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1.
Int J Mol Sci ; 23(15)2022 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-35955429

RESUMO

DNA methylation is an epigenetic modification of the genome involved in the regulation of gene expression and modulation of chromatin structure. Plant genomes are widely methylated, and the methylation generally occurs on the cytosine bases through the activity of specific enzymes called DNA methyltransferases. On the other hand, methylated DNA can also undergo demethylation through the action of demethylases. The methylation landscape is finely tuned and assumes a pivotal role in plant development and evolution. This review illustrates different molecular aspects of DNA methylation and some plant physiological processes influenced by this epigenetic modification in model species, crops, and ornamental plants such as orchids. In addition, this review aims to describe the relationship between the changes in plant DNA methylation levels and the response to biotic and abiotic stress. Finally, we discuss the possible evolutionary implications and biotechnological applications of DNA methylation.


Assuntos
Metilação de DNA , Regulação da Expressão Gênica de Plantas , DNA de Plantas/genética , DNA de Plantas/metabolismo , Epigênese Genética , Genoma de Planta , Plantas/genética , Plantas/metabolismo
2.
Plant Cell ; 34(10): 3899-3914, 2022 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-35775944

RESUMO

In eukaryotes, the STRUCTURAL MAINTENANCE OF CHROMOSOME 5/6 (SMC5/6) complex is critical to maintaining chromosomal structures around double-strand breaks (DSBs) in DNA damage repair. However, the recruitment mechanism of this conserved complex at DSBs remains unclear. In this study, using Arabidopsis thaliana as a model, we found that SMC5/6 localization at DSBs is dependent on the protein scaffold containing INVOLVED IN DE NOVO 2 (IDN2), CELL DIVISION CYCLE 5 (CDC5), and ALTERATION/DEFICIENCY IN ACTIVATION 2B (ADA2b), whose recruitment is further mediated by DNA-damage-induced RNAs (diRNAs) generated from DNA regions around DSBs. The physical interactions of protein components including SMC5-ADA2b, ADA2b-CDC5, and CDC5-IDN2 result in formation of the protein scaffold. Further analysis indicated that the DSB localization of IDN2 requires its RNA-binding activity and ARGONAUTE 2 (AGO2), indicating a role for the AGO2-diRNA complex in this process. Given that most of the components in the scaffold are conserved, the mechanism presented here, which connects SMC5/6 recruitment and small RNAs, will improve our understanding of DNA repair mechanisms in eukaryotes.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Reparo do DNA/genética , DNA de Plantas/metabolismo , RNA/genética , Fatores de Transcrição/metabolismo
3.
Cells ; 11(11)2022 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-35681443

RESUMO

Modifications of DNA nucleobases are present in all forms of life. The purpose of these modifications in eukaryotic cells, however, is not always clear. Although the role of 5-methylcytosine (m5C) in epigenetic regulation and the maintenance of stability in plant genomes is becoming better understood, knowledge pertaining to the origin and function of oxidized nucleobases is still scarce. The formation of 5-hydroxymetylcytosine (hm5C) in plant genomes is especially debatable. DNA modifications, functioning as regulatory factors or serving as DNA injury markers, may have an effect on DNA structure and the interaction of genomic DNA with proteins. Thus, these modifications can influence plant development and adaptation to environmental stress. Here, for the first time, the changes in DNA global levels of m5C, hm5C, and 8-oxo-7,8-dihydroguanine (8-oxoG) measured by ELISA have been documented in recalcitrant embryonic axes subjected to desiccation and accelerated aging. We demonstrated that tissue desiccation induces a similar trend in changes in the global level of hm5C and 8-oxoG, which may suggest that they both originate from the activity of reactive oxygen species (ROS). Our study supports the premise that m5C can serve as a marker of plant tissue viability whereas oxidized nucleobases, although indicating a cellular redox state, cannot.


