolation. Transcript accumulation was analyzed by RT–qPCR. The eIF4a gene was used as an internal control. Biological triplicates were averaged. Different letters represent a significant difference at P < 0.05 (one-way ANOVA with Fisher’s post-hoc test). Bars indicate the SEM. To rule out the possibility that reduced TAG accumulation in myb96-2 mutants may be attributable to increased TAG catabolic activities, expression of TAG catabolic genes including SUGAR-DEPENDENT 1 (SDP1), SDP1L, MYZUS PERSICAE-INDUCED LIPASE 1 (MPL1), LIPOIC ACID SYNTHASE 1 (LIP1) and DAD1-LIKE LIPASE 5 (DALL5) was also examined in myb96-misexpressing seeds. These genes are involved in TAG degradation and β-oxidation during seed maturation and seedling establishment (He and Gan 2002, El-Kouhen et al. 2005, Seo et al. 2009, Slocombe et al. 2009, Kelly et al. 2013, Fan et al. 2014, Kim et al. 2014, Thazar-Poulot et al. 2015). As expected, all genes examined were uninfluenced in MYB96-misexpressing seeds, whereas transcript accumulation of DGAT1 and PDAT1 was changed accordingly (Supplementary Fig. S9), supporting the role of MYB96 in TAG biosynthesis. Overexpression of MYB96 leads to increased TAG accumulation in seeds To strengthen our results further, we also evaluated the effects of overexpression of MYB96 on seed TAG accumulation. We generated transgenic plants expressing the MYB96 gene under the control of the Cauliflower mosaic virus (CaMV) 35S or the seed-specific phaseolin (Phas) promoter. T3 generation seeds of 10 randomly selected lines for each genotype were used to determine total FA contents. GC analysis revealed that a majority of transgenic lines showed a significant increase of FA levels in dry seeds compared with the wild type (Fig. 3A;Supplementary Fig. S10). Consistent with the increase in seed total FA levels, the seed dry weight and size were also increased (Supplementary Fig. S11). Notably, the large increase in the relative content of total FAs by weight supported the high oil density of transgenic seeds (Fig. 3A). Transcript levels of DGAT1 and PDAT1 were also significantly elevated in selected transgenic lines with high seed oil contents (Fig. 3B). Fig. 3 View largeDownload slide Total fatty acid levels in transgenic seeds overexpressing MYB96. In (A) and (C), the abundance of total fatty acids in dry seeds was determined by GC. At least 300 seeds per independent T3 transgenic line were measured in each replicate. Triplicate experiments were averaged. Statistically significant differences between the wild-type and transgenic seeds are indicated by asterisks (Student’s t-test, *P < 0.05, **P < 0.01). Bars indicate the SEM. (A) Total fatty acid contents. (B) Expression of DGAT1 and PDAT1 genes in siliques of 35S:MYB96 and Phas:MYB96 transgenic plants. Developing siliques were used to analyze transcript accumulation. The eIF4a gene was used as an internal control. Biological triplicates were averaged. Statistically significant differences between wild-type and transgenic plants are indicated by asterisks (*P < 0.05, Student’s t-test). Bars indicate the SEM. (C) Fatty acid profiles. Fatty acid content is expressed as the percentage of the amount present in dry seeds of the indicated genotypes relative to wild-type seeds (100%). Fig. 3 View largeDownload slide Total fatty acid levels in transgenic seeds overexpressing MYB96. In (A) and (C), the abundance of total fatty acids in dry seeds was determined by GC. At least 300 seeds per independent T3 transgenic line were measured in each replicate. Triplicate experiments were averaged. Statistically significant differences between the wild-type and transgenic seeds are indicated by asterisks (Student’s t-test, *P < 0.05, **P < 0.01). Bars indicate the SEM. (A) Total fatty acid contents. (B) Expression of DGAT1 and PDAT1 genes in siliques of 35S:MYB96 and Phas:MYB96 transgenic plants. Developing siliques were used to analyze transcript accumulation. The eIF4a gene was used as an internal control. Biological triplicates were averaged. Statistically significant differences between wild-type and transgenic plants are indicated by asterisks (*P < 0.05, Student’s t-test). Bars indicate the SEM. (C) Fatty acid profiles. Fatty acid content is expressed as the percentage of the amount present in dry seeds of the indicated genotypes relative to wild-type seeds (100%). FA composition analysis demonstrated that all FA species were increased in the transgenic lines (Fig. 3C), which may account for the substantial accumulation of TAG in MYB96-overexpressing seeds. In particular, an increase in the levels of C18:1, which is known to be a preferred substrate of DGAT1, was prominent in all transgenic lines examined. It was also notable that the employed promoters have different effects on the FA composition of seeds, as exemplified by C20:1 accumulation (Fig. 3C). These results indicate that seed TAG accumulation is positively regulated by MYB96. MYB96 directly binds to the PDAT1 promoter We next asked whether the MYB96 transcription factor binds directly to the promoters of TAG biosynthetic genes. Sequence analysis revealed that each gene promoter contains conserved sequence motifs that are analogous to the R2R3-type MYB-binding consensus sequence (Borg et al. 2011, Seo et al. 2011) (Fig. 4A, B). Fig. 4 View largeDownload slide MYB96 binding to the PDAT1 promoter. In (A) and (B), putative R2R3-MYB-binding sites are indicated by arrowheads. Black underbars indicate regions of PCR amplification after chromatin immunoprecipitation (ChIP). Total protein extracts from pMYB96:MYB96-MYC transgenic seeds were immunoprecipitated with an anti-MYC antibody. Enrichment of putative MYB-binding regions was analyzed by qPCR analysis. Biological triplicates were averaged, and the statistical significance of measurements was determined using a Student’s t-test (*P < 0.05). Bars indicate the SEM. In each experiment, the measurement values in pBA002 were set to 1 after normalization against eIF4a for qPCR analysis. (A) Binding of MYB96 to the PDAT1 promoter. (B) Binding of MYB96 to the DGAT1 promoter. (C) Transient expression assays. The core element (B; CGAATAGTTACGGA) and mutated version of the core sequence (mB; CGAAAAAAAACGGA) were inserted into the reporter plasmid. Recombinant reporter and effector constructs were co-expressed transiently in Arabidopsis protoplasts, and GUS activity was determined fluorimetrically. Luciferase gene expression was used to normalize GUS activity. The normalized values in control protoplasts were set to 1 and are represented as relative activation. Three independent measurements were averaged. Statistical significance was determined by a Student’s t-test (*P < 0.05). Bars indicate the SEM. Fig. 4 View largeDownload slide MYB96 binding to the PDAT1 promoter. In (A) and (B), putative R2R3-MYB-binding sites are indicated by arrowheads. Black underbars indicate regions of PCR amplification after chromatin immunoprecipitation (ChIP). Total protein extracts from pMYB96:MYB96-MYC transgenic seeds were immunoprecipitated with an anti-MYC antibody. Enrichment of putative MYB-binding regions was analyzed by qPCR analysis. Biological triplicates were averaged, and the statistical significance of measurements was determined using a Student’s t-test (*P < 0.05). Bars indicate the SEM. In each experiment, the measurement values in pBA002 were set to 1 after normalization against eIF4a for qPCR analysis. (A) Binding of MYB96 to the PDAT1 promoter. (B) Binding of MYB96 to the DGAT1 promoter. (C) Transient expression assays. The core element (B; CGAATAGTTACGGA) and mutated version of the core sequence (mB; CGAAAAAAAACGGA) were inserted into the reporter plasmid. Recombinant reporter and effector constructs were co-expressed transiently in Arabidopsis protoplasts, and GUS activity was determined fluorimetrically. Luciferase gene expression was used to normalize GUS activity. The normalized values in control protoplasts were set to 1 and are represented as relative activation. Three independent measurements were averaged. Statistical significance was determined by a Student’s t-test (*P < 0.05). Bars indicate the SEM. To examine whether MYB96 is targeted to TAG biosynthetic gene promoters, chromatin immunoprecipitation (ChIP) assays were performed using pMYB96:MYB96-MYC transgenic seeds. Total protein extracts from pBA002 control and pMYB96:MYB96-MYC transgenic seeds were immunoprecipitated with an anti-MYC-antibody. DNA bound to epitope-tagged MYB96 proteins was analyzed by quantitative real-time