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1.
Food Chem ; 335: 127582, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-32739806

RESUMO

To improve genetically modified product labelling legislation and promote the development of genetically modified foods, the mass variations of genomic DNA and length distributions of DNA fragments in non-genetically and genetically modified soybean (Roundup Ready) and the variations in transgenic contents during soybean protein concentrate (SPC) and soybean protein isolate (SPI) preparation were monitored. The material masses throughout the process conformed to the law of mass conservation, and amounts of DNA recovered decreased with SPC and SPI preparation. The successive steps of ethanol extraction led to a decrease in the size distribution of the recovered DNA. For the LECTIN and CP4 EPSPS targets investigated, longer fragments (>800 bp) were more affected than smaller fragments (<200 bp), and both targets degraded slowly upon degradation into small fragments. DNA distribution and degradation thereby affected GMO quantification. After preparation procedures, the transgenic contents of SPC and SPI products were higher than that of raw soybean.


Assuntos
DNA de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Proteínas de Soja/metabolismo , Soja/genética , Proteínas de Soja/genética , Proteínas de Soja/isolamento & purificação
2.
Plant Mol Biol ; 104(6): 575-582, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33000386

RESUMO

KEY MESSAGE: In the ros1-defective mutant, DREB1A repression by the transgene-induced promoter methylation of ice1-1 became inheritable across generations even in the absence of the causative transgene NICE1. Transgene silencing (TGS) is a widely observed event during plant bioengineering, which is presented as a gradual decrease in ectopic gene expression across generations and occasionally coupled with endogenous gene silencing based on DNA sequence similarity. TGS is known to be established by guided DNA methylation machinery. However, the machinery underlying gene recovery from TGS has not been fully elucidated. We previously reported that in ice1-1 outcross descendants, the expressional repression and recovery of DREB1A/CBF3 were instantly achieved by a newly discovered NICE1 transgene, instead of the formerly proposed ice1-1 mutation in the ICE1 gene. The plants harboring NICE1 produced small RNAs targeting and causing the DREB1A promoter to be hypermethylated and silenced. To analyze the role of the plant-specific active DNA demethylase REPRESSOR OF SILENCING 1 (ROS1) in instant DREB1A recovery, we propagated the NICE1-segregating population upon ros1 dysfunction and evaluated the gene expression and DNA methylation levels of DREB1A through generations. Our results showed that the epigenetic DREB1A repression was substantially sustained in subsequent generations even without NICE1 and stably inherited across generations. Consistent with the gene expression results, only incomplete DNA methylation removal was detected in the same generations. These results indicate that a novel inheritable epiallele emerged by the ros1 dysfunction. Overall, our study reveals the important role of ROS1 in the inheritability of TGS-associated gene repression.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Proteínas Nucleares/fisiologia , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Metilação de DNA , DNA de Plantas/metabolismo , Padrões de Herança
3.
Nucleic Acids Res ; 48(17): 9637-9648, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32890394

RESUMO

The MADS transcription factors (TF), SEPALLATA3 (SEP3) and AGAMOUS (AG) are required for floral organ identity and floral meristem determinacy. While dimerization is obligatory for DNA binding, SEP3 and SEP3-AG also form tetrameric complexes. How homo and hetero-dimerization and tetramerization of MADS TFs affect genome-wide DNA-binding and gene regulation is not known. Using sequential DNA affinity purification sequencing (seq-DAP-seq), we determined genome-wide binding of SEP3 homomeric and SEP3-AG heteromeric complexes, including SEP3Δtet-AG, a complex with a SEP3 splice variant, SEP3Δtet, which is largely dimeric and SEP3-AG tetramer. SEP3 and SEP3-AG share numerous bound regions, however each complex bound unique sites, demonstrating that protein identity plays a role in DNA-binding. SEP3-AG and SEP3Δtet-AG share a similar genome-wide binding pattern; however the tetrameric form could access new sites and demonstrated a global increase in DNA-binding affinity. Tetramerization exhibited significant cooperative binding with preferential distances between two sites, allowing efficient binding to regions that are poorly recognized by dimeric SEP3Δtet-AG. By intersecting seq-DAP-seq with ChIP-seq and expression data, we identified unique target genes bound either in SEP3-AG seq-DAP-seq or in SEP3/AG ChIP-seq. Seq-DAP-seq is a versatile genome-wide technique and complements in vivo methods to identify putative direct regulatory targets.