Assuntos
Dessecação , Sementes , Dano ao DNA , DNA de Plantas/genética , DNA de Plantas/metabolismo , Epigênese Genética , Genômica , Sementes/metabolismo
4.
Science ; 375(6586): 1281-1286, 2022 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-35298257

RESUMO

The tail of replication-dependent histone H3.1 varies from that of replication-independent H3.3 at the amino acid located at position 31 in plants and animals, but no function has been assigned to this residue to demonstrate a unique and conserved role for H3.1 during replication. We found that TONSOKU (TSK/TONSL), which rescues broken replication forks, specifically interacts with H3.1 via recognition of alanine 31 by its tetratricopeptide repeat domain. Our results indicate that genomic instability in the absence of ATXR5/ATXR6-catalyzed histone H3 lysine 27 monomethylation in plants depends on H3.1, TSK, and DNA polymerase theta (Pol θ). This work reveals an H3.1-specific function during replication and a common strategy used in multicellular eukaryotes for regulating post-replicative chromatin maturation and TSK, which relies on histone monomethyltransferases and reading of the H3.1 variant.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Reparo do DNA , Replicação do DNA , DNA de Plantas/metabolismo , Histonas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Quebras de DNA de Cadeia Dupla , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Genoma de Planta , Instabilidade Genômica , Histonas/química , Lisina/metabolismo , Metilação , Metiltransferases/genética , Mutação , Domínios e Motivos de Interação entre Proteínas
5.
Sci Rep ; 12(1): 2688, 2022 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-35177666

RESUMO

Shoot development in maize begins when meristematic, non-pigmented cells at leaf base stop dividing and proceeds toward the expanded green cells of the leaf blade. During this transition, promitochondria and proplastids develop into mature organelles and their DNA becomes fragmented. Changes in glycation damage during organelle development were measured for protein and DNA, as well as the glycating agent methyl glyoxal and the glycation-defense protein DJ-1 (known as Park7 in humans). Maize seedlings were grown under normal, non-stressful conditions. Nonetheless, we found that glycation damage, as well as defenses against glycation, follow the same developmental pattern we found previously for reactive oxygen species (ROS): as damage increases, damage-defense measures decrease. In addition, light-grown leaves had more glycation and less DJ-1 compared to dark-grown leaves. The demise of maize organellar DNA during development may therefore be attributed to both oxidative and glycation damage that is not repaired. The coordination between oxidative and glycation damage, as well as damage-response from the nucleus is also discussed.


Assuntos
DNA de Plantas/metabolismo , Organelas/metabolismo , Proteínas de Plantas/metabolismo , Proteína Desglicase DJ-1/metabolismo , Plântula/crescimento & desenvolvimento , Zea mays/crescimento & desenvolvimento , DNA de Plantas/genética , Organelas/genética , Proteínas de Plantas/genética , Proteína Desglicase DJ-1/genética , Plântula/genética , Zea mays/genética
6.
Plant Cell ; 34(2): 852-866, 2022 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-34791445

RESUMO

Base excision repair and active DNA demethylation produce repair intermediates with DNA molecules blocked at the 3'-OH end by an aldehyde or phosphate group. However, both the physiological consequences of these accumulated single-strand DNAs break with 3'-blocked ends (DNA 3'-blocks) and the signaling pathways responding to unrepaired DNA 3'-blocks remain unclear in plants. Here, we investigated the effects of DNA 3'-blocks on plant development using the zinc finger DNA 3'-phosphoesterase (zdp) AP endonuclease2 (ape2) double mutant, in which 3'-blocking residues are poorly repaired. The accumulation of DNA 3'-blocked triggered diverse developmental defects that were dependent on the ATM and RAD3-related (ATR)-suppressor of gamma response 1 (SOG1) signaling module. SOG1 mutation rescued the developmental defects of zdp ape2 leaves by preventing cell endoreplication and promoting cell proliferation. However, SOG1 mutation caused intensive meristematic cell death in the radicle of zdp ape2 following germination, resulting in rapid termination of radicle growth. Notably, mutating FORMAMIDOPYRIMIDINE DNA GLYCOSYLASE (FPG) in zdp ape2 sog1 partially recovered its radicle growth, demonstrating that DNA 3'-blocks generated by FPG caused the meristematic defects. Surprisingly, despite lacking a functional radicle, zdp ape2 sog1 mutants compensated the lack of root growth by generating anchor roots having low levels of DNA damage response. Our results reveal dual roles of SOG1 in regulating root establishment when seeds germinate with excess DNA 3'-blocks.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Reparo do DNA/fisiologia , Fatores de Transcrição/metabolismo , Arabidopsis/citologia , Proteínas de Arabidopsis/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Morte Celular/genética , Proliferação de Células/genética , DNA de Plantas/genética , DNA de Plantas/metabolismo , DNA-Formamidopirimidina Glicosilase/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Regulação da Expressão Gênica de Plantas , Pleiotropia Genética , Germinação/genética , Meristema/citologia , Meristema/genética , Células Vegetais , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Sementes/fisiologia , Transdução de Sinais , Fatores de Transcrição/genética
7.
Plant Cell Rep ; 41(1): 175-194, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34623476

