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.

Guard-cell phytochromes impact seedling photomorphogenesis and rosette leaf morphology.

Using a previously established transgenic approach to inactivate phytochrome chromophore synthesis in specific organs or tissues, we used a guard cell-specific promoter to induce phytochrome deficiencies in guard cells of Arabidopsis thaliana. Analyses of multiple homozygous lines depleted of phytochromes in stomatal guard cells indicated elongated hypocotyls specifically in red and far-red growth conditions. Furthermore, rosette leaves of adult plants with guard cell-specific phytochrome deficiencies showed enhanced serration compared to the wild-type Col-0 parent. Thus, we demonstrate that guard cell-localized phytochromes impact the inhibition of hypocotyl elongation, as well as leaf margin morphology of adult rosette leaves in A. thaliana.

Functional Antagonism of WRI1 and TCP20 Modulates GH3.3 Expression to Maintain Auxin Homeostasis in Roots.

Auxin is a well-studied phytohormone, vital for diverse plant developmental processes. The GH3 genes are one of the major auxin responsive genes, whose expression changes lead to modulation of plant development and auxin homeostasis. However, the transcriptional regulation of these GH3 genes remains largely unknown. WRI1 is an essential transcriptional regulator governing plant fatty acid biosynthesis. Recently, we identified that the expression of GH3.3 is increased in the roots of wri1-1 mutant. Nevertheless, in this study we found that AtWRI1 did not activate or repress the promoter of GH3.3 (proGH3.3) despite of its binding to proGH3.3. Cross-family transcription factor interactions play pivotal roles in plant gene regulatory networks. To explore the molecular mechanism by which WRI1 controls GH3.3 expression, we screened an Arabidopsis transcription factor library and identified TCP20 as a novel AtWRI1-interacting regulator. The interaction between AtWRI1 and TCP20 was further verified by several approaches. Importantly, we found that TCP20 directly regulates GH3.3 expression via binding to TCP binding element. Furthermore, AtWRI1 repressed the TCP20-mediated transactivation of proGH3.3. EMSAs demonstrated that AtWRI1 antagonized TCP20 from binding to proGH3.3. Collectively, we provide new insights that WRI1 attenuates GH3.3 expression through interaction with TCP20, highlighting a new mechanism that contributes to fine-tuning auxin homeostasis.

The surface morphology and dynamic impact properties with rebounding and splashing of water droplet on phase separation and breath figure assisted electrospinning films.

Electrospinning provides a versatile, efficient and low-cost method for the preparation of continuous nanofibres from various polymers. In this study, the polyhedral oligomeric silsesquioxanes (POSS) block copolymer was synthesized via atom transfer radical polymerization. The smooth fiber, porous fiber or hierarchically porous microspheres were prepared by electrospinning from POSS block copolymer, poly(vinylidene fluoride) (PVDF) and aluminium oxide (Al2O3). The influence of copolymer concentration, the ratio of the solvents, the diameter and concentration of the Al2O3 on the surface morphology were investigated. Porous fibers and porous microspheres were prepared by regulating the ratio of the solvents from the phase separation and breath figure methods. The dynamic behavior of the water droplet with the constant volume impacting on the electrospinning films were reported. The morphology evolution, restitution coefficient, the change of energy of the water droplets are examined. The droplet bounces several times on the superhydrophobic surface, while the droplet remains pinned and does not rebound when the contact angles was lower than 150°. On the other hand, the water droplets were splashed on the Al2O3 based electrospinning films. Finally, the mechanical properties of the electrospinning films were investigated.

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.

Wide-detector CT combined iterative reconstruction in pediatric low-dose scan of the paranasal sinus.

BACKGROUND: Computed tomography (CT) is considered a standard modality for imaging the paranasal sinus (PS), but increasingly radiation dose is of concern, especially in children. OBJECTIVE: This study aims to investigate the feasibility of using a 320-detector CT scanner with a 16 cm wide-detector combined with iterative reconstruction (IR) algorithm to further reduce radiation dose when scanning the PS. METHODS: A total of 90 children who underwent CT of the PS were randomly allocated into three groups namely, (1) the experimental group using low-dose wide-detector scan (n = 30, 9±4 years); (2) low-dose helical group (n = 30, 9±4 years); and (3) pediatric conventional group (n = 30, 8±4 years). Statistical software SPSS 19.0 was used for one-way ANOVA analysis of the general data (age, BMI), image quality, and radiation dose. Multiple comparisons of data without homogeneity of variance were analyzed by Bonferroni test and Tamhane's test. RESULTS: All patients underwent successful CT examinations. No significant differences in the general data and image quality evaluation were detected between three groups (all P values > 0.05). CTDIvol and DLP were 2.87 mGy and 32.58 mGy·cm in the experimental group, 4.92 mGy and 70.84 mGy·cm in the low-dose helical group, and 9.95 mGy and 131.83 mGy·cm in the conventional group, respectively, which were significantly different among these three groups as indicated by multiple comparisons (all P values < 0.05). In the experimental group, the effective radiation dose was 0.07 mSv, which was reduced by 76% and 56% comparing to the conventional group and the low-dose helical group, respectively. CONCLUSIONS: The 320-detector CT scanner equipped with the wide-detector combined with IR can further reduce radiation dose, while maintaining good image quality comparing to the low-dose helical or pediatric modes.