Assuntos
Proteína AGAMOUS de Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Homeodomínio/metabolismo , Análise de Sequência de DNA/métodos , Fatores de Transcrição/metabolismo , Proteína AGAMOUS de Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sítios de Ligação , Proteínas de Transporte/genética , DNA de Plantas/genética , DNA de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Proteínas de Homeodomínio/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Multimerização Proteica , Fatores de Transcrição/genética
4.
Plant Mol Biol ; 104(4-5): 483-498, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32813232

RESUMO

The phytohormone ethylene is widely involved in many developmental processes and is a crucial regulator of defense responses against biotic and abiotic stresses in plants. Ethylene-responsive element binding protein, a member of the APETALA2/ethylene response factor (AP2/ERF) superfamily, is a transcription factor that regulates stress-responsive genes by recognizing a specific cis-acting element of target DNA. A previous study showed only the NMR structure of the AP2/ERF domain of AtERF100 in complex with a GCC box DNA motif. In this report, we determined the crystal structure of AtERF96 in complex with a GCC box at atomic resolution. We analyzed the binding residues of the conserved AP2/ERF domain in the DNA recognition sequence. In addition to the AP2/ERF domain, an N-terminal α-helix of AtERF96 participates in DNA interaction in the flanking region. We also demonstrated the structure of AtERF96 EDLL motif, a unique conserved motif in the group IX of AP2/ERF family, might involve in the transactivation of defense-related genes. Our study establishes the structural basis of the AtERF96 transcription factor in complex with the GCC box, as well as the DNA binding mechanisms of the N-terminal α-helix and AP2/ERF domain.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/química , Arabidopsis/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sítios de Ligação , Cristalografia por Raios X , DNA de Plantas/metabolismo , Modelos Moleculares , Mutação , Conformação Proteica , Protoplastos , Fatores de Transcrição/genética
5.
PLoS One ; 15(7): e0236273, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32722687

RESUMO

Creating a homologous restorer line for Ogura cytoplasmic male sterility (Ogu-CMS) in Brassica napus is meaningful for the wider application of Ogu-CMS system in rapeseed production. Previously, an independent development of a new Ogu-CMS restorer line (CLR650) was reported locally from crossing between Raphanobrassica (AACCRR, 2n = 56) and B. napus and a new version of Ogu CMS lines CLR6430 derived from CLR650 was characterized in this study. The results showed that the fertility restoration gene in CLR6430 presented a distorted segregation in different segregating populations. However, the majority of somatic cells from roots had a regular chromosome number (2n = 38) and no radish signal covered a whole chromosome was detected using GISH. Thirty-two specific markers derived from the introgressed radish fragments were developed based on the re-sequencing results. Unique radish insertions and differences between CLR6430 and R2000 were also identified through both radish-derived markers and PCR product sequences. Further investigations on the genetic behaviors, interactions between the fertility restoration and other traits and specific molecular markers to the introgression in CLR6430 were also conducted in this study. These results should provide the evidence of nucleotide differences between CLR6430 and R2000, and the specific markers will be helpful for breeding new Ogura restore lines in future.


Assuntos
Brassica napus/genética , Marcadores Genéticos/genética , Infertilidade das Plantas/genética , Brassica rapa/genética , Mapeamento Cromossômico , Cromossomos de Plantas , DNA de Plantas/química , DNA de Plantas/isolamento & purificação , DNA de Plantas/metabolismo , Repetições de Microssatélites/genética , Raphanus/genética
6.
Sci Rep ; 10(1): 12188, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32699274

RESUMO

The rose is one of the most important ornamental woody plants because of its extensive use and high economic value. Herein, we sequenced a complete chloroplast genome of the miniature rose variety Rosa 'Margo Koster' and performed comparative analyses with sequences previously published for other species in the Rosaceae family. The chloroplast genome of Rosa 'Margo Koster', with a size of 157,395 bp, has a circular quadripartite structure typical of angiosperm chloroplast genomes and contains a total of 81 protein-coding genes, 30 tRNA genes and 4 rRNA genes. Conjunction regions in the chloroplast genome of Rosa 'Margo Koster' were verified and manually corrected by Sanger sequencing. Comparative genome analysis showed that the IR contraction and expansion events resulted in rps19 and ycf1 pseudogenes. The phylogenetic analysis within the Rosa genus showed that Rosa 'Margo Koster' is closer to Rosa odorata than to other Rosa species. Additionally, we identified and screened highly divergent sequences and cpSSRs and compared their power to discriminate rose varieties by Sanger sequencing and capillary electrophoresis. The results showed that 15 cpSSRs are polymorphic, but their discriminating power is only moderate among a set of rose varieties. However, more than 150 single nucleotide variations (SNVs) were discovered in the flanking region of cpSSRs, and the results indicated that these SNVs have a higher divergence and stronger power for profiling rose varieties. These findings suggest that nucleotide mutations in the chloroplast genome may be an effective and powerful tool for rose variety discrimination and DNA profiling. These molecular markers in the chloroplast genome sequence of Rosa spp. will facilitate population and phylogenetic studies and other related studies of this species.