RESUMO

KEY MESSAGE: Combination of UBIQUITIN10 promoter-directed CAS9 and tRNA-gRNA complexes in gene-editing assay induces 80% mutant phenotype with a knockout of the four allelic copies in the T0 generation of allotetraploid tobaccos. While gene-editing methodologies, such as CRISPR-Cas9, have been developed and successfully used in many plant species, their use remains challenging, because they most often rely on stable or transient transgene expression. Regrettably, in all plant species, transformation causes epigenetic effects such as gene silencing and variable transgene expression. Here, UBIQUITIN10 promoters from several plant species were characterized and showed their capacity to direct high levels of transgene expression in transient and stable transformation assays, which in turn was used to improve the selection process of regenerated transformants. Furthermore, we compared various sgRNAs delivery systems and showed that the combination of UBIQUITIN10 promoters and tRNA-sgRNA complexes produced 80% mutant phenotype with a complete knockout of the four allelic copies, while the remaining 20% exhibited weaker phenotype, which suggested partial allelic knockout, in the T0 generation of the allotetraploid Nicotiana tabacum. These data provide valuable information to optimize future designs of gene editing constructs for plant research and crop improvement and open the way for valuable gene editing projects in non-model Solanaceae species.


Assuntos
DNA de Plantas/genética , Edição de Genes/métodos , Genoma de Planta , Proteínas de Plantas/genética , RNA de Plantas/genética , Tabaco/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas/genética , RNA Guia/genética , RNA Guia/metabolismo , RNA de Plantas/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Tetraploidia , Ubiquitinas/genética , Ubiquitinas/metabolismo
8.
Plant Physiol ; 187(4): 2763-2784, 2021 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-34890461

RESUMO

Jasmonic acid (JA) and ethylene (ET) signaling modulate plant defense against necrotrophic pathogens in a synergistic and interdependent manner, while JA and ET also have independent roles in certain processes, e.g. in responses to wounding and flooding, respectively. These hormone pathways lead to transcriptional reprogramming, which is a major part of plant immunity and requires the roles of transcription factors. ET response factors are responsible for the transcriptional regulation of JA/ET-responsive defense genes, of which ORA59 functions as a key regulator of this process and has been implicated in the JA-ET crosstalk. We previously demonstrated that Arabidopsis (Arabidopsis thaliana) GDSL LIPASE 1 (GLIP1) depends on ET for gene expression and pathogen resistance. Here, promoter analysis of GLIP1 revealed ERELEE4 as the critical cis-element for ET-responsive GLIP1 expression. In a yeast one-hybrid screening, ORA59 was isolated as a specific transcription factor that binds to the ERELEE4 element, in addition to the well-characterized GCC box. We found that ORA59 regulates JA/ET-responsive genes through direct binding to these elements in gene promoters. Notably, ORA59 exhibited a differential preference for GCC box and ERELEE4, depending on whether ORA59 activation is achieved by JA and ET, respectively. JA and ET induced ORA59 phosphorylation, which was required for both activity and specificity of ORA59. Furthermore, RNA-seq and virus-induced gene silencing analyses led to the identification of ORA59 target genes of distinct functional categories in JA and ET pathways. Our results provide insights into how ORA59 can generate specific patterns of gene expression dynamics through JA and ET hormone pathways.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Ciclopentanos/metabolismo , Etilenos/metabolismo , Oxilipinas/metabolismo , Imunidade Vegetal/genética , Fatores de Transcrição/genética , Arabidopsis/imunologia , Proteínas de Arabidopsis/metabolismo , DNA de Plantas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo
9.
Nat Commun ; 12(1): 7040, 2021 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-34857773