Application of 640-slice CT wide-detector volume scan in low-dose CT pulmonary angiography.

BACKGROUND: Computed tomography (CT) pulmonary angiography (CTPA) examination has been frequently applied in detecting suspected pulmonary embolism (PE). How to reduce radiation dose to patients is also of concern. OBJECTIVE: To assess the value of using 640-slice CT wide-detector volume scan with adaptive statistical iterative reconstruction (ASIR) algorithm in low-dose CTPA. METHODS: Fifty-eight patients who performed with CTPA were divided into two groups randomly. In the first experimental group (n = 30), ASIR combined with volume scan were performed on the patients, while in the second conventional group (n = 28), patients received ASIR combined with conventional spiral scan. General data including age and body mass index, image quality, pulmonary arterial phase, and radiation dose were analyzed by t test in the two groups. RESULTS: In both groups, all images revealed the 5-order or higher pulmonary arterial branches and fully met the needs for clinical diagnosis. There was no statistical difference in general data between the two groups. In terms of pulmonary phase accuracy, compared with the conventional group, images at pulmonary arterial phase could be captured more accurately in the experimental group. CTDI in the experimental group decreased by 30% compared with that in the conventional group. The actual radiation dose in the experimental group was 1.5 mSv, which is reduced by 53% compared to that in the conventional group. CONCLUSIONS: Compared with the conventional spiral scan, using 640-slice CT volume scan with ASIR in CTPA is more accurate in scanning phase and has lower radiation dose. There is no significant difference in image quality between the two groups.

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.

WRINKLED1 transcription factor: How much do we know about its regulatory mechanism?

Many plant species produce and build up triacylglycerol (TAG) in their seeds as a main resource to provide carbon and energy during seedling development. Plant seed oils are important not only for human diets but also as renewable feedstock of industrial uses. WRINKLED1 (WRI1), an APETALA2 (AP2) transcription factor, plays an essential role in the transcriptional regulation of TAG biosynthesis as WRI1 regulates the expression of key genes in the glycolytic and fatty acid biosynthetic pathways. Recent work has identified intrinsic structural disorder in WRI1 that may affect the stability of the protein. Furthermore, WRI1 activity is modulated by post-translational modifications and interacting partners. These progresses shed light on regulatory functions of WRI1 at the molecular levels, paving new paths to the use of WRI1 for bioengineering of TAG in plants.

The Arabidopsis WRINKLED1 transcription factor affects auxin homeostasis in roots.

WRINKLED1 (WRI1) is a key transcriptional regulator of fatty acid biosynthesis genes in diverse oil-containing tissues. Loss of function of Arabidopsis WRI1 leads to a reduction in the expression of genes for fatty acid biosynthesis and glycolysis, and concomitant strong reduction of seed oil content. The wri1-1 loss-of-function mutant shows reduced primary root growth and decreased acidification of the growth medium. The content of a conjugated form of the plant growth hormone auxin, indole-3-acetic acid (IAA)-Asp, was higher in wri1-1 plants compared with the wild-type. GH3.3, a gene encoding an enzyme involved in auxin degradation, displayed higher expression in the wri1-1 mutant. EMSAs demonstrated that AtWRI1 bound to the promoter of GH3.3. Specific AtWRI1-binding motifs were identified in the promoter of GH3.3. In addition, wri1-1 displayed decreased auxin transport. Expression of some PIN genes, which encode IAA carrier proteins, was reduced in wri1-1 plants as well. Correspondingly, AtWRI1 bound to the promoter regions of some PIN genes. It is well known that auxin exerts its maximum effects at a specific, optimal concentration in roots requiring a finely balanced auxin homeostasis. This process appears to be disrupted when the expression of WRI1 and in turn a subset of its target genes are misregulated, highlighting a role for WRI1 in root auxin homeostasis.

Dynamic effects and adhesion of water droplet impact on hydrophobic surfaces: bouncing or sticking.

This work reported the dynamic effects of water droplet impact on flat, porous and pincushion structure films of star shaped polyhedral oligomeric silsesquioxane (POSS) fluorinated acrylates, POSS-poly(trifluoroethyl methacrylate)8 (POSS-(PTFEMA)8) and POSS-(poly(trifluoroethyl methacrylate)-b-poly(methyl methacrylate))8 (POSS-(PTFEMA-b-PMMA)8), using the breath figure method. The porous and pincushion structure films with different surface chemical compositions were obtained by controlling the copolymer structure and temperature and by stripping of the surface. The water contact angles on the different films were measured, and the water droplets on the pincushion structure films when reversed at 45°, 90°, 135° and 180° were also studied. It was found that the pincushion structure films revealed a water adhesion ability. Furthermore, the water droplet impact behavior on these films was investigated. The morphology variations of water droplets, spreading diameter of the droplets, energy conversion, restitution coefficient and adhesion force were examined. Finally, the schematic illustration of water droplets under the static and dynamic states in contact with the pincushion and porous structure surfaces was proposed. It is critical to materialize various applications such as microdroplet transportation, soil erosion, spray painting, anti-icing surface and antifouling agents for textiles.

Correction: Genome, Functional Gene Annotation, and Nuclear Transformation of the Heterokont Oleaginous Alga Nannochloropsis oceanica CCMP1779.

[This corrects the article DOI: 10.1371/journal.pgen.1003064.].

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.