Assuntos
Cloroplastos/genética , Genoma de Cloroplastos , Rosa/genética , Hibridização Genômica Comparativa , DNA de Plantas/química , DNA de Plantas/genética , DNA de Plantas/metabolismo , Eletroforese Capilar , Genótipo , Repetições de Microssatélites/genética , Filogenia , Polimorfismo de Nucleotídeo Único , Rosa/classificação , Análise de Sequência de DNA
7.
PLoS One ; 15(6): e0234088, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32559183

RESUMO

Bacterial blight (BB) and fungal blast diseases are the major biotic constraints that limit rice productivity. To sustain yield improvement in rice, it is necessary to developed yield potential of the rice varieties by incorporation of biotic stress resistance genes. Tellahamsa is a well-adapted popular high yielding rice variety in Telangana state, India. However, the variety is highly susceptible to BB and blast. In this study, simultaneous stepwise transfer of genes through marker-assisted backcross breeding (MABB) strategy was used to introgress two major BB (Xa21 and xa13) and two major blast resistance genes (Pi54 and Pi1) into Tellahamsa. In each generation (from F1 to ICF3) foreground selection was done using gene-specific markers viz., pTA248 (Xa21), xa13prom (xa13), Pi54MAS (Pi54) and RM224 (Pi1). Two independent BC2F1 lines of Tellahamsa/ISM (Cross-I) and Tellahamsa/NLR145 (Cross-II) possessing 92% and 94% recurrent parent genome (RPG) respectively were intercrossed to develop ICF1-ICF3 generations. These gene pyramided lines were evaluated for key agro-morphological traits, quality, and resistance against blast at three different hotspot locations as well as BB at two locations. Two ICF3 gene pyramided lines viz., TH-625-159 and TH-625-491 possessing four genes exhibited a high level of resistance to BB and blast. In the future, these improved Tellahamsa lines could be developed as mega varieties for different agro-climatic zones and also as potential donors for different pre-breeding rice research.


Assuntos
Resistência à Doença/genética , Genoma de Planta , Oryza/genética , Doenças das Plantas/genética , DNA de Plantas/metabolismo , Grão Comestível/fisiologia , Marcadores Genéticos , Genótipo , Oryza/crescimento & desenvolvimento , Doenças das Plantas/microbiologia
8.
PLoS One ; 15(6): e0235073, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32584862

RESUMO

The 'Fuji' line includes many varieties with a similar genetic background and consistent inducement factors with epigenetic occurrence, thus it may be considered an ideal candidate for epigenetic research. In this study, 91 bud mutations of 'Fuji' apple were used as the test materials. Using the genetic variation within 'Fuji' as the control, the characteristics of epigenetic variation at different levels in both varieties and mutant groups were examined. The results showed that: (1) the global genomic DNA methylation level of the 91 bud mutants of 'Fuji' ranged from 29.120%-45.084%, with an average of 35.910%. Internal cytosine methylation was the main DNA methylation pattern. Regarding the variation of methylation patterns of 'Fuji' mutants, the vast majority of loci maintained the original methylation pattern existed in 'Fuji'. CHG methylation variation was the main type of variation; (2) the variation in methylation patterns between the mutant groups was greater than that of methylation levels. Among these patterns, the variation in CHG methylation patterns (including CHG hypermethylation and CHG demethylation) was expected to be dominant. The observed variation in methylation levels was more important in the Color mutant group; however, the variation in methylation patterns was more obvious in both the early maturation and Spur mutant groups. Moreover, the range of variation in the Early-maturation group was much wider than that in the Spur mutant group; (3) epigenetic diversity and genetic diversity were both low between the mutant groups. In the 'Fuji' mutant groups, there was few correlation between genetic and epigenetic variation, and epigenetic differentiation resulted in more loci with moderate or greater differentiation; (4) the purifying selection seemed to play a major role in the differentiation of different groups of 'Fuji' mutants (65.618%), but epigenetic diversity selection still occurred at nearly 35% of loci. Sixteen epigenetic outlier loci were detected.