RESUMO

Phosphate, a key plant nutrient, is perceived through inositol polyphosphates (InsPs) by SPX domain-containing proteins. SPX1 an inhibit the PHR2 transcription factor to maintain Pi homeostasis. How SPX1 recognizes an InsP molecule and represses transcription activation by PHR2 remains unclear. Here we show that, upon binding InsP6, SPX1 can disrupt PHR2 dimers and form a 1:1 SPX1-PHR2 complex. The complex structure reveals that SPX1 helix α1 can impose a steric hindrance when interacting with the PHR2 dimer. By stabilizing helix α1, InsP6 allosterically decouples the PHR2 dimer and stabilizes the SPX1-PHR2 interaction. In doing so, InsP6 further allows SPX1 to engage with the PHR2 MYB domain and sterically block its interaction with DNA. Taken together, our results suggest that, upon sensing the surrogate signals of phosphate, SPX1 inhibits PHR2 via a dual mechanism that attenuates dimerization and DNA binding activities of PHR2.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , DNA de Plantas/química , Fosfatos de Inositol/metabolismo , Proteínas Nucleares/química , Oryza/metabolismo , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA de Plantas/genética , DNA de Plantas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Fosfatos de Inositol/química , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nutrientes/química , Nutrientes/metabolismo , Oryza/química , Oryza/genética , Plantas Geneticamente Modificadas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais
10.
Cells ; 10(12)2021 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-34943918

RESUMO

Experiments on Vicia faba root meristem cells exposed to 150 µM cadmium chloride (CdCl2) were undertaken to analyse epigenetic changes, mainly with respect to DNA replication stress. Histone modifications examined by means of immunofluorescence labeling included: (1) acetylation of histone H3 on lysine 56 (H3K56Ac), involved in transcription, S phase, and response to DNA damage during DNA biosynthesis; (2) dimethylation of histone H3 on lysine 79 (H3K79Me2), correlated with the replication initiation; (3) phosphorylation of histone H3 on threonine 45 (H3T45Ph), engaged in DNA synthesis and apoptosis. Moreover, immunostaining using specific antibodies against 5-MetC-modified DNA was used to determine the level of DNA methylation. A significant decrease in the level of H3K79Me2, noted in all phases of the CdCl2-treated interphase cell nuclei, was found to correspond with: (1) an increase in the mean number of intranuclear foci of H3K56Ac histones (observed mainly in S-phase), (2) a plethora of nuclear and nucleolar labeling patterns (combined with a general decrease in H3T45Ph), and (3) a decrease in DNA methylation. All these changes correlate well with a general viewpoint that DNA modifications and post-translational histone modifications play an important role in gene expression and plant development under cadmium-induced stress conditions.


Assuntos
Cádmio/toxicidade , Replicação do DNA/genética , Epigênese Genética , Meristema/citologia , Meristema/genética , Estresse Fisiológico/genética , Vicia faba/genética , 5-Metilcitosina/metabolismo , Acetilação/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Cromatina/metabolismo , Replicação do DNA/efeitos dos fármacos , DNA de Plantas/metabolismo , Epigênese Genética/efeitos dos fármacos , Histonas/metabolismo , Lisina/metabolismo , Meristema/efeitos dos fármacos , Metilação/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Vicia faba/citologia , Vicia faba/efeitos dos fármacos
11.
Science ; 374(6575): 1579-1586, 2021 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-34941388

RESUMO

DNA methylation affects gene expression and maintains genome integrity. The DNA-dependent RNA polymerase IV (Pol IV), together with the RNA-dependent RNA polymerase RDR2, produces double-stranded small interfering RNA precursors essential for establishing and maintaining DNA methylation in plants. We determined the cryo­electron microscopy structures of the Pol IV­RDR2 holoenzyme and the backtracked transcription elongation complex. These structures reveal that Pol IV and RDR2 form a complex with their active sites connected by an interpolymerase channel, through which the Pol IV­generated transcript is handed over to the RDR2 active site after being backtracked, where it is used as the template for double-stranded RNA (dsRNA) synthesis. Our results describe a 'backtracking-triggered RNA channeling' mechanism underlying dsRNA synthesis and also shed light on the evolutionary trajectory of eukaryotic RNA polymerases.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Arabidopsis/genética , RNA Polimerases Dirigidas por DNA/química , RNA de Cadeia Dupla/biossíntese , RNA de Plantas/biossíntese , RNA Polimerase Dependente de RNA/química , Motivos de Aminoácidos , Proteínas de Arabidopsis/metabolismo , Domínio Catalítico , Microscopia Crioeletrônica , Metilação de DNA , DNA de Plantas/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Holoenzimas/química , Modelos Moleculares , Complexos Multiproteicos/química , Conformação Proteica , Domínios Proteicos , RNA Polimerase II/química , RNA Interferente Pequeno/biossíntese , RNA Polimerase Dependente de RNA/metabolismo , Elongação da Transcrição Genética , Fatores de Transcrição/metabolismo
12.
Nat Commun ; 12(1): 6751, 2021 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-34799578

