Transcription factor bZIP52 modulates Arabidopsis seed oil biosynthesis through interaction with WRINKLED1.

Transcriptional regulation mediated by combinatorial interaction of transcription factors (TFs) is a key molecular mechanism modulating plant development and metabolism. Basic leucine zipper (bZIP) TFs play important roles in various plant developmental and physiological processes. However, their involvement in fatty acid biosynthesis is largely unknown. Arabidopsis (Arabidopsis thaliana) WRINKLED1 (WRI1) is a pivotal TF in regulation of plant oil biosynthesis and interacts with other positive and negative regulators. In this study, we identified two bZIP TFs, bZIP21 and bZIP52, as interacting partners of AtWRI1 by yeast-two-hybrid (Y2H)-based screening of an Arabidopsis TF library. We found that coexpression of bZIP52, but not bZIP21, with AtWRI1 reduced AtWRI1-mediated oil biosynthesis in Nicotiana benthamiana leaves. The AtWRI1-bZIP52 interaction was further verified by Y2H, in vitro pull-down, and bimolecular fluorescence complementation assays. Transgenic Arabidopsis plants overexpressing bZIP52 showed reduced seed oil accumulation, while the CRISPR/Cas9-edited bzip52 knockout mutant exhibited increased seed oil accumulation. Further analysis revealed that bZIP52 represses the transcriptional activity of AtWRI1 on the fatty acid biosynthetic gene promoters. Together, our findings suggest that bZIP52 represses fatty acid biosynthesis genes through interaction with AtWRI1, resulting in a reduction of oil production. Our work reports a previously uncharacterized regulatory mechanism that enables fine-tuning of seed oil biosynthesis.

Molecular basis of the key regulator WRINKLED1 in plant oil biosynthesis.

Vegetable oils are not only major components of human diet but also vital for industrial applications. WRINKLED1 (WRI1) is a pivotal transcription factor governing plant oil biosynthesis, but the underlying DNA-binding mechanism remains incompletely understood. Here, we resolved the structure of Arabidopsis WRI1 (AtWRI1) with its cognate double-stranded DNA (dsDNA), revealing two antiparallel ß sheets in the tandem AP2 domains that intercalate into the adjacent major grooves of dsDNA to determine the sequence recognition specificity. We showed that AtWRI1 represented a previously unidentified structural fold and DNA-binding mode. Mutations of the key residues interacting with DNA element affected its binding affinity and oil biosynthesis when these variants were transiently expressed in tobacco leaves. Seed oil content was enhanced in stable transgenic wri1-1 expressing an AtWRI1 variant (W74R). Together, our findings offer a structural basis explaining WRI1 recognition and binding of DNA and suggest an alternative strategy to increase oil yield in crops through WRI1 bioengineering.

Transcriptional regulation of oil biosynthesis in seed plants: Current understanding, applications, and perspectives.

Plants produce and accumulate triacylglycerol (TAG) in their seeds as an energy reservoir to support the processes of seed germination and seedling development. Plant seed oils are vital not only for the human diet but also as renewable feedstocks for industrial use. TAG biosynthesis consists of two major steps: de novo fatty acid biosynthesis in the plastids and TAG assembly in the endoplasmic reticulum. The latest advances in unraveling transcriptional regulation have shed light on the molecular mechanisms of plant oil biosynthesis. We summarize recent progress in understanding the regulatory mechanisms of well-characterized and newly discovered transcription factors and other types of regulators that control plant fatty acid biosynthesis. The emerging picture shows that plant oil biosynthesis responds to developmental and environmental cues that stimulate a network of interacting transcriptional activators and repressors, which in turn fine-tune the spatiotemporal regulation of the pathway genes.

Sunflower WRINKLED1 Plays a Key Role in Transcriptional Regulation of Oil Biosynthesis.