Assuntos
Metilação de DNA , DNA de Plantas , Epigênese Genética , Loci Gênicos , Malus , Mutação , DNA de Plantas/genética , DNA de Plantas/metabolismo , Malus/genética , Malus/metabolismo
9.
Food Chem ; 326: 126986, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32407998

RESUMO

In the present work, a barcode-DNA analysis method is described for the detection of plant oil adulteration in milk and dairy products. The method relies on the fact that plant DNA should not be present in readily detectable amounts in a dairy product unless it contains undeclared plant material. Thus, a universal plant barcode is chosen as the target to be amplified from dairy samples. Accordingly, barcode PCR-CE (PCR-capillary electrophoresis) assays are described, which do not require preliminary information on the species source of the adulterant oil type. Two PCR-CE assays, one operating on the plastid trnL (UAA) intron and the other targeting its inner P6 loop in nested format, were shown to detect corn, soybean, rapeseed and sunflower oils in clarified butter, milk and yogurt. Both barcodes are robustly amplified with extremely conserved primers. While the intron provides the species discrimination ability, the P6 loop provides superior detection sensitivity.


Assuntos
DNA de Plantas/análise , Laticínios/análise , Eletroforese Capilar/métodos , Leite/química , Óleos Vegetais/química , Animais , Código de Barras de DNA Taxonômico , DNA de Plantas/genética , DNA de Plantas/metabolismo , Óleos Vegetais/metabolismo , Plastídeos/genética , Reação em Cadeia da Polimerase , Soja/genética , Iogurte/análise , Zea mays/genética
10.
Sci Rep ; 10(1): 8701, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32457375

RESUMO

DNA barcoding is a valuable tool to support species identification with broad applications from traditional taxonomy, ecology, forensics, food analysis, and environmental science. We introduce Microfluidic Enrichment Barcoding (MEBarcoding) for plant DNA Barcoding, a cost-effective method for high-throughput DNA barcoding. MEBarcoding uses the Fluidigm Access Array to simultaneously amplify targeted regions for 48 DNA samples and hundreds of PCR primer pairs (producing up to 23,040 PCR products) during a single thermal cycling protocol. As a proof of concept, we developed a microfluidic PCR workflow using the Fluidigm Access Array and Illumina MiSeq. We tested 96 samples for each of the four primary DNA barcode loci in plants: rbcL, matK, trnH-psbA, and ITS. This workflow was used to build a reference library for 78 families and 96 genera from all major plant lineages - many currently lacking in public databases. Our results show that this technique is an efficient alternative to traditional PCR and Sanger sequencing to generate large amounts of plant DNA barcodes and build more comprehensive barcode databases.


Assuntos
Código de Barras de DNA Taxonômico/métodos , DNA de Plantas/química , Plantas/genética , Cycadopsida/genética , DNA de Plantas/genética , DNA de Plantas/metabolismo , Magnoliopsida/genética , Microfluídica , Reação em Cadeia da Polimerase
11.
Mol Phylogenet Evol ; 150: 106845, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32360706

RESUMO

The subclass Corallinophycidae is the only group of red algae characterized by the presence of calcite crystals in their cell walls. Except for the Rhodogorgonales, the remaining orders - collectively called corallines - are diverse and widely distributed, having calcified cell walls and highly variable morphology. Corallines constitute the group with the richest fossil record among marine algae. In the present study, we investigate the evolutionary history of the subclass Corallinophycidae and provide a time-calibrated phylogeny to date the radiation of the crown group and its main lineages. We use a multi-locus dataset with an extensive taxon sampling and comprehensive collection of fossil records, carefully assigned to corallines, to reconstruct a time-calibrated phylogeny of this subclass. Our molecular clock analyses suggest that the onset of crown group diversification of Corallinophycidae started in the Lower Jurassic and sped up in the Lower Cretaceous. The divergence time of the oldest order Sporolithales is estimated in the Lower Cretaceous followed by the remaining orders. We discuss the long period of more than 300 million years between the early Paleozoic records attributed to the stem group of Corallinophycidae and the radiation of the crown group. Our inferred phylogeny yields three highly-supported suprageneric lineages for the order Corallinales; we confirm the family Mastophoraceae and amend circumscription of the families Corallinaceae and Lithophyllaceae. These three families are distinguished by a combination of vegetative and reproductive features. In light of the phylogeny, we discuss the evolutionary trends of eleven morphological characters. In addition, we also highlight homoplasious characters and selected autapomorphies emerging in particular taxa.