RESUMO

Single-stranded oligodeoxynucleotides (ssODNs) are widely used as DNA repair templates in CRISPR/Cas precision genome editing. However, the underlying mechanisms of single-strand templated DNA repair (SSTR) are inadequately understood, constraining rational improvements to precision editing. Here we study SSTR at CRISPR/Cas12a-induced DNA double-strand breaks (DSBs) in the eukaryotic model green microalga Chlamydomonas reinhardtii. We demonstrate that ssODNs physically incorporate into the genome during SSTR at Cas12a-induced DSBs. This process is genetically independent of the Rad51-dependent homologous recombination and Fanconi anemia pathways, is strongly antagonized by non-homologous end-joining, and is mediated almost entirely by the alternative end-joining enzyme polymerase θ. These findings suggest differences in SSTR between C. reinhardtii and animals. Our work illustrates the promising potentially of C. reinhardtii as a model organism for studying nuclear DNA repair.


Assuntos
Chlamydomonas reinhardtii/genética , Reparo do DNA por Junção de Extremidades , DNA de Plantas/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Quebras de DNA de Cadeia Dupla , DNA de Plantas/genética , DNA de Cadeia Simples/genética , DNA Polimerase Dirigida por DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Edição de Genes/métodos , Instabilidade Genômica , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , RNA Guia/metabolismo
13.
Nat Commun ; 12(1): 5976, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34645826

RESUMO

In plants, cytosine DNA methylations (5mCs) can happen in three sequence contexts as CpG, CHG, and CHH (where H = A, C, or T), which play different roles in the regulation of biological processes. Although long Nanopore reads are advantageous in the detection of 5mCs comparing to short-read bisulfite sequencing, existing methods can only detect 5mCs in the CpG context, which limits their application in plants. Here, we develop DeepSignal-plant, a deep learning tool to detect genome-wide 5mCs of all three contexts in plants from Nanopore reads. We sequence Arabidopsis thaliana and Oryza sativa using both Nanopore and bisulfite sequencing. We develop a denoising process for training models, which enables DeepSignal-plant to achieve high correlations with bisulfite sequencing for 5mC detection in all three contexts. Furthermore, DeepSignal-plant can profile more 5mC sites, which will help to provide a more complete understanding of epigenetic mechanisms of different biological processes.


Assuntos
Arabidopsis/genética , Citosina/metabolismo , DNA de Plantas/genética , Epigênese Genética , Genoma de Planta , Oryza/genética , Arabidopsis/metabolismo , Ilhas de CpG , Metilação de DNA , DNA de Plantas/metabolismo , Aprendizado Profundo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Nanoporos , Oryza/metabolismo , Análise de Sequência de DNA , Sulfitos/química
15.
Int J Mol Sci ; 22(17)2021 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-34502465

RESUMO

Due to their sessile lifestyle, plants are especially exposed to various stresses, including genotoxic stress, which results in altered genome integrity. Upon the detection of DNA damage, distinct cellular responses lead to cell cycle arrest and the induction of DNA repair mechanisms. Interestingly, it has been shown that some cell cycle regulators are not only required for meristem activity and plant development but are also key to cope with the occurrence of DNA lesions. In this review, we first summarize some important regulatory steps of the plant cell cycle and present a brief overview of the DNA damage response (DDR) mechanisms. Then, the role played by some cell cycle regulators at the interface between the cell cycle and DNA damage responses is discussed more specifically.