Sunflower (Helianthus annuus) is one of the most important oilseed crops worldwide. However, the transcriptional regulation underlying oil accumulation in sunflower is not fully understood. WRINKLED1 (WRI1) is an essential transcription factor governing oil accumulation in plant cells. Here, we identify and characterize a sunflower ortholog of WRI1 (HaWRI1), which is highly expressed in developing seeds. Transient production of HaWRI1 stimulated substantial oil accumulation in Nicotiana benthamiana leaves. Dual-luciferase reporter assay, electrophoretic mobility shift assay, fatty acid quantification, and gene expression analysis demonstrate that HaWRI1 acts as a pivotal transcription factor controlling the expression of genes involved in late glycolysis and fatty acid biosynthesis. HaWRI1 directly binds to the cis-element, AW-box, in the promoter of biotin carboxyl carrier protein isoform 2 (BCCP2). In addition, we characterize an 80 amino-acid C-terminal domain of HaWRI1 that is crucial for transactivation. Moreover, seed-specific overexpression of HaWRI1 in Arabidopsis plants leads to enhanced seed oil content as well as upregulation of the genes involved in fatty acid biosynthesis. Taken together, our work demonstrates that HaWRI1 plays a pivotal role in the transcriptional control of seed oil accumulation, providing a potential target for bioengineering sunflower oil yield improvement.

The function of the WRI1-TCP4 regulatory module in lipid biosynthesis.

The plant-specific TCP transcription factors play pivotal roles in various processes of plant growth and development. However, little is known regarding the functions of TCPs in plant oil biosynthesis. Our recent work showed that TCP4 mediates oil production via interaction with WRINKLED1 (WRI1), an essential transcription factor governing plant fatty acid biosynthesis. Arabidopsis WRI1 (AtWRI1) physically interacts with multiple TCPs, including TCP4, TCP10, and TCP24. Transient co-expression of AtWRI1 with TCP4, but not TCP10 or TCP24, represses oil accumulation in Nicotiana benthamiana leaves. Increased TCP4 in transgenic plants overexpressing a miR319-resistant TCP4 (rTCP4) decreased the expression of AtWRI1 target genes. The tcp4 knockout mutant, the jaw-D mutant with significant reduction of TCP4 expression, and a tcp2 tcp4 tcp10 triple mutant, display increased seed oil contents compared to the wild-type Arabidopsis. The APETALA2 (AP2) transcription factor WRI1 is characterized by regulating fatty acid biosynthesis through cross-family interactions with multiple transcriptional, post-transcriptional, and post-translational regulators. The interacting regulator modules control the range of AtWRI1 transcriptional activity, allowing spatiotemporal modulation of lipid production. Interaction of TCP4 with AtWRI1, which results in a reduction of AtWRI1 activity, represents a newly discovered mechanism that enables the fine-tuning of plant oil biosynthesis.

TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR4 Interacts with WRINKLED1 to Mediate Seed Oil Biosynthesis.

Cross-family transcription factor (TF) interactions play critical roles in the regulation of plant developmental and metabolic pathways. WRINKLED1 (WRI1) is a key TF governing oil biosynthesis in plants. However, little is known about WRI1-interacting factors and their roles in oil biosynthesis. We screened a TF library using Arabidopsis (Arabidopsis thaliana) WRI1 (AtWRI1) as bait in yeast two-hybrid assays and identified three TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) family TFs, namely TCP4, TCP10, and TCP24, as AtWRI1-interacting partners. The physical interaction between AtWRI1 and TCPs was further validated using bimolecular fluorescence complementation assays. TCPs play important roles in various plant developmental processes; however, their involvement in fatty acid biosynthesis was not previously known. Coexpression of TCP4, but not TCP10 or TCP24, with AtWRI1 reduced AtWRI1-mediated oil biosynthesis in Nicotiana benthamiana leaves. Transcriptomic analysis in transgenic Arabidopsis plants with enhanced TCP4 activity engineered by expressing rTCP4 (i.e. miR319-resistant TCP4) revealed that AtWRI1 target genes were significantly repressed. TCP4 expression is strongly correlated with AtWRI1 during embryo development. A tcp4 loss-of-function mutant, the jaw-D mutant with a strong reduction of TCP4 expression, and a tcp2 tcp4 tcp10 triple mutant accumulated more seed oil than wild-type Arabidopsis. In addition, TCP4 repressed the AtWRI1-mediated transactivation of the promoters of fatty acid biosynthetic genes. Collectively, our findings suggest that TCP4 represses fatty acid biosynthetic gene expression through interaction with AtWRI1, leading to a reduction of AtWRI1-mediated seed oil accumulation.