Assuntos
Rodófitas/classificação , Teorema de Bayes , Evolução Biológica , Parede Celular/química , DNA de Plantas/química , DNA de Plantas/metabolismo , Fósseis , Ligação Genética , Filogenia , Rodófitas/genética
12.
PLoS One ; 15(4): e0231973, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32343710

RESUMO

Potato psyllid (Bactericera cockerelli Sulc)-transmitted "Candidatus Liberibacter solanacearum" (Lso) has been negatively impacting the potato industry in the United States as well as other potato-producing countries. Lso has been linked to a condition known as zebra chip (ZC) that affects yield and quality of potato tubers. Efforts to find sources of resistance to ZC have primarily focused on greenhouse evaluations based on a single inoculation time prior to harvest. Plant response to infection, however, could be influenced by the developmental stage of the host plant, and ZC may continue to develop after harvest. The objectives of this study were to quantify Lso inoculation success, Lso titer, ZC severity and Lso development during storage in eight potato genotypes. These evaluations were conducted on plants infested with Lso-positive psyllids at 77, 12, and 4 days before vine removal (DBVR). The evaluated genotypes were categorized according to their relative resistance to Lso and tolerance to ZC symptoms. Lso inoculation success in the genotype family A07781, derived from Solanum chacoense, was lower than that of the susceptible control ('Russet Burbank'). A07781-4LB and A07781-3LB genotypes were characterized relatively resistant to the pathogen and highly tolerant to ZC symptoms, while A07781-10LB was categorized as susceptible to Lso but relatively tolerant to symptom expression. In stored potatoes, increase in Lso concentrations was observed for all infestation times. However, significantly higher Lso titer was detected in tubers infested 12 DBVR and the effect was similar across genotypes. Overall, the A07781 family can be considered as a promising source of resistance or tolerance to ZC.


Assuntos
Hemípteros/fisiologia , Doenças das Plantas/etiologia , Solanum tuberosum/genética , Animais , DNA de Plantas/isolamento & purificação , DNA de Plantas/metabolismo , Resistência à Doença , Suscetibilidade a Doenças , Genótipo , Doenças das Plantas/genética , Doenças das Plantas/parasitologia , Tubérculos/genética , Tubérculos/crescimento & desenvolvimento
13.
Plant Mol Biol ; 103(1-2): 225-234, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32140819

RESUMO

DNA N6-methyladenine (6 mA) is one of the most vital epigenetic modifications and involved in controlling the various gene expression levels. With the avalanche of DNA sequences generated in numerous databases, the accurate identification of 6 mA plays an essential role for understanding molecular mechanisms. Because the experimental approaches are time-consuming and costly, it is desirable to develop a computation model for rapidly and accurately identifying 6 mA. To the best of our knowledge, we first proposed a computational model named i6mA-Fuse to predict 6 mA sites from the Rosaceae genomes, especially in Rosa chinensis and Fragaria vesca. We implemented the five encoding schemes, i.e., mononucleotide binary, dinucleotide binary, k-space spectral nucleotide, k-mer, and electron-ion interaction pseudo potential compositions, to build the five, single-encoding random forest (RF) models. The i6mA-Fuse uses a linear regression model to combine the predicted probability scores of the five, single encoding-based RF models. The resultant species-specific i6mA-Fuse achieved remarkably high performances with AUCs of 0.982 and 0.978 and with MCCs of 0.869 and 0.858 on the independent datasets of Rosa chinensis and Fragaria vesca, respectively. In the F. vesca-specific i6mA-Fuse, the MBE and EIIP contributed to 75% and 25% of the total prediction; in the R. chinensis-specific i6mA-Fuse, Kmer, MBE, and EIIP contribute to 15%, 65%, and 20% of the total prediction. To assist high-throughput prediction for DNA 6 mA identification, the i6mA-Fuse is publicly accessible at https://kurata14.bio.kyutech.ac.jp/i6mA-Fuse/.