Assuntos
Ciclo Celular , Dano ao DNA , DNA de Plantas/metabolismo , Plantas/metabolismo , DNA de Plantas/genética , Plantas/genética
16.
J Mol Biol ; 433(22): 167269, 2021 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-34571015

RESUMO

VERNALIZATION1 (VRN1) is a transcriptional repressor involved in plant vernalization that undergoes liquid-liquid phase separation (LLPS) with DNA. The naturally occurring VRN1-like proteins contain two B3 DNA binding domains connected by an intrinsically disordered region (IDR). The IDR length in VRN1-like proteins has a broad distribution, while the charge segregation pattern is largely conserved. We studied the effect of IDR length and charge segregation on DNA-induced VRN1 phase separation. When only neutral residues (Pro-Ser repeats) were used, the phase separation behavior is sensitive to IDR length, changing from gel-like aggregates (L = 40) to liquid-like droplets (L = 100-120) and clear solution (L = 160). When a pair of continuous patches of positive and negative residues were added to the IDRs, all the VRN1 variants formed robust and durable droplets with DNA independent of the IDR length. To test how robust the system is, we introduced folded green fluorescent protein or the enzyme GPX4 into VRN1 variants with charge segregation in IDR, the resulting proteins form LLPS with DNA as well. Our study implies that VRN1-like proteins use conserved charge segregation pattern to retain functional LLPS during evolution, and demonstrates the possibility of using this system to design novel biosensors or bio-factories by introducing various functional modules.


Assuntos
Proteínas de Arabidopsis/química , DNA de Plantas/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Repressoras/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , DNA de Plantas/química , Difusão Dinâmica da Luz , Polarização de Fluorescência , Recuperação de Fluorescência Após Fotodegradação , Transferência Ressonante de Energia de Fluorescência , Proteínas Intrinsicamente Desordenadas/metabolismo , Simulação de Dinâmica Molecular , Domínios Proteicos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/química
17.
PLoS One ; 16(8): e0256373, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34428237

RESUMO

The family Arecaceae is distributed throughout tropical and subtropical regions of the world. Among the five subfamilies, Arecoideae is the most species-rich and still contains some ambiguous inter-generic relationships, such as those within subtribes Attaleinae and Bactridineae. The hypervariable regions of plastid genomes (plastomes) are interesting tools to clarify unresolved phylogenetic relationships. We sequenced and characterized the plastome of Bactris gasipaes (Bactridinae) and compared it with eight species from the three Cocoseae sub-tribes (Attaleinae, Bactridinae, and Elaeidinae) to perform comparative analysis and to identify hypervariable regions. The Bactris gasipaes plastome has 156,646 bp, with 113 unique genes. Among them, four genes have an alternative start codon (cemA, rps19, rpl2, and ndhD). Plastomes are highly conserved within tribe Cocoseae: 97.3% identity, length variation of ~2 kb, and a single ~4.5 kb inversion in Astrocaryum plastomes. The LSC/IR and IR/SSC junctions vary among the subtribes: in Bactridinae and Elaeidinae the rps19 gene is completely contained in the IR region; in the subtribe Attaleinae the rps19 gene is only partially contained in the IRs. The hypervariable regions selected according to sequence variation (SV%) and frequency of parsimony informative sites (PIS%) revealed plastome regions with great potential for molecular analysis. The ten regions with greatest SV% showed higher variation than the plastid molecular markers commonly used for phylogenetic analysis in palms. The phylogenetic trees based on the plastomes and the hypervariable regions (SV%) datasets had well-resolved relationships, with consistent topologies within tribe Cocoseae, and confirm the monophyly of the subtribes Bactridinae and Attaleinae.


Assuntos
Arecaceae/genética , Evolução Molecular , Plastídeos/genética , Arecaceae/classificação , Hibridização Genômica Comparativa , DNA de Plantas/química , DNA de Plantas/genética , DNA de Plantas/metabolismo , Genomas de Plastídeos , Filogenia , Plastídeos/classificação , Análise de Sequência de DNA
18.
Int J Biol Macromol ; 189: 72-83, 2021 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-34411617