PCR (qPCR) assays. Analysis showed that the B and C regions of the PDAT1 promoter were enriched as a result of ChIP (Fig. 4A). In contrast, resin alone did not bind to the PDAT1 promoter (Supplementary Fig. S12). However, direct binding of MYB96 to the DGAT1 promoter was not detected (Fig. 4B). These results indicate that MYB96 specifically binds to the PDAT1 promoter and indirectly influences DGAT1. To support further direct binding of MYB96 to the PDAT1 promoter, we conducted a transient expression analysis using Arabidopsis protoplasts. The core element in the B region (B; CGAATAGTTACGGA) or a mutated element (mB; CGAAAAAAAACGGA) was fused to the 35S minimal reporter promoter. A recombinant reporter plasmid and the effector p35S:MYB96 plasmid were co-transformed into Arabidopsis protoplasts. Co-transformation with the reporter B construct increased β-glucuronidase (GUS) activity by approximately 4-fold, but co-transformation with the reporter mB did not stimulate reporter gene expression (Fig. 4C). These results indicate that MYB96 binds to the PDAT1 promoter and transcriptionally activates expression. MYB96 has been reported to bind to the promoter of ABI4 in seeds (Lee et al. 2015c), which directly regulates DGAT1 (Yang et al. 2011, Kong et al. 2013). Therefore, we speculated that MYB96 might regulate DGAT1 through ABI4. To test this hypothesis, we employed myb96-ox/abi4-1 plants, in which the myb96-ox mutant was genetically crossed with the abi4-1 loss-of-function mutant allele (Lee et al. 2015c), and compared transcript accumulation of DGAT1 with myb96-ox. The elevated expression of DGAT1 in myb96-ox was suppressed in myb96-ox/abi4-1 developing seeds, while PDAT1 expression was unaffected by ABI4 activity (Supplementary Fig. S13). Furthermore, transient expression assays also supported that MYB96 regulation of DGAT1 depends on ABI4. A reporter construct, in which the DGAT1 promoter sequence was fused with the minimal 35S promoter, was co-expressed with an effector construct overexpressing MYB96 in Arabidopsis protoplasts isolated from wild-type and abi4-1 leaves. Reporter GUS activity measurement revealed that MYB96 significantly increased DGAT1 promoter activity in the wild-type background, but this function was impaired in the abi4-1 background (Supplementary Fig. S14). These observations provide support that ABI4 mediates MYB96 regulation of DGAT1 to control TAG biosynthesis. The MYB96-regulated pathway is independent of WRI1 Seed oil accumulation is co-ordinated by FA biosynthesis and TAG assembly (Baud et al. 2007, Baud et al. 2008). While it was evident that MYB96 is mainly involved in TAG assembly and that genes involved in FA biosynthesis were uninfluenced in MYB96-misexpressing seeds (Fig. 1), we wanted to know whether FA levels reciprocally influence MYB96 expression. Based on the essential role of WRI1 in FA accumulation (Baud et al. 2007, To et al. 2012), we analyzed transcript accumulation of MYB96 in wri1-3 mutant seeds. RT–qPCR analysis revealed that MYB96 expression was unaffected in wri1-3 (Fig. 5A), which shows a substantial decrease in seed FA levels (Vanhercke et al. 2013). Consistently, DGAT1 and PDAT1 transcript accumulation was also marginally affected in wri1-3 (Fig. 5B, C). In addition, WRI1 expression was unchanged in dgat1-1 and pdat1-1 mutant seeds (Fig. 5D), which show a reduction in TAG accumulation. Fig. 5 View largeDownload slide Independent transcriptional control of fatty acid biosynthesis and TAG assembly. In (A–D), transcript accumulation was analyzed by RT–qPCR. The eIF4a gene was used as an internal control. Biological triplicates were averaged. Bars indicate the SEM. (A) Expression of MYB96 in wri1-3 seeds. (B) Expression of DGAT1 in wri1-3 seeds. (C) Expression of PDAT1 in wri1-3 seeds. (D) Transcript accumulation of WRI1 in dgat1-1 and pdat1-1 seeds. (E) Proposed working diagram. FA biosynthesis and TAG assembly are under independent transcriptional control. During seed maturation, WRI1 plays a crucial role in early FA biosynthesis, whereas MYB96 is primarily involved in TAG assembly. The two transcription factors fine-tune stepwise metabolic processes in immature seeds. Fig. 5 View largeDownload slide Independent transcriptional control of fatty acid biosynthesis and TAG assembly. In (A–D), transcript accumulation was analyzed by RT–qPCR. The eIF4a gene was used as an internal control. Biological triplicates were averaged. Bars indicate the SEM. (A) Expression of MYB96 in wri1-3 seeds. (B) Expression of DGAT1 in wri1-3 seeds. (C) Expression of PDAT1 in wri1-3 seeds. (D) Transcript accumulation of WRI1 in dgat1-1 and pdat1-1 seeds. (E) Proposed working diagram. FA biosynthesis and TAG assembly are under independent transcriptional control. During seed maturation, WRI1 plays a crucial role in early FA biosynthesis, whereas MYB96 is primarily involved in TAG assembly. The two transcription factors fine-tune stepwise metabolic processes in immature seeds. Taken together, MYB96 regulates TAG accumulation in developing seeds by activating DGAT1 and PDAT1, which are responsible for catalyzing the rate-limiting step of TAG biosynthesis (Zhang et al. 2009, Xu et al. 2012). The trio of MYB96, DGAT1 and PDAT1 were uninfluenced by WRI1, and vice versa, indicating that FA biosynthesis and TAG assembly are under independent transcriptional control. During seed maturation, WRI1 plays a crucial role in FA supply, whereas MYB96 is primarily involved in TAG assembly (Fig. 5E). The sequential actions of transcriptional regulators may account for the control of seed maturation and lipid storage. Discussion Diverse roles of MYB96 in seeds The MYB96 transcription factor is known as a central mediator of ABA signaling in a variety of physiological processes, such as lateral root development, hormone metabolism, stomatal opening and cuticular wax biosynthesis (Seo et al. 2009, Seo and Park 2010, Seo et al. 2011, Lee et al. 2015a, Lee et al. 2015b, Lee et al. 2015c, Lee et al. 2016). In addition to its roles in vegetative tissues, MYB96 is also highly expressed in seeds (Lee et al. 2015b, Lee et al. 2015c) and participates in the processes of seed maturation, dormancy and germination (Lee et al. 2015a, Lee et al. 2015c). During seed maturation, MYB96 is implicated in carbon metabolic processes. The gene encoding the seed-specific FATTY ACID ELONGATION 1 (FAE1) enzyme, which triggers chain elongation of C18–C20 and C22 (James et al. 1995, Roscoe et al. 2001), is up-regulated in myb96-ox seeds (Lee et al. 2015b), contributing to long chain FA accumulation in maturing seeds. The FAD3 gene was also affected in myb96-ox (Supplementary Fig. S1), which implies an additional role for MYB96 in lipid desaturation during seed development. MYB96 establishes primary seed dormancy by co-ordinating ABA and gibberellic acid metabolism during seed maturation, and inhibits viviparous germination (Lee et al. 2015b). Furthermore, this transcription factor also suppresses lipid degradation, which fuels seed germination by supplying an energy source, in order to determine the proper timing for germination (Lee et al. 2015c). MYB96 primarily regulates ABI4 transcription in the control of lipid mobilization-dependent seed germination and confers ABA sensitivity specifically in the seed embryo (Lee et al. 2015c). Close connections between ABA signaling and TAG metabolism have been extensively proposed. During seed maturation, high levels of ABA ensure TAG and oil body accumulation as well as inhibition of lipid breakdown (Crowe et al. 2000, Brocard-Gifford et al. 2003). Consistent with these observations, this study demonstrates that the ABA-inducible MYB96 transcription factor contributes to TAG accumulation, largely through the transcriptional control of DGAT1 and PDAT1. Large amounts of TAG accumulate in myb96-ox seeds, whereas myb96 mutation leads to reduced TAG accumulation. Increased TAG accumulation by MYB96 was independent of FA metabolic gene activities in that MYB96 overexpression did not affect any genes involved in FA biosynthesis and degradation pathways. Intrinsic FA biosynthetic enzyme activities might be sufficient to support TAG accumulation in myb96-ox. Otherwise, FA biosynthetic enzymes may be modulated at another level, such as changes in biochemical activities or protein turnover. For instance, the acetyl-CoA carboxylase in FA biosynthesis may not be feedback inhibited by acyl-ACP products due to an efficient incorporation of acyl-ACP into TAG (Andre et al. 2012). Taken together, MYB96 has