RESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica de Plantas , Ácidos Graxos/metabolismo , Óleos de Plantas/metabolismo , Sementes/genética , Sementes/metabolismo , Plantas Geneticamente Modificadas/metabolismoRESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Helianthus , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica de Plantas , Helianthus/genética , Helianthus/metabolismo , Óleos de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Sementes/genética , Sementes/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Ácidos Graxos/biossíntese , Sementes/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis , Sementes/crescimento & desenvolvimento , NicotianaRESUMO
[This corrects the article DOI: 10.1371/journal.pgen.1003064.].
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Óleos de Plantas , Sementes/genética , Sementes/metabolismo , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas/metabolismoRESUMO
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.
Assuntos
Seios Paranasais/diagnóstico por imagem , Doses de Radiação , Interpretação de Imagem Radiográfica Assistida por Computador/métodos , Tomografia Computadorizada por Raios X/instrumentação , Adolescente , Algoritmos , Criança , Pré-Escolar , Humanos , Proteção Radiológica , Tomografia Computadorizada por Raios X/métodosRESUMO
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 (nâ=â30), ASIR combined with volume scan were performed on the patients, while in the second conventional group (nâ=â28), 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.
Assuntos
Angiografia por Tomografia Computadorizada/métodos , Artéria Pulmonar/diagnóstico por imagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Angiografia por Tomografia Computadorizada/instrumentação , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Embolia Pulmonar/diagnóstico por imagem , Doses de RadiaçãoRESUMO
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.
Assuntos
Proteínas 14-3-3/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Óleos de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Sementes/metabolismo , Fatores de Transcrição/metabolismo , Proteínas 14-3-3/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Plantas Geneticamente Modificadas/genética , Ligação Proteica , Estabilidade Proteica , Sementes/genética , Nicotiana/genética , Nicotiana/metabolismo , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Raízes de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sítios de Ligação , Transporte Biológico/genética , Regulação da Expressão Gênica de Plantas , Homeostase , Proteínas de Membrana Transportadoras/genética , Mutação , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Óleos de Plantas/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Dados de Sequência Molecular , Mutação , Fosforilação , Plantas Geneticamente Modificadas , Estabilidade Proteica , Estrutura Terciária de Proteína , Nicotiana/genética , Fatores de Transcrição/genéticaRESUMO
Triacylglycerol (TAG) is a storage lipid used for food purposes and as a renewable feedstock for biodiesel production. WRINKLED1 (WRI1) is a transcription factor that governs fatty acid (FA) synthesis and, indirectly, TAG accumulation in oil-storing plant tissues, and its ectopic expression has led to TAG accumulation in vegetative tissues of different dicotyledonous plants. The ectopic expression of BdWRI1 in the grass Brachypodium distachyon induced the transcription of predicted genes involved in glycolysis and FA biosynthesis, and TAG content was increased up to 32.5-fold in 8-week-old leaf blades. However, the ectopic expression of BdWRI1 also caused cell death in leaves, which has not been observed previously in dicotyledonous plants such as Arabidopsis (Arabidopsis thaliana). Lipid analysis indicated that the free FA content was 2-fold elevated in BdWRI1-expressing leaf blades of B. distachyon. The transcription of predicted genes involved in ß-oxidation was induced. In addition, linoleic FA treatment caused cell death in B. distachyon leaf blades, an effect that was reversed by the addition of the FA biosynthesis inhibitor cerulenin. Taken together, ectopic expression of BdWRI1 in B. distachyon enhances FA biosynthesis and TAG accumulation in leaves, as expected, but also leads to increased free FA content, which has cytotoxic effects leading to cell death. Thus, while WRI appears to ubiquitously affect FA biosynthesis and TAG accumulation in diverse plants, its ectopic expression can lead to undesired side effects depending on the context of the specific lipid metabolism of the respective plant species.
Assuntos
Brachypodium/fisiologia , Ácidos Graxos/biossíntese , Óleos de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Brachypodium/genética , Expressão Ectópica do Gene , Regulação da Expressão Gênica de Plantas , Homeostase , Lipídeos/análise , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sementes/genética , Sementes/fisiologia , Fatores de Transcrição/genética , Triglicerídeos/metabolismoRESUMO
The maize R2R3-MYB regulator C1 cooperates with the basic helix-loop-helix (bHLH) factor R to activate the expression of anthocyanin biosynthetic genes coordinately. As is the case for other bHLH factors, R harbors several protein-protein interaction domains. Here we show that not the classical but rather a briefly extended R bHLH region forms homodimers that bind canonical G-box DNA motifs. This bHLH DNA-binding activity is abolished if the C-terminal ACT (aspartokinase, chorismate, and TyrA) domain is licensed to homodimerize. Then the bHLH remains in the monomeric form, allowing it to interact with R-interacting factor 1 (RIF1). In this configuration, the R-RIF1 complex is recruited to the promoters of a subset of anthocyanin biosynthetic genes, such as A1, through the interaction with its MYB partner C1. If, however, the ACT domain remains monomeric, the bHLH region dimerizes and binds to G-boxes present in several anthocyanin genes, such as Bz1. Our results provide a mechanism by which a dimerization domain in a bHLH factor behaves as a switch that permits distinct configurations of a regulatory complex to be tethered to different promoters. Such a combinatorial gene regulatory framework provides one mechanism by which genes lacking obviously conserved cis-regulatory elements are regulated coordinately.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Vias Biossintéticas/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Modelos Moleculares , Proteínas Nucleares/química , Proteínas de Plantas/química , Zea mays/química , Antocianinas/biossíntese , Vias Biossintéticas/genética , Imunoprecipitação da Cromatina , Dimerização , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica de Plantas/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogen-depleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica-specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus.
Assuntos
Genoma , Anotação de Sequência Molecular , Estramenópilas/genética , Sequência de Bases , Genômica , Nitrogênio/administração & dosagem , Nitrogênio/metabolismo , Análise de Sequência de DNA , Análise de Sequência de RNA/métodos , Especificidade da Espécie , Estramenópilas/crescimento & desenvolvimento , Transformação GenéticaRESUMO
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.
RESUMO
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.
RESUMO
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.
Assuntos
Regulação da Expressão Gênica de Plantas , Óleos de Plantas , Plantas , Ácidos Graxos/metabolismo , Óleos de Plantas/metabolismo , Plantas/genética , Plantas/metabolismo , Sementes/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Triglicerídeos/metabolismoRESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Humanos , Óleos de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Flores/genética , Nicotiana/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Antocianinas/biossíntese , Arabidopsis/genética , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Flores/citologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genoma de Planta/genética , Redes e Vias Metabólicas/genética , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Fenótipo , Filogenia , Pigmentação/genética , Proteínas de Plantas/química , Plantas Geneticamente Modificadas , Propanóis/metabolismo , Ligação Proteica , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Supressão Genética , Nicotiana/citologia , Regulação para Cima/genéticaRESUMO
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.