RESUMO

The basic helix-loop-helix (bHLH) transcription factors play important roles in many processes such as plant growth, metabolism and response to biotic/abiotic stresses. Sunflower (Helianthus annuus) is a major oil crop, cultivated throughout the world. However, no systematic characterization of bHLH gene members in sunflower (HabHLH) and their functions involved in drought, cadmium tolerance and Orobanche cumana resistance has been reported yet. In this study, 183 HabHLH genes were identified and named according to their chromosomal locations. We classified these proteins into 21 subfamilies by phylogenetic tree analysis. Subsequently, DNA-binding patterns, sequence analysis, duplication analysis and gene structures were analyzed. All of the HabHLH genes were randomly distributed on 17 chromosomes, and 10 pairs of tandem duplicated genes and one pair of segmental duplicated genes were detected in the HabHLH family. Among the duplicated gene pairs, eight pairs of HabHLH genes suffer from positive selection. Moreover, qRT-PCR results revealed significant up-regulated expression of HabHLH024 gene in response to both abiotic (cadmium, drought) and biotic (Orobanche cumana) stresses, suggesting its important functions in response to different stresses. Therefore, HabHLH024 would be the potential candidate gene for the sunflower tolerance breeding.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Helianthus/genética , Helianthus/fisiologia , Estresse Fisiológico/genética , Motivos de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Cádmio/toxicidade , DNA de Plantas/metabolismo , Secas , Perfilação da Expressão Gênica , Genes de Plantas , Variação Genética , Helianthus/efeitos dos fármacos , Helianthus/microbiologia , Família Multigênica , Especificidade de Órgãos/genética , Orobanche/fisiologia , Filogenia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Ligação Proteica , Duplicações Segmentares Genômicas
19.
Plant J ; 108(2): 509-527, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34382710

RESUMO

Transition to the reproductive phase, inflorescence formation and flower development are crucial elements that ensure maximum reproductive success in a plant's life cycle. To understand the regulatory mechanisms underlying correct flower development in barley (Hordeum vulgare), we characterized the multiovary 5 (mov5.o) mutant. This mutant develops abnormal flowers that exhibit mosaic floral organs typified by multiple carpels at the total or partial expense of stamens. Genetic mapping positioned mov5 on the long arm of chromosome 2H, incorporating a region that encodes HvLFY, the barley orthologue of LEAFY from Arabidopsis. Sequencing revealed that, in mov5.o plants, HvLFY contains a single amino acid substitution in a highly conserved proline residue. CRISPR-mediated knockout of HvLFY replicated the mov5.o phenotype, suggesting that HvLFYmov5 represents a loss of function allele. In heterologous assays, the HvLFYmov5 polymorphism influenced protein-protein interactions and affinity for a putative binding site in the promoter of HvMADS58, a C-class MADS-box gene. Moreover, molecular analysis indicated that HvLFY interacts with HvUFO and regulates the expression of floral homeotic genes including HvMADS2, HvMADS4 and HvMADS16. Other distinct changes in expression differ from those reported in the rice LFY mutants apo2/rfl, suggesting that LFY function in the grasses is modulated in a species-specific manner. This pathway provides a key entry point for the study of LFY function and multiple ovary formation in barley, as well as cereal species in general.


Assuntos
Flores/crescimento & desenvolvimento , Hordeum/fisiologia , Proteínas de Plantas/genética , Substituição de Aminoácidos , Proteínas de Arabidopsis/genética , Sítios de Ligação , Mapeamento Cromossômico , Cromossomos de Plantas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA de Plantas/metabolismo , Flores/fisiologia , Regulação da Expressão Gênica de Plantas , Genes Homeobox , Hordeum/crescimento & desenvolvimento , Inflorescência/genética , Mutação , Proteínas de Plantas/metabolismo , Polimorfismo de Nucleotídeo Único , Fatores de Transcrição/genética
20.
DNA Res ; 28(3)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34254113

RESUMO

We have established a high-quality, chromosome-level genome assembly for the hexaploid common wheat cultivar 'Fielder', an American, soft, white, pastry-type wheat released in 1974 and known for its amenability to Agrobacterium tumefaciens-mediated transformation and genome editing. Accurate, long-read sequences were obtained using PacBio circular consensus sequencing with the HiFi approach. Sequence reads from 16 SMRT cells assembled using the hifiasm assembler produced assemblies with N50 greater than 20 Mb. We used the Omni-C chromosome conformation capture technique to order contigs into chromosome-level assemblies, resulting in 21 pseudomolecules with a cumulative size of 14.7 and 0.3 Gb of unanchored contigs. Mapping of published short reads from a transgenic wheat plant with an edited seed-dormancy gene, TaQsd1, identified four positions of transgene insertion into wheat chromosomes. Detection of guide RNA sequences in pseudomolecules provided candidates for off-target mutation induction. These results demonstrate the efficiency of chromosome-scale assembly using PacBio HiFi reads and their application in wheat genome-editing studies.


Assuntos
Edição de Genes , Genoma de Planta , Triticum/genética , Agrobacterium tumefaciens/metabolismo , Cromossomos de Plantas , DNA de Plantas/metabolismo , Análise de Sequência de DNA , Triticum/microbiologia
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