Molecular Basis of Plant Oil Biosynthesis: Insights Gained From Studying the WRINKLED1 Transcription Factor.

Most plant species generate and store triacylglycerol (TAG) in their seeds, serving as a core supply of carbon and energy to support seedling development. Plant seed oils have a wide variety of applications, from being essential for human diets to serving as industrial renewable feedstock. WRINKLED1 (WRI1) transcription factor plays a central role in the transcriptional regulation of plant fatty acid biosynthesis. Since the discovery of Arabidopsis WRI1 gene (AtWRI1) in 2004, the function of WRI1 in plant oil biosynthesis has been studied intensively. In recent years, the identification of WRI1 co-regulators and deeper investigations of the structural features and molecular functions of WRI1 have advanced our understanding of the mechanism of the transcriptional regulation of plant oil biosynthesis. These advances also help pave the way for novel approaches that will better utilize WRI1 for bioengineering oil production in crops.

WRINKLED1, a "Master Regulator" in Transcriptional Control of Plant Oil Biosynthesis.

A majority of plant species generate and accumulate triacylglycerol (TAG) in their seeds, which is the main resource of carbon and energy supporting the process of seedling development. Plant seed oils have broad ranges of uses, being not only important for human diets but also renewable feedstock of industrial applications. The WRINKLED1 (WRI1) transcription factor is vital for the transcriptional control of plant oil biosynthetic pathways. Since the identification of the Arabidopsis WRI1 gene (AtWRI1) fifteen years ago, tremendous progress has been made in understanding the functions of WRI1 at multiple levels, ranging from the identification of AtWRI1 target genes to location of the AtWRI1 binding motif, and from discovery of intrinsic structural disorder in WRI1 to fine-tuning of WRI1 modulation by post-translational modifications and protein-protein interactions. The expanding knowledge on the functional understanding of the WRI1 regulatory mechanism not only provides a clearer picture of transcriptional regulation of plant oil biosynthetic pathway, but also helps generate new strategies to better utilize WRI1 for developing novel oil crops.

WRINKLED1 as a novel 14-3-3 client: function of 14-3-3 proteins in plant lipid metabolism.

The conserved plant 14-3-3 proteins (14-3-3s) function by binding to phosphorylated client proteins to regulate their function. Previous studies indicate that 14-3-3s are involved in the regulation of plant primary metabolism; however, not much is known regarding the functions of 14-3-3s in plant oil biosynthesis. Our recent work shows that 14-3-3 plays a role in mediating plant oil biosynthesis through interacting with the transcription factor, WRINKLED1 (WRI1). WRI1 is critical for the transcriptional control of plant oil biosynthesis. Arabidopsis WRI1 physically interacts with 14-3-3s. Transient co-expression of AtWRI1 with 14-3-3s enhances plant oil biosynthesis in leaves of Nicotiana benthamiana. Transgenic plants overexpressing of a 14-3-3 show enhanced seed oil content. Co-expression of a 14-3-3 with AtWRI1 results in increased transcriptional activity and protein stability of AtWRI1. Our transcriptional regulation model supports a concept that interaction of a 14-3-3 with a transcription factor enhances the transcriptional activity through protein stabilization.

14-3-3 protein mediates plant seed oil biosynthesis through interaction with AtWRI1.

Plant 14-3-3 proteins are phosphopeptide-binding proteins, belonging to a large family of proteins involved in numerous physiological processes including primary metabolism, although knowledge about the function of 14-3-3s in plant lipid metabolism is sparse. WRINKLED1 (WRI1) is a key transcription factor that governs plant oil biosynthesis. At present, AtWRI1-interacting partners remain largely unknown. Here, we show that 14-3-3 proteins are able to interact with AtWRI1, both in yeast and plant cells. Transient co-expression of 14-3-3- and AtWRI1-encoding cDNAs led to increased oil biosynthesis in Nicotiana benthamiana leaves. Stable transgenic plants overproducing a 14-3-3 protein also displayed increased seed oil content. Co-production of a 14-3-3 protein with AtWRI1 enhanced the transcriptional activity of AtWRI1. The 14-3-3 protein was found to increase the stability of AtWRI1. A possible 14-3-3 binding motif was identified in one of the two AP2 domains of AtWRI1, which was also found to be critical for the interaction of AtWRI1 with an E3 ligase linker protein. Thus, we hypothesize a regulatory mechanism by which the binding of 14-3-3 to AtWRI1 interferes with the interaction of AtWRI1 and the E3 ligase, thereby protecting AtWRI1 from degradation. Taken together, our studies identified AtWRI1 as a client of 14-3-3 proteins and provide insights into a role of 14-3-3 in mediating plant oil biosynthesis.