Assuntos
Adenina/análogos & derivados , DNA de Plantas/metabolismo , Rosaceae/metabolismo , Adenina/metabolismo , Algoritmos , Sítios de Ligação , Biologia Computacional , Conjuntos de Dados como Assunto , Aprendizado de Máquina , Modelos Genéticos , Rosaceae/genética
14.
Nat Commun ; 11(1): 1417, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32184398

RESUMO

Holliday junctions (HJs) are key DNA intermediates in genetic recombination and are eliminated by nuclease, termed resolvase, to ensure genome stability. HJ resolvases have been identified across all kingdoms of life, members of which exhibit sequence-dependent HJ resolution. However, the molecular basis of sequence selectivity remains largely unknown. Here, we present the chloroplast resolvase MOC1, which cleaves HJ in a cytosine-dependent manner. We determine the crystal structure of MOC1 with and without HJs. MOC1 exhibits an RNase H fold, belonging to the retroviral integrase family. MOC1 functions as a dimer, and the HJ is embedded into the basic cleft of the dimeric enzyme. We characterize a base recognition loop (BR loop) that protrudes into and opens the junction. Residues from the BR loop intercalate into the bases, disrupt the C-G base pairing at the crossover and recognize the cytosine, providing the molecular basis for sequence-dependent HJ resolution by a resolvase.


Assuntos
Arabidopsis/enzimologia , Cloroplastos/enzimologia , DNA Cruciforme/metabolismo , Oryza/enzimologia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Recombinases/química , Recombinases/metabolismo , Soja/enzimologia , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/metabolismo , Sequência de Bases , Cloroplastos/química , Cloroplastos/genética , DNA Cruciforme/química , DNA Cruciforme/genética , DNA de Plantas/química , DNA de Plantas/genética , DNA de Plantas/metabolismo , Oryza/química , Oryza/genética , Oryza/metabolismo , Recombinases/genética , Soja/química , Soja/genética , Soja/metabolismo
15.
Proc Natl Acad Sci U S A ; 117(12): 6901-6909, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32165537

RESUMO

The Evening Complex (EC), composed of the DNA binding protein LUX ARRHYTHMO (LUX) and two additional proteins EARLY FLOWERING 3 (ELF3) and ELF4, is a transcriptional repressor complex and a core component of the plant circadian clock. In addition to maintaining oscillations in clock gene expression, the EC also participates in temperature and light entrainment, acting as an important environmental sensor and conveying this information to growth and developmental pathways. However, the molecular basis for EC DNA binding specificity and temperature-dependent activity were not known. Here, we solved the structure of the DNA binding domain of LUX in complex with DNA. Residues critical for high-affinity binding and direct base readout were determined and tested via site-directed mutagenesis in vitro and in vivo. Using extensive in vitro DNA binding assays of LUX alone and in complex with ELF3 and ELF4, we demonstrate that, while LUX alone binds DNA with high affinity, the LUX-ELF3 complex is a relatively poor binder of DNA. ELF4 restores binding to the complex. In vitro, the full EC is able to act as a direct thermosensor, with stronger DNA binding at 4 °C and weaker binding at 27 °C. In addition, an excess of ELF4 is able to restore EC binding even at 27 °C. Taken together, these data suggest that ELF4 is a key modulator of thermosensitive EC activity.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Ritmo Circadiano , DNA de Plantas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Arabidopsis/genética , Proteínas de Arabidopsis/genética , DNA de Plantas/genética , Proteínas de Ligação a DNA/genética
16.
ACS Synth Biol ; 9(4): 864-882, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32163700

RESUMO

We present the OpenPlant toolkit, a set of interlinked resources and techniques to develop Marchantia as testbed for bioengineering in plants. Marchantia is a liverwort, a simple plant with an open form of development that allows direct visualization of gene expression and dynamics of cellular growth in living tissues. We describe new techniques for simple and efficient axenic propagation and maintenance of Marchantia lines with no requirement for glasshouse facilities. Marchantia plants spontaneously produce clonal propagules within a few weeks of regeneration, and lines can be amplified million-fold in a single generation by induction of the sexual phase of growth, crossing, and harvesting of progeny spores. The plant has a simple morphology and genome with reduced gene redundancy, and the dominant phase of its life cycle is haploid, making genetic analysis easier. We have built robust Loop assembly vector systems for nuclear and chloroplast transformation and genome editing. These have provided the basis for building and testing a modular library of standardized DNA elements with highly desirable properties. We have screened transcriptomic data to identify a range of candidate genes, extracted putative promoter sequences, and tested them in vivo to identify new constitutive promoter elements. The resources have been combined into a toolkit for plant bioengineering that is accessible for laboratories without access to traditional facilities for plant biology research. The toolkit is being made available under the terms of the OpenMTA and will facilitate the establishment of common standards and the use of this simple plant as testbed for synthetic biology.