roles in seed development and germination and also controls metabolic processes to link these biological programs. Molecular web underlying MYB96-regulated TAG accumulation TAG accumulation is regulated by a multitude of stepwise metabolic processes. In particular, the last acylation step is important for seed TAG assembly, which is catalyzed by DGAT1 and PDAT1, and many regulatory actions target this step for proper TAG accumulation (Zhang et al. 2009, Bates et al. 2013). Considering their biological and biotechnological importance, identification of upstream regulator(s) of DGAT1 and/or PDAT1 has been an important challenge in plant lipid metabolic engineering. Notably, the MYB96 transcription factor co-ordinates expression of the two core TAG biosynthetic genes and ensures proper levels of TAG biosynthesis in maturing seeds. Transcript accumulation of DGAT1 and PDAT1 was increased in myb96-ox, but suppressed in myb96-deficient mutant seeds. However, the mechanisms underlying the transcriptional regulation of DGAT1 and PDAT1 differ. The MYB96 transcription factor binds directly to the PDAT1 promoter, but not to the DGAT1 promoter. However, MYB96 regulation of DGAT1 is still relevant, because MYB96 regulates DGAT1 expression through ABI4. MYB96 binds to the R2R3-MYB-binding cis-elements on the ABI4 promoter and activates expression (Lee et al. 2015c). Then, ABI4 activates DGAT1 expression by binding directly to its promoter (Yang et al. 2011, Kong et al. 2013). Consistently, the elevated DGAT1 expression in myb96-ox was compromised in myb96-ox/abi4-1, providing support that the MYB96-ABI4 module is important for proper DGAT1 expression. Collectively, MYB96 is a pivotal regulator of TAG biosynthesis by regulating key genes that encode rate-limiting enzymes. FA biosynthesis and TAG assembly are co-ordinated to ensure elaborate lipid metabolism. Our results suggest that cross-talk between the metabolic processes is not observed, at least at the transcriptional level. MYB96 regulation of TAG accumulation was independent of a WRI1-regulated FA biosynthetic pathway, and vice versa. MYB96 and WRI1 are likely to play independent and separate roles in lipid metabolism: WRI1 mainly regulates the glycolytic and late FA biosynthetic pathways, whereas MYB96 stimulates the TAG assembly process. However, it is very likely that another regulatory layer is involved to ensure balanced metabolism between FA biosynthesis and TAG assembly, and further studies should yield a comprehensive view of the mechanisms of seed oil accumulation. Seed-derived TAG is a valuable source of food and biofuel. Therefore, metabolic engineering to boost TAG production in plants would be widely considered for commercial benefit. Given the strong effects of MYB96 on TAG accumulation, MYB96 could be a powerful genetic resource that could be applied to oilseed crops to enhance seed oil accumulation. Given that MYB96, DGAT1 and PDAT1 are also highly expressed in vegetative tissues, MYB96 can also be used for higher TAG accumulation in vegetative tissues. Furthermore, considering the independent transcriptional control, efficient lipid metabolic engineering could be achieved by simultaneous expression of key transcriptional regulators of FA biosynthesis and TAG assembly. Materials and Methods Plant materials and growth conditions Arabidopsis thaliana (Columbia-0 ecotype) was used for all experiments unless otherwise specified. Plants were grown under long-day conditions (16 h light/8 h dark cycles) with cool white fluorescent light (150 µmol photons m–2 s–1) at 22–23°C. The myb96-ox and myb96-2 (SALK_111645) mutants were previously reported (Seo et al. 2009, Lee et al. 2015c). The dgat1-1 (CS3861, Zou et al. 1999), pdat1-1 (SALK_032261, Mhaske et al. 2005) and wri1-3 (SALK_085693) mutants were obtained from the Arabidopsis Biological Resource Center (http://abrc.osu.edu/). Quantitative real-time RT–PCR analysis Total RNA was extracted using TRI reagent (TAKARA BIO INC.) according to the manufacturer’s recommendations. Reverse transcription was performed using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Dr. Protein) with oligo(dT)18 to synthesize first-strand cDNA from 2 µg of total RNA. Total RNA samples were pre-treated with RNase-free DNase. cDNAs were diluted to 100 µl with TE buffer, and 1 µl of diluted cDNA was used for PCR amplification. RT–qPCRs were performed in 96-well blocks using the Step-One Plus Real-Time PCR System (Applied Biosystems). The PCR primers used are listed in Supplementary Table S1. The values for each set of primers were normalized relative to the EUKARYOTIC TRANSLATION INITIATION FACTOR 4A1 (eIF4A) gene (At3g13920). All RT–qPCRs were performed with biological triplicates using total RNA samples extracted from three independent replicate samples. The comparative ΔΔCT method was employed to evaluate the relative quantities of each amplified product in the samples. The threshold cycle (CT) was automatically determined for each reaction with the analysis software set using default parameters. The specificity of the RT–qPCRs was determined by melt curve analysis of the amplified products using the standard method employed by the software. Fatty acid analysis The total FA content and their composition in seeds were measured by GC analysis with a known amount of glyceryl triheptadecanoate (Sigma-T2151) as an internal standard. Samples were transmethylated at 90°C for 90 min in 0.3 ml of toluene and 1 ml of 5% H2SO4 (v/v methanol). After transmethylation, 1.5 ml of 0.9% NaCl solution was added, and the FA methyl esters (FAMEs) were transferred to a new tube for three sequential extractions with 1.5 ml of n-hexane. FAMEs were analyzed by GC using a GC-2010 plus instrument (Shimadzu with a 30 m×0.25 mm (25 µm film thickness) DB-23 column (Agilent), during which the oven temperature was maintained for 10 min at 190°C, followed by an increase of 5°C min–1 to 230°C, and maintained for 10 min at 230°C. Chromatin immunoprecipitation ChIP assay was performed as previously described (Schoppee Bortz and Wamhoff 2011). pMYB96:MYB96-MYC transgenic seeds, anti-MYC antibodies (Millipore) and salmon sperm DNA/protein A agarose beads (Millipore) were used for ChIP. DNA was purified using phenol/chloroform/isoamyl alcohol and sodium acetate (pH 5.2). The level of precipitated DNA fragments was quantified by qPCR using specific primer sets (Supplementary Table S2). Values were normalized to the input DNA level. Values for control plants were set to 1 after normalization against eIF4a for quantitative PCR analysis. Transient expression analysis For transient expression assays using Arabidopsis protoplasts, reporter and effector plasmids were constructed. The reporter plasmid contains a minimal 35S promoter sequence and the GUS gene. The core elements on the DGAT1 and PDAT1 promoters were inserted into the reporter plasmid. To construct the p35S:MYB96 effector plasmid, MYB96 cDNA was inserted into the effector vector containing the CaMV 35S promoter. Recombinant reporter and effector plasmids were co-transformed into Arabidopsis protoplasts by polyethylene glycol-mediated transformation (Yoo et al. 2007). GUS activities were measured by a fluorometric method. A CaMV 35S promoter–luciferase construct was also co-transformed as an internal control. The luciferase assay was performed using the Luciferase Assay System kit (Promega). Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the National Research Foundation of Korea [Basic Research Laboratory grant program (NRF-2017R1A4A1015620 to M.C.S. and P.J.S.) and the MidCareer Researcher grant program (NRF-2017R1A2B4007096, H.U.K.); the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry, and Fisheries (IPET) [116079-03 and 316087-4 to H.U.K.]; and the Rural Development Administration [Cooperative Research Program for Agriculture Science and Technology Development (PJ01261303 to P.J.S.)]. Disclosures The authors have no conflicts of interest to declare. References Andre C. , Haslam R.P. , Shanklin J. ( 2012 ) Feedback regulation of plastidic acetyl-CoA carboxylase by 18:1-acyl carrier protein in Brassica napus . Proc. Natl. Acad. Sci. USA 109 : 10107 – 10112 . Angeles-Nunez J.G. , Tiessen A. 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Abbreviations Abbreviations ABI ABSCISC ACID INSENSITIVE BCCP biotin carboxyl carrier protein CaMV Cauliflower mosaic virus ChIP chromatin immunoprecipitation DAG diacylglycerol DGAT acyl-CoA:diacylglycerol acyltransferase FA fatty acid GC gas chromatography GUS β-glucuronidase KAS 3-ketoacyl-ACP synthetase LEC LEAFY COTYLEDON PDAT phospholipid:diacylglycerol acyltransferase qPCR quantitative real-time PCR RT–qPCR quantitative real-time reverse transcription–PCR TAG triacylglycerol WRI1 WRINKLED 1 © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant and Cell Physiology Oxford University Press