Deletion of a C-terminal intrinsically disordered region of WRINKLED1 affects its stability and enhances oil accumulation in Arabidopsis.

WRINKLED1 (WRI1) is a key transcription factor governing plant oil biosynthesis. We characterized three intrinsically disordered regions (IDRs) in Arabidopsis WRI1, and found that one C-terminal IDR of AtWRI1 (IDR3) affects the stability of AtWRI1. Analysis by bimolecular fluorescence complementation and yeast-two-hybrid assays indicated that the IDR3 domain does not determine WRI1 stability by interacting with BTB/POZ-MATH proteins connecting AtWRI1 with CULLIN3-based E3 ligases. Analysis of the WRI1 sequence revealed that a putative PEST motif (proteolytic signal) is located at the C-terminal region of AtWRI1(IDR) (3). We also show that a 91 amino acid domain at the C-terminus of AtWRI1 without the PEST motif is sufficient for transactivation. We found that removal of the PEST motif or mutations in putative phosphorylation sites increased the stability of AtWRI1, and led to increased oil biosynthesis when these constructs were transiently expressed in tobacco leaves. Oil content was also increased in the seeds of stable transgenic wri1-1 plants expressing AtWRI1 with mutations in the IDR3-PEST motif. Taken together, our data suggest that intrinsic disorder of AtWRI1(IDR3) may facilitate exposure of the PEST motif to protein kinases. Thus, phosphorylation of the PEST motif in the AtWRI1(IDR) (3) domain may affect AtWRI1-mediated plant oil biosynthesis. The results obtained here suggest a means to increase accumulation of oils in plant tissues through WRI1 engineering.

Wrinkled1, a ubiquitous regulator in oil accumulating tissues from Arabidopsis embryos to oil palm mesocarp.

Wrinkled1 (AtWRI1) is a key transcription factor in the regulation of plant oil synthesis in seed and non-seed tissues. The structural features of WRI1 important for its function are not well understood. Comparison of WRI1 orthologs across many diverse plant species revealed a conserved 9 bp exon encoding the amino acids "VYL". Site-directed mutagenesis of amino acids within the 'VYL' exon of AtWRI1 failed to restore the full oil content of wri1-1 seeds, providing direct evidence for an essential role of this small exon in AtWRI1 function. Arabidopsis WRI1 is predicted to have three alternative splice forms. To understand expression of these splice forms we performed RNASeq of Arabidopsis developing seeds and queried other EST and RNASeq databases from several tissues and plant species. In all cases, only one splice form was detected and VYL was observed in transcripts of all WRI1 orthologs investigated. We also characterized a phylogenetically distant WRI1 ortholog (EgWRI1) as an example of a non-seed isoform that is highly expressed in the mesocarp tissue of oil palm. The C-terminal region of EgWRI1 is over 90 amino acids shorter than AtWRI1 and has surprisingly low sequence conservation. Nevertheless, the EgWRI1 protein can restore multiple phenotypes of the Arabidopsis wri1-1 loss-of-function mutant, including reduced seed oil, the "wrinkled" seed coat, reduced seed germination, and impaired seedling establishment. Taken together, this study provides an example of combining phylogenetic analysis with mutagenesis, deep-sequencing technology and computational analysis to examine key elements of the structure and function of the WRI1 plant transcription factor.

Site-directed mutagenesis and saturation mutagenesis for the functional study of transcription factors involved in plant secondary metabolite biosynthesis.