Assuntos
Edição de Genes/métodos , Regulação da Expressão Gênica de Plantas/genética , Marchantia , Software , Biologia Sintética/métodos , Cloroplastos/genética , DNA de Plantas/genética , DNA de Plantas/metabolismo , Genes de Plantas/genética , Marchantia/genética , Marchantia/crescimento & desenvolvimento , Marchantia/fisiologia , Transcriptoma/genética
17.
PLoS One ; 15(2): e0228776, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32032368

RESUMO

The Mediterranean Basin is a biodiversity hotspot, where islands play a key role because of their high biological diversity, degree of endemicity and human pressure. One of these islands, Sardinia, is a good evolutionary laboratory, especially for the study of complex genera, such as Centaurea. In particular, endemic species of Centaurea sect. Centaurea from Sardinia provides an interesting case study of plant evolution on continental islands. We attempted to clarify the processes leading to the diversification of Centaurea species on Sardinia using bi-parentally inherited nuclear markers and maternally inherited plastid markers. Our plastid results revealed the presence of five lineages of sect. Centaurea on the island. Three of them were defined as three species: C. ferulacea, C. filiformis and C. horrida. The other two lineages highlighted the complex evolutionary history of the two polyploids C. corensis and C. magistrorum. Multiple colonization events from the mainland involving the C. deusta and C. paniculata lineages among others, have led to the diversity of sect. Centaurea on Sardinia. One colonization event likely followed a southern path via the land connection between the mainland, the Calabrian Plate and Sardinia. A second pathway likely followed a northern connection, probably through the Tuscan Archipelago. Implications of these findings on conservation efforts for Centaurea endemics on Sardinia are also discussed.


Assuntos
Evolução Biológica , Centaurea/crescimento & desenvolvimento , Centaurea/classificação , Centaurea/genética , DNA de Plantas/química , DNA de Plantas/isolamento & purificação , DNA de Plantas/metabolismo , Ligação Genética , Ilhas , Itália , Filogenia , Plastídeos/genética , Poliploidia
18.
Mutat Res ; 819-820: 111689, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32004947

RESUMO

Because of their sessile lifestyle, plants are inescapably exposed to various kinds of environmental stresses throughout their lifetime. Therefore, to regulate their growth and development, plants constantly monitor the environmental signals and respond appropriately. However, these environmental stress factors, along with some endogenous metabolites, generated in response to environmental stress factors often induce various forms of DNA damage in plants and thus promote genome instability. To maintain the genomic integrity, plants have developed an extensive, sophisticated and coordinated cellular signaling mechanism known as DNA damage response or DDR. DDR evokes a signaling process which initiates with the sensing of DNA damage and followed by the subsequent activation of downstream pathways in many directions to repair and eliminate the harmful effects of DNA damages. SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), one of the newly identified components of DDR in plant genome, appears to play central role in this signaling network. SOG1 is a member of NAC [NO APICAL MERISTEM (NAM), ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR (ATAF), CUP-SHAPED COTYLEDON (CUC)] domain family of transcription factors and involved in a diverse array of function in plants, encompassing transcriptional response to DNA damage, cell cycle checkpoint functions, ATAXIA-TELANGIECTASIA-MUTATED (ATM) or ATAXIA TELANGIECTASIA AND RAD3-RELATED (ATR) mediated activation of DNA damage response and repair, functioning in programmed cell death and regulation of induction of endoreduplication. Although most of the functional studies on SOG1 have been reported in Arabidopsis, some recent reports have indicated diverse functions of SOG1 in various other plant species, including Glycine max, Medicago truncatula, Sorghum bicolour, Oryza sativa and Zea mays, respectively. The remarkable functional diversity shown by SOG1 protein indicates its multitasking capacity. In this review, we integrate information mainly related to functional aspects of SOG1 in the context of DDR in plants. Considering the important role of SOG1 in DDR and its functional diversity, in-depth functional study of this crucial regulatory protein can provide further potential information on genome stability maintenance mechanism in plants in the context of changing environmental condition.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Reparo do DNA , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Fatores de Transcrição/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Dano ao DNA , DNA de Plantas/metabolismo , Endorreduplicação , Redes Reguladoras de Genes , Instabilidade Genômica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo
19.
Plant Mol Biol ; 103(1-2): 33-50, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32048207