The MYB96 Transcription Factor Regulates Triacylglycerol Accumulation by Activating DGAT1 and PDAT1 Expression in Arabidopsis Seeds

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© The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com
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0032-0781
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1471-9053
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Abstract

Abstract Maturing seeds stimulate fatty acid (FA) biosynthesis and triacylglycerol (TAG) accumulation to ensure carbon and energy reserves. Transcriptional reprogramming is a key regulatory scheme in seed oil accumulation. In particular, TAG assembly is mainly controlled by the transcriptional regulation of two key enzymes, acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) and phospholipid:diacylglycerol acyltransferase 1 (PDAT1), in Arabidopsis seeds. However, the transcriptional regulators of these enzymes are as yet unknown. Here, we report that the R2R3-type MYB96 transcription factor regulates seed oil accumulation by activating the genes encoding DGAT1 and PDAT1, the rate-limiting enzymes of the last step of TAG assembly. Total FA levels are significantly elevated in MYB96-overexpressing transgenic seeds, but reduced in MYB96-deficient mutant seeds. Notably, MYB96 regulation of TAG accumulation is independent of WRINKLED 1 (WRI1)-mediated FA biosynthesis. Taken together, our findings indicate that FA biosynthesis and TAG accumulation are under independent transcriptional control, and MYB96 is mainly responsible for TAG assembly in seeds. Introduction Seeds are the products of plant sexual reproduction and enable the search for favorable places for germination. For long-term survival, seeds accumulate storage reserves, such as proteins, carbohydrates and lipids, mainly in the cotyledons during the maturation stage (Mansfield and Briarty 1991, Baud et al. 2008). Notably, oil constitutes 35–40% of the fresh weight of mature Arabidopsis seeds (Graham 2008), and triacylglycerol (TAG) is the most important storage form of seed oil (Kelly et al. 2011). Seed oil accumulation requires the co-ordination of de novo fatty acid (FA) biosynthesis and TAG assembly. FAs are synthesized in plastids using plastidial acetyl-CoA as the building block (Feria Bourrellier et al. 2010, Andre et al. 2012). The main pathway for generating acetyl-CoA is the glycolytic process mediated by the plastidial pyruvate dehydrogenase complex (Ke et al. 2000, Lin and Oliver 2008), which catalyzes oxidative decarboxylation of pyruvate to produce acetyl-CoA, CO2 and NADH (Sawers and Clark 2004). Then, formation of malonyl-CoA from acetyl-CoA and bicarbonate requires the acetyl-CoA carboxylase (ACC) multisubunit heteromeric enzyme complex with two catalytic sites (Baud et al. 2003, Li-Beisson et al. 2013): the biotin carboxylase domain of ACC catalyzes transfer of CO2 from bicarbonate to a biotin prosthetic group bound to biotin carboxyl carrier protein (BCCP) (Thelen and Ohlrogge 2002a, Li et al. 2011); the carboxyltransferase domain of ACC transfers the carboxyl group of carboxy-biotin to acetyl-CoA and yields malonyl-CoA (Shorrosh et al. 1996, Sasaki et al. 1997, Thelen and Ohlrogge 2002a, Thelen and Ohlrogge 2002b). The malonyl group of malonyl-CoA is then transferred to an acyl carrier protein by a malonyl-CoA:acyl carrier protein malonyltransferase (Thelen and Ohlrogge 2002a, Thelen and Ohlrogge 2002b). The production of 16- or 18-carbon acyl chains is catalyzed by FA synthase. Acetyl-CoA is used as a starting unit, and malonyl-ACP provides the two-carbon units at each step of chain elongation (Dehesh et al. 2001, Shockey et al. 2002, Thelen and Ohlrogge 2002b). The initial condensation reaction is catalyzed by 3-ketoacyl-ACP synthetase III (KASIII) (Dehesh et al. 2001, Lai and Cronan 2003), and KASI is responsible for subsequent elongation from six to 16 carbons (Pidkowich et al. 2007, Yang et al. 2016). KASII finally extends 16:0-ACP to 18:0-ACP (Pidkowich et al. 2007). In addition, two reductases, 3-ketoacyl-ACP reductase and enoyl-ACP reductase (Massengo-Tiasse and Cronan 2009, Wu and Xue 2010), and one dehydrase, hydroxyacyl-ACP dehydratase, are additionally required after each condensation step to obtain a saturated FA. The synthesis of long-chain FAs is terminated with the release of the acyl group from ACP by acyl-ACP thioesterases (Jones et al. 1995, Tjellstrom et al. 2013). FAs synthesized in plastids undergo conversion into acyl-CoA, and these acyl-CoAs are incorporated into TAG in the endoplasmic reticulum (Chapman and Ohlrogge 2012, Bates et al. 2013). FA chains are transferred from acyl-CoA to the glycerol-3-phosphate backbone at the sn-1 and sn-2 positions by the acyltransferase reactions of glycerol-3-phosphate acyltransferase and lysophosphatidic acid acyltransferase, respectively (Kim et al. 2005, Shockey et al. 2016, Singer et al. 2016). Then, lysophosphatidic acid at the sn-3 position is dephosphorylated by phosphatidate phosphatase to form diacylglycerol (DAG) (Reddy et al. 2010). A third FA is transferred to the sn-3 of DAG by acyl-CoA:diacylglycerol acyltransferase (DGAT; Durrett et al. 2010, Liu et al. 2012). Although DGAT1 is a major enzyme for TAG assembly in Arabidopsis (Zhang et al. 2009, Vanhercke et al. 2013), other enzymes also compensate for TAG biosynthesis. Phospholipid:diacylglycerol acyltransferase 1 (PDAT1) catalyzes an acyl-CoA-independent reaction, by which the sn-2 acyl group of phospholipids such as phosphatidylcholine and phosphatidylethanolamine is transferred to the sn-3 position of DAG (Banas et al. 2000, Dahlqvist et al. 2000, Stahl et al. 2004). Notably, the final acylation step in TAG biosynthesis, which converts DAG to TAG, is considered the rate-limiting event (Katavic et al. 1995, Routaboul et al. 1999, Zhang et al. 2009). Consistently, disruption of both DGAT1 and PDAT1 genes results in a 70–80% decrease in seed oil content with abnormal seed development (Zhang et al. 2009, Chapman and Ohlrogge 2012). Transcriptional reprogramming is a key molecular scheme for both FA biosynthesis and TAG assembly during seed maturation (Bates et al. 2013, Collakova et al. 2013). The APETALA2 (AP2)/ethylene-responsive element-binding protein (EREBP)-type WRINKLED 1 (WRI1) transcription factor regulates FA biosynthesis at the onset of seed maturation by binding directly to the promoters of FA biosynthetic genes, such as PLASTIDIC PYRUVATE KINASE BETA SUBUNIT 1 (PKp-β1) and BCCP2 (Baud et al. 2007, Baud and Lepiniec 2009, To et al. 2012). Disruption of WRI1 results in an approximately 80% decrease in total FA levels in Arabidopsis seeds (Focks and Benning 1998, Chapman and Ohlrogge 2012). In addition, the B3 domain-containing transcription factor LEAFY COTYLEDON 2 (LEC2) also unequivocally regulates seed oil accumulation, together with other master regulators of seed development, including LEC1, LEAFY COTYLEDON1-LIKE (LIL), FUSCA3 (FUS3) and ABSCISIC ACID INSENSITIVE 3 (ABI3) (Stone et al. 2001, Kagaya et al. 2005, Kim et al. 2013, Chen et al. 2015). These proteins control FA biosynthetic and TAG metabolic genes during seed maturation to ensure the accumulation of storage reserves (Baud et al. 2008). In Arabidopsis, ectopic expression of LEC2 leads to considerable FA and TAG accumulation even in vegetative tissues, whereas LEC2-deficient seeds have approximately 30% less oil (Angeles-Nunez and Tiessen 2011, Kim et al. 2013, Kim et al. 2015). Despite the importance of TAG assembly in seed oil accumulation, the transcriptional regulator(s) responsible for the process remains to be fully unraveled. In this study, we report that MYB96 is a crucial regulator of TAG accumulation during seed maturation, which transcriptionally activates DGAT1 and PDAT1. The MYB96 gene is highly expressed in developing seeds and positively regulates TAG biosynthesis in seeds. MYB96-mediated TAG accumulation is largely independent of a WRI1-mediated FA biosynthesis pathway, highlighting its specific role in TAG assembly. Results Expression of core TAG biosynthetic genes is down-regulated in myb96-deficient seeds The MYB96 transcription factor is known to regulate