Regulation of gene expression is largely coordinated by a complex network of interactions between transcription factors (TFs), co-factors, and their cognate cis-regulatory elements in the genome. TFs are multidomain proteins that arise evolutionarily through protein domain shuffling. The modular nature of TFs has led to the idea that specific modules of TFs can be re-designed to regulate desired gene(s) through protein engineering. Utilization of designer TFs for the control of metabolic pathways has emerged as an effective approach for metabolic engineering. We are interested in engineering the basic helix-loop-helix (bHLH, Myc-type) transcription factors. Using site-directed and saturation mutagenesis, in combination with efficient and high-throughput screening systems, we have identified and characterized several amino acid residues critical for higher transactivation activity of a Myc-like bHLH transcription factor involved in anthocyanin biosynthetic pathway in plants. Site-directed and saturation mutagenesis should be generally applicable to engineering of all TFs.

Isolation and functional characterization of a floral tissue-specific R2R3 MYB regulator from tobacco.

Tobacco is a commonly used heterologous system for studying combinatorial regulation of the flavonoid biosynthetic pathway by the bHLH-MYB transcription factor (TF) complex in plants. However, little is known about the endogenous tobacco bHLH and MYB TFs involved in the pathway. Ectopic expression in tobacco of heterologous bHLH TF genes, such as maize Lc, leads to increased anthocyanin production in the reproductive tissues, suggesting the presence of a reproductive tissue-specific MYB TF that interacts with the Lc-like bHLH TFs. We isolated a gene (NtAn2) encoding a R2R3 MYB TF from developing tobacco flowers. NtAn2 shares high sequence homology with other known flavonoid-related MYB TFs and is mostly expressed in developing flowers. Constitutive ectopic expression of NtAn2 induces whole-plant anthocyanin production in tobacco and Arabidopsis. In transgenic tobacco and Arabidopsis expressing NtAn2, both subsets of early and late flavonoid pathway genes are up-regulated. Suppression of NtAn2 by RNAi in tobacco resulted in a white-flowered phenotype and the inhibition of the late pathway genes. Yeast two-hybrid assays demonstrated that NtAn2 can interact with five heterologous bHLH TFs known to induce anthocyanin synthesis in other species including maize, perilla, snapdragon and Arabidopsis. Bimolecular fluorescent complementation using split YFP demonstrated that NtAn2 interacts with Lc in tobacco cells and that the complex is localized to nuclei. Transient co-expression of NtAn2 and Lc or Arabidopsis TT8 in tobacco protoplasts activated the promoters of two key flavonoid pathway genes, chalcone synthase and dihydroflavonol reductase. These results suggest that NtAn2 is a key gene controlling anthocyanin production in reproductive tissues of tobacco.

Directed evolution of plant basic helix-loop-helix transcription factors for the improvement of transactivational properties.

Myc-RP from Perilla frutescens and Delila from Antirrhinum majus, two plant basic helix-loop-helix transcription factors (bHLH TFs) involved in the flavonoid biosynthetic pathway, have been used for the improvement of transactivational properties by directed evolution. Through two rounds of DNA shuffling, Myc-RP variants with up to 70-fold increase in transcriptional activities have been identified using a yeast transactivation system. In a tobacco protoplast transient expression assay, one of the most improved variants, M2-1, also shows significant increase of transactivation. The majority of resulting mutations (approximately 53%) are localized in the acidic (activation) domains of the improved Myc-RP variants. In variant M2-1, three of the four mutations (L301P/N354D/S401F) are in the acidic domain. The fourth mutation (K157M) is localized to a helix within the N-terminal interaction domain. Combinatorial site-directed mutagenesis reveals that, while the acidic domain mutations contribute modestly to the increase in activity, the K157M substitution is responsible for 80% of the improvement observed in variant M2-1. The transactivation activity of the K157M/N354D double mutant is equal to that of M2-1. These results suggest that the interaction domain plays a critical role in transactivation of these bHLH TFs. Delila variants have also been screened for increased activities toward the Arabidopsis chalcone synthase (CHS) promoter, a pathway promoter that responds weakly to the bHLH TFs. Variants with increased activity on the CHS promoter, while maintaining wildtype-level activities on the naturally responsive dihydroflavonol reductase promoter, have been obtained. This study demonstrates that functional properties of TFs can be modified by directed evolution.