RESUMO

KEY MESSAGE: The Taguchi method and metAFLP analysis were used to optimise barley regenerants towards maximum and minimum levels of tissue culture-induced variation. The subtle effects of symmetric and asymmetric methylation changes in regenerants were identified. Plant tissue cultures (PTCs) provide researchers with unique materials that accelerate the development of new breeding cultivars and facilitate studies on off-type regenerants. The emerging variability of regenerants derived from PTCs may have both genetic and epigenetic origins, and may be desirable or degrade the value of regenerated plants. Thus, it is crucial to determine how the PTC variation level can be controlled. The easiest way to manipulate total tissue culture-induced variation (TTCIV) is to utilise appropriate stress factors and suitable medium components. This study describes the optimisation of in vitro tissue culture-induced variation in plant regenerants derived from barley anther culture, and maximizes and minimizes regenerant variation compared with the source explants. The approach relied on methylation amplified fragment length polymorphism (metAFLP)-derived TTCIV characteristics, which were evaluated in regenerants derived under distinct tissue culture conditions and analysed via Taguchi statistics. The factors that may trigger TTCIV included CuSO4, AgNO3 and the total time spent on the induction medium. The donor plants prepared for regeneration purposes had 5.75% and 2.01% polymorphic metAFLP loci with methylation and sequence changes, respectively. The level of TTCIV (as the sum of all metAFLP characteristics analyzed) identified in optimisation and verification experiments reached 7.51 and 10.46%, respectively. In the trial designed to produce a minimum number of differences between donor and regenerant plants, CuSO4 and AgNO3 were more crucial than time, which was not a significant factor. In the trial designed to produce a maximum number of differences between donor and regenerant plants, all factors had comparable impact on variation. The Taguchi method reduced the time required for experimental trials compared with a grid method and suggested that medium modifications were required to control regenerant variation. Finally, the effects of symmetric and asymmetric methylation changes on regenerants were identified using novel aspects of the metAFLP method developed for this analysis.


Assuntos
Hordeum/fisiologia , Técnicas de Cultura de Tecidos , Metilação de DNA , DNA de Plantas/metabolismo , Variação Genética , Hordeum/genética , Regeneração
20.
Plant Cell ; 32(4): 1240-1269, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32001503

RESUMO

COMPROMISED HYDROLYSIS OF TRIACYLGLYCEROLS7 (CHT7) in Chlamydomonas (Chlamydomonas reinhardtii) was previously shown to affect the transcription of a subset of genes during nitrogen (N)-replete growth and following N refeeding. Here, we show that an extensive derepression of genes involved in DNA metabolism and cell cycle-related processes, as well as downregulation of genes encoding oxidoreductases and nutrient transporters, occurs in the cht7 mutant during N deprivation. Cellular mutant phenotypes are consistent with the observed transcriptome misregulation, as cht7 cells fail to properly arrest growth, nuclear replication, and cell division following N deprivation. Reduction in cht7 colony formation following N refeeding is explained by its compromised viability during N deprivation and by the occurrence of abortive divisions during N refeeding. Surprisingly, the largely unstructured C-terminal half of CHT7 with predicted protein binding domains, but not the canonical CXC DNA binding domain, is essential for the ability of CHT7 to form stable complexes and reverse the cellular phenotypes and transcription levels in the cht7 mutant. Hence, although lacking the presumed DNA binding domain, CHT7 modulates the expression of cell cycle genes in response to N availability, which is essential for establishing an effective quiescent state and the coordinated resumption of growth following N refeeding.


Assuntos
Ciclo Celular/genética , Chlamydomonas/citologia , Chlamydomonas/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Sequência de Aminoácidos , Biomarcadores/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Rastreamento de Células , DNA de Plantas/metabolismo , Meiose/genética , Modelos Biológicos , Mutação/genética , Nitrogênio/farmacologia , Fenótipo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Deleção de Sequência , Transcriptoma/genética
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