diverse aspects of plant growth and development. In particular, lipid metabolic processes, including very long chain fatty acid biosynthesis, FA elongation and modification, and lipid breakdown, are under the control of MYB96 (Seo et al. 2011, Lee et al. 2015a, Lee et al. 2015b, Lee et al. 2015c). Thus, we hypothesized that additional lipid metabolic processes might be affected by MYB96. To examine this possibility, we widely analyzed expression of lipid metabolic genes in MYB96-overexpressing activation-tagging myb96-ox, MYB96-deficient myb96-2 and wild-type seedlings. Quantitative real-time reverse transcription–PCR (RT–qPCR) analysis revealed that transcript accumulation of DGAT1, PDAT1 and FATTY ACID DESATURASE 3 (FAD3) was elevated in myb96-ox seedlings, whereas DGAT1 and PDAT1 were significantly down-regulated in myb96-2 mutant seedlings (Supplementary Fig. S1). Considering the expression patterns of myb96-ox and myb96-2 mutants, the DGAT1 and PDAT1 genes appeared likely to be key regulatory targets of MYB96, possibly involved in the control of TAG biosynthesis. Since TAG is a major carbon storage form in seeds (Baud et al. 2008, Graham 2008), we hypothesized that MYB96 may mainly be involved in TAG accumulation in the seeds. In support of this, the MYB96 gene is highly expressed in seeds and regulates a variety of seed developmental and metabolic processes, including seed dormancy, germination and FA modification (Seo et al. 2009, Seo et al. 2011, Lee et al. 2015a, Lee et al. 2015c). Our RT–qPCR analysis also provided support for MYB96 being highly expressed in developing siliques containing immature seeds, with an expression level equivalent to leaf tissues (Supplementary Fig. S2). DGAT1 and PDAT1 were also highly expressed in developing seeds and vegetative tissues (Supplementary Fig. S2). These observations were also supported by a web-based Arabidopsis gene expression database (eFP browser; http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi) (Supplementary Fig. S3). Hence, while we performed expression profiling of lipid metabolic genes in seedlings (Supplementary Fig. S1), it was necessary to test the transcriptional regulation of the TAG assembly genes by MYB96 in developing seeds. To this end, we analyzed expression of key genes influencing FA and TAG metabolism in developing siliques of wild-type, myb96-ox and myb96-2 genotypes (Fig. 1). We harvested silique tissues at mid-developmental stages (Supplementary Fig. S4), during which seed oil accumulation actively occurs (Kwong et al. 2003, Fatihi et al. 2013, Li et al. 2017). Consistent with the results in seedlings (Supplementary Fig. S1), the glycolytic and late FA biosynthetic genes, including PKp2, PDH-E1a, BCCP2, MAT, KASI, KASIII, ENR1 and FATA (To et al. 2012), were negligibly affected in MYB96-misexpressing seeds (Fig. 1). Expression of WRI1, a gene encoding a transcriptional regulator of FA metabolism, was also independent of MYB96 activity (Fig. 1). Moreover, glycerol-3-phosphate dehydrogenase genes (GPDH and GPDHc1), which are responsible for the glycerol backbone supply (Shen et al. 2006, To et al. 2012), were also uninfluenced (Fig. 1). Fig. 1 View largeDownload slide Expression of genes involved in lipid metabolism in developing siliques of the wild-type, myb96-ox and myb96-2 genotypes. Long green mature siliques were harvested for each genotype (Stage 2 in Supplementary Fig. S4). Transcript accumulation was analyzed by quantitative real-time reverse transcription–PCR (RT–qPCR). The EUKARYOTIC TRANSLATION INITIATION FACTOR 4A1 (eIF4a) gene (At3g13920) was used as an internal control. Biological triplicates were averaged. Statistically significant differences between the wild type and mutants are indicated by asterisks (*P < 0.05, Student’s t-test). Bars indicate the SEM. Fig. 1 View largeDownload slide Expression of genes involved in lipid metabolism in developing siliques of the wild-type, myb96-ox and myb96-2 genotypes. Long green mature siliques were harvested for each genotype (Stage 2 in Supplementary Fig. S4). Transcript accumulation was analyzed by quantitative real-time reverse transcription–PCR (RT–qPCR). The EUKARYOTIC TRANSLATION INITIATION FACTOR 4A1 (eIF4a) gene (At3g13920) was used as an internal control. Biological triplicates were averaged. Statistically significant differences between the wild type and mutants are indicated by asterisks (*P < 0.05, Student’s t-test). Bars indicate the SEM. However, the genes encoding DGAT1 and PDAT1, the rate-limiting enzymes for TAG assembly, were significantly repressed in myb96-2, but up-regulated in myb96-ox, especially at mid-stages of silique development (Fig. 1; Supplementary Fig. S4). In contrast, the two genes were not differentially expressed in dry seeds of myb96-ox and myb96-2 plants (Supplementary Fig. S5), possibly because TAG metabolic processes have been completed in dry seeds. These results suggest that MYB96 primarily regulates TAG accumulation in immature seeds, possibly by activating metabolic enzymes involved in the final acylation step of TAG assembly, rather than FA biosynthesis. TAG levels are reduced in myb96-deficient mutant seeds DGAT1 and PDAT1 are key rate-limiting enzymes in TAG biosynthesis (Katavic et al. 1995, Routaboul et al. 1999, Zhang et al. 2009). To assess the connection between MYB96 and TAG biosynthesis, we employed wild-type and MYB96-deficient myb96-2 mutant seeds (Lee et al. 2015b, Lee et al. 2016) and measured total FA contents. Total FAs were extracted from dry seeds of all genotypes and analyzed by gas chromatography (GC). Quantitative analysis of total FAs revealed that myb96-2 seeds had approximately 20% lower contents of total FAs compared with wild-type seeds (Fig. 2A;Supplementary Fig. S6), despite the dry weight of myb96-2 mutant seeds being comparable with that of wild-type seeds (Supplementary Fig. S7). FA composition was further analyzed, and the levels of most FA species significantly decreased in myb96-2 relative to wild-type seeds (Fig. 2B). To support this result, we also tested the additional myb96-1 mutant, which is a weak mutant allele possibly due to the T-DNA insertion in the intron (Fig. 2C;Seo et al. 2009). However, unexpectedly, although expression of DGAT1 and PDAT1 was reduced in myb96-1 seeds (Fig. 2C), total FA contents were negligibly influenced (Supplementary Fig. S8), which may be due to insufficient reduction of DGAT1 and PDAT1 in myb96-1 to influence TAG accumulation. Fig. 2 View largeDownload slide Quantification of total fatty acid levels in wild-type and myb96-2 mutant seeds. In (A) and (B), the abundance of total fatty acids in dry seeds was determined by gas chromatography (GC). At least 300 seeds per independent line were measured in each replicate. Triplicate experiments were averaged. Statistically significant differences between the wild type and the myb96-2 mutant are indicated by asterisks (Student’s t-test, *P < 0.05, **P < 0.01). Bars indicate the SEM. (A) Total fatty acid contents. (B) Fatty acid profiles. (C) Expression of MYB96, DGAT1 and PDAT1 in immature seeds of myb96 mutants. Developing siliques were harvested for total RNA isolation. Transcript accumulation was analyzed by RT–qPCR. The eIF4a gene was used as an internal control. Biological triplicates were averaged. Different letters represent a significant difference at P < 0.05 (one-way ANOVA with Fisher’s post-hoc test). Bars indicate the SEM. Fig. 2 View largeDownload slide Quantification of total fatty acid levels in wild-type and myb96-2 mutant seeds. In (A) and (B), the abundance of total fatty acids in dry seeds was determined by gas chromatography (GC). At least 300 seeds per independent line were measured in each replicate. Triplicate experiments were averaged. Statistically significant differences between the wild type and the myb96-2 mutant are indicated by asterisks (Student’s t-test, *P < 0.05, **P < 0.01). Bars indicate the SEM. (A) Total fatty acid contents. (B) Fatty acid profiles. (C) Expression of MYB96, DGAT1 and PDAT1 in immature seeds of myb96 mutants. Developing siliques were harvested for total RNA isolation. Transcript accumulation was analyzed by RT–qPCR. The eIF4a gene was used as an internal control. Biological triplicates were averaged. Different letters represent a significant difference at P < 0.05 (one-way ANOVA with Fisher’s post-hoc test). Bars indicate the SEM. To rule out the possibility that reduced TAG accumulation in myb96-2 mutants may be attributable to increased TAG catabolic activities, expression of TAG catabolic genes including SUGAR-DEPENDENT 1 (SDP1), SDP1L, MYZUS PERSICAE-INDUCED LIPASE 1 (MPL1), LIPOIC ACID SYNTHASE 1 (LIP1) and DAD1-LIKE LIPASE 5 (DALL5) was also examined in myb96-misexpressing seeds. These genes are involved in TAG degradation and β-oxidation during seed maturation and seedling establishment (He and Gan 2002, El-Kouhen et al. 2005, Seo et al. 2009, Slocombe et al. 2009, Kelly et al. 2013, Fan et al. 2014, Kim et al. 2014, Thazar-Poulot et al. 2015). As expected, all genes examined were uninfluenced in MYB96-misexpressing seeds, whereas transcript accumulation of DGAT1 and PDAT1 was changed accordingly (Supplementary Fig. S9), supporting the role of MYB96 in TAG biosynthesis. Overexpression of MYB96 leads to increased TAG accumulation in seeds To strengthen our results further, we also evaluated the effects of overexpression of MYB96 on seed TAG accumulation. We generated transgenic plants expressing the MYB96 gene under the control of the Cauliflower mosaic virus (CaMV) 35S or the seed-specific phaseolin (Phas) promoter. T3 generation seeds of 10 randomly selected lines for each genotype were used to determine total FA contents. GC analysis revealed that a majority of transgenic lines showed a significant increase of FA levels in dry seeds compared with the wild type (Fig. 3A;Supplementary Fig. S10). Consistent with the increase in seed total FA levels, the seed dry weight and size were also increased (Supplementary Fig. S11). Notably, the large increase in the relative content of total FAs by weight supported the high oil density of transgenic seeds (Fig. 3A). Transcript levels of DGAT1 and PDAT1 were also significantly elevated in selected transgenic lines with high seed oil contents (Fig. 3B). Fig. 3 View largeDownload slide Total fatty acid levels in transgenic seeds overexpressing MYB96. In (A) and (C), the abundance of total fatty acids in dry seeds was determined by GC. At least 300 seeds per independent T3 transgenic line were measured in each replicate. Triplicate experiments were averaged. Statistically significant differences between the wild-type and transgenic seeds are indicated by asterisks (Student’s t-test, *P < 0.05, **P < 0.01). Bars indicate the SEM. (A) Total fatty acid contents. (B) Expression of DGAT1 and PDAT1 genes in siliques of 35S:MYB96 and Phas:MYB96 transgenic plants. Developing siliques were used to analyze transcript accumulation. The eIF4a gene was used as an internal control. Biological triplicates were averaged. Statistically significant differences between wild-type and transgenic plants are indicated by asterisks (*P < 0.05, Student’s t-test). Bars indicate the SEM. (C) Fatty acid profiles. Fatty acid content is expressed as the percentage of the amount present in dry seeds of the indicated genotypes relative to wild-type seeds (100%). Fig. 3 View largeDownload slide Total fatty acid levels in transgenic seeds overexpressing MYB96. In (A) and (C), the abundance of total fatty acids in dry seeds was determined by GC. At least 300 seeds per independent T3 transgenic line were measured in each replicate. Triplicate experiments were averaged. Statistically significant differences between the wild-type and transgenic seeds are indicated by asterisks (Student’s t-test, *P < 0.05, **P < 0.01). Bars indicate the SEM. (A) Total fatty acid contents. (B) Expression of DGAT1 and PDAT1 genes in siliques of 35S:MYB96 and Phas:MYB96 transgenic plants. Developing siliques were used to analyze transcript accumulation. The eIF4a gene was used as an internal control. Biological triplicates were averaged. Statistically significant differences between wild-type and transgenic plants are indicated by asterisks (*P < 0.05, Student’s t-test). Bars indicate the SEM. (C) Fatty acid profiles. Fatty acid content is expressed as the percentage of the amount present in dry seeds of the indicated genotypes relative to wild-type seeds (100%). FA composition analysis demonstrated that all FA species were increased in the transgenic lines (Fig. 3C), which may account for the substantial accumulation of TAG in MYB96-overexpressing seeds. In particular, an increase in the levels of C18:1, which is known to be a preferred substrate of DGAT1, was prominent in all transgenic lines examined. It was also notable that the employed promoters have different effects on the FA composition of seeds, as exemplified by C20:1 accumulation (Fig. 3C). These results indicate that seed TAG accumulation is positively regulated by MYB96. MYB96 directly binds to the PDAT1 promoter We next asked whether the MYB96 transcription factor binds directly to the promoters of TAG biosynthetic genes. Sequence analysis revealed that each gene promoter contains conserved sequence motifs that are analogous to the R2R3-type MYB-binding consensus sequence (Borg et al. 2011, Seo et al. 2011) (Fig. 4A, B). Fig. 4 View largeDownload slide MYB96 binding to the PDAT1 promoter. In (A) and (B), putative R2R3-MYB-binding sites are indicated by arrowheads. Black underbars indicate regions of PCR amplification after chromatin immunoprecipitation (ChIP). Total protein extracts from pMYB96:MYB96-MYC transgenic seeds were immunoprecipitated with an anti-MYC antibody. Enrichment of putative MYB-binding regions was analyzed by qPCR analysis. Biological triplicates were averaged, and the statistical significance of measurements was determined using a Student’s t-test (*P < 0.05). Bars indicate the SEM. In each experiment, the measurement values in pBA002 were set to 1 after normalization against eIF4a for qPCR analysis. (A) Binding of MYB96 to the PDAT1 promoter. (B) Binding of MYB96 to the DGAT1 promoter. (C) Transient expression assays. The core element (B; CGAATAGTTACGGA) and mutated version of the core sequence (mB; CGAAAAAAAACGGA) were inserted into the reporter plasmid. Recombinant reporter and effector constructs were co-expressed transiently in Arabidopsis protoplasts, and GUS activity was determined fluorimetrically. Luciferase gene expression was used to normalize GUS activity. The normalized values in control protoplasts were set to 1 and are represented as relative activation. Three independent measurements were averaged. Statistical significance was determined by a Student’s t-test (*P < 0.05). Bars indicate the SEM. Fig. 4 View largeDownload slide MYB96 binding to the PDAT1 promoter. In (A) and (B), putative R2R3-MYB-binding sites are indicated by arrowheads. Black underbars indicate regions of PCR amplification after chromatin immunoprecipitation (ChIP). Total protein extracts from pMYB96:MYB96-MYC transgenic seeds were immunoprecipitated with an anti-MYC antibody. Enrichment of putative MYB-binding regions was analyzed by qPCR analysis. Biological triplicates were averaged, and the statistical significance of measurements was determined using a Student’s t-test (*P < 0.05). Bars indicate the SEM. In each experiment, the measurement values in pBA002 were set to 1 after normalization against eIF4a for qPCR analysis. (A) Binding of MYB96 to the PDAT1 promoter. (B) Binding of MYB96 to the DGAT1 promoter. (C) Transient expression assays. The core element (B; CGAATAGTTACGGA) and mutated version of the core sequence (mB; CGAAAAAAAACGGA) were inserted into the reporter plasmid. Recombinant reporter and effector constructs were co-expressed transiently in Arabidopsis protoplasts, and GUS activity was determined fluorimetrically. Luciferase gene expression was used to normalize GUS activity. The normalized values in control protoplasts were set to 1 and are represented as relative activation. Three independent measurements were averaged. Statistical significance was determined by a Student’s t-test (*P < 0.05). Bars indicate the SEM. To examine whether MYB96 is targeted to TAG biosynthetic gene promoters, chromatin immunoprecipitation (ChIP) assays were performed using pMYB96:MYB96-MYC transgenic seeds. Total protein extracts from pBA002 control and pMYB96:MYB96-MYC transgenic seeds were immunoprecipitated with an anti-MYC-antibody. DNA bound to epitope-tagged MYB96 proteins was analyzed by quantitative real-time PCR (qPCR) assays. Analysis showed that the B and C regions of the PDAT1 promoter were enriched as a result of ChIP (Fig. 4A). In contrast, resin alone did not bind to the PDAT1 promoter (Supplementary Fig. S12). However, direct binding of MYB96 to the DGAT1 promoter was not detected (Fig. 4B). These results indicate that MYB96 specifically binds to the PDAT1 promoter and indirectly influences DGAT1. To support further direct binding of MYB96 to the PDAT1 promoter, we conducted a transient expression analysis using Arabidopsis protoplasts. The core element in the B region (B; CGAATAGTTACGGA) or a mutated element (mB; CGAAAAAAAACGGA) was fused to the 35S minimal reporter promoter. A recombinant reporter plasmid and the effector p35S:MYB96 plasmid were co-transformed into Arabidopsis protoplasts. Co-transformation with the reporter B construct increased β-glucuronidase (GUS) activity by approximately 4-fold, but co-transformation with the reporter mB did not stimulate reporter gene expression (Fig. 4C). These results indicate that MYB96 binds to the PDAT1 promoter and transcriptionally activates expression. MYB96 has been reported to bind to the promoter of ABI4 in seeds (Lee et al. 2015c), which directly regulates DGAT1 (Yang et al. 2011, Kong et al. 2013). Therefore, we speculated that MYB96 might regulate DGAT1 through ABI4. To test this hypothesis, we employed myb96-ox/abi4-1 plants, in which the myb96-ox mutant was genetically crossed with the abi4-1 loss-of-function mutant allele (Lee et al. 2015c), and compared transcript accumulation of DGAT1 with myb96-ox. The elevated expression of DGAT1 in myb96-ox was suppressed in myb96-ox/abi4-1 developing seeds, while PDAT1 expression was unaffected by ABI4 activity (Supplementary Fig. S13). Furthermore, transient expression assays also supported that MYB96 regulation of DGAT1 depends on ABI4. A reporter construct, in which the DGAT1 promoter sequence was fused with the minimal 35S promoter, was co-expressed with an effector construct overexpressing MYB96 in Arabidopsis protoplasts isolated from wild-type and abi4-1 leaves. Reporter GUS activity measurement revealed that MYB96 significantly increased DGAT1 promoter activity in the wild-type background, but this function was impaired in the abi4-1 background (Supplementary Fig. S14). These observations provide support that ABI4 mediates MYB96 regulation of DGAT1 to control TAG biosynthesis. The MYB96-regulated pathway is independent of WRI1 Seed oil accumulation is co-ordinated by FA biosynthesis and TAG assembly (Baud et al. 2007, Baud et al. 2008). While it was evident that MYB96 is mainly involved in TAG assembly and that genes involved in FA biosynthesis were uninfluenced in MYB96-misexpressing seeds (Fig. 1), we wanted to know whether FA levels reciprocally influence MYB96 expression. Based on the essential role of WRI1 in FA accumulation (Baud et al. 2007, To et al. 2012), we analyzed transcript accumulation of MYB96 in wri1-3 mutant seeds. RT–qPCR analysis revealed that MYB96 expression was unaffected in wri1-3 (Fig. 5A), which shows a substantial decrease in seed FA levels (Vanhercke et al. 2013). Consistently, DGAT1 and PDAT1 transcript accumulation was also marginally affected in wri1-3 (Fig. 5B, C). In addition, WRI1 expression was unchanged in dgat1-1 and pdat1-1 mutant seeds (Fig. 5D), which show a reduction in TAG accumulation. Fig. 5 View largeDownload slide Independent transcriptional control of fatty acid biosynthesis and TAG assembly. In (A–D), transcript accumulation was analyzed by RT–qPCR. The eIF4a gene was used as an internal control. Biological triplicates were averaged. Bars indicate the SEM. (A) Expression of MYB96 in wri1-3 seeds. (B) Expression of DGAT1 in wri1-3 seeds. (C) Expression of PDAT1 in wri1-3 seeds. (D) Transcript accumulation of WRI1 in dgat1-1 and pdat1-1 seeds. (E) Proposed working diagram. FA biosynthesis and TAG assembly are under independent transcriptional control. During seed maturation, WRI1 plays a crucial role in early FA biosynthesis, whereas MYB96 is primarily involved in TAG assembly. The two transcription factors fine-tune stepwise metabolic processes in immature seeds. Fig. 5 View largeDownload slide Independent transcriptional control of fatty acid biosynthesis and TAG assembly. In (A–D), transcript accumulation was analyzed by RT–qPCR. The eIF4a gene was used as an internal control. Biological triplicates were averaged. Bars indicate the SEM. (A) Expression of MYB96 in wri1-3 seeds. (B) Expression of DGAT1 in wri1-3 seeds. (C) Expression of PDAT1 in wri1-3 seeds. (D) Transcript accumulation of WRI1 in dgat1-1 and pdat1-1 seeds. (E) Proposed working diagram. FA biosynthesis and TAG assembly are under independent transcriptional control. During seed maturation, WRI1 plays a crucial role in early FA biosynthesis, whereas MYB96 is primarily involved in TAG assembly. The two transcription factors fine-tune stepwise metabolic processes in immature seeds. Taken together, MYB96 regulates TAG accumulation in developing seeds by activating DGAT1 and PDAT1, which are responsible for catalyzing the rate-limiting step of TAG biosynthesis (Zhang et al. 2009, Xu et al. 2012). The trio of MYB96, DGAT1 and PDAT1 were uninfluenced by WRI1, and vice versa, indicating that FA biosynthesis and TAG assembly are under independent transcriptional control. During seed maturation, WRI1 plays a crucial role in FA supply, whereas MYB96 is primarily involved in TAG assembly (Fig. 5E). The sequential actions of transcriptional regulators may account for the control of seed maturation and lipid storage. Discussion Diverse roles of MYB96 in seeds The MYB96 transcription factor is known as a central mediator of ABA signaling in a variety of physiological processes, such as lateral root development, hormone metabolism, stomatal opening and cuticular wax biosynthesis (Seo et al. 2009, Seo and Park 2010, Seo et al. 2011, Lee et al. 2015a, Lee et al. 2015b, Lee et al. 2015c, Lee et al. 2016). In addition to its roles in vegetative tissues, MYB96 is also highly expressed in seeds (Lee et al. 2015b, Lee et al. 2015c) and participates in the processes of seed maturation, dormancy and germination (Lee et al. 2015a, Lee et al. 2015c). During seed maturation, MYB96 is implicated in carbon metabolic processes. The gene encoding the seed-specific FATTY ACID ELONGATION 1 (FAE1) enzyme, which triggers chain elongation of C18–C20 and C22 (James et al. 1995, Roscoe et al. 2001), is up-regulated in myb96-ox seeds (Lee et al. 2015b), contributing to long chain FA accumulation in maturing seeds. The FAD3 gene was also affected in myb96-ox (Supplementary Fig. S1), which implies an additional role for MYB96 in lipid desaturation during seed development. MYB96 establishes primary seed dormancy by co-ordinating ABA and gibberellic acid metabolism during seed maturation, and inhibits viviparous germination (Lee et al. 2015b). Furthermore, this transcription factor also suppresses lipid degradation, which fuels seed germination by supplying an energy source, in order to determine the proper timing for germination (Lee et al. 2015c). MYB96 primarily regulates ABI4 transcription in the control of lipid mobilization-dependent seed germination and confers ABA sensitivity specifically in the seed embryo (Lee et al. 2015c). Close connections between ABA signaling and TAG metabolism have been extensively proposed. During seed maturation, high levels of ABA ensure TAG and oil body accumulation as well as inhibition of lipid breakdown (Crowe et al. 2000, Brocard-Gifford et al. 2003). Consistent with these observations, this study demonstrates that the ABA-inducible MYB96 transcription factor contributes to TAG accumulation, largely through the transcriptional control of DGAT1 and PDAT1. Large amounts of TAG accumulate in myb96-ox seeds, whereas myb96 mutation leads to reduced TAG accumulation. Increased TAG accumulation by MYB96 was independent of FA metabolic gene activities in that MYB96 overexpression did not affect any genes involved in FA biosynthesis and degradation pathways. Intrinsic FA biosynthetic enzyme activities might be sufficient to support TAG accumulation in myb96-ox. Otherwise, FA biosynthetic enzymes may be modulated at another level, such as changes in biochemical activities or protein turnover. For instance, the acetyl-CoA carboxylase in FA biosynthesis may not be feedback inhibited by acyl-ACP products due to an efficient incorporation of acyl-ACP into TAG (Andre et al. 2012). Taken together, MYB96 has roles in seed development and germination and also controls metabolic processes to link these biological programs. Molecular web underlying MYB96-regulated TAG accumulation TAG accumulation is regulated by a multitude of stepwise metabolic processes. In particular, the last acylation step is important for seed TAG assembly, which is catalyzed by DGAT1 and PDAT1, and many regulatory actions target this step for proper TAG accumulation (Zhang et al. 2009, Bates et al. 2013). Considering their biological and biotechnological importance, identification of upstream regulator(s) of DGAT1 and/or PDAT1 has been an important challenge in plant lipid metabolic engineering. Notably, the MYB96 transcription factor co-ordinates expression of the two core TAG biosynthetic genes and ensures proper levels of TAG biosynthesis in maturing seeds. Transcript accumulation of DGAT1 and PDAT1 was increased in myb96-ox, but suppressed in myb96-deficient mutant seeds. However, the mechanisms underlying the transcriptional regulation of DGAT1 and PDAT1 differ. The MYB96 transcription factor binds directly to the PDAT1 promoter, but not to the DGAT1 promoter. However, MYB96 regulation of DGAT1 is still relevant, because MYB96 regulates DGAT1 expression through ABI4. MYB96 binds to the R2R3-MYB-binding cis-elements on the ABI4 promoter and activates expression (Lee et al. 2015c). Then, ABI4 activates DGAT1 expression by binding directly to its promoter (Yang et al. 2011, Kong et al. 2013). Consistently, the elevated DGAT1 expression in myb96-ox was compromised in myb96-ox/abi4-1, providing support that the MYB96-ABI4 module is important for proper DGAT1 expression. Collectively, MYB96 is a pivotal regulator of TAG biosynthesis by regulating key genes that encode rate-limiting enzymes. FA biosynthesis and TAG assembly are co-ordinated to ensure elaborate lipid metabolism. Our results suggest that cross-talk between the metabolic processes is not observed, at least at the transcriptional level. MYB96 regulation of TAG accumulation was independent of a WRI1-regulated FA biosynthetic pathway, and vice versa. MYB96 and WRI1 are likely to play independent and separate roles in lipid metabolism: WRI1 mainly regulates the glycolytic and late FA biosynthetic pathways, whereas MYB96 stimulates the TAG assembly process. However, it is very likely that another regulatory layer is involved to ensure balanced metabolism between FA biosynthesis and TAG assembly, and further studies should yield a comprehensive view of the mechanisms of seed oil accumulation. Seed-derived TAG is a valuable source of food and biofuel. Therefore, metabolic engineering to boost TAG production in plants would be widely considered for commercial benefit. Given the strong effects of MYB96 on TAG accumulation, MYB96 could be a powerful genetic resource that could be applied to oilseed crops to enhance seed oil accumulation. Given that MYB96, DGAT1 and PDAT1 are also highly expressed in vegetative tissues, MYB96 can also be used for higher TAG accumulation in vegetative tissues. Furthermore, considering the independent transcriptional control, efficient lipid metabolic engineering could be achieved by simultaneous expression of key transcriptional regulators of FA biosynthesis and TAG assembly. Materials and Methods Plant materials and growth conditions Arabidopsis thaliana (Columbia-0 ecotype) was used for all experiments unless otherwise specified. Plants were grown under long-day conditions (16 h light/8 h dark cycles) with cool white fluorescent light (150 µmol photons m–2 s–1) at 22–23°C. The myb96-ox and myb96-2 (SALK_111645) mutants were previously reported (Seo et al. 2009, Lee et al. 2015c). The dgat1-1 (CS3861, Zou et al. 1999), pdat1-1 (SALK_032261, Mhaske et al. 2005) and wri1-3 (SALK_085693) mutants were obtained from the Arabidopsis Biological Resource Center (http://abrc.osu.edu/). Quantitative real-time RT–PCR analysis Total RNA was extracted using TRI reagent (TAKARA BIO INC.) according to the manufacturer’s recommendations. Reverse transcription was performed using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Dr. Protein) with oligo(dT)18 to synthesize first-strand cDNA from 2 µg of total RNA. Total RNA samples were pre-treated with RNase-free DNase. cDNAs were diluted to 100 µl with TE buffer, and 1 µl of diluted cDNA was used for PCR amplification. RT–qPCRs were performed in 96-well blocks using the Step-One Plus Real-Time PCR System (Applied Biosystems). The PCR primers used are listed in Supplementary Table S1. The values for each set of primers were normalized relative to the EUKARYOTIC TRANSLATION INITIATION FACTOR 4A1 (eIF4A) gene (At3g13920). All RT–qPCRs were performed with biological triplicates using total RNA samples extracted from three independent replicate samples. The comparative ΔΔCT method was employed to evaluate the relative quantities of each amplified product in the samples. The threshold cycle (CT) was automatically determined for each reaction with the analysis software set using default parameters. The specificity of the RT–qPCRs was determined by melt curve analysis of the amplified products using the standard method employed by the software. Fatty acid analysis The total FA content and their composition in seeds were measured by GC analysis with a known amount of glyceryl triheptadecanoate (Sigma-T2151) as an internal standard. Samples were transmethylated at 90°C for 90 min in 0.3 ml of toluene and 1 ml of 5% H2SO4 (v/v methanol). After transmethylation, 1.5 ml of 0.9% NaCl solution was added, and the FA methyl esters (FAMEs) were transferred to a new tube for three sequential extractions with 1.5 ml of n-hexane. FAMEs were analyzed by GC using a GC-2010 plus instrument (Shimadzu with a 30 m×0.25 mm (25 µm film thickness) DB-23 column (Agilent), during which the oven temperature was maintained for 10 min at 190°C, followed by an increase of 5°C min–1 to 230°C, and maintained for 10 min at 230°C. Chromatin immunoprecipitation ChIP assay was performed as previously described (Schoppee Bortz and Wamhoff 2011). pMYB96:MYB96-MYC transgenic seeds, anti-MYC antibodies (Millipore) and salmon sperm DNA/protein A agarose beads (Millipore) were used for ChIP. DNA was purified using phenol/chloroform/isoamyl alcohol and sodium acetate (pH 5.2). The level of precipitated DNA fragments was quantified by qPCR using specific primer sets (Supplementary Table S2). Values were normalized to the input DNA level. Values for control plants were set to 1 after normalization against eIF4a for quantitative PCR analysis. Transient expression analysis For transient expression assays using Arabidopsis protoplasts, reporter and effector plasmids were constructed. The reporter plasmid contains a minimal 35S promoter sequence and the GUS gene. The core elements on the DGAT1 and PDAT1 promoters were inserted into the reporter plasmid. To construct the p35S:MYB96 effector plasmid, MYB96 cDNA was inserted into the effector vector containing the CaMV 35S promoter. Recombinant reporter and effector plasmids were co-transformed into Arabidopsis protoplasts by polyethylene glycol-mediated transformation (Yoo et al. 2007). GUS activities were measured by a fluorometric method. A CaMV 35S promoter–luciferase construct was also co-transformed as an internal control. The luciferase assay was performed using the Luciferase Assay System kit (Promega). Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the National Research Foundation of Korea [Basic Research Laboratory grant program (NRF-2017R1A4A1015620 to M.C.S. and P.J.S.) and the MidCareer Researcher grant program (NRF-2017R1A2B4007096, H.U.K.); the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry, and Fisheries (IPET) [116079-03 and 316087-4 to H.U.K.]; and the Rural Development Administration [Cooperative Research Program for Agriculture Science and Technology Development (PJ01261303 to P.J.S.)]. Disclosures The authors have no conflicts of interest to declare. References Andre C. , Haslam R.P. , Shanklin J. ( 2012 ) Feedback regulation of plastidic acetyl-CoA carboxylase by 18:1-acyl carrier protein in Brassica napus . Proc. Natl. Acad. Sci. USA 109 : 10107 – 10112 . Angeles-Nunez J.G. , Tiessen A. 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Abbreviations Abbreviations ABI ABSCISC ACID INSENSITIVE BCCP biotin carboxyl carrier protein CaMV Cauliflower mosaic virus ChIP chromatin immunoprecipitation DAG diacylglycerol DGAT acyl-CoA:diacylglycerol acyltransferase FA fatty acid GC gas chromatography GUS β-glucuronidase KAS 3-ketoacyl-ACP synthetase LEC LEAFY COTYLEDON PDAT phospholipid:diacylglycerol acyltransferase qPCR quantitative real-time PCR RT–qPCR quantitative real-time reverse transcription–PCR TAG triacylglycerol WRI1 WRINKLED 1 © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Plant and Cell PhysiologyOxford University Press

Published: Apr 5, 2018

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